Method for preparing induced paraxial mesoderm progenitor (IPAM) cells and their use

Abstract

The present invention relates to an ex vivo method for preparing induced paraxial mesoderm progenitor (iPAM) cells, said method comprising the step of culturing pluripotent cells in an appropriate culture medium comprising an effective amount of an activator of the Wnt signaling pathway and an effective amount of an inhibitor of the Bone Morphogenetic Protein (BMP) signaling pathway.

Claims

1. An ex vivo method for preparing a cell population comprising induced human paraxial mesoderm progenitor (iPAM) cells, the method comprising: culturing human pluripotent cells in a basal culture medium comprising an effective amount of a R-spondin protein or a GSK-3 inhibitor, wherein the culturing results in the production of a human cell population comprising iPAM cells expressing Msgn1 and Tbx6.

2. An ex vivo method for preparing a cell population comprising induced human paraxial mesoderm progenitor (iPAM) cells, the method comprising: culturing human pluripotent cells selected from the group consisting of human embryonic stem cells or human induced pluripotent stem (iPS) cells, in a basal culture medium comprising an effective amount of a BMP4 inhibitor, and an effective amount of a R-spondin protein or a GSK-3 inhibitor, wherein the culturing results in the production of a human cell population comprising iPAM cells expressing Msgn1 and Tbx6.

3. The ex vivo method according to claim 1, wherein the R-spondin protein is selected from the group consisting of R-spondin 3, R-spondin 2, and a combination of R-spondin 3 and R-spondin 2.

4. The ex vivo method according to claim 3, wherein the R-spondin 3 protein is human R-spondin 3 protein encoded by SEQ ID NO: 1, or human R-spondin 3 isoform 2 protein, encoded by SEQ ID NO: 5.

5. The ex vivo method according to claim 3, wherein the R-spondin 2 protein is human R-spondin 2 encoded by SEQ ID NO: 3, human R-spondin 2 isoform 2, encoded by SEQ ID NO: 6, or human R-spondin 2 isoform 3, encoded by SEQ ID NO: 7.

6. The ex vivo method according to claim 2, wherein the BMP4 inhibitor is a protein selected from the group consisting of Noggin, Follistatin and Dorsomorphin.

7. The ex vivo method according to claim 6, wherein the BMP4 inhibitor is Noggin.

8. The ex vivo method according to claim 2, wherein the BMP4 inhibitor is a chemical inhibitor.

9. The ex vivo method according to claim 1, wherein the culture medium further comprises DMSO.

10. The ex vivo method according to claim 2, wherein the basal culture medium further comprises DMSO.

11. The ex vivo method according to claim 1, wherein the GSK-3 inhibitor is a chemical inhibitor.

12. The ex vivo method according to claim 11, wherein the GSK-3 inhibitor is CHIR99021 or LiCl.

13. The ex vivo method according to claim 2, wherein the GSK-3 inhibitor is a chemical inhibitor.

14. The ex vivo method according to claim 13, wherein the GSK-3 inhibitor is CHIR99021 or LiCl.

15. The ex vivo method according to claim 1, wherein the basal culture medium further comprises serum or a serum substitute.

16. The ex vivo method according to claim 2, wherein the basal culture medium further comprises serum or a serum substitute.

