WO2014146013A1 - Selective paraxial or lateral mesoderm differentiation of pluripotent stem cells - Google Patents

Abstract

The present disclosure provides for growth factors and cytokines that influence the outcome of paraxial mesoderm and paraxial mesoderm lineage differentiation from human pluripotent cells. As a part of this disclosure, an in vitro method is provided that promotes differentiation of human pluripotent cells into skeletal myocytes.

Description

SELECTIVE PARAXIAL OR LATERAL MESODERM DIFFERENTIATION

OF PLURIPOTENT STEM CELLS

CROSS REFERENCE TO RELATED APPLICATION

This application is a conversion of provisional application US 61/794,195 filed on March 15, 2013. This application is herein incorporated by reference.

BACKGROUND OF THE INVENTION

One of the major tasks of gastrulation is the formation of mesoderm. As the embryo elongates, specialized lateral plate and paraxial mesoderm subtypes become distinct. Lateral plate mesoderm eventually gives rise to cells of the heart, blood vessels, and blood cells of the circulatory system. Cells that form the axial and appendicular skeleton, skeletal muscles and other connective tissue components arise from the paraxial mesoderm. While a variety of markers exist that can be used to identify mesoderm subtypes and derivatives at various stages of development, little is known about the signaling factors that influence or induce paraxial mesoderm specialization events. The present invention was focused to identify these factors, knowledge of which has facilitated the development of in vitro strategies that uniquely allow for the selective induction of paraxial mesoderm from human pluripotent stem cells. As disclosed, paraxial mesoderm differentiation can be further optimized to significantly increase the proportion of one or more lineage specific cell types such as skeletal myocytes. It is anticipated that this work will ultimately provide unique access to an unlimited number of specialized progenitor populations and derivatives that may be highly beneficial for regenerative and pharmaceutical applications while enabling more in-depth investigations of disease states.

BRIEF SUMMARY OF THE INVENTION

During the earliest stages of embryonic development, events of gastrulation result in the production of the primary germ layers: mesoderm, endoderm and ectoderm. Germ layer mesoderm cells dissociate, migrate and compact into regions of more specialized lateral plate and paraxial mesoderm progenitors that eventually provide for cells of the myocardium, blood vessels and blood circulatory system, portions of the dermis, skeleton and skeletal muscle, and other connective tissue components such as fat and cartilage. Based on their capacity to differentiate into lineages from all three germ layers, pluripotent stem cells provide a unique in vitro opportunity to investigate fundamental processes of human development and disease. Human pluripotent stem cells (hPSC) could also serve as a potentially unlimited source of specialized progenitors and lineage specific cell types for regenerative and pharmaceutical applications.

Embryonic stem cells (ESC) are pluripotent cells derived from early embryos and characterized by their ability to proliferate indefinitely in vitro in an essentially undifferentiated state. Induced pluripotent stem cells (iPSC) are typically derived from non-pluripotent somatic cells via genetic reprogramming wherein fully reprogrammed iPSC are believed to be nearly identical to ESC in most respects including the expression of sternness genes, methylation patterns, proliferation rates, embryoid body (EB) and teratoma formation. Despite differences in their origins, hPSC types can be maintained under similar in vitro culture conditions. Upon removal of pluripotent factors that sustain the undifferentiated state, hPSC

spontaneously differentiate as embryoid bodies (EBs); however, due to the

uncontrolled nature and limited extent of gastrulation-like events that occur in the EB, the proportion of mesoderm lineages such as cardiac and skeletal myocytes derived from hPSC is generally low. Given the promise of hPSC as a renewable cell source for research and therapeutic purposes, development of protocols that would enable enrichment of specialized mesoderm progenitors and their derivatives from hPSC is of critical importance but this would ultimately require a better understanding of the signaling factors and conditions required for mesoderm and mesoderm lineage differentiation.

