# Patent

## Title
Methods of treating inflammatory colon diseases

## Abstract

A method of treating ulcerative colitis or Crohn's disease in a subject in need thereof is disclosed. The method comprising administering to the subject a therapeutically effective amount of adherent cells from a placenta or adipose tissue, thereby treating the ulcerative colitis or Crohn's disease.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Phase Application of International Application Number PCT/IL2009/000527, filed May 26, 2009, and claims the benefit of U.S. Provisional Application No. 61/071,944, filed May 27, 2008, the content of all of which are incorporated herein by reference.


FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to methods of treating inflammatory colon diseases using adherent cells from adipose or placenta tissues and, more particularly, but not exclusively, to methods of treating ulcerative colitis or Crohn's disease using the adherent cells.
In the developing medical world a growing need exists for large amounts of adult stem cells for the purpose of cell engraftment and tissue engineering. In addition, adult stem cell therapy is continuously developing for treating and curing various conditions such as hematopoietic disorders, heart disease, Parkinson's disease, Alzheimer's disease, stroke, burns, muscular dystrophy, autoimmune disorders, diabetes and arthritis.
In recent years, considerable activity has focused on the therapeutic potential of mesenchymal stromal cells (MSCs) for various medical applications including tissue repair of damaged organs such as the brain, heart, bone and liver and in support of bone marrow transplantations (BMT). MSCs, a heterogeneous population of cells obtained from e.g. bone marrow, adipose tissue, placenta, and blood, is capable of differentiating into different types of mesenchymal mature cells (e.g. reticular endothelial cells, fibroblasts, adipocytes, osteogenic precursor cells) depending upon influences from various bioactive factors. Accordingly, MSCs have been widely studied in regenerative medicine as the foundation to build new tissues such as bone, cartilage and fat for the repair of injury or replacement of pathologic tissues and as treatment for genetic and acquired diseases. Furthermore, the multipotent ability of MSCs, their easy isolation and culture, as well as their high ex vivo expansion potential make them an attractive therapeutic tool.
Inflammatory bowel disease (IBD), a group of inflammatory conditions of the large intestine and small intestine, includes Crohn's disease and ulcerative colitis and is a chronic, relapsing, and remitting condition of an unknown origin which affects at least 1 in 1,000 people in Western countries.
Crohn's disease (also known as granulomatous colitis and regional enteritis), an autoimmune disease caused by the immune system's attacking the gastrointestinal tract and producing inflammation in the gastrointestinal tract, is an inflammatory disease that may affect any part of the gastrointestinal tract from mouth to anus, causing a wide variety of symptoms. It primarily causes abdominal pain, diarrhea, vomiting and weight loss, but may also cause complications outside of the gastrointestinal tract such as skin rashes, arthritis and inflammation of the eye. There is currently no known drug or surgical cure for Crohn's disease and treatment options are restricted to controlling symptoms, maintaining remission and preventing relapse (e.g. 5-aminosalicylic acid (5-ASA) formulations, corticosteroids such as prednisone and hydrocortisone, and immunomodulators such as azathioprine and mercaptopurine).
Ulcerative colitis, a form of colitis, is a disease of the intestine, specifically the large intestine or colon that includes characteristic ulcers, or open sores, in the colon. The main symptom of active disease is usually constant diarrhea mixed with blood. Current treatment of ulcerative colitis includes anti-inflammatory drugs, immunosuppression, and biological therapy targeting specific components of the immune response. Colectomy (partial or total removal of the large bowel through surgery) is occasionally necessary, and is considered to be a cure for the disease.
Okamoto et al. [Okamoto et al., supra] and Matsumoto et al. [Matsumoto et al., Gastroenterology (2005) 128: 1851-1867] reported that bone-marrow-derived cells (BMDCs) can repopulate the epithelia of the human gastrointestinal tract after graft-versus-host disease or gastric ulcer formation following irradiation and bone marrow transplantation. Komori et al. 2005 [Komori et al., J Gastroenterol (2005) 40: 591-599] also reported transient increases in bone-marrow-derived mucosal epithelial cells and myofibroblasts during the healing process of gastric ulcers and trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. In addition, Osiris therapeutics (www.osiris.com) is evaluating Prochymal, a product derived from bone marrow MSCs, for the treatment of Crohn's disease. Osiris is currently conducting a multi-center trial to evaluate the safety and efficacy of Prochymal for Crohn's disease.
PCT Publication No. WO 2008/100498 discloses methods of treating immune-related diseases (e.g. inflammatory bowel disease, graft-versus-host disease) using placental stem cells or umbilical cord stem cells. The stem cells disclosed are derived from a mammalian placenta, regardless of morphology, cell surface markers or the number of passages after a primary culture, and adhere to a tissue culture substrate (e.g., tissue culture plastic or a fibronectin-coated tissue culture plate).
U.S. Publication No. 20080213227 discloses methods of treating autoimmune diseases and inflammatory diseases (e.g. inflammatory bowel disease and Crohn's disease) by administering mesenchymal stem cells in an effective amount. The mesenchymal cells disclosed may be obtained from adherent marrow or periosteal cells or alternatively from blood, skin, cord blood, muscle, fat, bone, or perichondrium.
PCT Publication No. WO 2007/108003 discloses methods of cell expansion, which comprise culturing adherent cells from placenta or adipose tissue under three-dimensional culturing conditions, which support cell expansion. Also provided are cells generated thereby and uses of same.
SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a method of treating ulcerative colitis or Crohn's disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of adherent cells from a placenta or adipose tissue, thereby treating the ulcerative colitis or Crohn's disease.
According to an aspect of some embodiments of the present invention there is provided a use of adherent cells from a placenta or adipose tissue for the manufacture of a medicament identified for treating ulcerative colitis or Crohn's disease.
According to an aspect of some embodiments of the present invention there is provided an article of manufacture comprising a packaging material which comprises a label for use in treating ulcerative colitis or Crohn's disease, the packaging material packaging a pharmaceutically effective amount of adherent cells from a placenta or adipose tissue.
According to some embodiments of the invention, the adherent cells comprise a positive marker expression selected from the group consisting of CD73, CD90, CD29 and CD105.
According to some embodiments of the invention, the adherent cells comprise a negative marker expression selected from the group consisting of CD3, CD4, CD45, CD80, HLA-DR, CD11b, CD14, CD19, CD34 and CD79.
According to some embodiments of the invention, the adherent cells are capable of suppressing an immune reaction.
According to some embodiments of the invention, suppressing an immune reaction comprises suppressing a T cell activity.
According to some embodiments of the invention, the adherent cells are obtained from a three-dimensional (3D) culture.
According to some embodiments of the invention, the three-dimensional (3D) culture comprises a 3D bioreactor.
According to some embodiments of the invention, culturing of the adherent cells in the 3D culture is effected under perfusion.
According to some embodiments of the invention, culturing of the adherent cells is effected for at least 3 days.
According to some embodiments of the invention, culturing of the adherent cells is effected until at least 10% of the adherent cells are proliferating.
According to some embodiments of the invention, the adherent cells comprise a gene expression profile as described in Table 11.
According to some embodiments of the invention, the adherent cells comprise cells cultured from the placenta or adipose tissue under 2 dimensional (2D) culturing conditions.
According to some embodiments of the invention, at least 12% of the adherent cells are at a S and/or G2/M proliferative phase.
According to some embodiments of the invention, the adherent cells comprise a gene expression profile as described in Table 8.
According to some embodiments of the invention, the adherent cells are less committed to an osteogenic lineage as compared to adherent cells from bone marrow grown and allowed to differentiate under the same conditions.
According to some embodiments of the invention, the adherent cells are less committed to an adipogenic lineage as compared to adherent cells from bone marrow grown and allowed to differentiate under the same conditions.
According to some embodiments of the invention, the article of manufacture further comprises an additional drug for treatment of colon inflammation.
According to some embodiments of the invention, the article of manufacture further comprises an immunosuppressant agent.
According to some embodiments of the invention, the article of manufacture further comprises an anti-inflammatory agent.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.



BRIEF DESCRIPTION OF THE DRAWINGS
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
FIGS. 1A-B are figures depicting cell cycle analysis of 2D adherent cells of placenta suitable for use in accordance with the present teachings (FIG. 1A) or adherent cells manufactured according to the teachings of WO/2007/108003, designated PLX (FIG. 1B). Cells were fixed in 70% EtOH O.N, centrifuged and re-suspended in a Propidium Iodide (PI) solution and then analyzed by FACS.
FIG. 2 is a bar graph depicting marker expression on 2D adherent cells of placenta suitable for use in accordance with the present teachings. Of note, negative expression was recorded for CD11b, CD34, HLA-DR, CD14, CD19 and CD45, while positive expression was noted for CD29, CD73, CD90 and CD105.
FIG. 3 is a bar graph depicting reduction of lymphocyte cell response by 2D adherent cells of placenta suitable for use in accordance with the present teachings. Peripheral blood (PB) derived mononuclear cells (MNCs) were stimulated with PHA (10 μg/ml). One of four different batches of 2D adherent cells were added to the stimulated MNCs. Three replicates of each group were seeded in 96-well plates.
FIGS. 4A-F are photographs depicting growth of bone marrow and placenta cells under osteogenesis or adipogenesis differentiation conditions. Bone marrow derived cells (FIGS. 4A-C) or placenta derived cells (FIGS. 4D-F) were plated in growth medium (FIGS. 4A and 4D), osteogenesis differentiation medium (FIGS. 4B and 4E) or adipogenesis differentiation medium (FIGS. 4C and 4F) in a 24 well plate coated with vitronectin and collagen. Medium was replaced every 3-4 days. At the end of growth period cells were fixed, stained and pictured as described in detail the Examples section which follows.
FIGS. 5A-F are photographs depicting growth of bone marrow and placenta cells under modified osteogenesis or adipogenesis differentiation conditions. Bone marrow derived cells (FIGS. 5A-C) or placenta derived cells (FIGS. 5D-F) were plated in growth medium (FIGS. 5A and 5D), osteogenesis differentiation medium (FIGS. 5B and 5E) or adipogenesis differentiation medium (FIGS. 5C and 5F) in a 24 well plate coated with vitronectin and collagen. Medium was replaced every 3-4 days. At the end of growth period cells were fixed, stained and pictured as described in detail the Examples section which follows.
FIGS. 6A-B depict cell cycle analysis of 3D adherent cells manufacture by Plurix (designated PLX, FIG. 6B) and by Celligen (PLX-C, FIG. 6A). Cells were fixed in 70% EtOH O.N, centrifuged and re-suspended in a Propidium Iodide (PI) solution and then analyzed by FACS.
FIGS. 7A-C depict expression of fibroblast-typical markers but not expression of endothelial typical markers on PLX-C. FIG. 7A depicts negative expression of the endothelial marker CD31; FIG. 7B depicts negative expression of the endothelial marker KDR; and FIG. 7C depicts positive expression of the human fibroblast marker (D7-FIB). Of note, the red histograms for Isotype IgG1 (FITC) represent the negative control while the blue histograms represent the positively stained cells.
FIGS. 8A-D depict expression of stimulatory and co-stimulatory molecules on PLX-C cells. FIG. 8A depicts PLX-C expression of CD80; FIG. 8B depicts PLX-C expression of CD86; FIG. 8C depicts PLX-C expression of CD40; and FIG. 8D depicts PLX-C expression of HLA-A/B/C. Negative controls were prepared with relevant isotype fluorescence molecules. Of note, red histograms indicate PLX-C marker-expressing population of cells, blue histograms indicate bone marrow (BM) marker-expressing population of cells, and green histograms indicate mononuclear cell (MNC) marker expressing population of cells.
FIGS. 9A-B depict inhibition of lymphocyte proliferation by PLX-C. FIG. 9A depicts Mixed Lymphocyte Reaction (MLR) tests performed with 2×105 peripheral blood (PB) derived mononuclear cells (MNC, donor A) stimulated with equal amount of irradiated (3000 Rad) PB derived MNCs (donor B) followed by addition of increasing amounts of PLX-C cells to the cultures. Three replicates of each group were seeded in 96-well plates. Proliferation rate was measured by [3H]thymidine incorporation; FIG. 9B depict peripheral blood (PB) derived MNCs stimulated with ConA (1.5 mg/ml). Increasing amounts of PLX-C cells were added to the cultures. Three replicates of each group were seeded in 96-well plates. Proliferation rate was measured by [3H]thymidine incorporation.
FIGS. 10A-C depict PLX-C regulation of pro-inflammatory and anti-inflammatory cytokine secretion following co-culture with peripheral blood cells. FIGS. 10A-B depict secretion of IFNγ (FIG. 10A) and TNFα (FIG. 10B) following co-culture of human derived MNCs (isolated from peripheral blood) stimulated with ConA with PLX-C; FIG. 10C depicts secretion of IFNγ, TNFα and IL-10 following co-culture of human derived MNCs (isolated from peripheral blood) stimulated with LPS with PLX-C. Supernatants were collected and subjected to cytokines analysis using ELISA.
FIG. 11 is a graph depicting macroscopic evaluation of colon tissues of colitic mice as represented by the Wallace score. TNBS (colitis model mice), TNBS+5-ASA (colitic mice who received the gold standard treatment), TNBS+2D adherent cells (batch 1) ip, TNBS+3D adherent cells (PLX-C, batch 2) ip, TNBS+2D adherent cells (batch 1) iv and TNBS+3D adherent cells (PLX-C, batch 2) iv. Macroscopic assessments were conducted blindly by two investigators.
FIG. 12 is a graph depicting microscopic evaluation of colon tissues of colitic mice as represented by the Ameho score. TNBS (colitis model mice), TNBS+5-ASA (colitic mice who received the gold standard treatment), TNBS+2D adherent cells (batch 1) ip, TNBS+3D adherent cells (PLX-C, batch 2) ip, TNBS+2D adherent cells (batch 1) iv and TNBS+3D adherent cells (PLX-C, batch 2) iv. Histological assessments were conducted blindly by two investigators.
FIG. 13 is a graph depicting IL-1β mRNA expression level in colonic tissues of colitic mice. Mice were rendered colitic by intrarectal administration of TNBS and were administered 2D or 3D (PLX-C) adherent cells via intraperitoneal or intravenous routes. Total RNA was isolated from colonic tissues of the different experimental groups and IL-1β expression levels were evaluated by RT-PCR.
FIG. 14 is a graph depicting microscopic evaluation of colon tissues of colitic rats. Rats were rendered colitic by intracolonic administration of TNBS and were administered PLX-C cells via intraperitoneal (ip) or intravenous (iv) routes.



DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to methods of treating inflammatory colon diseases using adherent cells from adipose or placenta tissues and, more particularly, but not exclusively, to methods of treating ulcerative colitis or Crohn's disease using the adherent cells.
The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
While reducing the present invention to practice, the present inventor has surprisingly uncovered that adherent cells from placental tissues may be used effectively to treat uleceritive colitis and Crohn's disease.
As is shown hereinbelow and in the Examples section which follows, the present inventor has uncovered through laborious experimentation that adherent cells obtained from placenta or adipose tissues and cultured under 2D (Example 2) or 3D culturing conditions (Examples 1 and 3) may be effectively used to treat colon inflammation, such as ulcerative colitis, as depicted in both mouse (Example 4) and rat (Example 5) experimental models. The present inventor has shown that intravenous (iv) or intraperitoneal (ip) administration of the 2D or 3D adherent cells of the present invention resulted in a major improvement in the inflammatory condition of the colon tissue as determined by both macroscopic and microscopic evaluations of the colon (FIGS. 11, 12 and 14). This anti-inflammatory effect was as efficient as the 5-ASA gold standard treatment. Taken together the present teachings portray an anti-inflammatory value for the adherent cells of the present invention and suggest the use of same for the treatment of inflammatory colon diseases such as ulcerative colitis and Crohn's disease.
Thus, according to one aspect of the present invention there is provided a method of treating ulcerative colitis or Crohn's disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of adherent cells from a placenta or adipose tissue, thereby treating the ulcerative colitis or Crohn's disease.
As used herein the term “treating” refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of ulcerative colitis or Crohn's disease. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
The term “ulcerative colitis” as used herein refers to a medical condition of the intestine, a form of inflammatory bowel disease (IBD), specifically of the large intestine or colon, that includes characteristic ulcers, or open sores, in the colon. Ulcerative colitis disease is usually diagnosed following recurrent symptoms of constant diarrhea mixed with blood, of gradual onset. Ulcerative colitis according to the present teachings refers to any stage or severity of ulcerative colitis (e.g. disease remission or acute disease).
The term “Crohn's disease” as used herein refers to an inflammatory condition that may affect any part of the gastrointestinal tract from mouth to anus, also known as granulomatous colitis or regional enteritis, and is a form of inflammatory bowel disease (IBD). Crohn's disease is a type of autoimmune disease and is usually diagnosed following recurrent symptoms of abdominal pain, diarrhea (which may be bloody), vomiting, weight loss, skin rashes, arthritis and inflammation of the eye. Crohn's disease according to the present teachings refers to any stage or severity of Crohn's disease (e.g. disease remission, acute disease, relapse).
As used herein the phrase “a subject in need thereof” refers to a mammal, preferably a human subject, male or female of any age, who has been diagnosed with probable or definite ulcerative colitis or Crohn's disease, e.g., a subject who experienced inflammatory colon disease. The diagnosis of ulcerative colitis or Crohn's disease may include any diagnosis test as, for example, laboratory tests, endoscopic evaluation, biopsies of the mucosa (for ulcerative colitis), barium follow-through x-ray (for Crohn's disease), and CT or MRI scans (for Crohn's disease).
It will be appreciated that the present invention also envision treatment of other colon inflammatory conditions, including, but are not limited to, chronic inflammatory intestinal diseases (Garcia Herola A. et al., Gastroenterol. Hepatol. 2000 January; 23 (1):16), celiac disease (Landau Y E. and Shoenfeld Y. Harefuah 2000 Jan. 16; 138 (2):122) and ileitis using the adherent cells of the present invention.
As mentioned hereinabove, the method, according to this aspect of the present invention, is effected by administering to the subject a therapeutically effective amount of adherent cells from a placenta or adipose tissue.
As used herein the phrase “adherent cells” refers to a homogeneous or heterogeneous population of cells which are anchorage dependent, i.e., require attachment to a surface in order to grow in vitro.
As used herein the phrase “adipose tissue” refers to a connective tissue which comprises fat cells (adipocytes).
As used herein the term “placenta tissue” refers to any portion of the mammalian female organ which lines the uterine wall and during pregnancy envelopes the fetus, to which it is attached by the umbilical cord. Following birth, the placenta is expelled (and is referred to as a post partum placenta). In an exemplary embodiment, placenta refers to whole placenta.
According to the present teachings, placenta or adipose tissue derived adherent cells can be propagated using two dimensional (2D) or three dimensional (3D) culturing conditions.
As used herein the phrase “two dimensional culture” refers to a culture in which the cells are disposed to conditions which are compatible with cell growth while allowing the cells to grow in one plane. The conditions in the two dimensional culture of the invention are designed to enable expansion of the adherent cells.
As used herein the phrase “three dimensional culture” refers to a culture in which the cells are disposed to conditions which are compatible with cell growth while allowing the cells to grow in more than one layer. It is well appreciated that the in situ environment of a cell in a living organism (or a tissue) is in a three dimensional architecture. Cells are surrounded by other cells. They are held in a complex network of extra cellular matrix nanoscale fibers that allows the establishment of various local microenvironments. Their extra cellular ligands mediate not only the attachment to the basal membrane but also access to a variety of vascular and lymphatic vessels. Oxygen, hormones and nutrients are ferried to cells and waste products are carried away. The conditions in the three dimensional culture of the invention are designed to mimic such an environment as is further exemplified below.
It will be appreciated that the conditions of the two-dimensional and three-dimensional cultures are such that enable expansion of the adherent cells.
As used herein the terms “expanding” and “expansion” refer to substantially differentiation-less maintenance of the cells and ultimately cell growth, i.e., increase of a cell population (e.g., at least 2 fold) without differentiation accompanying such increase.
As used herein the terms “maintaining” and “maintenance” refer to substantially differentiation-less cell renewal, i.e., substantially stationary cell population without differentiation accompanying such stationarity.
As mentioned, the adherent cells of this aspect of the invention are retrieved from an adipose or placental tissue.
Placental cells may be obtained from a full-term or pre-term placenta. Placenta is preferably collected once it has been ex blooded. The placenta is preferably perfused for a period of time sufficient to remove residual cells. The term “perfuse” or “perfusion” used herein refers to the act of pouring or passaging a fluid over or through an organ or tissue. The placental tissue may be from any mammal; for example, the placental tissue is human. A convenient source of placental tissue is from a post partum placenta (e.g., 1-6 hours), however, the source of placental tissue or cells or the method of isolation of placental tissue is not critical to the invention.
Placenta derived adherent cells may be obtained from both fetal (i.e., amnion or inner parts of the placenta, see Example 1) and maternal (i.e., decidua basalis, and decidua parietalis) parts of the placenta. Tissue specimens are washed in a physiological buffer [e.g., phosphate-buffered saline (PBS) or Hank's buffer]. Single-cell suspensions are made by treating the tissue with a digestive enzyme (see below) or/and mincing and flushing the tissue parts through a nylon filter or by gentle pipetting (Falcon, Becton, Dickinson, San Jose, Calif.) with washing medium.
Adipose tissue derived adherent cells may be isolated by a variety of methods known to those skilled in the art. For example, such methods are described in U.S. Pat. No. 6,153,432. The adipose tissue may be derived from omental/visceral, mammary, gonadal, or other adipose tissue sites. One source of adipose tissue is omental adipose. In humans, the adipose is typically isolated by liposuction.
Isolated adherent cells from placenta or adipose tissue may be derived by treating the tissue with a digestive enzyme such as collagenase, trypsin and/or dispase; and/or effective concentrations of hyaluronidase or DNAse; and ethylenediaminetetra-acetic acid (EDTA); at temperatures between 25-50° C., for periods of between 10 minutes to 3 hours. The cells may then be passed through a nylon or cheesecloth mesh filter of between 20 microns to 1 mm. The cells are then subjected to differential centrifugation directly in media or over a Ficoll or Percoll or other particulate gradient. Cells are centrifuged at speeds of between 100 to 3000×g for periods of between 1 minutes to 1 hour at temperatures of between 4-50° C. (see U.S. Pat. No. 7,078,230).
In addition to placenta or adipose tissue derived adherent cells, the invention also envisages the use of adherent cells from other cell sources which are characterized by stromal stem cell phenotype (as will be further described herein below). Tissue sources from which adherent cells can be retrieved include, but are not limited to, cord blood, scalp, hair follicles [e.g. as described in Us Pat. App. 20060172304], testicles [e.g., as described in Guan K., et al., Nature. 2006 Apr. 27; 440(7088):1199-203], human olfactory mucosa [e.g., as described in Marshall, C T., et al., Histol Histopathol. 2006 June; 21(6):633-43], embryonic yolk sac [e.g., as described in Geijsen N, Nature. 2004 Jan. 8; 427(6970):148-54] and amniotic fluid [Pieternella et al. (2004) Stem Cells 22:1338-1345], all of which are known to include mesenchymal stem cells. Adherent cells from these tissue sources can be isolated by culturing the cells on an adherent surface, thus isolating adherent cells from other cells in the initial population.
Regardless of the origin (e.g., placenta or adipose tissue), cell retrieval is preferably effected under sterile conditions. Once isolated cells are obtained, they are allowed to adhere to an adherent material (e.g., configured as a surface) to thereby isolate adherent cells. Culturing then proceeds under 2D conditions (as described in Example 2 of the Examples section) and cells may be further transferred to 3D conditions (as described in Examples 1 and 3 of the Examples section).
As used herein “an adherent material” refers to a synthetic, naturally occurring or a combination of same of a non-cytotoxic (i.e., biologically compatible) material having a chemical structure (e.g., charged surface exposed groups) which may retain the cells on a surface.
Examples of adherent materials which may be used in accordance with this aspect of the invention include, but are not limited to, a polyester, a polypropylene, a polyalkylene, a polyfluorochloroethylene, a polyvinyl chloride, a polystyrene, a polysulfone, a cellulose acetate, a glass fiber, a ceramic particle, a matrigel, an extra cellular matrix component (e.g., fibronectin, chondronectin, laminin), a collagen, a poly L lactic acid and an inert metal fiber.
It will be appreciated that seeding of placenta or adipose cells is typically effected at a culture density of 3±0.2×103 cells/cm2. Following seeding, cell cultures are usually cultured in a tissue culture incubator under humidified conditions with 5% CO2 at 37° C.
Further steps of purification or enrichment for stromal stem cells may be effected using methods which are well known in the art (such as by FACS using stromal stem cell marker expression, as further described herein below).
Non-limiting examples of base media useful in culturing according to the invention include Minimum Essential Medium Eagle, ADC-1, LPM (Bovine Serum Albumin-free), F10 (HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton-Jackson Modification), Basal Medium Eagle (BME-with the addition of Earle's salt base), Dulbecco's Modified Eagle Medium (DMEM-without serum), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, Medium M199 (M199E-with Earle's sale base), Medium M199 (M199H-with Hank's salt base), Minimum Essential Medium Eagle (MEM-E-with Earle's salt base), Minimum Essential Medium Eagle (MEM-H-with Hank's salt base) and Minimum Essential Medium Eagle (MEM-NAA with non essential amino acids), among numerous others, including medium 199, CMRL 1415, CMRL 1969, CMRL 1066, NCTC 135, MB 75261, MAB 8713, DM 145, Williams' G, Neuman & Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, MDBC 153. A preferred medium for use in the invention is DMEM. These and other useful media are available from GIBCO, Grand Island, N.Y., USA and Biological Industries, Bet HaEmek, Israel, among others. A number of these media are summarized in Methods in Enzymology, Volume LVIII, “Cell Culture”, pp. 62 72, edited by William B. Jakoby and Ira H. Pastan, published by Academic Press, Inc.
The medium may be supplemented such as with serum such as fetal serum of bovine or other species, and optionally or alternatively, growth factors, vitamins (e.g. ascorbic acid), cytokines, salts (e.g. B-glycerophosphate), steroids (e.g. dexamethasone) and hormones e.g., growth hormone, erythropoeitin, thrombopoietin, interleukin 3, interleukin 6, interleukin 7, macrophage colony stimulating factor, c-kit ligand/stem cell factor, osteoprotegerin ligand, insulin, insulin like growth factors, epidermal growth factor, fibroblast growth factor, nerve growth factor, cilary neurotrophic factor, platelet derived growth factor, and bone morphogenetic protein at concentrations of between picogram/ml to milligram/ml levels.
It is further recognized that additional components may be added to the culture medium. Such components may be antibiotics, antimycotics, albumin, amino acids, and other components known to the art for the culture of cells. Additionally, components may be added to enhance the differentiation process when needed (see further below).
It will be appreciated that in case the adherent cells of the invention are administered to a human subject, the cells and the culture medium (e.g., with the above described medium additives) should be substantially xeno-free, i.e., devoid of any animal contaminants e.g., mycoplasma. For example, the culture medium can be supplemented with a serum-replacement, human serum and/or synthetic or recombinantly produced factors.
As mentioned, once adherent cells are at hand they may be passaged to 2D or 3D settings (see Examples 1, 2 and 3 of the Examples section which follows). It will be appreciated though, that the cells may be transferred to a 3D-configured matrix immediately after isolation or alternatively, may be passaged to 3D settings following 2D conditions (as mentioned hereinabove).
It will be appreciated that during the 2D culturing conditions, the adherent cells may be continuously passaged. According to an embodiment of the present invention, the cells may be passaged for at least 4 passages, at least 5 passages, at least 6 passages, at least 7 passages or at least 8 passages. It will be appreciated that cells are typically passaged when the culture reaches about 70-80% confluence, typically after 3-5 days (1.5-2 doublings). Moreover, under the 2D culturing conditions, the cells may be grown in a culture medium devoid of antibiotic supplements from at least passage 2, at least passage 3, or at least passage 4.
Thus, during the 2D culture, culturing is effected for at least about 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, a month or even more. Passaging may also be effected to increase cell number. It will be appreciated that culture medium may be changed in order to prolong and improve culturing conditions.
The 2D adherent cells can be harvested when at least about 12% of cells are proliferating while avoiding uncontrolled differentiation and senescence.
2D adherent cells of some embodiments of the present invention comprise at least about 10%, 28%, 30%, 50%, 80% or more proliferative cells (as can be assayed by FACS monitoring S and/or G2/M phases).
As mentioned, the adherent cells may be transferred to 3D settings.
Thus, the adherent material of this aspect of the invention is configured for 3D culturing thereby providing a growth matrix that substantially increases the available attachment surface for the adherence of the cells so as to mimic the infrastructure of the tissue (e.g., placenta).
For high scale production, culturing can be effected in a 3D bioreactor.
Examples of such bioreactors include, but are not limited to, a plug flow bioreactor, a continuous stirred tank bioreactor, a stationary-bed bioreactor, a CelliGen Plus® bioreactor system (New Brunswick Scientific (NBS) or a BIOFLO 310 bioreactor system (New Brunswick Scientific (NBS).
As shown Example 3 of the Examples section, the Celligen bioreactor is capable of 3D expansion of adherent cells under controlled conditions (e.g. pH, temperature and oxygen levels) and with constant cell growth medium perfusion. Furthermore, the cell cultures can be directly monitored for concentration levels of glucose, lactate, glutamine, glutamate and ammonium. The glucose consumption rate and the lactate formation rate of the adherent cells enable to measure cell growth rate and to determine the harvest time.
Other 3D bioreactors that can be used with the invention include, but are not limited to, a continuous stirred tank bioreactor, where a culture medium is continuously fed into the bioreactor and a product is continuously drawn out, to maintain a time-constant steady state within the reactor. A stirred tank bioreactor with a fibrous bed basket is available for example at New Brunswick Scientific Co., Edison, N.J.), A stationary-bed bioreactor, an air-lift bioreactor, where air is typically fed into the bottom of a central draught tube flowing up while forming bubbles, and disengaging exhaust gas at the top of the column], a cell seeding perfusion bioreactor with Polyactive foams [as described in Wendt, D. et al., Biotechnol Bioeng 84: 205-214, (2003)] tubular poly-L-lactic acid (PLLA) porous scaffolds in a Radial-flow perfusion bioreactor [as described in Kitagawa et al., Biotechnology and Bioengineering 93(5): 947-954 (2006). Other bioreactors which can be used in accordance with the invention are described in U.S. Pat. Nos. 6,277,151, 6,197,575, 6,139,578, 6,132,463, 5,902,741 and 5,629,186.
Cell seeding is preferably effected 100,000-1,500,000 cells/mm at seeding. In an exemplary embodiment a total of 150±30×106 cells are seeded, 3-5×106 cell/gr carrier are seeded, or 0.015-0.1×106 cell/ml are seeded.
Culturing is effected for at least about 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, a month or even more. It will be appreciated that culturing in a bioreactor may prolong this period. Culturing of the adherent cells in the 3D culture can be effected under a continuous flow of a culture medium. Passaging may also be effected to increase cell number. It will be appreciated that culture medium may be changed in order to prolong and improve culturing conditions.
According to an embodiment of the present invention, culturing the adherent cells in a 3D culture may be effected under perfusion of the culture medium. Typically, the perfusion rate is determined by the glucose concentration in the culture medium of the adherent cells. Thus, according to the present teachings, the culture medium may be changed when the glucose concentration is about 500 mg/L, about 550 mg/L, or about 600 mg/L.
The 3D adherent cells can be harvested when at least about 10% of cells are proliferating while avoiding uncontrolled differentiation and senescence.
3D adherent cells of some embodiments of the present invention comprise at least about 10%, 28%, 30%, 50%, 80% or more proliferative cells (as can be assayed by FACS monitoring S and/or G2/M phases).
Adherent cells of some embodiments of the invention may comprise at least one “stromal stem cell phenotype”.
As used herein “a stromal stem cell phenotype” refers to a structural or functional phenotype typical of a bone-marrow derived stromal (i.e., mesenchymal) stem cell
As used herein the phrase “stem cell” refers to a cell which is not terminally differentiated.
Thus for example, the cells may have a spindle shape. Alternatively or additionally the cells may express a marker or a collection of markers (e.g. surface marker) typical to stromal stem cells. Examples of stromal stem cell surface markers (positive and negative) include but are not limited to CD105+, CD29+, CD44+, CD73+, CD90+, CD3−, CD4−, CD34−, CD45−, CD80−, CD19−, CD5−, CD20−, CD11B−, CD14−, CD19−, CD79−, HLA-DR−, and FMC7−. Other stromal stem cell markers include but are not limited to tyrosine hydroxylase, nestin and H—NF.
According to a specific embodiment of the present invention, the adherent cells do not express Oct-4.
It will be appreciated that the 2D adherent cells of placenta tissue generated according to the present teachings have a gene expression profile essentially as described in Table 8 of the Examples section which follows. While, the 3D adherent cells of placenta tissue generated according to the present teachings have a gene expression profile essentially as described in Table 11 of the Examples section which follows.
According to an exemplary embodiment, the 2D and 3D adherent cells of the present invention are less committed to differentiation into osteogenic or adipogenic lineages as compared to adherent cells from the bone marrow grown and differentiated under the same conditions.
Examples of functional phenotypes typical of stromal stem cells include, but are not limited to, T cell suppression activity (they don't stimulate T cells and conversely suppress same) and hematopoietic stem cell support activity.
According to one embodiment of the invention, the adherent cells of the invention are capable of suppressing immune reaction in a subject.
As used herein the phrase “suppressing immune reaction in a subject” refers to decreasing or inhibiting the immune reaction occurring in a subject in response to an antigen (e.g., a foreign cell or a portion thereof). The immune response which can be suppressed by the adherent cells include the humoral immune responses, and cellular immune responses, which involve specific recognition of pathogen antigens via antibodies and T-lymphocytes (proliferation of T cells), respectively.
As is shown in Examples 4-5 of the Examples section which follows, the 2D and 3D adherent cells of the present invention were found to induce an anti-inflammatory effect in colon inflammatory conditions. It will be further appreciated that this effect may be mediated by the cells per se or by a factor secreted thereby having an anti-inflammatory effect even in the absence of cells. Thus, the adherent cells of the present invention may be preferentially used in treating intestinal inflammation, such as in conditions of ulcerative colitis and Crohn's disease.
The phrase “administering to the subject” refers to the introduction of the cells of the invention to target tissue. The cells can be derived from the recipient or from an allogeneic or xenogeneic donor. This phrase also encompasses “transplantation”, “cell replacement” or “grafting” of the cells of the invention into the subject.
According to specific embodiments of the invention, the adherent cells may be administered to the subject by any means known to one of ordinary skill in the art, for example, by intravenous (iv), intramuscular (im), or intraperitoneal (ip) administration.
Cells which may be administered in accordance with this aspect of the invention include the above-described adherent cells which may be cultured in three-dimensional or two dimensional settings as well as mesenchymal and-non mesenchymal partially or terminally differentiated derivatives of same.
Methods of deriving lineage specific cells from the stromal stem cells of the invention are well known in the art. See for example, U.S. Pat. Nos. 5,486,359, 5,942,225, 5,736,396, 5,908,784 and 5,902,741.
The cells may be naïve or genetically modified such as to derive a lineage of interest (see U.S. Pat. Appl. No. 20030219423).
The cells may be of autologous or non-autologous source (i.e., allogeneic or xenogeneic) of fresh or frozen (e.g., cryo-preserved) preparations.
Since non-autologous cells may induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells in immunoisolating, semipermeable membranes before transplantation.
Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).
Methods of preparing microcapsules are known in the arts and include for example those disclosed by Lu M Z, et al., Cell encapsulation with alginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., A novel cell encapsulation method using photosensitive poly(allylamine alpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.
For example, microcapsules are prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 μm. Such microcapsules can be further encapsulated with additional 2-5 μm ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S. M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56).
Other microcapsules are based on alginate, a marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Technol. Ther. 2003, 5: 665-8) or its derivatives. For example, microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.
It will be appreciated that cell encapsulation is improved when smaller capsules are used. Thus, the quality control, mechanical stability, diffusion properties, and in vitro activities of encapsulated cells improved when the capsule size was reduced from 1 mm to 400 μm (Canaple L. et al., Improving cell encapsulation through size control. J Biomater Sci Polym Ed. 2002; 13:783-96). Moreover, nanoporous biocapsules with well-controlled pore size as small as 7 nm, tailored surface chemistries and precise microarchitectures were found to successfully immunoisolate microenvironments for cells (Williams D. Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).
Examples of immunosuppressive agents which may be used include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF.alpha. blockers, a biological agent that targets an inflammatory cytokine, and Non-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.
Depending on the medical condition, the subject may be administered with additional chemical drugs (e.g., immunomodulatory, chemotherapy, anti-inflammatory etc.) or cells.
For the treatment of inflammatory colon conditions, including ulcerative colitis and Crohn's disease, any treatment known to one of ordinary skill in the art may be employed, including for example, Aminosalicylates (e.g. Sulfasalazine, Mesalazine, Balsalazide, Olsalazine), Corticosteroids (e.g. Cortisone, Prednisone, Prednisolone, Cortifoam, Hydrocortisone, Methylprednisolone, Beclometasone, Budesonide), Immunosuppressive drugs (e.g. Mercaptopurine, Azathioprine, Methotrexate, Tacrolimus), Biological treatments (e.g. Infliximab, Visilizumab), Low Molecular Weight Heparin (LMWH), dietary modifications (e.g. fibers) and surgery.
The subject may also be administered an anti-inflammatory agent such as, but not limited to, Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; Halopredone Acetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol; llonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lomoxicam; Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate; Momiflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride; Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin; Zomepirac Sodium.
In any of the methods described herein, the cells can be administered either per se or, preferably as a part of a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier.
As used herein a “pharmaceutical composition” refers to a preparation of the adherent cells of the invention (i.e., adherent cells from placenta or adipose tissue, which are obtained from a 2D or 3D culture), with other chemical components such as pharmaceutically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the cells to a subject.
Hereinafter, the term “pharmaceutically acceptable carrier” refers to a carrier or a diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compound. Examples, without limitations, of carriers are propylene glycol, saline, emulsions and mixtures of organic solvents with water.
Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
According to a preferred embodiment of the invention, the pharmaceutical carrier is an aqueous solution of saline.
Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.
One may administer the pharmaceutical composition in a systemic manner (as detailed hereinabove). Alternatively, one may administer the pharmaceutical composition locally, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.
Pharmaceutical compositions of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, physiological salt buffer, or freezing medium containing cryopreservents. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. Preferably, a dose is formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition, (see e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).
Dosage amount and interval may be adjusted individually to levels of the active ingredient which are sufficient to effectively regulate the neurotransmitter synthesis by the implanted cells. Dosages necessary to achieve the desired effect will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or diminution of the disease state is achieved.
The amount of a composition to be administered will, of course, be dependent on the individual being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. The dosage and timing of administration will be responsive to a careful and continuous monitoring of the individual changing condition.
Models for inflammatory colon diseases include animal models of ulcerative colitis such as, but are not limited to, trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats and mice [Komori et al., J Gastroenterol (2005) 40: 591-599; and Examples 4-5 hereinbelow].
Compositions including the preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
Compositions of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
The adherent cells of the invention can be suitably formulated as pharmaceutical compositions which can be suitably packaged as an article of manufacture. Such an article of manufacture comprises a label for use in treating ulcerative colitis or Crohn's disease, the packaging material packaging a pharmaceutically effective amount of adherent cells from a placenta or adipose tissue.
It will be appreciated that the article of manufacture may further comprise additional drugs for the treatment of colon inflammatory conditions including, for example, anti-inflammatory agents, immunomodulatory agents, anti-inflammatory agents, and other drugs for the treatment of inflammatory colon conditions (as described in further detail hereinabove).
As used herein the term “about” refers to ±10%.
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
The term “consisting of means” including and limited to”.
The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
Example 1
Methods of Generating Placenta Derived 3D Adherent Cells
Adherent cells were produced as was previously described (see WO/2007/108003) in a bioreactor system containing 3D carriers to produce 3D-adherent cells (designated herein as PLX).
Materials and Experimental Procedures
Placenta Derived Adherent Cells
Inner parts of a full-term delivery placenta (Bnei Zion medical center, Haifa, Israel) were cut under sterile conditions, washed 3 times with Hank's Buffer and incubated for 3 hours at 37° C. with 0.1% Collagenase (1 mg/ml tissue; Sigma-Aldrich, St. Lewis, Mo.). Using gentle pipetting, suspended cells were then washed with DMEM supplemented with 10% FCS, Pen-Strep-Nystatin mixture (100 U/ml:100 μg/ml:1.25 un/ml) and 2 mM L-glutamine, seeded in 75 cm2 flasks and incubated at 37° C. in a tissue culture incubator under humidified condition with 5% CO2.
Two Dimensional (2D) Cell Growth
Cells were allowed to adhere to a plastic surface for 72 hours after which the media was changed every 3-4 days. After 2-3 passages, the cells were cryopreserved, thawed and seeded for a secondary growth in flasks. When reaching 60-80% confluence cells were detached from the growth flask using 0.25% trypsin-EDTA and seeded into new flasks (usually every 3-5 days), for another 2-5 passages. Cultured cells were thereafter collected for analysis or for culturing in bioreactors.
PluriXTM Plug Flow Bioreactor
The PluriXTM Plug Flow bioreactor (Pluristem, Haifa, Israel; see U.S. Pat. No. 6,911,201 and WO/2007/108003), was loaded with 1-100 ml packed 3D porrosive carriers (4 mm in diameter) made of a non woven fabric matrix of polyester. These carriers enable the propagation of large cell numbers in a relatively small volume. Glassware was designed and manufactured by Pluristem (Pluristem, Haifa, Israel). The bioreactor was maintained in an incubator of 37° C., with flow rate regulated and monitored by a valve, and peristaltic pump. The bioreactor contains a sampling and injection point, allowing the sequential seeding of cells. Culture medium was supplied at pH 6.7-7.4 from a reservoir. The reservoir was supplied by a filtered gas mixture, containing air/CO2/O2 at differing proportions, depending on cell density in the bioreactor. The O2 proportion was suited to the level of dissolved O2 at the bioreactor exit, determined by a monitor. The gas mixture was supplied to the reservoir via silicone tubes or diffuser (Degania Bet, Emek Hayarden, Israel). The culture medium was passed through a separating container which enables collection of circulating, nonadherent cells. Circulation of the medium was obtained by a peristaltic pump. The bioreactor was further equipped with an additional sampling point and containers for continuous medium exchange.
Production of 3D-Adherent Cells (PLX)
Non-confluent primary human adherent 2D cell cultures, grown as described above, were trypsinized, washed, resuspended in DMEM supplemented with 10% FBS, Pen-Strep-Nystatin mixture (100 U/ml:100 ug/ml:1.25 un/ml) and 2 mM L-glutamine, and seeded (103-105 cells/ml) via an injection point onto the 3D carriers in a sterile Plug Flow bioreactor. Prior to inoculation, bioreactor was filled with PBS-Ca—Mg (Biological Industries, Beit Ha'emek, Israel), autoclaved (120° C., 30 min) and washed with Dulbecco's growth medium containing 10% heat-inactivated fetal calf serum and a Pen-Strep-Nystatin mixture (100 U/ml:100 ug/ml:1.25 un/ml). Flow was kept at a rate of 0.1-5 ml/min. Seeding process involved cease of circulation for 2-48 hrs, thereby allowing the cells to settle on the carriers. Bioreactor was kept under controlled temperature (37° C.) and pH conditions (pH=6.7-7.4); using an incubator supplied with sterile air and CO2 as needed. Growth medium was replaced 2-3 times a week. Circulation medium was replaced with fresh DMEM media, every 4 hr to 7 days. At a density of 1×106-1×107 cells/ml (following 12-40 days of growth), total medium volume was removed from the bioreactor and bioreactor and carriers were washed 3-5 times with PBS. 3D-adherent cells were then detached from the carriers with Trypsin-EDTA; (Biological Industries, Beit Ha'emek, Israel; 3-15 minutes with gentle agitation, 1-5 times), and were thereafter resuspended in DMEM and cryopreserved.
Example 2
Methods of Generating 2D Adherent Cells Suitable for Use in Accordance with the Present Teachings and the 2D Adherent Cells Generated Thereby
2D adherent cells were produced which exhibit different characteristics then the above described 3D adherent cells (PLX, Example 1). Next, 2D adherent cells from bone marrow or placenta origin were grown under osteocyte or adipocyte differentiation stimulating conditions.
Materials and Experimental Procedures
Manufacturing Process of 2D Adherent Cells
Receipt of Human Tissue
All placentas obtained were received from the maternity ward under approval of the Helsinki Committee of the medical facility. Accordingly, all placenta donors signed an informed consent and Donor Screening and Donor Testing was performed (IPC1). Immediately after taking the placenta from the donor (during the caesarean procedure), it was placed in a sterile plastic bag and then in a Styrofoam box with ice packs. The placenta was delivered and immediately placed in a quarantine area until released to use by Quality Control (QC) and Quality Assurance (QA). All the following production steps were performed in a quarantine, clean room facility until QC approval of mycoplasma test results arrived and the cells were release for 2D cell growth.
Recovery and Processing of Adherent Cells
To initiate the process, the placenta was cut into pieces under aseptic conditions under laminar flow hood, washed with Hank's buffer solution and incubated for 3 hours at 37° C. with 0.1% Collagenase (1 mg Collagenase/ml tissue). 2D cell medium (2D-Medium comprising DMEM supplemented with 10% FBS, fungizone 0.25 μg/ml and gentamycine 50 μg/ml) was added and the digested tissue was roughly filtered through a sterile metal strainer, collected in a sterile beaker and centrifuged (10 minutes, 1200 RPM, 4° C.). Using gentle pipeting, suspended cells were then washed with 2D-Medium supplemented with antibiotics, seeded in 80 cm2 flasks and incubated at 37° C. in a tissue culture incubator under humidified condition supplemented with 5% CO2. Following 2-3 days, in which the cells were allowed to adhere to the flask surface, they were washed with PBS and 2D-Medium was added.
Two Dimensional (2D) Cell Growth
Prior to the first passage, growth medium samples of 10% of the total flask number in quarantine was pooled and taken for mycoplasma testing (IPC2). If cells were found to be negative for Mycoplasma (EZ-PCR Mycoplasma kit, Biological Industries, Israel), cells were released from quarantine. After 1-2 additional passages, cells were transferred to the 2D production clean room (2DP). Once in Room 2DP, culture was continued for another 3-5 passages (of note, cells were grown in 2D-Medium supplemented with antibiotics until passage 2, thereafter cells were grown in 2D-Medium without antibiotics). IPC-3 sample was taken for immune phenotype after passage 4. Throughout the process, cultures were grown in a tissue culture incubator under humidified conditions with 5% CO2 at 37° C. After a total of 6-8 passages (9-16 cell doublings), cells were collected and cryopreserved as the 2D-Cell Stock (2DCS).
The first passage was usually carried out after 10-15 days. Beginning at passage 2 and continuing until passage 6-8, cells were passaged when the culture reached 70-80% confluence, usually after 3-5 days (1.5-2 doublings). The cells were detached from the flasks using 0.25% trypsin-EDTA (4 minutes at 37° C.) and seeded in a culture density of 3±0.2×103 cells/cm2. The size of the tissue culture flasks raised as the passages proceed. The culturing process started in 80 cm2 tissue culture flask, continued in 175 cm2, then in 500 cm2 (Triple flask) and finally the cells were seeded into Cell Factory 10 tray (6320 cm2).
Prior to cryopreservation, at the end of 2DCS growth period, the growth medium was collected and the sample was prepared to be sent to an approved GLP laboratory for Mycoplasma test (IPC 4).
Cryopreservation Procedure for 2D-Cell-Stock Product
For 2DCS cryopreservation, 2D-cultured cells were collected under aseptic conditions using 0.25% trypsin-EDTA. The cells were centrifuged (1200 RPM, 10′, 4° C.), counted and re-suspended in 2D-Medium.
For freezing, cell suspensions were diluted. 1:1 with 2D-Freezing Mixture (final concentrations was 10% DMSO, 40% FBS and 50% 2D-Medium). Approximately 1.5-2.5×109 cells were manufactured from one placenta. 4 ml of the cells were stored at a final concentration of 10×106/ml in 5 ml cryopreservation polypropylene vials. The vials were labeled and transferred to a controlled rate freezer for a graduated temperature reducing process (1° C./min), after which they were transferred to storage in gas-phase of a liquid nitrogen freezer located in the Cold Storage Room. This material was referred to as the 2D-Cell Stock (2DCS) batch.
Cell Cycle Analysis
2D adherent cells and PLX cells were fixed with 70% EtOH O.N, centrifuged and re-suspended in a Propidium Iodide (PI) solution containing 2 μg/ml PI (Sigma), 0.2 mg/ml Rnase A (Sigma) and 0.1% (v/v) Triton (Sigma) for 30 minutes. Cell cycle was analyzed by FACS.
Gene Expression Array (Microarray)
Adherent cells were obtained from human full term placentas and were expanded by 2D cultures or according to the teachings of WO/2007/108003 (as described in detail in Examples 1-2). Three different batches of cells were obtained from each of the expansion methods for further examination.
RNA was extracted from the cells (Qiagen-Rneasy micro kit) and applied to an Affymetrix whole genome expression array GeneChip® Human Exon 1.0 ST Array (Affymetrix, Santa Clara, Calif., USA).
FACS Analysis of Membrane Markers
Cells were stained with monoclonal antibodies as previously described. In short, 400,000-600,000 cells were suspended in 0.1 ml flow cytometer buffer in a 5 ml test tube and incubated for 15 minutes at room temperature (RT), in the dark, with each of the following monoclonal antibodies (MAbs): FITC-conjugated anti-human CD29 MAb (eBioscience), PE conjugated anti human CD73 MAb (Becton Dickinson), PE conjugated anti human CD105 MAb (eBioscience), PE conjugated anti human CD90 MAb (Becton Dickinson), FITC-conjugated anti-human CD45 MAb (IQProducts), PE-conjugated anti-human CD19 MAb (IQProducts), PE conjugated anti human CD14 MAb (IQProducts), FITC conjugated anti human HLA-DR MAb (IQProduct), PE conjugated anti human CD34 MAb (IQProducts), FITC conjugated anti human CD31 MAb (eBioscience), FITC conjugated anti human KDR MAb (R&D systems), anti human fibroblasts marker (D7-FIB) MAb (ACRIS), FITC-conjugated anti-human CD80 MAb (BD), FITC-conjugated anti-human CD86 MAb (BD), FITC-conjugated anti-human CD40 MAb (BD), FITC-conjugated anti-human HLA-ABC MAb (BD), Isotype IgG1 FITC conjugated (IQ Products), Isotype IgG1 PE conjugated (IQ Products).
Cells were washed twice with flow cytometer buffer, resuspended in 500 μl flow cytometer buffer and analyzed by flow cytometry using FC-500 Flow Cytometer (Beckman Coulter). Negative controls were prepared with relevant isotype fluorescence molecules.
Immunomodulation Assay
Human derived mononuclear cells (MNCs) were isolated from peripheral blood. Suspension of 200,000 MNCs per 200 μl medium (RPMI 1640 medium containing 20% FBS per 96 well) were stimulated with 10 μg PHA/ml (SIGMA) in the presence of 20,000 2D adherent cells for 5 days under humidified 5% CO2 at 37° C. Four different batches of 2D adherent cells were used. Three replicates of each group were seeded in 96-well plated. During the last 18 hrs of the 5-day culture, cells were pulsed with 1 μC 3H-thymidine and further harvested over fiberglass filter. Thymidine uptake was quantified by a scintillation counter.
Induction of Osteogenesis in 2D Adherent Cells
Osteogenesis was carried out according to Chemicon osteogenesis kit (cat no. scr028, Millipore, Mass., USA)
Osteogenesis Induction Medium
Osteogenesis induction medium was freshly made prior to each medium exchange using the kit components (See Table 1, below).







