PATENT ABSTRACT
The present disclosure relates to a method for obtaining human brain-like endothelial cells by contacting a population of cells isolated from stem cells with a differentiation medium to obtain endothelial cells and co-culturing said endothelial cells with pericytes, with cells of the neurovascular unit or with a pericytes conditioned medium, to obtain brain-like endothelial cells. The present disclosure also relates to the use of the brain-like endothelial cells as an in vitro model of human blood-brain barrier and a kit for measuring blood-brain barrier permeability of a substance, comprising in vitro human endothelial cells.

PATENT DESCRIPTION
TECHNICAL FIELD 
       [0001]    The present disclosure relates to a method to generate a population of endothelial cells showing a brain endothelial cells-like phenotype. 
         [0002]    The present disclosure further relates to a population of endothelial cells, said cells showing phenotype similar to brain endothelial cells. 
         [0003]    The present disclosure also relates to a stable and reproducible in vitro human brain-blood barrier model. 
       BACKGROUND 
       [0004]    Blood-brain barrier (BBB) models can provide a valuable tool for studying mechanistic aspects related to the transport of drugs at the brain, as well as biological and pathological processes related to the BBB (Cecchelli, R., et al. Modelling of the blood-brain barrier in drug discovery and development; Nat Rev Drug Discov 6, 650-661 (2007)). Although several in vitro models were established using murine and bovine cells, the establishment of a stable human BBB model is very important to account for differences between species (Cecchelli, R., et al. Modelling of the blood-brain barrier in drug discovery and development; Nat Rev Drug Discov 6, 650-661 (2007)). Primary human brain endothelial cells (hBECs) and immortalized human cells have been used as in vitro models; however, several issues prevent their general use including constraints in obtaining human tissue, loss of hBEC phenotype during immortalized cell culture, or lack of important tight junctions and low TEER values as shown in human cell lines (Weksler, B. B., et al. Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19, 1872-1874 (2005); Sano, Y., et al. Establishment of a new conditionally immortalized human brain microvascular endothelial cell line retaining an in vivo blood-brain barrier function. J Cell Physiol 225, 519-528 (2010)). Recently, hBECs have been differentiated from induced pluripotent stem cells (iPSCs); however, the low stability and high variability in the BBB system formed by different iPSC lines might preclude its general use (Lippmann, E. S., et al. Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells. Nat Biotechnol (2012)). 
         [0005]    For example, document WO/2006/056879 A1 relates to an immortalized human brain endothelial cell line that is useful as an in vitro model of the blood brain barrier. The document describes the generation of an immortalized human brain endothelial cell line (hCMEC/D3). The cell line was derived from primary brain endothelial cells transfected with a lentiviral vector system leading to the production of telomerase and SV40 Large T antigen. The cell line retained morphological characteristics of primary brain endothelial cells and expressed specific brain endothelial markers and cell surface adhesion molecules. Furthermore, the cell line expressed chemokine receptors and ATP binding cassette (ABC)-transporters. However, the expression level of ABCA2, MDR1, MRP4, BCRP, GUT1, 4F2hc, MCT1 and insulin receptor is 4 times lower in hCMEC/D3 cell line than in human brain microvessels (Ohtsoki et al., Molecular Pharmaceutics 2013, 10(1), 289-296). Furthermore, the cells show deficiency in typical and important brain endothelial properties such as low TEER value and relative high permeability towards small tracer molecules indicating paracellular leakiness and suboptimal formation of tight junctions. 
         [0006]    Document WO/2007/072953 A1 intends to provide a screening system for a drug which passes the blood-brain barrier and acts on the center, a drug which acts on the blood-brain barrier per se, or a drug which is not expected to act in the center but migrates into the brain. This in vitro BBB model is formed by a co-culture of primary brain endothelial cells, pericytes and astrocytes in a three-dimensional culture device. The invention is not related to the use of human brain endothelial cells and therefore does not take into account with inter-species differences in terms of metabolism and physiology. Furthermore, even if a human BBB model could be proposed, it requires the isolation of human brain endothelial cells from an autopsy tissue or freshly resected brain specimens derived from brain tumor or epilepsy patients. The issue here is that brain endotheial cells are not available in enough number for this purpose, and do not have the enough stability to act as a reliable in vitro BBB model. 
         [0007]    Document US/2012/0015395 A1 describes a method for producing brain specific endothelial cells, preferably comprising the steps of growing human pluripotent stem cells inducing differentiation of the cells by culturing the cells in unconditioned medium, and further expanding the endothelial cells in endothelial cell medium, wherein the expanded cells are GLUT-1 + , PECAM-1 + , claudin-5 + , occludin + , ZO-1 + , and p-glycoprotein + . The invention also claimed a method comprising the step of co-culturing the cells with a cell type selected from the group of astrocytes, neural progenitor cells, and pericytes. The brain endothelial cells derived from human pluripotent stem cells yielded TEER with an average value of 860±260 Ωcm2 (Lippmann et. al., Nature Biotechnology 2012). Yet, the TEER values fluctuated over time. For example, the TEER values in a co-culture of brain endothelial cells with astrocytes changed 200% during the first 50 h. Furthermore, it is unclear the stability of the system overtime and its reproducibility. 
         [0008]    Document WO/2007/140340 A2 related to methods for providing CD34+ cells from embryoid bodies and stimulating these cells to give rise to endothelial-like and/or smooth muscle-like cells. However, the object of the invention was not linked to the specification of the endothelial cells into brain endothelial cells. 
       SUMMARY 
     Definitions 
       [0009]    “CD34 +  cells” refers to cells expressing CD34 antigen. This antigen is a single-chain transmembrane glycoprotein expressed in several cells including human hematopoietic stem and progenitor cells, vascular endothelial cells, embryonic fibroblasts and some cells in fetal and adult nervous tissue. 
         [0010]    “Brain-like endothelial cells” refers to cells that share properties (gene, protein and functional) of fully functional brain endothelial cells, including the expression of at least one of the following markers, low density lipoprotein receptor, insulin receptor, leptin receptor, transferrin receptor, receptor for advanced glycation endproducts, retinol binding protein, SLC7A5, SLC2A1, SLC38A5, SLC16A1, ABCB1, ABCG2, ABCC1, ABCC2, ABCC4, ABCC5, claudin 1, claudin 3, ZO-1, occludin, JAM-A and claudin-5. 
         [0011]    “Hematopoietic stem cells” refers to cells that can themselves or whose progeny can form myeloid, erythroid, and/or megakaryocyte colonies as described in Eaves, et al.,  Atlas of Human Hematopoietic Colonies,  1995, StemCell Technologies, Vancouver; Coutinho, et al, in  Hematopoiesis: A Practical Approach , Testa, et al, eds., 1993, Oxford Univ. Press, NY, pp 75-106, and Kaufman, et al.,  PNAS,  2001, 98:10716-10721. 
         [0012]    “Pericytes” refers to cells that express one of the following markers: vimentin, neuro-glial 2 (NG2), platelet-derived growth factor receptor beta (PDGFR-β), α-smooth muscle actin (α-SMA), cells that express one of the following markers: viet al., Current Neurovascular Research 2011, Modelling the neurovascular unit and the BBB with the unique function of Pericytes). 
         [0013]    In view of the drawbacks to the prior art, one of the problems was to develop an in vitro human BBB system that could be stable for more than 15 days and be reproducible for different stem cells and could be implemented in different laboratories. The system described is the first human in vitro BBB system with high reproducibility—evaluated in three different laboratories; and in stem cells collected from more than 4 different donors—and stable more than 20 days. This has not ever been addressed by previous technologies described in the literature. 
         [0014]    An aspect of disclosed subject matter relates to human brain-like endothelial cells wherein at least a portion of the cells express at least one of the following markers: ZO-1, occludin, JAM-A, claudin-5, claudin-3, claudin-1, preferably express ZO-1 and claudin-1. 
         [0015]    In embodiments of the disclosure, the brain-like endothelial cells further express at least one of the following transporters or receptors: aminoacid—SLC7A5, SLC16A1, glucose—SLC2A1. 
         [0016]    In embodiments of the disclosure, the brain-like endothelial cells further express a portion of at least one of the following molecules: CD40, VCAM-1. 
         [0017]    Others embodiments of the disclosure, at least a portion of the brain-like endothelial cells express at least one of the following transcripts of key efflux transporters as P-glycoprotein, breast cancer resistance protein and multidrug resistance protein. 
         [0018]    In others embodiments of the disclosure, at least a portion of the brain-like endothelial cells expresses at least one of the following genes up-regulated: SLC44A5, SLC25A27 the endothelial cells, SLC23A3. 
         [0019]    In others embodiments of the disclosure, at least a portion of the brain-like endothelial cells further express at least one of the following markers: lipoprotein receptor, insulin receptor, leptin receptor, transferrin receptor, receptor for advanced glycation endproducts, retinol binding protein, SLC38A5, ABCB1, ABCG2, ABCC1, ABCC2, ABCC4, ABCC5. 
         [0020]    In others embodiments of the disclosure, endothelial cells derived from stem cells, can be preferably from hematopoietic stem/progenitor cells (CD34 +  cells) derived from human cord blood, but can be extended to endothelial cells derived from hematopoietic stem/progenitor cells derived from human peripheral blood, or other type of endothelial cells. 
         [0021]    A further aspect of the disclosure relates to a method of inducing a blood brain barrier phenotype in endothelial cells derived from stem cells, preferably from hematopoietic stem/progenitor cells (CD34 +  cells) derived from human cord blood, but can be extended to endothelial cells derived from hematopoietic stem/progenitor cells derived from human peripheral blood, or other type of endothelial cells. 
         [0022]    An aspect of disclosed subject matter relates to a method for obtaining in vitro human brain-like endothelial cells comprising the following steps:
       contacting a population of stem cells with a differentiation medium to form endothelial cells;   co-culturing the said endothelial cells with pericytes or with cells of the neurovascular unit or with a pericytes conditioned medium, to obtain brain like endothelial cells, preferably during at least 4 days, more preferably during 5-7 days, namely 5, 6, 7 days.       
 
         [0025]    In others embodiments of the method for obtaining in vitro human brain-like endothelial cells wherein the said stem cells may be CD34+ derived from human cord blood, or cells from human peripheral blood. 
         [0026]    In others embodiments of the a method for obtaining in vitro human brain-like endothelial cells wherein the cells are grown on solid support, preferably transwell systems or well plates. In preferred embodiments, the pericytes can be placed in the bottom of each plate. 
         [0027]    In others embodiments of the method for obtaining in vitro human brain-like endothelial the differentiation medium may be EGM-2 medium with 20% (v/v) FBS and 50 ng/mL of VEGF165. 
         [0028]    In others embodiments of the method for obtaining in vitro human brain-like endothelial cells the pericytes may express at least one of the following markers: vimentin, PDGF-β, NG-2, α-SMA; γ-GT. 
         [0029]    In others embodiments of the method for obtaining in vitro human brain-like endothelial cells the pericytes may be seeded at a density of 40×10 3 -50×10 3 , preferably 45×10 3  cells. 
         [0030]    In others embodiments of the method, the pericytes might be replaced by a cell line that secrete Wnt3a or Wnt7a. 
         [0031]    In others embodiments of the method for obtaining in vitro human brain-like endothelial cells the pericytes-conditioned medium (obtained from 45×10 3  cells of pericytes cultured for 6 days in a well of a 12-well plate) may be replaced every day. 
         [0032]    A further aspect of aspect of disclosed subject matter relates to a method for evaluating blood-brain barrier permeability of a substance, cell or protein comprising exposing the said test substance, cell or protein to the in vitro endothelial cells, said substance can be any synthetic or natural compound. 
         [0033]    In others embodiments a method for evaluating blood-brain barrier permeability of a substance, cell or protein by the measurement of efflux transport, preferably in the presence or absence of inhibitors of the efflux pumps. Preferably, the inhibitors may be at least one of the following: cyclosporin-A, PSC-833, MK-571, KO-143, verapamil or elacridar. 
         [0034]    A further aspect of aspect of disclosed subject matter relates to a method for evaluating blood-brain barrier metabolism of a test substance, cell or protein which comprises the following steps:
       contacting a test substance, cell or protein to the brain endothelial cells previously described;   analysing the metabolic degradation of the said test substance, cell or protein.       
 
         [0037]    In others embodiments of the method for evaluating blood-brain barrier toxicity of a test substance comprises the culturing the said brain endothelial cells in the presence of the said test substance. 
         [0038]    The viability can be determined by a live/dead assay, preferably using calcein and propidium iodide as reagents, ATP production, cell membrane damage by the release of lactate dehydrogenase, cell replication by a BrdU assay. 
         [0039]    Any changes in the BBB functionality (e.g: permeability to a non permeant marker) in vitro could be used as an alternative toxicological endpoint. 
         [0040]    In others embodiments of the method for evaluating blood-brain barrier metabolism of a test substance comprises the culturing the said brain endothelial cells in the presence of the said test substance. 
         [0041]    A further aspect of the disclosure relates to a kit for measuring blood-brain barrier permeability of a substance, comprising the in vitro human endothelial cells previously described. 
         [0042]    In various embodiments, pericytes are preferably derived from bovine, but they can be isolated from any other species. They are characterized by a set of different markers including PDGF-β in other species. They are characterized by a set of different markers including PDGF-β isolated from endothelial cell culture, α-smooth muscle actin (α-SMA), γ-glutamyl-transpeptidase and P-glycoprotein (P-gp) and others that someone skilled in the art will identify. So far, there is no single marker that differentiates pericytes from other cells. In one embodiment, the pericytes are placed in the bottom of the plate, but they can be also applied in one of the sides of the transwell filter. In a preferable embodiment, the pericytes are seeded at a density of 45×10 3  cells into each well of 12-well plates. The cells are cultured on Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 20% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 50 μg/mL gentamycin and 1 ng/mL basic fibroblast growth factor. 
         [0043]    In a preferred embodiment, the pericytes are cultured in the presence of the endothelial cells to induce BBB properties. Conditioned medium obtained from pericytes might have the same inductive effect on endothelial cells if medium is replaced every day, and at suitable concentrations. 
         [0044]    In one embodiment, the BBB properties of endothelial cells can be achieved by supplementing the culture medium with Wnt3a, Wnt7a or a mixture of Wnt3a and Wnt7a. In a preferable embodiment, the concentration of Wnt3a and Wnt7a is 6.25 ng/mL. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0045]    Without intent to limit the disclosure herein, this application presents attached drawings of illustrated embodiments for an easier understanding. 
           [0046]      FIG. 1 : Expression of BBB markers, stability and functional properties of a monolayer of human BLECs;
   (A) BLECs were obtained by the co-culture of CD34+-derived ECs with pericytes for 6 days in a Transwell™ system.   (B-D) Paracellular permeability to lucifer yellow of EC monolayers either cultured alone or with pericytes. Results are Mean±SEM (n≧4);   (E) Expression of endothelial and BBB markers in BLECs as obtained by immunofluorescence;   (F) Electron micrographs of ECs cultured alone (2,3) or with pericytes (1);
       (1) In the intercellular cleft, WGA-HRP penetrates from the luminal compartment (asterisks) to the tight junction, which occludes the cleft (arrows). From this point, the intercellular space is free of the electron-dense reaction product;   (2) When ECs are cultured alone, there is no occlusion of the intercellular space between the ECs in 84% of the cases, and the tracer penetrates from the luminal compartment (asterisks) trough the entire intercellular cleft and is deposited in the underlying matrix (arrowheads);   
       (G) BLEC gene expression of tight junctions and influx transporters. Results are Mean±SEM (n=3);   (H) BLEC gene expression of efflux transporters and large molecule receptors;   (I) Expression of P-gp and RAGE as evaluated by immunofluorescence. In E and I, bar corresponds to 50 μm. *P&lt;0.05, **P&lt;0.01, ***P&lt;0.001.     
           [0056]      FIG. 2 : Functional properties of BLECs and mechanism for the in the induction of BBB properties in CD34+-derived ECs;
   (A) Effect of P-gp protein inhibition on active transport of drugs;   (B) Efflux ratio of small (sucrose) and large (HSA and IgG) molecules. In A and B: results±SEM (n=3-7);   (C) Unbound brain-to-plasma or CSF-to-plasma concentration ratio&#39;s for human and rat;   (D) Expression of BBB markers as evaluated by whole genome microarrays of monocultures or co-cultures of CD34+-derived ECs with pericytes at day 3 and 6;   (E) Effect of Wnt3a, Wnt7a and BIO in the expression of β-catenin (after 1 day) as well as in the paracellular permeability (at days 1 and 5) of monocultures of CD34+-derived ECs. Results are Mean±SEM (n=3-6). The dashed line represents the paracellular permeability of ECs in co-culture with pericytes for 6 days. For permeability results the concentrations of Wnt3a, Wnt7a and BIO were 6.25 ng/mL, 6.25 ng/mL, and 0.5 μM;   (F) Expression and localization of claudin-1 (at day 6) and total β-catenin (day 3) in monoculture of CD34+-derived ECs cultured in medium supplemented with BIO (0.5 μM) or Wnt3a (6.25 ng/mL). Arrow-heads indicate nuclear accumulation of β-catenin. Bar corresponds to 50 μm. *P&lt;0.05, **P&lt;0.01, ***P&lt;0.001.     
           [0063]      FIG. 3 : Differentiation of human umbilical cord CD34+ cells into ECs and evaluation of their paracellular permeability;
   (A) Schematic representation of the differentiation of hematopoietic stem cells (CD34+CD45+CD31+KDR-vWF-CD14-) into ECs (2-3 weeks of differentiation) and evaluation of their paracellular permeability (Pe) using a Transwell™ system;   (B) ECs immediately after differentiation (before culture in the Transwell™ system) express typical EC markers including CD31, VE-cadherin (VECAD), vWF and are able to incorporate AcLDL;   (C) ECs after culture in the Transwell™ system have typical cobblestone morphology, express vWF and markers associated to hBECs such as claudin-5, ZO-1, and occludin; however, the expression of all these markers is discontinuous and cells do not express claudin-1 at cell-cell contacts. Bar corresponds to 50 μm;   (D) Paracellular permeability of human ECs in monoculture and bovine ECs in co-culture with astrocytes for 12 days.     
           [0068]      FIG. 4 : (A) Paracellular permeability of CD34+-derived ECs after co-culture with different types of cells in EGM-2 supplemented with 2% fetal calf serum FCS). Results are Mean±SEM (n=6); (B) Characterization of bovine pericytes by phase contrast and Immunocytochemistry for the expression of vimentin, neuro-glial 2 (NG2), platelet-derived growth factor receptor beta (PDGFR-β), and α-smooth muscle actin (α-SMA). Scale bar corresponds to 50 μm. CM stands for conditioned media; (C) The induction of BBB properties on CD34+-derived ECs requires the presence of pericytes in the co-culture system since pericyte-conditioned medium does not have the same BBB-inductive properties. 
           [0069]      FIG. 5 : (A) Double immunostaining for anti-human receptor for advanced glycation endproducts (RAGE) and anti-human organic cation/carnitine transporter (OCTN2; also known as SLC22A5) in monoculture (A.1) or in co-culture of CD34+-derived ECs with pericytes (A.2) at day 6. In the co-culture system, RAGE is present essentially in the luminal side of endothelial cells and OCTN2 in the abluminal side, while in mono-culture, both markers seem to be located in the same plane. Bar corresponds to 10 μm;
   (B) Paracellular permeability in a co-culture of CD34+-derived ECs with pericytes at day 6 obtained from different donors. Results are Mean±SEM (n=4);   (C) Interlaboratory reproducibility. The BBB was generated in two different laboratories. The paracellular permeability to lucifer yellow was not statistical significant. Results are Mean±SEM (n≧4);   (D) Stability of the BBB properties after removal of the pericytes. CD34+-derived ECs were in co-culture with pericytes for 14 days (1) or in co-culture for 6 days and then 8 days in monoculture (2).   
 
