WO2014198720A1

WO2014198720A1 - Method for cell differentiation

Info

Publication number: WO2014198720A1
Application number: PCT/EP2014/062027
Authority: WO
Inventors: Jianliang Li
Original assignee: GE Healthcare UK Ltd
Current assignee: GE Healthcare UK Ltd
Priority date: 2013-06-11
Filing date: 2014-06-10
Publication date: 2014-12-18
Anticipated expiration: 2015-12-11
Also published as: GB201310354D0

Abstract

The present invention relates to the field of differentiated stem cell production, particularly to the production of stem cell derived cardiomyocytes. The invention provides a method that uses follistatin as a biomarker to select for stem cells for stem cell differentiation in a simple streamlined process of cell production. The invention has particular application in the large scale production of populations of stem cell derived cardiomyocytes.

Description

Method for Cell Differentiation

Field of Invention The present invention relates to the field of differentiated stem cell production, particularly to the production of stem cell derived cardiomyocytes. The invention provides a method that uses follistatin as a biomarker to select for stem cells for stem cell differentiation in a simple streamlined process of cell production. The invention has particular application in the large scale production of populations of stem cell derived cardiomyocytes.

Background to the Invention

There are two main sources of pluripotent human stem cells; one source is from the isolation of human embryonic stem (hES) cells directly from the embryo, the other method involves the generation of induced pluripotent stem (IPS) cells by

reprogramming differentiated cells.

Stem cell based therapy has revealed considerable potential, especially in the field of regenerative medicine. Pluripotent stem cell can differentiate into multiple lineages, thereby offering the potential of developing cell compositions that can help reconstitute biological function in certain medical conditions. In addition, the differentiation of hES cells and/or IPS cells into specific cell types, such as heptaocytes and cardiomyocytes offers a novel cell source for toxicological screening and in vitro modelling of genetic disorders. "Pluripotent stem cells" are pluripotent cells which have the characteristic of being capable under appropriate conditions of producing progeny of different cell types that are derivatives of all of the three germinal layers (endoderm, mesoderm and ectoderm) or the ability to form identifiable cells of all three germ layers in tissue culture. Included in the definition of pluripotent stem cells are embryonic cells of various types, exemplified by human embryonic stem cells, described by Thomson et al. (Science, 1998, vol 282, 1 145-47), induced Pluripotent Stem (iPS) cells, described by Takahashi et al. (Cell, 2007, vol 131 , 861 -872) which involves the reprogramming of differentiated cells and parthenogenetic human embryonic stem cells (phESC), described by

Revazova et al. (Cloning Stem Cells, 2007, vol 9 (3), 432-449), which involves the growth and development of the embryo without fertilisation.

Recently Kilmanskaya et al. (Nature, 2006, vol 444, 481 -485) described a single blastomere biopsy method for isolating hES cells from single blastomeres without destroying the embryo. Furthermore Chung et al. (Cell Stem Cell, February 2008, vol 2(2): 1 13-7) demonstrated the derivation of five hES cell lines without embryo

destruction, including one without hES cell co-culture. The blastomeres were removed using a technique similar to preimplantation genetic diagnosis and the procedure did not appear to interfere with subsequent blastocyst development of the parent embryo.

Follistatin, also known as activin binding protein, is involved in the nearly irreversible binding and neutralization of activin. Follistatin was originally isolated from ovarian follicular fluid and shown to be capable of suppressing FSH (follicle stimulating hormone) secretion from pituitary cells. Follistatin is found in the pituitary gland and gonads, it is widely distributed among all tissues in which activin is present and a substantial amount is also found in the blood. There are three known isoforms of follistatin; FS-288, FS-303 and FS-315 (Schneyer et al., Differential Distribution of Follistatin Isoforms: Application of a New FS315-Specific Immunoassay, The Journal of Clinical Endocrinology & Metabolism 89(10); 5067-5075). The homo sapien follistatin protein sequence (Asscession number: AAH04107) was described in Proc. Natl. Acad. Sci. U.S.A. 99 (26), 16899-16903 (2002).

Follistatin and activin interactions are associated with the developmental process;

interactions take place during the processes of cartilage maturation, endochondral bone and osteoblast formation and bone remodelling. In addition a role for follistatin has been identified for such processes as erythroid/myeloid precursor differentiation, pancreatic islet cell function, hepatocellular regeneration and placental function during parturition (Welt et al., Activins, Inhibins, and Follistatins: From Endocrinology to Signaling. A Paradigm for the New Millennium, Experimental Biology and medicine 2002, 227:723- 752).