Paraxial mesoderm differentiation is not recapitulated in EBs and, prior to the filing of this application, little was known about the signaling factors that are required to increase the proportion of paraxial mesoderm progenitors and lineages in the hPSC system. To overcome these deficiencies, the present disclosure was initially focused to biochemically identify (via human antibody arrays and mass spectometry) and compare factor secretions of lateral versus more medial regions of various staged avian embryonic mesoderm explants. This enabled the identification of a subset of heretofore unknown factors that could be tested for their ability to differentiate paraxial cell types from hPSC. Despite the multitude of proteins contained in serum, the inventor hypothesized that a subset of serum factors may also be critical for paraxial mesoderm specialization, expansion and/or survival which could, in part, be identified as those significantly depleted from serum-containing explant conditioned medium. In one embodiment of the present invention, a unique collection of growth factors and cytokines that selectively influence the outcome of paraxial mesoderm and paraxial mesoderm lineage differentiation from hPSC is disclosed. In yet another embodiment, an in vitro method is provided that promotes skeletal myogenesis.

The present disclosure's enabling differentiation of paraxial mesoderm cell types from hPSC will assist in meeting the cellular demands for research, pharmaceutical and clinical applications. Other features and advantages of the present invention will become apparent after study of the specification and claims that follow. It should be understood, however, that the detailed description and the specific examples, while indicating preferred methods of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

As defined in the art, human "pluripotent stem cells" and "pluripotent cells" refer to human embryonic stem cells (e.g., hESC) or human pluripotent cells derived from non-pluripotent sources (e.g., iPSC). "Pluripotent cells" are capable of differentiating to one or more lineages derived from all three primary germ layers and it is anticipated that virtually any pluripotent stem cell line or clone that meets the defining criteria of pluripotency as established by the art may be used with the present invention.

For purposes of performing the disclosed methods, hPSC can be obtained from a variety of sources. For example, the International Stem Cell Registry provides a comprehensive, searchable database that includes current published and validated unpublished information on all known human pluripotent cell lines including human ESC and human iPSC lines. This includes cell lines approved by the National Institute of Health (NIH) for federal funding and those that were derived through other public or private funding sources such as non-profit institutions, academic centers, research enterprises, stem cell banks and industry based in the United States and abroad (for further information, see http://www.umassmed.edu/iscr/index.aspx). Use of alternative or non-registered hPSC does not depart from the spirit and scope of the present invention.

Terms describing "expansion of or "expanded" or "maintained" pluripotent cells refer to any method or condition used to support the growth and proliferation of hPSC in an essentially undifferentiated state without any restriction as to the mode of action. Under appropriate conditions, undifferentiated hPSC are routinely dissociated into individual cells or small clumps via enzymatic or manual methods, then re-plated into fresh expansion medium and allowed to proliferate as essentially undifferentiated cell population. A variety of methods and components for expansion and identification of undifferentiated pluripotent cells are known; therefore, it is appreciated that additional methods for the culture and maintenance of hPSC may be used with the present invention. It is also clearly understood that expanding colonies of essentially undifferentiated pluripotent cells can be surrounded by neighboring cells that are differentiated wherein the undifferentiated cell population persists. As noted, hPSC are often expanded in the presence of one or more matrix or substrate components that support the essentially undifferentiated state.

For purposes of the present invention, hPSC are expanded in serum- and feeder-free culture conditions that include fibroblast growth factor (FGF), wherein this preferred condition can be uniformly applied across hPSC lines and types.

As used herein, the terms "induction medium" or "differentiation medium" refer to any medium that induces, promotes or influences the differentiation of hPSC to paraxial mesoderm and one or more paraxial mesoderm lineages in the absence of feeder cells without any restriction as to the mode of action. In vitro differentiation requires exposing the hPSC to an environment that is, in at least one respect, distinct from the expansion condition(s). It is further appreciated that differentiation of hPSC is not necessarily an "all or nothing" event but encompasses a transition of cells to a non-pluripotent state. While the rate by which cells respond to the differentiation condition is known to vary from one hPSC line or type to another, which may be further dependent upon the concentration of one or more factors, there are no known overall differences in the mechanism(s) of hPSC differentiation. DETAILED DESCRIPTION OF THE INVENTION