Description

FIGURES AND TABLES

(1) TABLE-US-00002 TABLE1 Sequencesoftheinvention Bank Reference Proteins number SEQID Sequences hRspondin3 NP-116173.2 SEQID MHLRLISWLFIILNFMEYIGSQNASRGRRQ (CAI20141.1) NO:1 RRMHPNVSQGCQGGCATCSDYNGCLSCKPR or LFFALERIGMKQIGVCLSSCPSGYYGTRYP Q9BXY4-1 DINKCTKCKADCDTCFNKNFCTKCKSGFYL HLGKCLDNCPEGLEANNHTMECVSIVHCEV SEWNPWSPCTKKGKTCGFKRGTETRVREII QHPSAKGNLCPPTNETRKCTVQRKKCQKGE RGKKGRERKRKKPNKGESKEAIPDSKSLES SKEIPEQRENKQQQKKRKVQDKQKSVSVST VH mRspondin3 NP-082627.3 SEQID MHLRLISCFFIILNFMEYIGSQNASRGRRQ NO:2 RRMHPNVSQGCQGGCATCSDYNGCLSCKPR LFFVLERIGMKQIGVCLSSCPSGYYGTRYP DINKCTKCKVDCDTCFNKNFCTKCKSGFYL HLGKCLDSCPEGLEANNHTMECVSIVHCEA SEWSPWSPCMKKGKTCGFKRGTETRVRDIL QHPSAKGNLCPPTSETRTCIVQRKKCSKGE RGKKGRERKRKKLNKEERKETSSSSDSKGL ESSIETPDQQENKERQQQQKRRARDKQQKS VSVSTVH hRspondin2 NP-848660.3 SEQID MQFRLFSFALIILNCMDYSHCQGNRWRRSK or NO:3 RASYVSNPICKGCLSCSKDNGCSRCQQKLF Q6UXX9-1 FFLRREGMRQYGECLHSCPSGYYGHRAPDM NRCARCRIENCDSCFSKDFCTKCKVGFYLH RGRCFDECPDGFAPLEETMECVEGCEVGHW SEWGTCSRNNRTCGFKWGLETRTRQIVKKP VKDTILCPTIAESRRCKMTMRHCPGGKRTP KAKEKRNKKKKRKLIERAQEQHSVFLATDR ANQ mRspondin2 NP-766403.1 SEQID MRFCLFSFALIILNCMDYSQCQGNRWRRNK NO:4 RASYVSNPICKGCLSCSKDNGCSRCQQKLF FFLRREGMRQYGECLHSCPSGYYGHRAPDM NRCARCRIENCDSCFSKDFCTKCKVGFYLH RGRCFDECPDGFAPLDETMECVEGCEVGHW SEWGTCSRNNRTCGFKWGLETRTRQIVKKP AKDTIPCPTIAESRRCKMAMRHCPGGKRTP KAKEKRNKKKRRKLIERAQEQHSVFLATDR VNQ hRspondin3 CAI20142.1 SEQID MHLRLISWLFIILNFMEYIGSQNASRGRRQ isoform2 or NO:5 RRMHPNVSQGCQGGCATCSDYNGCLSCKPR Q9BXY4-2 LFFALERIGMKQIGVCLSSCPSGYYGTRYP DINKCTKCKADCDTCFNKNFCTKCKSGFYL HLGKCLDNCPEGLEANNHTMECVSIVHCEV SEWNPWSPCTKKGKTCGFKRGTETRVREII QHPSAKGNLCPPTNETRKCTVQRKKCQKGE RGKKGRERKRKKPNKGESKEAIPDSKSLES SKEIPEQRENKQQQKKRKVQDKQKSGIEVT LAEGLTSVSQRTQPTPCRRRYL hRspondin2 Q6UXX9.2 SEQID MRQYGECLHSCPSGYYGHRAPDMNRCARCR isoform2 NO:6 IENCDSCFSKDFCTKCKVGFYLHRGRCFDE CPDGFAPLEETMECVEGCEVGHWSEWGTCS RNNRTCGFKWGLETRTRQIVKKPVKDTILC PTIAESRRCKMTMRHCPGGKRTPKAKEKRN KKKKRKLIERAQEQHSVFLATDRANQ hRspondin2 Q6UXX9-3 SEQID