WO2011106676A2 - Methods of human embryonic stem cell growth - Google Patents

Abstract

The present invention is partly based on the seminal discovery that human embryonic stem cell (hESC) growth can be supported using a conditioned medium (CM) from SDEC cells, where the CM has reduced level of sFRP-1. In addition, such hESC growth can be supported using a Type I collagen (COL I) biomatrix. Provided are compositions for hESC growth including condition medium with reduced level of secreted frizzled-related protein 1 (sFRP-1). Also provided are methods to enhance hESC growth and methods to reduce sFRP-1 protein levels. Methods of screening cell lines for reduced sFRP-1 levels and compounds that can specifically overcome sFRP-1 antagonism or promote wnt/ß-catenin agonism are also provided.

Description

METHODS OF HUMAN EMBRYONIC STEM CELL GROWTH BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0001] The present invention relates generally to the field of human embryonic stem cells (hESCs) and more specifically to proliferation and pluripotency of human embryonic stem cells maintained on type I collagen.

BACKGROUND INFORMATION

[0002] Therapies developed using human embryonic stem cells (hESCs) have the potential to transform the treatment for a wide variety of diseases, including Parkinson's, diabetes, and heart disease. These cells can also provide a source of cellular therapies for the replacement of damaged or destroyed tissues, and tissues derived from hESCs can be used in drug discovery and toxicology studies. The success of these applications, however, depends on our capacity to provide an adequate supply of hESCs for research and development purposes. Critical to this goal is the specification of well-defined and reproducible culture conditions that will allow for large-scale expansion of these cells while providing efficient self-renewal, maintenance of pluripotency, and chromosomal stability.

[0003] Currently, hESCs are typically co-cultured with feeder layers of mouse embryonic fibroblasts (MEF). Cells grown in this manner are not ideal for transplantation into humans due to the risk of xenogenic-based rejection by the immune system as well as the potential for cross-species transfer of viruses or pathogens. MEF feeder layers can be replaced with human-derived cell lines to avoid hESC contact with murine cells, but the presence of additional cell lines in hESC culture inevitably contributes to variability and increased cost during the expansion and scale-up of stem cells. As a result, direct co-culture has been replaced by media conditioning, where hESC cells are grown on an acellular support matrix in medium that has been conditioned by a separate, supporting, cell line. [0004] hESCs grown in feeder-free culture require a solid growth matrix on which to proliferate, the most common being Matrigel™ (MAT), a complex basement membrane extract derived from Engelbreth-Holm-Swarm mouse sarcoma whose composition is not well defined. MAT is widely used because most hESCs do not grow well on more defined matrices like fibronectin, laminin, or collagen. A simple, completely defined matrix that could support hESC growth and maintenance of pluripotency would provide significant advantages over the use of MAT or feeder layers in terms of reproducibility, scale-up, cost, and applicability to human therapies. Type I collagen (COL I) offers significant potential as a hESC growth biomatrix because it is well defined, widely available, and FDA approved for several applications.

SUMMARY OF THE INVENTION

[0005] The present invention is partly based on the seminal discovery that human embryonic stem cell (hESC) growth can be supported using a conditioned medium (CM) from SDEC cells, where the CM has reduced level of sFRP-1. In addition, such hESC growth can be supported using a Type I collagen (COL I) biomatrix. Provided are compositions for hESC growth including condition medium with reduced level of secreted frizzled-related protein 1 (sFRP-1). Also provided are methods to enhance hESC growth and methods to reduce sFRP-1 protein levels. Methods of screening cell lines for reduced sFRP-1 levels and compounds that can specifically overcome sFRP-1 antagonism or promote wnt/ -catenin agonism are also provided.

[0006] In one embodiment, the present invention provides a composition for supporting growth of human embryonic stem cells (hESCs). The composition includes a first conditioned medium (CM) with a reduced level of secreted frizzled-related protein (sFRP-1) as compared to a second conditioned medium from fetal foreskin fibroblast cells and/or fetal lung fibroblast cells.

[0007] In one aspect, the first CM is from cells of human origin. In various aspects, the fetal foreskin fibroblast cells include human fetal foreskin fibroblast line Detroit 551. In various aspects, the fetal lung fibroblast cells include human fetal lung fibroblast line WI38. [0008] In various aspects, the sFRP-1 level of the second CM is at least 4 times greater than the sFRP-1 level of the first CM. In various aspects, the sFRP-1 level of the second CM is at least 8 times greater than the sFRP-1 level of the first CM. In various aspects, the sFRP- 1 level of the second CM is at least 20 times greater than the sFRP-1 level of the first CM. In various aspect, the the sFRP-1 level of the first CM is less than 17 ng/L. In various aspect, the the sFRP-1 level of the first CM is less than 300 ng/L. In various aspect, the the sFRP-1 level of the first CM is less than 3 ng/mL.

[0009] In one aspect, the first conditioned medium (CM) has an elevated level of

prostaglandin E2 (PGE2) and/or 6-keto-prostagIandin F l a (6-k-PGF l ) as compared to a second conditioned medium from fetal foreskin fibroblast cells and/or fetal lung fibroblast cells, or a based medium. In another aspect, the first conditioned medium (CM) is derived from human SDEC cells. In another aspect, the growth of hESCs is on Type I collagen (COL I). In various aspects, the PGE2 level of the first CM is at least 7 times greater than the PGE2 level of the second CM. In various aspects, the PGE2 level of the first CM is at least 10 times greater than the PGE2 level of the second CM. In various aspects, the PGE2 level of the first CM is about 7-40 times greater than the PGE2 level of the second CM. In various aspect, the PGE2 level of the first CM is more than 20 ng/L.

[0010] In an additional aspect, the 6-k-PGFla level of the first CM is at least 30 times greater than the 6-k-PGFla level of the second CM. In another aspect, the 6-k-PGFl a level of the first CM is at least 50 times greater than the 6-k-PGFla level of the second CM. In another aspect, the 6-k-PGFla level of the first CM is about 30-60 times greater than the 6-k-PGFla level of the second CM. In various aspect, the 6-k-PGFl a level of the first CM is more than 40 ng/L.

[0011] In various aspects, the first conditioned medium (CM) has an elevated level of at least one cytokine as listed in Table 3, 4, or 5 as as compared to a second conditioned medium from fetal foreskin fibroblast cells and/or fetal lung fibroblast cells, or a based medium. In various aspects, the elevated level of the at least one cytokine is at least 1.5 fold, 3 fold, 6 fold, 20 fold, 100 fold, 500 fold, or 2000 fold. [0012] In another embodiment, the present invention provides a method to enhance human embryonic stem cells (hESCs) growth on Type I collagen (COL I). The method includes contacting the hESCs with a first conditioned medium (CM) with a reduced level of secreted frizzled-related protein (sFRP- 1 ) as compared to a second conditioned medium from fetal foreskin fibroblast cells and/or fetal lung fibroblast cells.

[0013] In one aspect, the first conditioned medium (CM) has an elevated level of

prostaglandin E2 (PGE2) and/or 6-keto-prostaglandin Fl (6-k-PGFla) as compared to a second conditioned medium from fetal foreskin fibroblast cells and/or fetal lung fibroblast cells. In another aspect, the first conditioned medium (CM) is derived from human SDEC cells.

[0014] In one aspect, the first CM is from cells of human origin. In various aspects, the fetal foreskin fibroblast cells include human fetal foreskin fibroblast line Detroit 551. In various aspects, the fetal lung fibroblast cells include human fetal lung fibroblast line WI38.

[0015] In various aspects, the sFRP-1 level of the second CM is at least 4 times greater than the sFRP-1 level of the first CM. In various aspect, the the sFRP- 1 level of the first CM is less than 17 ng/L. In various aspect, the the sFRP-1 level of the first CM is less than 300 ng/L. In various aspect, the the sFRP-1 level of the first CM is less than 3 ng/mL. In various aspects, the PGE2 level of the first CM is at least 7 times greater than the PGE2 level of the second CM. In various aspect, the PGE2 level of the first CM is more than 20 ng/L. In an additional aspect, the 6-k-PGFl a level of the first CM is at least 30 times greater than the 6-k-PGF l level of the second CM. In various aspect, the 6-k-PGFla level of the first CM is more than 40 ng/L.

[0016] In another embodiment, the present invention provides a method to reduce secreted frizzled-related protein (sFRP-1) in a conditioned medium for supporting hESC growth. The method includes silencing gene expression of sFRP-1 using an agent comprising a siRNA, a miRNA, or an antisense nucleic acid. [0017] In another embodiment, the present invention provides a method to reduce secreted frizzled-related protein (sFRP-1) in a conditioned medium for supporting hESC growth. The method includes capturing sFRP-1 protein in the condition medium using an agent specifically binds to the sFRP-1 protein. In one aspect, the agent comprising an antibody specifically binds to the sFRP-1 protein. In another aspect, the agent comprising an affinity

chromatography comprising an antibody specifically binds to the sFRP-1 protein.

[0018] In another embodiment, the present invention provides a method for screening cell lines for reduced level of secreted frizzled-related protein (sFRP-1 ) in conditioned medium. The method includes (a) measuring sFRP-1 protein level in a first conditioned medium from at least one cell line of human origin; and (b) comparing the sFRP-1 protein level measured in step (a) to a second conditioned medium of cells selected from the group consisting of human SDEC cells, fetal foreskin fibroblast cells, and fetal lung fibroblast cells, thereby identifying a cell line with reduced level of sFRP-1.

[0019] In another embodiment, the present invention provides a method for screening cell lines for reduced level of secreted frizzled-related protein (sFRP- 1 ) in conditioned medium. The method includes (a) measuring sFRP-1 mRNA level in at least one cell line of human origin; and (b) comparing the sFRP- 1 mRNA level measured in step (a) to sFRP-1 mRNA levels selected from the group consisting of human SDEC cells, fetal foreskin fibroblast cells, and fetal lung fibroblast cells, thereby identifying a cell line with reduced level of sFRP-1. In various aspects, the sFRP-1 mRNA level is measured using quantitative reverse transcription polymerase chain reaction (RT-QPCR or QRT-PCR) or microarray.

[0020] In another embodiment, the present invention provides a method for screening test agents or compounds for reducing level of secreted frizzled-related protein (sFRP-1) in conditioned medium. The method includes (a) contacting at least one cell line of human origin with at least one test agent or compound; and (b) measuring sFRP-1 protein or mRNA levels in the at least one cell line of human origin before and after step (a), thereby identifying the test agent or compound for reducing level of sFRP-1. [0021] In another embodiment, the present invention provides a method for screening cell lines for elevated expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and/or prostaglandin 12 synthase (PGIS). The method includes (a) measuring protein or mRNA levels of PTGS2 and/or PGIS in at least one cell line of human origin; and (b) comparing the protein or mRNA levels of PTGS2 and/or PGIS measured in step (a) to protein or mRNA levels of PTGS2 and/or PGIS of human SDEC cells, fetal foreskin fibroblast cells, and/or fetal lung fibroblast cells, thereby identifying a cell line with elevated expression of PTGS2 and/or PGIS.

[0022] In another embodiment, the present invention provides a method for screening test agents or compounds for elevated level of PGE2 and/or 6-k-PGF l a in conditioned medium. The method includes (a) contacting at least one cell line of human origin with at least one test agent or compound; and (b) measuring protein or mRNA levels of PTGS2 and/or PGIS in the at least one cell line of human origin before and after step (a), thereby identifying the test agent or compound for elevated level of PGE2 and/or 6-k-PGFl .

[0023] In various aspects, the mRNA level is measured using quantitative reverse

transcription polymerase chain reaction (RT-QPCR or QRT-PCR) or microarray. In various aspects, the protein level is measured using Western blotting or ELISA. In various aspects, the fetal foreskin fibroblast cells include human fetal foreskin fibroblast line Detroit 551. In various aspects, the fetal lung fibroblast cells include human fetal lung fibroblast line WI38.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 shows hESC proliferation and OCT4 expression for cells grown in SDEC, MEF, WI38, and DET CM on Matrigel. Figure 1 A shows population doubling (mean ± SE, n = 3) of hESCs over 3 weeks. Figure I B shows percentage of OCT4 positive cells (mean ± SE, n = 3) after 3 weeks. Immunofluorescent staining of hESCs can be observed with OCT4 after 3 weeks, where nuclei can be stained with DAPI. DAPI = 4',6-diamidino-2-phenylindole; DET = Detroit 551 ; hESCs = human embryonic stem cells; MEF = mouse embryonic fibroblast; SE = standard error. Significance:*, PO.05; ***, P<0.001. [0025] Figure 2 shows hESC proliferation and expression of pluripotency markers for cells grown in SDEC or MEF CM on MAT or COL. Figure 2A shows percentage of OCT4 positive cells (mean ± SE, n = 4) after one month. Figure 2B shows population doubling (mean ± SE, n = 3) of hESC at the end of one month. Immunofluorescent staining of hESC s grown in SDEC CM on COL after 1 month can be observed where nuclei, OCT4, NANOG, SSEA-4, AP, and/or TRA-1 -60 can be stained. Phase microscopy of hESC colonies growing on COL and MAT can be observed.

[0026] Figure 3 shows expression of secreted frizzled-related protein (sFRP-1) and prostaglandin-endoperoxide synthase 2 (PTGS2) and prostaglandins by DET, WI38, and SDEC cell lines. Quantitative RT-PCR is done to determine the fold-difference in mRNA expression level (mean ± SE, n = 3) for s-FRPl (Figure 3 A) and PTGS2 (Figure 3B). mRNA levels are normalized using PPIA mRNA (a housekeeping gene). Semi-quantitative Western blots are performed on intercellular PTGS2 (Figure 3C; using β-actin to control for protein loading) and sFRP- 1 recovered from CM (Figure 3D; 180 ml from SDEC and DET, 150 ml from WI38). sFRP-1 recovered from MEF CM (180 ml) was also included in this western blot. The indicated amounts of recombinant human sFRP-1 are included for comparison. Figure 3E shows levels (mean ± SE, n = 3, ng/ml) of 6-keto-prostaglandin F l a (6-k-PGF la; black) and prostaglandin E2 (PGE2; white) in CM. Significance:*, P<0.05; **, P<0.01 ; ***, P<0.001.

[0027] Figure 4 shows cell proliferation assay of H9 hESC grown on COL or MAT matrices in SDEC CM supplemented with various concentrations of sFRP-1. Proliferation of control (no added sFRP-1) is set to 100% and the percent of control proliferation (mean ± SE, n = 4) for each sFRP-1 concentration is reported. Significance:*, P<0.05; **, PO.01 ; ***, PO.001.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention is partly based on the seminal discovery that human embryonic stem cell (hESC) growth can be supported using a conditioned medium (CM) from SDEC cells, where the CM has reduced level of sFRP- 1. In addition, such hESC growth can be supported using a Type I collagen (COL I) biomatrix. Provided are compositions for hESC growth including condition medium with reduced level of secreted frizzled-related protein 1 (sFRP-1). Also provided are methods to enhance hESC growth and methods to reduce sFRP-1 protein levels. Methods of screening cell lines for reduced sFRP-1 levels and compounds that can specifically overcome sFRP-1 antagonism or promote wnt/p-catenin agonism are also provided.