           [0073]      FIG. 6 : (A) Transendothelial electrical resistance (TEER) of monocultures of CD34+-derived ECs or co-cultures of ECs with pericytes for 6 days. The TEER of the co-culture of ECs was compared with the gold standard of bovine brain microvascular endothelial cells co-cultured with bovine astrocytes for 12 days on insert filters 30 mm diameter. Values are Mean±SEM, n=4. ***P&lt;0.001; ns means P&gt;0.05; (B) Expression of adhesion molecules by ECs in co-culture with pericytes. The expression of the adhesion molecules was assessed by flow cytometry analysis on untreated and treated ECs by TNFα (10 ng/mL) for 24 h. 
           [0074]      FIG. 7 : (A-C) Western blot for the expression of Shh (A), Wnt7a (B), Wnt3a (C) and total β-catenin (D) in CD34+-derived ECs in monoculture (1) or in co-culture with pericytes (2), or pericytes in monoculture (3) or pericytes in co-culture with ECs (4), for 6 days. Human recombinant Wnt3a, Wnt7a and Shh were used as a positive control. Data shown are representative of n=2. In D: results±SEM, n=2. 
           [0075]      FIG. 8 : qRT-PCR results showing changes on Wnt signaling (A-C), tight junctions (D) and BBB transporters (E) genes on CD34+-derived ECs co-cultured with pericytes for 1, 3 and 6 days. Values are Mean±SEM, n=4. Our results show that Wnt3a transcript increased significantly at day 1 followed by a decrease at day 6 to baseline levels. Genes of canonical Wnt ligands Wnt7a and Wnt7b, which have been reported to be involved in BBB development, increased slightly at day 1 and then decreased at day 3 to baseline levels. The expression of genes encoding Wnt receptor frizzled 4 (FZD4) and frizzled 6 (FZD6) were not affected by the co-culture system; however, Wnt receptor frizzled 7 (FZD7) was significantly up-regulated up to 6 days. The expression of LEFT, the β-catenin-associated transcription factor, peaked at day 1 matching the profile observed for Wnt3a and FZD7. The expression of APCDD1, an antagonist of Wnt signaling and highly expressed in adult brain endothelial cells, peaked at day 3, at the time that Wnt3a drops significantly. Finally, genes related to tight junctions such as claudin 1 and ZO-1 and the transporters SLC7A5 and SLC16A1 are upregulated overtime. 
           [0076]      FIG. 9 : Modulation of Wnt signaling activates the barrier properties of ECs in monoculture;
   (A) Schematic representation of the methodology used to assess the modulation of Wnt signaling. CD34+-derived ECs were seeded in a Transwell™ insert coated with Matrigel at a density of 80,000 cells. Wnt ligands were added in the culture medium at the basolateral side;   (B) qRT-PCR results showing differences in expression of claudin-1 and Lefl genes on CD34+-derived ECs cultured with or without Wnt3a. Values are Mean±SEM, n=4;   (C-D) Paracellular permeability of untreated ECs or ECs treated with different concentrations of human recombinant protein Wnt3a (C) or Wnt7a (D) for 5 days. Results are Mean±SEM (n=4).     
           [0080]      FIG. 10 : Abrogation of Wnt signaling in ECs during co-culture with pericytes affect their paracellular permeability;
   (A) Schematic representation of the methodology used to assess the effect of abrogation of Wnt signaling. CD34+-derived ECs were seeded in a Transwell™ insert coated with Matrigel at a density of 80,000 cells and cultured in medium supplemented with XAV 939 (0.1 and 1 μM). In the bottom of the Transwell™ was seeded 45,000 bovine pericytes. After 4 days of coculture, the paracellular permeability and cell organization were evaluated;   (B) Fluorescence microscopy images showing the expression of ZO-1 in untreated ECs or ECs treated with XAV 939 (1 μM) for 4 days;   (C) Paracellular permeability of untreated ECs or ECs treated with 0.1 or 1 μM XAV939 for 4 days. Results are Mean±SEM (n=4).     
       
    
    