FRLFSFALIILNCMDYSHCQGNRWRRSK isoform3 NO:7 RGCRIENCDSCFSKDFCTKCKVGFYLHRGR CFDECPDGFAPLEETMECVGCEVGHWSEWG TCSRNNRTCGFKWGLETRTRQIVKKPVKDT ILCPTIAESRRCKMTMRHCPGGKRTPKAKE KRNKKKKRKLIERAQEQHSVFLATDRANQ mMsgn1 NM.019544.1 SEQID ATGGACAACCTGGGTGAGACCTTCCTCAGC NO:8 CTGGAGGATGGCCTGGACTCTTCTGACACC GCTGGTCTGCTGGCCTCCTGGGACTGGAAA AGCAGAGCCAGGCCCTTGGAGCTGGTCCAG GAGTCCCCCACTCAAAGCCTCTCCCCAGCT CCTTCTCTGGAGTCCTACTCTGAGGTCGCA CTGCCCTGCGGGCACAGTGGGGCCAGCACA GGAGGCAGCGATGGCTACGGCAGTCACGAG GCTGCCGGCTTAGTCGAGCTGGATTACAGC ATGTTGGCTTTTCAACCTCCCTATCTACAC ACTGCTGGTGGCCTCAAAGGCCAGAAAGGC AGCAAAGTCAAGATGTCTGTCCAGCGGAGA CGGAAGGCCAGCGAGAGAGAGAAACTCAGG ATGCGGACCTTAGCCGATGCCCTCCACACG CTCCGGAATTACCTGCCGCCTGTCTACAGC CAGAGAGGCCAACCGCTCACCAAGATCCAG ACACTCAAGTACACCATCAAGTACATCGGG GAACTCACAGACCTCCTCAACAGCAGCGGG AGAGAGCCCAGGCCACAGAGTGTGTGA hMsgn1 NM00110556 SEQID ATGGACAACCTGCGCGAGACTTTCCTCAGC 9.1 NO:9 CTCGAGGATGGCTTGGGCTCCTCTGACAGC CCTGGCCTGCTGTCTTCCTGGGACTGGAAG GACAGGGCAGGGCCCTTTGAGCTGAATCAG GCCTCCCCCTCTCAGAGCCTTTCCCCGGCT CCATCGCTGGAATCCTATTCTTCTTCTCCC TGTCCAGCTGTGGCTGGGCTGCCCTGTGAG CACGGCGGGGCCAGCAGTGGGGGCAGCGAA GGCTGCAGTGTCGGTGGGGCCAGTGGCCTG GTAGAGGTGGACTACAATATGTTAGCTTTC CAGCCCACCCACCTTCAGGGCGGTGGTGGC CCCAAGGCCCAGAAGGGCACCAAAGTCAGG ATGTCTGTCCAGCGGAGGCGGAAAGCCAGC GAGAGGGAGAAGCTCAGGATGAGGACCTTG GCAGATGCCCTGCACACCCTCCGGAATTAC CTGCCACCTGTCTACAGCCAGAGAGGCCAG CCTCTCACCAAGATCCAGACACTCAAGTAC ACCATCAAGTACATCGGGGAACTCACAGAC CTCCTTAACCGCGGCAGAGAGCCCAGAGCC CAGAGCGCGTGA mNoggin NP_032737 SEQID MERCPSLGVTLYALVVVLGLRAAPAGGQHY NO:10 LHIRPAPSDNLPLVDLIEHPDPIFDPKEKD LNETLLRSLLGGHYDPGFMATSPPEDRPGG GGGPAGGAEDLAELDQLLRQRPSGAMPSEI KGLEFSEGLAQGKKQRLSKKLRRKLQMWLW SQTFCPVLYAWNDLGSRFWPRYVKVGSCFS KRSCSVPEGMVCKPSKSVHLTVLRWRCQRR GGQRCGWIPIQYPIISECKCSC hNoggin EAW94528 SEQID MERCPSLGVTLYALVVVLGLRATPAGGQHY NO:11 LHIRPAPSDNLPLVDLIEHPDPIFDPKEKD LNETLLRSLLGGHYDPGFMATSPPEDRPGG GGGAAGGAEDLAELDQLLRQRPSGAMPSEI KGLEFSEGLAQGKKQRLSKKLRRKLQMWLW SQTFCPVLYAWNDLGSRFWPRYVKVGSCFS KRSCSVPEGMVCKPSKSVHLTVLRWRCQRR GGQRCGWIPIQYPIISECKCSC

