WO2009055868A1 - Process and compositions for culturing cells - Google Patents

Abstract

The present invention relates generally to the field of cell biology. For example, the present invention relates to a method of maintaining stem cells (for example, embryonic stem cells or adult stem cells or induced pluripotent cells), including pluripotent or multipotent stem cells, preferably human stem cells in an undifferentiated state and to a cell culture media composition for the maintenance of human stem cells in an undifferentiated state.

Description

PROCESS AND COMPOSITIONS FOR CULTURING CELLS

RELATED APPLICATION DATA

The present application claims priority from Australian Patent Application Nos. 2007905979 and 2007905966 filed in the Australian Patent Office on October 31, 2007 each entitled "Cell culture media and methods of use", the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates generally to the field of cell biology. For example, the present invention relates to a method of maintaining stem cells (for example, embryonic stem cells or adult stem cells or induced pluripotent cells), including pluripotent or multipotent stem cells, preferably human stem cells in an undifferentiated state and to a cell culture media composition for the maintenance of human stem cells in an undifferentiated state.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge or state of the art in the field of endeavour to which this specification relates in Australia or that the prior publication is relevant to the present invention.

Various types of stem cells differentiate when they divide, maturing into cells that can carry out one or more unique functions of particular tissues or organs, such as the heart, the liver, or the brain. It is now recognised that these stem cells may be a source of cells and/or tissue to replace or supplement cells and/or tissues that have been damaged in the course of or as a result of trauma, disease, infection, or as a result of a congenital abnormality.

There are several broad categories of stem cells including, such as those that naturally occur in an animal, for example, embryonic stem cells, which are derived from blastocysts, stem cells from adult and/or embryonic tissues, such as mesenchymal stem cells or cord blood stem cells which are found in the umbilical cord. In a developing embryo, stem cells can differentiate into all the specialised embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialised cells. Another source of stem cells is induced pluripotent stem (iPS) cells, which are adult cells that have been induced to become pluripotent, for example, de-differentiate by introducing nucleic acid encoding particular proteins and/or contacting the cell with one or more compounds that induce pluripotency. Exemplary iPS cells are described in Takahashi et α/.,(2007), Cell, 131: 861-872, or Yu et al, (2007), Science, 318: 1917-1920.

A type of stem cell of particular interest to the research community and researchers developing cell-based therapies is human embryonic stem (hES) cells. These cells are derived from a human embryonic source, generally a blastocyst stage embryo. hES cells are pluripotent meaning that they have the potential to differentiate into any or each of the three germ layers, i.e., endoderm, ectoderm or mesoderm and, as a consequence an ability to differentiate into most, if not all, of the differentiated cell types of a mature subject. Desirable characteristics of hES cells are that they are capable of proliferation in vitro in an undifferentiated state when cultured under the correct conditions, and importantly, that they are pluripotent.

Isolation and in vitro propagation of stem cell lines, for example, hES cell lines, has facilitated study of early stages of human development, generation of in vitro models for human diseases, in vitro testing of pharmaceuticals and other therapeutic products, and production of transplantable cells for tissue repair and/or regeneration. Whilst stem cells, such as hES cells offer such desirable outcomes, to achieve these outcomes it is important to be capable of maintaining and growing stem cells in such a manner to retain their pluripotent or multipotent state. If the stem cells are not grown or cultured under such conditions, they will merely differentiate, generally in an uncontrolled manner. This generally results in a mixed cell population that is not useful for therapeutic, screening or research purposes.

The environment in which a stem cell grows plays an important role in the regulation of cell differentiation. Without optimal culture conditions or genetic manipulation stem cells will rapidly differentiate and lose their pluripotent or multipotent potential. As used herein, the term "multipotent" shall be taken to mean that a cell is capable of differentiating into at least two distinct cell types, for example, at least two cells of one lineage (such as a megakaryocyte and an erythrocyte of the haematological lineage) or at least two cells of different lineages.

The composition of the culture medium in which stem cells are grown or maintained is therefore a critical factor influencing the ability to maintain stem cells in an undifferentiated state. Traditional methods for maintaining hES cells and/or iPS cells in an undifferentiated state involve co-culturing those cells with "feeder" cells (usually fibroblasts) derived from mouse or human. The feeder cells form a feeder layer on which the stem cells can be cultured and this feeder layer provides secreted factors, extracellular matrix, and cellular contacts for maintaining the hES cells in an undifferentiated state. The feeder cells act through an as yet incompletely understood mechanism to encourage the stem cells to remain in an undifferentiated state. The same phenomenon can also be achieved by exposing the stem cells to "conditioned media". Conditioned media is a stem cell culture medium in which feeder cells, such as mouse embryonic fibroblast (MEF) cells, have been previously cultured.

However, there are limitations and drawbacks to these procedures. Culturing stem cells in the presence of a feeder cell or in conditioned medium, raises a concern that one or more agents such as a virus could be transmitted from the feeder cells to the stem cells or cells differentiated therefrom. In addition, exposure of human pluripotent cells to animal cell products can induce molecular or physiological changes that may be undesirable in a therapeutic setting. For example, most existing hES cell lines which have been exposed directly to mouse cells or to a medium in which mouse cells have been cultured previously exhibit properties not normally seen in human cells, for example the expression of the sialic residue Neu5Gc.

ES cells and iPS cells are also often cultured in the presence of serum, for example, fetal bovine serum. Again, a disadvantage of such culture techniques is that cells that may be used in therapeutic applications are exposed to animal products that may be infected with a pathogen.

A further disadvantage of culturing in the presence of feeder cells or serum is that the culture conditions are uncharacterized, i.e., all of the components of serum or all of the factors secreted from feeder cells are not known. This is an undesirable situation in the case of cells being produced for therapeutic purposes both from a safety and a regulatory point of view, when, for example, it is important to know what factors cells have been exposed to before administering them to a subject. Moreover, the uncharacterized nature of the culture conditions makes it difficult to study ES cell or iPS cell biology because it is difficult to control the composition of media in which cells are grown. For example, when determining factors that control ES cell or iPS cell biology it is difficult to determine the effect of test factors without being able to control all factors in culture media or even to determine whether or not there is a component of a media that is inhibiting or enhancing the activity of a test compound.

It is clear from the foregoing that there is a need in the art for factors or agents capable of maintaining pluripotent cells or multipotent cells in an undifferentiated state, preferably in the absence of feeder cells and/or serum.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a culture medium for maintaining or culturing a stem cell in a substantially undifferentiated state said culture medium comprising a compound that enhances or activates signal transduction in a stem cell mediated by a growth and differentiation factor (GDF) comprising a sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO: 2.

In another aspect, the present invention provides a culture medium for maintaining or culturing a stem cell in a substantially undifferentiated state said culture medium comprising a growth and differentiation factor (GDF) comprising a sequence at least about 90% or 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in any one of SEQ ID NOs: 1 to 13, or an active fragment, derivative, analog or mimetic of said GDF.

Yet another aspect of the present invention provides a method for maintaining or culturing a stem cell in a substantially undifferentiated state, the method comprising contacting or culturing said stem cell with a compound that enhances or activates signal transduction in a stem cell mediated by a growth and differentiation factor (GDF) comprising a sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO: 2, for a time and under conditions sufficient for the stem cell to proliferate.

In another aspect, the present invention provides a method for maintaining or culturing a stem cell in a substantially undifferentiated state, the method comprising contacting or culturing the stem cell with a growth and differentiation factor (GDF) comprising a sequence at least about 90% or 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in any one of SEQ ID NOs: 1 to 13, or an active fragment, derivative, analog or mimetic of said GDF, for a time and under conditions sufficient for the stem cell to proliferate.

Yet another aspect of the present invention provides a method for producing a population of stem cells in a substantially undifferentiated state, said method comprising performing a method as described herein according to any embodiment to culture or maintain a stem cell for a time and under conditions sufficient for said stem cell to proliferate and produce a population of stem cells.

A further aspect of the present invention provides a method for producing a population of differentiated cells, said method comprising performing a method as described herein according to any embodiment to produce a population of stem cells and differentiating those stem cells to produce a population of differentiated cells.

A still further aspect of the present invention provides a method for producing a population of differentiated cells, said method comprising obtaining a population of stem cells produced by performing a method as described herein according to any embodiment and differentiating those stem cells to produce a population of differentiated cells.

Another aspect of the present invention provides a population of substantially undifferentiated human stem cells or differentiated cells produced by performing a method as described herein according to any embodiment.

In yet another aspect the present invention provides a kit for maintaining or culturing human stem cells in a substantially undifferentiated state, said kit comprising a cell culture medium as described herein according to any embodiment.

GENERAL

This specification contains nucleotide and amino acid sequence information prepared using Patentln Version 3.4, presented herein after the claims. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (for example <210>l, <210>2, <210>3, etc). The length and type of sequence

(DNA, protein (PRT), etc), and source organism for each nucleotide sequence, are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the term "SEQ ID NO:", followed by the sequence identifier (for example SEQ ID NO: 1 refers to the sequence in the sequence listing designated as <400>l).

The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue. As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

Each embodiment described herein is to be applied mutatis mutandis to each and every other embodiment unless specifically stated otherwise.

Each embodiment directed methods for culturing or maintaining a stem cell in an undifferentiated state shall be taken to apply mutatis mutandis to a method for culturing or maintaining a substantial portion of a population of stem cells in an undifferentiated state. The term "substantial portion" shall be taken to mean at least about 60% or 70% or 75% or 80% or 85% or 90% or 95% of a population of stem cells.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

BRIEF DESCRIPTION OF THE FIGURES Figure IA shows an alignment of active or mature domains of Homo sapiens GDF-8; Mus musculus GDF-8; Rattus norvegenicus GDF-8; Pan troglodytes GDF-8; Danio rerio GDF- 8; H. sapiens GDF-I l; M musculus GDF-I l; R. norvegenicus GDF-Il and Danio rerio GDF-Il. '*' indicates positions which have a single, fully conserved residue. ':' indicates that one of the following groups is fully conserved: STA NEQK NHQK NDEQ QHRK MILV MILF HY FYW. '.' indicates that one of the following groups is fully conserved: CSA ATV SAG STNK STPA SGND SNDEQK NDEQHK NEQHRK FVLIH FYM. Bold text indicates a conserved sequence in GDFs.