     DETAILED DESCRIPTION 
       [0084]    In the present disclosure is described a method to generate a human blood-brain barrier model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into endothelial cells followed by the induction of blood-brain barrier (BBB) properties by co-culture with pericytes. The brain-like endothelial cells (BLECs) express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. 
         [0085]    To differentiate stem cells into endothelial cells, CD34+CD45+CD31+KDR-vWF-CD14-cells isolated from cord blood were initially cultured for 15-20 days in EGM-2 medium with 20% (v/v) FBS and 50 ng/mL of VEGF165 (Supplementary  FIG. 1A ). At this stage, cells have a cobblestone-like morphology and express high levels of endothelial cells markers, including CD31, VE-cadherin and vWF (Supplementary  FIG. 1B ). When these cells were grown to confluence on filters for 6 days they show discontinuous expression of ZO-1, occludin and claudin-5, do not express claudin-1 at cell-cell contacts and have high permeability to Lucifer yellow (2.0×10−3 cm/min) as compared to bovine BECs (Supplementary  FIGS. 1C and 1D ). 
         [0086]    To induce BBB properties in CD34+-derived endothelial cells, cells were seeded in a Transwell™ system and co-cultured with pericytes ( FIG. 1A ). Pericytes were selected after a screening of different cell types from the neurovascular unit (Supplementary  FIGS. 2A and 2B ) and because of their role in the stabilization/maturation of BBB (Armulik, A., et al. Pericytes regulate the blood-brain barrier. Nature 468, 557-561 (2010); Daneman, R., Zhou, L., Kebede, A. A. &amp; Barres, B. A. Pericytes are required for blood-brain barrier integrity during embryogenesis. Nature 468, 562-566 (2010)). Under these conditions, the permeability of endothelial cells decreases during the first 3 days until it reaches a stationary phase at day 4 ( FIG. 1B ), maintaining its stability up to 20 days ( FIG. 1C ). At day 6, the cells had low permeability values (0.61×10−3 cm/min) similarly to the values found in other BBB models (Deli, M. A., et al. Permeability studies on in vitro blood-brain barrier models: physiology, pathology, and pharmacology. Cell Mol Neurobiol 25, 59-127 (2005)) ( FIG. 1D ), they showed a continuous expression of ZO-1, occludin, JAM-A and claudin-5 at cell-cell contacts ( FIG. 1E ) and they were able to block the passage of wheat germ agglutinin (WGA)-horseradish peroxidase (HRP) in contrast with monolayers of CD34+-derived endothelial cells where WGA-HRP reached the underlying matrix ( FIG. 1F ). Importantly, the induction of BBB properties in CD34+-derived endothelial cells is highly reproducible since similar permeability results were obtained for cells derived from multiple human donors (Supplementary  FIG. 3B ) and in 3 different laboratories (Supplementary  FIG. 3C ). Furthermore, the BBB properties of CD34+-derived endothelial cells are lost if the pericytes are removed from the co-culture system (Supplementary  FIG. 3D ) showing that the crosstalk between the two cells is important to maintain the BBB properties. Cells co-cultured with pericytes for 6 days express transcripts encoding tight junctions such as ZO-1 and claudin-1 higher than in endothelial cells in monoculture, while the expression of claudin-3 and occludin was similar ( FIG. 1G ). Importantly, the expression of influx transporters, specifically the expression of aminoacid (SLC7A5, SLC16A1) and glucose (SLC2A1) transporters and receptors (e.g. transferrin receptor; TFRC) was increased when the cells were co-cultured with pericytes relatively to cells cultured alone. In addition, endothelial cells co-cultured with pericytes for 6 days express transcripts of key efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance protein (MRP; subfamily of the ATP-binding cassette (ABC) transporters) family ( FIG. 1H ). As in hBECs, the receptor for advanced glycation end products (RAGE) and P-gp protein were expressed as confirmed by immunofluorescence ( FIG. 1I ), being RAGE located at the luminal side of cells (Supplementary  FIG. 3A ). Overall, endothelial cells co-cultured with pericytes for 6 days have BBB properties at gene, protein and permeability levels, and from now on are named as brain endothelial-like cells (BLECs). 
         [0087]    BLECs have the ability to act as an active barrier. The inhibition of P-gp protein by verapamil or elacridar, and the concomitant blocking of the active transport of drugs to outside the cell, it leads to a significant increase in the accumulation of the antitumor drug vincristine ( FIG. 2A ). This result demonstrates that P-gp is functionally active in BLECs. The higher efflux ratio of IgG as compared to human serum albumin shows receptor-mediated transport of macromolecules across the polarized monolayer ( FIG. 2B ). In addition, BLECs have the ability to form a monolayer that has a transendothelial electric resistance (TEER) similar to monolayers of bovine BECs (Supplementary  FIG. 4A ) and higher than monolayers of human hCMEC/D3 cell line (&lt;40 Ωcm2) (Weksler, B. B., et al. Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19, 1872-1874 (2005)). Moreover, BLECs express constitutively the adhesion molecule ICAM-2, typically found in hBECs (Bo, L., et al. Distribution of immunoglobulin superfamily members ICAM-1, -2, -3, and the beta 2 integrin LFA-1 in multiple sclerosis lesions. J Neuropathol Exp Neurol 55, 1060-1072 (1996)), and show an up-regulation in the expression of ICAM-1, ICAM-2, CD40 and VECAM-1 after stimulation with 10 ng/mL TNF-α for 24 h, as hBECs (Weksler, B. B., et al. Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19, 1872-1874 (2005) (Supplementary  FIG. 4B ). Finally, the in vitro ratio of concentrations of unbound drug in brain and plasma for atenolol, bupropion, rifampicin and verapamil were closer to the in vivo ratio of concentrations of unbound drugs in cerebrospinal fluid (CSF) and plasma reported in humans than in rats (Friden, M., Gupta, A., Antonsson, M., Bredberg, U. &amp; Hammarlund-Udenaes, M. In vitro methods for estimating unbound drug concentrations in the brain interstitial and intracellular fluids. Drug Metab Dispos 35, 1711-1719 (2007)) ( FIG. 2C ). Together, these results indicate that brain-like endothelial cells can be used to predict accurately in humans the in vivo transport of drugs with different properties. 
         [0088]    To study the induction of BBB properties in CD34+-derived endothelial cells, these cells cultured alone or with pericytes for 3 or 6 days were characterized by whole genome microarrays. Gene expression analyses at 6 days show that 84 and 2 genes are up- and down-regulated in CD34+-derived endothelial cells in co-culture, respectively, relatively to CD34+-derived endothelial cells in monoculture (Supplementary Tables 3 and 4). From the overall up-regulated genes, 3 genes were related with influx transporters including SLC44A5, SLC25A27 and SLC23A3, and 2 genes were related with Wnt signaling (Wnt inhibitory factor 1 and disheveled associated activator of morphogenesis (Cecchelli, R., et al. Modelling of the blood-brain barrier in drug discovery and development. Nat Rev Drug Discov 6, 650-661 (2007)) (Daam 1)) ( FIG. 2D ). Yet, the expression of most markers associated to BBB (tight junctions and transporters) was not significantly different in CD34+-derived endothelial cells in monoculture and co-culture, which indicates that pericytes exert a discrete influence on endothelial BBB-specific genes. Gene expression on CD34+-derived endothelial cells in co-culture at day 6 and 3 was significantly different regarding BBB markers, specifically for efflux transporters including solute carrier family members SLC2A3, SLC6A6 and SLC47A1 (downregulated at day 6), and members SLC30A3, SLC26A10, SLC13A3 and SLC44A5 (upregulated at day 6) and non-BBB markers such as channels and extracellular matrix (Supplementary Tables 3 and 4). Together these results show that the induction process is a dynamic process affecting the expression of transporters, channels and extracellular matrix components. 
         [0089]    Two major pathways regulating the formation of BBB are the canonical Wnt/wingless pathway acting via β-catenin stabilization and Sonic hedgehog (Shh) pathway (Liebner, S., et al. Wnt/beta-catenin signaling controls development of the blood-brain barrier. J Cell Biol 183, 409-417 (2008); Alvarez, J. I., et al. The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence. Science 334, 1727-1731 (2011); Daneman, R., et al. Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis. Proc Natl Acad Sci USA 106, 641-646 (2009). Protein analyses show that pericytes do not express Shh but do express Wnt ligands such as Wnt3a and Wnt7a (Supplementary  FIG. 5 ). By other hand, endothelial cells express at gene level Wnt receptors such as frizzled receptor 4, 6 and 7 (FZD4, FZD6 and FZD7), and in co-culture with pericytes, they show an up-regulation in the expression of Wnt3a and FZD7 receptor during the first day followed by a decrease in the next 5 days (Supplementary  FIG. 6 ). This was accompanied by an increase of APCDD1, an antagonist of Wnt signaling, that peaked at day 3, and an increase of the tight junctions ZO-1 and claudin-1 (Supplementary  FIG. 6 ). To determine whether the activation of Wnt is required for the induction of barrier properties in CD34+-derived endothelial cells, these cells were cultured alone for 5 days and then exposed them to Wnt ligands/agonists. Endothelial cells respond rapidly to BIO, a specific pharmacological inhibitor of glycogen synthase kinase-3 (GSK-3) and thus an activator of Wnt signaling, or Wnt3a by increasing the expression of active β-catenin ( FIG. 2E ). The paracellular permeability of Wnt3a-treated endothelial cells to Lucifer Yellow was statistical lower (P&lt;0.01, n=4) for short-term (1 day) and long-term (5 days) as compared to untreated cells ( FIG. 2E  and Supplementary  FIG. 7 ). The effect of Wnt7a and BIO was only observed at day 5. During the induction process by Wnt 3a or BIO, there is an increase in the expression and nuclear localization of total β-catenin ( FIG. 2F  and Supplementary  FIG. 5 ) and the localization of claudin-1 at the cell-cell contacts ( FIG. 2F ). The localization of claudin-1 at the periphery of the cells might explain the restrictive permeability of endothelial cells in co-culture with pericytes. Overall, results indicate that Wnt pathway contributes, at least in part, for the induction of BBB properties in CD34+-derived endothelial cells. 
         [0090]    To further confirm the role of Wnt pathway in the induction of BBB properties, was abrogated the Wnt signaling in endothelial cells co-cultured with pericytes. Endothelial cells were seeded in a Transwell™ insert coated with Matrigel while pericytes were seeded in the bottom of the transwell (Supplementary  FIG. 8 ). Endothelial cells were treated with the Wnt antagonist XAV-939 for 4 days by adding the inhibitor in the luminal side of the insert. The abrogation of Wnt pathway, in conditions that did not affect cell viability, increased the paracellular permeability of the endothelial monolayer to lucifer yellow. These results again indicate that Wnt signaling is required for the BBB properties in CD34+-derived endothelial cells co-cultured with pericytes. 
         [0091]    In summary, was generated a human in vitro BBB model from endothelial cells derived from cord blood hematopoietic stem cells that is highly reproducible and stable for at least 20 days after its derivation. Is provided in vitro evidence for a role of pericytes in the induction of BBB formation through the canonical Wnt pathway. Due to the relative easy access to cord blood stem cells, this model can be adopted by the research community to improve the delivery of therapeutic agents into the central nervous compartment for the treatment of stroke, multiple sclerosis and brain tumors. 
         [0092]    In one embodiment of the present disclosure can be used as a method to measure BBB permeability to a test substance. The test substance may be any synthetic or natural compound, with variable molecular weight and hydrophilicity/hydrophobicity ratio. The method of the disclosure can measure passive diffusion or active transport, as appreciated by those skilled in the art. Efflux transport can be measured wherein measuring permeability values is performed in the presence or absence of inhibitors of the efflux pumps such as, but not limited to, cyclosporin-A, PSC-833, MK-571, KO-143. The methods of the present disclosure can also be used to measure blood brain barrier metabolism of a substance by measuring permeability values and profiling the metabolic degradation of compounds of interest as a function of time using quantitative analytical techniques such as high pressure liquid chromatography and mass spectrometry. Test substances that prove to pass our BBB in vitro model may be further analyzed for their pharmacological profile. 
         [0093]    In another embodiment, the in vitro BBB model of the present disclosure may be useful as a method for determining the toxicity of a test substance or vector towards the BBB. In this case, the method comprises the culture of the brain endothelial-like cells in the presence of the test substance and assessing its viability after a certain time. A range of concentrations of the test substance can be used to determine the IC50. Cell viability can be determined by a live/dead assay using calcein and propidium iodide as reagents, ATP production, cell membrane damage by the release of lactate dehydrogenase, cell replication by a BrdU assay. 
         [0094]    In another embodiment, the in vitro BBB model can be used to design more effective vectors to target or delivery drugs into the brain. This might be useful for the treatment of vascular dysfunction in patients with Alzheimer&#39;s. Neudegeneration is likely a consequence of altered drug transport across the BBB and abnormal cerebral blood flow due to amyloid peptide deposition. Our in vitro BBB model can be very useful for testing drug candidates for the treatment of Alzheimer. 
         [0000]    Isolation and Differentiation of CD34+ Cells from UCB 
         [0095]    In a preferred embodiment CD34+ cells may be isolated from human umbilical cord blood and differentiated into endothelial cells according to a protocol previously reported by us (Pedroso, D. C., et al. Improved survival, vascular differentiation and wound healing potential of stem cells co-cultured with endothelial cells. PLoS One 6, e16114 (2011)) Briefly, isolated CD34+ cells were cultured in EGM-2 medium (preferably Lonza) supplemented with 20% (v/v) fetal bovine serum (preferably FBS; Life Technologies) and 50 ng/mL of VEGF165 (preferably PeproTech Inc.), on 1% gelatin-coated 24-well plates (2×10 5  cells/well). After 15-20 days endothelial cells are seen in the culture dish. For each experiment, the cells were expanded in 1% (w/v) gelatin-coated 100 mm Petri dishes (preferably BD Falcon) in EGM-2 medium (with all the supplements except FBS and gentamycin/amphotericin) supplemented with 2% (v/v) FBS, 50 μg/mL gentamycin (preferably Biochrom AG) and 1 ng/mL home-made bFGF. 
         [0000]    Differenciation of CD34+ Derived Endothelial Cells into Brain Like Endothelial Cells by Pericytes 
       Isolation of Pericytes 
       [0096]    In a preferred embodiment pericytes may be extracted from freshly collected bovine brain capillaries. Brain capillaries were collected on a 60 μm nylon sieve (preferably Blutex®, Saati, France) as described by Méresse et al. (1989) and suspended in Hanks Balanced Salt Solution (preferably HBSS, Sigma-Aldrich) containing 10 mM HEPES and 0.1% BSA. This suspension was centrifuged at 1000 g for 7 min at room temperature. The pellet was then digested with 2 mg/mL collagenase-dispase (preferably Roche Diagnostics), 10 μg/mL DNaseI (preferably Roche Diagnostics) and 0.147 μg/mL TLCK (preferably Sigma-Aldrich), for 30 minutes at 37° C. in a shaking water bath. After washes, the digested capillaries were seeded onto growth factor reduced Matrigel (preferably BD Biosciences)-coated dishes (preferably Corning) containing pericyte growth culture medium: DMEM (preferably Life Technologies) supplemented with 20% fetal calf serum (preferably Integro), 2 mM L-glutamine (preferably Merck Chemicals), 50 μg/mL gentamicin (preferably Biochrom AG) and 1 ng/mL bFGF (preferably Sigma-Aldrich). The medium was changed every other day. Pericytes and endothelial cells migrated from the vessels walls. Pericytes rapidly overgrew from capillaries and invaded the whole surface of the dishes. Confluent cultures consisting almost exclusively of pericytes, were dissociated using trypsin/EDTA saline solution (preferably 0.05%/0.02% Biochrom AG), and cells were frozen in liquid nitrogen. For experiments, each pericyte vial was rapidly thawed and seeded in gelatin (preferably sigma-Aldrich)-coated 60-mm Petri dishes containing pericyte culture medium. After thawing, there were no endothelial cells left in cultures. Pericytes were subcultured at a split ratio 1/3, and were used at passages ≦3. 
         [0000]    Co-Culture of CD34+ Derived Endothelial Cells with Pericytes. 
         [0097]    In a preferred embodiment for co-culture experiments, pericytes may be initially seeded on 60-mm gelatin-coated petri dishes and cultured in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) (preferably Life Technologies) supplemented with 20% (v/v) fetal bovine serum (FBS) (preferably Life Technologies), 2 mM L-glutamine, 50 μg/mL gentamycin and 1 ng/mL basic fibroblast growth factor (bFGF). The cells reached confluency after 2 days. 45×10 3  cells were seeded into each well of 12-well plates (preferably Costar). CD34+-endothelial cells growing on gelatin-coated 100 mm petri dishes in EGM-2 (with all the supplements except FBS and gentamycin/amphotericin) supplemented with 2% (v/v) FBS, 50 μg/mL gentamycin (preferably Biochrom AG) and 1 ng/mL home-made bFGF were trypsinized and cells were seeded at a density of 8×10 4 /insert onto the Matrigel-coated (preferably BD Biosciences) Transwell™ inserts (preferably Costar). After 6 days in co-culture, the experiments were carried out. 
         [0098]    Reverse transcription and quantitative real time polymerase chain reaction (qRT-PCR) analysis. CD34+-endothelial cells cultured in different conditions were homogenized in Trizol reagent (preferably Life Technologies) and total RNA was extracted using the RNeasy Mini Kit (preferably Qiagen), according to manufacturer&#39;s instructions. In all cases, cDNA was prepared from 1 μg total RNA using Taqman Reverse transcription reagents (preferably Applied Biosystems). Non-quantitative RT-PCR was performed using the conditions described in Sano et al. (2010) and DNA migrated on a agarose gel electrophoresis (1.5%) with a low range DNA molecular weight marker (preferably Euromedex) to visualize the sizes. Gels were then stained with gel red nucleic acid gel stain (preferably Interchim) and visualized on a UV light transilluminator (preferably Bio-Rad). Quantitative real time PCR (qRT-PCR) was performed using Power SYBR Green PCR Master Mix (preferably Applied Biosystems) and the detection was carried out in a 7500 Fast Real-Time PCR System (preferably Applied Biosystems). Quantification of target genes was performed relatively to the reference GAPDH gene: relative expression=2[−(Ctsample-CtGADPH)]. Primer sequences are given as supporting information (Table S2). 
       Multidrug Resistance Accumulation Assay 
       [0099]    In a preferred embodiment cell monolayers may be washed with pre-warmed HEPES-buffered Ringer&#39;s (RH) solution (NaCl 150 mM, KCl 5.2 mM, CaCl2 2.2 mM, MgCl2 0.2 mM, NaHCO3 6 mM, Glucose 2.8 mM, HEPES 5 mM, water for injection). Cells were incubated with RH solution containing [3H]-vincristine sulphate at a final concentration of 66.5 nM with or without P-gp inhibitor (25 μM of verapamil (preferably Sigma) or 0.5 μM elicridar). After 2 h, Transwell™ filter with monolayer cells were placed on ice and the cells were washed five times with ice-cold HEPES-buffered Ringer&#39;s solution. Cells were then lysed with 1% (v/v) Triton X-100 in RH solution for 5 min at 37° C. and transferred to scintillation vials. Samples (100 μL) were diluted in liquid scintillation cocktail Ultima Gold M.V (preferably 4 mL, Perkin Elmer) and analyzed by a liquid scintillation analyzer, TRI-CARB 2100 TR (preferably Perkin Elmer). 
       Characterization of CD34+ Derived Brain Like Endothelial Cells 
     Ultrastructural Analysis of Cell Monolayers by Transmission Electron Microscopy (TEM) 
       [0100]    In a preferred embodiment wheat germ agglutinin conjugated horseradish peroxidase (WGAHRP) (preferably Sigma-Aldrich) was used for ultrastructural analysis of endothelial cells monolayers. Filter inserts with endothelial cells were transferred into plates containing 1.5 mL of HEPES-buffered Ringer&#39;s solution (150 mM NaCl, 5.2 mM KCl, 2.2 mM CaCl2, 0.2 mM MgCl2-6H2O, 6 mM NaHCO3, 5 mM HEPES, 2.8 mM glucose, pH 7.4) (lower compartment), and 0.5 mL of HEPES-buffered Ringer&#39;s solution supplemented with 0.1 mg/mL WGA-HRP was applied to the upper compartment. After 10 min incubation at 37° C. in a 5% CO2/95% air atmosphere, the WGA-HRP solution was removed and the specimens were washed twice with HEPES-buffered Ringer&#39;s solution and fixed for 1 h at room temperature with 2.5% glutaraldehyde and 1% paraformaldehyde in 0.1 M sodium cacodylate (pH 7.4). After washing with 0.1 M sodium cacodylate, the fixed endothelial cell monolayers were incubated for 30 min at room temperature with the HRP substrate 3,3′-diaminobenzidine tetrahydrochloride (preferably 1.5 mg/mL; Sigma-Aldrich) and 0.02% H2O2 (v/v) in a TRIS-imidazol buffer (0.1 M imidazol, 0.05 M TRIS/HCl, pH 7.0). After washing with 0.1 M sodium cacodylate, cells were fixed again for 1 h at RT with 2.5% glutaraldehyde and 1% paraformaldehyde in cacodylate buffer. Specimens were washed twice with 0.1 M sodium cacodylate buffer, postfixed with 1% OsO4 in 0.1 M cacodylate buffer. After dehydration in graded ethanol, samples were embedded in Epon 812. Ultrathin sections were cut on Ultracut UCT (preferably Leica), contrasted with uranyl acetate and lead citrate, and examined with a Jeol 1011 TEM at an accelerating voltage of 100 Kv. 
       Microarray Studies 
       [0101]    In a preferred embodiment CD34+-endothelial cells were cultured in monoculture or in co-culture with pericytes for 3 days and 6 days in the same culture conditions described in the CD34+-endothelial cells co-culture experiments section. At days 3 and 6, the CD34+-endothelial cells were homogenized in Trizol reagent (preferably Life Technologies) and the total amount of RNA was extracted with RNeasy Mini Kit (preferably Qiagen), according to manufacturer&#39;s instructions. RNA quality was assessed preferably by an Agilent 2100 Bioanalyser (G2943CA), using preferably an Agilent RNA 6000 Nano Kit (5067-1511). Gene expression was evaluated by a whole human genome (4×44K) microarray (preferably G4112F from Agilent Technologies). The microarrays were scanned preferably by an Agilent B Scanner (G2565BA). The raw data were analyzed using preferably BRB-ArrayTools v3.4.0 developed by Dr. Richard Simon and BRB-ArrayTools Development Team (Simon et al., 2007). This analysis generated a median normalized dataset that was subjected to a statistical study and clustering using preferably MeV software (Saeed et al., 2006). The differential expressed genes obtained from MeV were used to calculate the M-value and Fold-change variation. It was considered as differentially expressed gene a variation equal or higher than 2× between the different conditions. 
       Wnt Signaling Experiments 
       [0102]    In a preferred embodiment for Wnt signaling experiments, mono- and co-culture systems were used. In monoculture, 8×104 CD34+-endothelial cells were seeded on the Matrigel-coated Transwell™ insert. The cells were then incubated with agonists/ligands (6.25 ng/mL-100 ng/mL Wnt3A (R&amp;D Systems), 6.25 ng/mL-250 ng/mL Wnt7A (preferably Peprotech) or 0.5-5 μM BIO (Sigma)) for 1 or 5 days. Co-cultures were prepared as described before. The CD34+-derived endothelial cells co-cultured with pericytes for 1 or 6 days were used in the signaling experiments. Agonist (preferably 0.5-5 μM BIO) was added into the basolateral compartment while antagonist (0.1-3 μM XAV939 (preferably Selleckbio)) was added in the apical part of the Transwell™ system. 
       FACS Analysis 
       [0103]    In a preferred embodiment cells were dissociated from the culture plate by exposure to Cell Dissociation Buffer (preferably Life Technologies) for 3-5 minutes and gentle pipetting, centrifuged and finally resuspended in PBS supplemented with 5% (v/v) FBS. The single cell suspensions were aliquoted (2.0′105 cells per condition), fixed with 4% (v/v) paraformaldehyde (PFA; EMS) or ice-cold absolute methanol and permeabilized with 0.1% (w/v) Triton X-100 (preferably Fluka) when necessary. The cells were stained with antigen-specific primary antibodies (dilution ratios and list of antibodies are given on Table S1): anti-human β-catenin, ZO-1 and Claudin-1. After the incubation with primary antibodies, cells were incubated with phycoerytrin (PE)-conjugated anti-rabbit (preferably R&amp;D Systems), and PE-conjugated anti-mouse (preferably Santa Cruz) secondary antibodies. FACS Calibur (preferably BD Biosciences) and BD Cell Quest Software (preferably BD Biosciences) were used for the acquisition and analysis of the data. 
       Characterization of the CD34+ Derived Human In Vitro BBB Model 
     Endothelial Permeability (Pe) Measurements 
       [0104]    In a preferred embodiment prior to the experiments, RH solution (in some cases EBM-2 medium) was added to empty wells of a 12-well plate (preferably Costar). Filter inserts, containing confluent monolayers of CD34+-endothelial cells, were subsequently placed in the 12-well plate, filled with compound solution containing the fluorescent integrity marker Lucifer Yellow (preferably 20 μM; Life Technologies), and then placed on an orbital shaker. After 1 h, filter inserts were withdrawn from the receiver compartment. Aliquots from the donor solution were taken at the beginning and at the end of the experiments and the fluorescence was quantified. At least three inserts with cells and three without cells were tested in each permeability measurement. Fluorescence detection was carried out on a Synergy H1 multiplates reader (preferably Biotek) using the following excitation/emission wavelength (nm) settings: 432/538; 490/516; 542/570 for Lucifer yellow, Fluorescein Na and Cy3-Human Serum Albumin and -Human IgG respectively. 
         [0105]    To obtain a concentration-independent transport parameter, the clearance principle was used. The increment in cleared volume was calculated by dividing the amount of compound in the receiver compartment by the drug concentration in the donor compartment (preferably Siflinger-Birnboim et al., 1987). The volume cleared was plotted versus time and the slope estimated by linear regression analysis. The slope of the clearance curve with inserts alone and inserts with cells is equal to PSf and PSt, respectively, where PS (microliters/minute) is the permeability surface area (square centimeter) product. The PS-value for endothelial monolayer (PSe) was calculated. To generate the endothelial permeability coefficient, Pe (cm/min), the PSe value was divided by the surface area of the filter (A in cm2) insert using the following equation: Pe=[1/PSt □1/PSf]−1/A. To assess possible adsorption to plastics and non-specific binding to cells, the mass balance (%) was calculated from the amount of compound recovered in both compartments at the end of the experiment divided by the total amount added in the donor compartment at time zero. For Pe determination, mass balance value should be between 80% and 120%. 
       Immunostaining 
       [0106]    In a preferred embodiment cells may be fixed in cold methanol/acetone (50%/50% v/v) for 1 min or 4% (v/v) paraformaldehyde (preferably Electron Microscopy Sciences, EMS) for 10 min at room temperature (see supplementary Table 1). After permeabilizing the cells with 0.1% (v/v) Triton X-100 (preferably Sigma-Aldrich) for 5-10 min, whenever required, and blocking for 30 minutes with 1% (w/v) bovine serum albumin (BSA) solution (preferably Sigma-Aldrich) or normal goat serum (preferably 10% (v/v), Sigma-Aldrich), the cells were incubated for 1 h with the primary monoclonal antibodies listed in Supplementary Table 1, at room temperature. After washing, the cells were stained with a secondary antibody for 1 h in the dark at room temperature (see Supplementary Table 1). In each immunofluorescence experiment, an isotype-matched IgG control was used. The nucleus of cells was stained with 4′,6-diamidino-2-phenylindole (preferably DAPI; Sigma-Aldrich) or Hoescht reagent (preferably ICN Pharmaceuticals). Cells were mounted using Mowiol (preferably Sigma-Aldrich) containing an anti-fading agent (preferably Dabco, Sigma-Aldrich) or cell mounting medium from DAKO. Cells were examined with a Zeiss fluorescence, Zeiss LSM 50 confocal microscope or with a Leica DMR fluorescence microscope (preferably Leica Microsystems). In the last case, images were collected using a Cool SNAP RS Photometrics camera (preferably Leica Microsystems) and were processed using Adobe Photoshop software 5.5 (preferably Adobe systems). 
       Transendothelial Electrical Resistance (TEER) 
       [0107]    In a preferred embodiment TEER (Ohm·cm 2 ) of human endothelial cells on Transwell™ filters was measured using the Millicell-ERS (preferably Electrical Resistance System). The resistance of Matrigel-coated inserts was subtracted from the resistance obtained in the presence of the endothelial cultures according to the followed equation: TEER=[(TEER, cells)−(TEER, insert)×A], where A is the area of the filter (cm2). 
       In Vitro Free Brain/Plasma Ratios 
       [0108]    In a preferred embodiment atenolol, bupropion, diazepam, rifampicin and verapamil (preferably AstraZeneca, Local Discovery Research Area CNS &amp; Pain Control, Södertälje, Sweden) at 10 mM in DMSO. 
       Preparation of Rat Glial Cell Cultures 
       [0109]    In a preferred embodiment primary cultures of glial cells may be isolated from newborn rat cerebral cortex (preferably Booher &amp; Sensenbrenner, 1972). After the meninges have been cleaned off, the brain tissue was forced gently through a nylon sieve. DMEM supplemented with 10% (v/v) FBS, 2 mM glutamine, and 50 μg/mL of gentamycin was used for the dissociation of cerebral tissue and development of glial cells. The glial cells were plated at a concentration of 5.5×10 4  cells on 12-well plates. The medium was changed every second day. Three weeks after seeding, glial cultures were stabilized and composed of astrocytes (˜60%), oligodendrocytes and microglial cells (Descamps et al., 2003). 
         [0110]    Prior experiments, rat glial cells were rinsed 3 times with HEPES-buffered Ringer&#39;s solution. 1.5 mL of RH solution was added to these receiver compartments. Inserts with human brain-like endothelial cells were also rinsed and placed in rat glial cell wells. 0.5 mL of tested drugs at 2 μM in HEPES-Buffered Ringer&#39;s solution with 0.5% human serum albumin was added to the donor compartment. After 1 h of incubation, aliquots from the donor and receiver compartment were taken and analyzed (see below). The in vitro free brain/plasma ratios (Cu,b/Cu,p) were calculated using the free drug concentration in the receiver compartment and in the donor compartment after 1 h. These experimental data were computed into the in vitro Cu, donor/Cu, receiver calculator (v0.1) (http://www.blue-norna.com) to generate in vitro steady-state Cu,br/Cu,pl ratios. 
         [0111]    All samples were analyzed using tandem mass spectrometry. Instruments that were used included: Mass spectrometer, Quattro Premier XE (preferably Waters); autosampler, Acquity sample manager; UPLC pump, Acquity Binary solvent manager (preferably Waters); robot for sample preparation, Biomek FX (preferably Beckman-Coulter). The following chemicals and reagents were used: Ammonium acetate (preferably Merck), acetonitrile gradient grade (preferably Merck), Methanol gradient grade (preferably Merck), laboratory deionised water, further purified with a Milli-Q water purifying system and ammonium acetate 1 mol/L in Milli-Q water. Samples were stored in a freezer (−20° C.) In order to minimize contamination of analysis instruments, protein precipitation was carried out on samples containing HSA; aliquots of samples were transferred to a deep well plate (1 mL), precipitated with acetonitrile and centrifuged (4000 rpm at 4° C. for 20 min). The supernatant was then transferred to a new deep well plate and RH buffer added. For chromatography the following system was used: analytical column, acquity UPLC BEH C18 1.7 μm 2.1×30 mm (Waters); mobile phase A, 2% acetonitrile, 10 mM ammonium acetate and B, 80% acetonitrile in 10 mM ammonium acetate; gradient, 2% B for 0.2 min, 2-100% B in 0.3 min, held at 100% B for 0.2 min and returned to initial condition in one step; solvent delay 0.4 min, time between injections 1.5 min; flow rate 0.6 ml/min; loop: 10 μL; injection volume: 5-10 μL. The quantification of unknown samples was performed, using preferably QuanLynx software. Response factors were constructed by plotting peak area of the analyte against concentration of each analyte using an average response factor of the donor (D0/C0) sample injections. The average RF function without weighting was used. 
       Bidirectional Transport Assay 
       [0112]    In a preferred embodiment sodium fluorescein 1 μM or Cy3-human serum albumin 500 nM or Cy3-human immunoglobulin G 100 nM (preferably Jackson ImmunoResearch) may applied on the apical or basolateral compartment of insert with endothelial cells. The opposite compartment was filled with RH solution. After 120 minutes, the fluorescence was quantified on a Synergy H1 multiplate reader (preferably Biotek) at an excitation/emission wavelength (nm) of 490/516 and 542/570 for sodium fluorescein and Cy3-human serum albumin/Cy3-human IgG, respectively. The efflux ratio was calculated using the equation: ER=(Papp,A&gt;B)/Papp,B&gt;A), where A&gt;B and B&gt;A denotes the transport direction in which Papp was determined. The apparent permeability coefficient (preferably Papp) in cm/sec was calculated according to the following equation: Papp=(k×Vr)/(A×60), where k is the transport rate (min−1) defined as the slope obtained by linear regression of cumulative fraction absorbed (FAcum) as a function of time (min), Vr is the volume in the receiver chamber (cm3), and A is the area of the filter (cm2). Determination of the cumulative fraction absorbed (amount permeated), FAcum, versus time. FAcum was calculated from the equation: FAcum=ΣCRi/CDi, where CRi was the receiver concentration at the end of the interval i and CDi was the donor concentration at the beginning of interval i. 
       TNF-α Experiments 
       [0113]    In a preferred embodiment adhesion molecule expression by BLECs was determined by FACS. For these experiments, CD34+-ECs were cultured with pericytes for 6 days. After co-culture, Transwell™s with BLEC monolayers were transferred to a new 12-well plate. BLECs were treated with 10 ng/mL TNF-α (preferably Peprotech) for 24 hours. Untreated BLECs were used as control. Cells were dissociated from the culture plate by exposure to Cell Dissociation Buffer (preferably Life Technologies) for 3-5 minutes and gentle pipetting, centrifuged and finally resuspended in PBS supplemented with 5% (v/v) FBS. The single cell suspensions were aliquoted (2.0′105 cells per condition) and incubated with primary antibodies against human CD40, ICAM1, ICAM2, VCAM1, PECAM1 (Table S1). After the incubation with primary antibodies, cells were incubated with phycoerytrin (PE)-conjugated anti-rabbit (preferably R&amp;D Systems), and PE-conjugated anti-mouse (preferably Santa Cruz) secondary antibodies. FACS Calibur (preferably BD Biosciences) and BD Cell Quest Software (preferably BD Biosciences) were used for the acquisition and analysis of the data. 
       Western Blot Analysis 
       [0114]    In a preferred embodiment total protein was isolated from CD34+-ECs and pericytes in mono-culture or co-culture preferably with RadioImmuno Precipitation Assay buffer [RIPA buffer; 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) and 1 mM ethylenediaminetetraacetic acid (EDTA)] supplemented with protease inhibitor cocktail (preferably Sigma-Aldrich), 1 mM sodium orthovanadate (preferably Sigma), 1 mM phenylmethanesulfonylfluoride (PMSF), 1 mM sodium fluoride (NaF) and 1 mM dithiothreitol (DTT). The protein samples were centrifuged at 14,000 g for 15 min at 4° C., the supernatants were collected into a new eppendorf tubes and stored at −20° C. until use. 50 μg of total protein was separated by 8-12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to polyvinylidene difluoride (PVDF) membranes using preferably the Trans-Blot® Turbo™ Transfer System (preferably Bio-Rad). After blocking for 1 h at room temperature with PBS— 0.1% Tween® (preferably Sigma)—5% low fat milk, the membranes were incubated overnight at 4° C. with antibodies against: Wnt3, Wnt7A, sonic hedgehog (Shh) (preferably all from Santa Cruz Biotechnology), rabbit anti-β-Catenin total (Abcam) or α-tubulin (preferably Sigma) followed by incubation with specific secondary antibodies for 1 h at room temperature (Table S1). The protein bands were revealed using enhanced chemiofluorescence [(ECF); preferably GE Healthcare Life Sciences] reagent on the Biorad FX Molecular Imager (preferably Bio-Rad). 
       Statistical Analysis 
       [0115]    In a preferred embodiment for analysis involving three or more groups, ANOVA was used, followed by a Bonferroni post test. For analysis of two groups, a paired t-test was used. Statistical analysis was performed using preferably GraphPad Prism software (preferably San Diego, Calif., USA). Results were considered significant when P≦0.05. 
         [0116]    The disclosure is of course not in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof without departing from the basic idea of the disclosure as defined in the appended claims. 
         [0117]    The above described particular embodiments are obviously combinable. The following claims set out particular embodiments of the disclosure. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 SUPPLEMENTARY TABLE 1 
               
             
             
               
                   
               
               
                 Antibodies used for immunofluorescence □ , flow cytometry ★  and Western blot ∘ . 
               