[0029] Human embryonic stem cells (hESCs) require a delicate balance of growth factors and signaling molecules to proliferate and retain pluripotency. The present invention provides that conditioned medium (CM) from a human embryonic germ cell-derived cell culture, SDEC, can support the growth of hESCs on type I collagen (COL I) as well as on Matrigel (MAT). After one month, the population doubling of hESCs grown in SDEC CM on COL I is equivalent to that of hESCs grown in mouse embryonic fibroblast (MEF) CM on MAT.

hESCs grown in SDEC CM on COL I express OCT4, NANOG, SSEA-4, alkaline phosphatase and TRA-1 -60, retain a normal karyotype, and are capable of forming teratomas. DNA microarray analysis is used to compare the transcriptional profiles of SDEC and the less supportive, WI38 and Detroit 551 human cell lines. Secreted frizzled-related protein (sFRP- 1), a known antagonist of the WNT/p-catenin signaling pathway, is significantly under- expressed by SDEC as compared to the other two cell lines, while two genes in the prostaglandin synthesis pathway, prostaglandin-endoperoxide synthase 2 (PTGS2 or COX-2) and prostaglandin 12 synthase (PGIS), are significantly over-expressed by SDEC. The level of sFRP-1 is significantly reduced and levels of two prostaglandins that are downstream products of PTGS2 and PGIS, prostaglandin E2 and 6-keto-prostaglandin F l a, are significantly elevated in SDEC CM compared to the other lines. Furthermore, addition of purified sFRP-1 to SDEC CM reduces the proliferation of hESCs grown on COL I as well as MAT in a dose dependent manner.

[0030] Using comparative transcription analyses and quantitative reverse transcription polymerase chain reaction (RT-PCR) assays, the present invention provides the relative under expression of mRNA coding secreted frizzled-related protein 1 (sFRP-l) by SDEC as compared to two other human cell lines (Detroit-551 and WI-38) reported to be supportive of hESC. The present invention provides that relatively low levels of sFRP-1 protein in SDEC CM is observed as compared to Detroit-551 and WI-38 CM. sFRP-1 is a known antagonist of the WNT/p-catenin signaling pathway, which has been shown to play a role in maintaining hESC proliferation and pluripotency. Addition of exogenous sFRP-1 to SDEC CM

significantly inhibits hESC proliferation on both Matrigel™ or type I collagen matrices. [0031] Human embryonic stem cells (hESCs) are a powerful and commonly used tool in basic science research and for development of future cell therapies. Although many methods have been described for maintaining hESCs in chemically defined conditions, no methods have been able to combine a defined growth media with a defined and clinically relevant biomatrix. SDEC cells are of human origin, which avoids concerns associated with clinical utility of hESC-derived cells and allow hESC to be grown on a matrix of type 1 collagen. Matrices composed of type I collagen are advantageous due to their fully defined nature, low cost and human clinical utility.

[0032] The present provides that secreted frizzled-related protein 1 (sFRP-1) is associated with ability of cells used to support hESC growth, and removal of this compound alone can be beneficial to many different hESC growth methods, as would addition of compounds that can overcome sFRPl antagonism or promote wnt/p-catenin agonism.

[0033] This invention pertains to improvements on existing methods for culture of hESCs. Data presented does not distinguish between hESC lines. The present invention is the identification of a human cell type that can support the undifferentiated proliferation of hESC. Furthermore, this invention identifies that some hESC support cells (feeder layers) produce sFRP-1 and that this is undesirable, as sFRP- 1 is known to antagonize wnt/p-catenin signaling and presence of sFRP- 1 is demonstrated to inhibit hESC cell proliferation. One embodiment of this invention is SDEC cells, which are presently demonstrated to be able to support hESC. Additional embodiments are other related cell cultures derived from human embryonic germ (EG) cells.

[0034] In an additional embodiment, methods that reduce sFRP-1 protein levels, including affinity chromatography or methods to reduce gene expression levels including siRNA or other genetic methods to disrupt sFRP-1 gene translation in cell to be used for hESC support cells are provided by this invention.

[0035] In an additional embodiment, screening cell lines for the mRNA or protein expression level of sFRP-1 are provided by this invention as it is predictive of the ability of cells to support hESC. [0036] In an additional embodiment, addition of compounds that specifically overcome sFRP- 1 antagonism or promote wnt/ -catenin agonism are provided by this invention.

[0037] All references cited herein are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Throughout this description, the preferred embodiment and examples shown should be considered as exemplars, rather than as limitations on the present invention.

[0038] There have been several attempts to eliminate the undefined and animal-derived elements in both the hESC liquid medium and solid growth matrix. Replacing the undefined MAT matrix often requires a complex and expensive combinatorial matrix composed of multiple constituents, including collagen IV, fibronectin, laminin, and vitronectin. The inconvenience in using such a complex matrix is noted as a drawback to the system, as are its expense and its potential as a source of contamination. The inventors conclude that development of the support matrix is one area of stem cell culture that requires improvement. Even with the use of a complex matrix, the hESCs eventually become karyotypically abnormal, a fact that may be due, at least in part, to the absence of a proper balance of growth factors.

[0039] It has been found that MEF conditioned media (CM) can support undifferentiated hESC growth on MAT or laminin surfaces, but that hESCs undergo spontaneous

differentiation when added to a fibronectin or type IV collagen matrix. Laminin and fibronectin, along with defined supplements, can sometimes support stem cell growth but these matrices may not be as effective as growth on MAT or direct co-culture with MEF. It has also been found that fibroblast growth factor 2 (FGF2) and Noggin can help support hESC growth on MAT, but the use of a laminin matrix is subject to significant batch-to-batch variability and an inability to support clonal hESC growth. Similar growth conditions have been tried and it has been found that another hESC line remains undifferentiated on MAT for up to 15 passages but only for three confirmed passages on laminin. Although hESCs can be maintained on a fibronectin matrix using transforming growth factor β, FGF2, and leukemia inhibitory factor (LIF) along with the addition of 15% serum replacement, growth on fibronectin exhibits lower growth rates, reduced cloning efficiency, and a higher rate of spontaneous differentiation compared to growth on MEF. These more defined culture systems often require conditioned medium, serum replacement products and/or added growth factors. Due to the difficulties involved in using more defined matrices, the great majority of experiments, including the derivation of embryonic stem cell-like iPS cells from adult fibroblasts, utilize the undefined MAT matrix along with MEF CM to maintain the cells in an undifferentiated state.

[0040] The present invention provides the ability of CM from a human embryonic germ cell- derived cell culture, SDEC, to support the growth of hESC on both MAT and COL I. Fifteen different embryonic germ cell-derived cultures are screened and SDEC is found to be the most supportive of hESC growth. In order to begin to identify the factors that allow CM from SDEC to support hESC growth on COL I and to obtain insights into the roles that these factors play in maintaining robust hESC self-renewal, the inventors compare the transcriptional profiles of SDEC and two other non-supportive feeder cell lines - human fetal foreskin fibroblast line Detroit 551 and human fetal lung fibroblast line WI38. These two cell lines were previously reported to be supportive, and non-supportive, respectively, of hESC growth when used as direct feeder layers; however, the inventors confirm that CM from neither cell line can support long term hESC growth.

[0041] Microarray data indicate that secreted frizzled-related protein (sFRP-1) expression is significantly down-regulated in SDEC compared to the other two cell lines. sFRP-1 is a known antagonist of the WNT/p-catenin signaling pathway, which has been shown to play a role in maintaining hESC proliferation and pluripotency. Two genes in the prostaglandin synthesis pathway, prostaglandin-endoperoxide synthase 2 (PTGS2) and prostaglandin 12 synthase (PGIS), are significantly up-regulated in the SDEC culture, and the levels of prostaglandin E2 (PGE2) and 6-keto-prostaglandin Fla (6-k-PGFla) are significantly elevated in SDEC CM compared to WI38 and Detroit 551 CM, suggesting that this pathway is activated in SDEC. The microarray data is confirmed by quantitative RT-PCR as well as Western blot analysis. The possible role of sFRP-1 in affecting stem cell growth is investigated further. SDEC media is supplemented with varying concentrations of purified sFRP-1 and the effect on hESC proliferation is monitored. Analysis of the factors present in SDEC CM may aid in the development of a defined cell culture system that permits hESC growth and expansion in a chemically defined liquid medium on a simple, fully defined matrix including COL I.

[0042] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0043] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described.

[0045] As used herein, "conditioned medium" refers to a growth medium that is further supplemented by factors derived from media obtained from cultures of cells including, foe example, feeder cells on which hESCs can be cultured. Suitable media for culturing hESCs have been previously described in, for example, U.S. Patent Nos. 6,331 ,406 and 6,245,566, the contents of which are incorporated by reference in their entireties.

[0046] The term "embryoid bodies" or "EBs" refers to collections of cells formed from the aggregation or clustering of cultured hESCs in culture. EBs have a three dimensional morphology, e.g., they can be a solid or a cystic embryoid body.

[0047] The term "embryonic germ cells" or "EG cells" refers to cells isolated or derived from primordial germ cells (PGCs). EG cells include cells derived from PGCs, including cell lines derived from these EG cells and all progeny. [0048] The term "primordial germ cells" (PGCs) refers to undifferentiated embryonic germ cells isolated from post-fertilization from anlagen or from yolk sac, mesenteries, or gonadal ridges of an embryo or a fetus. PGCs can be harvested from the mesenteric or genital ridges of embryos or as gonocytes of later testicular tissues. EG and PGC cells are described in further detail in U.S. Pat. No. 6,090,622.

[0049] The term "embryonic stem cells" or "ES cells" refers to cells that are derived from the inner cell masses of pre-implantation embryos.

[0050] As used herein, pluripotent cells include cells that have the potential to divide in vitro for an extended period of time (greater than one year) and have the unique ability to differentiate into cells derived from all three embryonic germ layers, including the endoderm, mesoderm and ectoderm.

[0051] hESCs are human cells that can be cultured indefinitely in an undifferentiated state, yet retain the ability to be differentiated into a variety of cell and tissue types. In accordance with the present invention, the terms "pluripotent" and "pluripotential cells" refer to those cells which retain the developmental potential to differentiate into a wide range of cell lineages including the germ line. The terms "embryonic stem cell phenotype" and "embryonic stemlike cell" also are used interchangeably herein to describe cells which are undifferentiated and thus are pluripotent cells and which are visually distinguished from other adult cells of the same animal.

[0052] The ability of hESCs to differentiate in vitro into a wide variety of cell types including the ability to differentiate into embryonic and more highly differentiated cell types which can easily be tested by means common to those in the art. For example, to induce differentiation in monolayer cultures hESCs are cultured for 2 weeks without passage onto a fresh feeder layer. To induce differentiation in suspension culture, the cells are passed onto a gelatinized plate to eliminate possible contamination by fibroblasts. After 4 to 7 days in culture, colonies are gently dislodged from the plate by mouth pipette and disaggregated after incubation in 0.25% trypsin-EDTA for 10-15 min. Dissociated cells are cultured in a microdrop of hESC culture medium containing 0.3 μΜ retinoic acid (Sigma) on a 35-mm nonadhesive petridish (Falcon). Suspension cultures are monitored daily for embryoid body formation which is indicative of a differentiated phenotype (similar experiments testing for differentiation of attached hESCs are well known to those in the art). Cell culture media is changed every other day. Based on the resulting differentiated morphological types putative hESCs can be tested for their pluripotency.

[0053] The term "STO cell" refers to mouse embryonic fibroblast (MEF) cells such as are commercially available and include those deposited as ATCC CRL 1503, and ATCC 56-X as feeder layer for hESC growth. Methods of growth and maintenance of cells are also well known in the art, (see Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., current edition, incorporated herein by reference).

[0054] The term "culture medium" means a suitable medium capable of supporting growth of hESCs. Examples of suitable culture media useful in practicing the present invention are a variety of hESC growth media prepared with a base of Dulbecco's minimal essential media (DMEM) supplemented with 15% fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, or glucose and phosphate free modified human tubal fluid media (HTF) supplemented with 15% fetal calf serum, 0.2 mM glutamine, 0.5 mM taurine, and 0.01 mM each of the following amino acids; asparagine, glycine, glutamic acid, cysteine, lysine, proline, serine, histidine, and aspartic acid (McKieman, S. M. Clayton, and B. Bavister, Molecular Reproduction and Development 42: 188-199, 1995). An effective amount of factors are then added daily to either of these base solutions to prepare hESC growth media. The phrase "effective amount" as used herein is the amount of such described factor as to permit a beneficial effect on hESC growth and viability of hESCs using judgement common to those in the art of cell culturing and by the teachings supplied herein.

[0055] "Non-essential Amino acids" refers to the amino acids L-alanine, L-asparagine, L- aspartic acid, L-glutamic acid, glycine, L-proline, and L-serine.

[0056] As used herein, "expression" refers to the production of a material or substance as well as the level or amount of production of a material or substance. Thus, determining the expression of a specific marker refers to detecting either the relative or absolute amount of the marker that is expressed or simply detecting the presence or absence of the marker. As used herein, "marker" refers to any molecule that can be observed or detected. For example, a marker can include, but is not limited to, a nucleic acid, such as a transcript of a specific gene, a polypeptide product of a gene, a non-gene product polypeptide, a glycoprotein, a

carbohydrate, a glycolipid, a lipid, a lipoprotein or a small molecule.

[0057] Detection and analysis of a variety of genes known in the art to be associated with pluripotent stem cells may include analysis of genes such as, but not limited to OCT4, NANOG, SALL4, SSEA-1 , SSEA-3, SSEA-4, TRA-1 -60, TRA-1 -81 , or a combination thereof. Pluripotent stem cells may express any number of pluripotent cell markers, including: alkaline phosphatase (AP); ABCG2; stage specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1 -60; TRA-1-81 ; Tra-2-49/6E; ERas ECAT5, E-cadherin; β-tubulin III; a- smooth muscle actin (a-SMA); fibroblast growth factor 4 (FGF4), Cripto, Daxl ; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Natl); ES cell associated transcript 1 (ECAT1); ESG 1 /DPP A5/ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1 ; ECAT15-2; Fthll7; Sall4; undifferentiated embryonic cell transcription factor (Utfl); Re l ; p53; G3PDH; telomerase, including TERT; silent X chromosome genes; Dnmt3a; Dnmt3b; TRJM28; F-box containing protein 15 (Fbxl 5); Nanog/ECAT4; Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1 ; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1 ; developmental pluripotency- associated 2 (DPPA2); T-cell lymphoma breakpoint 1 (Tel l ); DPPA3/Stella; DPPA4; as well as other general markers for pluripotency.

[0058] "Detectably-labeled" refers to any means for marking and identifying the presence of a cell or part thereof, i.e., an oligonucleotide probe or primer, an antibody or fragment thereof, a protein or fragment thereof, a gene or fragment thereof, or a cDNA molecule. Methods for detectably-labeling cells or molecules are well known in the art and include, without limitation, radioactive labeling (e.g., with an isotope such as ³²P or ³⁵S) and nonradioactive labeling (e.g., chemiluminescent labeling, fluorescent labeling, enzymatic reaction products coded by genes, i.e., CAT).

[0059] As used herein, "differentiation" refers to a change that occurs in cells to cause those cells to assume certain specialized functions and to lose the ability to change into certain other specialized functional units. Cells capable of differentiation may be any of totipotent, pluripotent or multipotent cells. Differentiation may be partial or complete with respect to mature adult cells. [0060] "Differentiated cell" refers to a non-embryonic, non-parthenogenetic or non- pluripotent cell that possesses a particular differentiated, i.e., non-embryonic, state. The three earliest differentiated cell types are endoderm, mesoderm, and ectoderm.

[0061] Pluripotency can also be confirmed by injecting the cells into a suitable animal, e.g., a SCID mouse, and observing the production of differentiated cells and tissues. Still another method of confirming pluripotency is using the subject pluripotent cells to generate chimeric animals and observing the contribution of the introduced cells to different cell types. Methods for producing chimeric animals are well known in the art and are described in U.S. Pat. No. 6,642,433, incorporated by reference herein.

[0062] Yet another method of confirming pluripotency is to observe cell differentiation into embryoid bodies and other differentiated cell types when cultured under conditions that favor differentiation (e.g., removal of fibroblast feeder layers).

[0063] The nucleic acid construct of the present invention can be introduced into cells by any viral or non-viral based transfection known in the art, such as, but not limited to

electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion. Other methods of transfection include proprietary transfection reagents such as Lipofectamine™, Dojindo Hilymax™, Fugene™, jetPEI™, Effectene™ and DreamFect™.

