US20160271184A1

US20160271184A1 - Methods of treating inflammatory colon diseases

Info

Publication number: US20160271184A1
Application number: US15/082,917
Authority: US
Inventors: Moran Meiron
Original assignee: Pluristem Ltd
Current assignee: Pluri Biotech Ltd
Priority date: 2008-05-27
Filing date: 2016-03-28
Publication date: 2016-09-22
Also published as: US9393273B2; EP2641606A1; RU2010153362A; RU2515156C2; AU2009252722A1; US20110129486A1; BRPI0909541A2; AU2009252722A2; EP2303293A1; ZA201009030B; CN102123717A; WO2009144720A1; CA2725637A1

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.

Description

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 PDC (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-AB/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 2×10⁵peripheral 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±0.2×10³cells/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±30×10⁶cells are seeded, 3-5×10⁶cell/gr carrier are seeded, or 0.015-0.1×10⁶cell/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; Ilonidap; 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 cm²flasks 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.
PluriX™ Plug Flow Bioreactor
The PluriX™ 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/O₂at differing proportions, depending on cell density in the bioreactor. The O₂proportion was suited to the level of dissolved O₂at 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 (10³-10⁵cells/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 CO₂as 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 1×10⁶-1×10⁷cells/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 cm²flasks 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±0.2×10³cells/cm². The size of the tissue culture flasks raised as the passages proceed. The culturing process started in 80 cm²tissue 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 1.5-2.5×10⁹cells 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% CO₂at 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, MA, 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-	0.1	M	20	μl	0.2	mM
Phosphate solution
Glycerol-2-Phosphate	1	M	100	μL	10	Mm
Solution

L-glutamine	×100	100	μl	×1
Pen & Strep	×100	100	μl	×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	×100	100	μl	×1
Serum (heat inactivated)		1	ml	10%

Dexamethasone (Chemicon)	10	mM	10	μl	10	μM
Asorbic Acid-2-Phosphate solution	0.1	M	20	μl	0.2	mM

(Chemicon)

Glycerol-2-Phosphate Solution

1

M

100

μL

10

Mm

(Chemicon)

Vitamin D (Sigma)

10

μM

10

μL

10

nM

Gentamycin (Biological Industries,

×100

100

μl

×1

Bet 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, MA, 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)	0.5	M	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	×100	50	μl	×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	×100	50	μl	×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)	0.5	M	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 2 D adherent cells suitable for use according to the
present teachings to those produced for PLX in WO/2007/108003

		2 D adherent cells of the
Parameter	WO/2007/108003	present teachings

Tissue culture flask	80 cm²and 175 cm²	175 cm², triple flasks and
		Multi Tray
Medium	In all stages of the	Up to passage 2 (included)
supplemented with	process
antibiotics
Cryopreservation of	After 2-3 passages,	After 5-8 passages, then
2DCS	then cryopreserved,	cryopreserved and thawed
	thawed and seeded	prior to use
	for a 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 asses the molecular mechanism underlying phenotypic variation between cells obtained by these different growth methods (see Table 8, below).

TABLE 8

Gene expression in 2 D adherent cells suitable for use according to the
present teachings compared to those expressed by PLX of WO/2007/108003