In the preferred methods, mesoderm differentiation is initiated by exposing the expanded cells to FGF, and most preferably bFGF, for a time period of at least one day but most preferably for at least 3-4 days. In keeping within the spirit and scope of the present invention, exposure of cells to FGF, or one or more additional factors that may be present in the medium, can occur via any route including, for example, direct addition of a recombinant factor to the culture condition, manipulation of a cell or cell population that results in the forced expression or secretion of one or more factors or factor receptors, etc. Culture conditions that initiate differentiation can be further optimized using serum-free culture conditions that may comprise a culture medium composition similar to the culture medium composition used for expansion. To this end, hPSC are sensitive to changes in the overall culture environment that initiates the differentiation process. These changes include, but are not limited to, changes in the concentration (or complete removal) of a factor or factors (e.g., FGF) used to maintain pluripotency, changes in the composition of a medium, substrate or matrix component, and changes in the density or method by which cells are grown. Based on these and other considerations, it is expected that culture conditions for paraxial mesoderm and paraxial mesoderm lineage differentiation will be optimized for a particular hPSC type, line or clone, technique or end application. In certain aspects, differentiation of the hPSC may be initiated using feeder-free adherent or non-adherent cell culture conditions. Regardless of the type of culture (e.g., plating, suspension or rotary shaker), pluripotent as well as non-pluripotent cell types tend to form aggregates (i.e., formation of 3-D cell-cell contact) as a general survival mechanism. With regard to the present methods, aggregate formation in suspension culture is used to optimize for the differentiation and survival of mesoderm and paraxial progenitors. While aggregate formation can be initiated using completely dissociated cells, which may delay the timing of differentiation, the preferred methods comprise brief enzymatic dissociation of hPSC to generate small clumps of expanded cells. In certain embodiments, fibronectin is added to the system. Fibronectin is a preferred matrix component herein discovered to enhance paraxial mesoderm differentiation, cell viability and skeletal myogenesis, and can be introduced into the culture condition(s) via any route. Substitution or combination of fibronectin with one or more components such as, for example, another matrix component (e.g., vitronectin), biological or synthetic substrate, or other survival factor or compound does not depart from the scope and spirit of the present disclosure.

The concentration of factors used in the present disclosure may be optimized for a preferred hPSC line or type, culture condition, technique or end application. As presently disclosed, the concentration of a factor or factors can be used to adjust for the proportion of skeletal myocytes that differentiate from hPSC.

As used herein, "paraxial mesoderm associated factors," or "paraxial differentiation factors," refers to one or more factors, used alone or in combination, capable of inducing or influencing the outcome of hPSC differentiation into target paraxial mesoderm cell types. These factors include, but are not limited to, inducers, enhancers, expansion, maturation and survival factors, and functional equivalents of the same. Paraxial mesoderm-associated factors identified in the present invention include, but are not limited to, growth factors and cytokines (and their isoforms) such as Fibroblast Growth Factor (FGF), Insulin-like Growth Factor (IGF), Insulin-like Growth Factor-Binding Protein (IGFBP), Angiogenin (ANG), Angiopoietin

(ANGPT), Epidermal Growth Factor (EGF), Heparin Binding EGF (HB-EGF), Osteoblast Differentiation Promoting Factor, Colony Stimulating Factor (CSF), Tumor Necrosis Factor (TNF), TNF Inducing Protein, Growth Differentiation Factor (GDF), Interleukin (IL), Follistatin, Neurotrophin (NT), Matrix Metalloproteinase (MMP), and Leukemia Inhibitor Factor (LIF). Other factors such as, for example, Transforming Growth Factors (TGFs) and Bone Morphogenetic Protein -4 (BMP-4) were abundant in conditioned mediums but were not specific to medial explants.

Under conditions employed in the present disclosure, preferred isoform subsets of select families of paraxial mesoderm factors include, but are not limited to: FGFs -2 (bFGF), -4, -6 and -9; IGF -1; IGFBPs -1, -2, -4 and -6; TNF -alpha and -beta; IL -8; ANGPT -1 and -2; NT -3; and MMP -2. Paraxial mesoderm factors that preferably induce paraxial mesoderm and skeletal myocytes with high efficiency include, but are not limited to FGF, IGF, and IGFBP; and most preferably FGF-2 (bFGF) and IGF-1, FGF-2 and IGFBP-6, or FGF-2, IGF- 1 and IGFBP-6. As used herein, "target paraxial mesoderm cell types" refers to paraxial mesoderm progenitors and lineages known to be derived from paraxial mesoderm. Paraxial lineages include, but are not limited to, skeletal myocytes, endothelial cells, mesenchymal cells, adipocytes, chondrocytes, osteocytes, and other cells that can be induced from paraxial mesoderm. Unless specified otherwise, "target paraxial mesoderm cell types" also refers to cells at any stage of early, intermediate and late development ranging from multipotent and lineage- specific progenitors to terminally differentiated cell types. Methods to identify paraxial lineages included

morphological, nucleation, protein expression and functional criteria whereas paraxial mesoderm was characterized by the expression of markers such as, for example,

TBX6 and MSGNl. For purposes of the present disclosure, target paraxial mesoderm cell types may contain one or more transgenes that would enable the user to, for example, select or screen for one or more cell types or lineages during or subsequent to the differentiation process. All publications and patents mentioned in this application are herein incorporated by reference for any purpose.