[0064] The term "polynucleotide" or "nucleotide sequence" or "nucleic acid molecule" is used broadly herein to mean a sequence of two or more deoxyribonucleotides or ribonucleotides that are linked together by a phosphodiester bond. As such, the terms include RNA and DNA, which can be a gene or a portion thereof, a cDNA, a synthetic polydeoxyribonucleic acid sequence, or the like, and can be single stranded or double stranded, as well as a DNA/RNA hybrid. Furthermore, the terms as used herein include naturally occurring nucleic acid molecules, which can be isolated from a cell, as well as synthetic polynucleotides, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by the polymerase chain reaction (PCR). It should be recognized that the different terms are used only for convenience of discussion so as to distinguish, for example, different components of a composition.

[0065] In general, the nucleotides comprising a polynucleotide are naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to 2'-deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose. Depending on the use, however, a polynucleotide also can contain nucleotide analogs, including non- naturally occurring synthetic nucleotides or modified naturally occurring nucleotides.

Nucleotide analogs are well known in the art and commercially available, as are

polynucleotides containing such nucleotide analogs. The covalent bond linking the nucleotides of a polynucleotide generally is a phosphodiester bond. However, depending on the purpose for which the polynucleotide is to be used, the covalent bond also can be any of numerous other bonds, including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides.

[0066] A polynucleotide or oligonucleotide comprising naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template. In comparison, a polynucleotide comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally will be chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template.

[0067] In various embodiments antisense oligonucleotides or RNA molecules include oligonucleotides containing modifications. A variety of modification are known in the art and contemplated for use in the present invention. For example oligonucleotides containing modified backbones or non-natural intemucleoside linkages are contemplated. As used herein, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides. [0068] In various aspects modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkyIene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates,

phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters,

selenophosphates and borano-phosphates having normal 3 -5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Certain oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof)- Various salts, mixed salts and free acid forms are also included.

[0069] In various aspects modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0070] In various aspects, oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. In various aspects, oligonucleotides may include phosphorothioate backbones and oligonucleosides with heteroatom backbones. Modified oligonucleotides may also contain one or more substituted sugar moieties. In some embodiments oligonucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-0- alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C\ to Cio alkyl or C₂ to Cio alkenyl and alkynyl. Particularly preferred are 0[(CH₂)_nO]mCH₃,

0(CH₂)_nOCH₃, 0(CH₂)_nNH₂, 0(CH₂)_nCH₃, 0(CH₂)_nONH₂ and 0(CH₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C] to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, CI, Br, CN, CF₃, OCF₃, SOCH₃, S0₂CH₃, 0N0₂, N0₂, N3, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Another modification includes 2'- methoxyethoxy(2'OCH₂CH₂OCH₃, also known as 2'-0-(2-methoxyethyl) or 2'-MOE).

[0071] In related aspects, the present invention includes use of Locked Nucleic Acids (LNAs) to generate antisense nucleic acids having enhanced affinity and specificity for the target polynucleotide. LNAs are nucleic acid in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (-CH₂-)_n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2.

[0072] Other modifications include 2'-methoxy(2'-0-CH₃), 2'-aminopropoxy(2'- OCH₂CH₂CH₂NH₂), 2'-allyl (2*-CH-CH-CH₂), 2'-0-allyl (2'-0-CH₂-CH-CH₂), 2'-fluoro (2'-F), 2'-amino, 2'-thio, 2'-Omethyl, 2'-methoxymethyl, 2'-propyl, and the like. The 2'- modification may be in the arabino (up) position or ribo (down) position. A preferred 2'- arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. [0073] Oligonucleotides may also include nucleobase modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine, 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8- hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (lH-pyrimido[5,4- b][l ,4]benzoxazi-n-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b][l,4]benzothiazin- 2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H- pyrimido[5,4-b][l ,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2- one), pyridoindole cytidine (H-pyrimido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2- pyridone. Further nucleobases are known in the art. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds described herein. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 C and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.

[0074] Another modification of the antisense oligonucleotides described herein involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The antisense oligonucleotides can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with R A. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., dihexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O-hexadecyl-rac- glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylaminocarbonyloxycholesterol moiety.

[0075] A test agent or compound according to the methods of the invention can be, for example, a polynucleotide, a peptide, a peptidomimetic, peptoids such as vinylogous peptoids, a small organic molecule, or the like, and can act in any of various ways to alter a function of a cell. For example, the test agent or compound can act extracellularly by binding to a cell surface receptor, thereby altering a function mediated by binding of a ligand that generally binds to and acts via the receptor. Alternatively, the test agent or compound can be one that traverses cell membrane, either passively or via an active transport mechanism, and acts within a cell to alter a function.

[0076] A peptide test agent according to the methods of the invention can include from about two to four residues to hundreds or thousands amino acids. The term "peptide," as used herein, does not suggest a particular size or number of amino acids comprising the molecule, and that a peptide test agent can contain up to several amino acid residues or more. Peptide test agents can be prepared, for example, by a method of chemical synthesis, or using methods of protein purification, followed by proteolysis and, if desired, further purification by chromatographic or electrophoretic methods, or can be expressed from an encoding polynucleotide. Further, a peptide test agent can be based on a known peptide, for example, a naturally occurring peptide, but can vary from the naturally occurring sequence, for example, by containing one or more D-amino acids in place of a corresponding L-amino acid; or by containing one or more amino acid analogs, for example, an amino acid that has been derivatized or modified at its reactive side chain.

[0077] Similarly, one or more peptide bonds in the peptide test agent can be modified, or a reactive group at the amino terminus or the carboxy terminus or both can be modified. Such peptides can have improved stability to a protease, an oxidizing agent or other reactive material the peptide test agent may encounter in a biological environment. Such peptide test agents also can be modified to have decreased stability in a biological environment where the period of time the peptide is active in the environment is reduced.

[0078] A polynucleotide test agent according to the methods of the invention can include from about two to four residues to hundreds or thousands nucleotides. The term "polynucleotide," as used herein, is not limited to a sequence of two or more deoxyribonucleotides or ribonucleotides that are linked together by a phosphodiester bond. Linkages other than a phosphodiester bond can also be used.

[0079] The covalent bond linking the nucleotides of a polynucleotide generally is a phosphodiester bond. However, the covalent bond also can be any of numerous other bonds, including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides (see, for example, Tarn et al. (1994) Nucl. Acids Res. 22:977-86; Ecker and Crooke (1995) BioTechnology 13:351-60, each of which is incorporated herein by reference). The incorporation of non-naturally occurring nucleotide analogs or bonds linking the nucleotides or analogs can be particularly useful where the polynucleotide is to be exposed to an environment that can contain a nucleolytic activity, including, for example, a tissue culture medium or upon administration to a living subject, since the modified polynucleotides can be less susceptible to degradation.

[0080] A polynucleotide test agent can be contacted with or introduced into a cell using methods as disclosed herein or otherwise known in the art. Generally, but not necessarily, the polynucleotide is introduced into the cell, where it effects its function either directly, or following transcription or translation or both. For example, the polynucleotide can encode a peptide test agent, which is expressed in a cell and alters a function of the cell. A polynucleotide test agent also can be, or can encode, an antisense molecule, an interfering RNA, a micro R A, a ribozyme or a triplexing agent, which can be designed to target one or more specific target nucleic acid molecules.

[0081] Antisense polynucleotides, ribozymes and triplexing agents generally are designed to be complementary to a target sequence, which can be a DNA or RNA sequence, for example, mRNA, and can be a coding sequence, a nucleotide sequence comprising an intron-exon junction, a regulatory sequence such as a Shine-Delgarno sequence, or the like. The degree of complementarity is such that the polynucleotide, for example, an antisense polynucleotide, can interact specifically with the target sequence in a cell. Depending on the total length of the antisense or other polynucleotide, one or a few mismatches with respect to the target sequence can be tolerated without losing the specificity of the polynucleotide for its target sequence. Thus, few if any mismatches would be tolerated in an antisense molecule consisting, for example, of 20 nucleotides, whereas several mismatches will not affect the hybridization efficiency of an antisense molecule that is complementary, for example, to the full length of a target mRNA encoding a cellular polypeptide. The number of mismatches that can be tolerated can be estimated, for example, using well known formulas for determining hybridization kinetics (see Sambrook, J.; Fritsch, E.F. and Maniatis, T. "Molecular Cloning: A Laboratory Manual," vol. I. 2^nd edition. Cold Spring Harbor Laboratory Press, 1989) or can be determined empirically using methods as disclosed herein or otherwise known in the art, particularly by determining that the presence of the antisense polynucleotide, ribozyme, or triplexing agent in a cell decreases the level of the target sequence or the expression of a polypeptide encoded by the target sequence in the cell.

[0082] A polynucleotide useful as an antisense molecule, a ribozyme or a triplexing agent can inhibit translation or cleave the nucleic acid molecule, thereby altering a function of a cell. An antisense molecule, for example, can bind to an mRNA to form a double stranded molecule that cannot be translated in a cell. Antisense oligonucleotides of at least about 15 to 25 nucleotides are preferred since they are easily synthesized and can hybridize specifically with a target sequence, although longer antisense molecules can be expressed from a polynucleotide introduced into the target cell. Specific nucleotide sequences useful as antisense molecules can be identified using well known methods, for example, gene walking methods (see, for example, Seimiya et al. (1997) J. Biol. Chez. 272:4631-36, which is incorporated herein by reference). Where the antisense molecule is contacted directly with a target cell, it can be operatively associated with a chemically reactive group such as iron-linked EDTA, which cleaves a target RNA at the site of hybridization. A triplexing agent, in comparison, can stall transcription (Maher et al. (1991) Antisense Res. Devel. 1 :227; Helene (1991) Anticancer Drug Design 6:569).

[0083] A screening method of the invention provides the advantage that it can be adapted to high throughput analysis and, therefore, can be used to screen combinatorial libraries of test agents in order to identify those agents that can alter a function of a pluripotent or multipotent cell. Methods for preparing a combinatorial library of molecules that can be tested for a desired activity are well known in the art and include, for example, methods of making a phage display library of peptides, which can be constrained peptides (see, for example, U. S. Patent No. 5,622, 699; U. S. Patent No. 5,206, 347; Scott and Smith (1992) Science 249:386- 90; Markland et al. (1991) Gene 109: 13-19; each of which is incorporated herein by reference); a peptide library (U. S. Patent No. 5,264, 563, which is incorporated herein by reference); a peptidomimetic library (Blondelle et al. (1995) Trends Anal. Chem. 14:83-92; a nucleic acid library (O'Connell et al. (1996) Proc. Natl. Acad. Set, USA 93:5883-87; Tuerk and Gold (1990) Science 249:505-10; Gold et al. (1995) Ann. Rev. Biochem. 64:763-97; each of which is incorporated herein by reference); an oligosaccharide library (York et al. (1996) Carb. Res. 285:99-128; Liang et al. (1996) Science 274: 1520-22; Ding et al. (1995) Adv. Expt. Med. Biol. 376:261 -69; each of which is incorporated herein by reference); a lipoprotein library (de ruif et al. (1996) FEBS Lett. 399:232-36, which is incorporated herein by reference); a glycoprotein or glycolipid library (Karaoglu et al. (1995) J Cell Biol. 130:567-77, which is incorporated herein by reference); or a chemical library containing, for example, drugs or other pharmaceutical agents (Gordon et al. (1994J J. Med. Chem. 37: 1385-1401 ; Ecker and Crooke (1995) BioTechnology 13:351-60; each of which is incorporated herein by reference).

[0084] Polynucleotides can be particularly useful as agents that can alter a function of cells because nucleic acid molecules having binding specificity for cellular targets, including cellular polypeptides, exist naturally, and because synthetic molecules having such specificity can be readily prepared and identified (see, for example, U. S. Patent No. 5,750, 342, which is incorporated herein by reference). [0085] For a high throughput format, cells of the invention can be introduced into wells of a multiwell plate or of a glass slide or microchip, and can be contacted with the test agent. Generally, the cells are organized in an array, particularly an addressable array, such that robotics conveniently can be used for manipulating the cells and solutions and for monitoring the cells of the invention, particularly with respect to the function being examined. An advantage of using a high throughput format is that a number of test agents can be examined in parallel, and, if desired, control reactions also can be run under identical conditions as the test conditions. As such, the methods of the invention provide a means to screen one, a few, or a large number of test agents in order to identify an agent that can alter a function of cells.

[0086] The antibodies used in the methods of the invention can be prepared via techniques well known to those having ordinary skill in the art (see, e.g., Harlow and Lane (eds.) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratories, 1 88). Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules or those portions of an immunoglobulin molecule that contain the antigen binding site, including Fab, F(ab)₂, and F(v). Polyclonal or monoclonal antibodies may be produced by methods known in the art. The antibodies or active fragments thereof may also be produced by genetic engineering including chimeric antibody, single chain antibody. The antibody or an active fragment thereof may be used as an immunotherapeutic. The antibody or an active fragment thereof may be administered alone, or in combination with

chemotherapeutics or immunosuppressive agents as are known in the art.

[0087] Methods of producing polyclonal and monoclonal antibodies are known to those of skill in the art and described in the scientific and patent literature, see, e.g., Coligan, Current Protocols In Immunology, Wiley/Greene, N.Y., 1991 ; Stites (eds.) Basic And Clinical Immunology (7th ed.) Lange Medical Publications, Los Altos, Calif. ("Stites"); Goding, Monoclonal Antibodies Principles And Practice (2d ed.) Academic Press, New York, N.Y., 1986; and Harlow, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, 1988.

[0088] The monoclonal antibodies of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the monoclonal antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize monoclonal antibodies of the invention are competitive and noncompetitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of the antigens using the monoclonal antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Alternatively, antibody of the invention can be used to detect CTAs present in electrophoretically dispersed gel protocols such as Western blots and 2-dimensional gels. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.

[0089] The monoclonal antibodies of the invention can be bound to many different carriers and used to detect the presence of specific antigens. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such using routine experimentation.

[0090] Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Huse, Science 246: 1275, 1989; Ward, Nature 341 :544, 1989; Hoogenboom, Trends Biotechnol. 15:62-70, 1997; Katz, Annu. Rev. Biophys. Biomol. Struct. 26:27-45, 1997.

Human antibodies can be generated in mice engineered to produce only human antibodies, as described by, e.g., U.S. Patent Nos. 5,877,397; 5,874,299; 5,789,650; and 5,939,598. B-cells from these mice can be immortalized using standard techniques (e.g., by fusing with an immortalizing cell line such as a myeloma or by manipulating such B-cells by other techniques to perpetuate a cell line) to produce a monoclonal human antibody-producing cell. See, e.g., U.S. Patent Nos. 5,916,771 ; 5,985,615.

[0091] "Purified antibody" means an antibody that is at least 60%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated.

Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody, e.g., an anti-SSEA-1 specific antibody. A purified antibody may be obtained, for example, by affinity chromatography using recombinantly-produced protein or conserved motif peptides and standard techniques. The invention can employ not only intact monoclonal or polyclonal antibodies, but also an immunologically-active antibody fragment, such as a Fab, Fab' or (Fab')₂ fragments, or a genetically engineered Fv fragment (Ladner et al., U.S. Pat. No. 4,946,788).

[0092] The following examples are provided to further illustrate the embodiments of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLE 1

Feeder cell derivation and culture

[0093] The SDEC cell culture is derived from human embryonic germ cells. Briefly, cells are derived and maintained on a matrix of type I bovine collagen (Collaborative Biomedical Products, Bedford, MA, 10 μg/cm²) in EGM-2MV media (Clonetics) supplemented with 5% fetal bovine serum, hydrocortisone, human basic fibroblast growth factor, human vascular endothelial growth factor, insulin-like growth factor I, ascorbic acid, human epidermal growth factor, gentamycin, and amphotericin. All experiments are carried out using SDEC cells on passage 8 through 10. Detroit 551 (ATCC, CCL-110) and WI38 (ATCC, CCL-75) cell lines are obtained from ATCC. Detroit 551, WI38 and mouse embryonic fibroblasts (MEF, strain CF1, Chemicon) are maintained at 37 °C, 5% C0₂, 95% humidity in DMEM media

(Invitrogen) supplemented with 10% fetal bovine serum (Hyclone), 100 U/ml Penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine.