	2 D vs.
	Plurix
	(fold	p-value
Gene	change)	(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 55kDa	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, 38kDa	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 22kDa 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, 37kDa	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 170kDa	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 70kDa 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, Hib	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 70kDa protein 1A	2.42	0.00734287
heat shock 70kDa protein 1A	2.42	0.00674816
carnitine pahnitoyltransferase 1A (liver)	2.41	0.00170894
neurofilament, medium polypeptide 150kDa	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 16kDa	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 70kDa protein 1B	2.28	0.0048959
heat shock 70kDa protein 1B	2.28	0.0054404
heat shock 70kDa 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.	2.25	0.00578953
coli)
metallothionein 1 L (gene.	2.25	0.00709646
RRS1 ribosome biogenesis regulator homolog (S.	2.24	0.0120061
cerevisiae)
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, 40kDa	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
(32kD)
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, 55kDa	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 27kDa	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.	2.13	0.00264664
cerevisia
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
ecotropic 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.	2.09	0.00294705
cerev
overexpressed in colon carcinoma-1	2.09	0.015322
hypothetical protein FL120425	2.09	0.0174225
primase, polypeptide 1, 49kDa	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 155kDa	2.07	0.00218453
FLJ20105 protein	2.06	0.0127979
CD44 molecule (Indian blood group)	2.06	0.000651436
polymerase (DNA directed), alpha 2 (70kd) 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 60kDa 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 34kDa, 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.	2.02	0.0199824
cerevisia
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 37kDa	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, 58kDa	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, 180kDa	−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, 72kDa 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 90kDa 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, 29kDa (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 (35kDa 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-11-			Plc
BM109	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
Celligen™ Plug Flow Bioreactor
The production of 3D adherent cells for use in accordance with the present invention by Celligen™ (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 gentamycin 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 cm²flasks 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±0.2×10³cells/cm². The size of the tissue culture flasks raised as the passages proceed. The culturing process started in 80 cm²tissue 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 1.5-2.5×10⁹cells 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±30×10⁶cells 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, N₂and 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)
2×10⁵peripheral 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
Parameter	WO/2007/108003	present 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 × 10⁶cell/gr	5 × 10⁶cell/gr	Better cell to cell
concentration	carrier	carrier	interaction in the
at seeding			present teachings
(cell/gr
carrier)
Cell	0.015 × 10⁶cell/ml	0.1 × 10⁶cell/ml	Better cell to cell
concentration			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 ± 50 mg/L)	removal of toxic
			agents produced
			by the cells.
			In batch mode -
			lower
			concentration of
			essential
			nutrients
			(limiting factors)
			Less cell debris
Harvest	Harvesting in 50 ml	Harvesting	Present teachings -
procedure	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, O₂and	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 asses 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*10⁶cells)
3) 10 control mice+PLX-C-I batch1 by iv (1*10⁶cells)
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*10⁶cells)
7) 10 TNBS mice+PLX-C-I batch 1 by ip (2*10⁶cells)
8) 10 TNBS mice+PLX-C-I batch 2 by iv (1*10⁶cells)
9) 10 TNBS mice+PLX-C-I batch 2 by ip (2*10⁶cells)
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 5×10⁶PLX-C-I cells
2) 4 rats were administered iv with 5×10⁶PLX-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 (m)
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

1-21. (canceled)

22. A dispenser device comprising:

a dispenser for intramuscular or intravascular administration of cells, wherein the dispenser contains a composition comprising a population of adherent cells derived from placenta, wherein said adherent 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.

23. The dispenser device of claim 22, wherein said adherent cells express one or more of CD73, CD90, CD29, or CD105.

24. The dispenser device of claim 22, wherein said adherent cells do not express CD3, CD4, CD45, CD80, HLA-DR, CD11b, CD14, CD19, or CD79.

25. The dispenser device of claim 22, wherein said adherent cells suppress an immune reaction by suppressing T cell activity.

26. The dispenser device of claim 22, wherein said adherent cells are obtained from a three-dimensional (3D) culture.

27. The dispenser device of claim 26, wherein said three-dimensional (3D) culture comprises a 3D bioreactor.

28. The dispenser device of claim 26, wherein culturing of said adherent cells in said 3D culture comprises perfusion of the adherent cells with a continuous flow of a culture medium.

29. The dispenser device of claim 26, wherein culturing of said adherent cells in said 3D culture occurs for at least 3 days.

30. The dispenser device of claim 26, wherein culturing of said adherent cells in said 3D culture is performed until at least 10% of said adherent cells are proliferating.

31. The dispenser device of claim 26, wherein said adherent cells are cultured from the placenta under 2 dimensional (2D) culturing conditions, prior to culturing of said adherent cells in said 3D culture.

32. The dispenser device of claim 22, 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.

33. The dispenser device of claim 22, wherein said adherent cells do not express CD34.

34. The dispenser device of claim 22, wherein the composition further comprises albumin.

35. The dispenser device of claim 22, wherein the composition further comprises a cryopreservent.