TABLE 1







Osteogenesis medium components











Stock




Component
concentration
Amount
Final con





DMEM low glucose

 8.7 ml
87%


(Invitrogen, Gibco)


Serum (heat inactivated)

 1 ml
10%


dexamethasone
1 mM
 1 μl
0.1 μM


Asorbic Acid-2-Phosphate solution
0.1M
 20 μl
0.2 mM


Glycerol-2-Phosphate Solution
  1M
100 μL
 10 Mm


L-glutamine
X 100
100 μl
X 1


Pen & Strep
X 100
100 μl
X 1









To arrive at 1 mM dexamethasone solution, 900 μl ethanol was added to 100 μl dexamethasone 10 mM solution. Stock solution was stored with the rest of the kit's components at −20° C. A 50 ml serum vial was heat inactivated, divided into 5 ml aliquots and kept at −20° C. until use.
Coating 24-Well Tissue Culture Plates
A coating mixture comprising 12 μg/ml vitronectin and 12 μg/ml collagen (both included in the kit) was prepared by diluting each with 1×PBS.
The coating mixture was then added to the wells to cover the well surfaces (5 wells×2 plates were prepared). Plates were incubated overnight at room temperature. The coating mixture was then removed and the wells were rinsed once with PBS. Plates were aspirated right before use.
Cell Growth
Placenta derived cells (plc11-3-1) or bone marrow derived cells (BM108) were plated (200,000 cells per well) in 1 ml growth medium comprising DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 Mm L-glutamine (Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and 0.25 μg/ml Fungizone (Invitrogen, Gibco). Placenta derived cells (4 wells×2 plates) or bone marrow derived cells (1 well×2 plates) were grown until 100% confluent (typically overnight) before initiating osteogenic differentiation.
When cells reached 100% confluence, growth medium was aspirated and replaced with 1 ml osteogenesis induction medium (differentiation day 1). Osteogenesis induction medium was replaced with fresh medium every 2-3 days for a total of 14-17 days.
As a control, one of the two plates (for each of the cell types) was not incubated with osteogenesis differentiation medium but rather with the growth medium (described hereinabove).
On day 17, osteocytes were fixed and stained with Alizarin Red Solution as depicted in detail below.
Staining Protocol
Osteocyte staining was performed by first carefully aspirating the medium from each well (carefully as to not aspirate the cells). Cells were then fixed by incubating in iced cold 70% ethanol for 1 hour at room temperature. The alcohol was then carefully aspirated and the cells were rinsed twice with water (5-10 minutes each wash). The water was then aspirated and alizarin red solution (500-1000 μl) was added to the cells. Cells were incubated with alizarin red solution at room temperature for 30 minutes. Alizarin red was removed and the cells were washed 4 times with 1 ml water and aspirated after each wash. Finally, 1-1.5 ml water was added to each well to prevent cell drying. The plates were microscopically visualized by an inverted Nikon microscope.
Induction of Osteogenesis in Modified Osteogenesis Induction Medium (2D Adherent Cells)
Osteogenesis induction medium was freshly made prior to each medium exchange using the components listed in Table 2, below, along with Vitamin D.







TABLE 2







Osteogenesis medium components










Component
Stock con
Amount
Final con





DMEM high glucose (Biological

 8.7 ml
87%


Industries, Bet Haemek, Israel)


L-glutamine
X 100
100 μl
X 1


Serum (heat inactivated)

 1 ml
10%


Dexamethasone (Chemicon)
10 mM
 10 μl
 10 μM


Asorbic Acid-2-Phosphate solution
0.1M
 20 μl
0.2 mM


(Chemicon)


Glycerol-2-Phosphate Solution
  1M
100 μL
 10 Mm


(Chemicon)


Vitamin D (Sigma)
10 μM
 10 μL
 10 nM


Gentamycin (Biological Industries, Bet
X 100
100 μl
X 1


Haemek, Israel)









A 50 ml serum vial was heat inactivated, divided into 5 ml aliquots and kept at −20° C. until use.
Coating 48-Well Tissue Culture Plates
A coating mixture comprising 12 μg/ml vitronectin and 12 μg/ml collagen (both from Chemicon) was prepared by diluting each with 1×PBS.
The coating mixture was then added to the wells to cover the well surfaces (5 wells×2 plates were prepared). Plates were incubated overnight at room temperature. The coating mixture was then removed and the wells were rinsed once with PBS. Plates were aspirated right before use.
Cell Growth
Placenta derived cells (PLC 8-2-1, PLC 15 3-4-2 or PLC 19-4-3-1 fetal cells) were plated (100,000 cells per well) in 0.5 ml growth medium comprising DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 Mm L-glutamine (Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and 0.25 μg/ml Fungizone (Invitrogen, Gibco) (4 wells×2 plates). Bone marrow derived cells (BM109) were plated (150,000 cells per well) in 0.5 ml growth medium (as described above) (1 well×2 plates). Cells were grown until 100% confluent (typically overnight) before initiating osteogenic differentiation.
When cells reached 100% confluence, growth medium was aspirated and replaced with 0.5 ml osteogenesis induction medium (differentiation day 1). Osteogenesis induction medium was replaced with fresh medium every 2-3 days for a total of 26 days.
As a control, one of the two plates (for each of the cell types) was not incubated with osteogenesis differentiation medium but rather with the growth medium (described hereinabove).
On day 26, osteocytes were fixed and stained with Alizarin Red Solution as depicted in detail below.
Staining Protocol
Osteocyte staining was performed by first carefully aspirating the medium from each well (carefully as to not aspirate the cells). Cells were then fixed by incubating in iced cold 70% ethanol for 1 hour at room temperature. The alcohol was then carefully aspirated and the cells were rinsed twice with water (5-10 minutes each wash). The water was then aspirated and alizarin red solution (500-1000 μl) was added to the cells. Cells were incubated with alizarin red solution at room temperature for 30 minutes. Alizarin red was removed and the cells were washed 4 times with 1 ml water and aspirated after each wash. Finally, 1-1.5 ml water was added to each well to prevent cell drying. The plates were microscopically visualized by an inverted Nikon microscope.
Induction of Adipogenesis in 2D Adherent Cells
Adipogenesis was carried out according to Chemicon adipogenesis kit (Chemicon adipogenesis kit, cat no. scr020, Millipore, Mass., USA)
Adipogenesis Induction Medium
Adipogenesis induction or maintenance mediums were freshly prepared prior to every medium exchange using the components depicted in Tables 3 and 4, below.







TABLE 3







Adipogenesis induction medium components










Component
Stock con
Amount
Final con





DMEM low glucose (Biological

4.4 ml
90%


Industries, Bet Haemek, Israel)


Serum (heat inactivated)

0.5 ml
10%


Dexamethasone (Sigma)
10 mM
0.5 μl
 1 μM


IBMX (Sigma)
0.5M
  5 μl
 0.5 mM


Insulin (Sigma)
10 mg/ml
  5 μL
 10 μg/ml


Indomethacin (Sigma)
10 mM
 50 μl
100 μM


Pen & Strep
X 100
 50 μl
X 1
















TABLE 4







Adipogenesis maintenance medium components












Component
Stock con
Amount
Final con







DMEM low glucose

4.4 ml
90%



Serum (heat inactivated)

0.5 ml
10%



Insulin
10 mg/ml
  5 μL
10 μg/ml



Pen & Strep
X 100
 50 μl
X 1










Cell Growth
Placenta derived cells (plc11-3-1) or bone marrow derived cells (BM108) were plated (200,000 cells per well) in 1 ml growth medium comprising DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 Mm L-glutamine (Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and 0.25 μg/ml Fungizone (Invitrogen, Gibco). Placenta derived cells (4 wells×2 plates) or bone marrow derived cells (1 well×2 plates) were grown until 100% confluent (typically overnight) before initiating adipogenesis differentiation.
When cells reached 100% confluence, growth medium was aspirated and replaced with 1 ml adipogenesis induction medium (differentiation day 1). Adipogenesis induction medium was replaced with fresh medium every 2-3 days for a total of 25 days (as depicted in detail in Table 5, hereinbelow). Of note, monolayers of adipogenic cells were extremely fragile and could easily dislodged from plates, therefore, medium changes were performed with gentle medium changes to avoid disruption of the lipid droplets.
As a control, one of the two plates (for each of the cell types) was not incubated with adipogenesis differentiation medium but rather with the growth medium (described hereinabove).







TABLE 5







Adipogenesis differentiation schedule








Day
Medium











1
Adipogenesis Induction medium


3
Adipogenesis Induction medium


5
Adipogenesis Induction medium


7
Adipogenesis Maintenance medium


9
Adipogenesis Induction medium


11
Adipogenesis Induction medium


13
Adipogenesis Induction medium


15
Adipogenesis Maintenance medium


17
Adipogenesis Induction medium


19
Adipogenesis Induction medium


21
Adipogenesis Induction medium









On day 25, adipocytes were fixed and stained with oil red solution as depicted in detail below.
Staining Protocol
Adipocyte staining was performed by first carefully aspirating the medium from each well (carefully as to not aspirate the cells). Cells were then fixed by incubating in 4% Para formaldehyde for 30-40 minutes at room temperature. The fixative was then carefully aspirated and the cells were rinsed three times with PBS (5-10 minutes each wash). Next, the PBS was aspirated and the cells were rinsed twice in water. The water was then aspirated and oil red solution (500-1000 μl) was added to the cells. Cells were incubated with oil red solution at room temperature for 50 minutes. Oil red solution was removed and the cells were washed 4 times with 1 ml water and aspirated after each wash. Finally, 1-1.5 ml water was added to each well to prevent cell drying. The plates were microscopically visualized by an inverted Nikon microscope.
Preparation of Oil Red Solution
Stock of 0.25 g oil red (Sigma) was used which was dissolved in 50 ml iso-propanol by incubating 10-15 min in 37° C. bath.
For use, 30 ml of the stock stain was mixed with 20 ml DDW (left to stand for 10 minutes and then filtered with coffee filter paper). The oil red solution was prepared fresh for each use.
Induction of Adipogenesis in Modified Adipogenesis Induction Medium (2D Adherent Cells)
Adipogenesis induction medium was freshly prepared prior to every medium exchange using the components depicted in Table 6, below.







TABLE 6







Adipogenesis induction medium components










Component
Stock con
Amount
Final con





DMEM low glucose

 4.4 ml
90%


Serum (heat inactivated)

 0.5 ml
10%


Dexamethasone (Sigma)
 1 mM
 5 μl
 1 μM


IBMX (Sigma)
0.5M
 5 μl
 0.5 mM


Insulin (Sigma)
10 mg/ml
 5 μL
 10 μg/ml


Indomethacin (Sigma)
10 mM
200 μl
100 μM


Gentamycine (Biological Industries)

 10 μl









Cell Growth
Placenta derived cells (PLC 8-2-1, PLC 15 3-4-2 or PLC 19-4-3-1 fetal cells) were plated (100,000 cells per well) in 0.5 ml growth medium comprising DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 Mm L-glutamine (Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and 0.25 μg/ml Fungizone (Invitrogen, Gibco) (5 wells×2 plates).
Bone marrow derived cells (BM109) were plated (100,000 cells per well) in 0.5 ml growth medium comprising DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 Mm L-glutamine (Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and 0.25 μg/ml Fungizone (Invitrogen, Gibco) (4 well×2 plates). Cells were grown until 100% confluent (typically overnight) before initiating adipogenesis differentiation.
When cells reached 100% confluence, growth medium was aspirated and replaced with 0.5 ml adipogenesis induction medium (differentiation day 1). Adipogenesis induction medium was replaced with fresh medium every 2-3 days for a total of 3-4 weeks.
As a control, one of the two plates (for each of the cell types) was not incubated with adipogenesis differentiation medium but rather with the growth medium (described hereinabove).
On day 26, adipocytes were fixed and stained with oil red solution as depicted in detail below.
Staining Protocol
Adipocyte staining was performed by first carefully aspirating the medium from each well (carefully as to not aspirate the cells). Cells were then fixed by incubating in 4% Para formaldehyde for 30-40 minutes at room temperature. The fixative was then carefully aspirated and the cells were rinsed three times with PBS (5-10 minutes each wash). Next, the PBS was aspirated and the cells were rinsed twice in water. The water was then aspirated and oil red solution (500-1000 μl) was added to the cells. Cells were incubated with oil red solution at room temperature for 50 minutes. Oil red solution was removed and the cells were washed 3 times with 1 ml double distilled water and aspirated after each wash. Finally, 1-1.5 ml water was added to each well to prevent cell drying. The plates were microscopically visualized by an inverted Nikon microscope.
Preparation of Oil Red Solution
Stock of 0.25 g oil red (Sigma) was used which was dissolved in 50 ml iso-propanol by incubating 10-15 min in 37° C. bath.
For use, 30 ml of the stock stain was mixed with 20 ml DDW (left to stand for 10 minutes and then filtered with coffee filter paper). The oil red solution was prepared fresh for each use.
Results
As is illustrated in Table 7, below, processing of the 2D adherent cells suitable for use according to the present teachings differed from the 2D stage of PLX (WO/2007/108003) in a few aspects. First, the new 2D adherent cell's culture medium was supplemented with antibiotics only during the initial culturing stage (up to passage 2). Also, the new 2D adherent cells were cryopreserved only after 5-8 passages (i.e. at the end of culture) and not, as in the PLX process, during intermediate stages of 2D growth.







TABLE 7







Comparison of the 2D adherent cells suitable for use according to the


present teachings to those produced for PLX in WO/2007/108003











2D adherent cells of the


Parameter
WO/2007/108003
present teachings





Tissue culture flask
80 cm2 and 175 cm2
175 cm2, triple flasks and




Multi Tray


Medium
In all stages of the
Up to passage 2


supplemented with
process
(included)


antibiotics


Cryopreservation of
After 2-3 passages, then
After 5-8 passages, then


2DCS
cryopreserved, thawed
cryopreserved and



and seeded for a
thawed prior to use



secondary growth in



flasks for 2-5 passages,



prior to seeding in



bioreactor


Freezing container
2 ml cryogenic vials
5 ml cryogenic vials


Freezing volume
1-1.5 ml
4 ml


Freezing method
Freezing container
Controlled rate freezer



(contains isopropyl



alcohol)









Changes in the manufacturing process of the new 2D adherent cells resulted in changes in characteristics of the obtained cells. These differences are summarized hereinbelow.
Cell cycle analysis of 2D adherent cells compared to 3D adherent cells of WO/2007/108003—2D adherent cells were compared to 3D adherent cells in order to examine the distribution of the cells between the different phases of the cell cycle. As is clear from FIGS. 1A-B, 2D adherent cells exhibited typical proliferating profile (distribution of cells between the different phases of cell cycle). Specifically, 28% of cells were in S and G2/M phases (FIG. 1A). These results indicated that cells were harvested during proliferation and that the culturing conditions supported cell growth.
Conversely, 3D adherent cells exhibited lower rates of proliferating cells. Less than 8% of cells were in S and G2/M phases (FIG. 1B). These results indicated that cells were harvested while low levels of proliferation were taking place and suggest that conditions in the bioreactor were suboptimal to support cell growth.
Microarray comparison between 2D cells suitable for use according to the present teachings to those obtained by the teachings of WO/2007/108003—gene expression arrays enabled to simultaneously monitor genome-wide expression profiles of adherent cells derived from human full term placentas expanded by 2D cultures or according to the teachings of WO/2007/108003 (PLX, see Example 1, hereinabove). These results enabled to assess the molecular mechanism underlying phenotypic variation between cells obtained by these different growth methods (see Table 8, below).