Figure IB shows an alignment of active or mature domains of Homo sapiens GDF-8; Mus musculus GDF-8; Rattus norvegenicus GDF-8; Pan troglodytes GDF-8; and Danio rerio GDF-8. '*' indicates positions which have a single, fully conserved residue. ':' indicates that one of the following groups is fully conserved: STA NEQK NHQK NDEQ QHRK MILV MILF HY FYW. '.' indicates that one of the following groups is fully conserved: CSA ATV SAG STNK STPA SGND SNDEQK NDEQHK NEQHRK FVLIH FYM.

Figure 1C shows an alignment of active or mature domains of H. sapiens GDF-I l; M musculus GDF-I l; R. norvegenicus GDF-I l and Danio rerio GDF-I l. '*' indicates positions which have a single, fully conserved residue. ':' indicates that one of the following groups is fully conserved: STA NEQK NHQK NDEQ QHRK MILV MILF HY FYW. '.' indicates that one of the following groups is fully conserved: CSA ATV SAG STNK STPA SGND SNDEQK NDEQHK NEQHRK FVLIH FYM.

Figure 2 is an image showing that GDF-11 maintains hES2. Bright field pictures show that hES2 grown in UCM supplemented with GDF-Il maintain a cell morphology similar to that of cell grown in CM. Cells grown in UCM alone show significant differentiation and loss of characteristics common to undifferentiated hES cells. Figure 3 is a graphical representation showing GDF-I l maintenance activity in hES2. GDF-Il dose response curve illustrates that GDF-Il maintains Oct3/4 and NANOG expression equal to that of CM at a concentration of 20 ng/ml. Maintenance drops off at 10 ng/ml and again at 40 ng/ml. Whilst maintenance is still occurring at these concentrations the optimal concentration appears to be 20 ng/ml. Cells grown in UCM show a 60-70 % reduction in their Oct3/4 and NANOG transcripts over the 7 day culture period. The UCM control demonstrates that the cells are capable of differentiating when not exposed to maintenance factors either from the CM or GDF-I l. Western blot (lower panel) also demonstrates maintenance of Oct3/4 expression in GDF-Il treated cells

Figure 4 is an image showing that GDF-Il maintains hES4. Bright Field pictures reveal the GDF-11 at 20 ng/ml is able to maintain a cell and colony morphology in hES4 similar to that of cells grown in CM. Cells grown un UCM show wide spread differentiation.

Figure 5 is a graphical representation showing that GDF-11 maintenance activity in hES4. GDF-I l also maintains Oct3/4 and NANOG expression in hES4 at a concentration of 20 ng/ml. Expression is approximately twice that of UCM.

Figure 6 is an image showing that GDF-Il maintains TRA-1-60 expression in hES2. Cells treated with CM or GDF-I l at 20 ng/ml maintain expression of TRA-1-60 (right panel) over 7 days while cells grown in UCM significantly downregulate expression of TRA- 160. DAPI staining (Left Panel) demonstrated that cells grown in CM or GDF at 20 ng/ml maintain a dense colony morphology while those growing in UCM become less densely packed and cells start to migrate away from each other.

Figure 7 is an image showing that GDF-I l maintains Oct3/4 and TRA-1-60 expression in hES4. HES4 treated with either CM or GDF-Il maintains expression of Oct3/4 and TRA- 1-60 over 7 days. Cells grown in UCM significantly downregulate Oct3/4 and TRA-1-60 expression over 7 days.

Figure 8 is an image showing that GDF-I l maintains expression of SSEA-4 in hES4. HES4 grown in CM or in GDF-I l supplemented medium maintain strong expression of SSEA-4 over 7 days. HES4 grown in UCM rapidly downregulate expression of SSEA-4 over 7 days.

Figure 9 is an image showing that the ALK 4/5/7 SB-431542 inhibiter, inhibits GDF-I l. HES2 grown in CM or GDF-I l + SB-431542 (bottom) exhibit a cell and colony morphology similar to that of differentiated hES cells. CM and GDF-I l without SB- 431542 (top) show cell and colony morphologies consistent with that of undifferentiated hES cells.

Figure 10 is a graphical representation showing that SB-431542 inhibits the activity of GDF-Il. Real-time PCR demonstrates that SB-431542 inhibits the maintenance activity of GDF-Il and CM in hES2. When CM or GDF-I l are treated with SB-431542 CM and GDF-I l are unable to maintain Oct3/4 and NANOG expression levels and cells differentiate.

Figure 11 is an image showing that SB-431542 inhibits expression of TRA- 1-60 in hES2. CM and GDF-Il supplemented media maintain expression of TRA-1-60 without SB- 431542 (left panel). With the inhibitor (right panel) CM and GDF-I l lose their ability to maintain expression of TRA-1-60.

Figure 12 is an image showing that SB-431542 inhibits CM and GDF-Il activity in hES4. SB-431542 inhibits the activity of CM and GDF-I l in hES4 and over 7-days cells become morphologically similar to differentiated hES cells

Figure 13 is a graphical representation showing that SB-431542 inhibits the activity of CM and GDF-11. Real-time PCR (upper) demonstrates that CM and GDF-11 are unable to maintain mRNA levels of Oct3/4 and NANOG in the presence of the SB-431542 inhibitor. Western blot (lower) also demonstrates the loss of Oct3/4 and NANOG protein levels when SB-431542 is added to CM and GDF-Il samples.

Figure 14 is an image showing that SB-431542 inhibits expression of TRA-1-60 in hES4. When cells are grown in the presence of SB-431542 (right) CM and GDF-11 are unable to maintain the expression of the pluripotent hES cell marker TRA- 1-60. Cells show normal expression of TRA-1-60 when grown in GDF-Il without SB-431542 (left),

Figure 15 is an image showing that SB-431542 inhibits SSEA-4 expression in hES4. When cells are grown in the presence of SB-431542 CM and GDF-Il are unable to maintain the expression of the pluripotent hES cell marker SSEA-4.

Figure 16 is an image showing that SB-431542 inhibits Oct3/4 and TRA-1-60 expression in hES4. SB-431542 inhibits the activity of CM and GDF-Il causing hES4 to downregulate Oct3/4 (red) and TRA-1-60 (green) in 7 days. Cells in CM or GDF-11 only maintain expression of these pluripotent markers.

Figure 17 is a graphical representation showing that long term maintenance ability of hES2 by GDF-Il. GDF-I l is able to maintain expression of TRA-1-60 as determined by FACS analysis over 2 weeks of consecutive passaging in hES2. GDF-11 (green) maintains TRA-1-60 expression at level similar to that of CM (blue) over two weeks while TRA-1- 60 expression in cells grown in UCM drops to approximately 60% after 1 week and were unable to be passaged for week 2 of analysis.

Figure 18 depicts a FACS profile of cells grown for 1 week in GDF-11. The FACS profile of hES2 grown under GDF-I l conditions is very similar to. that of cells grown in CM. Cells grown in UCM show a differentiated profile of very few TRA-1-60 positiye cells.

Figure 19 is an image showing GDF-8 maintains HES2. HES2 cultures supplemented with GDF-8 display growth characteristics and morphologies similar to that of cells that grown in CM, tightly packed colonies, reduced cytoplasmic space and large nuclei. This effect is most obvious at the range of 20-40 ng/ml. Cells growing in UCM show typical characteristics of differentiation such as less dense colonies with individual cells showing a large cytoplasmic space.

Figure 20 is a graphical representation showing GDF-8 maintain Oct3/4 and NANOG expression in HES2. HES2 grown in the presence of GDF-8 maintain levels of Oct3/4 and NANOG that are higher than cells grown in UCM as determined by real-time PCR. Levels of Oct3/4 and NANOG expression begin to approach levels of CM at 20-40 ng/ml. This result confirms the results of figure 19 indicating that at >20 ng/ml of GDF-8 must be used for maintenance of hES cells. n=3

Figure 21 is an image showing GDF-8 maintains TRA- 1-60 and Oct3/4 expression in HES2. Cells grown in the presence of GDF-8 at a concentration of 20 ng/ml maintain expression of TRA- 1-60 and Oct3/4 when compared to cells grown CM. Cells growing in UCM rapidly downregulate expression of both TRA- 1-60 and Oct3/4 and show morphological characteristics of differentiated cells. Loss of Oct3/4 expression as visualised by immunostaining confirms the results observed using real-time PCR (Fig. 20).

Figure 22 is an image showing GDF-8 maintains HES4. HES4 grown in GDF-8 supplemented medium show a morphology similar to that of cells growing in CM over 7 days. Cells growing in UCM display a typical differentiated morphology.

Figure 23 is a graphical representation showing GDF-8 maintains HES4 over 7 days of growth. HES4 grown in the presence of GDF-8 at 20 ng/ml show increased expression of Oct3/4 and NANOG when compared to cells grown in UCM. Levels of Oct3/4 and NANOG are almost double that of UCM. n = 4.

Figure 24 is an image showing GDF-8 maintains TRA-I -60 and Oct3/4 expression in HES4. GDF-8 at 20 ng/ml is sufficient to maintain Oct3/4 and TRA-1-60 expression over 7 days of culture. Maintenance of Oct3/4 expression in cells growing in CM or GDF-8 medium confirms observation made using realtime PCR as does the loss of Oct3/4 in UCM cells.

Figure 25 is an image showing GDF-8 maintains SSEA-4 expression in HES4. GDF-8 at 20 ng/ml is sufficient to maintain expression of SSEA-4 in HES4. Cells grown in UCM rapidly downregulate expression of SSEA-4 and display a morphology characteristic of differentiated hES cells. Figure 26 is an image showing SB431542 inhibits GDF-8 activity. When inhibitor SB431542 is added to cultures supplemented with 20 ng/ml GDF-8 all previously observed maintenance ability is inhibited. When the inhibitor is added cell colonies become less dense and increase their cytoplasmic space, characteristics of differentiated hES cells. HES4 n=3.

Figure 27 is a graphical representation showing SB431542 inhibits Oct3/4 and NANOG expression. Cells grown in the presence of GDF-8 at 20 ng/ml maintain levels of Oct3/4 and NANOG similar to that of Cm as shown previously. Addition of SB431542 to cultures inhibits all maintenance activity of GDF-8 HES4 n=3.