Interestingly Beattie et al., (Activin A Maintains Pluripotency of Human Embryonic Stem Cells in the Absence of Feeder Layers, Stem Cells, 2005, Vol 23, 4, 489-496) showed that activin and other TGF-β family members signalling through Smad2 and 3 were found to maintain pluripotency and self-renewal. Culture medium enriched with Activin A was capable of maintaining hES cells in an undifferentiated state for more than 20 passages without the need for feeder layers, conditioned medium from mouse embryonic feeder layers or STAT3 activation.

Albano and Smith (Follistatin expression in ES and F9 cells and in preimplantation mouse embryos, International Journal of Developmental Biology, 1994, volume 38, 543- 547) reported that expression of activin mRNA decreased in differentiated mouse ES cells and postulated that these decreasing levels of functional activin may be due to the simultaneous increase in follistatin.

Vallier et al. (Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells, Journal of Cell Science, 2005, volume 1 18, 19, 4495- 4509) demonstrated that blocking activin signalling resulted in hES cell differentiation. Inhibition of Activin/Nodal signalling by follistatin and by overexpression of Lefty or Cerberus-Short or by the Activin receptor inhibitor SB431542, precipitates hES cell differentiation.

WO2005/083438 (Monash University) describes a method of prognoses or monitoring the development or progress of a moderate grade cancer to a higher grade cancer in a mammal by screening for the level of one or more of activin pa, activin βο, inhibin a or follistatin protein or gene expression, wherein the increase in the level of the protein or gene expression is indicative of a neoplasm progressing to a higher grade.

US2006/0166214 (Yukio Kato et al) describes markers for detecting and separating mesenchymal stem cells and one of those markers is follistatin. The applicant measured the gene expression of follistatin in mesenchymal stems cells compared to fibroblasts, the expression ratio was 7.6.

US2009/0075380 (Smith et al) discloses methods for predicting the embryo

transplantation potential of human embryos by determining expression levels of follastatin. Thus, for example, oocytes and embryos with increased levels of

endogenous follastatin are preferentially selected for nuclear transfer and/or in vitro fertilization procedures.

Although advances have been made in improving differentiation strategies or directing differentiation of stem cells, such processes are still time consuming and expensive. Therefore it would be beneficial to know in advance of committing to the stem cell differentiation process if the stem cell culture is likely to efficiently produce a

differentiated cell population. There is therefore a need for an improved and simplified process for performing large scale production of cell populations derived from human pluripotent stem cells. The present invention addresses this problem and provides methods that use an early biomarker, follistatin, to indicate the differentiation potential of human pluripotent stem cells.

Summary of the Invention

The present invention provides methods which can be used to determine the differentiation efficiency or potential of a pluripotent stem cell population.

According to a first aspect of the present invention, there is provided a method for differentiating stem cells into mesoderm cells comprising the steps of i) measuring the level of follistatin in one or more populations of stem cells prior to differentiation, and

ii) selecting a population for differentiation into mesoderm cells on the basis of said level of follistatin.

The advantage of the method described is follistatin can be used as an early marker in stem cell production for determining whether or not to proceed with cell differentiation, thereby saving time on cell production and money that may be spent on differentiation reagents.

In one aspect, said mesoderm cells are cardiomyocytes.

In a further aspect, step i) involves measuring the level of expression of follistatin. In another aspect, step i) is performed between 1 to 7 days before cell differentiation is induced.

In one aspect, the level of follistatin is measured using a method selected from the group consisting of western blotting, ELISA and PCR.

In another aspect, the level of follistatin measured in the media is greater than 1 C^g/ml in a cell population of between 1x10⁵ and 1x10⁷ cells. In a further aspect, the level of follistatin is measured in the media after at least 24 hours after media refreshment.

In another aspect, the level of follistatin expression is measured in the media less than 24 hours after media refreshment and is defined as greater than 1 C^g/ml in a cell population of between 1 x10⁵ and 1x10⁷ cells.

In a further aspect, the expression level of a house keeping gene is measured and compared with the level of follistatin expression.

In one aspect, the house keeping gene is selected from the group consisting of GAPDH, beta-actin, 18s RNA and HPRT.