             
          
           
               
                   
                 Antibody 
                 Dilution 
                 Reference 
                 Supplier 
                 Fixation 
               
               
                   
                   
               
             
          
           
               
                 Endothelial 
                 Rabbit anti- 
                 1/200 □   
                 71-500 
                 Life Technologies 
                 4% PFA 
               
               
                 cells 
                 occludin 
               
               
                   
                 Rabbit anti-ZO1 
                 1/200 □   
                 61-7300 
                 Life Technologies 
                 4% PFA 
               
               
                   
                 Rabbit anti- 
                 1/100 □   
                 34-1600 
                 Life Technologies 
                 Methanol/acetone 
               
               
                   
                 claudin5 
               
               
                   
                 Rabbit anti- 
                 1/10 □   
                 71-7800 
                 Life Technologies 
                 4% PFA 
               
               
                   
                 claudin1, 3 
               
               
                   
                 Rabbit anti- 
                 1/25 □   
                 Ab15098 
                 Abcam 
                 4% PFA □   
               
               
                   
                 claudin1 
               
               
                   
                 Mouse anti-JAM1 
                 1/100 □   
                 552147 
                 Becton Dickinson 
                 Methanol/acetone 
               
               
                   
                 Mouse anti-Pgp 
                 1/10 □   
                 GTX23364 
                 GeneTex 
                 4% PFA 
               
               
                   
                 Goat anti-RAGE 
                 1/100 □   
                 Sc-8230 
                 Santa Cruz 
                 4% PFA 
               
               
                   
                   
                   
                   
                 Biotechnology 
               
               
                   
                 Rabbit anti- 
                 1/500 ∘   
                 Sc-28824 
                 Santa Cruz 
                 N/A 
               
               
                   
                 Wnt3 
                   
                   
                 Biotechnology 
               
               
                   
                 Goat anti-Wnt7A 
                 1/250 ∘   
                 Sc-26361 
                 Santa Cruz 
                 N/A 
               
               
                   
                   
                   
                   
                 Biotechnology 
               
               
                   
                 Goat anti-Shh 
                 1/250 ∘   
                 Sc-1194 
                 Santa Cruz 
                 N/A 
               
               
                   
                   
                   
                   
                 Biotechnology 
               
               
                   
                 Rabbit anti- 
                 1/50 □   
                 Sc-98373 
                 Santa Cruz 
                 4% PFA 
               
               
                   
                 AHNAK 
                   
                   
                 Biotechnology 
               
               
                   
                 Mouse anti- 
                 1/50 ★,□   
                 M0823 
                 DAKO 
                 4% PFA 
               
               
                   
                 PECAM1 
               
               
                   
                 Mouse anti-VE- 
                 1/50 □   
                 Sc-9989 
                 Santa Cruz 
                 4% PFA 
               
               
                   
                 cadherin 
                   
                   
                 Biotechnology 
               
               
                   
                 Mouse anti-von 
                 1/50 □   
                 M0616 
                 DAKO 
                 4% PFA 
               
               
                   
                 Willebrand 
               
               
                   
                 Factor 
               
               
                   
                 FITC mouse 
                 1/50 ★   
                 551146 
                 BD Biosciences 
                 N/A 
               
               
                   
                 anti-CD106 
               
               
                   
                 (VCAM-1) 
               
               
                   
                 Mouse anti-CD40 
                 1/50 ★   
                 Sc-65264 
                 Santa Cruz 
                 N/A 
               
               
                   
                   
                   
                   
                 Biotechnology 
               
               
                   
                 Mouse anti- 
                 1/50 ★   
                 Sc-107 
                 Santa Cruz 
                 N/A 
               
               
                   
                 ICAM1 
                   
                   
                 Biotechnology 
               
               
                   
                 Mouse anti- 
                 1/50 ★   
                 Sc-23935 
                 Santa Cruz 
                 N/A 
               
               
                   
                 ICAM2 
                   
                   
                 Biotechnology 
               
               
                   
                 Mouse anti- 
                 1/300 ★   
                 05-665 
                 Millipore 
                 4% PFA 
               
               
                   
                 active beta 
               
               
                   
                 catenin 
               
               
                   
                 Rabbit anti- 
                 1/2000 □ , 
                 Ab6302 
                 Abcam 
                 4% PFA 
               
               
                   
                 total beta 
                 1/4000 ∘   
               
               
                   
                 catenin 
               
               
                   
                 Rabbit Anti- 
                 1/50 □   
                 Home-made 
                 It was kindly supplied 
                 4% PFA 
               
               
                   
                 OCTN2 
                   
                 antibody 
                 by Dr Nalecz KA, Nencki 
               
               
                   
                   
                   
                   
                 Institute of 
               
               
                   
                   
                   
                   
                 Experimental Biology, 
               
               
                   
                   
                   
                   
                 Warsaw, Poland. 
               
               
                   
                 Goat Anti-RAGE 
                 1/100 □   
                 Sc-8230 
                 Santa Cruz 
                 4% PFA 
               
               
                   
                   
                   
                   
                 Biotechnology 
               
               
                   
                 Mouse anti- 
                 1/1000 ∘   
                 T6199 
                 Sigma 
                 N/A 
               
               
                   
                 alpha tubulin 
               
               
                 Pericytes 
                 Rabbit anti- 
                 1/100 □   
                 Ab51092 
                 Abcam 
                 4% PFA 
               
               
                   
                 PDGFR-beta 
               
               
                   
                 Rabbit anti- 
                 1/200 □   
                 M0851 
                 DAKO 
                 4% PFA 
               
               
                   
                 alpha SMA 
               
               
                   
                 Rabbit anti-NG2 
                 1/200 □   
                 Ab5320 
                 Millipore 
                 4% PFA 
               
               
                 Secondary 
                 Alexa Fluor 488 
                 1/200 □   
                 A11034 
                 Molecular Probes 
                 4% PFA 
               
               
                 antibodies 
                 anti rabbit 
               
               
                   
                 Alexa Fluor 568 
                 1/200 □   
                 A11036 
                 Molecular Probes 
                 4% PFA 
               
               
                   
                 anti rabbit 
               
               
                   
                 Alexa Fluor 568 
                 1/200 □   
                 A11031 
                 Molecular Probes 
                 4% PFA 
               
               
                   
                 anti mouse 
               
               
                   
                 Alexa Fluor 568 
                 1/200 □   
                 A11057 
                 Molecular Probes 
                 4% PFA 
               
               
                   
                 anti goat 
               
               
                   
                 Cy3 anti mouse 
                 1/100 □   
                 C2181 
                 Sigma 
                 4% PFA 
               
               
                   
                 Phycoerythrin 
                 1/20 ★   
                 F0110 
                 R&amp;D Systems 
                 4% PFA, Methanol 
               
               
                   
                 anti rabbit 
               
               
                   
                 Cy3 anti rabbit 
                 1/100 □   
                 111-165-144 
                 Jackson Immunoresearch 
                 4% PFA 
               
               
                   
                 Phycoerythrin 
                 1/100 ★   
                 Sc-358926 
                 Santa Cruz 
                 N/A 
               
               
                   
                 anti mouse 
                   
                   
                 Biotechnology 
               
               
                   
                 Alkaline 
                 1/5000 ∘   
                 RPN5781 
                 GE Healthcare 
                 N/A 
               
               
                   
                 phosphatase 
               
               
                   
                 anti mouse 
               
               
                   
                 Alkaline 
                 1/5000 ∘   
                 RPN5783 
                 GE Healthcare 
                 N/A 
               
               
                   
                 phosphatase 
               
               
                   
                 anti rabbit 
               
               
                   
                 Alkaline 
                 1/3000 ∘   
                 705-055-003 
                 Jackson Immunoresearch 
                 N/A 
               
               
                   
                 phosphatase 
               
               
                   
                 anti goat 
               
               
                 Other 
                 Hoechst 33258 
                 4 mg/mL □   
                 190304 
                 ICN 
                 4% PFA 
               
               
                 reagents 
                 DAPI 
                 2 μg/mL □   
                 D9542 
                 Sigma 
                 4% PFA 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Supplementary Table 2 
               
               
                 Primers used for quantitative real time-PCR and non-quantitative PCR*. 
               
             
          
           
               
                   
                 SEQ ID 
                   
                 SEQ ID 
                   
               
               
                 Gene 
                 NO: 
                 Forward sequence 
                 NO: 
                 Reverse sequence 
               
               
                   
               
               
                 GAPDH 
                  1 
                 AGCCACATCGCTCAGACACC 
                 31 
                 GTACTCAGCGCCAGCATCG 
               
               
                   
               
               
                 CLDN-1 
                  2 
                 GAAAGACTACGTGTGACA 
                 32 
                 GGTCCTAATGTTAATGATAGTATC 
               
               
                   
               
               
                 CLDN-3 
                  3 
                 ATCACGTCGCAGAACATC 
                 33 
                 TACACCTTGCACTGCATCTG 
               
               
                   
               
               
                 CLDN-5 
                  4 
                 TTAACAGACGGAATGAAGTT 
                 34 
                 AAGCGAAATCCTCAGTCT 
               
               
                   
               
               
                 OCLDN 
                  5 
                 TTCTGGATCTCTATATGGTTCA 
                 35 
                 CCACAACACAGTAGTGATAC 
               
               
                   
               
               
                 ZO-1 
                  6 
                 CCTGAACCAGTATCTGATAA 
                 36 
                 AATCTTCTCACTCCTTCTG 
               
               
                   
               
               
                 SLC6A8 
                  7 
                 TGAGAGAATGAGATTTCTGCTTGT 
                 37 
                 TAGGGCTCACAGGGATGG 
               
               
                   
               
               
                 SLC3A2 
                  8 
                 TTGGCTCCAAGGAAGATT 
                 38 
                 GAGTAAGGTCCAGAATGACA 
               
               
                   
               
               
                 SLC2A1 
                  9 
                 GAGACACTTGCCTTCTTC 
                 39 
                 GCTTTGTAGTTCATAGTTCG 
               
               
                   
               
               
                 SLC7A5 
                 10 
                 TTGACACCACTAAGATGAT 
                 40 
                 GTAGCAATGAGGTTCCAA 
               
               
                   
               
               
                 SLC7A1 
                 11 
                 CCTCCTGAGACATCTTTG 
                 41 
                 CTGGAATATGACGGGAAG 
               
               
                   
               
               
                 SLC16A1 
                 12 
                 ACACAAAGCCAATAAGAC 
                 42 
                 ACAGAATCCAACATAGGTA 
               
               
                   
               
               
                 TFRC 
                 13 
                 ATGCTGACAATAACACAA 
                 43 
                 CCAAGTAGCCAATCATAA 
               
               
                   
               
               
                 WNT3A 
                 14 
                 ATCCTCTGCCTCAAATTCT 
                 44 
                 TTCGTCTAACTCCGTTGG 
               
               
                   
               
               
                 WNT7A 
                 15 
                 CGGGAGATCAAGCAGAATG 
                 45 
                 CGTGGCACTTACATTCCAG 
               
               
                   
               
               
                 WNT7B 
                 16 
                 GCTTCGTCAAGTGCAACA 
                 46 
                 GGAGTGGATGTGCAAAATG 
               
               
                   
               
               
                 FZD4 
                 17 
                 TACCTCACAAAACCCCCATCC 
                 47 
                 GGCTGTATAAGCCAGCATCAT 
               
               
                   
               
               
                 FZD6 
                 18 
                 TCGTCAGTACCATATCCCATG 
                 48 
                 CCCATTCTGTGCATGTCTTTT 
               
               
                   
               
               
                 FZD7 
                 19 
                 GATGATAACGGCGATGTGA 
                 49 
                 AACAAAGCAGCCACCGCAGAC 
               
               
                   
               
               
                 APCDD1 
                 20 
                 GGAGTCACAGTGCCATCACAT 
                 50 
                 CCTGACCTTACTTCACAGCCT 
               
               
                   
               
               
                 LEF1 
                 21 
                 AAGGAACACTGACATCAATT 
                 51 
                 TTTGGAACTTGGCTCTTG 
               
               
                   
               
               
                 P-GP* 
                 22 
                 GCCTGGCAGCTGGAAGACAAATA 
                 52 
                 CAGACAGCAGCTGACAGTCCAAGAAC 
               
               
                   
                   
                 CACAAAATT 
                   
                 AGGACT 
               
               
                   
               
               
                 BCRP* 
                 23 
                 TGGCTGTCATGGCTTCAGTA 
                 53 
                 GCCACGTGATTCTTCCACAA 
               
               
                   
               
               
                 MRP1* 
                 24 
                 ACCAAGACGTATCAGGTGGCC 
                 54 
                 CTGTCTGGGCATCCAGGAT 
               
               
                   
               
               
                 MRP2* 
                 25 
                 CCAATCTACTCTCACTTCAGCGA 
                 55 
                 AGATCCAGCTCAGGTCGGTACC 
               
               
                   
                   
                 GA 
                   
                   
               
               
                   
               
               
                 MRP4* 
                 26 
                 AAGTGAACAACCTCCAGTTCCA 
                 56 
                 CCGGAGCTTTCAGAATTGAC 
               
               
                   
               
               
                 MRP5* 
                 27 
                 AGTGGCACTGTCAGATCAAATT 
                 57 
                 TTGTTCTCTGCAGCAGCAAAC 
               
               
                   
               
               
                 hTRF* 
                 28 
                 CTGCTATGGGACTATTGCTGTG 
                 58 
                 CCGACAACTTTCTCTTCAGGTC 
               
               
                   
               
               
                 RAGE* 
                 29 
                 CTCGAATGGAAACTGAACAC 
                 59 
                 CTGGTAGTTAGACTTGGTCTC 
               
               
                   
               
               
                 LRP1* 
                 30 
                 GCATCCTGATCGAGCACCTG 
                 60 
                 GCCAATGAGGTAGCTGGTGG 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
           
               
                 SUPPLEMENTARY TABLE 3 
               
             
             
               
                   
               
               
                 Down-regulated genes in the microarray. 
               
             
          
           
               
                 Unique ID 
                 Target ID 
                 Gene Symbol 
                 Gene Name 
                 M Value 
               
               
                   
               
             
          
           
               
                 Co-culture 6 days versus Mono-culture 6 days 
               
             
          
           
               
                 A_23_P328740 
                 BC012317 
                 LINCR 
                 likely ortholog of mouse lung-inducible 
                 −2.3787753 
               
               
                   
                   
                   
                 Neutralized-related C3HC4 RING domain 
               
               
                   
                   
                   
                 protein 
               
               
                 A_24_P659122 
                 AK125790 
                 LOC401357 
                 hypothetical LOC401357 
                 −2.383352 
               
             
          
           
               
                 Co-culture 6 days versus Co-culture 3 days 
               
             
          
           
               
                 A_23_P259314 
                 NM_001008 
                 RPS4Y1 
                 ribosomal protein S4, Y-linked 1″ 
                 −12.6156694 
               
               
                 A_23_P324384 
                 NM_001039567 
                 RPS4Y2 
                 ribosomal protein S4, Y-linked 2 
                 −11.6134898 
               
               
                 A_23_P254944 
                 NM_000853 
                 GSTT1 
                 glutathione S-transferase theta 1 
                 −9.3861013 
               
               
                 A_23_P217797 
                 AF000984 
                 DDX3Y 
                 DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, 
                 −8.8324792 
               
               
                   
                   
                   
                 Y-linked″ 
               
               
                 A_23_P73848 
                 NR_001544 
                 CYorf14 
                 chromosome Y open reading frame 14 
                 −6.8855352 
               
               
                 A_24_P325205 
                 NM_003471 
                 KCNAB1 
                 potassium voltage-gated channel, shaker- 
                 −6.6730979 
               
               
                   
                   
                   
                 related subfamily, beta member 1″ 
               
               
                 A_23_P364792 
                 NM_001005852 
                 CYorf15A 
                 chromosome Y open reading frame 15A 
                 −6.5728944 
               
               
                 A_24_P237511 
                 NM_004681 
                 EIF1AY 
                 eukaryotic translation initiation factor 
                 −6.5474298 
               
               
                   
                   
                   
                 1A, Y-linked″ 
               
               
                 A_23_P121441 
                 NM_014893 
                 NLGN4Y 
                 neuroligin 4, Y-linked″ 
                 −6.5044705 
               
               
                 A_23_P152002 
                 NM_004049 
                 BCL2A1 
                 BCL2-related protein A1 
                 −6.238545 
               
               
                 A_23_P113613 
                 NM_022842 
                 CDCP1 
                 CUB domain containing protein 1 
                 −6.0755863 
               
               
                 A_23_P44494 
                 NM_003471 
                 KCNAB1 
                 potassium voltage-gated channel, shaker- 
                 −6.025817 
               
               
                   
                   
                   
                 related subfamily, beta member 1″ 
               
               
                 A_23_P149345 
                 NM_015967 
                 PTPN22 
                 protein tyrosine phosphatase, non-receptor 
                 −5.8213383 
               
               
                   
                   
                   
                 type 22 (lymphoid)″ 
               
               
                 A_24_P319001 
                 NM_000853 
                 GSTT1 
                 glutathione S-transferase theta 1 
                 −5.7133297 
               
               
                 A_24_P182929 
                 NM_003471 
                 KCNAB1 
                 potassium voltage-gated channel, shaker- 
                 −5.677885 
               
               
                   
                   
                   
                 related subfamily, beta member 1″ 
               
               
                 A_23_P33903 
                 NM_014893 
                 NLGN4Y 
                 neuroligin 4, Y-linked″ 
                 −5.4683848 
               
               
                 A_24_P942743 
                 NM_003411 
                 ZFY 
                 zinc finger protein, Y-linked 
                 −5.3774487 
               
               
                 A_23_P139881 
                 NM_001759 
                 CCND2 
                 cyclin D2 
                 −5.3197285 
               
               
                 A_23_P150457 
                 NM_006691 
                 LYVE1 
                 lymphatic vessel endothelial hyaluronan 
                 −5.106593 
               