[0094] Preparation of CM: Feeder cells are grown to confluence in their respective growth medias, then media is changed to huES media consisting of Knockout DMEM (Invitrogen), 10% knockout serum replacement (Invitrogen), 10% Plasmanate® (Bayer), 2 mM L-glutamine (Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), 1 mM sodium pyruvate

(Invitrogen), 0.1 mM 2-mercaptoethanol, and 8 ng/ml FGF2 (R&D systems). After 24 hours the conditioned media is collected, filtered over a 0.22 μιη filter and stored at -80 °C for up to 2 months. CM harvesting is repeated for up to 2 weeks or until feeder layers degraded and pooled prior to use to insure uniformity. CM is supplemented with an additional 8 ng/ml FGF2 immediately prior to use.

[0095] hESC culture: For long term maintenance, hESC lines HI and H9 (WiCell) are maintained in huES media on irradiated MEF and passaged with 1 mg/ml collagenase. Prior to initiation of CM experiments, hESC lines are passaged three times using 0.05% trypsin- EDTA and plated on growth factor reduced MAT (BD Biosciences) coated plates (1 :20 dilution in base media) in the presence of MEF conditioned huES media. For routine passage, cells are digested with 0.05% trypsin-EDTA for 5 minutes at 37 °C then trypsin neutralizing solution is added and cells are triturated gently to form small clumps of cells. Cells are centrifuged at 100 x g for 5 minutes and resuspended in media. For cell counts, the same procedure is followed except cells are triturated until a single-cell suspension is formed. In between passages, the media is replaced with fresh CM everyday and 8 ng/ml FGF2 is added immediately prior to use. For CM experiments, hESC lines are passaged 1 :3 every 3-5 days using 0.05% trypsin-EDTA and plated on dishes coated with either growth factor reduced MAT or COL I. Equal numbers of cells are plated each passage. Cell counting is performed on a Nucleocounter (New Brunswick Scientific) on triplicate wells. Population doubling is calculated as 3.32 * (LoglO cell countfinal-LoglO cell countstarting). Statistical significance is calculated using the heteroscedastic Student's t-test.

EXAMPLE 2

Characterization of hESC cells

[0096] Cell proliferation assay: hESCs previously passaged on MAT are plated at 5x10³ cells/ well in a 96 well dish coated with COL I or MAT in SDEC CM. After 24 hours, SDEC CM is supplemented with various concentrations of sFRP-1 (R&D Systems) and used to replace the cell media daily (n=4 wells per dosage). Control wells are supplemented with sFRP- 1 resuspension buffer (PBS with 0.1% bovine serum albumin). After a total of 3 days, CellTiter 96 AQueous (Promega) MTS reagent is added, the plate is incubated for 1 hour and the optical density at 490 nm is determined. A standard curve of cell number/well is done in parallel to insure readings are within the linear range of the assay. Statistical significance is calculated directly from the optical density data using the heteroscedastic Student's t-test. [0097] Immunocytochemistry: hESCs are fixed in 4% paraformaldehyde for 5 minutes at room temperature, blocked in 5% donkey serum, and 1 % BSA for 30 minutes at room temperature, then incubated with antibodies to SSEA-4 (Chemicon), OCT4 (Chemicon), Nanog (Chemicon), or Tra-1 -60 (Chemicon). Staining for OCT4 and Nanog are carried out in the presence of 0.1% Triton-X l OO. Secondary antibodies are donkey anti-mouse conjugated to either Alexafluor-488 or Alexafluor-594 (Invitrogen). Cells are counterstained with 4',6- diamidino-2-phenylindole (DAPI) to detect the nuclei. The percentage of cells expressing each marker is determined by viewing 5 randomly acquired fields with a total of > 1000 nuclei per well using a microscope equipped frame capturing software. Each percentage is calculated by dividing the number of cells with positive staining for the marker by the total number of cells (determined via DAPI staining). Data is reported as a mean of triplicate wells ± standard error. Statistical significance is calculated using the heteroscedastic Student's t-test.

[0098] Karyotype analysis: hESCs prepared for cytogenetic analysis are incubated in growth media with 0.1 mg/ml of Colcemid for 1 hour, washed in PBS, trypsinized, resuspended in 0.075 M KC1, and incubated for 20 minutes at 37 °C, then fixed in 3: 1 methanol/acetic acid. A minimum of 5 metaphase spreads are analyzed manually.

[0099] Teratoma formation: hESCs are passaged in SDEC CM on COL I for at least 20 population doublings over a 1 month period, then approximately 3x10⁶ hESCs are injected into the calf muscle of NOD/SCID mice. After one month, tumors are fixed in 4%

paraformaldehyde in PBS then processed for paraffin embedding. Sections are stained with Hematoxylin and Eosin following transplantation of hESCs into NOD/SCID mice.

EXAMPLE 3

Detection of Nucleic Acids and Proteins

[0100] RNA isolation and DNA microarray analysis: Total RNA is isolated from confluent 10 cm dishes of SDEC, WI38, and Detroit 551 cells (maintained in huES media for at least 48 hours) using the RNeasy Total RNA isolation kit (Qiagen) according to the manufacturer's instructions. Three biological replicate dishes of each cell type are used. Using 5 μg of total RNA, cDNA is synthesized using the GeneChip® One-Cycle cDNA Synthesis Kit (Manufactured by Invitrogen for Affymetrix). The cDNA is used as a template to synthesize biotin-labeled cR A using the GeneChip® IVT Labeling Kit (Affymetrix). Hybridization to the Human Genome U133 Plus 2.0 DNA microarrays (Affymetrix), washing, and scanning are completed according to the manufacturer's instructions. The program dChip is used to normalize and compare the data from the nine samples with minimal criteria set as +/- fold change of 2, a p-value of 0.02, and a signal presence percentage of 30%. Fold change values are recalculated with respect to the supportive feeder layer, SDEC.

[0101] Quantitative RT-PCR: Total RNA isolated from SDEC, WI38 and Detroit 551 is reverse transcribed into cDNA using oligo(dT) primers and MMLV (Moloney Murine Leukemia Virus) Reverse Transcriptase (Invitrogen). Quantitative RT-PCR is carried out using TaqMan reagents and primer/probe sets (Applied Biosystems) for sFRP-1

(Hs00610060_ml) and PTGS2 (Hs01573471_ml) using a 7900HT sequence detector (Applied Biosystems), standard cycling parameters and the Ct quantification method. Levels of cyclophilin A (PPIA, Applied Biosystems Endogenous Control) are used to normalize expression data. Standard curves are created by serial dilution of pooled samples. Data is reported as a mean of triplicate cultures ± standard error. Statistical significance is calculated using the heteroscedastic Student's t-test.

[0102] Western blot analysis of PTGS2: Confluent dishes of SDEC, WI38, and Detroit 551 cells are used to condition huES media for 24 hours. The conditioned media is collected and cell cytoplasmic lysates are obtained using the NE-PER cell lysis kit (Pierce Scientific). The total protein in the lysate is quantified using the BCA™ Protein Assay Kit (Pierce). Equal amounts (~20-30 μg) of protein are separated on 4-12% NuPAGE Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes. Membranes are blocked in 5% non-fat dried milk in Tris-buffered saline supplemented with 0.1% Tween-20 (TTBS), then incubated with antibodies to PTGS2 (Calbiochem) or β-actin (Abeam) in blocking buffer and visualized by using chemiluminescence (Supersignal West Pico, Pierce).

[0103] Analysis of sFRP- 1 in conditioned media: Briefly, frozen CM from SDEC, Detroit 551 , WI38, and MEF cells are thawed, pre-cleared by centrifugation and loaded on separate 1 ml HiTrap Heparin HP columns (GE Healthcare) that had been equilibrated with 0.05 M phosphate buffer, pH 7.4 containing 0.2 M NaCl. After washing the columns with 30 ml of equilibration buffer, retained protein is eluted with 10 ml each of 0.05 M phosphate buffer, pH 7.4 solutions containing increasing concentrations of NaCl (0.3, 0.7, 1.0 and 1.2 M). During the elution steps, 1 ml fractions are collected and aliquots of each fraction (24 μΐ) are resolved on 10% polyacrylamide-SDS Tris-HCl gels (Criterion Precast Gel, Bio-Rad) under reducing conditions. Proteins are transferred to Immobilon-P membranes (Millipore) that are blocked in 5% non-fat dried milk dissolved in TTBS. Membranes are then incubated with 2.5 μg/ml of an sFRP-1 amino-terminal peptide antibody in blocking buffer, followed by donkey anti-rabbit IgG (1 : 10,000; GE Healthcare) and sFRP- 1 protein is visualized with chemiluminescent reagents (Supersignal West Femto, Pierce). Subsequently, aliquots of the peak fractions from each cell type are pooled, resolved by SDS-PAGE and immunoblotted alongside

corresponding pools from the other cell types. Varying amounts of recombinant human sFRP- 1 are included in all blots to facilitate quantitative analysis of sFRP-1 concentration.

[0104] Analysis of Prostaglandins in conditioned media: Levels of 6-keto-prostaglandin Fla (6-k-PGFl a, Oxford Biomedical Research) and prostaglandin E2 (PGE2, Cayman Chemical) in CM are determined by commercially prepared ELISA from CM collected from 3 independent cultures of each cell type. Data are reported as a mean of triplicate wells ± standard error. Significance is determined by one way ANOVA with Bonferroni-Holm posthoc testing.

EXAMPLE 4

SPEC CM Supports hESC Maintenance and Proliferation

[0105] CM from the human cell types SDEC, WI38 and Detroit 551 are compared to MEF CM for their capacity to support hESC self-renewal and pluripotency over a three week period (5 passages) when plated on MAT coated plates. There are no significant differences in hESC line HI population doublings (PD) during the first week of growth in the various CM (Figure 1A). However, by week 2, HI cells proliferate significantly less in WI38 (PD=1.1) or Detroit 551 (PD=0.9) CM compared to SDEC (PD=2.2) or MEF (PD=2.3) CM. By week 3, hESC numbers decrease in WI38 (PD= -1.1) and Detroit 551 (PD= -1.3) CM while maintaining the week 2 PD in SDEC and MEF CM. There is no significant difference between PD in SDEC and MEF CM (Figure 1 A). [0106] After the third week of growth under these conditions, hESC are immunostained to determine the percentage of cells expressing OCT4. There is a small but significant difference (P<0.05) between the percentage of OCT4+ hESC grown in SDEC CM (94 ± 2.3) compared to MEF CM (84 ± 2.4)(Figure I B). However, there is a large significant difference (PO.001) between the percentage of OCT4+ cells in SDEC and either Detroit 551 (49 ± 1.5) or WI38 (23 ± 1.5) CM. OCT4 immunostains of hESC grown in each of the conditioned media types can be observed. The experiment is terminated after 3 weeks because of insufficient stem cell numbers in the WI38 and Detroit 551 CM treatments. The PD and OCT4 protein expression data suggest that SDEC CM is capable of supporting hESC proliferation and maintenance on MAT at least as well as MEF CM. In contrast, WI38 and Detroit 551 CM are not capable of maintaining long term hESC growth. The data presented here represents a single experiment but these general findings are seen in multiple experiments and are replicated using hESC line H9.

[0107] SDEC CM allows for hESC growth on COL I: In order to investigate the ability of SDEC CM to support hESC growth and maintain hESC pluripotency on different growth matrices, the inventors compare HI hESC grown in either SDEC or MEF CM on both MAT and COL I over a one month period (at least 20 population doublings). There are no statistically significant differences between the percentage of cells expressing OCT4 for MEF CM on MAT (89 ± 4.2), SDEC CM on MAT (98.4 ± 0.9) and SDEC CM on COL I (100 ± 0) (Figure 2A). The population doubling per week after 1 month in MEF or SDEC CM on MAT is 1.3, while the population doubling for the SDEC CM on COL I is 1.7, but this difference is not statistically significant (Figure 2B). Surprisingly, unlike SDEC CM, MEF CM is completely unable to support the growth of hESC on COL I. After 1 month, hESCs growing in SDEC CM on COL I also expresse pluripotency markers Nanog, SSEA-4, alkaline phosphatase (AP) and TRA- 1 -60. hESC colonies grown on COL I resemble those grown on MAT, with typical cellular morphology and clearly defined colony borders. The data presented here represents a single experiment but these general findings are seen in four replica experiments and are also replicated using hESC line H9. At the end of this one month period, both hESC lines HI and H9 retain a normal karyotype and are capable of forming teratomas containing differentiated tissues from all three embryonic germ layers, such as: bone, cartilage, muscle, neuroepithelial cells and intestinal mucosal cells, following transplantation into immunocompromised mice. EXAMPLE 5

Transcriptional Profiling of Supportive and Non-supportive Feeder Cells

[0108] Based on the finding that SDEC CM is supportive of hESC growth and plunpotency, the inventors compare the transcriptional profile of this cell culture to those of the non- supportive human feeder cell lines, WI38 and Detroit 551 , using DNA microarray analysis. MEF cells are not included in this comparison due to their non-human origin. The microarray data has been deposited in NCBl's Gene Expression Omnibus and is accessible through GEO Series accession number GSE 15400 on world wide web at ncbi.nlm.nih.gov/

geo/query/acc.cgi?acc=GSE 15400.

Table 1. Top 20 genes overexpressed by the non-supportive feeder layers.

Fold Di Terence

Accession DET/ WI38/

Gene

No. SDEC SDEC

Secreted frizzled-related protein 1 AI332407 55.35 153.14

SIX homeobox 1 NM 005982 13.47 81.76

Hepatocyte growth factor X I 6323 22.90 62.37

Matrix metallopeptidase 3 NM 002422 58.88 13.44

Pentraxin-related gene, rapidly induced by IL- 1

NM_002852 16.12 44.43 beta

Fibulin 1 NM 006486 45.72 12.31

Endothelin receptor type A NM 001957 46.85 7.91

Cell adhesion molecule 1 NM 014333 1 1.52 31.76

Forkhead box F2 NM 001452 1 1.57 29.07

Sema domain, seven thrombospondin repeats,

TM and short cytoplasmic domain (semaphorin) NM_003966 30.36 9.06 5A

Paired related homeobox 1 AA775472 28.41 10.80

PR domain containing 1 , with ZNF domain AI692659 28.58 7.98

Homeobox B5 NM 002147 7.94 21.07

Hephaestin NM 014799 17.84 5.14

Plasminogen activator, urokinase NM 002658 6.67 14.96

Forkhead box F 1 NM 001451 5.87 14.49

Syndecan 1 NM 002997 14.37 3.89

Sema domain, immunoglobulin domain (Ig),

NM_006080 3.78 13.43 short basic domain, secreted (semaphorin) 3A

Runt-related transcription factor 1 ; translocated

NM_004349 13.08 3.55 to, 1 (cyclin D-related)

Suppressor of cytokine signaling 2 NM 003877 3.53 12.21 [0109] For initial analysis of the data, fold change in gene expression level is calculated with respect to the SDEC culture. These values are ranked and 20 genes are identified as the most highly over-expressed by the non-supportive cell lines compared to SDEC (Table 1) and also 20 genes are identified as the most highly over-expressed by SDEC compared to the non- supportive lines (Table 2). sFRP- 1 is the most highly over-expressed gene in WI38 and Detroit 551 as compared to SDEC. Conversely, two enzymes in the prostaglandin synthetic pathway, PTGS2 and PGIS, are both highly over-expressed by SDEC compared to the non- supportive feeder lines. Interestingly, the gene for leukemia inhibitory factor (LIF) is also up- regulated in SDEC, although unlike in mouse embryonic stem cell culture, this protein in not sufficient to prevent differentiation in hESC culture.