In a further aspect, the level of follistatin expression is defined as a Act greater than -8.0, wherein Act is the difference in the ct level between follistatin and the house keeping gene. In a further aspect, the method comprises an additional step iii) of measuring the level of myosin heavy chain positive cells.

In another aspect, said stem cells are primate pluripotent stem cells.

In one aspect, the primate pluripotent stem cells are selected from the group consisting of human embryonic stem cells, induced pluripotent stem cells and mesenchymal derived stem cells.

According to a second aspect of the present invention there is provided a method for predicting the ability of stem cells to differentiate into mesoderm cells, said method comprising: i) measuring the levels of follistatin in a population of stem cells, wherein, said level of follistatin is indicative of the ability of said population of stem cells to differentiate into mesoderm cells.

In one aspect, said mesoderm cells are cardiomyocytes.

In a further aspect, step i) involves measuring the level of expression of follistatin.

In another aspect, step i) is performed between 1 to 7 days before cell differentiation is induced. In one aspect, the level of follistatin is measured using a method selected from the group consisting of western blotting, ELISA and PCR.

In another aspect, the level of follistatin is measured in the media and is greater than 1 C^g/ml in a cell population of between 1x10⁵ and 1x10⁷ cells.

In a further aspect, the level of follistatin expression is measured in the media after at least 24 hours after media refreshment.

In a further aspect, additionally comprising the step of measuring the expression level of a house keeping gene and comparing said level with the level of follistatin expression.

In another aspect, the house keeping gene is selected from the group consisting of GAPDH, beta-actin, 18S rRNA and HRPT.

In a further aspect, the level of follistatin expression is defined as a Act greater than -8.0, wherein Act is the difference in the ct level between follistatin and the house keeping gene.

In one aspect, the method comprises an additional step ii) of measuring the level of myosin heavy chain positive cells. In another aspect, said stem cells are primate pluripotent stem cells.

In a further aspect, the primate pluripotent stem cells are selected from the group consisting of human embryonic stem cells, induced pluripotent stem cells and mesenchymal derived stem cells.

In another aspect, wherein the level of follistatin expression is measured by measuring the level of follistatin RNA production.

In a further aspect, wherein the level of follistatin expression is measured using an anti- follistatin antibody.

According to a third aspect of the present invention there is provided a use of follistatin as a biomarker for determining the ability of a population of stem cells to differentiate.

In one aspect, wherein said population of stems cells have the ability to differentiate into mesoderm cells.

In another aspect, wherein said mesoderm cells are cardiomyocyte cells.

According to a third aspect of the present invention there is provided a kit for predicting the ability of stem cells to differentiate into mesoderm cells comprising means for measuring the level of follistatin and instructions for carrying out the method as hereinbefore described.

Brief Description of the Figures

Figure 1 (a to g) shows the level of relative RNA expression of seven different markers (ACVR1 , ACVR2A, ACVR2B, BMPR1A, BMPR2, CER1 and FST) on day 12 of undifferentiated stem cell production versus the amount of myosin heavy chain RNA expression on day 28 of cardiomyocyte cell production.

Figure 2 shows the level of follistatin (FST) protein production (x-axis) on day 12 of stem cell production versus the percentage of myosin heavy chain positive cells (y-axis) on day 28 of cardiomyocyte production.

Figure 3 shows how the standard curve for the ELISA assay was prepared by several water based dilutions of the standard.

Detailed Description of the Invention

Chemicals and Materials Used

A list of the chemicals used in the experimental studies and their sources is given below: X-VIVO 10™ (Lonza, 04-743Q).

100ug/ml Recombinant Fibroblast Growth Factor β (rhFGFpl ) (Invitrogen, PGH0263). ^g/ml Recombinant Transforming Growth Factor β (rhTGFpi ) (R&D Systems, 240-B- 010).

200U/ml Collagenase (Gibco, 17104).

Growth Factor Reduced (GFR) Matrigel coated Flasks (BD Biosciences, 356231 ).