               
                   
                   
                   
                 receptor 1 
               
               
                 A_23_P400449 
                 NM_020927 
                 VAT1L 
                 vesicle amine transport protein 1 homolog 
                 −5.0158587 
               
               
                   
                   
                   
                 ( T. californica )-like 
               
               
                 A_24_P306443 
                 NM_001033515 
                 LOC100132288 
                 hypothetical protein LOC100132288 
                 −5.0061309 
               
               
                 A_23_P383009 
                 NM_000599 
                 IGFBP5 
                 insulin-like growth factor binding protein 5 
                 −4.994765 
               
               
                 A_23_P80570 
                 NM_001086 
                 AADAC 
                 arylacetamide deacetylase (esterase) 
                 −4.8682788 
               
               
                 A_23_P138524 
                 NM_198148 
                 CPXM2 
                 carboxypeptidase X (M14 family), member 2″ 
                 −4.849019 
               
               
                 A_23_P56505 
                 NM_000885 
                 ITGA4 
                 integrin, alpha 4 (antigen CD49D, alpha 4 
                 −4.6922746 
               
               
                   
                   
                   
                 subunit of VLA-4 receptor)″ 
               
               
                 A_32_P231179 
                 NM_144705 
                 TEKT4 
                 tektin 4 
                 −4.66489 
               
               
                 A_23_P96658 
                 ENST00000382832 
                 CYorf15B 
                 chromosome Y open reading frame 15B 
                 −4.6436677 
               
               
                 A_23_P66798 
                 NM_002276 
                 KRT19 
                 keratin 19 
                 −4.519458 
               
               
                 A_24_P160401 
                 NM_178181 
                 CDCP1 
                 CUB domain containing protein 1 
                 −4.4753457 
               
               
                 A_24_P216625 
                 NR_001544 
                 CYorf14 
                 chromosome Y open reading frame 14 
                 −4.4421405 
               
               
                 A_23_P314755 
                 NM_003155 
                 STC1 
                 stanniocalcin 1 
                 −4.3293859 
               
               
                 A_23_P1682 
                 NM_138788 
                 TMEM45B 
                 transmembrane protein 45B 
                 −4.2804522 
               
               
                 A_24_P49260 
                 NM_018327 
                 SPTLC3 
                 serine palmitoyltransferase, long chain 
                 −4.2327729 
               
               
                   
                   
                   
                 base subunit 3 
               
               
                 A_23_P74609 
                 NM_015714 
                 G0S2 
                 G0/G1switch 2 
                 −4.173994 
               
               
                 A_23_P89871 
                 NM_018355 
                 ZNF415 
                 zinc finger protein 415 
                 −4.1417134 
               
               
                 A_32_P224302 
                 NM_003436 
                 ZNF135 
                 zinc finger protein 135 
                 −4.0770383 
               
               
                 A_32_P94199 
                 BC068588 
                 LOC653071 
                 similar to CG32820-PA, isoform A 
                 −4.021826 
               
               
                 A_24_P307993 
                 BC035312 
                 CYorf15B 
                 chromosome Y open reading frame 15B 
                 −3.9682909 
               
               
                 A_23_P121987 
                 NM_033035 
                 TSLP 
                 thymic stromal lymphopoietin 
                 −3.9430309 
               
               
                 A_23_P201181 
                 NM_012411 
                 PTPN22 
                 protein tyrosine phosphatase, non-receptor 
                 −3.9010589 
               
               
                   
                   
                   
                 type 22 (lymphoid)″ 
               
               
                 A_32_P55840 
                 ENST00000377186 
                 LOC730405 
                 hypothetical protein LOC730405 
                 −3.862708 
               
               
                 A_24_P245379 
                 NM_002575 
                 SERPINB2 
                 serpin peptidase inhibitor, clade B 
                 −3.852809 
               
               
                   
                   
                   
                 (ovalbumin), member 2″ 
               
               
                 A_23_P4953 
                 NM_018215 
                 PNMAL1 
                 PNMA-like 1 
                 −3.8066147 
               
               
                 A_23_P421664 
                 NM_006366 
                 CAP2 
                 CAP, adenylate cyclase-associated protein, 
                 −3.709496 
               
               
                   
                   
                   
                 2 (yeast)″ 
               
               
                 A_23_P419714 
                 NM_001018072 
                 BTBD11 
                 BTB (POZ) domain containing 11 
                 −3.6847212 
               
               
                 A_23_P329835 
                 NM_007125 
                 UTY 
                 ubiquitously transcribed tetratricopeptide 
                 −3.674434 
               
               
                   
                   
                   
                 repeat gene, Y-linked″ 
               
               
                 A_24_P389415 
                 NM_007257 
                 PNMA2 
                 paraneoplastic antigen MA2 
                 −3.635188 
               
               
                 A_23_P371039 
                 NM_002531 
                 NTSR1 
                 neurotensin receptor 1 (high affinity) 
                 −3.5936048 
               
               
                 A_23_P361448 
                 NM_144665 
                 SESN3 
                 sestrin 3 
                 −3.5924191 
               
               
                 A_32_P34844 
                 NM_199355 
                 ADAMTS18 
                 ADAM metallopeptidase with thrombospondin 
                 −3.567005 
               
               
                   
                   
                   
                 type 1 motif, 18″ 
               
               
                 A_24_P296808 
                 NM_018215 
                 PNMAL1 
                 PNMA-like 1 
                 −3.5341394 
               
               
                 A_23_P422911 
                 NM_153456 
                 HS6ST3 
                 heparan sulfate 6-O-sulfotransferase 3 
                 −3.5049952 
               
               
                 A_32_P114003 
                 NR_024360 
                 LOC100192378 
                 hypothetical LOC100192378 
                 −3.4973402 
               
               
                 A_23_P166109 
                 NM_198391 
                 FLRT3 
                 fibronectin leucine rich transmembrane 
                 −3.461738 
               
               
                   
                   
                   
                 protein 3 
               
               
                 A_23_P348227 
                 NM_003436 
                 ZNF135 
                 zinc finger protein 135 
                 −3.4533736 
               
               
                 A_23_P350001 
                 NM_000855 
                 GUCY1A2 
                 guanylate cyclase 1, soluble, alpha 2″ 
                 −3.4059908 
               
               
                 A_23_P420863 
                 NM_022162 
                 NOD2 
                 nucleotide-binding oligomerization domain 
                 −3.4018255 
               
               
                   
                   
                   
                 containing 2 
               
               
                 A_23_P353865 
                 AB041269 
                 KRT19P2 
                 keratin 19 pseudogene 2 
                 −3.3981624 
               
               
                 A_23_P64539 
                 NM_000559 
                 HBG1 
                 hemoglobin, gamma A″ 
                 −3.3592979 
               
               
                 A_23_P97402 
                 NM_020439 
                 CAMK1G 
                 calcium/calmodulin-dependent protein 
                 −3.3406473 
               
               
                   
                   
                   
                 kinase IG 
               
               
                 A_23_P15004 
                 NM_199355 
                 ADAMTS18 
                 ADAM metallopeptidase with thrombospondin 
                 −3.338263 
               
               
                   
                   
                   
                 type 1 motif, 18″ 
               
               
                 A_23_P117104 
                 NM_001651 
                 AQP5 
                 aquaporin 5 
                 −3.320025 
               
               
                 A_23_P137238 
                 NM_004653 
                 JARID1D 
                 jumonji, AT rich interactive domain 1D″ 
                 −3.27148 
               
               
                 A_32_P80245 
                 NM_001109809 
                 ZFP57 
                 zinc finger protein 57 homolog (mouse) 
                 −3.2573022 
               
               
                 A_23_P395438 
                 NM_053044 
                 HTRA3 
                 HtrA serine peptidase 3 
                 −3.230038 
               
               
                 A_23_P217379 
                 NM_033641 
                 COL4A6 
                 collagen, type IV, alpha 6″ 
                 −3.206919 
               
               
                 A_32_P181222 
                 NM_002247 
                 KCNMA1 
                 potassium large conductance calcium- 
                 −3.1611311 
               
               
                   
                   
                   
                 activated channel, subfamily M, alpha 
               
               
                   
                   
                   
                 member 1″ 
               
               
                 A_23_P53137 
                 NM_000559 
                 HBG1 
                 hemoglobin, gamma A″ 
                 −3.1572033 
               
               
                 A_23_P154037 
                 NM_001159 
                 AOX1 
                 aldehyde oxidase 1 
                 −3.156966 
               
               
                 A_23_P112698 
                 NM_007257 
                 PNMA2 
                 paraneoplastic antigen MA2 
                 −3.1515593 
               
               
                 A_23_P49376 
                 NM_000078 
                 CETP 
                 cholesteryl ester transfer protein, 
                 −3.1096273 
               
               
                   
                   
                   
                 plasma″ 
               
               
                 A_23_P378555 
                 NM_152615 
                 PARP15 
                 poly (ADP-ribose) polymerase family, 
                 −3.0923947 
               
               
                   
                   
                   
                 member 15 
               
               
                 A_23_P160004 
                 NM_182660 
                 UTY 
                 ubiquitously transcribed tetratricopeptide 
                 −3.086247 
               
               
                   
                   
                   
                 repeat gene, Y-linked″ 
               
               
                 A_23_P318881 
                 NM_170601 
                 SIAE 
                 sialic acid acetylesterase 
                 −3.074874 
               
               
                 A_23_P112482 
                 NM_004925 
                 AQP3 
                 aquaporin 3 (Gill blood group) 
                 −3.0721986 
               
               
                 A_23_P434398 
                 NM_153235 
                 TXLNB 
                 taxilin beta 
                 −3.023871 
               
               
                 A_23_P48414 
                 NM_003914 
                 CCNA1 
                 cyclin A1 
                 −3.0115854 
               
               
                 A_23_P369899 
                 NM_015444 
                 TMEM158 
                 transmembrane protein 158 
                 −3.0086165 
               
               
                 A_24_P39919 
                 NM_023926 
                 ZSCAN18 
                 zinc finger and SCAN domain containing 18 
                 −3.002447 
               
               
                 A_23_P69171 
                 NM_033050 
                 SUCNR1 
                 succinate receptor 1 
                 −3.0015972 
               
               
                 A_32_P100830 
                 NM_153209 
                 KIF19 
                 kinesin family member 19 
                 −2.9953816 
               
               
                 A_24_P917819 
                 DQ179139 
                 C21orf99 
                 chromosome 21 open reading frame 99 
                 −2.9928046 
               
               
                 A_24_P290709 
                 CR593166 
                 TOM1L1 
                 target of myb1 (chicken)-like 1 
                 −2.990506 
               
               
                 A_23_P10542 
                 NM_053044 
                 HTRA3 
                 HtrA serine peptidase 3 
                 −2.9631009 
               
               
                 A_32_P215700 
                 NM_181643 
                 C1orf88 
                 chromosome 1 open reading frame 88 
                 −2.9483571 
               
               
                 A_24_P339429 
                 NM_021012 
                 KCNJ12 
                 potassium inwardly-rectifying channel, 
                 −2.945337 
               
               
                   
                   
                   
                 subfamily J, member 12″ 
               
               
                 A_23_P84063 
                 NM_016522 
                 NTM 
                 neurotrimin 
                 −2.939568 
               
               
                 A_32_P83098 
                 NM_000336 
                 SCNN1B 
                 sodium channel, nonvoltage-gated 1, beta″ 
                 −2.9331773 
               
               
                 A_23_P57658 
                 NM_020386 
                 HRASLS 
                 HRAS-like suppressor 
                 −2.909311 
               
               
                 A_23_P114084 
                 NM_000444 
                 PHEX 
                 phosphate regulating endopeptidase 
                 −2.887279 
               
               
                   
                   
                   
                 homolog, X-linked″ 
               
               
                 A_23_P39550 
                 NM_030923 
                 TMEM163 
                 transmembrane protein 163 
                 −2.8791509 
               
               
                 A_23_P153185 
                 NM_002575 
                 SERPINB2 
                 serpin peptidase inhibitor, clade B 
                 −2.873689 
               
               
                   
                   
                   
                 (ovalbumin), member 2″ 
               
               
                 A_23_P404016 
                 BC026362 
                 KIF19 
                 kinesin family member 19 
                 −2.8724893 
               
               
                 A_23_P136116 
                 NM_001004320 
                 TMEM195 
                 transmembrane protein 195 
                 −2.8718006 
               
               
                 A_32_P68103 
                 NM_012409 
                 PRND 
                 prion protein 2 (dublet) 
                 −2.8585701 
               
               
                 A_24_P66233 
                 NR_001543 
                 TTTY14 
                 testis-specific transcript, Y-linked 14″ 
                 −2.8562426 
               
               
                 A_32_P204795 
                 ENST00000299997 
                 LOC100128252 
                 similar to MGC9913 protein 
                 −2.8463146 
               
               
                 A_23_P28948 
                 NM_014012 
                 REM1 
                 RAS (RAD and GEM)-like GTP-binding 1 
                 −2.846291 
               
               
                 A_23_P324754 
                 NM_018689 
                 KIAA1199 
                 KIAA1199 
                 −2.8399187 
               
               
                 A_23_P144746 
                 NM_182594 
                 ZNF454 
                 zinc finger protein 454 
                 −2.8371706 
               
               
                 A_23_P106405 
                 NM_002487 
                 NDN 
                 necdin homolog (mouse) 
                 −2.834055 
               
               
                 A_23_P118493 
                 NM_005486 
                 TOM1L1 
                 target of myb1 (chicken)-like 1 
                 −2.8302737 
               
               
                 A_23_P54100 
                 NM_001437 
                 ESR2 
                 estrogen receptor 2 (ER beta) 
                 −2.8253245 
               
               
                 A_23_P361085 
                 NR_003038 
                 SNHG5 
                 small nucleolar RNA host gene 5 (non- 
                 −2.820832 
               
               
                   
                   
                   
                 protein coding) 
               
               
                 A_23_P205164 
                 NM_006237 
                 POU4F1 
                 POU class 4 homeobox 1 
                 −2.8180525 
               
               
                 A_32_P225472 
                 XM_001125792 
                 LOC727834 
                 hypothetical LOC727834 
                 −2.7696979 
               
               
                 A_23_P371145 
                 NM_138430 
                 ADPRHL1 
                 ADP-ribosylhydrolase like 1 
                 −2.7319997 
               
               
                 A_23_P161439 
                 NM_006829 
                 C10orf116 
                 chromosome 10 open reading frame 116 
                 −2.714516 
               
               
                 A_24_P357847 
                 BC030813 
                 IGK@ 
                 immunoglobulin kappa locus 
                 −2.7117672 
               
               
                 A_23_P397285 
                 NM_017527 
                 LY6K 
                 lymphocyte antigen 6 complex, locus K″ 
                 −2.7007713 
               
               
                 A_24_P33982 
                 NM_001085423 
                 C17orf60 
                 chromosome 17 open reading frame 60 
                 −2.699036 
               
               
                 A_23_P55682 
                 NM_023926 
                 ZSCAN18 
                 zinc finger and SCAN domain containing 18 
                 −2.692261 
               
               
                 A_23_P150394 
                 NM_022003 
                 FXYD6 
                 FXYD domain containing ion transport 
                 −2.6878983 
               
               
                   
                   
                   
                 regulator 6 
               
               
                 A_23_P17663 
                 NM_002462 
                 MX1 
                 myxovirus (influenza virus) resistance 1, 
                 −2.687721 
               
               
                   
                   
                   
                 interferon-inducible protein p78 (mouse)″ 
               
               
                 A_23_P142075 
                 NM_001611 
                 ACP5 
                 acid phosphatase 5, tartrate resistant″ 
                 −2.673893 
               
               
                 A_32_P449517 
                 NM_001033515 
                 LOC100132288 
                 hypothetical protein LOC100132288 
                 −2.663306 
               
               
                 A_24_P324883 
                 AK097143 
                 FLJ39824 
                 hypothetical LOC441173 
                 −2.6557348 
               
               
                 A_23_P127220 
                 NM_021800 
                 DNAJC12 
                 DnaJ (Hsp40) homolog, subfamily C, member 
                 −2.655188 
               
               
                   
                   
                   
                 12″ 
               
               
                 A_23_P501010 
                 NM_000494 
                 COL17A1 
                 collagen, type XVII, alpha 1″ 
                 −2.6486354 
               
               
                 A_23_P150768 
                 NM_007256 
                 SLC02B1 
                 solute carrier organic anion transporter 
                 −2.6455366 
               
               
                   
                   
                   
                 family, member 2B1″ 
               
               
                 A_32_P70315 
                 NM_003256 
                 TIMP4 
                 TIMP metallopeptidase inhibitor 4 
                 −2.6388437 
               
               
                 A_23_P120125 
                 NM_199235 
                 COLEC11 
                 collectin sub-family member 11 
                 −2.5952846 
               
               
                 A_23_P8640 
                 NM_001039966 
                 GPER 
                 G protein-coupled estrogen receptor 1 
                 −2.576825 
               
               
                 A_23_P115726 
                 NM_194298 
                 SLC16A9 
                 solute carrier family 16, member 9 
                 −2.5672209 
               
               
                   
                   
                   
                 (monocarboxylic acid transporter 9)″ 
               
               
                 A_24_P48204 
                 NM_003004 
                 SECTM1 
                 secreted and transmembrane 1 
                 −2.5662345 
               
               
                 A_32_P107876 
                 NM_025074 
                 FRAS1 
                 Fraser syndrome 1 
                 −2.5492005 
               
               
                 A_32_P148118 
                 XM_001717196 
                 LOC642424 
                 similar to hCG1742442 
                 −2.537786 
               
               
                 A_23_P357101 
                 NM_145298 
                 APOBEC3F 
                 apolipoprotein B mRNA editing enzyme, 
                 −2.532397 
               
               
                   
                   
                   
                 catalytic polypeptide-like 3F″ 
               
               
                 A_23_P360754 
                 NM_005099 
                 ADAMTS4 
                 ADAM metallopeptidase with thrombospondin 
                 −2.527619 
               
               
                   
                   
                   
                 type 1 motif, 4″ 
               
               
                 A_24_P196658 
                 NM_005486 
                 TOM1L1 
                 target of myb1 (chicken)-like 1 
                 −2.5056678 
               
               
                 A_23_P38630 
                 NM_001050 
                 SSTR2 
                 somatostatin receptor 2 
                 −2.505605 
               
               
                 A_23_P119886 
                 NM_001486 
                 GCKR 
                 glucokinase (hexokinase 4) regulator 
                 −2.4917698 
               
               
                 A_23_P120931 
                 NM_014508 
                 APOBEC3C 
                 apolipoprotein B mRNA editing enzyme, 
                 −2.486985 
               
               
                   
                   
                   
                 catalytic polypeptide-like 3C 
               
               
                 A_23_P212050 
                 NM_000055 
                 BCHE 
                 butyrylcholinesterase 
                 −2.485476 
               
               
                 A_24_P339858 
                 NR_026547 
                 C21orf90 
                 chromosome 21 open reading frame 90 
                 −2.4830492 
               
               
                 A_23_P417261 
                 NM_144715 
                 EFHB 
                 EF-hand domain family, member B″ 
                 −2.4813671 
               
               
                 A_24_P402242 
                 NM_000090 
                 COL3A1 
                 collagen, type III, alpha 1″ 
                 −2.457087 
               
               
                 A_24_P323148 
                 NM_182573 
                 LYPD5 
                 LY6/PLAUR domain containing 5 
                 −2.4320639 
               
               
                 A_23_P60210 
                 NM_006911 
                 RLN1 
                 relaxin 1 
                 −2.4130411 
               
               
                 A_23_P43095 
                 NM_024721 
                 ZFHX4 
                 zinc finger homeobox 4 
                 −2.4117598 
               