TABLE 8







Gene expression in 2D adherent cells suitable for use according to the


present teachings compared to those expressed by PLX of WO/2007/108003










2D vs. Plurix



Gene
(fold change)
p-value (treat)












interferon-induced protein with tetratricopeptide repeats
21.82
0.0401812


leukocyte-derived arginine aminopeptidase
14.56
3.88E−06


signal peptide, CUB domain, EGF-like 3
10.82
0.0255115


dickkopf homolog 1 (Xenopus laevis)
6.84
3.06E−07


integrin, alpha 6
6.76
0.0411667


keratin 27 pseudogene 27
6.39
0.000224998


similar to Keratin, type I cytoskeletal 18 (Cytokerati
6.24
0.000304949


aldehyde dehydrogenase 1 family, member A1
5.84
0.00145807


G protein-coupled receptor, family C, group 5, member A
5.75
3.39E−05


coagulation factor III (thromboplastin, tissue factor)
5.55
0.012192


cyclin-dependent kinase inhibitor 3 (CDK2-associated
5.51
0.000732492


dual


G protein-coupled receptor 126
5.50
0.00197635


DEP domain containing 1
5.41
0.000370513


SHC SH2-domain binding protein 1
4.96
0.00430878


centrosomal protein 55 kDa
4.78
0.0021952


interferon-induced protein with tetratricopeptide repeats
4.66
0.0139777


NUF2, NDC80 kinetochore complex component, homolog
4.61
0.00276524


(S. cere


mal, T-cell differentiation protein-like
4.44
0.00664216


interferon-induced protein with tetratricopeptide repea
4.42
0.00357376


kinesin family member 18A
4.33
0.00134108


cholinergic receptor, muscarinic 2
4.07
0.0320078


cell division cycle 2, G1 to S and G2 to M
4.06
0.0017111


non-SMC condensin I complex, subunit G
4.06
0.00537097


denticleless homolog (Drosophila)
4.06
0.00141153


shugoshin-like 1 (S. pombe)
4.00
0.00101318


chromosome 13 open reading frame 3
3.98
0.000548296


PDZ binding kinase
3.97
0.00784983


lymphocyte cytosolic protein 1 (L-plastin)
3.97
0.0049584


WAS
3.96
0.00178153


cyclin E2
3.94
0.000203389


cathepsin C
3.93
0.00532262


integrin, alpha 4 (antigen CD49D, alpha 4 subunit of
3.91
0.0158411


VLA-4


KIAA0101
3.90
0.0105909


kinesin family member 20A
3.90
0.00582352


opioid growth factor receptor-like 1
3.87
0.00114551


anillin, actin binding protein
3.83
0.010923


catenin (cadherin-associated protein), alpha-like 1
3.76
7.46E−05


cell division cycle 20 homolog (S. cerevisiae)
3.70
0.00514206


diaphanous homolog 3 (Drosophila)
3.69
0.00107709


family with sequence similarity 111, member B
3.69
0.000125819


aurora kinase A
3.66
0.00632571


fibroblast growth factor 7 (keratinocyte growth factor)
3.64
0.0328983


maternal embryonic leucine zipper kinase
3.63
0.00908391


Rho GDP dissociation inhibitor (GDI) beta
3.63
0.00200066


centromere protein N
3.62
0.000540143


MAD2 mitotic arrest deficient-like 1 (yeast)
3.62
0.00488102


thymidylate synthetase
3.61
0.00685584


cyclin B2
3.60
0.016544


regulator of G-protein signalling 4
3.59
0.00781061


chromosome 6 open reading frame 173
3.58
0.00222408


hyaluronan-mediated motility receptor (RHAMM)
3.55
0.00467816


BUB1 budding uninhibited by benzimidazoles 1 homolog
3.54
0.0108258


(yeast


SPC25, NDC80 kinetochore complex component,
3.53
0.00568662


homolog (S. ce


establishment of cohesion 1 homolog 2 (S. cerevisiae)
3.52
0.000773033


cyclin A2
3.51
0.00965934


CDC28 protein kinase regulatory subunit 2
3.51
0.0128024


keratin 18
3.47
0.000514523


ribonucleotide reductase M2 polypeptide
3.46
0.00834059


arylacetamide deacetylase-like 1
3.44
0.000902645


kinesin family member 11
3.43
0.00915145


Rho GTPase activating protein 11A
3.41
0.00834174


GINS complex subunit 1 (Psf1 homolog)
3.39
0.00104515


discs, large homolog 7 (Drosophila)
3.38
0.0317074


TTK protein kinase
3.38
0.0112171


deleted in lymphocytic leukemia, 2
3.38
0.0109528


replication factor C (activator 1) 3, 38 kDa
3.37
0.00109668


solute carrier family 7, (cationic amino acid transporte
3.36
0.00688017


dual-specificity tyrosine-(Y)-phosphorylation regulated ki
3.34
0.0234606


kinesin family member 2C
3.34
0.0059888


heat shock 22 kDa protein 8
3.32
0.0219583


polo-like kinase 1 (Drosophila)
3.30
0.0140309


v-myb myeloblastosis viral oncogene homolog (avian)-lik
3.28
0.0043878


trypsinogen C
3.28
0.00416276


thymidine kinase 1, soluble
3.27
0.00124134


NAD(P)H dehydrogenase, quinone 1
3.27
0.000282423


high-mobility group box 2
3.24
0.0196872


cell division cycle associated 2
3.24
0.0122226


apolipoprotein B mRNA editing enzyme, catalytic
3.23
0.00308692


polypep


serpin peptidase inhibitor, clade B (ovalbumin), member
3.22
0.0190218


guanine nucleotide binding protein (G protein), gamma 11
3.22
0.00140559


chromosome 15 open reading frame 23
3.21
0.000147331


kinesin family member 14
3.19
0.00947901


transmembrane protein 154
3.18
0.0045589


glycerol kinase
3.16
2.66E−05


KIAA1524
3.15
0.0380688


coagulation factor XIII, B polypeptide
3.14
0.0294465


tight junction protein 2 (zona occludens 2)
3.13
0.00012562


nei endonuclease VIII-like 3 (E. coli)
3.12
0.00115606


pleckstrin 2
3.11
0.0304429


kinesin family member 23
3.09
0.00790585


Rac GTPase activating protein 1
3.09
0.00381613


keratinocyte growth factor-like protein 1
3.07
0.0300588


keratinocyte growth factor-like protein 1
3.07
0.0300588


keratinocyte growth factor-like protein 1
3.07
0.0300588


transcription factor 19 (SC1)
3.07
0.00109627


OCIA domain containing 2
3.07
0.00122147


lung cancer metastasis-associated protein
3.06
0.00148024


transcription factor 19 (SC1)
3.05
0.00124327


transcription factor 19 (SC1)
3.05
0.00124327


Rho GTPase activating protein 29
3.05
0.0466211


glucosaminyl (N-acetyl) transferase 1, core 2 (beta-1,6-N-
3.05
0.0197148


replication factor C (activator 1) 4, 37 kDa
3.04
0.00164152


protein regulator of cytokinesis 1
3.01
0.0325664


transforming, acidic coiled-coil containing protein 3
2.98
0.0014577


cancer susceptibility candidate 5
2.96
0.0330594


nucleolar and spindle associated protein 1
2.96
0.00520875


cyclin B1
2.96
0.0103092


transmembrane protein 48
2.96
0.00458248


ZW10 interactor
2.95
1.88E−05


endonuclease domain containing 1
2.95
0.000429245


hypoxanthine phosphoribosyltransferase 1 (Lesch-Nyhan
2.94
0.000634057


synd


fucosidase, alpha-L-2, plasma
2.94
0.00540929


ubiquitin-conjugating enzyme E2T (putative)
2.93
0.00741886


lipase A, lysosomal acid, cholesterol esterase (Wolman
2.92
0.0167385


dise


villin 2 (ezrin)
2.92
0.0131934


glycerol kinase
2.90
3.37E−06


WD repeat domain 76
2.89
0.0023531


CD97 molecule
2.89
0.00994045


chromosome 18 open reading frame 24
2.89
0.00347442


topoisomerase (DNA) II alpha 170 kDa
2.89
0.0321109


integrin, alpha 3 (antigen CD49C, alpha 3 subunit of
2.87
0.00574148


VLA-3


family with sequence similarity 29, member A
2.85
0.00111165


kinesin family member 4A
2.85
0.0114203


BRCA1 associated RING domain 1
2.85
0.000540414


serum
2.84
0.0387246


RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)
2.83
0.000854739


Fanconi anemia, complementation group I
2.83
0.00464532


dihydrofolate reductase
2.82
0.00178879


claspin homolog (Xenopus laevis)
2.81
0.00683624


ornithine decarboxylase 1
2.81
0.00144868


sperm associated antigen 5
2.80
0.00906321


histone cluster 1, H3b
2.80
0.0304598


ATPase family, AAA domain containing 2
2.79
0.00415258


KIAA0286 protein
2.79
0.00130563


guanine nucleotide binding protein (G protein), alpha inhi
2.76
0.00184597


BUB1 budding uninhibited by benzimidazoles 1 homolog
2.74
0.0166047


beta


dihydrofolate reductase pseudogene
2.74
0.00141306


brix domain containing 1
2.73
0.00471977


cytoskeleton associated protein 2
2.72
0.0030499


mitochondrial ribosomal protein S28
2.72
0.00298194


polymerase (DNA directed), epsilon 2 (p59 subunit)
2.72
0.00479612


family with sequence similarity 72, member A
2.72
0.00143248


EBNA1 binding protein 2
2.70
0.00296292


similar to 40S ribosomal protein SA (P40) (34
2.70
0.0385298


adipose differentiation-related protein
2.70
0.000331751


thioredoxin reductase 1
2.70
0.000197486


minichromosome maintenance complex component 5
2.69
0.00475504


von Hippel-Lindau binding protein 1
2.69
0.00329061


SCL
2.68
0.00390288


Fanconi anemia, complementation group D2
2.68
0.0281405


NIMA (never in mitosis gene a)-related kinase 2
2.68
0.00289469


PHD finger protein 19
2.68
0.000177604


microsomal glutathione S-transferase 1
2.68
0.041701


breast cancer 2, early onset
2.68
0.00586847


non-SMC condensin I complex, subunit H
2.67
0.0216752


chromosome 13 open reading frame 27
2.67
0.0234588


histone cluster 1, H2bg
2.67
0.000180822


non-SMC condensin II complex, subunit G2
2.66
0.0130322


centromere protein I
2.64
0.0106816


stomatin
2.64
0.00387095


glutathione S-transferase omega 1
2.63
0.000648379


protein tyrosine phosphatase-like A domain containing
2.62
0.0419644


calcyclin binding protein
2.62
0.00524566


KIT ligand
2.61
0.00641955


ubiquitin-conjugating enzyme E2L 3
2.61
0.00343347


serpin peptidase inhibitor, clade B (ovalbumin), member
2.60
0.0030439


ATPase, Ca++ transporting, plasma membrane 4
2.60
0.023011


TPX2, microtubule-associated, homolog (Xenopus laevis)
2.60
0.0253137


thyroid hormone receptor interactor 13
2.59
0.0118319


H2A histone family, member Z
2.59
0.0129697


CDC28 protein kinase regulatory subunit 1B
2.57
0.0107391


cell division cycle associated 3
2.57
0.006289


minichromosome maintenance complex component 8
2.57
0.000841489


E2F transcription factor 2
2.55
0.0496479


TIMELESS interacting protein
2.55
0.00771062


minichromosome maintenance complex component 4
2.54
0.00342054


polo-like kinase 4 (Drosophila)
2.53
0.00209633


kinesin family member C1
2.53
0.00821937


dihydrofolate reductase
2.52
0.00307793


glycerol-3-phosphate dehydrogenase 2 (mitochondrial)
2.52
0.00211969


TGF beta-inducible nuclear protein 1
2.51
0.0365579


integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2
2.51
0.0210165


receptor


MLF1 interacting protein
2.51
0.0177203


heat shock 70 kDa protein 2
2.50
0.0215102


hairy and enhancer of split 1, (Drosophila)
2.50
0.000283509


ATP-binding cassette, sub-family C (CFTR
2.49
0.00382491


serglycin
2.48
0.0443487


sema domain, immunoglobulin domain (Ig), short basic
2.47
0.008548


doma


ankyrin repeat domain 1 (cardiac muscle)
2.47
0.00911953


transporter 1, ATP-binding cassette, sub-family B (MDR
2.47
0.00859077


transporter 1, ATP-binding cassette, sub-family B (MDR
2.47
0.00859077


transporter 1, ATP-binding cassette, sub-family B (MDR
2.47
0.00859077


histone cluster 1, H1b
2.46
0.0470898


family with sequence similarity 72, member A
2.46
0.00165234


membrane bound O-acyltransferase domain containing 1
2.46
0.01185


epidermal growth factor receptor pathway substrate 8
2.45
0.0194949


ASF1 anti-silencing function 1 homolog B (S. cerevisiae)
2.45
0.00543408


dedicator of cytokinesis 11
2.44
0.00697577


family with sequence similarity 72, member A
2.44
0.00162905


actin related protein 2
2.44
0.000288443


CTP synthase
2.43
8.80E−05


M-phase phosphoprotein 1
2.43
0.0271814


CDC28 protein kinase regulatory subunit 1B
2.43
0.0145263


histone cluster 1, H2ai
2.43
0.0161621


high-mobility group nucleosomal binding domain 2
2.42
0.0030536


heat shock 70 kDa protein 1A
2.42
0.00734287


heat shock 70 kDa protein 1A
2.42
0.00674816


carnitine palmitoyltransferase 1A (liver)
2.41
0.00170894


neurofilament, medium polypeptide 150 kDa
2.41
0.0190611


transmembrane protein 62
2.41
0.00761064


vaccinia related kinase 1
2.40
0.0233182


geminin, DNA replication inhibitor
2.40
0.00167629


phosphoglucomutase 2
2.40
0.00818204


lamin B1
2.40
0.0477748


keratin 18
2.40
0.000112551


deafness, autosomal dominant 5
2.39
0.00235481


proteasome (prosome, macropain) subunit, beta type, 9
2.39
0.0202595


(lar


proteasome (prosome, macropain) subunit, beta type, 9
2.39
0.0202595


(lar


proteasome (prosome, macropain) subunit, beta type, 9
2.39
0.0202595


(lar


chromosome 12 open reading frame 31
2.39
0.0173089


isocitrate dehydrogenase 3 (NAD+) alpha
2.39
0.00297129


forkhead box M1
2.38
0.0203154


transmembrane protein 106C
2.38
0.000214223


hypothetical protein LOC729012
2.38
0.000446087


PHD finger protein 1
2.37
0.010191


mitochondrial ribosomal protein L15
2.37
0.0306092


elastin microfibril interfacer 2
2.37
0.0192072


hypothetical protein DKFZp762E1312
2.37
0.00726778


retinoblastoma-like 1 (p107)
2.36
0.00319946


tissue factor pathway inhibitor (lipoprotein-associated
2.36
0.0356227


epithelial cell transforming sequence 2 oncogene
2.36
0.000571152


crystallin, zeta (quinone reductase)
2.36
0.0370884


hect domain and RLD 4
2.36
0.00679184


high-mobility group nucleosomal binding domain 2
2.36
0.00384071


cell division cycle 25 homolog A (S. pombe)
2.36
0.000341692


thymopoietin
2.35
0.0223176


interferon-induced protein with tetratricopeptide repeats
2.34
0.0177928


Bloom syndrome
2.34
0.0209259


dual specificity phosphatase 1
2.34
0.00211272


elongation factor, RNA polymerase II, 2
2.34
0.0130017


small nuclear ribonucleoprotein D1 polypeptide 16 kDa
2.34
0.0334665


CDC45 cell division cycle 45-like (S. cerevisiae)
2.33
0.00735977


exonuclease 1
2.33
0.00739393


ribosomal protein L39-like
2.33
0.00429384


histone cluster 1, H2bh
2.33
0.0377748


ribonucleotide reductase M1 polypeptide
2.33
0.000170076


sulfiredoxin 1 homolog (S. cerevisiae)
2.32
5.14E−05


multiple coagulation factor deficiency 2
2.31
0.0116892


proteasome (prosome, macropain) subunit, alpha type, 3
2.31
0.0195874


ribonuclease H2, subunit A
2.30
0.00669936


minichromosome maintenance complex component 10
2.29
0.0037925


heat shock 70 kDa protein 1B
2.28
0.0048959


heat shock 70 kDa protein 1B
2.28
0.0054404


heat shock 70 kDa protein 1B
2.28
0.0054404


ATPase, Na+
2.28
0.000381464


hypothetical protein LOC201725
2.28
0.000313319


cathepsin L1
2.27
0.0314419


cell division cycle associated 5
2.27
0.01021


RAB8B, member RAS oncogene family
2.27
0.00417066


SPC24, NDC80 kinetochore complex component,
2.27
0.00287227


homolog (S. ce


gamma-glutamyl hydrolase (conjugase,
2.26
0.0195219


folylpolygammaglutamyl


cell division cycle 25 homolog C (S. pombe)
2.25
0.0169914


mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)
2.25
0.00578953


metallothionein 1L (gene
2.25
0.00709646


RRS1 ribosome biogenesis regulator homolog (S. cerevisiae)
2.24
0.0120061


cell division cycle associated 8
2.24
0.00619878


shugoshin-like 2 (S. pombe)
2.24
0.000852557


mRNA turnover 4 homolog (S. cerevisiae)
2.24
0.00373104


ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,
2.24
0.00830766


v-ets erythroblastosis virus E26 oncogene homolog 2
2.23
0.0364123


(avian)


replication factor C (activator 1) 2, 40 kDa
2.23
0.00768959


NIMA (never in mitosis gene a)-related kinase 7
2.23
0.00159114


basic leucine zipper and W2 domains 2
2.23
0.0190782


histone cluster 1, H2bf
2.23
0.0124279


eukaryotic translation initiation factor 1A, X-linked
2.23
0.00330183


transporter 1, ATP-binding cassette, sub-family B (MDR
2.22
0.0164234


transporter 1, ATP-binding cassette, sub-family B (MDR
2.22
0.0164234


transporter 1, ATP-binding cassette, sub-family B (MDR
2.22
0.0164234


polymerase (RNA) III (DNA directed) polypeptide G
2.22
0.0298794


(32 kD)


phosphatidylinositol-4-phosphate 5-kinase, type II, alph
2.22
0.00964099


proteasome (prosome, macropain) 26S subunit, ATPase, 6
2.22
0.024269


pituitary tumor-transforming 1
2.21
0.0485166


histone cluster 2, H3d
2.21
0.0102932


sulfide quinone reductase-like (yeast)
2.21
0.0473641


serglycin
2.20
0.00880325


ribosomal protein L22-like 1
2.20
0.00335381


membrane protein, palmitoylated 1, 55 kDa
2.20
0.000396285


solute carrier family 24 (sodium
2.20
0.0328774


STAM binding protein-like 1
2.20
0.0181743


WD repeat and HMG-box DNA binding protein 1
2.20
0.0034833


CSE1 chromosome segregation 1-like (yeast)
2.20
0.0013662


origin recognition complex, subunit 6 like (yeast)
2.20
0.00182466


transcription factor A, mitochondrial
2.19
0.0110092


exosome component 8
2.19
0.00132017


mitochondrial ribosomal protein L1
2.19
0.0361058


sphingomyelin synthase 2
2.19
0.0020701


deoxycytidine kinase
2.18
0.00101444


family with sequence similarity 29, member A
2.18
0.00469407


chromosome 6 open reading frame 167
2.18
0.0011095


dual specificity phosphatase 11 (RNA
2.18
0.00426788


F-box protein 45
2.18
0.00510098


ras-related C3 botulinum toxin substrate 2 (rho family,
2.17
0.0292466


sma


FK506 binding protein 5
2.17
0.0193805


breast cancer 1, early onset
2.17
0.0180553


nuclear factor I
2.17
0.0010313


thioredoxin
2.17
0.009636


SH2 domain containing 4A
2.16
0.0323646


TGF beta-inducible nuclear protein 1
2.16
0.00285964


PSMC3 interacting protein
2.16
0.00766442


chromosome 3 open reading frame 14
2.15
0.0377617


polycomb group ring finger 5
2.15
0.000294142


centrosomal protein 27 kDa
2.15
0.00931602


family with sequence similarity 64, member A
2.14
0.0019785


acidic (leucine-rich) nuclear phosphoprotein 32 family, m
2.14
0.0300263


sterol O-acyltransferase (acyl-Coenzyme A: cholesterol
2.14
0.0193637


acy


TATA box binding protein (TBP)-associated factor, RNA
2.13
0.00514451


poly


origin recognition complex, subunit 5-like (yeast)
2.13
0.049697


Rac GTPase activating protein 1 pseudogene
2.13
0.000269488


LSM5 homolog, U6 small nuclear RNA associated (S. cerevisia
2.13
0.00264664


minichromosome maintenance complex component 7
2.13
0.0457691


met proto-oncogene (hepatocyte growth factor receptor)
2.13
0.0318147


tripartite motif-containing 25
2.13
0.0456344


chromosome 13 open reading frame 34
2.13
0.000702936


patatin-like phospholipase domain containing 4
2.13
0.0168306


minichromosome maintenance complex component 6
2.12
0.0161279


intraflagellar transport 80 homolog (Chlamydomonas)
2.12
0.0247286


peptidylprolyl isomerase F (cyclophilin F)
2.12
0.00093846


UTP15, U3 small nucleolar ribonucleoprotein, homolog
2.12
0.00482559


(S. c


TAF9B RNA polymerase II, TATA box binding protein
2.12
0.0170365


(TBP)-as


TAF9B RNA polymerase II, TATA box binding protein
2.12
0.0170365


(TBP)-as


esotropic viral integration site 2B
2.12
0.0171408


3′-phosphoadenosine 5′-phosphosulfate synthase 2
2.12
1.43E−05


proteasome (prosome, macropain) activator subunit 2
2.12
0.00609885


(PA28


ADAM metallopeptidase with thrombospondin type 1
2.12
0.0102751


motif,


flap structure-specific endonuclease 1
2.12
0.006882


S100 calcium binding protein A3
2.12
0.0324073


RAD18 homolog (S. cerevisiae)
2.11
0.0016685


minichromosome maintenance complex component 3
2.11
0.0018389


exosome component 3
2.11
0.0249115


cysteinyl-tRNA synthetase 2, mitochondrial (putative)
2.11
0.00564558


glutamate-cysteine ligase, modifier subunit
2.11
0.00378868


brix domain containing 1
2.11
0.00981178


kinesin family member 22
2.11
0.0192406


UTP11-like, U3 small nucleolar ribonucleoprotein, (yeast)
2.10
0.0132794


v-ral simian leukemia viral oncogene homolog B (ras
2.10
0.012225


related


meiotic nuclear divisions 1 homolog (S. cerevisiae)
2.10
0.00164447


phenylalanyl-tRNA synthetase, beta subunit
2.10
0.000245973


similar to Ubiquitin-conjugating enzyme E2S (Ubiqui
2.10
0.000415822


coiled-coil domain containing 68
2.10
0.00227586


lamin B receptor
2.10
0.000151784


Niemann-Pick disease, type C1
2.10
0.0108117


hydroxysteroid dehydrogenase like 2
2.09
3.71E−05


RMI1, RecQ mediated genome instability 1, homolog (S. cerev
2.09
0.00294705


overexpressed in colon carcinoma-1
2.09
0.015322


hypothetical protein FLJ20425
2.09
0.0174225


primase, polypeptide 1, 49 kDa
2.09
0.00801018


chromosome 20 open reading frame 121
2.09
0.0146323


microtubule associated serine
2.08
0.00536974


endothelial differentiation, sphingolipid G-protein-coupled
2.08
0.0132848


homeobox A9
2.08
0.00520942


centromere protein L
2.08
0.000880856


nucleolar complex associated 3 homolog (S. cerevisiae)
2.07
0.000373346


fibroblast growth factor 7 (keratinocyte growth factor)
2.07
0.0173208


cysteine rich transmembrane BMP regulator 1 (chordin-
2.07
0.0267286


like)