Harvesting Medium:

- 90ml DMEM (Gibco, 41965);

- 10ml FCS (Foetal calf Serum) [USDA] (PAA, A15-351 ). Adaptation Medium:

- 800ml Knockout D-MEM (Invitrogen, 10829-018);

- 200ml Knockout Serum Replacement for ES Cells (Invitrogen, 10828-028);

- 10ml MEM Non-Essential Amino Acids Solution 10 mM (100X) (NEAA) (Invitrogen, 1 1 140-050);

- 70μΙ of 1 in 10 dilution of 2-mercaptoethanol (Invitrogen, 31350-010);

- 80ng/ml 100 g/ml rhbFGF (Invitrogen, PGH0263);

- 0.5ng/ml 1 g/ml rhTGFpl (R&D Systems, 240-B-010).

Trypsin-EDTA (0.25%) (Invitrogen, 25200).

Trypsin-EDTA (0.05%) (PAA, L1 1 -004).

RPMI-B27:

- 1000ml RMPI Medium 1640 (Gibco, 21875);

- 20ml B27 Supplement (Gibco, 17504-044).

RMPI-B27 + BMP4 (1 mg):

Human Bone Morphogenetic Protein 4 (BMP4) (R&D Systems, 314-BP/CF).

RPMI-B27 + Activin A (1 mg):

Human Activin A (R&D Systems, 338-AC/CF). Counting Medium:

- 500ml DMEM (Gibco, 41965);

- 55ml FBS Gold (PAA, A15-151 ). DMSO (Sigma, D2650).

Reagents used in FACs analysis:

PBS (PAA, H15-002).

FBS (PAA, A15-151 ).

DMEM (Invitrogen, 41965).

Human IgG (Sigma, 14506).

I OXPWB (BD, 554723).

4% PFA (USB, 19443).

Mouse anti-MHC1 (Abeam, ab15).

Mouse anti-Oct 4 (Chemicon, MAB4401 ).

mlgG1 isotype control (BD, 554121 ).

Alexa Fluor 488 Goat Anti-Mouse lgG1 (BD, A21 121 ). Reagents used in PCR analysis: lllustra RNAspin Mini Isolation Kit (GE Healthcare, 25-0500-70). High Capacity cDNA Reverse Transcription Kits (Life Technologies, 4374966). Primer Synthesis Service desalt-grade (Sigma-Aldrich, N/A). SYBR® Green JumpStart™ Taq ReadyMix™ (Sigma-Aldrich, S4438-500RXN). MicroAmp® Optical 96-Well Reaction Plate (Life Technologies, 4316813). MicroAmp Optical Adhesive Film (Life Technologies, 431 1971 ). PTC-200 Thermal Cycler (MJ Research, BC-MJPC200). 7900HT Fast Real-Time PCR System (Life Technologies, 4351405).

Reagents used in follistatin ELISA assay:

Human Follistatin Immunoassay kit (R&D Solutions, DFN00)

Primer sequences: SEQUENCE No. 1 :

GAPDH Forward: gagtcaacggatttggtcgt

SEQUENCE No. 2:

GAPDH Reverse: gacaagcttcccgttctcag

SEQUENCE No. 3:

FST Forward: ggaaaacctaccgcaatgaa

SEQUENCE No. 4:

FST Reverse: gagctgcctggacagaaaac

Experimental Results

3.75x10⁶ WiCell H7 undifferentiated human embryonic stem cells (provided by Geron Corporation and also available from the National Institute of Health (NIH) - Cell Code GE07) are seeded into matrigel coated T75 cell culture flasks in a final volume of 30ml culture medium. The cell culture vessels were placed in a humidified incubator at 37°C± 1°C and 5% ±1 %CO₂. 48 hours post plating the culture medium is exchanged for adaptation medium. The adaptation medium is composed of KO-SR and KO-DMEM supplemented with 80ng/ml rhbFGF and 0.5ng/ml rhTGF-βΙ . Cells were fed daily from days 2 to 6. On day 7 post seeding the embryonic cells were replated at a density 3 times the initial seeding density into fresh matrigel coated culture flasks. Between days 8 to 1 1 the cell culture was maintained in the same adaptation medium. The medium was exchanged on days 8, 10 and 1 1 . On day 12 supernatant from undifferentiated human embryonic stem cells was collected from 14 batches of cells. Separately up to 5x10⁶ cells were harvested by trypsin or mechanical scrapping for RNA analysis studies from 6 batches of cells. The cells were washed in IxDPBS and centrifuged at 1200rpm for 3 minutes, the supernatant was discarded and the cell pellet was frozen at -80°C or directly used for RNA extraction.

