Patent Application: US-201414780653-A

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 .

Description:
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 . 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 fig1 a ). at this stage , cells have a cobblestone - like morphology and express high levels of endothelial cells markers , including cd31 , ve - cadherin and vwf ( supplementary fig1 b ). 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 fig1 c and 1d ). to induce bbb properties in cd34 +- derived endothelial cells , cells were seeded in a transwell ™ system and co - cultured with pericytes ( fig1 a ). pericytes were selected after a screening of different cell types from the neurovascular unit ( supplementary fig2 a 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 ( fig1 b ), maintaining its stability up to 20 days ( fig1 c ). 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 )) ( fig1 d ), they showed a continuous expression of zo - 1 , occludin , jam - a and claudin - 5 at cell - cell contacts ( fig1 e ) 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 ( fig1 f ). 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 fig3 b ) and in 3 different laboratories ( supplementary fig3 c ). furthermore , the bbb properties of cd34 +- derived endothelial cells are lost if the pericytes are removed from the co - culture system ( supplementary fig3 d ) 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 ( fig1 g ). 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 ( fig1 h ). as in hbecs , the receptor for advanced glycation end products ( rage ) and p - gp protein were expressed as confirmed by immunofluorescence ( fig1 i ), being rage located at the luminal side of cells ( supplementary fig3 a ). 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 ). 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 ( fig2 a ). 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 ( fig2 b ). in addition , blecs have the ability to form a monolayer that has a transendothelial electric resistance ( teer ) similar to monolayers of bovine becs ( supplementary fig4 a ) 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 fig4 b ). 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 )) ( fig2 c ). 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 . 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 )) ( fig2 d ). 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 . 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 fig5 ). 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 fig6 ). 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 fig6 ). 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 ( fig2 e ). 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 ( fig2 e and supplementary fig7 ). 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 ( fig2 f and supplementary fig5 ) and the localization of claudin - 1 at the cell - cell contacts ( fig2 f ). 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 . 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 fig8 ). 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 . 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 . 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 . 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 . 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 . 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 . differenciation of cd34 + derived endothelial cells into brain like endothelial cells by pericytes 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 . 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 . 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 ). 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 ). 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 . 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 . 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 . 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 . 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 . 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 %. 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 ). 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 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 . 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 ). 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 . 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 . 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 . 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 . 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 ). 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 . 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 . the above described particular embodiments are obviously combinable . the following claims set out particular embodiments of the disclosure . antibodies used for immunofluorescence □ , flow cytometry ★ and western blot ∘ . 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 ). 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 ; 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