Figure 28 is an image showing SB431542 inhibits Oct3/4 and TRAl -60 expression in HES2. SB431542 inhibits the maintenance activity of GDF-8 causing the downregulation of TRA- 1-60 and Oct3/4 over 7 days of culture. As well as obvious downregulation of these markers cells also exhibit characteristics of differentiated cells. The immuno staining data for Oct3/4 also validates the real-time data on Oct3/4 expression both in the absence and presence of the inhibitor.

Figure 29 is an image showing SB431542 inhibits SSEA-4 and Oct3/4 expression in HES4. SB431542 effectively inhibits the maintenance activity of 20 ng/ml of GDF-8 and causes the downregulation of SSEA-4 and Oct3/4 over 7 days of culture. Again loss of Oct3/4 expression validates the real-time data showing reduced Oct3/4 expression over 7 days when using SB431542.

Figure 30 is a graphical representation showing that long term maintenance ability of liES2 by GDF-8. GDF-8 is able to maintain expression of TRA-1-60 as determined by FACS analysis over 2 weeks of consecutive passaging in hES2. GDF-8 (green) maintains TRA-1-60 expression at level similar to that of CM (blue) over two weeks while TRA-1- 60 expression in cells grown in UCM drops to approximately 60% after 1 week and were unable to be passaged for week 2 of analysis. Figure 31 shows that Myostatin and BMP-Il inhibit hES cell differentiation and maintain Nanog and Oct4 expression in short term cultures. Mouse embryonic fibroblast conditioned medium (CM) was diluted in KOSR medium and used to culture HES2 hESC for 7 days. A step wise reduction in MEF conditioned medium laeads to A) a progressive increase in porphologically differentiated hESC and B) a stepwise reduction in POUfI and Nanog mRNA expression. Addition of increasing amounts of either BMP-I l (C) or Myostatin (D) to unconditioned KOSR medium with 4 ng/ml b-FGF maintains the undifferentiated appearance of hESC similar to hESC grown in CM over 7 days. Real time PCR analysis shows that increasing the concentration of BMP-Il C) or Myostatin D) to 20 ng/ml progressively increased Oct3/4 and NANOG mRNA expression to levels similar to CM over a 7 day culture period. Maintenance of Oct3/4 and NANOG protein expression is detected in the presence of increasing concentrations of BMP-Il E) or myostatin F) to levels similar to hES cells grown in CM. RT-PCR is based on three independent experiments where solid bars indicate the average of the three experiments normalised to CM, error bars indicate standard error of the mean, statistical analysis was performed using ANOVA where each sample is compared to the UCM control for differentiation. * p=0.01- 0.05, ** p=0.001-0.01, *** pO.001. A representative Western blot experiment of three is shown.

Figure 32 is an image showing BMP-Il and myostatin Maintain Oct3/4, NANOG, TRA- 1-60 and SSEA-4 Expression.) HES2 hESC grown in KOSR + 4 ng/ml b-FGF supplemented with A) 20 ng/ml BMP-Il or B) 20 ng/ml myostatin over a 7 day culture period display strong expression of the pluripotency markers SSEA-4 and NANOG and TRA- 1-60 and Oct3/4 at levels similar to MEF conditioned medium C) Flow cytometric analysis shows that HES2 hESC cultured for 7 days in KOSR supplemented with BMP-11 (20ng/ml) or mysostatin (20 ng/ml) display similar TRA-1-60 expression to hESC cultured in CM. Flow cytometry frequency distribution profiles for secondary antibody controls (i) CM (ii) BMP-11 20 ng/ml (iii) and myostatin 20 ng/ml are shown with the UCM profile in grey. A representative experiment of three is shown.

Figure 33 is an image showing that BMP-I l induces SMAD2/3 phosphorylation. HES2 hESC were serum starved for 48 hrs using DMEM/F12, and then stimulated with fresh KOSR with or without 20 ng/ml BMP-I l for 2 hrs. BMP-Il addition caused a clear increase in the amount of pSMAD2/3 detected in the nuclei of Oct3/4 positive cells.

Figure 34 shows that BMP-Il and Myostatin maintain TRA-1-60 and POUfI and Nanog rnRNA expression for up to 10 passages in a feeder free and serum free conditions. HES2 hESC were cultured on matrigel and either MEF conditioned KOSR medium or KOSR + 20 ng/ml b-FGF supplemented with 20 ng/ml BMP-I l or 20 ng/ml Myostatin. A) Expression of the pluripotency marker TRA-1-60 was determined by flow cytometry at passage 1, 6 and 10. hESC cultured in myostatin or BMP-I l supplemented medium show similar expression of TRA-1-60 as hESC cultured in CM. A representative experiment of three is shown b) POUfI and Nanog mRNA expression was determined by real time PCR at passage 10, normalized to beta-actin mRNA expression and expressed relative to hESC cultured in CM. The results are the average of three independent experiments in triplicate.

Figure 35 shows the addition of SB431542 to hESC maintained by Myostatin and BMP- 11 leads to morpholocical differentiation and a loss of Tra-1-60, Nanog and Oct4 expression. HES2 hESC were cultured in CM, UCM or UCM supplemented with either A) 20 ng/ml BMP-I l or B) 20 ng/ml Myostatin in the absence or presence (+1) of 10 uM of the ALK4,5,7 inhibitor SB431542 for 7 days. Addition of 10 uM SB431542 to either BMP-I l or Myostatin supplemented cultures leads to a loss the undifferentiated morphology of hESC, a strong reduction in Tra-1-60 and Oct4 expression by in situ immunodetectionand a loss of Oct4 and Nanog mRNAand protein expression. A representative experiment of three is shown.

Figure 36 shows the efficacy of BMP-Il and Myostatin in inhibiting hESC differentiation is similar to Activin A. HES2 hESC cultured on Matrigel coated TC plates for 7 days in UCM supplemented with 20 ng/ml Activin- A, 20 ng/ml Myostatin , 20 ng/ml BMP-Il or bFGF 100 ng/ml display A) a similar undifferentiated morphology B) robust expression of the pluripotency markers TRA-1-60 and POUFl and C) maintenance of POUFl and Nanog mRNA expression with the exception of b-FGF. The results are the average of three independent experiments in triplicate. DETAILED DESCRIPTION OF THE INVENTION

The present have inventors identified a class of growth and differentiation factors (GDFs) that are capable of maintaining stem cells in an undifferentiated state. For example, the inventors have determined that a GDF comprising a sequence ESRCCRYPLTVDFEAFGWDWπAPKRYKANYCSG (SEQ ID NO: 1) and/or a sequence ANPRGSAGPCCTPTKMSPINMLYFN (SEQ ID NO: 2) and/or that comprises a sequence having at least about 90% identity to a sequence set forth in any one or more of SEQ ID NOs: 1 to 13 is capable of maintaining a stem cell in an undifferentiated state. This class of GDFs is exemplified by GDF-8 and GDF-11.

The inventors have also demonstrated that members of the identified class of GDFs are capable of maintaining stem cells in an undifferentiated state in the absence of feeder cells and/or serum. In doing so, the inventors have also produced media, for example, serum- free media that are capable of maintaining stem cells in an undifferentiated state.

The inventors have also produced a media composition that is capable of maintaining stem cells in an undifferentiated state without inducing genetic instability or expression of a marker of genetic instability, for example, CD30, in the stem cells. Such a media is important for ongoing culture of stem cells, particularly for therapeutic use. For example, use of genetically-unstable stem cells in therapy may result in tumor formation in a subject to whom they are administered.

These findings by the inventors form the basis for methods and compositions for growing stem cells without inducing those cells to differentiate and/or for maintaining stem cells in an undifferentiated state.

Accordingly, one aspect of the present invention provides a culture medium for maintaining or culturing a stem cell in a substantially undifferentiated state said culture medium comprising a compound that enhances or activates signal transduction in a stem cell mediated by a growth and differentiation factor (GDF) comprising a sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO: 2. In another aspect, the present invention provides a culture medium for maintaining or culturing a stem cell in a substantially undifferentiated state said culture medium comprising a growth and differentiation factor (GDF) comprising a sequence at least about 90% or 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in any one of SEQ ID NOs: 1 to 13, or an active fragment, derivative, analog or mimetic of said GDF.

As used herein, the term "culture medium" shall be understood to mean a mixture of compounds that are necessary and/or desirable for proliferation of a stem cell. Such a culture medium comprises, for example, nutrients, salts, a buffer, etc that is required for proliferation of a stem cell. Preferably, the culture medium does not comprise a compound that induces differentiation of a pluripotent stem cell when used in the context of a medium as described herein according to any embodiment (i.e., the compound may induce differentiation in its own right, however when used in a medium of the present invention such differentiation inducing activity is reduced or suppressed). In one embodiment, the medium lacks serum and/or does not comprise factors or proteins secreted by a feeder cell, for example, a mouse or human embryonic fibroblast. Exemplary culture media will be apparent to the skilled artisan and include for example, knockout serum replacement containing media (for example, comprising components listed in Table 2); mTeSR (StemCell Technologies Inc), StemPro (Invitrogen) and APEL (as described in Nature Protocols, 2008, 3(5):768-76). Compositions of additional media will be apparent to the skilled artisan and/or described herein.

As used herein, the term "stem cell" shall be taken to mean any cell which exhibits the potential to develop in any of the directions inherently possible, given its particular genetic constitution and thus to form a new organism or to regenerate any part of an organism. More particularly, the stem cell is any cell capable of self-renewal and differentiation into at least two different cell types. Exemplary stem cells are pluripotent stem cells, such as embryonic stem cells or iPS cells or multipotent stem cells, such as adult stem cells. Preferably, the stem cell is an embryonic stem cell. It is also preferred that the stem cell is a human stem cell. In one exemplified form of the present invention, the stem cell is a human embryonic stem cell (hES cell). As used herein, the term "substantially undifferentiated state" shall be taken to mean that a stem cell retains the ability to self renew and to differentiate into at least two different cell types.