(2) FIG. 1: R-spondin induces induced Paraxial Mesoderm progenitor (iPAM) cells fate.

(3) (A) Comparison of fluorescent Msgn1 Reporter activation (YFP positive cells (YFP+ cells)) after 4 days of differentiation of mES cells (Msgn1RepV), under default culture conditions in 15% FBS or 15% KSR medium, with or without recombinant mouse Rspo3 (10 ng/mL). YFP channel, 50. (B) Robustness of iPAM cells induction in response to mouse Rspo3 in 15% FBS medium. Triplicate wells measurements by flow cytometry. Error bar is s.e.m.

(4) FIG. 2: Flow-cytometry analysis of the induction of the Msgn1-YFP+ (induced Paraxial Mesoderm progenitor (iPAM) cells) population upon treatment with Rspo3.

(5) (A) Flow-cytometry analysis on the Msgn1RepV mES cells at day 0 of differentiation in 15% FBS medium. YFP+ population represents less than 1%. (B) Flow-cytometry analysis at day 4 of differentiation in 15% FBS medium supplemented with R-spondin3 10 ng/mL. YFP+ population represents more than 70% of the total population.

(6) FIG. 3: Paraxial mesoderm progenitors (induced Paraxial Mesoderm progenitor (iPAM) cells) characterization.

(7) (A) Differentiation of mouse Msgn1RepV reporter mES cells into iPAM cells after 4 days in culture labeled with an anti-YFP antibody and co-stained with Hoechst, 10. (B) qRT-PCR analysis of FACS sorted iPAM YFP positive population for the paraxial mesoderm progenitors specific genes Msgn1 and Tbx6, relative expression normalized to non iPAM YFP negative population expression level (fold enrichment).

(8) FIG. 4: R-spondin activity in induced Paraxial Mesoderm progenitor (iPAM) cells mediated by canonical Wnt signaling.

(9) (A) Comparison of the effect of different doses of Rspo3 on iPAM induction (% YFP positive cells) after 4 days of differentiation in 15% FBS medium in the presence or absence of the canonical Wnt inhibitor Dkk1. Concentrations in ng/mL. (B) Comparison of the efficiency of the four recombinant Rspo family members on iPAM induction (% YFP positive cells) after 4 days in differentiation in 15% FBS medium. Concentrations in ng/mL. (C) Luciferase detection in Msgn1RepV reporter mES cells transfected with a Batluc reporter construct for canonical Wnt signaling activation and cultured in the presence of Rspo3 (10 ng/mL), Dkk1 (50 ng/mL) and LiCl (5 mM) in low serum (1% FBS) containing medium. Treatment with Rspo3 strongly activates the canonical Wnt response in differentiating ES cells.

(10) FIG. 5: R-spondin activity can be mimicked by the GSK3beta inhibitor CHIR99021.

(11) Comparison of the efficiency of Rspo3 and CHIR99021 on iPAM induction (% YFP positive cells) after 3 and 4 days in differentiation 15% FBS medium. Concentrations are in ng/mL for Rspo3 or in M for CHIR99021.

(12) FIG. 6: DMSO has a positive effect on induced Paraxial Mesoderm progenitor (iPAM) cells induction.

(13) iPAM induction after 4 days of differentiation in 15% FBS medium containing 0.5% DMSO. Optimal iPAM induction is obtained by combining R-spondins and DMSO. Concentrations in ng/mL.

(14) FIG. 7: Rspo2 and 3 activity in defined medium.

(15) Analysis of the effect of recombinant mouse and human R-spondin 2 and 3 effect on iPAM induction (% of YFP positive cells) after 4 days of differentiation in 1% FBS (A) or 15% KSR (B) media. Concentrations in ng/mL.

(16) FIG. 8: Characterization of Populations of the Invention at day 18 of differentiation.

(17) From day 0 to day 4, mouse ES cells were differentiated in presence of Rspo3, followed by 15% FBS medium until day 18. Cell types were identified by tissue-specific antibody staining, namely Muscle (Desmin, green), Endothelium (PECAM1/CD31, green) and Cartilage (Alcian Blue).

(18) FIG. 9: Dermal and myogenic differentiation of the iPAM cells after 5 days of culture.

(19) ES cells were cultured for 4 days in FBS15%, DMSO 0.5% and 10 ng/ml Rspo3 and then switched to FBS 15% or FBS 1% or FBS1% plus Sonic Hedgehog (Shh) and Retinoic acid (F1ShhRA) or plus Shh, Noggin and LiCl (F1SNLi). Cells were harvested the next day and analyzed by qRT-PCR for the dermal marker Dermo1 (A) and the muscle marker Myf5 (B). Graphs show fold enrichment.