nucleoporin 155 kDa
2.07
0.00218453


FLJ20105 protein
2.06
0.0127979


CD44 molecule (Indian blood group)
2.06
0.000651436


polymerase (DNA directed), alpha 2 (70 kD subunit)
2.06
0.0033903


v-myb myeloblastosis viral oncogene homolog (avian)-
2.06
0.00989416


like 2


origin recognition complex, subunit 1-like (yeast)
2.06
0.00207753


hypothetical protein FLJ25416
2.06
0.000177531


kinesin family member 22
2.06
0.0242075


heat shock 60 kDa protein 1 (chaperonin)
2.06
0.0327412


minichromosome maintenance complex component 2
2.05
0.0021347


fumarylacetoacetate hydrolase (fumarylacetoacetase)
2.05
3.88E−05


glycerol kinase 3 pseudogene
2.05
0.0103203


retinitis pigmentosa 2 (X-linked recessive)
2.05
0.0264185


U2AF homology motif (UHM) kinase 1
2.05
0.0255167


chaperonin containing TCP1, subunit 5 (epsilon)
2.04
0.00125909


ATPase, H+ transporting, lysosomal 34 kDa, V1 subunit D
2.04
0.0317453


transcription termination factor, RNA polymerase II
2.04
0.000393489


succinate-CoA ligase, GDP-forming, beta subunit
2.04
0.0028167


cyclin-dependent kinase inhibitor 1B (p27, Kip1)
2.04
0.00183021


tyrosine 3-monooxygenase
2.04
0.00021508


cofactor required for Sp1 transcriptional activation, subu
2.04
0.00141809


glycosyltransferase 8 domain containing 3
2.03
0.022868


ribosomal RNA processing 15 homolog (S. cerevisiae)
2.03
0.0274884


glycogenin 1
2.03
0.0224317


hypothetical protein FLJ40869
2.03
0.00444509


proliferating cell nuclear antigen
2.03
0.0031727


sterile alpha motif domain containing 12
2.03
0.0232188


chromosome 16 open reading frame 59
2.03
0.00185191


cofilin 2 (muscle)
2.03
0.0459235


eukaryotic translation initiation factor 2, subunit 2 bet
2.03
0.0139947


chromatin assembly factor 1, subunit B (p60)
2.03
0.0119687


Zwilch, kinetochore associated, homolog (Drosophila)
2.02
0.000725107


ATP-binding cassette, sub-family E (OABP), member 1
2.02
0.00454751


LSM3 homolog, U6 small nuclear RNA associated (S. cerevisia
2.02
0.0199824


IQ motif containing GTPase activating protein 3
2.02
0.0495882


tubulin, alpha 1c
2.02
0.00862586


DBF4 homolog (S. cerevisiae)
2.01
0.0458795


amyloid beta precursor protein binding protein 1
2.01
0.000910538


suppressor of variegation 3-9 homolog 1 (Drosophila)
2.01
0.00224324


THO complex 7 homolog (Drosophila)
2.01
0.0047251


amyotrophic lateral sclerosis 2 (juvenile) chromosome re
2.01
0.0484466


nucleoporin 37 kDa
2.01
0.00652747


nucleolar protein 11
2.01
0.000852662


ATP synthase, H+ transporting, mitochondrial F0 complex
2.01
0.00866627


histone cluster 1, H2ai
2.01
0.0129155


phytoceramidase, alkaline
2.01
0.0157729


primase, polypeptide 2A, 58 kDa
2.01
0.00290097


similar to High mobility group protein B1 (High mobili
2.00
0.000363158


mastermind-like 3 (Drosophila)
−2.00
0.00386667


UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-
−2.01
0.0268634


acetylga


ring finger protein 122
−2.01
0.0236621


chromodomain helicase DNA binding protein 3
−2.01
6.39E−05


centaurin, gamma-like family, member 10 pseudogene
−2.01
8.70E−05


chromosome 7 open reading frame 10
−2.01
0.00738442


chromosome 6 open reading frame 111
−2.01
0.0104492


centaurin, gamma-like family, member 10 pseudogene
−2.01
0.000334818


Prader-Willi syndrome chromosome region 1
−2.01
0.0415526


KIAA1245
−2.01
0.0186309


peroxidasin homolog (Drosophila)
−2.01
0.00219049


melanoma antigen family D, 4
−2.02
0.0263076


melanoma antigen family D, 4
−2.02
0.0263076


glucosidase, alpha; acid (Pompe disease, glycogen storage
−2.02
0.000418401


di


phospholipase A2 receptor 1, 180 kDa
−2.03
0.00069343


glycosyltransferase 8 domain containing 2
−2.03
0.0173546


KIAA1546
−2.03
0.000255634


protocadherin beta 9
−2.03
0.0285124


TBC1 domain family, member 3B
−2.03
0.000414974


sushi, nidogen and EGF-like domains 1
−2.03
0.00161129


microtubule-actin crosslinking factor 1
−2.04
0.00216


region containing neuroblastoma breakpoint family,
−2.04
0.0213393


golgi autoantigen, golgin subfamily a-like pseudogene
−2.04
0.0182674


transducin-like enhancer of split 4 (E(sp1) homolog,
−2.04
0.0164153


Drosop


solute carrier family 22 (organic cation transporter),
−2.05
0.0137275


neighbor of Punc E11
−2.05
0.0184739


insulin-like growth factor binding protein 5
−2.05
0.011614


KIAA1245
−2.06
0.0185376


vitamin D (1,25-dihydroxyvitamin D3) receptor
−2.06
0.000192208


B-cell CLL
−2.06
0.00343507


KIAA1305
−2.06
0.00813727


KIAA1245
−2.06
0.0185609


centaurin, gamma-like family, member 10 pseudogene
−2.07
3.08E−05


TBC1 domain family, member 3B
−2.07
0.00141297


similar to TBC1 domain family member 3 (Rab GTPase-
−2.08
0.00105098


mannosidase, alpha, class 2B, member 1
−2.08
0.000353303


cysteine-rich PAK1 inhibitor
−2.08
0.000125336


midline 1 (Opitz
−2.08
0.00130803


small nucleolar RNA, H
−2.09
0.017124


urocortin 2
−2.09
0.00172263


neuroblastoma breakpoint family, member 11
−2.09
0.0138065


collagen, type VI, alpha 3
−2.09
2.09E−06


neuroblastoma breakpoint family, member 11
−2.09
0.0148372


hypothetical protein LOC646870
−2.09
0.0117625


calsyntenin 3
−2.09
0.00300887


cortactin binding protein 2
−2.09
2.28E−05


synaptic vesicle glycoprotein 2A
−2.10
0.00704212


similar to Dynamin-1 (D100) (Dynamin, brain) (B-dyn
−2.10
0.0190733


similar to Dynamin-1 (D100) (Dynamin, brain) (B-dyn
−2.10
0.0190733


similar to TBC1 domain family member 3 (Rab GTPase-
−2.10
0.00108467


Notch homolog 2 (Drosophila) N-terminal like
−2.10
0.0193058


matrix-remodelling associated 5
−2.11
0.000317637


complement component 1, s subcomponent
−2.11
0.0395863


cysteine sulfinic acid decarboxylase
−2.11
0.00428211


hypothetical protein FLJ36144
−2.11
0.00958437


hypothetical protein FLJ36144
−2.11
0.00958437


dihydropyrimidinase-like 3
−2.12
0.0165203


procollagen C-endopeptidase enhancer
−2.12
0.0039236


golgi autoantigen, golgin subfamily a-like pseudogene
−2.12
0.00720508


TBC1 domain family, member 3B
−2.12
0.00122924


collagen, type VII, alpha 1 (epidermolysis bullosa, dystr
−2.13
0.00109233


versican
−2.14
0.023885


mannose receptor, C type 2
−2.14
0.00012142


golgi autoantigen, golgin subfamily a-like pseudogene
−2.14
0.00767095


dynamin 1
−2.15
0.00139674


TBC1 domain family, member 3B
−2.16
0.00130459


PHD finger protein 21A
−2.17
0.00980401


centaurin, gamma-like family, member 10 pseudogene
−2.17
0.000180846


slit homolog 3 (Drosophila)
−2.17
0.02844


neuroepithelial cell transforming gene 1
−2.18
0.0109689


cyclin L2
−2.18
0.00093459


similar to dJ402H5.2 (novel protein similar to wo
−2.18
0.00621503


phospholipase D family, member 3
−2.18
1.17E−05


collagen, type VIII, alpha 1
−2.19
0.00187242


cyclin L2
−2.19
0.00109621


protocadherin beta 14
−2.20
0.0103892


matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase,
−2.20
5.59E−05


lysyl oxidase-like 4
−2.21
0.0120148


golgi autoantigen, golgin subfamily a-like pseudogene
−2.21
0.00977719


WW domain containing transcription regulator 1
−2.21
0.0379899


PDZ domain containing RING finger 3
−2.21
0.00931014


chromosome 14 open reading frame 37
−2.21
0.0182453


brain and acute leukemia, cytoplasmic
−2.22
0.0476919


calcium channel, voltage-dependent, L type, alpha 1C sub
−2.22
0.0189661


jun oncogene
−2.23
7.21E−05


interleukin 19
−2.23
0.0310328


centaurin, gamma-like family, member 10 pseudogene
−2.23
0.000595086


centaurin, gamma-like family, member 10 pseudogene
−2.23
0.000595086


—
−2.24
0.00666187


golgi autoantigen, golgin subfamily b, macrogolgin (with
−2.24
0.0164005


chromosome 15 open reading frame 51
−2.24
0.0123547


similar to Dynamin-1 (D100) (Dynamin, brain) (B-dyn
−2.24
0.0123547


similar to Dynamin-1 (D100) (Dynamin, brain) (B-dyn
−2.24
0.0123547


AE binding protein 1
−2.25
0.000105628


golgi autoantigen, golgin subfamily a-like pseudogene
−2.26
0.00770626


transmembrane protein 16A
−2.27
0.0481085


hypothetical LOC399844
−2.27
0.000491694


oculomedin
−2.27
0.00778869


low density lipoprotein-related protein 1 (alpha-2-
−2.28
4.26E−05


macroglo


fibronectin leucine rich transmembrane protein 2
−2.28
0.0135122


phospholipid transfer protein
−2.29
0.00999206


similar to Dynamin-1 (D100) (Dynamin, brain) (B-dyn
−2.29
0.0122573


SATB homeobox 2
−2.31
0.039781


similar to TBC1 domain family member 3 (Rab GTPase-
−2.32
0.000870285


tweety homolog 1 (Drosophila)
−2.32
0.00450824


CD24 molecule
−2.34
0.0340122


chimerin (chimaerin) 1
−2.35
0.0287031


AHA1, activator of heat shock 90 kDa protein ATPase
−2.37
0.00979472


homolog


bicaudal C homolog 1 (Drosophila)
−2.38
0.0347162


solute carrier family 6 (neurotransmitter transporter, ta
−2.38
0.00729635


milk fat globule-EGF factor 8 protein
−2.39
0.000987073


WNK lysine deficient protein kinase 1
−2.40
1.57E−05


small nucleolar RNA, H
−2.41
0.00843141


tweety homolog 3 (Drosophila)
−2.42
0.000165552


SH3 and PX domains 2B
−2.42
0.0244357


WD repeat and SOCS box-containing 1
−2.44
0.0387851


hypothetical protein PRO2012
−2.45
0.00756704


golgi autoantigen, golgin subfamily a-like pseudogene
−2.46
0.00320764


microfibrillar-associated protein 2
−2.47
0.0152901


collagen, type XII, alpha 1
−2.47
0.000204664


ST6 beta-galactosamide alpha-2,6-sialyltranferase 2
−2.47
0.0216987


thioredoxin interacting protein
−2.48
0.0135494


latent transforming growth factor beta binding protein 2
−2.49
4.08E−05


golgi autoantigen, golgin subfamily a-like pseudogene
−2.49
0.00603583


formin binding protein 1-like
−2.50
0.00290401


maternally expressed 3
−2.52
0.0112259


PTK7 protein tyrosine kinase 7
−2.54
0.000116114


ribonuclease P RNA component H1
−2.57
0.0156126


sushi-repeat-containing protein, X-linked 2
−2.58
0.0253856


sortilin-related VPS10 domain containing receptor 2
−2.58
0.00936311


similar to RIKEN cDNA 1110018M03
−2.59
0.00516476


pyridoxal-dependent decarboxylase domain containing 2
−2.60
0.00683647


Enah
−2.61
0.0077547


asporin
−2.62
0.000659873


small Cajal body-specific RNA 17
−2.63
0.0301336


nuclear pore complex interacting protein
−2.67
0.00988632


sushi, von Willebrand factor type A, EGF and pentraxin
−2.69
2.23E−05


dom


protein tyrosine phosphatase, receptor type, U
−2.69
0.0270428


collagen, type V, alpha 1
−2.70
0.0166427


nuclear pore complex interacting protein
−2.73
0.0018339


transformer-2 alpha
−2.74
0.012256


dystrophin related protein 2
−2.79
0.0137557


golgi autoantigen, golgin subfamily a, 8A
−2.80
0.0111179


collagen, type VI, alpha 2
−2.81
0.0149554


transforming growth factor, beta 3
−2.81
0.0287865


trophinin
−2.82
0.00298044


hypothetical protein MGC24103
−2.86
0.0346673


supervillin
−2.87
0.0412717


ADAM metallopeptidase with thrombospondin type 1
−2.90
0.0113968


motif,


kinesin family member 26B
−2.91
0.00363199


nuclear pore complex interacting protein
−2.91
0.00160273


trichorhinophalangeal syndrome I
−2.94
0.00557712


nuclear pore complex interacting protein
−2.96
0.00111223


small nucleolar RNA, C
−2.96
0.00666866


homeobox A2
−2.97
0.0435423


distal-less homeobox 5
−3.00
0.000640157


dachsous 1 (Drosophila)
−3.00
0.00697244


small nucleolar RNA, C
−3.06
0.0274043


small nucleolar RNA, C
−3.06
0.0274043


nuclear pore complex interacting protein
−3.09
0.00583397


small nucleolar RNA, C
−3.14
0.0104491


small nucleolar RNA, C
−3.14
0.0104491


sushi-repeat-containing protein, X-linked
−3.16
0.00370941


zinc finger protein 521
−3.17
0.00668815


nuclear pore complex interacting protein
−3.17
0.00117582


chromosome 9 open reading frame 3
−3.18
0.00410177


golgi autoantigen, golgin subfamily a, 8B
−3.18
0.0121417


hemicentin 1
−3.21
0.0461603


small nucleolar RNA, C
−3.24
0.00765575


Kallmann syndrome 1 sequence
−3.25
0.000548703


tenascin C (hexabrachion)
−3.26
8.26E−05


nuclear pore complex interacting protein
−3.29
0.00282604


nuclear pore complex interacting protein
−3.34
0.00263888


homeobox B2
−3.36
0.00665994


similar to nuclear pore complex interacting protein
−3.41
0.0187322


nuclear pore complex interacting protein
−3.46
0.00354416


cholesterol 25-hydroxylase
−3.51
0.0445558


ring finger protein 144
−3.52
0.0135334


nuclear pore complex interacting protein
−3.55
0.00316496


calbindin 2, 29 kDa (calretinin)
−3.56
0.0290743


nuclear pore complex interacting protein
−3.58
0.00032839


nuclear pore complex interacting protein
−3.60
0.000414309


nuclear pore complex interacting protein
−3.62
0.00283418


nuclear pore complex interacting protein
−3.64
0.000213956


nuclear pore complex interacting protein
−3.66
0.000377834


KIAA1641
−3.69
0.0191782


UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-
−3.72
0.00964109


acetylga


nuclear pore complex interacting protein
−3.73
0.000352007


leucine rich repeat containing 17
−3.75
0.0263961


chromosome 9 open reading frame 3
−3.80
0.0233723


nuclear pore complex interacting protein
−3.82
0.00368967


neurotrimin
−3.87
3.78E−06


protein tyrosine phosphatase, receptor type, N
−4.02
0.0294569


KIAA1641
−4.02
0.00659194


—
−4.06
0.00488845


KIAA1641
−4.16
0.0170531


integrin, alpha 11
−4.16
0.000390317


KIAA1641
−4.27
0.013175


odz, odd Oz
−4.28
0.00172671


transmembrane protein 119
−4.34
0.00801387


plexin domain containing 2
−4.44
0.031799


ras homolog gene family, member J
−4.59
0.00197982


homeobox B3
−4.60
0.0354368


similar to Protein KIAA0220
−4.72
0.0302619


raftlin family member 2
−4.79
0.0260454


WNT1 inducible signaling pathway protein 1
−5.99
0.000672342


clusterin
−6.40
0.0303973


serpin peptidase inhibitor, clade F (alpha-2 antiplasmi
−6.47
0.00362941


sulfatase 2
−6.58
5.88E−05


hephaestin
−6.74
0.0123141


junctional adhesion molecule 2
−7.33
0.0306758


fibronectin type III domain containing 1
−7.46
0.0334696


sarcoglycan, delta (35 kDa dystrophin-associated
−7.69
0.000881984


glycoprotei


cystatin SN
−8.27
0.0496433


microfibrillar-associated protein 4
−8.67
0.00155578


biglycan
−8.70
0.00161284


transmembrane, prostate androgen induced RNA
−10.54
0.000100935


carboxypeptidase E
−12.48
0.00738131









Characterization of membrane markers on 2D adherent cells suitable for use according to the present teachings—the surface antigens expressed by 2D adherent cells were examined using monoclonal antibodies. These cells were stable adhesive cells that were expanded in vitro without the loss of phenotype and without showing signs of karyotypic changes. Flow cytometric analysis of 2D adherent cells' membrane markers showed a high incidence of cells expressing CD105, CD73, CD90 and CD29. Furthermore, a high incidence of cells was lacking the expression of CD45, CD34 and CD19, CD11b, CD14 and HLA-DR surface markers (FIG. 2).
Immunomodulation by 2D adherent cells—The immunogenicity of the 2D adherent cells was investigated next. As shown in FIG. 3, four different batches of 2D adherent cells were capable of reducing lymphocyte proliferation, following mitogenic stimuli with Phytohemagglutinin (PHA), as was measured by Thymidine incorporation.
Osteocyte induction—osteocyte differentiation of placenta- or bone marrow-derived adherent cells in osteogenic induction medium resulted in differentiation of over 50% of the bone marrow cells, as demonstrated by positive alizarin red staining (FIG. 4B). On the contrary, none of the placental derived cells showed any signs of osteogenic differentiation (see FIGS. 4B and 4E and Table 9, below).