In the context of a pluripotent stem cell, the term "substantially undifferentiated state" means that the cell remains pluripotent, for example, is able to differentiate into endodermal cells, ectodermal cells and mesodermal cells. However, in some embodiments the substantially undifferentiated cell need not necessarily differentiate into each of these lineages equally or even to the same degree as a pluripotent cell cultured under different conditions. For example, a cell cultured or maintained according to a method of the present invention may be biased toward differentiating into one lineage over another lineage. Methods for determining the ability of a cell to differentiate into each of these cell types will be apparent to the skilled artisan and includes, for example, assessment of teratoma formation in a murine model, that is, the pluripotency of cells can be confirmed by injecting cells into SCID mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically for evidence of cell types from the three germ layers. Alternatively, or in addition, the pluripotency of a cell is assessed by detecting expression of a nucleic acid or protein expressed by a pluripotent stem cell, such as, for example, Oct3/4 and/or Nanog and/or Tra-1-60, SSEA-3 and SSEA-4, and markers detectable using antibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). Pluripotent cells typically have alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde, and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.). Additional methods of determining the pluripotency of cells will be well known to those skilled in the art. Cells can be karyotyped using a standard G-banding technique and compared to published karyotypes. It is desirable to obtain cells that have a "normal karyotype", which means that the cells are euploid, wherein all human chromosomes are present and not noticeably altered.

Methods of detection of the expression of tissue-specific antigens and/or markers will be well known to those skilled in the art including, for example, suitable immunological techniques, such as flow cytometry for membrane-bound markers, immunohistochemistry for extracellular or intracellular markers, and enzyme-linked immunoassay, for markers secreted into the medium. The expression of protein markers can also be detected at the inRNA level by reverse transcriptase-PCR using marker-specific primers. See U.S. Pat. No. 5,843,780 for further details.

In the context of a multipotent stem cell, the term "substantially undifferentiated state" shall be understood to mean that the stem cell retains an ability to generate another multipotent stem cell and to differentiate into at least two different cell types, however not into each of the three germ layers, i.e., endoderm, ectoderm and mesoderm.

As used herein, the term "culturing" means growing a stem cell in a culture medium, i.e., a composition comprising components, e.g., nutrients that are required for stem cells to proliferate. The skilled artisan will also be aware of other conditions required for culturing a stem cell, such as for example, temperature, oxygen, carbon dioxide and nitrogen.

As used herein, the term "maintaining" shall be taken to refer to a process in which stem cells are treated so as to continue to be pluripotent or multipotent.

The subject cells may have been freshly isolated from an individual or they may have been sourced from a non-fresh source, such as from a culture (for example, where cell numbers were expanded and/or the cells were cultured) or a frozen stock of cells (for example, an established embryonic stem cell line), which had been isolated at some earlier time point either from an individual or from another source. It should also be understood that the subject cells, may have undergone some other form of treatment or manipulation, such as but not limited to enrichment or purification, modification of cell cycle status or the formation of a cell line such as an embryonic stem cell line. Accordingly, the subject cell may be a primary cell or a secondary cell. A primary cell is one which has been isolated from an individual. A secondary cell is one which, following its isolation, has undergone some form of in vitro manipulation such as the preparation of an embryonic stem cell line, prior to the application of the invention.

To the extent that human embryonic stem cells are sought to be used, these cells may be derived from the inner cell mass of a blastocyst stage human embryo or an established cell line may be used such as, for example, those developed by Thomson and Odorico, Trends BiotechnoL, 18:53-57 (2002), namely, Hl, H7, H9.1, H9.2, H13 or H14); hES cells such as hES2, hES3 and hES4 and MEL cells such as MELl. However, it should be understood that any human embryonic stem cell line described in the literature can be used.

To generate human embryonic stem cell cultures de novo, cells from the inner cell mass are separated from the surrounding trophectoderm by microsurgery or by immunosurgery (which employs antibodies directed to the trophectoderm to break it down) and are plated in culture dishes containing growth medium supplemented with fetal bovine serum (alternatively, KnockOut Dulbecco's modified minimal essential medium containing basic .FGF can be supplemented with Serum Replacer (Life Technologies) and used without serum), usually on feeder layers of mouse embryonic fibroblasts that have been mitotically inactivated to prevent replication. Alternatively, a feeder-free culture system may be employed, such as that reported by Chunhui Xu, Melissa Carpenter and colleagues using Matrigel or laminin as a substrate, basic FGF, and conditioned medium from cultures of mouse embryo fibroblasts (Xu, et al, Nat BiotechnoL 2001 Oct; 19(10):971-4) and/or unconditioned medium. However, the skilled artesian will also be aware of other components that may be used, for example, nutrients that are required for the stem cells to proliferate.

As used herein, the term "compound that enhances or activates signalling of a growth and differentiation factor (GDF) comprising a sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO: 2" shall be taken to encompass a compound that modulates any component of signalling pathway mediated by a GDF comprising a sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO: 2 in a stem cell so as to enhance or activate said signalling pathway. Exemplary components of such a signaling pathway include an activin-like kinase (ALK) receptor 4 or 5 or 7 and/or a type II BMP receptor and/or a SMAD-2 and/or a SMAD-3 and/or a SMAD-4. Preferably, the compound enhances or activates signalling initiated or mediated by an ALK5 receptor. Preferably, the compound is a GDF comprising a sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO: 2. As used herein, the term "growth and differentiation factor" or "GDF" shall be taken to mean a class of proteins that belong to the transforming growth factor β (TGF-/3) family of signalling proteins that are capable of binding to a dimer between a BMP receptor and an ALK receptor and mediating signal transduction within a cell. Preferably, the GDF is a dimeric protein with each subunit linked by a disulfide bond. In the context of the present invention, a preferred GDF comprises a sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Preferably the GDF binds to an ALK receptor 4 or 5 or 7 and/or a type II BMP receptor, preferably an ALK5 receptor and/or the GDF activates or enhances phosphorylation of a SMAD-2 and/or a SMAD-3 and/or a SMAD-4. Preferably, the GDF comprises a sequence at least about 80%, or 84% or 85% or 87% or 88% or 90% or 94% or 95% or 96% or 97% or 98% or 99% identical to one or more sequences set forth in SEQ ID NOs: 3-11. Exemplary GDFs falling within the class of GDFs identified by the inventors as being capable of maintaining a stem cell in a substantially undifferentiated state are depicted in Figures lA-lC. In this respect, the sequences set forth in SEQ ID Nos: 3-11 and in Figures 1A-1C are sequences of an active or mature GDF. GDFs are also expressed as prepro proteins and/or contain a signalling domain, and the present invention also clearly contemplates such proteins. Exemplary sequences of full-length human GDFs are set forth in SEQ ID Nos: 12 and 13.

In one example, the GDF is a GDF-8 (syn. Myostatin). For the purposes of nomenclature and not limitation, the sequence of an active domain of a GDF-8 is set forth in any one or more of SEQ ID Nos: 3-7 or 12. Preferably, a GDF-8 comprises a sequence at least about 85% or 88% or 90% or 95% or 96% or 97% or 98% or 99% or 100% identical to a sequence set forth in any one or more of SEQ ID NOs: 3-7 or 12. In one example, the GDF-8 is a human GDF-8, for example, comprising a sequence set forth in SEQ ID NO: 3 or 12.

In another example, the GDF is a GDF-I l (syn. BMP-I l). For the purposes of nomenclature and not limitation, the sequence of an active domain of a GDF-11 is set forth in any one or more of SEQ ID Nos: 8-11 or 13. Preferably, a GDF-I l comprises a sequence at least about 85% or 90% or 95% or 96% or 97% or 98% or 99% or 100% identical to a sequence set forth in any one or more of SEQ ID NOs: 8-11 or 13. hi one example, the GDF-I l is a human GDF-Il, for example, comprising a sequence set forth in SEQ ID NO: 8 or 13.

As used herein, the term "analog" shall be taken to mean a peptide that is modified to comprise one or more non-naturally-occurring amino acids. Analogs of the molecules contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogs. Analogs may also comprise sterically similar compounds that mimic critical subdomains of a peptide, The specific form which such modifications can take will depend on whether the subject molecule is proteinaceous or non-proteinaceous. Such "peptidomimetics" are produced by modelling and chemical design processes known to those of skill in the art.

For example, examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norv aline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated herein is shown in Table 1.

TABLE 1

Crosslinkers can be used, for example, to stablise 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH₂)_n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety.

As used herein the term "derivative" shall be taken to mean a peptide that is derived from a GDF described and/or exemplified herein for example, a fragment or processed form of the peptide, or a molecule comprising one or more amino acid substitutions, or comprising additional amino acid residues or non-amino acid substituents, relative to the base peptide from which it is derived.

More particularly, "derivatives" of the GDF described and/or exemplified herein includes functional fragments, parts, portions, variants or mutants from either natural or non-natural sources. Non-natural sources include, for example, recombinant or synthetic sources. By "recombinant sources" is meant that the cellular source from which the subject molecule is harvested has been genetically altered. This may occur, for example, in order to increase or otherwise enhance the rate and volume of production by that particular cellular source. Parts or fragments include, for example, active regions of the molecule. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in a sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins, as detailed above. Derivatives also include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules. The term "derivative" also encompasses fusion proteins comprising a GDF described herein according to any embodiment, as well as a GDF described herein according to any embodiment which is immobilized into a solid surface.

By "active fragment" is meant a portion of a polypeptide that retains the biological activity of the polypeptide i.e. ability of that polypeptide to modulate GDF signaling. It should be understood that an active fragment or analog or derivative may have the same level of activity as the original protein or an enhanced or reduced level of activity compared to the level of activity of the original protein.

"Mimetics" should be understood as molecules exhibiting any one or more of the functional activities of the subject molecule, which mimetics may be derived from any source such as being chemically synthesised or identified via screening processes such as natural product screening. For example, mimetics can be designed and/or identified utilising well known methods such as combinatorial chemistry or high throughput screening of recombinant libraries or following natural product screening.

For example, libraries containing small organic molecules may be screened, wherein organic molecules having a large number of specific parent group substitutions are used. A general synthetic scheme may follow published methods (eg., Bunin et al. (1994) Proc. Natl. Acad. Sd. USA, 91:4708-4712; DeWitt et al. (1993) Proc. Natl. Acad. ScL USA, 90:6909-6913). Briefly, at each successive synthetic step, one of a plurality of different selected substituents is added to each of a selected subset of tubes in an array, with the selection of tube subsets being such as to generate all possible permutation of the different substituents employed in producing the library. One suitable permutation strategy is outlined in US. Patent No. 5,763,263.