(20) FIG. 10: R-spondin induces iPAM fate in human ES cells.

(21) Comparison of the expression of paraxial mesoderm progenitor markers Brachyury (A), PDGFRa (B), Tbx6 (C), Msgn1 (D) measured by Q RT-PCR in HUES1 undifferentiated or cultured in 15% FBS containing medium with or without Rspo3 for up to 10 days. Relative expression to undifferentiated HUES1 cells is shown (fold induction).

(22) FIG. 11: Noggin promotes iPAM fate by counteracting BMP4 activity.

(23) BMP4 expression at day3 (d3) and day4 (d4) of differentiation in the presence of 10 ng/ml Rspo3 and DMSO 0.5%, with (RDN) or without (RD) addition of Noggin (200 ng/ml) Data are shown as normalized expression value. Grey colored data point is considered non significant. Data points are means of biological triplicate samples.

(24) FIG. 12: Molecular characterization of early stages of paraxial mesoderm differentiation.

(25) (A). Venn diagram comparing gene signature lists (GSL method) of posterior and anterior PSM domains and of the in vitro differentiated Msgn1RepV Venus positive ES cells harvested at day3 and 4, respectively, in presence of 10 ng/ml Rspo3, DMSO 0.5% and 200 ng/ml Noggin. Key signature genes shared between the PSM in vivo and in vitro the Venus positive ES cells (induced Paraxial Mesoderm progenitor (iPAM) cells) are highlighted (black boxes). Red arrow shows the gene signature shift between day3 and day4 of ES cells differentiation.

(26) (B) Representative genes from the Signature Gene lists common to the PSM and to the Msgn1RepV cells differentiated in vitro for 3 and 4 days (induced Paraxial Mesoderm progenitor (iPAM) cells). The genes shown were validated as strongly expressed and paraxial mesoderm specific by in-situ hybridization (data not shown). Whereas genes activated at day 3 were mostly specific to the posterior PSM, genes activated at day 4 clearly showed acquisition of anterior PSM identity.

(27) FIG. 13: Pax3-positive PSM progenitors differentiated from ES cells in vitro can generate muscle fibers in vivo.

(28) (A) Tibialis anterior muscles were collected after 1 month post-transplantation with in vitro differentiated Pax3-positive cells (induced Paraxial Mesoderm progenitor (iPAM) cells) labeled with a CAG-GFP lentivirus (Transverse section). Control (Crtl, non-grafted area) and engrafted area were stained (red) with antibodies against Dystrophin (DYS) and Laminin (LAMA1). Engrafted progenitors produce large areas of muscle fibers expressing dystrophin and Laminin. Nuclei are counterstained with Hoechst. Scale bar, 100 m.

(29) (B) Tibialis anterior muscle collected after 1 month post-transplantation (Transverse section). Control (Crtl) and Engrafted area were stained with antibodies against Myogenin (MYOG) and PAX7 (red). Nuclei are counterstained with Hoechst. For PAX7 panels, insert panels show GFP distribution. Scale bar, 100 m.

(30) (C) Engrafted cells express the embryonic myosin MyHCemb, MyHCI (slow) and MyHCperi/MyHC II (fast) (Left panel), and overlay with corresponding GFP/Hoechst conterstaining is shown in Right panel. For each antibody, grafted tissue is shown on top and control tissue is shown below. Scale bar, 200 m.

EXAMPLES

Material & Methods

(31) Cell Culture

(32) Undifferentiated mouse ES cells Msgn1RepV (E14 derived) were maintained on gelatin-coated dishes in DMEM supplemented with 15% fetal bovine serum (FBS; from PAA), penicillin, streptomycin, 2 mM L-Glutamine, 0.1 mM non essential amino acids, 1 mM sodium pyruvate, 0.2% -mercaptoethanol and 1,500 U/mL LIF. ES cells were co-cultured with mytomicin-inactivated MEFs (feeders). Undifferentiated human ES cells were cultured on plates coated with matrigel (BD Biosciences) in mTeSR medium (STEMCELL Technologies). Cultures were maintained in an incubator in 5% CO2 at 37 C.