EXAMPLES

The following examples are provided as the preferred methods, wherein one or more of the methods or components described may be overlapping. However,

modifications to these methods or components can be made without undue experimentation. These include, but are not limited to, those that will optimize conditions for a particular hPSC line or clone, those that are required to improve the efficiency of a particular technique or meet the demands of an end application, and those that result in a preferred differentiation outcome. Modifications are normally expected for culture methods of this nature and do not depart from the spirit of the claimed invention.

Example 1: Expansion of hPSC.

In the preferred methods, FGF is employed to expand hPSC and then initiate differentiation. For best results, hPSC are expanded on Matrigel or Geltrex for at least one passage in serum- and feeder-free conditions comprising bFGF prior to initiating differentiation. One skilled will be familiar with the concentration of FGF (likely between 4 and 100 ng/ml) that is necessary to maintain pluripotency within the composition of a preferred expansion medium or condition. Use of fibroblast conditioned medium (CM) supplemented with about 4-40 ng/ml FGF also enables uniform hPSC expansion. More specifically, the preferred CM expansion medium includes 80% DMEM F-12, 20% KO Serum Replacement, 1% NEAA, 1.0 mmol/L L- glutamine, 0.1 mmol/L β-mercaptoethanol supplemented with 10 ng/ml bFGF. Cells are fed daily with the FGF- supplemented medium and passaged at a preferred confluence of approximately 60%.

Example 2: Use of FGF to initiate differentiation. To initiate mesoderm differentiation from hPSC, hPSC are enzymatically or mechanically removed from the expansion condition and immediately placed into serum-free differentiation medium comprising FGF. Cells are fed daily with an exchange of fresh FGF-containing medium for a time period of at least one day but preferably for at least three to four days. In the preferred conditions, differentiation is initiated by dissociating hPSC following a brief (approximately 3 minutes at 37°C) exposure to collagenase and placing semi- dissociated colonies into non-adherent suspension culture in the presence of serum- free CM plus 4-8 ng/ml bFGF for a time period of three days. Cells are fed daily with fresh bFGF- supplemented medium through Day 3 after which the medium is switched to unconditioned DMEM/F-12 basal medium.

In certain embodiments, mesoderm differentiation was also initiated by placing the expanded hPSC in serum-free bFGF-containing medium and further exposing the cells to fibronectin-coated plates wherein fibronectin supported robust differentiation of hPSC to paraxial mesoderm and cell viability. Evaluation of target paraxial mesoderm cell types after 7, 14 and/or 21 days of cells in culture was performed alone or in conjunction with expression analyses as described above. In all cases, treated and untreated cells or aggregates were fixed in 4% formaldehyde/phosphate-buffered saline and permeabilized with 0.1% Triton X-100 at 4°C prior to double or triple-labeled immuno staining. To minimize nonspecific binding of antibodies, cells were preincubated in blocking buffer consisting of 2% donkey serum/1% BSA in phosphate-buffered saline at 4°C. Immunostaining with monoclonal antibodies specific to sarcomeric actin required brief methanol fixation. Antibody incubations with aggregates were performed at 4°C overnight in a humidified chamber, followed by extensive washing with phosphate-buffered saline. In many cases, cells were counterstained with propidium iodide (PI). Induced cell populations were also evaluated based on observations of cell morphology, nucleation states, expression patterns of cytoskeletal proteins and the presence or absence of contractile activity.

Example 3: Discovery of paraxial mesoderm associated factors.

To elucidate heretofore unknown factors that may induce or influence paraxial mesoderm differentiation from hPSC, lateral and medial regions of various staged avian embryonic mesoderm explants were used to generate mesoderm-conditioned medium in the presence and absence of 2% serum. Mesoderm conditioned medium was collected on a daily basis and analyzed along with no-cell controls via

membrane-based human antibody arrays manufactured by Raybiotech Inc. MALDI- TOF was also performed. Although mass spectometry resulted in the identification of an overwhelming number of factors and fragments, it could be relied upon to confirm the antibody array data which was derived in accordance with the protocols available at http://Raybiotech.com.