On day 13 medium was replaced with RPMI-B27 medium containing 100ng/ml Activin A for the remaining cell cultures. On day 14 the medium was aspirated and replaced with 0.5ml RPMI-B27 medium with 10ng/ml BMP-4. No further medium exchange was performed until day 18.

Day 18 to Day 26 cells were maintained in RPMI-B27 medium and the medium was exchanged on Days 18, 20, 22, 24 and 26. At Day 28 the differentiated cell population was dissociated from the flask surface using trypsin. The cells were collected, centrifuged and washed. FACS analysis was used to analyse cardiomyocytes on day 28, an antibody to alpha myosin-heavy chain was used as a marker for cardiomyocyte positive cells and staining was measured using flow cytometry. RNA analysis and First strand cDNA synthesis was carried out as follows:

2x RT Master Mix was prepared by combining the reagents listed in Table 1 below.

Table 1

1 ug of RNA was added to the 10ul of RT Master Mix in a 0.2ul PCR tube and pipetted up and down twice. The PCR tubes were briefly centrifuged. The PCR tubes were added to a PTC-200 Thermal Cycler and the program used is listed in table 2 below.

Table 2

The subsequent cDNA sample produced was combined with Master Mix 1 and 2 described below in Table 3 for a total volume of 20μΙ.

Table 3 The PCR reaction mix with the cDNA sample was loaded into a 96-well PCR plate and run on the 7900HT Fast Real time PCR System and data were analysed via the relative expression route using GAPDH expression as the endogenous control.

The PCR thermocycler was set to:

40 cycles of: 2 minutes at 95°C, 15 seconds at 95°C, 1 minute at 60°C, 1 minute at 72°C. Finally 5 minutes at 72°C, 15 seconds at 95°C, 15 seconds at 60°C, 15 seconds at 95°C (2%ramp rate from 60 degree to 95 degree).

The PCR reactions that gave rise to a single peak in the melting curve were used as the criteria for acceptance. The results in figure 1 show the level of relative RNA expression of seven markers on day 12 of undifferentiated stem cell production versus the amount of myosin heavy chain RNA expression on day 28 of cardiomyocyte cell production. The gene expression shown in figure 1 is the relative expression to the house keeping gene GAPDH as an endogenous control. The error bar represents the standard variation of three technical replicates. The percentage of myosin heavy chain positive populations was shown in the bottom of the bar which was used as a quality marker for

cardiomyocyte production.

It was shown that follistatin (figure 1 G) was the marker with the best correlation in predicting the ability of the stem cells to differentiate into cardiomyocytes compared to the other six markers analysed.

ELISA analysis was carried out as follows:

The Human Follistatin Immunoassay kit from R&D Solutions (Catalogue number DFN00) was used. The reagents, except the conjugate, were brought to room temperature. The Wash Buffer was diluted from a 25x concentrated stock. The

Calibrator Diluent RD5L was diluted from 2x concentrated stock. The Follistatin

Standard was reconstituted into 160,000pg/ml with 1.0ml deionized or distilled water and remained at room temperature for 30 minutes. The standards were made as shown in figure 3.

15ul of cell culture supernatants were diluted in 135ul of RD5-21 or incubated in SR medium. Excess microplate strips were removed from the plate frame. 10Oul of assay diluent was added to each well. 10Oul of standard, control or sample was added to the respective wells. An adhesive strip was used to cover the plate and incubated for 3 hours at 2-8°C. Each well was aspirated and washed four times with 400ul of Wash Buffer. 200ul of cold Follistatin Conjugate was added to each well; the plate was covered with a new adhesive strip and incubated for 2 hours at 2-8°C. The plates were again washed four times with 400ul of Wash Buffer. 200ul of Substrate Solution (A+B, 1 :1 ) was added to each well, protected from light and incubated for 20 minutes at room temperature. 50ul of Stop Solution was added to each well, the colour in the wells changed from blue to yellow. If the colour in the wells was green or the colour change did not appear uniform, the plates were gently tapped to ensure thorough mixing. The optical density of each well was measured within 30 minutes using a microplate reader set to 450nm.

The results of the assay were only accepted when the standard curve was linear. The results in figure 2 show the level of follistatin (FST) protein production (x-axis) on day 12 of stem cell culture production versus the percentage of myosin heavy chain positive cells (y-axis) on day 28 of cardiomyocyte production.