               
                 A_23_P258612 
                 NM_016529 
                 ATP8A2 
                 ATPase, aminophospholipid transporter- 
                 −2.400947 
               
               
                   
                   
                   
                 like, class I, type 8A, member 2″ 
               
               
                 A_23_P397293 
                 NM_017527 
                 LY6K 
                 lymphocyte antigen 6 complex, locus K″ 
                 −2.389667 
               
               
                 A_23_P254816 
                 NM_004609 
                 TCF15 
                 transcription factor 15 (basic helix-loop- 
                 −2.37409 
               
               
                   
                   
                   
                 helix) 
               
               
                 A_32_P225092 
                 NM_019590 
                 KIAA1217 
                 KIAA1217 
                 −2.3737608 
               
               
                 A_24_P684186 
                 NR_003955 
                 LOC647121 
                 embigin homolog (mouse) pseudogene 
                 −2.3651279 
               
               
                 A_23_P133408 
                 NM_000758 
                 CSF2 
                 colony stimulating factor 2 (granulocyte- 
                 −2.3637897 
               
               
                   
                   
                   
                 macrophage) 
               
               
                 A_32_P138348 
                 NM_017527 
                 LY6K 
                 lymphocyte antigen 6 complex, locus K″ 
                 −2.3624065 
               
               
                 A_23_P155755 
                 NM_002993 
                 CXCL6 
                 chemokine (C-X-C motif) ligand 6 
                 −2.3263602 
               
               
                   
                   
                   
                 (granulocyte chemotactic protein 2) 
               
               
                 A_23_P71379 
                 NM_005672 
                 PSCA 
                 prostate stem cell antigen 
                 −2.320595 
               
               
                 A_23_P101193 
                 NM_001080467 
                 MYO5B 
                 myosin VB 
                 −2.320276 
               
               
                 A_23_P143713 
                 NM_021822 
                 APOBEC3G 
                 apolipoprotein B mRNA editing enzyme, 
                 −2.316103 
               
               
                   
                   
                   
                 catalytic polypeptide-like 3G″ 
               
               
                 A_23_P52323 
                 NM_000494 
                 COL17A1 
                 collagen, type XVII, alpha 1″ 
                 −2.3158622 
               
               
                 A_24_P40721 
                 NM_018327 
                 SPTLC3 
                 serine palmitoyltransferase, long chain 
                 −2.3155127 
               
               
                   
                   
                   
                 base subunit 3 
               
               
                 A_24_P142503 
                 NM_018242 
                 SLC47A1 
                 solute carrier family 47, member 1″ 
                 −2.3143373 
               
               
                 A_24_P81900 
                 NM_006931 
                 SLC2A3 
                 solute carrier family 2 (facilitated 
                 −2.31389 
               
               
                   
                   
                   
                 glucose transporter), member 3″ 
               
               
                 A_23_P376704 
                 NM_198289 
                 CIDEA 
                 cell death-inducing DFFA-like effector a 
                 −2.3045821 
               
               
                 A_24_P272146 
                 BC067092 
                 IGKC 
                 immunoglobulin kappa constant 
                 −2.3027578 
               
               
                 A_23_P161563 
                 NM_022337 
                 RAB38 
                 RAB38, member RAS oncogene family″ 
                 −2.2882872 
               
               
                 A_23_P121657 
                 NM_005114 
                 HS3ST1 
                 heparan sulfate (glucosamine) 3-O- 
                 −2.273005 
               
               
                   
                   
                   
                 sulfotransferase 1 
               
               
                 A_23_P48217 
                 NM_030817 
                 APOLD1 
                 apolipoprotein L domain containing 1 
                 −2.27031 
               
               
                 A_23_P86012 
                 NM_001017402 
                 LAMB3 
                 laminin, beta 3″ 
                 −2.252338 
               
               
                 A_23_P160968 
                 NM_018891 
                 LAMC2 
                 laminin, gamma 2″ 
                 −2.2487942 
               
               
                 A_23_P134734 
                 NM_017786 
                 GOLSYN 
                 Golgi-localized protein 
                 −2.2441505 
               
               
                 A_23_P217901 
                 NM_001126312 
                 RP11- 
                 KAT protein 
                 −2.219194 
               
               
                   
                   
                 544M22.4 
               
               
                 A_23_P88819 
                 NM_017458 
                 MVP 
                 major vault protein 
                 −2.210613 
               
               
                 A_23_P111126 
                 L06175 
                 HCP5 
                 HLA complex P5 
                 −2.203004 
               
               
                 A_23_P160720 
                 NM_018664 
                 BATF3 
                 basic leucine zipper transcription factor, 
                 −2.202483 
               
               
                   
                   
                   
                 ATF-like 3 
               
               
                 A_24_P173754 
                 NM_030806 
                 C1orf21 
                 chromosome 1 open reading frame 21 
                 −2.202426 
               
               
                 A_24_P388786 
                 NM_001369 
                 DNAH5 
                 dynein, axonemal, heavy chain 5 
                 −2.1940723 
               
               
                 A_23_P407096 
                 NM_152625 
                 ZNF366 
                 zinc finger protein 366 
                 −2.1921406 
               
               
                 A_23_P131935 
                 NM_017671 
                 FERMT1 
                 fermitin family homolog 1 ( Drosophila ) 
                 −2.1760775 
               
               
                 A_23_P381645 
                 NM_001005463 
                 EBF3 
                 early B-cell factor 3 
                 −2.1604936 
               
               
                 A_23_P125705 
                 NM_021963 
                 NAP1L2 
                 nucleosome assembly protein 1-like 2 
                 −2.159929 
               
               
                 A_23_P422350 
                 NM_000260 
                 MYO7A 
                 myosin VIIA 
                 −2.1547 
               
               
                 A_23_P398476 
                 NM_022658 
                 HOXC8 
                 homeobox C8 
                 −2.1503644 
               
               
                 A_23_P210581 
                 NM_002237 
                 KCNG1 
                 potassium voltage-gated channel, subfamily 
                 −2.144751 
               
               
                   
                   
                   
                 G, member 1″ 
               
               
                 A_32_P206415 
                 NM_001008781 
                 FAT3 
                 FAT tumor suppressor homolog 3 
                 −2.1277075 
               
               
                   
                   
                   
                 ( Drosophila ) 
               
               
                 A_23_P149121 
                 NM_004675 
                 DIRAS3 
                 DIRAS family, GTP-binding RAS-like 3 
                 −2.125239 
               
               
                 A_24_P944588 
                 NM_033196 
                 ZNF682 
                 zinc finger protein 682 
                 −2.1232018 
               
               
                 A_23_P171132 
                 NM_021783 
                 EDA2R 
                 ectodysplasin A2 receptor 
                 −2.1180549 
               
               
                 A_23_P104555 
                 NM_020349 
                 ANKRD2 
                 ankyrin repeat domain 2 (stretch 
                 −2.112261 
               
               
                   
                   
                   
                 responsive muscle) 
               
               
                 A_23_P69206 
                 NM_003043 
                 SLC6A6 
                 solute carrier family 6 (neurotransmitter 
                 −2.110186 
               
               
                   
                   
                   
                 transporter, taurine), member 6″ 
               
               
                 A_23_P145054 
                 NM_001085480 
                 FAM162B 
                 family with sequence similarity 162, 
                 −2.1101041 
               
               
                   
                   
                   
                 member B″ 
               
               
                 A_23_P40217 
                 NM_018431 
                 DOK5 
                 docking protein 5 
                 −2.109531 
               
               
                 A_23_P139123 
                 NM_000062 
                 SERPING1 
                 serpin peptidase inhibitor, clade G (C1 
                 −2.098983 
               
               
                   
                   
                   
                 inhibitor), member 1″ 
               
               
                 A_24_P95059 
                 NM_007155 
                 ZP3 
                 zona pellucida glycoprotein 3 (sperm 
                 −2.097852 
               
               
                   
                   
                   
                 receptor) 
               
               
                 A_23_P48438 
                 NM_199162 
                 ADPRHL1 
                 ADP-ribosylhydrolase like 1 
                 −2.097145 
               
               
                 A_23_P94319 
                 NM_014867 
                 KBTBD11 
                 kelch repeat and BTB (POZ) domain 
                 −2.087171 
               
               
                   
                   
                   
                 containing 11 
               
               
                 A_23_P160214 
                 NM_001080494 
                 TTC39A 
                 tetratricopeptide repeat domain 39A 
                 −2.084316 
               
               
                 A_24_P291231 
                 NM_016831 
                 PER3 
                 period homolog 3 ( Drosophila ) 
                 −2.0800868 
               
               
                 A_23_P101623 
                 NM_022103 
                 ZNF667 
                 zinc finger protein 667 
                 −2.0795908 
               
               
                 A_23_P207221 
                 NM_018242 
                 SLC47A1 
                 solute carrier family 47, member 1″ 
                 −2.0635317 
               
               
                 A_24_P81789 
                 NM_019034 
                 RHOF 
                 ras homolog gene family, member F (in 
                 −2.055134 
               
               
                   
                   
                   
                 filopodia) 
               
               
                 A_23_P85952 
                 NM_024901 
                 DENND2D 
                 DENN/MADD domain containing 2D 
                 −2.0337004 
               
               
                 A_23_P16953 
                 NM_000867 
                 HTR2B 
                 5-hydroxytryptamine (serotonin) receptor 2B 
                 −2.033488 
               
               
                 A_23_P76538 
                 NM_017899 
                 TESC 
                 tescalcin 
                 −2.0306527 
               
               
                 A_23_P381431 
                 NM_030769 
                 NPL 
                 N-acetylneuraminate pyruvate lyase 
                 −2.0289607 
               
               
                   
                   
                   
                 (dihydrodipicolinate synthase) 
               
               
                 A_23_P145024 
                 NM_000024 
                 ADRB2 
                 adrenergic, beta-2-, receptor, surface″ 
                 −2.0257697 
               
               
                 A_23_P169351 
                 NM_003026 
                 SH3GL2 
                 SH3-domain GRB2-like 2 
                 −2.0247844 
               
               
                 A_23_P152047 
                 NM_138967 
                 SCAMP5 
                 secretory carrier membrane protein 5 
                 −2.023881 
               
               
                 A_23_P388168 
                 NM_002867 
                 RAB3B 
                 RAB3B, member RAS oncogene family″ 
                 −2.0154913 
               
               
                 A_23_P201497 
                 NM_182663 
                 RASSF5 
                 Ras association (RalGDS/AF-6) domain 
                 −2.0139146 
               
               
                   
                   
                   
                 family member 5 
               
               
                 A_24_P925342 
                 AB209275 
                 MAN1C1 
                 mannosidase, alpha, class 1C, member 1 
                 −2.004328 
               
               
                 A_23_P130376 
                 NM_022068 
                 FAM38B 
                 family with sequence similarity 38, member B 
                 −2.000109 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
           
               
                 SUPPLEMENTARY TABLE 4 
               
             
             
               
                   
               
               
                 Up-regulated genes in the microarray. 
               
             
          
           
               
                 Unique ID 
                 Target ID 
                 Gene Symbol 
                 Gene Name 
                 M Value 
               
               
                   
               
             
          
           
               
                 Co-culture 6 days versus Mono-culture 6 days 
               
             
          
           
               
                 A_24_P940694 
                 AK091400 
                 SLC44A5 
                 solute carrier family 44, member 5 
                 2.0041642 
               
               
                 A_24_P171141 
                 AF130049 
                 LOC114227 
                 hypothetical protein LOC114227 
                 2.008274 
               
               
                 A_23_P393401 
                 NR_003610 
                 PDXDC2 
                 pyridoxal-dependent decarboxylase domain 
                 2.0142204 
               
               
                   
                   
                   
                 containing 2 
               
               
                 A_23_P80570 
                 NM_001086 
                 AADAC 
                 arylacetamide deacetylase 
                 2.0229129 
               
               
                 A_23_P81721 
                 NM_004277 
                 SLC25A27 
                 solute carrier family 25, member 27″ 
                 2.0271153 
               
               
                 A_24_P419087 
                 NM_006576 
                 AVIL 
                 advillin 
                 2.0292216 
               
               
                 A_23_P360542 
                 NR_023925 
                 C18orf2 
                 chromosome 18 open reading frame 2 
                 2.037899 
               
               
                 A_23_P346048 
                 NR_002824 
                 HERC2P2 
                 hect domain and RLD 2 pseudogene 2 
                 2.041409 
               
               
                 A_23_P60811 
                 NM_006252 
                 PRKAA2 
                 protein kinase, AMP-activated, alpha 2 
                 2.0519602 
               
               
                   
                   
                   
                 catalytic subunit″ 
               
               
                 A_24_P222237 
                 NR_002824 
                 HERC2P2 
                 hect domain and RLD 2 pseudogene 2 
                 2.065124 
               
               
                 A_23_P207493 
                 NM_016424 
                 CROP 
                 cisplatin resistance-associated 
                 2.070006 
               
               
                   
                   
                   
                 overexpressed protein 
               
               
                 A_32_P110550 
                 AB042555 
                 PDE4DIP 
                 phosphodiesterase 4D interacting protein 
                 2.0739637 
               
               
                 A_23_P340308 
                 NM_176888 
                 TAS2R48 
                 taste receptor, type 2, member 48 
                 2.0754272 
               
               
                 A_24_P60217 
                 AK055730 
                 SLC23A3 
                 solute carrier family 23 (nucleobase 
                 2.0777667 
               
               
                   
                   
                   
                 transporters), member 3″ 
               
               
                 A_24_P940725 
                 AL080186 
                 SFRS18 
                 splicing factor, arginine/serine-rich 18 
                 2.0808406 
               
               
                 A_24_P98161 
                 NM_194455 
                 KRIT1 
                 KRIT1, ankyrin repeat containing 
                 2.0865297 
               
               
                 A_23_P28246 
                 NM_144712 
                 SLC23A3 
                 solute carrier family 23 (nucleobase 
                 2.0946742 
               
               
                   
                   
                   
                 transporters), member 3″ 
               
               
                 A_23_P54447 
                 BC069765 
                 C15orf5 
                 chromosome 15 open reading frame 5 
                 2.0985755 
               
               
                 A_24_P453855 
                 AK126267 
                 PNPLA7 
                 patatin-like phospholipase domain 
                 2.108414 
               
               
                   
                   
                   
                 containing 7 
               
               
                 A_32_P169491 
                 AK098200 
                 LOC161527 
                 hypothetical protein LOC161527 
                 2.1091287 
               
               
                 A_24_P341985 
                 NM_031938 
                 BC02 
                 beta-carotene oxygenase 2 
                 2.1149247 
               
               
                 A_32_P468289 
                 AL162056 
                 DOPEY1 
                 dopey family member 1 
                 2.1163766 
               
               
                 A_24_P50368 
                 NM_001001786 
                 BLID 
                 BH3-like motif containing, cell death 
                 2.1165517 
               
               
                   
                   
                   
                 inducer″ 
               
               
                 A_24_P928522 
                 AK025142 
                 DST 
                 dystonin 
                 2.126594 
               
               
                 A_32_P216872 
                 BX647358 
                 PDXDC2 
                 pyridoxal-dependent decarboxylase domain 
                 2.1297436 
               
               
                   
                   
                   
                 containing 2 
               
               
                 A_23_P303851 
                 NM_176886 
                 TAS2R45 
                 taste receptor, type 2, member 45 
                 2.1319761 
               
               
                 A_32_P211080 
                 NR_002824 
                 HERC2P2 
                 hect domain and RLD 2 pseudogene 2 
                 2.141752 
               
               
                 A_23_P75071 
                 NM_016195 
                 KIF20B 
                 kinesin family member 20B 
                 2.1492891 
               
               
                 A_23_P354308 
                 AK025204 
                 ABI3BP 
                 ABI family, member 3 (NESH) binding 
                 2.1495505 
               
               
                   
                   
                   
                 protein″ 
               
               
                 A_24_P8200 
                 AB095943 
                 SHPRH 
                 SNF2 histone linker PHD RING helicase 
                 2.1648123 
               
               
                 A_24_P93754 
                 AB018323 
                 JMJD2C 
                 jumonji domain containing 2C 
                 2.166888 
               
               
                 A_23_P257164 
                 NM_000481 
                 AMT 
                 aminomethyltransferase 
                 2.169648 
               
               
                 A_32_P139738 
                 NR_002827 
                 HERC2P4 
                 hect domain and RLD 2 pseudogene 4 
                 2.171488 
               
               
                 A_24_P925158 
                 D26122 
                 SF1 
                 splicing factor 1 
                 2.1740255 
               
               
                 A_32_P78385 
                 BC094802 
                 DPY19L2P2 
                 dpy-19-like 2 pseudogene 2 ( C. elegans ) 
                 2.1745228 
               
               
                 A_23_P306511 
                 BC022302 
                 CMAH 
                 cytidine monophosphate-N-acetylneuraminic 
                 2.1938505 
               
               
                   
                   
                   
                 acid hydroxylase (CMP-N-acetylneuraminate 
               
               
                   
                   
                   
                 monooxygenase) pseudogene 
               
               
                 A_23_P36865 
                 NM_025114 
                 CEP290 
                 centrosomal protein 290 kDa 
                 2.1972585 
               
               
                 A_32_P48244 
                 ENST00000358296 
                 ZNF100 
                 zinc finger protein 100 
                 2.2173142 
               
               
                 A_24_P932416 
                 NM_001123228 
                 TMEM14E 
                 transmembrane protein 14E 
                 2.227468 
               
               
                 A_23_P31681 
                 AK074467 
                 C8orf38 
                 chromosome 8 open reading frame 38 
                 2.2299826 
               
               
                 A_23_P8961 
                 NM_000880 
                 IL7 
                 interleukin 7 
                 2.23187 
               
               
                 A_32_P195850 
                 NM_173812 
                 DPY19L2 
                 dpy-19-like 2 ( C. elegans ) 
                 2.2422097 
               
               
                 A_24_P910580 
                 NM_181077 
                 GOLGA8A 
                 golgi autoantigen, golgin subfamily a, 
                 2.2559056 
               
               
                   
                   
                   
                 8A″ 
               
               
                 A_24_P463973 
                 AK123878 
                 MEG3 
                 maternally expressed 3 (non-protein 
                 2.2698766 
               
               
                   
                   
                   
                 coding) 
               
               
                 A_32_P75559 
                 AK303593 
                 BST2 
                 bone marrow stromal cell antigen 2 
                 2.272775 
               
               
                 A_23_P253622 
                 AK024934 
                 ANKRD36B 
                 ankyrin repeat domain 36B 
                 2.291946 
               
               
                 A_23_P340312 
                 NM_176888 
                 TAS2R48 
                 taste receptor, type 2, member 48 
                 2.2991645 
               
               
                 A_23_P115192 
                 NM_031282 
                 FCRL4 
                 Fc receptor-like 4 
                 2.3092101 
               
               
                 A_24_P51067 
                 NM_025114 
                 CEP290 
                 centrosomal protein 290 kDa 
                 2.3149905 
               
               
                 A_24_P265177 
                 AK022791 
                 PHC3 
                 polyhomeotic homolog 3 ( Drosophila ) 
                 2.3190363 
               
               
                 A_32_P156373 
                 XM_001715393 
                 LOC100132218 
                 hypothetical protein LOC100132218 
                 2.3305676 
               
               
                 A_32_P208733 
                 AK055279 
                 UTP23 
                 UTP23, small subunit (SSU) processome 
                 2.3333396 
               
               
                   
                   
                   
                 component, homolog (yeast)″ 
               
               
                 A_24_P114249 
                 NM_004482 
                 GALNT3 
                 UDP-N-acetyl-alpha-D-galactosamine: 
                 2.3560943 
               
               
                   