[0110] Validation of Microarray Results: Quantitative RT-PCR is used to validate the differential expression of sFRP- 1 and PTGS2 in SDEC compared to WI38 and Detroit 551. agreement with the microarray results, the mRNA level of sFRP-1 in WI38 cells increase 1 ' ± 38-fold (P<0.01) over the level detected in SDEC and sFRP-1 expression in Detroit 551 cells increased 20 ± 1.1 -fold (P<0.05) over the level in SDEC (Figure 3 A). Conversely, the PTGS2 mRNA level is significantly lower (PO.001) in Detroit 551 (18.6 ± 0.03-fold) and in WI38 (51.2 ± 0.4-fold) relative to the SDEC cells (Figure 3B). Thus, the quantitative RT-PCR data for these two genes correlates with differences observed in the microarray data.

EXAMPLE 6

Expression of sFRP-1 and PTGS2 proteins

[0111] Semi-quantitative Western blot analysis is performed to examine the expression levels of sFRP-1 and PTGS2 at the protein level. The intracellular levels of PTGS2 in SDEC, WI38 and Detroit 551 cells are compared using cell cytoplasmic extracts. Equivalent amounts of total protein are used in each lane and β-actin is used as a loading control. As shown in Figure 3C, SDEC expresses the most PTGS2, followed by Detroit 551 , while the PTGS2 in WI38 is barely detectable.

[0112] To compare the amounts of sFRP-1 secreted into the CM by the three human cell types as well as MEF cells, heparin-Sepharose chromatography is used to purify sFRP-1 from similar quantities of CM from each cell type. sFRP-1 is recovered in the 1.0 M NaCl fractions. Quantitative analysis of Western blots indicates that the level of sFRP-1 in the WI38 CM is greater than the level in Detroit 551 CM, and that the CM from both of these lines contained 10-20 times more sFRP-1 than SDEC or MEF CM (Figure 3D). In agreement with the microarray data, SDEC expresses significantly less sFRP-1 and significantly more PTGS2 than the two less-supportive human feeder cell lines.

[0113] Levels of prostaglandins in CM: In order to determine whether the expression level of the intracellular protein PTGS2 would affect the levels of its soluble downstream products, PGE2 and prostacyclin (PGI2), in the conditioned media, ELISAs are performed to probe the concentrations of these compounds in CMs of SDEC, WI38, and Detroit 551. PGI2 is unstable (tl/2 = 2-3 minutes.) so its stable hydrolysis product, 6-k-PGFla, is typically monitored. The mean concentrations (n=3) of 6-k-PGF la are 44 ± 6 ng/ml, 1.4 ± 0.4 ng/ml and 0.7 ± 0.1 ng/ml for SDEC, Detroit 551 , and WI38 CM, respectively. Levels of PGE2 are 26.6 ± 0.9 ng/ml, 3.9 ± 0.3 ng/ml and 0.7 ± 0.1 ng/ml for SDEC, Detroit 551 and WI38 CM, respectively (Figure 3E). These data indicate that the levels of both PGE2 and 6-k-PGF l are significantly increased in the supportive SDEC CM compared with both the non-supportive CM and MEF CM.

[0114] Effect of sFRPl on hESC proliferation: A cell proliferation assay is performed to determine the impact of exogenous sFRP-1 on hESC line H9 growth on different biomatrices. Cells grown in SDEC CM on either MAT or COL I are exposed to different concentrations of sFRP-1 for 3 days and then hESC proliferation is measured. After the 3 day exposure to SDEC CM supplemented with 5 μg/ml sFRP-1 , the highest concentration tested, there is a significant reduction in the number of cells growing on MAT (82 ± 1.1 % of control, PO.01 ) or on COL I (74 ± 1.2% of control, P<0.001) when compared to CM without any added sFRP- 1 (Figure 4). Furthermore, there are significantly fewer cells on COL I than MAT (PO.01 ). At sFRPl concentrations of 1.0 μg/ml and 0.2 μg/ml, there is a small but significant difference (P<0.05) between the number of hESC growing in the presence versus absence of sFRP-1 but only for cells growing on COL I. Similar results can be seen in replica experiments on hESC HI cells.

EXAMPLE 7

Association Between sFRP-1 and the Supportive Layer

[0115] Maintaining human embryonic stem cells in culture requires a complex mixture of soluble and extracellular matrix components. Current hESC culture techniques often involve hESC contact with media components that are undefined or of animal origin, such as MEF, MAT, or conditioned media from feeder cell lines. Unfortunately, these incompletely defined culture conditions limit our ability to grow pluripotent hESC in a reproducible and scalable manner for both laboratory and clinical applications. There is significant interest in understanding the contributions of media composition, growth matrix, and feeder cell line to overall hESC growth and pluripotency, both to understand the mechanisms and functional roles of these components in hESC self-renewal, and as a means of developing a more defined culture environment that would be suitable for future clinical phase expansion of hESC.

[0116] Significant progress has been made in developing more defined media formulations and in eliminating undefined or xenogenic components such as animal serum and

contamination by mitotically inactivated but viable feeder cells. These defined media formulations sometimes require conditioning by supporting cells, such as MEF or human fibroblasts, for karyotypically stable long term maintenance of hESC. Furthermore, hESC growth in these defined, cell-conditioned media often still requires the use of complex and poorly defined xenogenic biomatricies, such as MAT. In a step towards identifying a more defined hESC growth environment, the present invention provides a human cell type, SDEC, that produces CM capable of maintaining hESC growth on a matrix of simple COL I. COL I is widely used in humans in health and beauty care applications. SDEC is one example of a cell type generically termed embryoid body derived (EBD). EBD cells including SDEC are typically capable of high rates of proliferation, have multi-lineage gene expression profiles, and have been used directly in cell transplantation studies. The hESCs grown in SDEC CM on MAT or COL I according to the present invention have equal or superior cell proliferation rates, maintenance of pluripotency, and karyotypic stability compared to hESC grown in MEF CM on MAT.

[0117] In order to begin to understand the complex nature of hESC supportive CM, the inventors compare the transcriptional profiles of the highly-supportive SDEC cell culture and two human fibroblast cell lines, WI38 and Detroit 551 , which do not produce supportive CM. A large number of genes are differentially regulated across the three cell types, and at least two of the most highly differentially expressed genes are provided in the present invention: PTGS2 and sFRP-1.

[0118] The protein sFRP-1 is a secreted protein that is a known antagonist of the Wnt/β- catenin signaling pathway. In this pathway, WNT binds to frizzled protein (Fzd) and activates the pathway by disrupting a complex of inhibitor proteins (including axin, CKIa, GSK-3 and APC), resulting in the stabilization of β-catenin. β-catenin can then enter the nucleus and interact with other proteins in order to promote transcription. sFRP-1 is homologous to Fzd and will bind to WNT, inhibiting WNT's interaction with Fzd and blocking activation of the β-catenin pathway. This pathway is known to have a key role in stem cell proliferation and maintenance of pluripotency through activation of NANOG and other factors such as OCT4. Thus, disruption of this pathway is a plausible mechanism for sFRP-1 's inhibitory activity in the hESC growth assays. However, sFRP-1 also modulates other signaling pathways and is known to interact with matrix molecules and other receptors, so it remains possible that its effect on hESC growth could involve additional mechanisms. [0119] The present invention provides high levels of sFRP-1 in the non-supportive WI38 and Detroit 551 CM relative to the supportive SDEC and MEF CM. These high levels of sFRP-1 can inhibit W T signaling and negatively impacting the growth of hESC grown in these CM. Indeed, a microarray study shows increased expression of sFRP- 1 (3.3 fold increase) and other inhibitors of WNT signaling in MEF feeder layers that are less supportive of hESC growth compared to those that are highly supportive of hESC growth. The present invention extends the inhibitory role of sFRP-1 on stem cell proliferation to human feeder layers. An even more dramatic change in sFRP-1 expression is observed with the non-supportive WI38 and Detroit 551 cell lines expressing 153- and 55-fold more sFRP-1 , respectively, than the SDEC culture.

[0120] To further investigate the role sFRP-1 plays in affecting hESC growth, the sFRP-1 concentration in SDEC CM growth media can be increased by supplementing the media with exogenous sFRP-1. These studies confirm the inhibitory role of sFRP-1 on stem cell proliferation on both COL I and MAT. The present invention also provides that the addition of sFRP family members to culture media caused a decrease in the percentage of

undifferentiated hESC colonies grown on MEF feeder layers. Unexpectedly, the negative effect of sFRP-1 on hESC proliferation provided in the present invention is more pronounced on COL I than it is on MAT. It is notable that heparin sulfate proteoglycan is one of the major components of MAT, along with laminin and type IV collagen. sFRP-1 is known to bind strongly to heparin, which is widely utilized to purify sFRP- 1 from culture media. Indeed, the procedure used to purify the sFRP-1 from the conditioned culture media according to the invention includes a heparin affinity column. The presence of the heparin sulfate proteoglycan in MAT may serve to bind to sFRP-1 in the CM, effectively sequestering it and reducing its inhibitory activity. In contrast, COL I lacks heparin sulfate and its potential capacity to sequester sFRP-1. One of the reasons that MAT is such a highly effective hESC growth matrix may be its capacity to absorb compounds, including sFRP-1 , that negatively impact stem cell self-renewal.

[0121] Surprisingly, the Western blot data also indicates low levels of sFRP-1 in the MEF CM that is supportive of hESC proliferation on MAT. The present invention provides that the sFRP-1 antiserum used in the Western blot analysis is generated against human sFRP-1 and that there might be a difference in its binding specificity with human versus mouse sFRP-1. However, the 14 amino acid synthetic peptide used to synthesize the antiserum differs by only 1 amino acid from the mouse sequence, and the entire sequences of the human and mouse proteins are 94% identical at the amino acid level. Therefore, the Western blot is likely to be an appropriate indicator of the level of sFRP-1 in the MEF CM used. Low levels of sFRP-1 in MEF CM would be expected according to the present invention since this media is also highly supportive of hESC growth.

[0122] The sFRP-1 concentrations in the non-supportive Detroit 551 and WI38 CM are estimated to be -25-50 ng/ml, while the sFRP-1 concentration required to negatively impact hESC growth in a 3-day assay is at least 200 ng/ml. The lower concentrations of sFRP-1 seen in the WI38 and Detroit 551 CM could still have the ability to affect hESC proliferation if the cells are exposed to it over a longer time period, such as the three to four week long. This idea is supported by the fact that the negative effect of W138 and Detroit 551 CM on hESC proliferation is only observed after 2-3 weeks of exposure (Figure 1). It is also likely that factors other than sFRP-1 in the CM can affect hESC proliferation.

EXAMPLE 8

Association between PTGS2 and the Non-supportive Layer

[0123] Western blot analysis also reveals an increase in the intracellular levels of PTGS2 in the SDEC culture compared to the non-supportive feeder lines. PTGS2 is not a secreted protein and is therefore unlikely to be present in CM, but it is involved in the biosynthesis of prostaglandins, which are secreted. Following PTGS2 (or homologous prostaglandin- endoperoxide synthase 1 ) activity, the precursor prostaglandin H2 can be converted to different prostaglandins including PGE2 and PGI2, which is generated by PGIS. The microarray results also detect very high expression of PGIS in the SDEC culture compared to the non-supportive feeder lines, suggesting that this prostaglandin synthesis pathway is elevated in SDEC. The present invention provides that SDEC CM contained 7- and 40-fold higher levels of PGE2 than Detroit 551 and WI38 CM, respectively. Likewise, SDEC CM contains 30- and 60-fold higher levels of 6-k-PGF l a (the stable hydrolysis product of PGI2) than Detroit 551 and WI38 CM, respectively. The present invention provides that increased expression of genes involved in prostaglandin synthesis, such as PTGS2 and PGIS, can indeed lead to increased secretion of certain prostaglandins into CM. Additionally, the present invention provides that PGE2 and 6-k-PGFl a levels are 3- and 10-fold higher in SDEC CM than in MEF CM, respectively.

[0124] The levels of PGE2 and PGI2 increase in the fallopian tubes (oviduct) following pregnancy and these two prostaglandins, also produced in embryos, enhance embryo cell numbers, hatching, and implantation. PGE2 is found to have a positive affect on the growth of hematopoietic stem cell progenitors obtained from Zebrafish and mouse embryonic stem cells. PTGS2 activity and PGE2 have also been associated with mouse embryonic stem cell proliferation and prevention of apoptosis. The present invention provides that increased PTGS2 activity and PGE2 secretion in cells that are supportive of hESC growth. The present invention also provides high expression of PGIS in cells that are supportive of hESC growth and obtain significantly higher concentrations of 6-k-PGFl in hESC supportive CM than in non-supportive CM. However, in a study on mouse embryonic stem cells, no PGIS is detected in the cell lysate and no 6-k-PGF l a is detected in the CM of undifferentiated cells. Increased levels of PGI2 have also been associated with inducing hESC to form cardiomyocytes indicating varying roles for PGI2 depending on the species and specific microenvironment. The present invention provides that PTGS2 and/or PGIS are associated with hESCs' self- renewal and/or survival.

[0125] Surprisingly, the down regulation of sFRP-1 and the increase in PTGS2 gene expression observed in the SDEC cells are related. A link between the prostaglandin and WNT signaling pathways can be established in mouse hematopoietic stem cells. PGE2 can regulate WNT signaling by direct phosphorylation of β-catenin and GSK-3 via cAMP PKA signaling. Increased levels of PTGS2 leads to the stabilization of β-catenin and consequently, to the enhanced formation and growth of hematopoietic stem cells. In addition, increased expression of WNT (increased WNT signaling) leads to increased levels of PTGS2 and increased PGE2 synthesis, suggesting that PTGS2 is a transcriptional target for β-catenin or that transcription of PTGS2 is regulated by β-catenin activated transcription factors. The present invention provides increased expression of genes involved in prostaglandin biosynthesis (PTGS2 and PGIS) and decreased expression of genes involved in inhibiting WNT signaling (sFRP-1) in the hESC-supportive SDEC cells. The present invention further provides that the coordinated down-regulation of WNT inhibitors (sFRP- 1 ) and up-regulation 6286

41

of factors which stimulate W T signaling (PTGS2) have an unexpected synergistic effect on human embryonic stem cell proliferation.

[0126] In terms of optimizing media conditions for hESC proliferation, the results presented here have begun to address the complexity of this issue. By eliminating direct contact with feeder cell lines and the use of the MAT, the present invention restricts the potential sources of variability to only those factors that are secreted by SDEC into the conditioned media. The insights provided in the present invention by gene expression profiling of supportive and non- supportive feeder layers suggest additional components that may facilitate hESC growth and may aid in the development of more defined media conditions in the future. In addition, implementation of a collagen growth matrix may represent a significant advance for the expansion of hESC, as this matrix is defined, inexpensive, and readily scalable for clinical development.

EXAMPLE 9

Cytokines Overexpressed in SDEC Cell Conditioned Media

[0127] The present invention provides analysis of cytokines secreted by SDEC cells. This example compares SDEC conditioned media from three different batches to the unconditioned based media. The present invention provides that these overexpressed cytokines are responsible for collagen permissiveness and/or proliferation of human embryonic stem cells (hESCs). The present invention further provides that listed inflammatory cytokines may play important roles in development, proliferation, and matintance of stem cell pools.