There is currently widespread interest in using combinational libraries of random organic molecules to search for biologically active compounds (see for example U.S. Patent No. 5,763,263). Ligands discovered by screening libraries of this type may be useful in mimicking or blocking natural ligands or interfering with the naturally occurring ligands of a biological target. In the present context, for example, they may be used as a starting point for determining GDF analogs which exhibit the biological properties of GDF. A GDF or a functional part thereof may according to the present invention be used in combination libraries formed by various solid-phase or solution-phase synthetic methods (see for example U.S. Patent No. 5,763,263 and references cited therein). By use of techniques, such as that disclosed in U.S. Patent No. 5,753,187, millions of new chemical and/or biological compounds may be routinely screened in less than a few weeks. Of the large number of compounds identified, only those exhibiting appropriate biological activity are further analysed.

With respect to high throughput library screening methods, oligomeric or small-molecule library compounds capable of interacting specifically with a selected biological agent, such as a biomolecule, a macromolecule complex, or cell, are screened utilising a combinational library device which is easily chosen by the person of skill in the art from the range of well-known methods, such as those described above. In such a method, each member of the library is screened for its ability to interact specifically with the selected agent, In practising the method, a biological agent is drawn into compound-containing tubes and allowed to interact with the individual library compound in each tube. The interaction is designed to produce a detectable signal that can be used to monitor the presence of the desired interaction. Preferably, the biological agent is present in an aqueous solution and further conditions are adapted depending on the desired interaction. Detection may be performed for example by any well-known functional or non-functional based method for the detection of substances.

Methods for determining the activity of GDF or an active fragment, or derivative, analog or mimetic of said GDF will be well known to those skilled in the art, for example, the culturing of cells in the presence of said GDF or an active fragment, or derivative, analog or mimetic of said GDF and using real-time PCR, FACS or immunostaining to compare the maintenance of Oct3/4, NANOG, TRA- 1-60 and/or SSEA-4 expression as hereinbefore described.

hi one example, the medium of the present invention comprises at least about 5ng/ml of GDF or an active fragment, or derivative, analog or mimetic of said GDF. Preferably, the medium comprises at least about 10 ng/ml or 20 lig/ml or 40ng/ml of GDF or an active fragment, or derivative, analog or mimetic of said GDF.

In one example, the medium of the present invention comprises an additional compound required for proliferation of a stem cell. For example, the medium comprises a basic fibroblast growth factor (bFGF) or an analog or derivative thereof. For example, the medium comprises a human bFGF, for example, comprising a sequence set forth in SEQ ID NO: 18. For example, the medium comprises at least about 2ng/ml or 4ng/ml or 5ng/ml of bFGF.

One exemplary form of a culture medium of the present invention maintains a stem cell in a substantially undifferentiated state without inducing genetic instability. In this respect, the inventors have identified a component of several commonly used culture media for stem cells that induces genetic instability, namely ascorbate or ascorbic acid. Without being bound by theory or mode of action, the inventors believe that the ascorbate or ascorbic acid induces signalling through kinase activity and genetic instability. Accordingly, in one example, the culture medium of the present invention does not comprise ascorbate or ascorbic acid, or comprises insufficient ascorbate or ascorbic acid to induce kinase activity or to induce or enhance genetic instability in a stem cell or to induce or enhance expression of CD30 or a variant thereof by a stem cell.

As used herein, the term "genetic instability" shall be taken to mean that a cell retains a karyotype normal for that cell type, for example, normal for the organism from which the cell is derived. An abnormal karyotype may comprise a deletion, an insertion, a translocation, an inversion, a duplication, etc. Methods for karyotypic analysis of the cell will be apparent to the skilled artisan and/or described herein. Alternatively, or in addition, genetic instability is detected by a surrogate marker, for example, detection of CD30 expression, hi this respect, CD30 expression by hES cells has been previously shown to be a marker of genetic instability and/or transformation in hES cells (Wolvetang et al, Nature Biotech., 24: 351-357, 2006). By "CD30" is meant a cell membrane protein of the tumor necrosis factor receptor family. This receptor is expressed by activated, but not by resting, T and B cells and by several pluripotent cell types. TRAF2 and TRAF5 can interact with this receptor, and mediate the signal transduction that leads to the activation of NF-κB. For the purposes of nomenclature and not limitation, a nucleic acid sequence of a CD30 cDNA is set forth in SEQ ID NO: 19 and protein in SEQ ID NO: 20. This term shall be taken to encompass detection of any splice variant of CD30, for example, expressing a nucleic acid comprising a sequence set forth in SEQ ID NO: 21 and/or a protein comprising a sequence set forth in SEQ ID NO: 22. CD30 expression or expression of a splice variant thereof can be detected at the nucleic acid or protein level by means well known in the art, such as, for example, those hereinbefore described.

hi one example, a culture medium of the present invention that maintains a stem cell in a substantially undifferentiated state without inducing genetic instability additionally comprises a compound required for proliferation of a stem cell. For example, the medium comprises a bFGF or an analog or derivative thereof. For example, the medium comprises a human bFGF, for example, comprising a sequence set forth in SEQ ID NO: 18. For example, the medium comprises at least about 20 ng/ml or 40 ng/ml or 50 ng/ml of bFGF.

In one example, a culture medium of the present invention that maintains a stem cell in a substantially undifferentiated state without inducing genetic instability comprises the components set forth in Table 3.

The present invention also provides a culture medium that does not induce genetic instability and/or CD30 expression in a stem cell, preferably a pluripotent stem cell, more preferably a liES cell, said medium comprising compounds necessary for proliferation of a stem cell and, preferably maintenance of a stem cell in an undifferentiated state, wherein said medium does not comprise ascorbate.

In one exemplified form of the invention, the medium comprises compounds listed in Table 4.

In one example, the medium additionally comprises a compound to induce or enhance proliferation of a stem cell, such as, for example, bFGF or an analog, derivative, or mimetic thereof, for example, a bFGF comprising a sequence set forth in SEQ ID NO: 18.

In another example, the medium additionally comprises a compound to reduce or prevent differentiation of a stem cell, such as, a GDF as defined herein or an activin-A, for example, comprising a sequence set forth in SEQ ID NO: 23 or an analog, derivative, or mimetic thereof.

In one example, the medium additionally comprises one or more factors secreted by a feeder cell, preferably a mouse or human embryonic fibroblast. In one example, the medium additionally comprises serum.

The present invention also provides a method for maintaining or culturing a stem cell in a substantially undifferentiated state, the method comprising contacting or culturing said stem cell with a compound that enhances or activates signal transduction in a stem cell mediated by a growth and differentiation factor (GDF) comprising a sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO: 2, for a time and under conditions sufficient for the stem cells to proliferate.

In one example, the present invention provides a method for maintaining or culturing a stem cell in a substantially undifferentiated state, the method comprising contacting or culturing said stem cell with a growth and differentiation factor (GDF) comprising a sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO: 2, for a time and under conditions sufficient for the stem cells to proliferate.

The present invention also provides a method for maintaining or culturing a stem cell in a substantially undifferentiated state, the method comprising contacting or culturing the stem cell with a growth and differentiation factor (GDF) comprising a sequence at least about 90% or 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in any one of SEQ ID NOs: 1 to 13, or an active fragment, derivative, analog or mimetic of said GDF, for a time and under conditions sufficient for the stem cells to proliferate.

Preferably, the stem cell is contacted with or cultured within a culture medium as described herein according to any embodiment.

In one exemplified form of the invention, the method comprises contacting or culturing said stem cell with a compound that enhances or activates signal transduction in a stem cell mediated by a growth and differentiation factor (GDF) comprising a sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO: 2 for a time and under conditions sufficient for the stem cells to proliferate, wherein said culturing or contacting is performed in the absence of ascorbate or ascorbic acid or in the presence of insufficient ascorbate or ascorbic acid to induce activin-like kinase signal transduction activity or in the presence of insufficient ascorbate or ascorbic acid to induce genetic instability.

The present invention also provides a method for culturing stem cells so as minimise or prevent the induction of genetic instability and/or so as not to induce expression of CD30 said method comprising maintaining a stem cell in a medium lacking ascorbate or ascorbic acid for a time and under conditions sufficient for the stem cell to proliferate.

In one example, the method additionally comprises determining the substantially undifferentiated state of a cell or population thereof derived from the stem cell. Methods for determining the differentiation state or undifferentiated state of a cell will be apparent to the skilled artisan and/or described herein, for example, detection of a nucleic acid or protein associated with a pluripotent cell as described supra, permitting cells to differentiate and detecting differentiated cells, or teratoma formation.

In another example, the method comprises detecting the genetic stability of cells derived from the stem cell, for example, by karyotype analysis and/or detecting expression of CD30 or a variant thereof.

The present invention also provides a method for producing a population of stem cells in a substantially undifferentiated state, said method comprising performing a method as described herein according to any embodiment to culture or maintain a stem cell for a time and under conditions sufficient for said stem cell to proliferate and produce a population of stem cells.

hi one example, the method of producing a population of stem cells additionally comprises isolating the stem cells from the culture, for example, using fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS).

The present invention also provides a method for producing a population of differentiated cells, said method comprising performing a method as described herein according to any embodiment to produce a population of stem cells and differentiating those stem cells to produce a population of differentiated cells.

Alternatively, the present invention provides a method for producing a population of differentiated cells, said method comprising obtaining a population of stem cells produced by performing a method as described herein according to any embodiment and differentiating those stem cells to produce a population of differentiated cells. In this respect, the population of stem cells may be produced by one party and obtained and differentiated by a different party.

The skilled artisan will be aware of differentiated cell populations that may be obtained from stem cells and methods for producing differentiated cell populations from stem cells. The present invention also provides a population of substantially undifferentiated human stem cells or differentiated cells produced by performing a method as described herein according to any embodiment.

The present invention also provides a kit for maintaining or culturing human stem cells in a substantially undifferentiated state, said kit comprising a cell culture medium as described herein according to any embodiment, hi one example, the GDF is packaged separately from other components of the medium. Optionally, the kit is packaged with instructions for use, for example, in a method as described herein according to any embodiment. It should be understood that the kit may also comprise other constituents such as, but not limited to, for example, growth factors, cells and/or antibodies to detect pluripotency markers as described herein.

The following Tables 2 to 4 represent examples of culture media which may be used in a medium of the present invention.

Table 3: Components of KOSR- supplemented with GDF and bFGF

The present invention is further described by reference to the following non-limiting Figures and Examples.