                   
                   
                 polypeptide N-acetylgalactosaminyltransferase 3 
               
               
                   
                   
                   
                 (GalNAc-T3) 
               
               
                 A_23_P374250 
                 NM_173812 
                 DPY19L2 
                 dpy-19-like 2 ( C. elegans ) 
                 2.3676346 
               
               
                 A_24_P687305 
                 NR_024583 
                 DKFZp434K191 
                 POM121 membrane glycoprotein-like 1 
                 2.3832968 
               
               
                   
                   
                   
                 pseudogene 
               
               
                 A_23_P426305 
                 NM_003734 
                 AOC3 
                 amine oxidase, copper containing 3 
                 2.4014171 
               
               
                   
                   
                   
                 (vascular adhesion protein 1)″ 
               
               
                 A_23_P331072 
                 BX647210 
                 LRRIQ3 
                 leucine-rich repeats and IQ motif 
                 2.4346236 
               
               
                   
                   
                   
                 containing 3 
               
               
                 A_23_P125748 
                 NM_032441 
                 ZMAT1 
                 zinc finger, matrin type 1″ 
                 2.438655 
               
               
                 A_23_P37623 
                 NM_181077 
                 GOLGA8A 
                 golgi autoantigen, golgin subfamily a, 
                 2.445613 
               
               
                   
                   
                   
                 8A″ 
               
               
                 A_24_P128442 
                 NM_152380 
                 TBX15 
                 T-box 15 
                 2.4838432 
               
               
                 A_32_P776863 
                 NM_173649 
                 C2orf61 
                 chromosome 2 open reading frame 61 
                 2.4990112 
               
               
                 A_23_P52121 
                 NM_002614 
                 PDZK1 
                 PDZ domain containing 1 
                 2.5010229 
               
               
                 A_23_P253524 
                 NM_001813 
                 CENPE 
                 centromere protein E, 312 kDa″ 
                 2.541844 
               
               
                 A_23_P385911 
                 ENST00000396791 
                 KIAA1712 
                 KIAA1712 
                 2.5994333 
               
               
                 A_23_P124805 
                 AK001243 
                 VPS13C 
                 vacuolar protein sorting 13 homolog C 
                 2.6165174 
               
               
                   
                   
                   
                 ( S. cerevisiae ) 
               
               
                 A_32_P122136 
                 AK057596 
                 LOC150759 
                 hypothetical protein LOC150759 
                 2.667798 
               
               
                 A_24_P246841 
                 NM_004277 
                 SLC25A27 
                 solute carrier family 25, member 27″ 
                 2.7717525 
               
               
                 A_23_P23611 
                 NM_001008219 
                 AMY1C 
                 amylase, alpha 1C (salivary) 
                 2.7829946 
               
               
                 A_23_P353614 
                 NM_152765 
                 C8orf46 
                 chromosome 8 open reading frame 46 
                 2.801072 
               
               
                 A_24_P916797 
                 AK000270 
                 AKAP9 
                 A kinase (PRKA) anchor protein (yotiao) 9 
                 2.8275501 
               
               
                 A_24_P303420 
                 AK126092 
                 LOC221442 
                 hypothetical LOC221442 
                 2.8380903 
               
               
                 A_24_P391591 
                 AK057596 
                 LOC150759 
                 hypothetical protein LOC150759 
                 2.871928 
               
               
                 A_24_P11100 
                 NM_032441 
                 ZMAT1 
                 zinc finger, matrin type 1″ 
                 2.8772666 
               
               
                 A_23_P51587 
                 NM_002924 
                 RGS7 
                 regulator of G-protein signaling 7 
                 2.9475363 
               
               
                 A_23_P414793 
                 NM_000096 
                 CP 
                 ceruloplasmin (ferroxidase) 
                 2.9993575 
               
               
                 A_24_P85258 
                 NM_001080484 
                 KIAA1751 
                 KIAA1751 
                 3.0383245 
               
               
                 A_24_P344890 
                 AK095605 
                 AMY2B 
                 amylase, alpha 2B (pancreatic) 
                 3.0388725 
               
               
                 A_23_P302060 
                 NM_176891 
                 IFNE 
                 interferon, epsilon″ 
                 3.0941112 
               
               
                 A_24_P53595 
                 NM_016592 
                 GNAS 
                 GNAS complex locus 
                 3.2041835 
               
               
                 A_24_P310256 
                 NM_139284 
                 LGI4 
                 leucine-rich repeat LGI family, member 4″ 
                 3.3231205 
               
               
                 A_24_P332081 
                 AL832756 
                 JAKMIP3 
                 janus kinase and microtubule interacting 
                 3.339777 
               
               
                   
                   
                   
                 protein 3 
               
               
                 A_23_P203191 
                 NM_000039 
                 APOA1 
                 apolipoprotein A-I 
                 3.3997842 
               
               
                 A_24_P341000 
                 AK092698 
                 FLJ35379 
                 similar to Alu subfamily J sequence 
                 3.597919 
               
               
                   
                   
                   
                 contamination warning entry 
               
               
                 A_32_P9941 
                 NM_007191 
                 WIF1 
                 WNT inhibitory factor 1 
                 3.8778475 
               
               
                 A_32_P197561 
                 NM_024007 
                 EBF1 
                 early B-cell factor 1 
                 4.4216269 
               
             
          
           
               
                 Co-culture 6 days versus Co-culture 3 days 
               
             
          
           
               
                 A_24_P500584 
                 NR_001564 
                 XIST 
                 X (inactive)-specific transcript (non- 
                 12.5931163 
               
               
                   
                   
                   
                 protein coding) 
               
               
                 A_23_P155786 
                 NM_005420 
                 SULT1E1 
                 sulfotransferase family 1E, estrogen- 
                 8.2046563 
               
               
                   
                   
                   
                 preferring, member 1″ 
               
               
                 A_23_P95790 
                 NM_017625 
                 ITLN1 
                 intelectin 1 (galactofuranose binding) 
                 8.0198263 
               
               
                 A_23_P60130 
                 NM_052886 
                 MAL2 
                 mal, T-cell differentiation protein 2″ 
                 7.5120488 
               
               
                 A_32_P179138 
                 NM_001130683 
                 GUCY1A3 
                 guanylate cyclase 1, soluble, alpha 3″ 
                 6.6513906 
               
               
                 A_23_P350005 
                 NM_173553 
                 TRIML2 
                 tripartite motif family-like 2 
                 5.6112492 
               
               
                 A_23_P43164 
                 NM_015170 
                 SULF1 
                 sulfatase 1 
                 5.589013 
               
               
                 A_24_P213161 
                 NM_017852 
                 NLRP2 
                 NLR family, pyrin domain containing 2″ 
                 5.5529553 
               
               
                 A_23_P129085 
                 NM_145658 
                 SPESP1 
                 sperm equatorial segment protein 1 
                 5.2953218 
               
               
                 A_23_P69573 
                 NM_000856 
                 GUCY1A3 
                 guanylate cyclase 1, soluble, alpha 3″ 
                 5.2366296 
               
               
                 A_24_P53778 
                 NM_080878 
                 ITLN2 
                 intelectin 2 
                 5.2284345 
               
               
                 A_24_P75917 
                 NM_182568 
                 CCDC144B 
                 coiled-coil domain containing 144B 
                 5.1691916 
               
               
                 A_32_P154911 
                 NM_175887 
                 PRR15 
                 proline rich 15 
                 5.15205 
               
               
                 A_23_P67847 
                 NM_024572 
                 GALNT14 
                 UDP-N-acetyl-alpha-D-galactosamine: 
                 4.879263 
               
               
                   
                   
                   
                 polypeptide N-acetylgalactosaminyltransferase 14 
               
               
                   
                   
                   
                 (GalNAc-T14) 
               
               
                 A_24_P13041 
                 NM_145307 
                 RTKN2 
                 rhotekin 2 
                 4.6814901 
               
               
                 A_23_P155688 
                 NM_021114 
                 SPINK2 
                 serine peptidase inhibitor, Kazal type 2 
                 4.66596 
               
               
                   
                   
                   
                 (acrosin-trypsin inhibitor) 
               
               
                 A_24_P288915 
                 AK093811 
                 CCDC144B 
                 coiled-coil domain containing 144B 
                 4.6400898 
               
               
                 A_23_P371729 
                 NM_005266 
                 GJA5 
                 gap junction protein, alpha 5, 40 kDa″ 
                 4.6273297 
               
               
                 A_23_P15450 
                 NM_018286 
                 TMEM100 
                 transmembrane protein 100 
                 4.5045573 
               
               
                 A_23_P36531 
                 NM_004616 
                 TSPAN8 
                 tetraspanin 8 
                 4.4025021 
               
               
                 A_23_P403445 
                 NM_006569 
                 CGREF1 
                 cell growth regulator with EF-hand domain 1 
                 4.239469 
               
               
                 A_23_P56746 
                 NM_004460 
                 FAP 
                 fibroblast activation protein, alpha″ 
                 4.1927154 
               
               
                 A_24_P288890 
                 NM_181709 
                 FAM101A 
                 family with sequence similarity 101, member A 
                 4.065982 
               
               
                 A_23_P52410 
                 NM_145307 
                 RTKN2 
                 rhotekin 2 
                 4.0503195 
               
               
                 A_23_P95029 
                 NM_021021 
                 SNTB1 
                 syntrophin, beta 1 (dystrophin-associated 
                 4.0177255 
               
               
                   
                   
                   
                 protein A1, 59 kDa, basic component 1)″ 
               
               
                 A_23_P406385 
                 NM_153350 
                 FBXL16 
                 F-box and leucine-rich repeat protein 16 
                 4.0115285 
               
               
                 A_23_P105144 
                 NM_020974 
                 SCUBE2 
                 signal peptide, CUB domain, EGF-like 2 
                 4.007575 
               
               
                 A_32_P200697 
                 NM_181709 
                 FAM101A 
                 family with sequence similarity 101, member A 
                 3.966588 
               
               
                 A_23_P215459 
                 NM_000501 
                 ELN 
                 elastin 
                 3.9293356 
               
               
                 A_23_P58676 
                 NM_024563 
                 C5orf23 
                 chromosome 5 open reading frame 23 
                 3.8902445 
               
               
                 A_23_P217917 
                 NM_147148 
                 GSTM4 
                 glutathione S-transferase mu 4 
                 3.8893845 
               
               
                 A_32_P181077 
                 NM_203447 
                 DOCK8 
                 dedicator of cytokinesis 8 
                 3.8843326 
               
               
                 A_23_P257649 
                 NM_002899 
                 RBP1 
                 retinol binding protein 1, cellular″ 
                 3.8630243 
               
               
                 A_23_P253536 
                 NM_000908 
                 NPR3 
                 natriuretic peptide receptor C/guanylate 
                 3.7955647 
               
               
                   
                   
                   
                 cyclase C (atrionatriuretic peptide 
               
               
                   
                   
                   
                 receptor C) 
               
               
                 A_23_P69497 
                 NM_003278 
                 CLEC3B 
                 C-type lectin domain family 3, member B″ 
                 3.7919 
               
               
                 A_23_P327451 
                 NM_000908 
                 NPR3 
                 natriuretic peptide receptor C/guanylate 
                 3.7844863 
               
               
                   
                   
                   
                 cyclase C (atrionatriuretic peptide 
               
               
                   
                   
                   
                 receptor C) 
               
               
                 A_23_P421401 
                 NM_002609 
                 PDGFRB 
                 platelet-derived growth factor receptor, 
                 3.7825935 
               
               
                   
                   
                   
                 beta polypeptide″ 
               
               
                 A_23_P19369 
                 NM_017640 
                 LRRC16A 
                 leucine rich repeat containing 16A 
                 3.7317835 
               
               
                 A_24_P164505 
                 BC098294 
                 FAM106A 
                 family with sequence similarity 106, member 
                 3.7136457 
               
               
                   
                   
                   
                 A″ 
               
               
                 A_24_P639679 
                 AK095831 
                 SNORD123 
                 small nucleolar RNA, C/D box 123″ 
                 3.660561 
               
               
                 A_24_P369232 
                 NM_031455 
                 CCDC3 
                 coiled-coil domain containing 3 
                 3.6385524 
               
               
                 A_23_P69738 
                 NM_023940 
                 RASL11B 
                 RAS-like, family 11, member B 
                 3.5668816 
               
               
                 A_24_P738168 
                 ENST00000329798 
                 FREM3 
                 FRAS1 related extracellular matrix 3 
                 3.5534517 
               
               
                 A_23_P31273 
                 NM_001635 
                 AMPH 
                 amphiphysin 
                 3.5149605 
               
               
                 A_23_P132956 
                 NM_004181 
                 UCHL1 
                 ubiquitin carboxyl-terminal esterase L1 
                 3.496619 
               
               
                   
                   
                   
                 (ubiquitin thiolesterase) 
               
               
                 A_23_P93737 
                 NM_004411 
                 DYNC1I1 
                 dynein, cytoplasmic 1, intermediate chain 1″ 
                 3.4706085 
               
               
                 A_23_P121926 
                 NM_005410 
                 SEPP1 
                 selenoprotein P, plasma, 1″ 
                 3.439935 
               
               
                 A_23_P63736 
                 BC007394 
                 MGC16291 
                 hypothetical protein MGC16291 
                 3.4054182 
               
               
                 A_23_P144911 
                 NM_152403 
                 EGFLAM 
                 EGF-like, fibronectin type III and laminin 
                 3.3878002 
               
               
                   
                   
                   
                 G domains 
               
               
                 A_23_P37727 
                 NM_002996 
                 CX3CL1 
                 chemokine (C-X3-C motif) ligand 1 
                 3.38521 
               
               
                 A_23_P49391 
                 NM_016212 
                 TP53TG3 
                 TP53 target 3 
                 3.3187182 
               
               
                 A_23_P79251 
                 NM_014600 
                 EHD3 
                 EH-domain containing 3 
                 3.2961135 
               
               
                 A_23_P386030 
                 AF028828 
                 SNTB1 
                 syntrophin, beta 1 (dystrophin-associated 
                 3.2353347 
               
               
                   
                   
                   
                 protein A1, 59 kDa, basic component 1)″ 
               
               
                 A_23_P83098 
                 NM_000689 
                 ALDH1A1 
                 aldehyde dehydrogenase 1 family, member A1″ 
                 3.212209 
               
               
                 A_23_P61967 
                 NM_014469 
                 RBMXL2 
                 RNA binding motif protein, X-linked-like 2″ 
                 3.1963621 
               
               
                 A_23_P47579 
                 NM_176822 
                 NLRP14 
                 NLR family, pyrin domain containing 14 
                 3.1881834 
               
               
                 A_24_P212021 
                 NM_000851 
                 GSTM5 
                 glutathione S-transferase mu 5 
                 3.18169 
               
               
                 A_23_P83134 
                 NM_002048 
                 GAS1 
                 growth arrest-specific 1 
                 3.1669307 
               
               
                 A_23_P79272 
                 NM_001003683 
                 PDE1A 
                 phosphodiesterase 1A, calmodulin-dependent″ 
                 3.161868 
               
               
                 A_23_P66827 
                 AK021862 
                 FAM106A 
                 family with sequence similarity 106, member A 
                 3.1335342 
               
               
                 A_24_P118196 
                 NM_001080393 
                 GLT8D4 
                 glycosyltransferase 8 domain containing 4 
                 3.1078747 
               
               
                 A_23_P92983 
                 NM_017614 
                 BHMT2 
                 betaine-homocysteine methyltransferase 2 
                 3.1073714 
               
               
                 A_32_P191840 
                 XM_933903 
                 LOC644662 
                 similar to hCG2042541 
                 3.0930806 
               
               
                 A_32_P16007 
                 NM_207355 
                 POTEB 
                 POTE ankyrin domain family, member B″ 
                 3.0563867 
               
               
                 A_32_P60065 
                 NM_004101 
                 F2RL2 
                 coagulation factor II (thrombin) receptor- 
                 3.017839 
               
               
                   
                   
                   
                 like 2 
               
               
                 A_23_P144718 
                 NM_004101 
                 F2RL2 
                 coagulation factor II (thrombin) receptor- 
                 2.988743 
               
               
                   
                   
                   
                 like 2 
               
               
                 A_23_P407497 
                 NM_013959 
                 NRG1 
                 neuregulin 1 
                 2.971679 
               
               
                 A_24_P270728 
                 NM_001042483 
                 NUPR1 
                 nuclear protein 1 
                 2.9712219 
               
               
                 A_23_P33326 
                 NM_000679 
                 ADRA1B 
                 adrenergic, alpha-1B-, receptor″ 
                 2.9130255 
               
               
                 A_23_P207003 
                 NM_004574 
                 38231 
                 septin 4 
                 2.9109967 
               
               
                 A_23_P420348 
                 NM_174981 
                 POTED 
                 POTE ankyrin domain family, member D″ 
                 2.9017281 
               
               
                 A_23_P167030 
                 NM_000316 
                 PTH1R 
                 parathyroid hormone 1 receptor 
                 2.8829803 
               
               
                 A_23_P54144 
                 NM_001202 
                 BMP 4 
                 bone morphogenetic protein 4 
                 2.87191 
               
               
                 A_23_P151805 
                 NM_006329 
                 FBLN5 
                 fibulin 5 
                 2.868258 
               
               
                 A_32_P471485 
                 BC025765 
                 RTKN2 
                 rhotekin 2 
                 2.8634051 
               
               
                 A_23_P333029 
                 NM_173549 
                 C8orf47 
                 chromosome 8 open reading frame 47 
                 2.8633264 
               
               
                 A_23_P26890 
                 NM_024302 
                 MMP28 
                 matrix metallopeptidase 28 
                 2.8536377 
               
               
                 A_23_P56328 
                 NM_031310 
                 PLVAP 
                 plasmalemma vesicle associated protein 
                 2.840474 
               
               
                 A_32_P109214 
                 NM_001004306 
                 MGC87631 
                 similar to hypothetical protein FLJ36492 
                 2.833538 
               
               
                 A_23_P113351 
                 NM_004684 
                 SPARCL1 
                 SPARC-like 1 (hevin) 
                 2.8273755 
               
               
                 A_23_P155596 
                 NM_001002294 
                 FMO3 
                 flavin containing monooxygenase 3 
                 2.8262236 
               
               
                 A_23_P114883 
                 NM_002023 
                 FMOD 
                 fibromodulin 
                 2.8169279 
               
               
                 A_24_P220485 
                 NM_182487 
                 OLFML2A 
                 olfactomedin-like 2A 
                 2.8071563 
               
               
                 A_24_P943588 
                 AF201385 
                 TXNRD2 
                 thioredoxin reductase 2 
                 2.7889967 
               
               
                 A_24_P246196 
                 NM_214675 
                 CLEC4M 
                 C-type lectin domain family 4, member M″ 
                 2.7829864 
               
               
                 A_23_P302672 
                 NM_145244 
                 DDIT4L 
                 DNA-damage-inducible transcript 4-like 
                 2.780097 
               
               
                 A_23_P75769 
                 NM_024021 
                 MS4A4A 
                 membrane-spanning 4-domains, subfamily A, 
                 2.7788735 
               
               
                   
                   
                   
                 member 4 
               
               
                 A_23_P64785 
                 NM_152320 
                 ZNF641 
                 zinc finger protein 641 
                 2.771647 
               
               
                 A_24_P292849 
                 AL137382 
                 LOC146429 
                 hypothetical protein LOC146429 
                 2.7697638 
               
               
                 A_32_P50066 
                 NM_001039580 
                 MAP9 
                 microtubule-associated protein 9 
                 2.764735 
               
               
                 A_23_P144916 
                 NM_005110 
                 GFPT2 
                 glutamine-fructose-6-phosphate transaminase 2 
                 2.745744 
               