Table 3. Cytokines overexpressed in SDEC conditioned media (ranked by fold changes for overexpression as compared to base media)

Rank Cytokine Fold Rank Cytokine Fold

# Overexpression # Overexpression

1 GM-CSF 2501.6 41 ICAM-3 6.7

2 IL-6 1360.2 42 TEC 6.1

3 GCP-2 1086.8 43 M-CSF 6.1

4 MCP-3 572.3 44 TGF-beta 3 5.9

5 Osteoprotegerin 490.0 45 sgpl30 5.8

6 IL-l beta 463.2 46 Leptin 5.7

7 Angiogenin 351.1 47 MIF 5.2 HGF 349.6 48 CCL-28 4.6

MCP-1 294.5 49 MIP-1 -delta 4.1

MIP-3-alpha 255.5 50 MCP-4 4.0

MCP-2 234.1 51 BTC 3.4

RANTES 220.7 52 Endoglin 3.3

GRO 208.7 53 lL-10 3.2

GRO-alpha 208.3 54 TIMP-1 3.1

MMP-1 199.6 55 IGFBP-3 3.1

IGFBP-2 165.8 56 sTNF-RI 3.0

TIMP-2 158.8 57 Axl 3.0

Activin A 98.6 58 Amphiregulin 2.9

IGFBP-4 90.4 59 TIMP-4 2.9

LAP 81.3 60 IGF-I SR 2.7

IL-8 63.0 61 Thrombopoietin 2.7

IGFBP-1 43.8 62 GITR-Ligand 2.7

ENA-78 42.6 63 GDNF 2.6

VEGF 39.5 64 VEGF-D 2.6

EGF 37.3 65 Lymphotactin 2.5

1-309 30.5 66 ICAM-2 2.5

IL-1 alpha 23.1 67 TRAIL R3 2.4 uPAR 22.2 68 SCF 2.1

PDGF AA 20.2 69 IL-7 2.1

IL-2 Ralpha 18.7 70 Acrp30 1.9

IGFBP-6 16.9 71 IL-2 Rapha 1.9

Eotaxin 15.1 72 MIP-1 beta 1.8

PDGF-BB 14.0 73 TGF-beta 1 1.7

IL-1 1 13.4 74 ALCAM 1.6

LIF 12.8 75 BDNF 1.5

FGF-7 1 1.7 76 TRAIL R4 1.5

GCSF 1 1.4 77 IL-12 p40 1.5

MIP-1 alpha 8.6 78 PDGF Ralpha 1.5

PIGF 8.6 79 SDF-1 1.5

Angiopoietin-2 7.0 [0128] The protein levels of various cytokines are measured from three experiments. The mean value (Mean) and strand error (SE) are caculated. The differences of protein levels for various cytokines are ranked based on either fold changes for overexpression or differences (delta). Table 3 shows cytokines with at least 1.5 fold increased expression level ranked by fold overexpression as compared to base media. Table 4 shows top 50 overexpressed cytokines ranked by difference to based media (delta). In addition, Table 4 provides brief background for known function of the listed cytokines. In one embodiment, the present invention provides homologs of listed cytokine with known functions can also have similar activities fo for collagen permissiveness and/or proliferation of human embryonic stem cells (hESCs).

Table 4. Top 50 cytokines overexpressed in SDEC conditioned media (ranked by differences to base media - delta)

Rank

Cytokine Delta Background

#

Chemokine CXCL1 or MGSA. Proinflamitory.

Induced in beta cells by ECM from rat bladder

1 GRO 62,617.0

carcinoma through bl integrin/laminin-5. Increased by IL-1. Promotes micorvascularization.

Proinflamitory. Secretion by human pancreatic periacinar myofibroblasts. IL-6 secretion is induced

2 IL-6 59,963.9

by IL-17, IL-1 beta, and TNF-a through NF-kappaB activation

CCL2. Proinflamitory. Binds receptors CCR2 and CCR4. Promotes monocyte recruitment, expression

3 CP-1 59,827.3

by insulin-producing cells can lead to insulitis and islet destruction

4 MCP-3 57,717.0 CCL7. Proinflamitory.

CSF2. Immunomodulation. GM-CSF dysregulates STATS binding at non-coding regulatory regions in

5 GM-CSF 43,024.5

NOD mice. GM-CSF was more effective in suppressing T1 D

6 GRO-alpha 39,535.6 SAME AS GRO

TGFb signaling mediator. TGFb 1 Inhibits acinar cell growth. Overexpression of TGFb 1 in islets leads to

7 LAP 30,452.1 fibrosis of the exocrine pancreas and expansion of fibroblastic cells. Overexpression Induces CP with decrease in beta cells

CCL20. Proinflamitory. Recptor CCR6. up-regulated

8 MIP-3-alpha 22,567.8 in Pane. Adenocarcinoma where it is expressed in ducts and islets

a matrix metalloproteinase inhibitor (MMP-1 is substrate) expressed by stellate cells. Overexpression

TIMP-1 2,016.5

in β-cells enhances the replication of β-cells and counteracts type 1 diabetes

GCP-2 1 ,916.1 CXCL6.

VEGF 1,785.4 VEGFA.

intercellular adhesion molecule 2 (not demonstrated

ICAM-2 1 ,715.0

in pancreas)

CSF3. works through JA /STAT, MAP , PI3K-

GCSF 1 ,348.3

AKT pathways

acts in a stimulatory capacity on the embryonic pancreatic epithelium, and in culture, initiates

EGF 1 ,312.9

epithelial precursor cell proliferation, endocrine cell differentiation is repressed

IL-2 Ralpha 1 ,270.8 JAK/STAT, MAPK, PI3K pathways

expressed in the periphery of fetal islets. Upregulated

TRAIL R3 1,031.5

in CP

CD166. activated Leukocyte adhesion molecule.

ALCAM 719.0 Expressed on all islet cells not ducts. R&D systems

$365

FGF10 and PDGFAA expressed in embryonic and

PDGF AA 713.4 regenerating adult pancreas. EGF and PDGFAA stimulated MAPK and Akt phosphorylation sgp l 30 567.7 selective inhibitor of IL6 signaling

Eotaxin 483.5 CCL 1 1. Proinflamatory

IL-1 1 467.5

Like FGF 10, expressed in the human embryonic pancreatic mesenchyme and promote the proliferation

FGF-7 421.6 of embryonic pancreatic epithelial cells, not an obligatory growth factor for pancreatic endocrine development

A subset of human beta-cells expresses functional CD14 receptor and thus is able to recognize LPS as

CD14 374.7

well as endogenous Iigands (e.g. glycolipids of beta- cell origin)

sTNF-RI 353.4 used to detect pancreatic inflammation

MIF macrophage migration inhibitory factor (glycosylation-inhibiting factor), growth factor

MIF 295.1

involved in cell cycle progression pane.

Adenocarcinoma

antagonist of angiopoietin 1 and endothelial TEK tyrosine kinase (TIE-2, TEK)and may induce

Angiopoietin-2 286.6

endothelial cell apoptosis. Plays a role in pancreas vasularization

MIP- 1 alpha 269.9 CCR1. Involved in TID

1-309 221.6 CCR8. Involved in TI D

Phosphatidylinositol-glycan biosynthesis class F

P1GF 202.8

protein enhances beta-cell mass and improve glucose control.

Transgenic expres. GDNF in pane led to increased

47 GDNF 174.3 Ngn3 -expressing cells and higher beta-cell mass at

El 8. influences Pdxl expression by enhancing Sox9 and neuroDl binding to Pdxl promoter

present in most ductal cells and acinar, islet cells, nerve fibers and ganglia cells. In CP intense immunostaining of BDNF was present in most cells

48 BDNF 169.3

of ductular complexes and in enlarged nerves.

Moderately in degenerating acinar cells and isletand intrinsic pancreatic ganglia cells in CP samples

49 ICAM-3 164.6

50 Amphiregulin 162.1

[0129] Table 5 listed selected cytokines of the invention, showing both proinflammatory cytokines and be cell mitogens.

Table 5. Selected cytokines of the invention.

Proinflammatory b cell mitogen

GRO HGF

IL-6 Activin A

MCP-1 ,3 LIF

GM-CSF TIMP-1

GRO-alpha GCSF

MIP- 1 ,3 alpha EGF

IL-lbeta PDGF AA

MMP- 1 FGF-7

IL-8 GDNF

RANTES VEGF

ENA-78

Osteoprotegerin

IL-1 alpha

MCP-2

GCP-2

IL-2 Ralpha

sgpl30

Eotaxin

IL-1 1

CD14 Table 5. Selected cytokines of the invention.

sTNF-RI

MIF

TRAIL R3

Angiopoietin-2

[0130] Table 6 shows the experimental data for all cytokines tested, where both delta and fold changes are shown. The levels in the based media are shown as Base and the cytokines are ranked by fold change overexpression, where Mean - Base = Delta.

Table 6. Levels of cytokines of SDEC cell condition media as compared to based media

(BASE).

# Cytokine Mean SE Base Delta Fold change

1 GM-CSF 43,041.7 7,309.1 17.2 43,024.5 2501.6388

2 IL-6 60,008.0 4, 1 12.3 44.1 59,963.9 1360.2203

3 GCP-2 1 ,917.9 319.4 1 .8 1 ,916.1 1086.8272

4 MCP-3 57,818.0 4,380.7 101.0 57,717.0 572.3047

5 Osteoprotegerin 8,522.2 2,135.0 17.4 8,504.8 489.99085

6 IL-lbeta 19,41 1.3 4,689.2 41.9 19,369.4 463.16021

7 Angiogenin 18,431.3 2,939.5 52.5 18,378.8 351.0817

8 HGF 15,202.0 3,242.8 43.5 15,158.6 349.62149

9 MCP-1 60,031.1 4,107.3 203.8 59,827.3 294.53323

10 MIP-3-alpha 22,656.4 6,140.7 88.7 22,567.8 255.50264

1 1 MCP-2 2,582.2 1,728.9 1 1.0 2,571.2 234.12453

12 RANTES 10,709.8 6,350.1 48.5 10,661.3 220.69309

13 GRO 62,918.5 5,310.0 301.5 62,617.0 208.70464

14 GRO-alpha 39,726.3 4,602.0 190.7 39,535.6 208.27626

15 MMP-1 16,175.5 381.1 81.0 16,094.4 199.57868

16 IGFBP-2 5,997.7 254.9 36.2 5,961.5 165.79459

17 TIMP-2 13,531.0 418.2 85.2 13,445.7 158.77004

18 Activin A 13,769.8 1 ,063.8 139.7 13,630.1 98.589859

19 IGFBP-4 1 19.7 26.2 1.3 1 18.3 90.418779

20 LAP 30,831.4 663.6 379.2 30,452.1 81.296999

21 IL-8 14,281.9 660.4 226.7 14,055.2 63.003621

22 IGFBP- 1 2,356.8 749.8 53.8 2,303.0 43.789313

23 ENA-78 9,951.8 696.1 233.6 9,718.1 42.594438

24 VEGF 1 ,831.8 219.6 46.4 1,785.4 39.495633

25 EGF 1 ,349.1 1 ,082.4 36.2 1 ,312.9 37.293662

26 1-309 229.1 149.9 7.5 221.6 30.549123

27 IL-1 alpha 3,083.3 231.6 133.7 2,949.6 23.066175

28 uPAR 2,216.3 519.1 99.7 2,116.6 22.225762 Table 6. Levels of cytokines of SDEC cell condition media as compared to based media (BASE).

# Cytokine Mean SE Base Delta Fold change

29 PDGF AA 750.6 279.7 37.2 713.4 20.171672

30 IL-2 Ralpha 1 ,342.6 364.5 71.9 1 ,270.8 18.680634

31 IGFBP-6 2,668.5 552.1 158.3 2,510.2 16.860262

32 Eotaxin 517.9 263.7 34.4 483.5 15.051053

33 PDGF-BB 1 11.5 30.6 7.9 103.5 14.039654

34 IL-1 1 505.2 145.0 37.7 467.5 13.406026

35 LIF 2,795.0 894.2 219.2 2,575.9 12.751928

36 FGF-7 460.9 141.3 39.3 421.6 1 1.737907

37 GCSF 1,477.6 546.6 129.3 1,348.3 1 1.429215

38 MIP-1 alpha 305.2 124.2 35.4 269.9 8.6310877

39 PIGF 229.5 23.2 26.7 202.8 8.6048237

40 Angiopoietin-2 334.1 137.5 47.5 286.6 7.0287539

41 ICAM-3 193.5 103.8 29.0 164.6 6.6766687

42 TECK 71.1 19.9 1 1.6 59.5 6.1307521

43 M-CSF 32.0 3.9 5.3 26.7 6.0504671

44 TGF-beta 3 20.8 2.6 3.5 17.3 5.8986835

45 sgpl 30 685.4 105.4 1 17.7 567.7 5.8239589

46 Leptin 10.1 2.7 1.8 8.3 5.7290843

47 MIF 364.7 59.8 69.6 295.1 5.2421424

48 CCL-28 87.1 18.9 19.1 68.0 4.5524156

49 MIP-1 -delta 3.6 2.3 0.9 2.7 4.1 1 13191

50 MCP-4 15.9 2.1 4.0 1 1.9 3.9995199

51 BTC 39.4 22.0 1 1.6 27.8 3.3942909

52 Endoglin 121.3 57.6 36.7 84.6 3.3052443

53 IL-10 97.1 48.7 30.0 67.1 3.2372388

54 TIMP-1 2,977.7 94.2 961.2 2,016.5 3.097803

55 IGFBP-3 70.9 25.8 23.2 47.7 3.0568093

56 sTNF-RI 530.2 47.8 176.8 353.4 2.9984428

57 Axl 161.6 14.6 53.9 107.7 2.9967293

58 Amphiregulin 246.2 25.5 84.1 162.1 2.9283079

59 TIMP-4 180.8 21.2 62.2 1 18.6 2.9069487

60 IGF-I SR 99.9 31.9 36.5 63.4 2.7356312

61 Thrombopoietin 78.7 20.1 29.0 49.8 2.7166467

62 GITR-Ligand 137.6 59.6 51.0 86.6 2.6980082

63 GDNF 281.5 108.2 107.2 174.3 2.6257046

64 VEGF-D 47.4 17.8 18.6 28.9 2.5569208

65 Lymphotactin 103.3 15.5 41.7 61.6 2.4748777

66 ICAM-2 2,880.3 1 ,368.0 1 , 165.3 1 ,715.0 2.4717877

67 TRAIL R3 1 ,791.5 358.0 760.1 1 ,031.5 2.3570779

68 SCF 131.7 1 1.6 61.3 70.4 2.148229

69 IL-7 64.8 1 1.1 31.3 33.5 2.0685518 Table 6. Levels of cytokines of SDEC cell condition media as compared to based media (BASE).

# Cytokine Mean SE Base Delta Fold change

70 Acrp30 68.3 27.5 36.5 31.8 1.8703326

71 IL-2 Rapha 109.5 12.6 58.6 51.0 1.870166

72 MIP-l beta 123.5 30.2 67.3 56.3 1.836953

73 TGF-beta 1 264.4 22.0 154.4 1 10.0 1.712335

74 ALCAM 1 ,868.5 384.7 1 ,149.5 719.0 1 .6255537

75 BDNF 477.2 94.4 307.9 169.3 1.5496804

76 TRAIL R4 37.9 12.1 24.9 13.0 1.522222

77 IL- 12 p40 65.9 22.8 43.5 22.4 1.5146404

78 PDGF Ralpha 43.1 3.0 28.5 14.5 1.5087637

79 SDF-1 43.1 3.0 28.7 14.4 1.5038985

80 b-NGF 99.8 29.3 66.7 33.2 1.4972152

81 IL-lra 48.9 9.7 32.6 16.2 1.4964147

82 IL-21R 37.8 4.2 25.5 12.3 1.4840065

83 FGF-4 85.5 9.8 58.6 26.9 1.4602337

84 IL-3 129.0 10.1 88.7 40.3 1.4546138

85 HCC-4 46.5 7.9 32.5 14.0 1.43171 1 1

86 ICAM-1 150.5 61.2 105.5 45.0 1.426085

87 NT-4 185.1 31.3 131.0 54.1 1.412538

88 Fas Ligand 389.9 28.8 277.3 1 12.6 1.4060521

89 PARC 466.2 200.4 335.3 130.9 1.3903822

90 IGF-I 127.0 6.4 94.9 32.2 1.3393829

91 MDC 43.1 5.1 32.2 10.9 1.3386023

92 PECAM-1 128.2 6.0 96.3 31.8 1.3302112

93 sTNF RII 566.2 1 10.7 430.8 135.5 1.3145273

94 IL-1 Rl 54.1 10.1 41.2 12.9 1.3141 147

95 Eotaxin-2 28.1 3.3 21.6 6.4 1.2978867

96 IL-6 R 123.5 24.5 96.8 26.7 1.2758824

97 CD14 1 ,839.1 221.1 1 ,464.5 374.7 1.2558451

98 L-Selectin 683.2 392.7 544.9 138.3 1.2538026

99 Fas/TNFRSF6 158.4 1 1.4 127.5 30.8 1.2418018

100 IL-1 R4/ST2 95.0 16.2 76.5 18.5 1.2414167

101 IL-17 93.4 9.6 76.5 16.9 1.2207316

102 Flt-3 Ligand 419.7 72.5 351.2 68.6 1.1952925

103 IL-5 Ralpha 100.1 16.2 84.1 16.0 1.1900035

104 IL-15 108.9 8.1 92.2 16.7 1.1815919

105 BMP-5 231.6 9.7 196.8 34.9 1.1771659

106 IL-13 96.3 7.8 82.1 14.3 1.1738654

107 I-TAC 45.5 2.2 38.8 6.6 1.1708846

108 B7-1(CD80) 149.4 10.6 129.0 20.5 1.1587251

109 FGF-6 19.4 1.9 16.8 2.6 1.1551696

1 10 DR6 (TNFRSF21) 125.0 17.6 109.1 16.0 1.1462955 Table 6. Levels of cytokines of SDEC cell condition media as compared to based media (BASE).