EXAMPLE 1

Methods

Bulk Culture

Human Embryonic Stem (hES) cell lines hES2 & 4 were cultured under standard bulk culture conditions using an irradiated mouse embryonic fibroblast (MEF) feeder layer and knockout serum replacement (KOSR) medium (Gibco) supplemented with 4 ng/ml recombinant bFGF (R&D Systems). Medium was replaced daily and cells were passaged weekly using Collagenase Type I (Worthingtons) and split approximately 1 :4-8.

Matrigel Culture

6-well plates were coated with Matrigel (BD Bioscience) according to the manufacturers specifications at a density of 0.0347 mg/cm2. Matrigel was allowed to polymerise and attach to plates for 24 hours at 37 ⁰C before gentle washing with sterile PBS to remove excess unpolymerised Matrigel. Combinations of conditioned medium (CM), unconditioned medium (UCM) and medium supplemented with GDF-8 (R&D SYSTEMS, INC. CAT NO: 788-G8) or GDF-I l (R&D SYSTEMS, INC. CAT NO: 1958-GD) were added to each well prior to transfer of bulk culture cells to Matrigel coated plates. To transfer bulk culture cells to Matrigel coated 6-well plates, day-7 bulk culture cells were treated with Collagenase Type I for 10-20 minutes, transferred to a 15 mL tube and collagenase inactivated with an equal volume of KOSR. Cells were centrifuged (1500 rpm, 1.5 min) and washed in KOSR before being split into 6-well plates at a ratio of 1 :6-10.

Feeder Free GDF-Il Cultures

T25 tissue culture flasks were coated with Matrigel as described previously. KOSR medium was supplemented with GDF-8 (R&D SYSTEMS, INC. CAT NO: 788-G8) or GDF-I l (R&D SYSTEMS, INC. CAT NO: 1958-GD) at 20 ng/ml. Collagenase Type I was used for all passaging of feeder free cells as described earlier.

RNA Isolation & cDNΛ synthesis

RNA was isolated using an RNeasy mini kit (QIAGEN) according to the manufacturers specifications. Cells were washed once with PBS prior to lysis and RLT lysis buffer was added directly to each well. RNA yields were determined using a NANOdrop spectrophotometer. cDNA was synthesised using a cDNA Archive Kit (Applied Biosystems) according to the manufacturers specifications using a total of 1 ug of total RNA in a 20 ul reaction.

Real-time PCR

Real-time PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems) according to the manufactures specifications. In a single 20 ul reaction 10 ul of master mix, 9 ul of cDNA template and 1 ul of probe was used. Oct3/4

(HsOl 895061_ul), NANOG (Hs02387400_gl) and Beta-Actin (Hs99999903_ml) probes (Applied Biosystems) were used for all real-time PCR reactions. AU samples were assayed in triplicate and relative expression calculated by Applied Biosystem Real-time PCR software. All data is expressed as an average of at least three independent experiments with error bars indicated the standard error of the mean.

Protein Isolation & Western Blot

Cells were washed once in PBS and then boiling hot Lamlei buffer (4% SDS, 20 % Glycerol, 10% Beta-mercaptoethanol, 0.0004% Bromophenol Blue, 0.125M Tris, pH8) was added to each well. Samples were transferred to 1 mL tubes and boiled for an additional 2 minutes and stored at -80°C until use. Protein samples were separated on a 12% Tris polyacrylamide gels and transferred to a nitrocellulose membrane. The membrane was blocked for 2 hours (TBST containing 5% skim milk powder) and then probed overnight at 4⁰C with the primary antibody (Oct3/4-Santa-Cruz, Nanog- eBioscience, Beta-rubulin). Membranes were probed with a secondary HRP antibody and bands visualised using enhanced chemiluminescent (ECL) solution (Pierce).

Immuno Staining and FA CS

For immuno staining cells were washed once with PBS and fixed with ice cold ethanol at - 20°C for 10 minutes. Plates were allowed to air dry and placed at -20⁰C until use. TRA-I- 60 (Millipore) was used at 1:100, SSEA-4 (Millipore) was used at 1 :100 and Oct3/4 (Santa Cruz) was used at 1:50. Cells were stained with primary antibodies diluted in KOSR overnight at 4°C. Secondary Alexa Flour antibodies in KOSR were used against the primary antibodies for 1 hour at RT. 4',6-diamidino-2-phenylindole (DAPI) was used to visualise the nucleus of fixed cells. For FACS analysis cells were harvested using cell dissociation buffer (Gibco) and stained with TRA- 1-60 in KOSR for 1 hour at RT. Cells were then incubated with an appropriate secondary Alexa flour antibody for 1 hour in KOSR at RT.

EXAMPLE 2 Results

GDF-8 maintains OctS/4 and NANOG mRNA expression over 7 days

Results demonstrate that GDF-8 (R&D SYSTEMS, INC. CAT NO: 788-G8) is able to maintain several important characteristics of undifferentiated hES cells. When observing the morphologies of cells growing under GDF-8 (R&D SYSTEMS, INC. CAT NO: 788- G8) supplemented conditions many similarities are obvious with cells being maintained by CM. hEC cells colonies are tightly packed and have well defined colony borders and individual cells display characteristics such as a large nucleus and small cytoplasmic space. This is consistent across two different hES cell lines HES2 and HES4.

Using real-time PCR it was demonstrated that 20 ng/ml of GDF-8 (R&D SYSTEMS, INC. CAT NO: 788-G8) is sufficient to maintain levels of Oct3/4 and NANOG at levels comparable to CM. Cells grown in UCM rapidly downregulate both Oct3/4 and NAOG after 7 days of culture. As Oct3/4 and NANOG are considered to be the major regulators of pluripotency, maintenance of expression of these two transcription factors is excellent evidence that GDF-8 may be able to maintain hES cells during prolonged feeder free culture. This data has been repeated in lines HES2 and HES4 so is not a cell line specific phenomenon.

Immunostaining using the markers of pluripotency TRA- 1-60, SSEA-4 and Oct3/4 also demonstrate maintenance of all these markers using GDF-8 (R&D SYSTEMS, INC. CAT NO: 788-G8). Maintenance of Oct3/4 expression by immunostaining validates the realtime data also showing maintenance of Oct3/4 expression. TRA- 1-60 and SSEA-4 are classical pluripotent stem cells markers and are maintenance of their expression is also good evidence that GDF-8 (R&D SYSTEMS, INC. CAT NO: 788-G8) will be able to maintain hES in a feeder free culture system for prolonged periods. Maintenance of TRA- 1-60 has also been demonstrated using FACS analysis and indicates that the percentage of cells positive for TRA-1-60 grown under GDF-8 (R&D SYSTEMS, INC. CAT NO: 788- G8) conditions is similar to that of cells grown in CM. The ALK4/5/7 inhibitor SB431542 inhibits the maintenance activity ofGDF-8

SB431542 is an inhibitor of the Activin type I receptors ALK4/5/7. SB431542 works by preventing the type I receptors phosphorylating the type II receptor and thus prevents the activation of an activin receptor signalling complex. The fact that SB431542 is able to inhibit the activity of GDF-8 (R&D SYSTEMS, INC. CAT NO: 788-G8) is strong evidence for the role of the Activin pathway in the maintenance of hES cells.

EXAMPLE 3

Results

GDF-Il maintains Oct3/4 and NANOG mRNA expression over 7 days

Oct3/4 and Nanog are considered the "master regulators" of pluripotency in human ES cells and their continued expression is an absolute requirement of the pluripotent state. Loss of Oct3/4 and Nanog expression causes spontaneous differentiation of the cells and renders them incapable of ongoing passaging. GDF-11 (R&D SYSTEMS, INC. CAT NO: 1958-GD) is sufficient to maintain the levels of Oct3/4 and Nanog in both hES2 and hES4 over 7 days in a feeder free Matrigel based culture system (Figures 2 and 4), indicating supplementation of UCM with GDF-Il is capable of maintain hES cells over long term feeder free culture.

GDF-Il Maintains Expression of hES cell Markers

TRA-1-60 and SSEA4 are both markers of pluripotent hES cells that are rapidly downregulated upon differentiation. GDF-I l (R&D SYSTEMS, INC. CAT NO: 1958-GD) is able to maintain expression of these markers over 7 days equal to that of CM (Figures 5, 6 and 7). Oct3/4 Expression as shown in Figure 6 is also maintained over a seven day period and validates the real-time PCR data indicating that GDF-I l (R&D SYSTEMS, INC. CAT NO: 1958-GD) is able to maintain expression of this pluripotent stem cell marker over 7 days. Taken together the immuno staining data indicate that GDF-Il (R&D SYSTEMS, INC. CAT NO: 1958-GD) like CM is able to maintain expression of 3 important markers of pluripotent hES cells as well as maintaining cell and colony morphologies identical to that of undifferentiated pluripotent hES cells.

Activin Receptor Inhibitor SB-431542 inhibits the activity of GDF-Il

SB 431542 inhibits the activity of TGF-/31 activin receptor-like kinases (ALKs). It is a selective and potent inhibitor of the phylo genetically related subset of ALK-4 (activin type I receptor), ALK-5 (TGFjS type I receptor), and ALK-7 (nodal type I receptor). In the presence of this inhibitor GDF-Il (R&D SYSTEMS, INC. CAT NO: 1958-GD) was unable to maintain hES2 cells in an undifferentiated state (Figure 8). mRNA levels of both were downregulated in the presence of this inhibitor, indicating the loss of the ability of GDF to maintain the expression of these markers (Figures 9, 12, 15). In addition, this inhibitor prevented GDF-I l (R&D SYSTEMS, INC. CAT NO: 1958-GD) from maintaining expression of the pluripotent hES cell marker TRA-1-60 Oct3/4 and Nanog (Figures 10, 13, 15, 16, 17).

Long Term Maintenance feeder free culture ofhESI using GDF-Il

After two weeks GDF-I l (R&D SYSTEMS, INC. CAT NO: 1958-GD) was able to maintain expression of TRA-1-60 at levels similar to that of CM.

EXAMPLE 4

Methods

HES Cell Culture

Human Embryonic Stem (hES) cell lines HES2 and HES4 were cultured under standard serum-free culture conditions using an irradiated mouse embryonic fibroblast (MEF) feeder layer and knockout serum replacement (KOSR) medium (Gibco) supplemented with 4 ng/ml recombinant bFGF (R&D Systems). Medium was replaced daily and cells were passaged weekly using Collagenase Type I (Worthingtons) and split approximately 1 :4- 1 :8. hES cell lines were only used up to weekly passage 8 when cultured under serum free conditions and then re-established from the stock cultures. Passage number of mechanically transferred hESC in 20% FCS stock cultures (P x) and subsequent weekly passage number in serum free culture (P y) is indicated as Px + y.