               
                 A_23_P422831 
                 NM_004816 
                 C9orf61 
                 chromosome 9 open reading frame 61 
                 2.739364 
               
               
                 A_23_P428080 
                 AB020701 
                 KIAA0894 
                 KIAA0894 protein 
                 2.7391762 
               
               
                 A_23_P302568 
                 NM_003459 
                 SLC30A3 
                 solute carrier family 30 (zinc 
                 2.7326405 
               
               
                   
                   
                   
                 transporter), member 3 
               
               
                 A_23_P214168 
                 NM_004370 
                 COL12A1 
                 collagen, type XII, alpha 1″ 
                 2.724168 
               
               
                 A_23_P122924 
                 NM_002192 
                 INHBA 
                 inhibin, beta A″ 
                 2.7137955 
               
               
                 A_23_P29124 
                 NM_002688 
                 38596 
                 septin 5 
                 2.713282 
               
               
                 A_23_P360534 
                 NR_023925 
                 C18orf2 
                 chromosome 18 open reading frame 2 
                 2.7031812 
               
               
                 A_23_P214803 
                 NM_014841 
                 SNAP91 
                 synaptosomal-associated protein, 91 kDa 
                 2.7022004 
               
               
                   
                   
                   
                 homolog (mouse) 
               
               
                 A_32_P32413 
                 NM_015559 
                 SETBP1 
                 SET binding protein 1 
                 2.695718 
               
               
                 A_24_P291814 
                 NM_004370 
                 COL12A1 
                 collagen, type XII, alpha 1″ 
                 2.6940175 
               
               
                 A_24_P208436 
                 NM_001003683 
                 PDE1A 
                 phosphodiesterase 1A, calmodulin-dependent 
                 2.6746507 
               
               
                 A_23_P51587 
                 NM_002924 
                 RGS7 
                 regulator of G-protein signaling 7 
                 2.6739832 
               
               
                 A_23_P414793 
                 NM_000096 
                 CP 
                 ceruloplasmin (ferroxidase) 
                 2.6516814 
               
               
                 A_23_P110473 
                 NM_004536 
                 NAIP 
                 NLR family, apoptosis inhibitory protein″ 
                 2.6419156 
               
               
                 A_23_P138655 
                 NM_057157 
                 CYP26A1 
                 cytochrome P450, family 26, subfamily A, 
                 2.641847 
               
               
                   
                   
                   
                 polypeptide 1″ 
               
               
                 A_23_P215744 
                 NM_033427 
                 CTTNBP2 
                 cortactin binding protein 2 
                 2.6312279 
               
               
                 A_24_P712271 
                 NM_207328 
                 LOC150763 
                 hypothetical protein LOC150763 
                 2.6251041 
               
               
                 A_24_P221414 
                 NM_004411 
                 DYNC1I1 
                 dynein, cytoplasmic 1, intermediate chain 1″ 
                 2.6146931 
               
               
                 A_23_P372974 
                 NM_152402 
                 TRAM1L1 
                 translocation associated membrane protein 
                 2.6134994 
               
               
                   
                   
                   
                 1-like 1 
               
               
                 A_24_P222237 
                 NR_002824 
                 HERC2P2 
                 hect domain and RLD 2 pseudogene 2 
                 2.599507 
               
               
                 A_23_P116642 
                 NM_133489 
                 SLC26A10 
                 solute carrier family 26, member 10″ 
                 2.5993607 
               
               
                 A_23_P87879 
                 NM_001781 
                 CD69 
                 CD69 molecule 
                 2.5931544 
               
               
                 A_23_P4551 
                 NM_015559 
                 SETBP1 
                 SET binding protein 1 
                 2.592962 
               
               
                 A_23_P8820 
                 NM_001442 
                 FABP4 
                 fatty acid binding protein 4, adipocyte″ 
                 2.589049 
               
               
                 A_32_P211080 
                 NR_002824 
                 HERC2P2 
                 hect domain and RLD 2 pseudogene 2 
                 2.584551 
               
               
                 A_23_P371495 
                 NM_175861 
                 TMTC1 
                 transmembrane and tetratricopeptide repeat 
                 2.5771015 
               
               
                   
                   
                   
                 containing 1 
               
               
                 A_24_P218805 
                 NM_017409 
                 HOXC10 
                 homeobox C10 
                 2.5694865 
               
               
                 A_23_P121564 
                 NM_000857 
                 GUCY1B3 
                 guanylate cyclase 1, soluble, beta 3 
                 2.558513 
               
               
                 A_23_P126075 
                 NM_002245 
                 KCNK1 
                 potassium channel, subfamily K, member 1″ 
                 2.5456856 
               
               
                 A_24_P356916 
                 NM_001011554 
                 SLC13A3 
                 solute carrier family 13 (sodium-dependent 
                 2.5359893 
               
               
                   
                   
                   
                 dicarboxylate transporter), member 3″ 
               
               
                 A_24_P53465 
                 NM_214675 
                 CLEC4M 
                 C-type lectin domain family 4, member M″ 
                 2.5326324 
               
               
                 A_23_P152305 
                 NM_001797 
                 CDH11 
                 cadherin 11, type 2, OB-cadherin 
                 2.519575 
               
               
                   
                   
                   
                 (osteoblast)″ 
               
               
                 A_24_P184803 
                 NM_004086 
                 COCH 
                 coagulation factor C homolog, cochlin 
                 2.5007844 
               
               
                   
                   
                   
                 ( Limulus polyphemus )″ 
               
               
                 A_32_P34444 
                 NM_025135 
                 FHOD3 
                 formin homology 2 domain containing 3 
                 2.5001057 
               
               
                 A_23_P357571 
                 NM_000854 
                 GSTT2 
                 glutathione S-transferase theta 2 
                 2.490856 
               
               
                 A_23_P346048 
                 NR_002824 
                 HERC2P2 
                 hect domain and RLD 2 pseudogene 2 
                 2.479499 
               
               
                 A_23_P346093 
                 NM_152468 
                 TMC8 
                 transmembrane channel-like 8 
                 2.4763698 
               
               
                 A_23_P166797 
                 NM_022147 
                 RTP4 
                 receptor (chemosensory) transporter protein 4 
                 2.4744943 
               
               
                 A_23_P419696 
                 NM_144586 
                 LYPD1 
                 LY6/PLAUR domain containing 1 
                 2.4715535 
               
               
                 A_23_P204286 
                 NM_000900 
                 MGP 
                 matrix Gla protein 
                 2.468325 
               
               
                 A_32_P139738 
                 NR_002827 
                 HERC2P4 
                 hect domain and RLD 2 pseudogene 4 
                 2.457105 
               
               
                 A_23_P106933 
                 NM_052956 
                 ACSM1 
                 acyl-CoA synthetase medium-chain family 
                 2.4475286 
               
               
                   
                   
                   
                 member 1 
               
               
                 A_23_P904 
                 NM_024603 
                 BEND5 
                 BEN domain containing 5 
                 2.4432084 
               
               
                 A_23_P115161 
                 NM_002036 
                 DARC 
                 Duffy blood group, chemokine receptor″ 
                 2.4372559 
               
               
                 A_23_P27013 
                 NM_024017 
                 HOXB9 
                 homeobox B9 
                 2.4330936 
               
               
                 A_23_P142239 
                 AK027130 
                 LOC541469 
                 hypothetical protein LOC541469 
                 2.4050138 
               
               
                 A_24_P221327 
                 BC020847 
                 LOC644246 
                 hypothetical protein LOC644246 
                 2.395498 
               
               
                 A_23_P28334 
                 NM_003853 
                 IL18RAP 
                 interleukin 18 receptor accessory protein 
                 2.333673 
               
               
                 A_23_P259442 
                 NM_001873 
                 CPE 
                 carboxypeptidase E 
                 2.330546 
               
               
                 A_23_P302634 
                 BC101016 
                 C12orf64 
                 chromosome 12 open reading frame 64 
                 2.3142719 
               
               
                 A_23_P50697 
                 NM_006905 
                 PSG1 
                 pregnancy specific beta-1-glycoprotein 1 
                 2.3101452 
               
               
                 A_24_P579826 
                 BC071681 
                 ARL17 
                 ADP-ribosylation factor-like 17 
                 2.3073995 
               
               
                 A_23_P258769 
                 NM_002121 
                 HLA-DPB1 
                 major histocompatibility complex, class II, 
                 2.3068786 
               
               
                   
                   
                   
                 DP beta 1″ 
               
               
                 A_23_P52761 
                 NM_002423 
                 MMP7 
                 matrix metallopeptidase 7 (matrilysin, 
                 2.3065215 
               
               
                   
                   
                   
                 uterine)″ 
               
               
                 A_23_P257993 
                 NM_004944 
                 DNASE1L3 
                 deoxyribonuclease I-like 3 
                 2.304541 
               
               
                 A_23_P34126 
                 NM_001711 
                 BGN 
                 biglycan 
                 2.295382 
               
               
                 A_23_P19020 
                 NM_005460 
                 SNCAIP 
                 synuclein, alpha interacting protein″ 
                 2.294381 
               
               
                 A_24_P331830 
                 NM_015209 
                 RP1-21O18.1 
                 kazrin 
                 2.286087 
               
               
                 A_23_P337346 
                 AK056484 
                 hCG_2009921 
                 hypothetical locus LOC441204 
                 2.2788632 
               
               
                 A_23_P13094 
                 NM_002425 
                 MMP10 
                 matrix metallopeptidase 10 (stromelysin 2) 
                 2.2774393 
               
               
                 A_23_P6818 
                 NM_020163 
                 SEMA3G 
                 sema domain, immunoglobulin domain (Ig), 
                 2.2771295 
               
               
                   
                   
                   
                 short basic domain, secreted, (semaphorin) 3G 
               
               
                 A_24_P128442 
                 NM_152380 
                 TBX15 
                 T-box 15 
                 2.2662993 
               
               
                 A_24_P246406 
                 NM_001006605 
                 FAM69A 
                 family with sequence similarity 69, member A″ 
                 2.2629583 
               
               
                 A_23_P356494 
                 NM_006846 
                 SPINK5 
                 serine peptidase inhibitor, Kazal type 5″ 
                 2.2532257 
               
               
                 A_24_P64401 
                 BC007394 
                 MGC16291 
                 hypothetical protein MGC16291 
                 2.2515278 
               
               
                 A_24_P940694 
                 AK091400 
                 SLC44A5 
                 solute carrier family 44, member 5 
                 2.250309 
               
               
                 A_23_P206920 
                 NM_001040114 
                 MYH11 
                 myosin, heavy chain 11, smooth muscle 
                 2.2377084 
               
               
                 A_23_P16252 
                 NM_002257 
                 KLK1 
                 kallikrein 1 
                 2.2310541 
               
               
                 A_24_P231829 
                 NM_017614 
                 BHMT2 
                 betaine-homocysteine methyltransferase 2 
                 2.2256352 
               
               
                 A_24_P59799 
                 NM_024781 
                 CCDC102B 
                 coiled-coil domain containing 102B 
                 2.2234418 
               
               
                 A_23_P109427 
                 NM_000854 
                 GSTT2 
                 glutathione S-transferase theta 2 
                 2.21082 
               
               
                 A_24_P245589 
                 NM_031310 
                 PLVAP 
                 plasmalemma vesicle associated protein 
                 2.2015346 
               
               
                 A_23_P256033 
                 NM_001958 
                 EEF1A2 
                 eukaryotic translation elongation factor 1 
                 2.1877389 
               
               
                   
                   
                   
                 alpha 2 
               
               
                 A_23_P357207 
                 NM_138409 
                 MRAP2 
                 melanocortin 2 receptor accessory protein 2 
                 2.182037 
               
               
                 A_32_P2452 
                 NM_175861 
                 TMTC1 
                 transmembrane and tetratricopeptide repeat 
                 2.174506 
               
               
                   
                   
                   
                 containing 1 
               
               
                 A_23_P30075 
                 NM_006095 
                 ATP8A1 
                 ATPase, aminophospholipid transporter 
                 2.1730165 
               
               
                   
                   
                   
                 (APLT), class I, type 8A, member 1″ 
               
               
                 A_23_P423074 
                 NM_015566 
                 FAM169A 
                 family with sequence similarity 169, member A″ 
                 2.1701266 
               
               
                 A_24_P208595 
                 NM_053034 
                 ANTXR1 
                 anthrax toxin receptor 1 
                 2.1540107 
               
               
                 A_24_P273799 
                 ENST00000301042 
                 ZNF641 
                 zinc finger protein 641 
                 2.14752 
               
               
                 A_23_P370027 
                 AK124788 
                 GGT7 
                 gamma-glutamyltransferase 7 
                 2.1393684 
               
               
                 A_24_P231010 
                 NM_018995 
                 MOV10L1 
                 Mov10l1, Moloney leukemia virus 10-like 1, 
                 2.1283951 
               
               
                   
                   
                   
                 homolog (mouse)″ 
               
               
                 A_23_P204847 
                 NM_002298 
                 LCP1 
                 lymphocyte cytosolic protein 1 (L-plastin) 
                 2.1257206 
               
               
                 A_23_P116898 
                 NM_000014 
                 A2M 
                 alpha-2-macro globulin 
                 2.124616 
               
               
                 A_32_P24122 
                 NM_015894 
                 STMN3 
                 stathmin-like 3 
                 2.118763 
               
               
                 A_24_P192485 
                 NM_002546 
                 TNFRSF11B 
                 tumor necrosis factor receptor superfamily, 
                 2.103257 
               
               
                   
                   
                   
                 member 11b″ 
               
               
                 A_23_P39202 
                 NM_033520 
                 C19orf33 
                 chromosome 19 open reading frame 33 
                 2.0971413 
               
               
                 A_24_P396662 
                 NM_147148 
                 GSTM4 
                 glutathione S-transferase mu 4 
                 2.093116 
               
               
                 A_23_P122906 
                 NM_015570 
                 AUTS2 
                 autism susceptibility candidate 2 
                 2.0852408 
               
               
                 A_23_P217319 
                 NM_004114 
                 FGF13 
                 fibroblast growth factor 13 
                 2.0801625 
               
               
                 A_24_P380734 
                 NM_002998 
                 SDC2 
                 syndecan 2 
                 2.077774 
               
               
                 A_24_P363408 
                 NM_012259 
                 HEY2 
                 hairy/enhancer-of-split related with YRPW 
                 2.0719206 
               
               
                   
                   
                   
                 motif 2 
               
               
                 A_23_P44794 
                 NM_138453 
                 RAB3C 
                 RAB3C, member RAS oncogene family″ 
                 2.0644617 
               
               
                 A_23_P305198 
                 NM_003151 
                 STAT4 
                 signal transducer and activator of 
                 2.0440189 
               
               
                   
                   
                   
                 transcription 4 
               
               
                 A_23_P203957 
                 NM_175861 
                 TMTC1 
                 transmembrane and tetratricopeptide repeat 
                 2.039811 
               
               
                   
                   
                   
                 containing 1 
               
               
                 A_23_P27795 
                 NM_021102 
                 SPINT2 
                 serine peptidase inhibitor, Kunitz type, 2″ 
                 2.037252 
               
               
                 A_23_P372308 
                 NM_020211 
                 RGMA 
                 RGM domain family, member A″ 
                 2.034106 
               
               
                 A_32_P216566 
                 NM_001009994 
                 C6orf159 
                 chromosome 6 open reading frame 159 
                 2.0275353 
               
               
                 A_24_P246573 
                 NM_015209 
                 RP1-21O18.1 
                 kazrin 
                 2.0188723 
               
               
                 A_23_P349321 
                 NM_022166 
                 XYLT1 
                 xylosyltransferase I 
                 2.0100237 
               
               
                 A_24_P920525 
                 AK022468 
                 SORBS1 
                 sorbin and SH3 domain containing 1 
                 2.007313 
               
               
                   
               
             
          
         
       
     
       REFERENCES 
       [0000]    
       
         1. Cecchelli, R., et al. Modelling of the blood-brain barrier in drug discovery and development. Nat Rev Drug Discov 6, 650-661 (2007). 
         2. Weksler, B. B., et al. Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19, 1872-1874 (2005). 
         3. Sano, Y., et al. Establishment of a new conditionally immortalized human brain microvascular endothelial cell line retaining an in vivo blood-brain barrier function. J Cell Physiol 225, 519-528 (2010). 
         4. Lippmann, E. S., et al. Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells. Nat Biotechnol (2012). 
         5. Pedroso, D. C., et al. Improved survival, vascular differentiation and wound healing potential of stem cells co-cultured with endothelial cells. PLoS One 6, e16114 (2011). 
         6. Armulik, A., et al. Pericytes regulate the blood-brain barrier. Nature 468, 557-561 (2010). 
         7. Daneman, R., Zhou, L., Kebede, A. A. &amp; Barres, B. A. 
       
     
         [0125]    Pericytes are required for blood-brain barrier integrity during embryogenesis. Nature 468, 562-566 (2010).
   8. Vandenhaute, E., et al. Brain pericytes from stress-susceptible pigs increase blood-brain barrier permeability in vitro. Fluids Barriers CNS 9, 11 (2012).   9. Liebner, S., et al. Wnt/beta-catenin signaling controls development of the blood-brain barrier. J Cell Biol 183, 409-417 (2008).   10. Tatsuta, T., Naito, M., Oh-para, T., Sugawara, I. &amp; Tsuruo, T. Functional involvement of P-glycoprotein in blood-brain barrier. J Biol Chem 267, 20383-20391 (1992).   11. Bo, L., et al. Distribution of immunoglobulin superfamily members ICAM-1, -2, -3, and the beta 2 integrin LFA-1 in multiple sclerosis lesions. J Neuropathol Exp Neurol 55, 1060-1072 (1996).   12. Friden, M., Gupta, A., Antonsson, M., Bredberg, U. &amp; Hammarlund-Udenaes, M. In vitro methods for estimating unbound drug concentrations in the brain interstitial and intracellular fluids. Drug Metab Dispos 35, 1711-1719 (2007).   13. Alvarez, J. I., et al. The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence. Science 334, 1727-1731 (2011).   14. Daneman, R., et al. Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis. Proc Natl Acad Sci USA 106, 641-646 (2009).   15. Booher J, Sensenbrenner M. Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures. Neurobiology. 1972; 2(3):97-105.   16. Descamps L, Coisne C, Dehouck B, Cecchelli R, Torpier G. Protective effect of glial cells against lipopolysaccharide-mediated blood-brain barrier injury. Glia. 2003 Apr. 1; 42(1):46-58.   17. Meresse S, Dehouck M P, Delorme P, Bensaid M, Tauber J P, Delbart C, Fruchart J C, Cecchelli R. Bovine brain endothelial cells express tight junctions and monoamine oxidase activity in long-term culture. J Neurochem 53(5):1363-1371.1989.   18. Saeed A I, Bhagabati N K, Braisted J C, Liang W, Sharov V, Howe E A, Li J, Thiagarajan M, White J A, Qackenbush J. TM4 microarray suite software. Methods Enzymol. 2006(411): 134-193.   19. Sano Y, Shimizu F, Abe M, Maeda T, Kashiwamura Y, Ohtsuki S, Terasaki T, Obinata M, Kajiwara K, Fujii M, Suzuki M, Kanda T. Establisment of a new conditionally immortalized human brain microvascular endothelial cell line retaining an in vivo blood-brain barrier function. J Cell Physiol. 2010 (225): 519-528.   20. Siflinger-Birnboim A, Del Vecchio P J, Cooper J A, Blumenstock F A, Shepard J M, Malik A B. Molecular sieving characteristics of the cultured endothelial monolayer. J Cell Physiol. 1987; 132(1):111-117.   21. Simon R, Lam A, Li M C, Ngan M, Menenzes S, Zhao Y. Analysis of gene expression data using BRB-array tools. Cancer Inform. 2007(3): 11-17.