The results in figure 2 confirm that the level of secreted FST protein in the culture medium on day 12 of stem cell culture production correlates with the percentage of cardiomyocytes, indicated by the percentage of myosin heavy chain positive cells on day 28 of cell production.

Claims

1 ) A method for differentiating stem cells into mesoderm cells comprising the steps of i) measuring the level of follistatin in one or more populations of stem cells prior to differentiation, and

2) The method according to claim 1 , wherein said mesoderm cells are

cardiomyocytes.

3) The method according to any preceding claim, wherein step i) involves

measuring the level of expression of follistatin.

4) The method according to any preceding claim, wherein step i) is performed

between 1 to 7 days before cell differentiation is induced.

5) The method according to any preceding claim, wherein the level of follistatin is measured using a method selected from the group consisting of western blotting, ELISA and PCR.

6) The method according to any preceding claim, wherein the level of follistatin measured in the media is greater than 1 C^g/ml in a cell population of between 1x10⁵ and 1x10⁷ cells.

7) The method according to claim 6, wherein the level of follistatin is measured in the media after at least 24 hours after media refreshment.

8) The method according to claim 3 to 7, wherein the expression level of a house keeping gene is measured and compared with the level of follistatin expression. 9) The method according to claim 8, wherein the house keeping gene is selected from the group consisting of GAPDH, beta-actin, 18s RNA and HPRT.

10) The method according to claims 8 and 9, wherein the level of follistatin

expression is defined as a Act greater than -8.0, wherein Act is the difference in the ct level between follistatin and the house keeping gene.

1 1 )The method according to any preceding claim, wherein the method comprises an additional step iii) of measuring the level of myosin heavy chain positive cells.

12) The method according to any preceding claim, wherein said stem cells are

primate pluripotent stem cells.

13) The method according to claim 12, wherein the primate pluripotent stem cells are selected from the group consisting of human embryonic stem cells, induced pluripotent stem cells and mesenchymal derived stem cells.

14) A method for predicting the ability of stem cells to differentiate into mesoderm cells, said method comprising:

i) measuring the levels of follistatin in a population of stem cells, wherein, said level of follistatin is indicative of the ability of said population of stem cells to differentiate into mesoderm cells.

15) The method according to claim 14, wherein said mesoderm cells are

cardiomyocytes.

16) The method according to claim 14 and 15, wherein step i) involves measuring the level of expression of follistatin. 17) The method according to claims 14 to 16, wherein step i) is performed between 1 to 7 days before cell differentiation is induced.

18) The method according to claims 14 to 17, wherein the level of follistatin is

measured using a method selected from the group consisting of western blotting, ELISA and PCR.

19) The method according to claims 14 to 18, wherein the level of follistatin is

measured in the media and is greater than 1 C^g/ml in a cell population of between 1x10⁵ and 1x10⁷ cells.

20) The method according to claims 19, wherein the level of follistatin expression is measured in the media after at least 24 hours after media refreshment.

21 ) The method according to claims 14 to 20, additionally comprising the step of measuring the expression level of a house keeping gene and comparing said level with the level of follistatin expression.

22) The method according to claim 21 , wherein the house keeping gene is selected from the group consisting of GAPDH, beta-actin, 18s rRNA and HRPT

23) The method according to claims 21 and 22, wherein the level of follistatin

24) The method according to claims 14 to 23, wherein the method comprises an additional step ii) of measuring the level of myosin heavy chain positive cells.

25) The method according to claims 14 to 25, wherein said stem cells are primate pluripotent stem cells. 26) The method according to claim 25, wherein the primate pluripotent stem cells are selected from the group consisting of human embryonic stem cells, induced pluripotent stem cells and mesenchymal derived stem cells.

27) The method according to claim 1 , wherein the level of follistatin expression is measured by measuring the level of follistatin RNA production.

28) The method according to claim 1 , wherein the level of follistatin expression is measured using an anti-follistatin antibody.

29) A use of follistatin as a biomarker for determining the ability of a population of stem cells to differentiate.

30) The use according to claim 29, wherein said population of stems cells have the ability to differentiate into mesoderm cells.

31 ) The use according to claim 30, wherein said mesoderm cells are cardiomyocyte cells.

32) A kit for predicting the ability of stem cells to differentiate into mesoderm cells comprising means for measuring the level of follistatin and instructions for carrying out the method of any of claims 1 to 28.