TABLE 9







Differentiation summary













BM 108 +



Plc



BM109
PLC-11-3-1
PLC-8-2-1
Plc-15-3-4-2
4-3-1
















Osteocytes
+++
−
−
−
−


Adipocytes
+++
−
−
−
−









Next, 2D adherent cells from bone marrow or placenta origin were stimulated to differentiate in a modified osteogenic medium comprising Vitamin D and higher concentrations of dexamethasone, a modification of the osteogenic differentiation protocol according to previous teachings [Parloni et al. (2008) Stem Cells 26(2): 300-11]. As evident from the results, over 50% of the bone marrow cells underwent differentiation into osteocytes, as demonstrated by positive alizarin red staining (see FIG. 5B). However, none of the placental derived cells showed any signs of osteogenic differentiation (see FIG. 5E and Table 9, hereinabove).
Adipocyte induction—adipocyte differentiation of placenta- or bone marrow-derived 2D adherent cells in adipocyte induction medium resulted in differentiation of over 50% of the bone marrow derived cells (see FIG. 4C), as demonstrated by positive oil red staining and by typical morphological changes (e.g. accumulation of oil droplets in the cytoplasm). In contrast, none of the placental derived cells differentiated into adipocytes (see FIG. 4F and Table 9, hereinabove).
Next, 2D adherent cells from bone marrow or placenta origin were stimulated to differentiate into adipocytes in a modified medium comprising a higher level of Indomethacine, a modification of the adipocyte differentiation protocol according to previous teachings [Parloni et al. (2007), supra]. As evident from the results, over 50% of the bone marrow derived cells underwent differentiation into adipocytes (see FIG. 5C), as demonstrated by positive oil red staining and by typical morphological changes (e.g. accumulation of oil droplets in the cytoplasm). In contrast, none of the placental derived cells exhibited morphological changes typical of adipocytes (see FIG. 5F and Table 9, hereinabove).
Example 3
Methods of Generating 3D Adherent Cells Suitable for Use in Accordance with the Present Teachings and the 3D Adherent Cells Generated Thereby
3D adherent cells (PLX-C) were produced which exhibit different characteristics then the above described 3D adherent cells (PLX, Example 1).
Materials and Experimental Procedures
CelligenTM Plug Flow Bioreactor
The production of 3D adherent cells for use in accordance with the present invention by CelligenTM (PLX-C cells) was composed of several major steps. The process started by collection of a placenta from a planned cesarean delivery at term.
Adherent cells were then isolated from whole placentas, grown in tissue culture flasks (2D cultures), harvested and stored in liquid nitrogen as 2D-Cell Stock (2DCS), the appropriate amount of 2DCS were thawed, washed and seeded onto carriers in bioreactors for further expansion as 3D-culture. After 4-21 days of growth in the bioreactors, cells were harvested and cryopreserved in gas phase of liquid nitrogen as PLX-C.
Receipt of Human Tissue
All placentas obtained were received from the maternity ward under approval of the Helsinki Committee of the medical facility. Accordingly, all placenta donors signed an informed consent and Donor Screening and Donor Testing was performed (IPC1). Immediately after taking the placenta from the donor (during the caesarean procedure), it was placed in a sterile plastic bag and then in a Styrofoam box with ice packs. The placenta was delivered and immediately placed in a quarantine area until released to use by Quality Control (QC) and Quality Assurance (QA). All the following production steps were performed in a quarantine, clean room facility until QC approval of mycoplasma test results arrived and the cells were release for 2D cell growth.
Recovery and Processing of Adherent Cells
To initiate the process, the whole placenta tissue was cut into pieces under aseptic conditions under laminar flow hood, washed with Hank's buffer solution and incubated for 3 hours at 37° C. with 0.1% Collagenase (1 mg Collagenase/ml tissue). 2D cell medium (2D-Medium comprising DMEM supplemented with 10% FBS, fungizone 0.25 μg/ml and gentamycine 50 μg/ml) was added and the digested tissue was roughly filtered through a sterile metal strainer, collected in a sterile beaker and centrifuged (10 minutes, 1200 RPM, 4° C.). Using gentle pippeting, suspended cells were then washed with 2D-Medium supplemented with antibiotics, seeded in 80 cm2 flasks and incubated at 37° C. in a tissue culture incubator under humidified condition supplemented with 5% CO2. Following 2-3 days, in which the cells were allowed to adhere to the flask surface, they were washed with PBS and 2D-Medium was added.
Two Dimensional (2D) Cell Growth
Prior to the first passage, growth medium samples of 10% of the total flask number in quarantine was pooled and taken for mycoplasma testing (IPC2). If cells were found to be negative for Mycoplasma (EZ-PCR Mycoplasma kit, Biological Industries, Israel), cells were released from quarantine. After 1-2 additional passages, cells were transferred to the 2D production clean room (2DP). Once in Room 2DP, culture was continued for another 3-5 passages (of note, cells were grown in 2D-Medium supplemented with antibiotics until passage 3, thereafter cells were grown in 2D-Medium without antibiotics). IPC-3 sample was taken for immune phenotype after passage 4. Throughout the process, cultures were grown in a tissue culture incubator under humidified conditions with 5% CO2 at 37° C. After a total of 6-8 passages (9-16 cell doublings), cells were collected and cryopreserved as the 2D-Cell Stock (2DCS).
The first passage was usually carried out after 10-15 days. Beginning at passage 2 and continuing until passage 6-8, cells were passaged when the culture reached 70-80% confluence, usually after 3-5 days (1.5-2 doublings). The cells were detached from the flasks using 0.25% trypsin-EDTA (4 minutes at 37° C.) and seeded in a culture density of 3±0.2×103 cells/cm2. The size of the tissue culture flasks raised as the passages proceed. The culturing process started in 80 cm2 tissue culture flask, continued in 175 cm2, then in 500 cm2 (Triple flask) and finally the cells were seeded into Cell Factory 10 tray (6320 cm2).
Prior to cryopreservation, at the end of 2DCS growth period, the growth medium was collected and the sample was prepared to be sent to an approved GLP laboratory for Mycoplasma test (IPC 4).
Cryopreservation Procedure for 2D-Cell-Stock Product
For 2DCS cryopreservation, 2D-cultured cells were collected under aseptic conditions using 0.25 trypsin-EDTA. The cells were centrifuged (1200 RPM, 10′, 4° C.), counted and re-suspended in 2D-Medium.
For freezing, cell suspensions were diluted 1:1 with 2D-Freezing Mixture (final concentrations was 10% DMSO, 40% FBS and 50% 2D-Medium). Approximately 1.5-2.5×109 cells were manufactured from one placenta. 4 ml of the cells were stored at a final concentration of 10×106/ml in 5 ml cryopreservation polypropylene vials. The vials were labeled and transferred to a controlled rate freezer for a graduated temperature reducing process (1° C./min), after which they were transferred to storage in gas-phase of a liquid nitrogen freezer located in the Cold Storage Room. This material was referred to as the 2D-Cell Stock (2DCS) batch.
Initiation of the Three Dimensional (3D) Culture Procedures
To begin 3D culture, an appropriate amount (150±30×106) of cells from 2DCS were thawed in the 2DP room and washed with 3D-Medium (DMEM with 10% FBS and 20 Mm Hepes) to remove DMSO prior to seeding in the prepared-in-advanced bioreactor systems. The content of each 2DCS vial was pipetted and diluted 1:9 with pre-warmed (37° C.) 3D-Medium. The cells were centrifuged (1200 RPM, 10′, 4° C.) and re-suspended again in 50-100 ml pre-warmed (37° C.) 3D-Medium in a 250 ml sterile bottle. A sample was taken and cells were counted using a Trypan Blue stain in order to determine cell number and viability. The cell suspension was transferred under a laminar flow hood into a 0.5 L seeding bottle. From the seeding bottle the cell suspension was transferred via sterile tubing to the bioreactor by gravitation.
Production of Adherent Cells in the Celligen Bioreactor (PLX-C)
Bioreactor Description
3D growth phase was performed using an automatic CelliGen Plus® or BIOFLO 310 bioreactor system [(New Brunswick Scientific (NBS)]. The bioreactor system was used for cultivation of cell culture, in which conditions were suitable for high cell concentrations. The cultivation process was carried out using a bioreactor in a perfusion mode. The lab scale bioreactor was constructed of two main systems—the control system and the bioreactor itself (vessel and accessories). The parameters of the process were monitored and controlled by a control console which included connectors for probes, motor and pumps, control loops for Dissolved Oxygen (DO), pH, perfusion and agitation (with a motor), a gases control system, water circulation and heating system for temperature control and an operator interface. The controlled process parameters (such as temperature, pH, DO etc.) could be displayed on the operator interface and monitored by a designated controller.
Cell Culture Growth Procedure in the Bioreactors
As noted in the section hereinabove, 150±30×106 cells from the cryopreserved 2DCS were thawed, washed and seeded in a sterile bioreactor. The bioreactor contained 30-50 gr carriers (FibraCel® disks, NBS), made of Polyester and Polypropylene and 1.5±0.1 L 3D-Medium. The growth medium in the bioreactor was kept at the following conditions: 37° C., 70% Dissolved Oxygen (DO) and pH 7.3. Filtered gases (Air, CO2, N2 and O2) were supplied as determined by the control system in order to keep the DO value at 70% and the pH value at 7.3. For the first 24 hours, the medium was agitated at 50 Rounds Per Minutes (RPM) and increased up to 200 RPM by day 2. For the first 2-3 days, the cells were grown in a batch mode. Perfusion was initiated when the medium glucose concentration decreased below 550 mg/liter. The medium was pumped from the feeding container to the bioreactor using sterile silicone tubing. All tubing connections were performed under laminar flow using sterile connectors. The perfusion was adjusted on a daily basis in order to keep the glucose concentration constant at approximately 550±50 mg/liter. A sample of the growth medium was taken every 1-2 days for glucose, lactate, glutamine, glutamate and ammonium concentration determination (BioProfile 400 analyzer, Nova Biomedical). The glucose consumption rate and the lactate formation rate of the cell culture enabled to measure cell growth rate. These parameters were used to determine the harvest time based on accumulated experimental data.
Harvest of the 3D Grown PLX-C Cells from the Bioreactor
The cell harvest process started at the end of the growth phase (4-10 days). Two samples of the growth medium were collected. One sample was prepared to be sent to an approved GLP laboratory for Mycoplasma testing according to USP and Eu standards, and the other one was transferred to a controlled rate freezer for a graduated temperature reducing process (1° C./min), after which they were transferred to storage in gas-phase of a liquid nitrogen freezer located in the Cold Storage Room, in case a repeat Mycoplasma testing was needed. These medium samples were considered as part of the Mycoplasma testing of the final product and the results were considered as part of the criteria for product release.
The 3D-grown culture was harvested in the Class-100 laminar area in room 3DP as follows:
The bioreactor vessel was emptied using gravitation via tubing to a waste container. The vessel was opened, by removing the head plate, and the carriers were aseptically transferred, using sterile forceps, from the basket to the upper basket net. The bioreactor vessel was then closed and refilled with 1.5 L pre-warmed PBS (37° C.). The agitation speed was increased to 150 RPM for 2 minutes. The PBS was drained via tubing by pressure or gravity to the waste bottle. The washing procedure was repeated twice.
In order to release the cells from the carriers, 1.5 L pre-warmed to 37° C. Trypsin-EDTA (Trypsin 0.25%, EDTA 1 mM) was added to the bioreactor vessel and carriers were agitated for 5 minutes in 150 RPM, 37° C. Cell suspension was collected to a 5 L sterile container containing 250 ml FBS. Cell suspension was divided to 4 500 ml sterile centrifuge tubes and a Mycoplasma test sample was withdrawn. Closed centrifuge tubes were transferred through the 3DP active pass-through into the class 10,000 filling room (FR1) in which the cells were aseptically filled and cryopreserved as PLX-C.
Cell Cycle Analysis
PLX-C cells obtained by Celligen and PLX cells obtained by Plurix were fixed with 70% EtOH O.N, centrifuged and re-suspended in a Propidium Iodide (PI) solution containing 2 μg/ml PI (Sigma), 0.2 mg/ml Rnase A (Sigma) and 0.1% (v/v) Triton (Sigma) for 30 minutes. Cell cycle was analyzed by FACS.
Gene Expression Array (Microarray)
Adherent cells were obtained from human full term placentas and were expanded Plurix or by Celligen. Three different batches of cells were obtained from each of the expansion methods for further examination.
RNA was extracted from the cells (Qiagen-Rneasy micro kit) and applied to an Affymetrix whole genome expression array. The chip used GeneChip® Human Exon 1.0 ST Array (Affymetrix, Santa Clara, Calif., USA).
FACS Analysis of Membrane Markers
Cells were stained with monoclonal antibodies as previously described. In short, 400,000-600,000 cells were suspended in 0.1 ml flow cytometer buffer in a 5 ml test tube and incubated for 15 minutes at room temperature (RT), in the dark, with each of the following monoclonal antibodies (MAbs): FITC-conjugated anti-human CD29 MAb (eBioscience), PE conjugated anti human CD73 MAb (Becton Dickinson), PE conjugated anti human CD105 MAb (eBioscience), PE conjugated anti human CD90 MAb (Becton Dickinson), FITC-conjugated anti-human CD45 MAb (IQProducts), PE-conjugated anti-human CD19 MAb (IQProducts), PE conjugated anti human CD14 MAb (IQProducts), FITC conjugated anti human HLA-DR MAb (IQProduct), PE conjugated anti human CD34 MAb (IQProducts), FITC conjugated anti human CD31 MAb (eBioscience), FITC conjugated anti human KDR MAb (R&D systems), anti human fibroblasts marker (D7-FIB) MAb(ACRIS), FITC-conjugated anti-human CD80 MAb (BD), FITC-conjugated anti-human CD86 MAb (BD), FITC-conjugated anti-human CD40 MAb (BD), FITC-conjugated anti-human HLA-ABC MAb (BD), Isotype IgG1 FITC conjugated (IQ Products), Isotype IgG1 PE conjugated (IQ Products).
Cells were washed twice with flow cytometer buffer, resuspended in 500 μl flow cytometer buffer and analyzed by flow cytometry using FC-500 Flow Cytometer (Beckman Coulter). Negative controls were prepared with relevant isotype fluorescence molecules.
Mixed Lymphocyte Reaction (MLR)
2×105 peripheral blood (PB) derived MNC (from donor A) were stimulated with equal amount of irradiated (3000 Rad) PB derived MNCs (from donor B). Increasing amounts of PLX-Cs were added to the cultures. Three replicates of each group were seeded in 96-well plates. Cells were cultured in RPMI 1640 medium containing 20% FBS. Plates were pulsed with 1 μC 3H-thymidine during the last 18 hrs of the 5-day culturing. Cells were harvested over a fiberglass filter and thymidine uptake was quantified with scintillation counter.
For CFSE staining, PB-MNC cells were stained for CFSE (Molecular Probes) for proliferation measurement before culturing. Cells were collected after 5 days and the intensity of CFSE staining was detected by Flow Cytometry.
ELISA
ELISA was carried out as was previously described. In short, MNCs (isolated from peripheral blood) were stimulated with 5 μg/ml ConA (Sigma), 0.5 μg/ml LPS (SIGMA), or 10 μg/ml PHA (SIGMA) in the presence of PLX-C under humidified 5% CO2 atmosphere at 37° C. Supernatants were collected and subjected to cytokine analysis using ELISA kits for IFNγ (DIACLONE), TNFα (DIACLONE) and IL-10 (DIACLONE).
Results
The changes in manufacturing with Celligen as compared to Plurix resulted in several major differences (summarized in Table 10, below).







TABLE 10







Comparison between Plurix system (WO/2007/108003) and


Celligen system (teachings of the present invention)












3D adherent





cells of the




present


Parameter
WO/2007/108003
teachings
Improvement





Working
280
1500
Scale up of the


volume (ml)


process.





Higher





production level





in the present





teachings (2-8





population





doubling)


Weight of
 1.4
 30
Scale up of the


carrier (gr)


process in the





present teachings.


Bed
Conic, 50 ml
Cylinder
The present


configuration
column
Packed bed
teachings -





Better flow of





medium and





nutrients.





WO/2007/108003 -





Inefficient





flow due to





narrow outlet





form the conic





structure





Better





homogeneity of





medium flow.





Channeling in the





plurix


Cell
3 × 106 cell/
5 × 106 cell/
Better cell to cell


concentration
gr carrier
gr carrier
interaction in the


at seeding


present teachings


(cell/gr


carrier)


Cell
0.015 × 106
0.1 × 106
Better cell to cell


concentration
cell/ml
cell/ml
interaction in the


at seeding


present teachings


(cell/ml)


Seeding
Seeding at low
Seeding at the
WO/2007/108003 -


procedure
medium volume
final working
Heterogenic



for 24 h followed
volume while
distribution of the



by addition of
agitating
cell culture inside



medium to final

the carrier bed



working volume

Insufficient





medium volume





in the first 24 h of





the run. Leading





to unsuitable





working





conditions (acidic





environment)


Production
14-21 days
4-10 days
Better product


phase


quality.


duration


Efficient harvest





process.





Better yield.





Lower cost





process in the





present teachings


Mode of
Repeated batch -
Perfusion
Present teachings -


operation
medium change
mode - rate
Moderate



twice a week
was adjusted
changes of the




according to
conditions




the glucose
regarding




concentration
medium




(the medium
composition




was changed
throughout the




at glucose
run




concentration
Continuous




of 550 ±
removal of toxic




50 mg/L)
agents produced





by the cells.





In batch mode -





lower





concentration of





essential





nutrients





(limiting factors)





Less cell debris


Harvest
Harvesting in
Harvesting
Present teachings -


procedure
50 ml tubes
inside the
More efficient



Trypsinization 3
bioreactor
process



cycles
Trypsinization
Harvest is carried




1 cycle
out in a close





system.





1 trypsinization





cycle - better





quality of the





cells.


Agitation
medium
Cell lift
Present teachings -



Circulation
impeller
Medium is



between reservoir

flowing through



container to the

the packed bed -



column using

Better supply of



peristaltic pump

nutrients and





oxygen to the





culture.





Homogeneity of





the medium





Improves other





control loops





(temp., DO, pH)


Temperature
The production
On-line direct
Present teachings -


control
was carried out
control.
more accurate



inside an
Heat transfer
measurement of



incubator.
via water
the culture



Indirect
jacket.
temperature.



temperature

Quick response.



control (of the

Short time to



incubator

reach set point.



chamber).



Heat transfer via



air interface


Temperature
Manually.
On-line direct
Present teachings -


monitoring
Indirect water
monitoring.
Better



temperature

monitoring and



monitoring.

control of the





process.





Quick response to





malfunctions.


DO
None
On-line
Present teachings -


monitoring

monitoring
Better





monitoring and





control of the





process.





Quick response to





malfunctions


DO control
None.
On-line direct
Present teachings -



Introduction of air
control of a
Better control



only
specific set
of DO level.




point using
Better




Air, O2 and
maintenance of a




N2.
specified working





conditions


pH
Only visual
On-line
Present teachings -


monitoring
monitoring
Control and
Better control


and control
(Phenol red as
monitoring
of pH level.



part of the

Better



medium)

maintenance of a





specified working





conditions


Aeration
Sparge only
Overlay
WO/2007/108003 -




(sparge as an
Aeration by




option)
sparge creates





foam that might





damage the cells.









The changes in the manufacturing process resulted in changes in characteristics of the obtained adherent cells. These differences are summarized below.
Cell Cycle Analysis of PLX Manufactured by Plurix Compared to PLX-C Manufactured by Celligen
PLX-C cells obtained by Celligen were compared to PLX cells obtained by Plurix in order to examine the distribution of the cells between the different phases of the cell cycle. As is clear from FIGS. 6A-B, PLX-C cells expanded by Celligen exhibited typical proliferating profile (distribution of cells between the different phases of cell cycle). Specifically, 28% of cells were in S and G2/M phases (FIG. 6A). These results indicated that cells were harvested during proliferation and that the Celligen bioreactor conditions supported cell growth.
Microarray Comparison Between Plurix and Celligen Obtained Cells
Gene expression arrays enabled to simultaneously monitor genome-wide expression profiles of adherent cells derived from human full term placentas expanded by Plurix (PLX) or by Celligen (PLX-C). These results enabled to assess the molecular mechanism underlying phenotypic variation between cells obtained by these different growth methods (see Table 11, below).







TABLE 11







Gene expression in Plurix cells (WO/2007/108003) compared


to Celligen cells (teachings of the present invention)










Celligen vs Plurix



Gene
(fold change)
p-value (treat)












interferon-induced protein with tetratricopeptide
17.52
0.0401812


repeats


aldehyde dehydrogenase 1 family, member A1
16.76
0.00145807


leukocyte-derived arginine aminopeptidase
13.99
3.88E−06


keratin 27 pseudogene 27
12.25
0.000224998


similar to Keratin, type I cytoskeletal 18 (Cytokerati
11.83
0.000304949


G protein-coupled receptor, family C, group 5,
10.35
3.39E−05


member A


integrin, alpha 6
9.84
0.0411667


G protein-coupled receptor 126
8.73
0.00197635


coagulation factor III (thromboplastin, tissue factor)
7.36
0.012192


Rho GDP dissociation inhibitor (GDI) beta
7.36
0.00200066


signal peptide, CUB domain, EGF-like 3
7.20
0.0255115


interferon-induced protein with tetratricopeptide
7.09
0.0139777


repeats


dickkopf homolog 1 (Xenopus laevis)
7.06
3.06E−07


NAD (P)H dehydrogenase, quinone 1
6.63
0.000282423


keratin 18
6.46
0.000514523


opioid growth factor receptor-like 1
5.96
0.00114551


mal, T-cell differentiation protein-like
5.95
0.00664216


neurofilament, medium polypeptide 150 kDa
5.86
0.0190611


DEP domain containing 1
5.82
0.000370513


cathepsin C
5.72
0.00532262


WAS
5.47
0.00178153


serpin peptidase inhibitor, clade B (ovalbumin),
5.44
0.0190218


member


solute carrier family 7, (cationic amino acid transporte
5.33
0.00688017


interferon-induced protein with tetratricopeptide repea
5.18
0.00357376


NUF2, NDC80 kinetochore complex component,
5.05
0.00276524


homolog (S. cere


SHC SH2-domain binding protein 1
4.95
0.00430878


thioredoxin reductase 1
4.86
0.000197486


lung cancer metastasis-associated protein
4.85
0.00148024


Rho GTPase activating protein 29
4.85
0.0466211


cell division cycle 20 homolog (S. cerevisiae)
4.80
0.00514206


family with sequence similarity 111, member B
4.63
0.000125819


PDZ binding kinase
4.54
0.00784983


establishment of cohesion 1 homolog 2 (S. cerevisiae)
4.53
0.000773033


guanylate binding protein 4
4.47
0.000215944


lipase A, lysosomal acid, cholesterol esterase
4.42
0.0167385


(Wolman dise


kinesin family member 20A
4.39
0.00582352


KIAA0101
4.28
0.0105909


cyclin-dependent kinase inhibitor 3 (CDK2-associated
4.25
0.000732492


dual


thymidylate synthetase
4.23
0.00685584


chromosome 13 open reading frame 3
4.18
0.000548296


aurora kinase A
4.16
0.00632571


nei endonuclease VIII-like 3 (E. coli)
4.14
0.00115606


centrosomal protein 55 kDa
4.13
0.0021952


oxidized low density lipoprotein (lectin-like) receptor 1
4.11
0.0205198


denticleless homolog (Drosophila)
4.05
0.00141153


anillin, actin binding protein
4.01
0.010923


ribonucleotide reductase M2 polypeptide
3.98
0.00834059


ankyrin repeat domain 1 (cardiac muscle)
3.93
0.00911953


transcription factor 19 (SC1)
3.89
0.00109627


keratin 18
3.89
0.000112551


non-SMC condensin I complex, subunit G
3.88
0.00537097


cyclin E2
3.87
0.000203389


trypsinogen C
3.86
0.00416276


small nucleolar RNA, C
3.81
0.0334484


tight junction protein 2 (zona occludens 2)
3.81
0.00012562


kinesin family member 18A
3.78
0.00134108


kinesin family member 2C
3.77
0.0059888


shugoshin-like 1 (S. pombe)
3.76
0.00101318


polo-like kinase 1 (Drosophila)
3.75
0.0140309


thymidine kinase 1, soluble
3.73
0.00124134


transcription factor 19 (SC1)
3.73
0.00124327


transcription factor 19 (SC1)
3.73
0.00124327


claspin homolog (Xenopus laevis)
3.71
0.00683624


GINS complex subunit 1 (Psf1 homolog)
3.69
0.00104515


microsomal glutathione S-transferase 1
3.67
0.041701


arylacetamide deacetylase-like 1
3.67
0.000902645


SPC25, NDC80 kinetochore complex component,
3.65
0.00568662


homolog (S. ce


integrin, alpha 4 (antigen CD49D, alpha 4 subunit of
3.62
0.0158411


VLA-4


catenin (cadherin-associated protein), alpha-like 1
3.57
7.46E−05


discs, large homolog 7 (Drosophila)
3.56
0.0317074


v-myb myeloblastosis viral oncogene homolog
3.55
0.0043878


(avian)-lik


serglycin
3.54
0.0443487


centromere protein N
3.53
0.000540143


cyclin A2
3.53
0.00965934


heat shock 22 kDa protein 8
3.52
0.0219583


sema domain, immunoglobulin domain (Ig), short
3.49
0.008548


basic doma


Rho GTPase activating protein 11A
3.49
0.00834174


Fanconi anemia, complementation group I
3.43
0.00464532


BUB1 budding uninhibited by benzimidazoles 1
3.42
0.0108258


homolog (yeast


ovary-specific acidic protein
3.42
0.00334641


cholinergic receptor, muscarinic 2
3.41
0.0320078


cell division cycle 2, G1 to S and G2 to M
3.41
0.0017111


protein regulator of cytokinesis 1
3.39
0.0325664


minichromosome maintenance complex component 5
3.38
0.00475504


sperm associated antigen 5
3.37
0.00906321


maternal embryonic leucine zipper kinase
3.34
0.00908391


small nucleolar RNA, C
3.33
0.0298703


carnitine palmitoyltransferase 1A (liver)
3.33
0.00170894


similar to Ubiquitin-conjugating enzyme E2S (Ubiqui
3.33
0.000415822


kinesin family member 11
3.33
0.00915145


NIMA (never in mitosis gene a)-related kinase 7
3.33
0.00159114


ADAM metallopeptidase with thrombospondin type 1
3.32
0.0102751


motif,


transforming, acidic coiled-coil containing protein 3
3.31
0.0014577


cyclin B1
3.29
0.0103092


MAD2 mitotic arrest deficient-like 1 (yeast)
3.28
0.00488102


dihydrofolate reductase
3.28
0.00178879


NIPA-like domain containing 3
3.27
0.00164708


cell division cycle associated 2
3.26
0.0122226


apolipoprotein B mRNA editing enzyme, catalytic
3.26
0.00308692


polypep


cyclin B2
3.25
0.016544


endonuclease domain containing 1
3.24
0.000429245


dihydrofolate reductase pseudogene
3.23
0.00141306


ATPase, Na+
3.23
0.000381464


replication factor C (activator 1) 3, 38 kDa
3.23
0.00109668


WD repeat domain 76
3.22
0.0023531


pleckstrin 2
3.17
0.0304429


Rac GTPase activating protein 1
3.17
0.00381613


PHD finger protein 19
3.17
0.000177604


deleted in lymphocytic leukemia, 2
3.15
0.0109528


centromere protein I
3.15
0.0106816


BRCA1 associated RING domain 1
3.14
0.000540414


regulator of G-protein signalling 4
3.13
0.00781061


STAM binding protein-like 1
3.11
0.0181743


sulfiredoxin 1 homolog (S. cerevisiae)
3.10
5.14E−05


chromosome 15 open reading frame 23
3.08
0.000147331


TTK protein kinase
3.08
0.0112171


non-SMC condensin II complex, subunit G2
3.08
0.0130322


villin 2 (ezrin)
3.07
0.0131934


stomatin
3.06
0.00387095


protein tyrosine phosphatase-like A domain containing
3.06
0.0419644


serpin peptidase inhibitor, clade B (ovalbumin),
3.05
0.0030439


member


kinesin family member 4A
3.05
0.0114203


hypothetical protein DKFZp762E1312
3.05
0.00726778


ubiquitin-conjugating enzyme E2S
3.04
0.00118205


hydroxysteroid dehydrogenase like 2
3.03
3.71E−05


ATPase family, AAA domain containing 2
3.01
0.00415258


TPX2, microtubule-associated, homolog (Xenopus
3.00
0.0253137


laevis)