Analyses of human antibody array and MALDI-TOF data revealed that the following growth factors and cytokines were most significantly present in or depleted from conditioned mediums generated from explants comprising more medial, presomitic regions of mesoderm: FGFs -2, -4 and -9; IGFs -1 and -2; IGFBPs -1, -2, and -4;

TGFs -alpha, -beta 2 and beta 3; BMP -4; ANG; ANGPTs -1 and -2; EGF; Osteoblast

Differentiation Promoting Factor , CSF, IL-8; TNF -alpha and -beta; TNF Inducing Protein, GDF; Latent TGF Binding Protein; Follistatin; NT -3; MMP -2, LIF and HB-

EGF. The most abundant of these were the FGFs, IGFs, TGFs, BMP-4 and IGFBPs; however, significant levels of TGFs and BMP-4 were also detected in conditioned mediums generated from lateral mesoderm explants.

Example 4 demonstrates that one or more of these factors can easily be tested, alone or in combination, for their ability to induce, influence or optimize for the outcome of hPSC differentiation to paraxial mesoderm and paraxial mesoderm lineages. Example 4: Assessing the functional role(s) of the most abundant paraxial mesoderm associated factors.

For purposes of assessing the functional role(s) of paraxial mesoderm factors identified in Example 3, differentiation of hPSC was initiated (Day 1) in serum-free differentiation medium comprising bFGF under non-adherent feeder-free culture conditions as described in Example 2. Due to the high number of factors and isoforms identified, the inventor's efforts were initially focused on examining the effects of FGF-2, IGFs -1 and -2, EGF and IGFBPs. Due to the abundance of IGFBPs in conditioned medium, the effects of IGFBP-6 were also examined. All factors were commercially available and purchased as relatively pure bioactive human

recombinant proteins. hPSC were exposed to varying concentrations (5-100 ng/ml) of these factors, used alone and in combination, for a period of between 4 and 7 days beginning on Days 3- 4 of the differentiation time period. Differentiated cell types were evaluated on Days 7, 14 and 21 via double or triple immunolabeling that included antibodies specific to skeletal and cardiac myocytes. Propidium iodide was used as a nuclear stain.

All cultures treated with FGF-2 alone on Days 1-3 (Example 2) contained a mix of mesoderm lineages that included foci of cardiomyocytes, skeletal myocytes and endothelial cells. The effects of adding IGF-2 alone or in combination with FGF-2 at Days 3-4 were undetected; surprisingly, however, IGF -1 or IGFBP -6, used alone or in combination, potently induced paraxial mesoderm and skeletal myogenesis which was initially detected via the presence of skeletal myoblasts that intermingled with elongated skeletal myotubes. Bands of skeletal myotubes and myocytes were often observed to twitch by Day 7. FGF -2 (50 ng/ml) enhanced the efficacies of IGF -1 and IGFBP -6 induction. All concentrations (5-100 ng/ml) of IGF - 1 and IGFBP -6 were effective to varying degrees; however, the most effective concentrations were 100 ng/ml for IGF-1 and 50 ng/ml for IGFBP-6. Under these conditions and in the additional presence of fibronectin, -71 and -81 , respectively, of total cells in culture were positively identified as skeletal myocytes (see Figure 1). The addition of IGF-1 at any concentration, in combination with FGF -2 at 50 ng/ml abrogated cardiomyocyte differentiation compared to approximately 30-40% of cultures that still contained cardiac cells when treated with IGF -1 alone. IGF -1 also induced cell spreading in cultures comprising fibronectin. Both IGFBPs -1 and -6 (w/FGF) were effective at reducing the cardiomyocyte population in a concentration- dependent manner: approximately 22% of cultures treated with 100 ng/ml of IGFBP - 1 still contained foci of cardiomyocytes vs. undetectable cardiomyocytes in all cultures treated with IGFBP -6 at the same concentration. These results strongly support a role for IGF -1 and IGFBPs as inducers of paraxial mesoderm and skeletal myocyte differentiation.

EGF, in combination with IGF or IGFBP, did not alter the overall inducing effects observed in the absence of EGF. Unexpectedly, EGF was observed to alter the proportion of skeletal myoblasts in a manner that strongly supports a role for this growth factor in myoblast fusion and/or skeletal myocyte maturation.