Short-term Feeder-Free Culture

6-well plates were coated with Matrigel (BD Bioscience) according to the manufacturers specifications at a density of 0.0347 mg/cni2. Matrigel was allowed to attach to plates for 24 hours at 37 °C before gentle washing with sterile PBS to remove unbound Matrigel. MEF conditioned KOSR medium was generated by adding 20 ml of KOSR medium with 4 ng/ml b-FGF to a T75 flask with 6 x 104 cells/crm MEFs and collected after 24 h. The MEF conditioned KOSR medium was next filtered through a 0.22 μm filter and an additional 4 ng/ml b-FGF was added before use on Matrigel cultures.

Either MEF-coήditioned medium (CM), unconditioned KOSR medium (UCM) or unconditioned medium supplemented with Myostatin or BMP-Il were added to each well prior to transfer of serum free cultured hESC to Matrigel coated plates. To transfer serum free cultured cells to Matrigel coated 6-well plates, day-7 serum free cultured cells were treated with Collagenase Type I for 10 minutes, transferred to a 15 mL tube followed by collagenase inactivation with an equal volume of KOSR. Cells were centrifuged (1500 rpm, 1.5 min) and washed in KOSR before being plated onto 6-well plates at a ratio of 1 :6- 1:10.

Long-term Feeder Free Cultures

T25 tissue culture flasks were coated with Matrigel for 24 hrs at 37⁰C. KOSR medium was supplemented with Myostatin or BMP-I l at the indicated concentrations with 20 ng/ml bFGF (R&D). Cells were passaged weekly using Collagenase Type I as described above. RNA Isolation & cDNA synthesis

RNA was isolated using an RNeasy mini kit (QIAGEN) according to the manufacturer's specifications. Cells were washed once with PBS prior to lysis and RLT lysis buffer was added directly to each well. RNA yields were determined using a NANO-drop spectrophotometer. cDNA was synthesised using a cDNA Archive Kit (Applied Biosystems) according to the manufacturer's specifications using a total of 1 ug of total RNA in a 20 ul reaction. cDNA was then diluted to a total volume of 400 μ\.

Real-time PCR

Real-time PCR was performed using TaqMan Universal PCR Master Mix (Applied Biosystems) according to the manufacture's specifications. In a single 20 ul reaction 10 μl of master mix, 9 μl of cDNA template and 1 μ\ of probe was used. Oct3/4 (Hs01895061_ul), NANOG (Hs02387400_gl), Brachyury (Hs00610080_ml), Pax2 (Hs00240858_ml), GATA4 (Hs00171403-al) were obtained from (Applied Biosystems) and a beta-Actin (Hs99999903_ml) probe was used to normalize real-time PCR data. Real time PCR assays were performed in triplicate on each sample and relative expression calculated by Applied Biosystem Real-time PCR software. All data are expressed as an average of at least three independent experiments with error bars indicating the standard error of the mean.

Protein Isolation & Western Blotting

Cells were washed once in PBS and harvested using RJDPA buffer (Pierce) containing Protease and Phosphatase inhibitors (Pierce) according to the manufacturer's specifications. Samples were transferred to 1 ml tubes, an equal volume of 2X Laemmli buffer (4% SDS, 20% Glycerol, 10% β-mercaptoethanol, 0.0004% Brome phenol blue, 0.125 M Tris, pH8) was added and samples heated to 100°C for 5 minutes. Samples were then stored at -80⁰C until used. Prior to SDS-PAGE samples were heated to 100°C for 5 minutes. Proteins were then separated by electrophoresis through 10% Tris-polyacrylamide gels and electro-transferred to a nitrocellulose membrane. The membrane was blocked for 2 hours (TBST containing 5% skim milk powder) and then probed overnight at 4⁰C with the primary antibodies to either (Oct3/4 1:50 (Santa-Cruz), Nanog 1:100 (eBioscience), Betatubulin dilution 1:2000 (Sigma- Aldrich) in TBST containing 5% BSA. Membranes were probed with the appropriate secondary HRP-conjugated antibodies (1:1000 DAKO (P0260)) and bands visualised using enhanced chemiluminescent (ECL) solution (Pierce). Images were acquired using the BioRad Gel ChemiDocxM XRS System.

Immunofluorescence staining

For immuno staining cells were washed once with PBS and fixed with ice cold ethanol at - 20°C for 10 minutes. Plates were allowed to air dry and placed at -2O⁰C until use. Fixed cultures were Blocked with KOSR for 1 hour at room temperature and incubated with TRA-1-60 (1:100, Millipore MAB4360), SSEA-4 (1:100, Millipore MAB4304) t,Oct3/4 (1:50, Santa Cruz sc-5279]) and Nanog (1:100, eBioscience 14-5769), pSMAD2/3 (1:100, Santa Cruz sc-11769). Cells were stained with primary antibodies diluted in KOSR overnight at 4° C. Cells were washed 3 times with PBS for 5 minutes. Secondary Alexa Fluor antibodies (1:1000 A21121, A21141, A21042, A21144) diluted in KOSR were added for 1 hour at RT to detect primary antibody binding. Cells were washed 3 times in PBS for 5 minutes per wash. Lastly cells were incubated with 0.1 μg/ml 4',6-diamidino-2- phenylindole (DAPI) for 2 minutes to visualise the nucleus of fixed cells.

Flow cytometry

For Flow cytometric analysis cells were harvested using cell dissociation buffer (Gibco) gently pipetted up and down to obtain a single cell suspension and incubated with TRA-1- 60 antibody (1:100 Millipore MAB4360) in KOSR for 1 hour at RT. Cells were washed 3 times in PBS for 5 minutes per wash. Cells were then incubated with the secondary antibody AlexaFluor IgM 488 (1 :200) Invitrogen A21042 ) for 1 hour in KOSR at RT and washed again in PBS in KOSR for 5 minutes. Finally PI (Sigma P4864) was added at 1 //g/ml for 2 min on ice for selection of live cells. Next data were collected using a Cytomics FC 500 series flow cytometry system (Beckman Coulter) and analysed with CXP software (Beckman Coulter). Gates for FACS analysis were set to the secondary only controls and only live cells were used for analysis.

Statistical Analysis

All graphs are displayed as averages of at least 3 experimental replicates. Error bars indicate standard error of the mean. AU statistical analysis was performed using a one-way ANOVA as calculated by the statistical program SigmaStat.

Results

Myostatin and BMP-Il maintain undifferentiated morphology of ItESC as well as POU5F1 and NANOG niRNA and protein expression over short term culture periods.

POU5fl (Oct4) and NANOG, together with SOX2, are considered part of the regulatory circuitry that control the pluripotent state in hES cells, hi order to rapidly and reliably quantify the ability of potential maintenance factors to promote undifferentiated growth of hESC a sensitive assay based on real-time PCR quantification of POU5fl and NANOG mRNA expression was developed. This assay was first validated by culturing hESC on progressively diluted MEF conditioned medium for 7 days followed by RNA extraction expression analysis of POU5fl (Oct4), Nanog and beta-Actin. As shown in Fig 31 A HES2 hESC cultured in MEF conditioned medium (CM) for 7 days display a high density of multilayered cells within each hES cell colony, well defined colony borders with few surrounding differentiated cells and cells within each colony displaying a high nucleus: cytoplasm ratio, typical of undifferentiated hES cells. Conversely, HES2 hESC grown in unconditioned medium (UCM) display obvious differentiation originating from the centre of each colony, a large proportion of flat cells that form a monolayer around the initial colony and that display a low nucleus: cytoplasm ratio. The colony borders of UCM cultured hES cells become less well defined as cells begin to migrate away from the edge of the colonies. The stepwise decrease in the percentage of CM in the culture medium leads to a progressive increase in differentiated hESC that is accompanied by a stepwise reduction in POU5fl and NANOG mRNA expression relative to beta-Actin (Fig 31B). In this and each subsequent experiment the data are expressed relative to the expression of these genes in parallel cultured hESC in MEF conditioned medium. This strategy reduces inter experimental variability brought about by variations in hESC background differentiation and variability in the quality of the MEF conditioned medium.

Prompted by a micro-array study comparing the transcriptomes of supportive and nonsupportive MEF 's the ability of the TGF-β family ligands Myostatin and BMP-I l to maintain hES cells in KOSR on feeder-free Matrigel coated dishes over 7 days was investigated. MEF conditioned medium (CM) and unsuppleniented KOSR with 4 ng/ml b- FGF were used as positive and negative controls, respectively. HES2 ITESC cultured in UCM supplemented with 20 ng/ml BMP-Il (Fig 31C) or 20 ng/ml Myostatin (Fig 31D) for 7 days display cell and colony morphologies similar to the cells grown in CM. Titration of these molecules indicated that a concentration >5 ng/ml of either BMP-I l (Fig 31C) or Myostatin (Fig 31D) was sufficient to produce a detectable maintenance effect based on morphological criteria. This same effect was also apparent in the HES4 hES cell line (data not shown), m agreement with the morphological differentiation assessment HES2 hES cells cultured in UCM supplemented with increasing concentrations of BMP-I l (Fig 31E) or Myostatin (Fig 31F) display progressively increased expression of POU5fl and NANOG mRNA . POU5fl and NANOG mRNA expression approached that of CM at approximately 20 ng/ml. Similar data were obtained for HES4 hESC. No significant increase in mRNA levels over that achieved at 20 ng/ml is observed with Myostatin or BMP-I l concentrations up to 80 ng/ml (not shown). Western blotting of protein extracts from BMP-I l (Fig 31G) and Myostatin (Fig 31H) cultured HES2 hESC shows maintenance of POU5fl and NANOG protein expression at levels similar to CM, confirming the real time PCR analyses.

Myostatin and BMP-Il maintain expression of the pluripotent markers SSEA4, TRA-I- 60, POUSfL and NANOG during short term passaging.