# Cytokine Mean SE Base Delta Fold change

111 TGF-alpha 73.9 6.8 64.7 9.1 1.1413406

1 12 PDGF-AB 191.9 7.2 171.8 20.1 1.1 170098

113 PDGF Rbeta 174.4 7.1 156.5 17.9 1.1142277

114 NT-3 58.8 6.5 53.8 5.0 1.0923937

1 15 TNF-alpha 120.5 7.7 1 12.9 7.6 1.0672603

1 16 MIG 91.3 7.4 85.6 5.7 1.0671446

1 17 IL-5 34.4 3.4 32.6 1.8 1.0539804

1 18 Cardiotrophin-1 107.5 17.7 102.5 5.0 1.0490257

1 19 IL-18 Rbeta 33.0 3.1 31.6 1.4 1.0439006

120 E-Selectin 289.3 27.9 277.8 1 1.5 1.0413126

121 Leptin R 200.3 8.1 193.7 6.6 1.0339309

122 CNTF 251.1 22.9 243.1 8.1 1.0331373

123 IFN-gamma 49.5 4.8 48.1 1.4 1.0291304

124 BLC 68.5 5.1 66.6 1.9 1.0279818

125 MMP-13 96.2 3.5 95.3 0.9 1.0096567

126 IL-2 1 13.1 1 1.8 1 13.4 -0.3 0.997735

127 Eotaxin-3 69.9 3.0 70.6 -0.6 0.9909349

128 Fractalkine 52.8 0.6 53.8 -1.0 0.9815873

129 CXCL- 16 193.7 33.7 198.3 -4.6 0.9767709

130 MSP-alpha 106.9 12.2 109.6 -2.7 0.9755838

131 MIP-3beta 85.3 13.6 87.5 -2.3 0.9741714

132 AgRP 219.9 47.6 226.7 -6.8 0.9700124

133 VEGF R2 252.4 31.4 261.0 -8.6 0.9669459

134 FGF-9 94.6 1 1.1 99.1 -4.5 0.9542185

135 NGF R 140.3 0.9 147.3 -7.0 0.952317

136 ErbB3 161.1 9.7 170.8 -9.6 0.9436615

137 LIGHT 63.8 9.8 67.9 -4.1 0.9395812

138 IL-9 222.9 20.2 240.1 - 17.2 0.9282202

139 Oncostatin 177.8 14.3 191.9 -14.1 0.9264947

140 TNF-beta 1 18.1 5.8 127.5 -9.4 0.9263465

141 VEGF R3 85.2 4.7 92.3 -7.1 0.9231564

142 IGF-II 14,786.1 1 ,766.4 16,039.9 -1 ,253.7 0.9218376

143 Tie-2 41.3 2.5 44.9 -3.5 0.9209905

144 SDF-lbeta 57.5 1.6 62.7 -5.2 0.9171889

145 TGF beta2 139.0 9.3 151.9 -12.9 0.9151532

146 IL-2 Rgamma 87.1 0.9 95.3 -8.3 0.9133667

147 BMP-7 107.2 4.6 1 19.3 -12.1 0.8988336

148 Tie-1 166.0 3.6 185.0 -19.0 0.8971863

149 CTACK 56.9 40.5 63.8 -6.9 0.8916213

150 EGF-R 1 ,125.8 402.2 1 ,266.8 -140.9 0.8887405

151 MMP-9 32.7 2.3 37.2 -4.5 0.8789951 Table 6. Levels of cytokines of SDEC cell condition media as compared to based media

(BASE).

# Cytokine Mean SE Base Delta Fold change

152 GITR 107.1 2.3 123.5 -16.4 0.8675678

153 IL-10 Rbeta 82.3 9.0 96.8 -14.5 0.8499417

154 Prolactin 23.8 3.8 28.0 -4.3 0.847943

155 BMP-4 32.1 4.5 38.4 -6.3 0.8358318

156 IP- 10 219.2 14.5 262.5 -43.3 0.8348837

157 IL-2 Rbeta 68.3 1.9 82.1 - 13.8 0.8320383

158 BMP-6 62.1 9.0 75.0 -12.9 0.8276388

159 IL-18 BPalpha 181.7 7.2 221.7 -40.1 0.8192651

160 M-CSF R 86.1 2.1 105.5 -19.4 0.8161 1 15

161 MPIF-1 215.7 13.2 266.1 -50.4 0.8106182

162 IL-1 R II 191.6 7.6 239.1 -47.5 0.8013421

163 Siglec-5 1 12.2 23.5 144.8 -32.6 0.7750023

164 IL-16 23.1 4.1 30.4 -7.3 0.7600337

165 Dtk 212.9 47.7 284.7 -71.8 0.7478833

166 CK beta 8-1 3.9 0.8 5.3 -1.4 0.7284177

167 IL-12 p70 104.0 15.1 143.2 -39.2 0.7263316

168 IL-13 Ralpha2 190.1 9.4 266.1 -75.9 0.7145832

169 TARC 22.8 1.4 32.2 -9.4 0.7065771

170 SCF R 1,508.6 719.2 2,715.4 -1,206.8 0.5555617

171 IL-4 17.0 2.7 32.6 -15.6 0.5213657

172 NAP-2 44.3 2.8 143.4 -99.0 0.3092128

173 VE-Cadherin 10.3 0.5 43.3 -33.0 0.2373047

174 bFGF 7,889.8 4,984.8 41 ,525.9 -33,636.1 0.1899972

[0131] Table 7 shows array data of cytokines tested (SDEC conditioned media internal control normalization without background), where data from three experiments are shown (10_27, 10_20, and 9_2). The most left column shows array number as 6, 7, or 8.

Table 7. Array data for cytokines tested in SDEC condition media as compared to base media

A # Cytokine 10 27 10 20 9_2 Mean SE Base Delta

6 Angiogenin 17,271.5 14,019.9 24,002.4 18,431.3 2,939.5 52.5 18,378.8

6 BDNF 630.0 304.8 496.8 477.2 94.4 307.9 169.3

6 BLC 78.5 61.6 65.3 68.5 5.1 66.6 1.9

6 BMP-4 33.0 23.8 39.4 32.1 4.5 38.4 -6.3

6 BMP-6 79.5 57.2 49.5 62.1 9.0 75.0 -12.9

6 CK beta 8-1 4.0 5.2 2.4 3.9 0.8 5.3 -1.4

6 CNTF 297.0 227.7 228.7 251.1 22.9 243.1 8.1

6 EGF 60.0 3,499.7 487.6 1,349.1 1 ,082.4 36.2 1 ,312.9

6 Eotaxin 974.0 60,4 519.3 517.9 263.7 34.4 483.5 Table 7. Array data for cytokines tested in SDEC condition media as compared to base media

A # Cytokine 10 27 10_20 9 2 Mean SE Base Delta

6 Eotaxin-2 31.0 21.5 31.7 28.1 3.3 21.6 6.4

6 Eotaxin-3 64.5 74.7 70.6 69.9 3.0 70.6 -0.6

6 FGF-6 23.0 18.3 16.8 19.4 1.9 16.8 2.6

6 FGF-7 562.5 181.6 638.5 460.9 141.3 39.3 421.6

6 Flt-3 Ligand 496.0 488.4 274.8 419.7 72.5 351.2 68.6

6 Fractalkine 53.5 51.7 53.3 52.8 0.6 53.8 - 1.0

6 GCP-2 2,130.0 2,333.7 1 ,290.0 1 ,917.9 319.4 1.8 1,916.1

6 GDNF 497.5 162.9 184.0 281.5 108.2 107.2 174.3

6 GM-CSF 56,903.5 32,091.5 40,130.1 43,041.7 7,309.1 17.2 43,024.5

6 1-309 156.0 13.9 517.4 229.1 149.9 7.5 221.6

6 IFN-gamma 57.0 40.5 50.9 49.5 4.8 48.1 1.4

6 IGFBP-1 3,038.0 859.2 3,173.3 2,356.8 749.8 53.8 2,303.0

6 IGFBP-2 6,025.0 5,543.3 6,424.8 5,997.7 254.9 36.2 5,961.5

6 IGFBP-4 170.0 81.9 107.1 1 19.7 26.2 1.3 1 18.3

6 IGF-I 120.5 120.8 139.8 127.0 6.4 94.9 32.2

6 IL-10 194.5 49.3 47.6 97.1 48.7 30.0 67.1

6 IL-13 105.0 80.7 103.3 96.3 7.8 82.1 14.3

6 IL-15 119.5 93.0 114.3 108.9 8.1 92.2 16.7

6 IL-16 19.5 18.7 31.2 23.1 4.1 30.4 -7.3

6 IL-1 alpha 3,511.0 2,715.6 3,023.4 3,083.3 231.6 133.7 2,949.6

6 IL-lbeta 26,854.5 10,748.7 20,630.7 19,411.3 4,689.2 41.9 19,369.4

6 IL-lra 61.5 29.8 55.2 48.9 9.7 32.6 16.2

6 IL-2 128.0 89.8 121.5 1 13.1 1 1.8 1 13.4 -0.3

6 IL-3 149.0 121.2 1 16.7 129.0 10.1 88.7 40.3

6 IL-4 18.0 1 1.9 21.1 17.0 2.7 32.6 -15.6

6 IL-5 40.5 28.6 34.1 34.4 3.4 32.6 1.8

6 IL-6 65,341.5 51 ,919.3 62,763.3 60,008.0 4, 1 12.3 44.1 59,963.9

6 IL-7 87.0 54.0 53.3 64.8 1 1.1 31.3 33.5

6 Leptin 14.0 1 1.5 4.8 10.1 2.7 1.8 8.3

6 LIGHT 79.5 45.7 66.3 63.8 9.8 67.9 -4.1

6 MCP-1 65,357.5 51,951.9 62,783.9 60,031.1 4,107.3 203.8 59,827.3

6 MCP-2 5,995.0 393.8 1,357.7 2,582.2 1,728.9 1 1.0 2,571.2

6 MCP-3 65,347.5 50,173.4 57,933.0 57,818.0 4,380.7 101.0 57,717.0

6 MCP-4 16.5 1 1.9 19.2 15.9 2.1 4.0 1 1.9

6 M-CSF 28.0 28.2 39.9 32.0 3.9 5.3 26.7

6 MDC 45.5 33.4 50.4 43.1 5.1 32.2 10.9

6 MIG 106.0 85.8 82.2 91.3 7.4 85.6 5.7

6 MIP-1 -delta 8.0 0.0 2.9 3.6 2.3 0.9 2.7

6 MIP-3-alpha 29,476.0 10,401.0 28,092.3 22,656.4 6,140.7 88.7 22,567.8

6 NAP-2 46.0 48.1 38.9 44.3 2.8 143.4 -99.0

6 NT-3 56.0 49.3 71.1 58.8 6.5 53.8 5.0

6 PARC 597.5 728.5 72.5 466.2 200.4 335.3 130.9 Table 7. Array data for cytokines tested in SDEC condition media as compared to base media

A # Cytokine 10_27 10 20 9_2 Mean SE Base Delta

6 PDGF-BB 127.5 154.6 52.4 1 1 1.5 30.6 7.9 103.5

6 RANTES 7,755.5 1 ,489.9 22,884.0 10,709.8 6,350.1 48.5 10,661.3

6 SCF 146.5 108.9 139.8 131.7 1 1.6 61.3 70.4

6 SDF-1 48.5 42.9 38.0 43.1 3.0 28.7 14.4

6 TARC 25.5 20.7 22.1 22.8 1.4 32.2 -9.4

6 TGF-beta 1 267.5 300.8 224.8 264.4 22.0 154.4 110.0

6 TGF-beta 3 26.0 18.7 17.8 20.8 2.6 3.5 17.3

6 TNF-alpha 132.5 106.1 123.0 120.5 7.7 1 12.9 7.6

6 TNF-beta 128.5 1 17.2 108.6 1 18.1 5.8 127.5 -9.4

7 Acrp30 47.0 35.0 123.0 68.3 27.5 36.5 31.8

7 AgRP 172.5 172.0 315.2 219.9 47.6 226.7 -6.8

7 Angiopoietin-2 165.0 230.9 606.5 334.1 137.5 47.5 286.6

7 Amphiregulin 196.5 260.6 281.4 246.2 25.5 84.1 162.1

7 Axl 144.5 190.7 149.6 161.6 14.6 53.9 107.7

7 bFGF 3,869.0 17,800.8 1 ,999.6 7,889.8 4,984.8 41 ,525.9 -33,636.1

7 b-NGF 73.5 67.5 158.4 99.8 29.3 66.7 33.2

7 BTC 25.0 10.5 82.5 39.4 22.0 1 1.6 27.8

7 CCL-28 62.0 75.2 124.1 87.1 18.9 19.1 68.0

7 CTACK 28.5 5.3 136.8 56.9 40.5 63.8 -6.9

7 Dtk 234.5 121.7 282.5 212.9 47.7 284.7 -71.8

7 EGF-R 1,256.0 1 ,748.1 373.3 1,125.8 402.2 1 ,266.8 -140.9

7 ENA-78 10,604.5 8,560.5 10,690.3 9,951.8 696.1 233.6 9,718.1

7 Fas/TNFRSF6 146.0 148.0 181.1 158.4 1 1.4 127.5 30.8

7 FGF-4 84.5 69.0 103.0 85.5 9.8 58. 26.9

7 FGF-9 94.0 75.7 114.1 94.6 1 1.1 99.1 -4.5

7 GCSF 2,385.0 495.8 1,552.0 1,477.6 546.6 129.3 1,348.3

7 GITR-Ligand 87.5 69.0 256.5 137.6 59.6 51.0 86.6

7 GITR 102.5 109.2 109.7 107.1 2.3 123.5 -16.4

7 GRO 53,877.5 62,613.6 72,264.2 62,918.5 5,310.0 301.5 62,617.0

7 GRO-alpha 47,128.0 31 ,287.7 40,763.3 39,726.3 4,602.0 190.7 39,535.6

7 HCC-4 41.0 36.4 62.0 46.5 7.9 32.5 14.0

7 HGF 13,439.5 10,678.0 21 ,488.6 15,202.0 3,242.8 43.5 15,158.6

7 ICAM-1 96.5 82.4 272.5 150.5 61.2 105.5 45.0

7 ICAM-3 103.5 76.7 400.5 193.5 103.8 29.0 164.6

7 IGFBP-3 52.0 38.8 121.9 70.9 25.8 23.2 47.7

7 IGFBP-6 2,664.0 1,714.6 3,626.9 2,668.5 552.1 158.3 2,510.2

7 IGF-I SR 77.5 59.4 162.8 99.9 31.9 36.5 63.4

7 IL- 1 R4/ST2 80.0 77.6 127.4 95.0 16.2 76.5 18.5

7 IL-1 RI 43.5 44.6 74.2 54.1 10.1 41.2 12.9

7 IL-1 1 478.0 268.8 768.8 505.2 145.0 37.7 467.5

7 IL-12 p40 46.0 40.2 1 1 1.3 65.9 22.8 43.5 22.4

7 IL-12 p70 77.5 104.9 129.6 104.0 15.1 143.2 -39.2 Table 7. Array data for cytokines tested in SDEC condition media as compared to base media