histone cluster 1, H4d
3.00
0.030183


kinesin family member 23
2.99
0.00790585


heat shock 70 kDa protein 2
2.99
0.0215102


origin recognition complex, subunit 1-like (yeast)
2.99
0.00207753


dihydrofolate reductase
2.98
0.00307793


hyaluronan-mediated motility receptor (RHAMM)
2.97
0.00467816


3′-phosphoadenosine 5′-phosphosulfate synthase 2
2.97
1.43E−05


glycerol-3-phosphate dehydrogenase 2 (mitochondrial)
2.95
0.00211969


nucleolar and spindle associated protein 1
2.95
0.00520875


diaphanous homolog 3 (Drosophila)
2.95
0.00107709


kinesin family member 14
2.94
0.00947901


histone cluster 1, H1b
2.93
0.0470898


guanine nucleotide binding protein (G protein), alpha
2.92
0.00184597


inhi


minichromosome maintenance complex component 8
2.92
0.000841489


cancer susceptibility candidate 5
2.92
0.0330594


leukotriene B4 12-hydroxydehydrogenase
2.92
0.000685452


glutamate-cysteine ligase, modifier subunit
2.91
0.00378868


forkhead box M1
2.91
0.0203154


adipose differentiation-related protein
2.90
0.000331751


membrane bound O-acyltransferase domain containing 1
2.90
0.01185


ubiquitin-conjugating enzyme E2T (putative)
2.90
0.00741886


cell division cycle associated 3
2.89
0.006289


integrin, alpha 3 (antigen CD49C, alpha 3 subunit of
2.88
0.00574148


VLA-3


coagulation factor XIII, B polypeptide
2.88
0.0294465


RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)
2.87
0.000854739


ATP-binding cassette, sub-family C (CFTR
2.87
0.00382491


family with sequence similarity 29, member A
2.85
0.00111165


SH2 domain containing 4A
2.84
0.0323646


membrane protein, palmitoylated 1, 55 kDa
2.84
0.000396285


CDC28 protein kinase regulatory subunit 1B
2.84
0.0107391


PSMC3 interacting protein
2.84
0.00766442


elastin microfibril interfacer 2
2.84
0.0192072


topoisomerase (DNA) II alpha 170 kDa
2.83
0.0321109


transmembrane protein 106C
2.82
0.000214223


histone cluster 1, H3b
2.80
0.0304598


chromosome 18 open reading frame 24
2.80
0.00347442


epidermal growth factor receptor pathway substrate 8
2.79
0.0194949


high-mobility group nucleosomal binding domain 2
2.78
0.0030536


SCL
2.78
0.00390288


hect domain and RLD 4
2.78
0.00679184


ASF1 anti-silencing function 1 homolog B (S. cerevisiae)
2.77
0.00543408


thyroid hormone receptor interactor 13
2.76
0.0118319


cell division cycle associated 8
2.75
0.00619878


kinesin family member C1
2.74
0.00821937


high-mobility group nucleosomal binding domain 2
2.73
0.00384071


ornithine decarboxylase 1
2.73
0.00144868


v-myb myeloblastosis viral oncogene homolog
2.71
0.00989416


(avian)-like 2


KIT ligand
2.70
0.00641955


dual-specificity tyrosine- (Y)-phosphorylation
2.70
0.0234606


regulated ki


intraflagellar transport 80 homolog (Chlamydomonas)
2.70
0.0247286


transmembrane protein 48
2.69
0.00458248


EBNA1 binding protein 2
2.69
0.00296292


ZW10 interactor
2.69
1.88E−05


exonuclease 1
2.68
0.00739393


transketolase (Wernicke-Korsakoff syndrome)
2.68
1.92E−05


somatostatin receptor 1
2.68
0.0144901


isocitrate dehydrogenase 3 (NAD+) alpha
2.67
0.00297129


cytoskeleton associated protein 2
2.67
0.0030499


minichromosome maintenance complex component 4
2.67
0.00342054


inhibitor of DNA binding 1, dominant negative helix-
2.66
0.036485


loop-hel


CDC28 protein kinase regulatory subunit 1B
2.66
0.0145263


keratin 18
2.66
8.40E−05


CD97 molecule
2.66
0.00994045


chromosome 6 open reading frame 173
2.64
0.00222408


BTB (POZ) domain containing 3
2.62
0.0166824


deafness, autosomal dominant 5
2.62
0.00235481


KIAA0286 protein
2.62
0.00130563


Fanconi anemia, complementation group D2
2.61
0.0281405


polo-like kinase 4 (Drosophila)
2.60
0.00209633


ribonucleotide reductase M1 polypeptide
2.60
0.000170076


malic enzyme 1, NADP (+)-dependent, cytosolic
2.59
0.0435444


non-SMC condensin I complex, subunit H
2.59
0.0216752


S100 calcium binding protein A3
2.58
0.0324073


ubiquitin-conjugating enzyme E2L 3
2.57
0.00343347


BUB1 budding uninhibited by benzimidazoles 1
2.56
0.0166047


homolog beta


glycerol kinase
2.55
2.66E−05


TAF9B RNA polymerase II, TATA box binding
2.54
0.0170365


protein (TBP)-as


TAF9B RNA polymerase II, TATA box binding
2.54
0.0170365


protein (TBP)-as


histone cluster 1, H2bg
2.52
0.000180822


high-mobility group box 2
2.52
0.0196872


NIMA (never in mitosis gene a)-related kinase 2
2.50
0.00289469


proline rich 11
2.50
0.0357125


myopalladin
2.49
0.0255088


brix domain containing 1
2.49
0.00471977


cell division cycle associated 5
2.49
0.01021


fucosidase, alpha-L-2, plasma
2.49
0.00540929


cyclin-dependent kinase 2
2.49
0.00250724


lamin B receptor
2.49
0.000151784


hypoxanthine phosphoribosyltransferase 1 (Lesch-
2.49
0.000634057


Nyhan synd


tripartite motif-containing 25
2.47
0.0456344


proteasome (prosome, macropain) subunit, beta type, 9
2.46
0.0202595


(lar


proteasome (prosome, macropain) subunit, beta type, 9
2.46
0.0202595


(lar


proteasome (prosome, macropain) subunit, beta type, 9
2.46
0.0202595


(lar


sphingomyelin synthase 2
2.46
0.0020701


transmembrane protein 62
2.45
0.00761064


glucose-6-phosphate dehydrogenase
2.44
0.00278311


PHD finger protein 1
2.44
0.010191


retinoblastoma-like 1 (p107)
2.44
0.00319946


KIAA1524
2.43
0.0380688


ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-
2.43
0.00830766


1,


cofilin 2 (muscle)
2.43
0.0459235


hypothetical protein LOC201725
2.42
0.000313319


cell division cycle 25 homolog A (S. pombe)
2.42
0.000341692


breast cancer 1, early onset
2.41
0.0180553


transaldolase 1
2.41
0.00199537


mRNA turnover 4 homolog (S. cerevisiae)
2.41
0.00373104


glucosaminyl (N-acetyl) transferase 1, core 2 (beta-
2.41
0.0197148


1,6-N-


cysteine rich transmembrane BMP regulator 1
2.41
0.0267286


(chordin-like)


tissue factor pathway inhibitor (lipoprotein-associated
2.40
0.0356227


chromosome 16 open reading frame 59
2.40
0.00185191


glycogenin 1
2.39
0.0224317


transmembrane protein 154
2.39
0.0045589


tubulointerstitial nephritis antigen-like 1
2.39
0.00510812


CTP synthase
2.38
8.80E−05


phenylalanyl-tRNA synthetase, beta subunit
2.38
0.000245973


geminin, DNA replication inhibitor
2.38
0.00167629


lamin B1
2.37
0.0477748


SPC24, NDC80 kinetochore complex component,
2.36
0.00287227


homolog (S. ce


glutathione reductase
2.36
0.00353875


ribosomal protein L22-like 1
2.36
0.00335381


fumarylacetoacetate hydrolase (fumarylacetoacetase)
2.36
3.88E−05


small nucleolar RNA, C
2.35
0.0188991


family with sequence similarity 64, member A
2.35
0.0019785


epithelial cell transforming sequence 2 oncogene
2.35
0.000571152


polymerase (DNA directed), epsilon 2 (p59 subunit)
2.34
0.00479612


glycerol kinase
2.34
3.37E−06


glutathione S-transferase M2 (muscle)
2.33
0.0402076


elongation factor, RNA polymerase II, 2
2.33
0.0130017


thioredoxin
2.33
0.009636


polymerase (DNA directed), alpha 2 (70 kD subunit)
2.32
0.0033903


breast cancer 2, early onset
2.32
0.00586847


CDC45 cell division cycle 45-like (S. cerevisiae)
2.32
0.00735977


H2A histone family, member Z
2.32
0.0129697


transporter 1, ATP-binding cassette, sub-family B
2.31
0.0164234


(MDR


transporter 1, ATP-binding cassette, sub-family B
2.31
0.0164234


(MDR


transporter 1, ATP-binding cassette, sub-family B
2.31
0.0164234


(MDR


nucleolar complex associated 3 homolog (S. cerevisiae)
2.30
0.000373346


ATPase, Ca++ transporting, plasma membrane 4
2.30
0.023011


minichromosome maintenance complex component 7
2.30
0.0457691


TIMELESS interacting protein
2.29
0.00771062


von Hippel-Lindau binding protein 1
2.28
0.00329061


ras-related C3 botulinum toxin substrate 2 (rho family,
2.28
0.0292466


sma


thymopoietin
2.28
0.0223176


peptidylprolyl isomerase F (cyclophilin F)
2.28
0.00093846


activated leukocyte cell adhesion molecule
2.27
0.00242163


polycomb group ring finger 5
2.27
0.000294142


Ran GTPase activating protein 1
2.27
9.68E−05


replication factor C (activator 1) 4, 37 kDa
2.26
0.00164152


tubulin, beta 2C
2.26
0.000346744


minichromosome maintenance complex component 10
2.26
0.0037925


H2B histone family, member S
2.25
0.000885505


gamma-glutamyl hydrolase (conjugase,
2.25
0.0195219


folylpolygammaglutamyl


transcription termination factor, RNA polymerase II
2.25
0.000393489


polymerase (DNA directed), delta 2, regulatory
2.25
0.0123823


subunit 50k


transporter 1, ATP-binding cassette, sub-family B
2.25
0.00859077


(MDR


transporter 1, ATP-binding cassette, sub-family B
2.25
0.00859077


(MDR


transporter 1, ATP-binding cassette, sub-family B
2.25
0.00859077


(MDR


histone cluster 1, H2bf
2.25
0.0124279


eukaryotic translation initiation factor 1A, X-linked
2.24
0.00330183


phosphoglucomutase 2
2.24
0.00818204


peroxisomal D3,D2-enoyl-CoA isomerase
2.24
0.00148722


interferon-induced protein with tetratricopeptide
2.24
0.0177928


repeats


G-2 and S-phase expressed 1
2.23
0.0241887


minichromosome maintenance complex component 2
2.23
0.0021347


family with sequence similarity 72, member A
2.23
0.00143248


RMI1, RecQ mediated genome instability 1, homolog
2.23
0.00294705


(S. cerev


FLJ20105 protein
2.23
0.0127979


multiple coagulation factor deficiency 2
2.22
0.0116892


phytoceramidase, alkaline
2.22
0.0157729


coiled-coil domain containing 68
2.22
0.00227586


dedicator of cytokinesis 11
2.21
0.00697577


platelet-derived growth factor alpha polypeptide
2.21
0.00176418


N-acylsphingosine amidohydrolase (non-lysosomal
2.20
0.00728536


cerami


S-phase kinase-associated protein 2 (p45)
2.20
0.00230153


polymerase (RNA) III (DNA directed) polypeptide G
2.20
0.0298794


(32 kD)


ADP-ribosylation factor-like 6 interacting protein 1
2.20
0.00139745


histone cluster 1, H2bh
2.19
0.0377748


origin recognition complex, subunit 5-like (yeast)
2.19
0.049697


CDC28 protein kinase regulatory subunit 2
2.19
0.0128024


histone cluster 1, H4c
2.19
0.0112695


hypothetical protein LOC729012
2.19
0.000446087


DEAD (Asp-Glu-Ala-Asp) box polypeptide 39
2.19
0.000340561


chromatin assembly factor 1, subunit B (p60)
2.18
0.0119687


MLF1 interacting protein
2.18
0.0177203


microtubule associated serine
2.18
0.00536974


MHC class I polypeptide-related sequence B
2.18
0.0165406


shugoshin-like 2 (S. pombe)
2.18
0.000852557


COP9 constitutive photomorphogenic homolog
2.18
0.000793512


subunit 6 (Arab


methylenetetrahydrofolate dehydrogenase (NADP+
2.18
0.00119726


dependent)


chromosome 6 open reading frame 167
2.18
0.0011095


pituitary tumor-transforming 1
2.17
0.0485166


ribonuclease H2, subunit A
2.17
0.00669936


X-ray repair complementing defective repair in
2.16
0.0369865


Chinese ham


membrane protein, palmitoylated 5 (MAGUK p55
2.16
0.00211873


subfamily memb


karyopherin alpha 2 (RAG cohort 1, importin alpha 1)
2.16
0.000650645


pleckstrin homology domain containing, family A
2.15
0.0256434


(phosphoi


ribosomal protein L39-like
2.15
0.00429384


karyopherin alpha 2 (RAG cohort 1, importin alpha 1)
2.15
0.000700649


amyloid beta (A4) precursor protein-binding, family
2.15
0.00201004


B, m


minichromosome maintenance complex component 3
2.14
0.0018389


histone cluster 1, H2ai
2.14
0.0129155


chromosome 13 open reading frame 34
2.14
0.000702936


RAD18 homolog (S. cerevisiae)
2.14
0.0016685


WD repeat and HMG-box DNA binding protein 1
2.13
0.0034833


sulfide quinone reductase-like (yeast)
2.13
0.0473641


chromosome 16 open reading frame 63
2.12
0.000804179


M-phase phosphoprotein 1
2.12
0.0271814


minichromosome maintenance complex component 6
2.12
0.0161279


homeobox A9
2.11
0.00520942


fibroblast growth factor 9 (glia-activating factor)
2.10
0.0475844


cell division cycle 25 homolog C (S. pombe)
2.10
0.0169914


chromosome 9 open reading frame 64
2.10
0.0265979


U2AF homology motif (UHM) kinase 1
2.09
0.0255167


replication factor C (activator 1) 2, 40 kDa
2.09
0.00768959


hypothetical protein LOC440894
2.09
0.0103358


small nuclear ribonucleoprotein D1 polypeptide
2.09
0.0334665


16 kDa


CSE1 chromosome segregation 1-like (yeast)
2.09
0.0013662


phosphatidylinositol glycan anchor biosynthesis, class W
2.09
0.0151967


centromere protein O
2.09
0.00397056


family with sequence similarity 20, member B
2.09
0.00460031


hypothetical protein FLJ40869
2.09
0.00444509


guanine nucleotide binding protein (G protein),
2.08
0.00140559


gamma 11


calcyclin binding protein
2.08
0.00524566


ATP-binding cassette, sub-family E (OABP), member 1
2.08
0.00454751


CD44 molecule (Indian blood group)
2.08
0.000651436


exosome component 8
2.08
0.00132017


family with sequence similarity 102, member B
2.08
0.025743


histone cluster 2, H3d
2.07
0.0102932


family with sequence similarity 33, member A
2.07
0.000318673


Fanconi anemia, complementation group B
2.07
0.000255109


kinesin family member 22
2.07
0.0192406


histone cluster 1, H2ai
2.07
0.0161621


vaccinia related kinase 1
2.06
0.0233182


integrator complex subunit 7
2.06
0.000841371


flap structure-specific endonuclease 1
2.06
0.006882


hypothetical protein FLJ25416
2.06
0.000177531


ecotropic viral integration site 2B
2.06
0.0171408


retinitis pigmentosa 2 (X-linked recessive)
2.05
0.0264185


centromere protein L
2.05
0.000880856


cofactor required for Sp1 transcriptional activation,
2.04
0.00141809


subu


chromosome 20 open reading frame 121
2.04
0.0146323


family with sequence similarity 72, member A
2.04
0.00162905


family with sequence similarity 72, member A
2.04
0.00165234


eukaryotic translation initiation factor 1A, X-linked
2.04
0.00520549


elongation factor, RNA polymerase II, 2
2.03
0.0458007


ATPase, Na+
2.03
0.0189108


histone cluster 1, H3a
2.03
0.0244273


brix domain containing 1
2.03
0.00981178


sushi domain containing 1
2.03
0.0258164


ectonucleoside triphosphate diphosphohydrolase 6
2.03
0.00423628


(putativ


fructosamine 3 kinase
2.03
0.00470972


Bloom syndrome
2.02
0.0209259


tubulin, alpha 1c
2.01
0.00862586


E2F transcription factor 2
2.01
0.0496479


exosome component 2
2.01
0.00649147


kinesin family member 22
2.01
0.0242075


LTV1 homolog (S. cerevisiae)
2.01
0.00812652


dihydrolipoamide S-acetyltransferase (E2 component
2.01
0.00179011


of pyruv


v-ral simian leukemia viral oncogene homolog B (ras
2.01
0.012225


related


ring finger and WD repeat domain 3
2.01
0.0013797


annexin A1
2.01
0.0173578


elaC homolog 2 (E. coli)
2.00
0.00266504


aldehyde dehydrogenase 9 family, member A1
2.00
0.00911609


tubulin, alpha 4a
2.00
0.0435427


nuclear pore complex interacting protein
−2.00
0.00111223


oculomedin
−2.01
0.00778869


similar to PI-3-kinase-related kinase SMG-1
−2.01
0.0356628


golgi autoantigen, golgin subfamily a-like pseudogene
−2.01
0.00770626


spectrin repeat containing, nuclear envelope 1
−2.01
0.00438469


nuclear pore complex interacting protein
−2.01
0.00117582


sushi, nidogen and EGF-like domains 1
−2.01
0.00161129


integrin, alpha V (vitronectin receptor, alpha
−2.02
0.00252702


polypeptide


cyclin-dependent kinase inhibitor 2B (p15, inhibits
−2.04
0.0150268


CDK4)


lysyl oxidase-like 4
−2.04
0.0120148


nuclear pore complex interacting protein
−2.04
0.000213956


calcium
−2.04
0.00657494


calsyntenin 3
−2.04
0.00300887


cell adhesion molecule 1
−2.05
0.0261129


solute carrier family 22 (organic cation transporter),
−2.05
0.0137275


RUN and FYVE domain containing 3
−2.05
0.00387265


glucosidase, alpha; acid (Pompe disease, glycogen
−2.05
0.000418401


storage di


nuclear pore complex interacting protein
−2.05
0.00988632


proline-rich nuclear receptor coactivator 1
−2.06
0.0039587


membrane metallo-endopeptidase
−2.06
0.0152684


PHD finger protein 21A
−2.06
0.00980401


Rho GTPase-activating protein
−2.06
0.00705186


homeobox B6
−2.06
0.00301714


nuclear pore complex interacting protein
−2.07
0.00032839


phospholipase A2 receptor 1, 180 kDa
−2.07
0.00069343


nuclear pore complex interacting protein
−2.08
0.000352007


slit homolog 3 (Drosophila)
−2.08
0.02844


nuclear pore complex interacting protein
−2.09
0.000414309


cyclin-dependent kinase 6
−2.09
0.0456892


dynamin 1
−2.09
0.00139674


jumonji, AT rich interactive domain 1B
−2.09
0.00861002


calcium binding and coiled-coil domain 1
−2.09
0.00370041


insulin-like growth factor 1 receptor
−2.09
0.00114467


nuclear pore complex interacting protein
−2.10
0.000377834


CD82 molecule
−2.10
0.0175517


bromodomain adjacent to zinc finger domain, 2B
−2.10
9.88E−05


—
−2.10
0.00666187


synaptotagmin XI
−2.11
0.0129428


KIAA1546
−2.11
0.000255634


jun B proto-oncogene
−2.12
0.0120169


CXXC finger 6
−2.12
0.0277527


nuclear pore complex interacting protein
−2.14
0.00282604


Cdon homolog (mouse)
−2.15
0.0350357


B-cell CLL
−2.15
0.00343507


nuclear pore complex interacting protein
−2.15
0.00263888


v-abl Abelson murine leukemia viral oncogene
−2.16
0.0136688


homolog 1


nuclear pore complex interacting protein
−2.16
0.00583397


FAT tumor suppressor homolog 1 (Drosophila)
−2.18
0.0158766


transformer-2 alpha
−2.18
0.012256


chimerin (chimaerin) 1
−2.18
0.0287031


milk fat globule-EGF factor 8 protein
−2.18
0.000987073


vitamin D (1,25-dihydroxyvitamin D3) receptor
−2.19
0.000192208


neuroblastoma, suppression of tumorigenicity 1
−2.20
0.00090639


jumonji domain containing 1A
−2.20
0.0188513


WNK lysine deficient protein kinase 1
−2.21
1.57E−05


protocadherin beta 14
−2.21
0.0103892


cortactin binding protein 2
−2.21
2.28E−05


WW domain containing transcription regulator 1
−2.22
0.0379899


cyclin L1
−2.22
0.00831474


nuclear factor of activated T-cells, cytoplasmic,
−2.22
0.00786451


calcine


pellino homolog 1 (Drosophila)
−2.23
0.00939357


golgi autoantigen, golgin subfamily a-like pseudogene
−2.24
0.00603583


chromosome 7 open reading frame 10
−2.26
0.00738442


golgi autoantigen, golgin subfamily a-like pseudogene
−2.27
0.00320764


small Cajal body-specific RNA 17
−2.27
0.0301336


latent transforming growth factor beta binding protein 2
−2.29
4.08E−05


golgi autoantigen, golgin subfamily a, 8A
−2.29
0.0111179


inhibin, beta A (activin A, activin AB alpha
−2.29
0.00877271


polypeptide)