To further assess the self renewal ability of Myostatin and BMP-11, one week feeder free cultured liES cell cultures were fixed with cold ethanol and probed with antibodies for the pluripotency markers SSEA-4, TRA-1-60, POU5fl and NANOG. As shown in Fig 32 colonies of HES2 hES cells cultured in both CM and in UCM supplemented with either 20 ng/ml BMP-I l (Fig 32A) or 20 ng/ml myostatin (Fig 32B) display virtually uniform SSEA4, TRA-I- 60, NANOG and POU5fl expression in almost all colonies in the culture dish while hES cells grown in UCM for 7 days showed extensive loss of SSEA4, TRA-1- 60, POU5fl and NANOG protein expression over the 7 days of culture. Similar data were obtained with the HES4 hES cell line (data not shown). Flow cytometric analysis (Figure 32C) shows that 80% of HES2 hES cells cultured for 7 days in UCM supplemented with 20 ng/ml Myostatin or 20 ng/ml BMP-I l maintain TRA-1-60 expression, similar to hES cells maintained in MEF-CM, While only 40% of hES cells cultured in UCM for 7 days displayed TRA-1-60 expression.

BMP-Il activates the SMAD2/3 intracellular signalling cascade

Having established that Myostatin and BMP-11 are able to maintain HESC over short term culture periods whether these molecules caused increased phosphorylation of their downstream signalling molecules SMAD2/3 was analysed. As shown in Figure 33, hES cells stimulated with 20 ng/ml BMP-11 for 2 hours display increased nuclear expression of phosphorylated SMAD2/3, when compared to the unstimulated control.

BMP-Il and Myostatin support long-term maintenance of pluripotent hES cell in a feeder free culture system

The ability of 20 ng/ml BMP-11 or Myostatin to maintain undifferentiated growth of hESC over a prolonged culture period of 10 weekly passages was analysed. It was observed that when using these factors in conjunction with only 4 ng/ml bFGF hESC cultures show a progressively reduced growth rate and could only be passaged for 4-5 weeks (data not shown). Increasing the bFGF concentration to 20 ng/ml, however, allowed us to passage these cells for 10 passages, although still at a reduced growth rate as compared to MEF CM. Cells cultured in BMP-Il or Myostatin supplemented UCM displayed TRA-1-60 expression levels similar to the CM positive control at Passage 1 , Passage 6 and Passage 10 (Figure 34A) and express Nanog and POUfI mRNA at levels similar to hESC cultured in CM (Fig 34B).

SB431542 inhibits Myostatin and BMP-U mediated self-renewal in ItES cells

In mouse ES-cells it was shown that BMP-I l signals predominantly through the ALK4 receptor, however signalling can also be mediated by ALK5 and to a lesser extent by ALK7. Similarly, Myostatin has also been shown to signal via the ALK4 and ALK5 type I receptors in the mouse. SB431542 (SB) inhibits the activity of the Type I receptors ALK4, ALK5 and ALK7 by preventing their phosphorylation by Type II receptors. Therefore hES cells were treated by culture in CM and UCM supplemented with 20 ng/ml Myostatin or 20 ng/ml BMP-11 in the absence or presence of 10 μM SB431542 for 7 days.

In agreement with previously published data suggesting that self-renewal signalling brought about by MEF conditioned medium is reliant on the activity of ALK4, ALK5 and

ALK7, the ability of MEF-CM to maintain hESC was abolished in the presence of

SB431542, as indicated by the differentiated morphology of the cells and the reduction in

POU5fl and NANOG mRNA and protein expression (Figure 35A and B). Addition of the

SB431542 inhibitor to hES cells cultured in UCM supplemented with 20ng/ml BMP-Il (Fig 35A)or 20 ng/ml Myostatin (Fig 35B) also resulted in morphological signs of differentiation, a reduced POU5fl and NANOG mRNA and protein expression, as well as reduced TRA- 1-60 expression (Figure 35D)

Myostatin and BMP-H Support POUSfI and NANOG expression to a level similar to that ofActivin-A and greater than that ofbFGF

The short-term maintenance ability of Activin-A and bFGF with that of Myostatin and BMP-Il was compared. hES cells cultured for 7 days in UCM supplemented with either Myostatin (20ng/ml), BMP-Il (20ng/ml), Activin-A (20ng/ml) or bFGF (100ng/ml) exhibit a cell and colony morphology similar to that of undifferentiated hES cells (Figure 36A) and show TRA-I -60 and POU5fl expression similar to cells maintained in CM over the 7 day growth period (Figure 36B). Quantitative RT-PCR analysis of POU5fl and NANOG expression in 7 day cultures supplemented with Activin-A (20ng/ml), Myostatin (20ng/ml) or BMP-11 (20ng/ml) indicate that Myostatin and BMP-11 are equally potent as Activin-A in maintaining undifferentiated hES cell growth (Figure 36C). Using this more sensitive assay each of the tested TGF-jS ligands was found to be superior to bFGF (100ng/ml) alone when comparing POU5fl and NANOG levels relative to the CM controls.

Claims

CLAIMS:

1. A culture medium for maintaining or culturing a stem cell in a substantially undifferentiated state said culture medium comprising a compound that enhances or activates signal transduction in a stem cell mediated by a growth and differentiation factor (GDF) comprising a sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO: 2.

2. A culture medium for maintaining or culturing a stem cell in a substantially undifferentiated state said culture medium comprising a growth and differentiation factor (GDF) comprising a sequence at least about 90% or 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in any one of SEQ ID NOs: 1 to 13, or an active fragment, derivative, analog or mimetic of said GDF.

3. The culture medium of claim 1 wherein said compound modulates one or more of the activin-like kinase (ALK) receptor 4 or 5 or 7 and/or a type II BMP receptor and/or a

SMAD-2 and/or a SMAD-3 and/or a SMAD-4.

4. The culture medium of claim 3 wherein said compound enhances or activates signalling initiated or mediated by an ALK5 receptor.

5. The culture medium of any one of claims 1, 3 or 4 wherein said compound is a GDF comprising a sequence at least about 90% or 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in any one of SEQ ID NOs: 1 to 13, or an active fragment, derivative, analog or mimetic of said GDF.

6. The culture medium of any one of claims 1, 3 or 4 wherein said compound is a GDF comprising a sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

7. The culture medium of claim 5 wherein said compound is a GDF comprising a sequence as set forth in SEQ ID NO: 12 and/or SEQ ID NO: 13.

8. The culture medium of claim 5 wherein said compound is a GDF-8 comprising a sequence at least about 90% or 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in any one or more of SEQ ID NOs: 3-7 or 12.

9. The culture medium of claim 8 wherein said GDF-8 is a human GDF-8, comprising a sequence set forth in SEQ ID NO: 3 or 12.

10. The culture medium of claim 5 wherein said compound is a GDF-Il comprising a sequence at least about 90% or 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in any one or more of SEQ ID NOs: 8-11 or 13.

11. The culture medium of claim 10 wherein said GDF-Il is a human GDF-I l comprising a sequence set forth in SEQ ID NO: 8 or 13.

12. The culture medium according to any one of claims 1 to 11 which comprises at least about 5ng/ml of said GDF or an active fragment thereof, or a derivative, analog or mimetic of said GDF.

13. A culture medium according to claim 12 which comprises at least about IOng/ml or 20ng/ml or 40ng/ml of said GDF or an active fragment thereof, or a derivative, analog or mimetic of said GDF.

14. The culture medium according to any one of claims 1 to 13 which comprises the components listed in Table 2.

15. The culture medium according to any one of claims 1 to 14 which comprises an additional compound required for proliferation of a stem cell.

16. The culture medium according to any one of claims 1 to 15 which does not comprise ascorbate or ascorbic acid or comprises insufficient ascorbate or ascorbic acid to induce kinase activity and/or to induce or enhance genetic instability in a stem cell and/or to induce or enhance expression of CD30 or a variant thereof by a stem cell.

17. The culture medium according to claim 16 wherein the culture medium does not contain ascorbate or ascorbic acid.

18. The culture medium according to any one of claims 15 to 17 wherein the medium comprises human bFGF.

19. The culture medium according to claim 18 wherein the amount of bFGF present in the medium is at least about 2ng/ml or 4ng/ml or 5ng/ml.

20. The culture medium of any one of claims 15 to 17 wherein said culture medium comprises compounds listed in Table 3.

21. The culture medium of any one of claims 1 to 20 which comprises an additional compound to reduce or prevent differentiation of a stem cell.

22. The culture medium of claim 21 wherein said compound is activin-A or an analog, derivative or mimetic thereof.

23. The culture medium according to any one of claims 1 to 22 which lacks serum.

24. The culture medium according to any one of claims 1 to 23 which comprises the components set forth in Table 4.

25. The culture medium of any one of claims 1 to 24 wherein said stem cell is a human embryonic stem cell or an induced pluripotent stem cell.

26. A method for maintaining or culturing a stem cell in a substantially undifferentiated state, the method comprising contacting or culturing said stem cell with a compound that enhances or activates signal transduction in a stem cell mediated by a growth and differentiation factor (GDF) comprising a sequence set forth in SEQ ID NO: 1 and/or SEQ TD NO: 2, for a time and under conditions sufficient for the stem cells to proliferate.

27. A method for maintaining or culturing a stem cell in a substantially undifferentiated state, the method comprising contacting or culturing the stem cell with a growth and differentiation factor (GDF) comprising a sequence at least about 90% or 95% or 96% or 97% or 98% or 99% identical to a sequence set forth in any one of SEQ ID NOs: 1 to 13, or an active fragment, derivative, analog or mimetic of said GDF, for a time and under conditions sufficient for the stem cells to proliferate.

28. The method according to claim 27 wherein the GDF has a sequence as set forth in SEQ ID NOs I to 13.

29. A method of culturing a stem cell in a substantially undifferentiated state, the method comprising culturing the stem cell in the culture media according to any one of claims 1 to 25.

30. A method for culturing stem cells so as to minimise or prevent induction of genetic instability and/or expression of CD30 said method comprising maintaining a stem cell in a medium lacking ascorbate or ascorbic acid for a time and under conditions sufficient for the stem cell to proliferate.

31. A method for producing a population of differentiated cells, said method comprising performing a method according to any one of claims 26 to 30 for a time and under conditions to produce a population of stem cells and differentiating those stem cells to produce a population of differentiated cells.

32. A population of substantially undifferentiated human stem cells or differentiated cells produced by performing a method according to any one of claims 26 to 31.

33. A kit for maintaining or culturing human stem cells in a substantially undifferentiated state, said kit comprising a cell culture medium as described herein according to any one of claims 1 to 25.