A # Cytokine 10 27 10 20 9 2 Mean SE Base Delta

7 IL-17 96.0 75.7 108.6 93.4 9.6 76.5 16.9

7 IL-2 Rapha 106.0 89.6 132.9 109.5 12.6 58.6 51.0

7 1L-6 R 87.0 1 13.5 170.0 123.5 24.5 96.8 26.7

7 IL-8 14,197.5 13, 182.6 15,465.5 14,281.9 660.4 226.7 14,055.2

7 I-TAC 46.5 41.2 48.7 45.5 2.2 38.8 6.6

7 Lymphotactin 96.5 80.5 132.9 103.3 15.5 41.7 61.6

7 MIF 381.0 253.9 459.2 364.7 59.8 69.6 295.1

7 MIP-1 alpha 373.5 64.2 478.0 305.2 124.2 35.4 269.9

7 MIP-lbeta 1 18.0 74.3 178.4 123.5 30.2 67.3 56.3

7 MIP-3beta 72.5 70.9 1 12.4 85.3 13.6 87.5 -2.3

7 MSP-alpha 96.0 93.4 131.3 106.9 12.2 109.6 -2.7

7 NT-4 150.5 157.1 247.6 185.1 31.3 131.0 54.1

7 Osteoprotegerin 8,252.5 4,966.5 12,347.6 8,522.2 2, 135.0 17.4 8,504.8

7 Oncostatin M 198.5 150.4 184.4 177.8 14.3 191.9 -14.1

7 P1GF 221.5 273.1 193.9 229.5 23.2 26.7 202.8

7 sgpl 30 529.5 640.5 886.2 685.4 105.4 1 17.7 567.7

7 sTNF RII 707.0 643.9 347.9 566.2 1 10.7 430.8 135.5

7 sTNF-RI 563.5 591.2 435.9 530.2 47.8 176.8 353.4

7 TECK 58.0 45.0 1 10.2 71.1 19.9 1 1.6 59.5

7 TIMP-1 3,1 19.0 2,799.2 3,014.9 2,977.7 94.2 961.2 2,016.5

7 TIMP-2 12,787.5 13,570.6 14,234.7 13,531.0 418.2 85.2 13,445.7

7 Thrombopoietin 72.5 47.4 1 16.3 78.7 20.1 29.0 49.8

7 TRAIL R3 2,046.5 2,243.5 1,084.5 1 ,791.5 358.0 760.1 1 ,031.5

7 TRAIL R4 24.5 27.3 62.0 37.9 12.1 24.9 13.0

7 uPAR 1,790.5 1 ,609.2 3,249.2 2,216.3 519.1 99.7 2,1 16.6

7 VEGF 1 ,609.5 1 ,614.9 2,271.0 1,831.8 219.6 46.4 1 ,785.4

7 VEGF-D 30.0 29.2 83.1 47.4 17.8 18.6 28.9

8 Activin A 14,721.5 11,646.1 14,941.8 13,769.8 1,063.8 139.7 13,630.1

8 ALCAM 2,451.5 2,011.9 1,142.1 1,868.5 384.7 1,149.5 719.0

8 B7-1(CD80) 148.0 168.4 131.9 149.4 10.6 129.0 20.5

8 BMP-5 250.0 216.9 227.9 231.6 9.7 196.8 34.9

8 BMP-7 1 14.0 109.2 98.5 107.2 4.6 1 19.3 -12.1

8 Cardiotro-phin- 1 141.0 100.4 81.0 107.5 17.7 102.5 5.0

8 CD14 2,242.5 1 ,480.5 1 ,794.4 1,839.1 221.1 1 ,464.5 374.7

8 CXCL- 16 206.5 244.6 130.0 193.7 33.7 198.3 -4.6

8 DR6 1 19.0 98.0 158.1 125.0 17.6 109.1 16.0

(TNFRSF21 )

8 Endoglin 34.0 230.0 99.9 121.3 57.6 36.7 84.6

8 ErbB3 171.5 141.7 170.2 161.1 9.7 170.8 -9.6

8 E-Selectin 341.0 281.4 245.4 289.3 27.9 277.8 1 1.5

8 Fas Ligand 425.0 412.0 332.7 389.9 28.8 277.3 1 12.6

8 ICAM-2 4,954.0 3,389.0 297.8 2,880.3 1,368.0 1,165.3 1 ,715.0 Table 7. Array data for cytokines tested in SDEC condition media as compared to base media

A# Cytokine 10_27 10 20 9_2 Mean SE Base Delta

8 IGF-II 17,824.0 14,828.9 11,705.5 14,786.1 1,766.4 16,039.9 -1,253.7

8 IL-1 RII 190.5 205.3 179.0 191.6 7.6 239.1 -47.5

8 IL-lORbeta 98.5 81.0 67.4 82.3 9.0 96.8 -14.5

8 IL-13 209.0 181.0 180.4 190.1 9.4 266.1 -75.9 Ralpha2

8 IL-18 185.5 191.7 167.8 181.7 7.2 221.7 -40.1 BPalpha

8 IL-18Rbeta 33.0 27.7 38.3 33.0 3.1 31.6 1.4

8 IL-2 Ralpha 1,981.0 1,328.1 718.7 1,342.6 364.5 71.9 1,270.8

8 IL-2 Rbeta 69.5 70.8 64.5 68.3 1.9 82.1 -13.8

8 IL-2 88.0 87.8 85.4 87.1 0.9 95.3 -8.3

Rgamma

8 IL-21R 31.0 36.9 45.6 37.8 4.2 25.5 12.3

8 IL-5 Ralpha 81.0 86.9 132.4 100.1 16.2 84.1 16.0

8 IL-9 263.0 199.0 206.6 222.9 20.2 240.1 -17.2

8 IP- 10 233.5 233.9 190.1 219.2 14.5 262.5 -43.3

8 LAP 31,036.5 29,593.3 31,864.3 30,831.4 663.6 379.2 30,452.1

8 Leptin R 187.5 198.0 215.3 200.3 8.1 193.7 6.6

8 LIF 2,747.0 1,270.9 4,367.3 2,795.0 894.2 219.2 2,575.9

8 L-Selectin 1,447.5 457.6 144.5 683.2 392.7 544.9 138.3

8 M-CSF R 90.0 85.4 82.9 86.1 2.1 105.5 -19.4

8 MMP-1 16,746.0 16,327.9 15,452.5 16,175.5 381.1 81.0 16,094.4

8 MMP-13 100.5 89.3 98.9 96.2 3.5 95.3 0.9

8 MMP-9 37.0 29.1 32.0 32.7 2.3 37.2 -4.5

8 MPIF-1 242.0 200.9 204.2 215.7 13.2 266.1 -50.4

8 NGFR 139.5 139.3 142.1 140.3 0.9 147.3 -7.0

8 PDGF AA 191.5 1,045.7 1,014.6 750.6 279.7 37.2 713.4

8 PDGF-AB 205.5 181.0 189.1 191.9 7.2 171.8 20.1

8 PDGF 38.0 48.5 42.7 43.1 3.0 28.5 14.5 Ralpha

8 PDGF Rbeta 184.0 160.6 178.5 174.4 7.1 156.5 17.9

8 PECAM-1 138.5 128.1 117.9 128.2 6.0 96.3 31.8

8 Prolactin 16.5 25.7 29.1 23.8 3.8 28.0 -4.3

8 SCFR 2,212.0 2,243.3 70.3 1,508.6 719.2 2,715.4 -1,206.8

8 SDF-lbeta 59.0 54.3 59.2 57.5 1.6 62.7 -5.2

8 Siglec-5 143.0 127.6 66.0 112.2 23.5 144.8 -32.6

8 TGF-alpha 64.0 70.8 86.8 73.9 6.8 64.7 9.1

8 TGF beta2 157.5 130.5 129.0 139.0 9.3 151.9 -12.9

8 Tie-1 173.0 164.0 161.0 166.0 3.6 185.0 -19.0

8 Tie-2 40.5 37.4 46.1 41.3 2.5 44.9 -3.5

8 TIMP-4 146.0 177.1 219.2 180.8 21.2 62.2 118.6

8 VE-Cadherin 9.5 11.2 10.2 10.3 0.5 43.3 -33.0 Table 7. Array data for cytokines tested in SDEC condition media as compared to base media

A # Cytokine 10 27 10 20 9 2 Mean SE Base Delta

8 VEGF R2 302.5 260.1 194.5 252.4 31.4 261.0 -8.6

8 VEGF R3 93.0 85.9 76.6 85.2 4.7 92.3 -7.1

[0132] Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. A composition comprising:

a first conditioned medium (CM) with a reduced level of secreted frizzled-related protein (sFRP-1) as compared to a second conditioned medium from fetal foreskin fibroblast cells and/or fetal lung fibroblast cells, wherein the first CM is from cells of human origin.

2. The composition of claim 1 , wherein the first conditioned medium (CM) has an

elevated level of prostaglandin E2 (PGE2) and/or 6-keto-prostaglandin F l a (6-k- PGF 1 a) as compared to a second conditioned medium from fetal foreskin fibroblast cells and/or fetal lung fibroblast cells, or a based medium.

3. The composition of claim 1 , wherein the fetal foreskin fibroblast cells comprises human fetal foreskin fibroblast line Detroit 551 .

4. The composition of claim 1 , wherein the fetal lung fibroblast cells comprises human fetal lung fibroblast line WI38.

5. The composition of claim 1 , wherein the first conditioned medium (CM) is derived from human SDEC cells.

6. The composition of claim 1 , wherein the sFRP-1 level of the second CM is at least 4 times greater than the sFRP- 1 level of the first CM.

7. The composition of claim 1 , wherein the sFRP-1 level of the first CM is less than 17 ng/L.

8. The composition of claim 2, wherein the PGE2 level of the first CM is at least 7 times greater than the PGE2 level of the second CM.

9. The composition of claim 2, wherein the PGE2 level of the first CM is more than 20 ng/mL.

10. The composition of claim 2, wherein the 6-k-PGF l level of the first CM is at least 30 times greater than the 6-k-PGFla level of the second CM.

1 1. The composition of claim 2, wherein the 6-k-PGFl a level of the first CM is more than 40 ng/mL.

12. A method to enhance human embryonic stem cells (hESCs) growth on Type I collagen (COL I) comprising:

contacting the hESCs with a first conditioned medium (CM) with a reduced level of secreted frizzled-related protein (sFRP-1) as compared to a second conditioned medium from fetal foreskin fibroblast cells and/or fetal lung fibroblast cells, wherein the first CM is from cells of human origin.

13. The method of claim 12, wherein the first conditioned medium (CM) has an elevated level of prostaglandin E2 (PGE2) and/or 6-keto-prostaglandin Fla (6-k-PGF la) as compared to a second conditioned medium from fetal foreskin fibroblast cells and/or fetal lung fibroblast cells.

14. The method of claim 12, wherein the fetal foreskin fibroblast cells comprises human fetal foreskin fibroblast line Detroit 551.

15. The method of claim 12, wherein the fetal lung fibroblast cells comprises human fetal lung fibroblast line W138.

16. The method of claim 12, wherein the first conditioned medium (CM) is derived from human SDEC cells.

17. The method of claim 12, wherein the sFRP-1 level of the second CM is at least 4 times greater than the sFRP-1 level of the first CM.

18. The method of claim 13, wherein the PGE2 level of the first CM is at least 7 times greater than the PGE2 level of the second CM.

19. The method of claim 13, wherein the 6-k-PGFl a level of the first CM is at least 30 times greater than the 6-k-PGFla level of the second CM.

20. A method to reduce secreted frizzled-related protein (sFRP-1) in a conditioned medium for supporting hESC growth comprising:

silencing gene expression of sFRP-1 using a nucleic acid agent comprising a siRNA, a miRNA, or an antisense nucleic acid; or

capturing sFRP-1 protein in the condition medium using an agent specifically binds to the sFRP- 1 protein.

21 . The method of claim 20, wherein the agent specifically binds to the sFRP-1 protein comprises an antibody specifically binds to the sFRP-1 protein.

22. The method of claim 20, wherein the agent specifically binds to the sFRP-1 protein comprises an affinity chromatography comprising an antibody specifically binds to the sFRP-1 protein.

23. A method for screening cell lines for reduced level of secreted frizzled-related protein (sFRP-1) in conditioned medium comprising:

(a) measuring sFRP-1 protein level in a first conditioned medium from at least one cell line of human origin; and

(b) comparing the sFRP-1 protein level measured in step (a) to a second conditioned medium of cells selected from the group consisting of human SDEC cells, fetal foreskin fibroblast cells, and fetal lung fibroblast cells, thereby identifying a cell line with reduced level of sFRP-1.

24. A method for screening cell lines for reduced level of secreted frizzled-related protein (sFRP-1) in conditioned medium comprising:

(a) measuring sFRP- 1 mRNA level in at least one cell line of human origin; and

(b) comparing the sFRP-1 mRNA level measured in step (a) to sFRP-1 mRNA levels selected from the group consisting of human SDEC cells, fetal foreskin fibroblast cells, and fetal lung fibroblast cells, thereby identifying a cell line with reduced level of sFRP- 1.

25. The method of claim 24, wherein the sFRP-1 mRNA level is measured using

quantitative reverse transcription polymerase chain reaction (RT-QPCR or QRT-PCR) or microarray.

26. A method for screening test agents for reducing level of secreted frizzled-related protein (sFRP-1) in conditioned medium comprising:

(a) contacting at least one cell line of human origin with at least one test agent; and

(b) measuring sFRP-1 protein or mRNA levels in the at least one cell line of human origin before and after step (a), or measuring protein or mRNA levels of PTGS2 and/or PGIS in the at least one cell line of human origin before and after step (a), thereby identifying the test agent for reducing level of sFRP-1.

27. A method for screening cell lines for elevated expression of prostaglandin- endoperoxide synthase 2 (PTGS2) and/or prostaglandin 12 synthase (PGIS) comprising:

(a) measuring protein or mRNA levels of PTGS2 and/or PGIS in at least one cell line of human origin; and

(b) comparing the protein or mRNA levels of PTGS2 and/or PGIS measured in step (a) to protein or mRNA levels of PTGS2 and/or PGIS of human SDEC cells, fetal foreskin fibroblast cells, and/or fetal lung fibroblast cells, thereby identifying a cell line with elevated expression of PTGS2 and/or PGIS.

28. The method of claim 26 or 27, wherein the mRNA level is measured using quantitative reverse transcription polymerase chain reaction (RT-QPCR or QRT-PCR) or microarray.

29. The method of claim 26 or 27, wherein the protein level is measured using Western blotting or ELISA.

30. The method of claim 23, 24, or 27, wherein the fetal foreskin fibroblast cells comprises human fetal foreskin fibroblast line Detroit 551.

31. The method of claim 23, 24, or 27, wherein the fetal lung fibroblast cells comprises human fetal lung fibroblast line WI38.

32. The composition of claim 1 , wherein the first conditioned medium (CM) has an

elevated level of at least one cytokine as listed in Table 3, 4, or 5 as as compared to a second conditioned medium from fetal foreskin fibroblast cells and/or fetal lung fibroblast cells, or a based medium.

33. The composition of claim 32, wherein the elevated level of the at least one cytokine is at least 1 .5 fold, 3 fold, 6 fold, 20 fold, 100 fold, 500 fold, or 2000 fold.