solute carrier family 41, member 2
−2.30
0.00453672


forkhead box P1
−2.30
0.0463138


matrix metallopeptidase 14 (membrane-inserted)
−2.31
1.93E−05


transcription factor 4
−2.31
0.0367869


jun oncogene
−2.32
7.21E−05


neuroepithelial cell transforming gene 1
−2.33
0.0109689


asporin
−2.33
0.000659873


v-fos FBJ murine osteosarcoma viral oncogene
−2.35
0.0138624


homolog


ephrin-B2
−2.36
0.00611474


WD repeat and SOCS box-containing 1
−2.36
0.0387851


similar to dJ402H5.2 (novel protein similar to wo
−2.36
0.00621503


PX domain containing serine
−2.38
0.000927628


collagen, type VII, alpha 1 (epidermolysis bullosa,
−2.38
0.00109233


dystr


AE binding protein 1
−2.39
0.000105628


peroxidasin homolog (Drosophila)
−2.40
0.00219049


calcium channel, voltage-dependent, L type, alpha 1C
−2.41
0.0189661


sub


Prader-Willi syndrome chromosome region 1
−2.45
0.0415526


midline 1 (Opitz
−2.45
0.00130803


nuclear pore complex interacting protein
−2.45
0.00354416


chromosome 1 open reading frame 54
−2.47
0.0186089


transmembrane protein 16A
−2.48
0.0481085


basic helix-loop-helix domain containing, class B, 2
−2.49
0.00270257


nuclear pore complex interacting protein
−2.50
0.00316496


runt-related transcription factor 1 (acute myeloid
−2.50
0.000607387


leukemi


zinc finger protein 292
−2.50
0.029832


fibronectin leucine rich transmembrane protein 2
−2.51
0.0135122


nuclear pore complex interacting protein
−2.51
0.00283418


potassium voltage-gated channel, subfamily G,
−2.54
0.0244306


member 1


interleukin 19
−2.54
0.0310328


transforming growth factor, beta 3
−2.54
0.0287865


dihydropyrimidinase-like 3
−2.55
0.0165203


golgi autoantigen, golgin subfamily a, 8B
−2.56
0.0121417


hypothetical protein PRO2012
−2.57
0.00756704


SATB homeobox 2
−2.57
0.039781


t-complex 11 (mouse)-like 2
−2.57
0.0324227


ring finger protein 122
−2.57
0.0236621


chromosome 8 open reading frame 57
−2.59
0.00261522


ADAM metallopeptidase with thrombospondin type 1
−2.60
0.0113968


motif,


sushi, von Willebrand factor type A, EGF and
−2.63
2.23E−05


pentraxin dom


ST6 beta-galactosamide alpha-2,6-sialyltranferase 2
−2.64
0.0216987


sortilin-related VPS10 domain containing receptor 2
−2.65
0.00936311


protocadherin beta 9
−2.66
0.0285124


chromosome 5 open reading frame 13
−2.67
0.00410172


Enah
−2.68
0.0077547


pyridoxal-dependent decarboxylase domain containing 2
−2.69
0.00683647


similar to nuclear pore complex interacting protein
−2.70
0.0187322


nuclear pore complex interacting protein
−2.70
0.00368967


transmembrane protein 119
−2.70
0.00801387


chromosome 14 open reading frame 37
−2.70
0.0182453


sushi-repeat-containing protein, X-linked 2
−2.71
0.0253856


PDZ domain containing RING finger 3
−2.71
0.00931014


collagen, type XII; alpha 1
−2.72
0.000204664


matrix-remodelling associated 5
−2.72
0.000317637


collagen, type V, alpha 1
−2.72
0.0166427


dystrophin related protein 2
−2.72
0.0137557


ATP-binding cassette, sub-family A (ABC1), member 1
−2.73
0.00131361


trophinin
−2.77
0.00298044


cornichon homolog 3 (Drosophila)
−2.78
0.0261738


formin binding protein 1-like
−2.78
0.00290401


brain and acute leukemia, cytoplasmic
−2.78
0.0476919


protein tyrosine phosphatase, receptor type, U
−2.80
0.0270428


hypothetical protein MGC24103
−2.82
0.0346673


interferon induced with helicase C domain 1
−2.83
0.0024839


phospholipid transfer protein
−2.84
0.00999206


immediate early response 3
−2.87
0.0152127


immediate early response 3
−2.87
0.0152127


ADAM metallopeptidase domain 12 (meltrin alpha)
−2.87
0.000870288


synaptic vesicle glycoprotein 2A
−2.88
0.00704212


chromosome 9 open reading frame 3
−2.88
0.00410177


thioredoxin interacting protein
−2.90
0.0135494


early growth response 1
−2.93
0.000425035


small nucleolar RNA, C
−2.94
0.00666866


small nucleolar RNA, C
−2.95
0.00765575


immediate early response 3
−2.99
0.0167309


low density lipoprotein-related protein 1 (alpha-2-
−2.99
4.26E−05


macroglo


bicaudal C homolog 1 (Drosophila)
−2.99
0.0347162


homeobox B2
−3.03
0.00665994


small nucleolar RNA, C
−3.10
0.0274043


small nucleolar RNA, C
−3.10
0.0274043


matrix metallopeptidase 2 (gelatinase A, 72 kDa
−3.13
5.59E−05


gelatinase,


KIAA1641
−3.14
0.00659194


collagen, type VI, alpha 3
−3.14
2.09E−06


homeobox A2
−3.15
0.0435423


SH3 and PX domains 2B
−3.15
0.0244357


collagen, type VI, alpha 2
−3.16
0.0149554


chromosome 9 open reading frame 3
−3.21
0.0233723


small nucleolar RNA, C
−3.24
0.0104491


small nucleolar RNA, C
−3.24
0.0104491


—
−3.27
0.00488845


UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-
−3.35
0.00964109


acetylga


cholesterol 25-hydroxylase
−3.38
0.0445558


KIAA1641
−3.40
0.013175


ring finger protein 144
−3.40
0.0135334


versican
−3.41
0.023885


angiopoietin-like 2
−3.42
0.0245161


KIAA1641
−3.44
0.0170531


FBJ murine osteosarcoma viral oncogene homolog B
−3.54
0.00025573


similar to RIKEN cDNA 1110018M03
−3.59
0.00516476


early growth response 2 (Krox-20 homolog,
−3.62
0.00821813


Drosophila)


dachsous 1 (Drosophila)
−3.63
0.00697244


kinesin family member 26B
−3.64
0.00363199


distal-less homeobox 5
−3.66
0.000640157


similar to Protein KIAA0220
−3.69
0.0302619


insulin-like growth factor 1 receptor
−3.71
3.42E−05


protein tyrosine phosphatase, receptor type, N
−3.77
0.0294569


KIAA1641
−3.85
0.0191782


sushi-repeat-containing protein, X-linked
−3.85
0.00370941


microfibrillar-associated protein 2
−3.91
0.0152901


complement component 1, s subcomponent
−3.97
0.0395863


CD24 molecule
−3.99
0.0340122


homeobox B3
−4.02
0.0354368


trichorhinophalangeal syndrome I
−4.02
0.00557712


Kallmann syndrome 1 sequence
−4.04
0.000548703


leucine rich repeat containing 17
−4.09
0.0263961


plexin domain containing 2
−4.32
0.031799


PTK7 protein tyrosine kinase 7
−4.42
0.000116114


supervillin
−4.43
0.0412717


zinc finger protein 521
−4.58
0.00668815


calbindin 2, 29 kDa (calretinin)
−4.77
0.0290743


ras homolog gene family, member J
−4.79
0.00197982


integrin, alpha 11
−4.80
0.000390317


odz, odd Oz
−5.05
0.00172671


F-box protein 32
−5.52
0.0212957


raftlin family member 2
−5.72
0.0260454


clusterin
−5.74
0.0303973


neurotrimin
−5.79
3.78E−06


WNT1 inducible signaling pathway protein 1
−5.86
0.000672342


insulin-like growth factor binding protein 5
−6.34
0.011614


sulfatase 2
−6.34
5.88E−05


microfibrillar-associated protein 4
−6.93
0.00155578


junctional adhesion molecule 2
−7.07
0.0306758


fibronectin type III domain containing 1
−7.29
0.0334696


sarcoglycan, delta (35 kDa dystrophin-associated
−7.37
0.000881984


glycoprotei


hephaestin
−7.53
0.0123141


serpin peptidase inhibitor, clade F (alpha-2 antiplasmi
−7.66
0.00362941


cystatin SN
−7.96
0.0496433


hemicentin 1
−8.18
0.0461603


tenascin C (hexabrachion)
−8.32
8.26E−05


biglycan
−8.62
0.00161284


transmembrane, prostate androgen induced RNA
−11.20
0.000100935


carboxypeptidase E
−11.22
0.00738131









Expression of Cellular Markers on PLX-C Cells
The surface antigens expressed by PLX-C were examined using monoclonal antibodies. Results indicated that PLX-C cells were characterized by the positive markers: CD73, CD29 and CD105 and the negative markers: CD34, CD45, CD19, CD14 and HLA-DR (data not shown). The immune phenotype test specifications were set as: ≧90% for all positive markers and ≦3% for all negative markers.
Furthermore, as shown in FIGS. 7A-B, PLX-C cultures did not express endothelial markers as shown by negative staining for the two endothelial markers CD31 and KDR. However, PLX-C expression of a fibroblast-typical marker was evident (expression of D7-fib, FIG. 7C).
Immunogenecity and Immunomodulatory Properties of PLX-C Cells
As PLX-C is comprised of adherent cells derived from placenta, it is expected to express HLA type I, which is expressed by all cells of the body and is known to induce an alloreactive immune response. HLA type II and other co-stimulatory molecules are typically expressed only on the surface of Antigen Presenting Cells (APCs).
In order to examine the immunogenicity of the obtained PLX-C cells, the expression of co-stimulatory molecules on the surface of these cell membranes were performed. FACS analysis demonstrated the absence of CD80, CD86 and CD40 on the PLX-C cell membranes (FIGS. 8A-C). Moreover, PLX-C expressed low levels HLA class I as detected by staining for HLA A/B/C (FIG. 8D). The expression of stimulatory and co-stimulatory molecules was similar to bone marrow (BM) derived MSCs (as shown in FIGS. 8A-D).
To further investigate the immunogenecity as well as the immunomodulation properties of PLX-C cells, Mix Lymphocyte Reaction (MLR) tests were performed. As shown in FIGS. 9A-B, PLX-C cells both escape allorecognition and reduce T cell response, as measured by Thymidine incorporation. Furthermore, the reduction in lymphocytes proliferation (evaluated by CPM measurement) was higher as the number of PLX-C cells increased (in a dose dependent manner). PLX-C also reduced lymphocyte proliferation following mitogenic stimuli, such as Concavalin A (Con A, FIG. 9B) and Phytohemagglutinin (PHA), and non-specific stimulation by anti-CD3, anti-CD28 (data not shown).
In order to investigate the mechanism of action by which PLX-C immunomodulate lymphocyte proliferation, and to see if this action is mediated via cell to cell interaction or cytokines secretion, PB derived Mononuclear cells (MNCs) were stimulated by PHA using the transwell method (which prevents cell to cell contact but enables the diffusion of cytokines between the two compartments). Results showed that the inhibition of proliferation maintained even when cell to cell contact was inhibited (data not shown).
Cytokines Secretion
As depicted hereinabove, PLX-C reduce the proliferation rate of lymphocytes, probably through soluble factors. Further investigation of the cytokines secreted by lymphocytes in response to PLX-C was performed to elucidate the mechanism of action of PLX-C. As depicted in FIGS. 10A-B, culturing of mononuclear cells with PLX-C slightly reduced the secretion of the pro-inflammatory cytokine INFγ and dramatically reduced the secretion of TNFα (even in the presence of low amounts of PLX-C). In addition, following lipopolysaccharide (LPS) stimulation, PB derived MNCs secretion of IL-10 increased in the presence of PLX-C, while the secretion level of TNFα decreased, in a dose dependent manner (FIG. 10C).
It will be appreciated that the PLX-C cells of the present teachings were also capable of homing to ischemic tissues following intramuscular or intravenous injection into mice (data not shown).
Example 4
The Anti-Inflammatory Effect of PLX-C Cells in an in vivo Murine Model of Acute Colitis
Materials and Experimental Procedures
TNBS Model of Intestinal Inflammation
Colitis was induced in susceptible strains of rodents by intrarectal instillation of the haptenating substance TNBS (Trinitrobenzyl sulphonic acid) in ethanol. The use of TNBS in Ethanol was based on previous reports that ethanol is required to break the mucosal barrier whereas TNBS haptenizes colonic autologous or microbiota proteins rendering them immunogenic to the host immune system [Wirtz et al., Nature Protocols (2007) 2(3): 541-546].
Briefly, for colitis induction, mice were anesthetized for 90-120 minutes and received an intrarectal administration of TNBS (40 μl, 150 mg/kg) dissolved in a 1:1 mixture of 0.9% NaCl in 100% ethanol. Control mice received a 1:1 mixture of 0.9% NaCl in 100% ethanol or a saline solution using the same technique.
Mice were sacrificed 5 days post TNBS administration to assess the anti-inflammatory effect of the therapeutic cells (PLX-C cells) of the present invention. PLX-C administration was evaluated by intravenous (iv) administration or by intraperitoneal (ip) injection of the cells 1 day post colitis induction.
Animals
C57b16 mice were used in these experiments. A total of 90 mice were used which were divided into 9 groups as follows:

    1) 10 control mice (received no treatment)
    2) 10 control mice+PLX-C-I batch 1 by ip (2*106 cells)
    3) 10 control mice+PLX-C-I batch 1 by iv (1*106 cells)
    4) 10 TNBS mice (colitis model mice)
    5) 10 TNBS mice+5-aminosalicylic acid (5-ASA)
    6) 10 TNBS mice+PLX-C-I batch 1 by iv (1*106 cells)
    7) 10 TNBS mice+PLX-C-I batch 1 by ip (2*106 cells)
    8) 10 TNBS mice+PLX-C-I batch 2 by iv (1*106 cells)
    9) 10 TNBS mice+PLX-C-I batch 2 by ip (2*106 cells)


Production of the 2D Placenta Derived Adherent Cells
As depicted in detail in Example 2, hereinabove.
Production of the 3D Placenta Derived Adherent Cells (PLX-C Cells)
As depicted in detail in Example 3, hereinabove.
Vehicle
PlasmaLyte containing 5% Albumin was used as a vehicle control.
Tests and Evaluation
Macroscopic and histological assessments of colitis were performed on colon specimens collected from the different mice experimental groups 5 days post TNBS administration. Macroscopic and histological assessments were conducted blindly by two investigators.
Macroscopical Analysis
The colon of each mouse was examined under a dissecting microscope (magnification, ×5) to evaluate the macroscopic lesions according to the Wallace criteria. The Wallace score rated the macroscopic lesions on a scale from 0 to 10 based on features reflecting inflammation, such as hyperemia, thickening of the bowel, and extent of ulceration.







TABLE 12







Wallace score








Score
Criteria of macroscopic evaluation











0
No Inflammation


1
Hyperemia without ulcerations


2
Hyperemia with thickening of the mucosa without



ulcerations


3
1 ulceration without thickening of the colonic wall


4
2 or more of ulcerative or inflammatory sites


5
2 or more of ulcerative or inflammatory sites with an



extent of more than 1 cm


6
1 ulcerative or inflammatory site of more than 2 cm


7
1 ulcerative or inflammatory site of more than 3 cm


8
1 ulcerative or inflammatory site of more than 4 cm


9
1 ulcerative or inflammatory site of more than 5 cm


10
1 ulcerative or inflammatory site of more than 6 cm









Histological Analysis
A colon specimen located precisely 2 cm above the anal canal was used for the histological evaluation according to the Ameho criteria. This grading (on a scale from 0 to 6) took into account the degree of inflammation infiltrate, the presence of erosion, ulceration, or necrosis, and the depth and surface extension of lesions.







TABLE 13







Ameho criteria








Score
Criteria of histological evaluation





0
No alterations


1
Middle mucosal and/or sub-mucosal inflammatory



infiltrates with oedema. Few mucosal erosions.



Integrity of the muscularis mucosae.


2
Same criteria as score 1 but more than 50% of the



section


3
Large inflammatory infiltrate with ulceration area



trough all the colonic


4
Same criteria as score 3 but more than 50% of the



section


5
Wide ulcerations with cellular necrosis


6
Wide ulcerations with cellular necrosis but more than



50% of the section









Molecular Analysis of Colitis
Quantification of mRNA Expression of IL-1 Beta
Total RNA was isolated from whole mice colonic tissues using Rneasy kit (Macherey Nagel, Hoerdt, France) according to the manufacturer's instructions. RNA quantification was performed using spectrophotometry. After treatment at 37° C. for 30 minutes with 20-50 units of RNase-free DNase I (Roche Diagnostics Corporation, Indianapolis, Ind., USA), oligo-dT primers (Roche Diagnostics Corporation, Indianapolis, USA) were used to synthesize single-stranded cDNA. mRNAs were quantified using SYBR green Master Mix (Applera, Courtaboeuf, France) with mouse specific oligonucleotides for IL-1β: S: 5′-gATCCACACTCTCCAgCTgCA-3′ (SEQ ID NO: 1) and AS: 5′-CAACCAACAAgTgATATTCTCCATg-3′ (SEQ ID NO: 2) in a GeneAmp Abiprism 7000 (Applera, Courtaboeuf, France). Each assay was calibrated and no-template controls were included. Each sample was run in triplicate. SYBR green dye intensity was analyzed using the Abiprism 7000 SDS software (Applera, Courtaboeuf, France). All results were normalized to the unaffected housekeeping gene β-actin (oligonucleotides for β-actin: S: 5′-gggTCAgAAggATTCCTATg-3′ SEQ ID NO: 3; AS: 5′ ggTCTCAAACATgATCTggg-3′ SEQ ID NO: 4).
Results
As described in detail hereinabove, mice were sacrificed on day 5 following induction of colitis by intra-rectal injection of TNBS on day 0 and adherent cell administration on day 1. Mice were administered with either 2D adherent cells (hereinafter batch 1) or PLX-C adherent cells (hereinafter batch 2) obtained from placenta 1 or placenta 2, respectively. After the mice were sacrificed, macroscopic and microscopic evaluations of the colon were performed.
As demonstrated in FIG. 11, mice treated by an iv injection of 2D and PLX-C cells (batch 1 or 2, respectively) exhibited a major improvement in the inflammatory condition of the colon tissue as represented by the Wallace score. This anti-inflammatory effect was as efficient as the 5-ASA gold standard treatment). It will be appreciated that ip administration of 2D adherent cells (batch 1) also resulted in a satisfactory improvement in the Wallace score of the colitis model mice.
Microscopic evaluation of the colon revealed that administration of PLX-C cells (batch 2) by either ip or iv routes significantly reduced colonic inflammation at the histological level compared to TNBS mice (as represented by the Ameho score, FIG. 12). A considerable improvement was also observed for TNBS mice who received 2D cells (batch 1) by iv administration (FIG. 12). The improvement in these treatment groups was significantly better compared to the 5-ASA treated TNBS group.
Furthermore, total RNA was isolated from colonic tissues and IL-1β expression levels were evaluated by RT-PCR (as described in detail hereinabove). As evident from the results (FIG. 13), administration of PLX-C cells (batch 2) by iv significantly reduced the expression level of IL-1β in colonic tissues. It will be appreciated that although IL-1β RNA expression levels were not significantly reduced by ip administration (2D or 3D adherent cells, batches 1 and 2, respectively) and iv administration (2D adherent cells, batch 1), administration of both PLX-C and 2D cells still resulted in significant reduction in inflammation based on macroscopic and microscopic evaluations in colitic mice.
Taken together these results demonstrated that administration of the placental adherent cells of the present invention (2D and 3D PLX-C cells) lead to a significant improvement in colon inflammation in a mouse model of acute colitis.
Example 5
The Anti-Inflammatory Effect of PLX-C Cells in an in-vivo Rat Model of Acute Colitis
Materials and Experimental Procedures
TNBS Model of Intestinal Inflammation
Rats were rendered colitic by intracolonic administration of 22 mg of TNBS dissolved in 1:1 mixture of EtOH and water. 24 hours post colitis induction rats were administered according to the study treatment.
The rats used in this study were divided into three study groups (as depicted in detail below). Eleven days following colitis induction, all rats were sacrificed and colonic damage was evaluated both microscopically and macroscopically.
Administration of TNBS was designated as day 0, PLX-C cells were administrated on day 1 and rats were sacrificed on day 11.
Animals
12 Female Lewis rats (100-120 g) were used in these experiments. A total of 12 rats were used which were divided into 4 groups as follows:

    1) 4 rats were administered ip with 5×106 PLX-C-I cells
    2) 4 rats were administered iv with 5×106 PLX-C-I cells
    3) 2 rats received PlasmaLyte by ip (control group)
    4) 2 rats received PlasmaLyte by iv (control group)


Production of the Placenta Derived Adherent Cells (PLX-C Cells)
Cells were produced as depicted in detail in Example 3, hereinabove.
Macroscopic Assessment of Colonic Damage
Macroscopic assessment of colonic damage was evaluated according to the following criteria:
0—No damage
1—Hyperemia but no ulcers
2—Fibrosis but no ulcers
3—Ulceration/necrosis less than 1 cm
4—Ulceration/necrosis less than 2 cm
5—Ulceration/necrosis more than 2 cm
Microscopic (Histological) Assessment of Colonic Damage
Microscopic assessment of colonic damage was evaluated according to all of the following criteria (A+B+C+D):
A. Extent of ulceration:
0—No ulcer
1-2—Small ulcers (less than 3 mm)
3-5—Large ulcers (more than 3 mm)
B. Submucosal infiltration:
0—None
1—Mild
2-3—Moderate
4-5—Severe
C. Crypt abscesses:
0—None
1-2—Rare
3-5—Diffuse
D. Wall thickness (μm)
0—less than 470
1—less than 600
2—less than 700
3—less than 800
4—less than 900
5—more than 900
Results
As evident from FIG. 14, administration of the 3D adherent cells of the present invention (PLX-C cells) lead to a significant improvement in the microscopic scores (histological assessment) of acute colitis in rats.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

## Claims

1. A method of treating inflammation of intestinal tissue in conditions of ulcerative colitis or Crohn's disease in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of adherent, non-autologous cells derived from placenta, thereby treating the inflammation of intestinal tissue in conditions of ulcerative colitis or Crohn's disease, wherein said adherent, non-autologous cells do not exhibit osteogenic differentiation as assessed by Alizarin Red staining, when subjected to conditions that induce bone marrow cells to undergo osteogenic differentiation.

2. The method of claim 1, wherein said adherent cells express one or more of CD73, CD90, CD29, or CD105.

3. The method of claim 1, wherein said adherent cells do not express CD3, CD4, CD45, CD80, HLA-DR, CD11b, CD14, CD19, or CD79.

4. The method of claim 1, wherein said adherent cells suppress an immune reaction by suppressing T cell activity.

5. The method of claim 1, wherein said adherent cells are obtained from a three-dimensional (3D) culture.

6. The method wherein said three-dimensional (3D) culture comprises a 3D bioreactor.

7. The method wherein culturing of said adherent cells in said 3D culture comprises perfusion of the adherent cells with a continuous flow of a culture medium.

8. The method wherein culturing of said adherent cells occurs for at least 3 days.

9. The method of claim 1, wherein said adherent cells comprise cells cultured from the placenta under 2 dimensional (2D) culturing conditions.

10. The method wherein at least 12% of said adherent cells are at a S and/or G2/M proliferative phase.

11. The method of claim 1, wherein said adherent cells are less committed to an adipogenic lineage as compared to adherent cells from bone marrow grown and allowed to differentiate under the same conditions.

12. The method of claim 1, further comprising co-administering an immunosuppressant agent or an anti-inflammatory agent.

13. The method of claim 1, wherein the adherent, non-autologous cells are administered via one of an intravenous, intramuscular, or intraperitoneal injection.

14. The method of claim 1, wherein said adherent, non-autologous cells do not express CD34.