Based on their method of discovery, paraxial mesoderm factors not assessed within the context of the present disclosure (e.g., ANG, ANGPT, Osteoblast Differentiation Promoting Factor , Colony Stimulating Factor, TNF and TNF Inducing Protein, GDF, IL -8, Follistatin, NT -3, MMP -2, LIF, and HB-EGF, or those without detectable effects (e.g., IGF-2) are hypothesized to function in one or more capacities that likely induce, inhibit, expand or promote the differentiation and/or survival paraxial mesoderm-derived cell types. It is notable that all cultures induced with one or more of IGF-1, IGFBP or both, in the presence and absence of FGF, contained varying proportions of mesenchymal and endothelial cells.

Claims

What is claimed is: 1. An in vitro method that differentiates paraxial mesoderm and paraxial mesoderm lineages including skeletal myocytes from human pluripotent cells, wherein the said method comprises:

a) culturing the human pluripotent cells in serum- and feeder-free conditions comprising FGF for a time period of between 1 and 4 days to initiate differentiation; and

b) exposing the cells from step a) to at least one factor known to influence the outcome of paraxial mesoderm differentiation.

2. The method of claim 1, wherein the outcome of the paraxial mesoderm

differentiation is further influenced by the concentration of the at least one factor.

3. The method of claim 1, wherein the human pluripotent cells comprise human embryonic stem cells (hESC) or human pluripotent cells derived from non-embryonic sources (hiPSC).

4. The method of claim 1, wherein the FGF is preferably FGF-2 (bFGF).

5. The method of claim 1, wherein the at least one factor comprises IGF.

6. The method of claim 5, wherein the IGF is IGF-1.

7. The method of claim 1, wherein the at least one factor comprises IGFBP.

8. The method of claim 7, wherein the IGFBP is preferably IGFBP-6.

9. The method of claim 1, wherein step b) further comprises exposing the cells from step a) to a factor selected from the group consisting of: Fibroblast Growth Factors - 2, -4, -6 and -9; Insulin-Like Growth Factor -2; Insulin-like Growth Factor Binding Proteins -1, -2, and -4; Epidermal Growth Factor; Angiogenin; Angiopoietins -1 and - 2; Osteoblast Differentiation Promoting Factor; Colony Stimulating Factor; Tumor Necrosis Factor Inducing Protein; Growth Differentiation Factor; IL-8; Tumor Necrosis Factors -alpha and -beta; Follistatin; Neurotrophin -3; Matrix

Metalloproteinase -2; Leukemia Inhibitory Factor; Heparin Binding-EGF; and combinations thereof.

10. The method of claim 1, wherein the paraxial mesoderm lineages further comprise endothelial cells, mesenchymal cells, adipocytes, chondrocytes, chondroblasts and other connective tissue cell types.

11. The method of claim 1, wherein step b) further comprises fibronectin.

12. The method of claim 1, wherein the skeletal myocytes further comprise skeletal myoblasts and myo tubes.

13. A defined culture medium composition that can be used to induce or promote differentiation of paraxial mesoderm and one or more paraxial mesoderm lineages from human pluripotent cells wherein the defined culture medium composition comprises at least one factor identified as a paraxial mesoderm associated factor.

14. A defined culture medium composition according to claim 13, wherein the at least one factor comprises FGF.

15. A defined culture medium composition according to claim 14, wherein the FGF is FGF-2.

16. A defined culture medium composition according to claim 13, wherein the at least one factor comprises IGF- 1.

17. A defined culture medium composition according to claim 13, wherein the at least one factor comprises IGFBP.

18. A defined culture medium composition according to claim 17, wherein the at least one factor comprises IGFBP -1, IGFBP-6 or both.

19. A defined culture medium composition according to claim 13, wherein the at least one factor further comprises a factor selected from the group consisting of: FGF -4, -

6 and -9; IGF -2; IGFBP -2, and -4; Angiogenin; Angiopoietin -1 and -2; EGF;

Osteoblast Differentiation Promoting Factor; Colony Stimulating Factor; TNF Inducing Protein; Growth Differentiation Factor; IL -8; TNF -alpha and -beta;

Follistatin; NT-3; MMP-2; LIF; HB-EGF; and combinations thereof.

20. A defined culture medium composition according to claim 13, wherein the composition further comprises fibronectin.