(33) Differentiation of ES Cells

(34) ES cells were trypsinized and plated at various densities in gelatin coated, feeder-free, 24 well plates directly in serum-based (15% FBS) or serum-replacement (15% KSR, Invitrogen) conditions supplemented with factors, and DMSO (Sigma). Recombinant proteins were obtained commercially (R&D) and stock solutions were prepared according to manufacturer's recommendation. The GSK-3 inhibitor CHIR99021 and the BMP type I receptors inhibitor Dorsomorphin were purchased from Stemgent and prepared according to the manufacturer's recommendations. Fluorescent reporter analysis and image acquisition were done on a Zeiss Axiovert system.

(35) FACS Analysis and Cell Sorting

(36) Cell cultures were dissociated by trypsinization, analyzed by flow cytometry on a FACScalibur (BD Biosciences) according to YFP expression. Data were further analyzed with MoFlo software (Beckman Coulter) and FlowJo software.

(37) DNA Microarrays

(38) Mouse E9.5 embryo PSMs were microdissected and processed as previously described (Krol et al, 2011), and prepared samples were run on Affymetrix GeneChip arrays. For differentiated cell cultures, iPAMs were sorted by flow-cytometry based on their Msgn1-YFP+ expression, and samples were prepared for microarrays. Experiments were conducted on biological triplicates. Datasets were processed using Affymetrix Microarray Suite (MAS) 5.0.

(39) Gene Signature Lists Method

(40) A gene expression reference was created by using all microarrays of wild-type mouse tissues deposited in GEO corresponding to Affymetrix Mouse Genome 430 2.0 Arrays (GEO platform id GPL1261). Normalization was done by calculating the mean values for each microarray. The median values for the distribution of those mean values across all microarrays were determined. This median is then used as a scaling factor for each value on each microarray. Once all microarrays have been normalized, the median expression value for each probeset is defined as the reference value. Gene signature list specific to one experimental condition was generated by normalizing the corresponding microarray data like the reference dataset. Probeset whose normalized expression value is 10 times higher than the corresponding reference value is considered to be a signature gene of that condition.

(41) Quantitative RT-PCR

(42) Total RNA was extracted from ES cell cultures using Trizol (Invitrogen) or with the Rnaeasy plus mini-kit (Qiagen). RT-PCR was performed on 5 ng total RNA using QuantiFast SYBR Green RT-PCR Kit (Qiagen), appropriate primers and run on a LightCycler 480II (Roche). GAPDH was used as the internal control.

(43) Differentiated Culture Phenotyping

(44) Cell cultures were fixed with PFA 4% overnight at 4 C. Cells were incubated 20 minutes with a blocking solution composed of 1% fetal bovine serum and 0.1% Triton in Tris Buffered Saline (TBS). Primary antibodies incubation was performed overnight at 4 C. and antibodies working dilutions were as follow: anti-GFP (Abcam) was 1:1,000, anti-Desmin (DSHB) was 1:100, anti-CD31 (BD Pharmingen) was 1:100. After TBS washes, cells were incubated with AlexaFluor488-conjugated secondary antibodies (Molecular probes) at 1:500 for 30 minutes, and counterstained with Hoechst. Alcian Blue staining was done according to standard protocol.

(45) Cells Preparation and Transplantations in Injured Tibialis Anterior Muscles

(46) Msgn1RepV ES cell cultures were trypsinized after 4 days of differentiation, and iPAMs were sorted based on YFP fluorescence using a FACS Aria, or Moflow Astrios (BD). iPAMS were permanently labeled by transduction overnight with a CAG-GFP lentivirus (MOI of 20-30). To remove non-integrated viral particles, cells were washed several time and reincubated, 1 hour in medium before preparation for transplantation. Grafted muscles were collected after 1 month and processed for immunohistochemistry. Dissected Tibialis Anterior muscles were prepared for cryosections (12 m) as described previously [B. Gayraud-Morel B. et al., 2012]. Antibodies used in this study are anti-Dystrophin (Sigma), anti-Laminin (Sigma), Myogenin (Dako), Pax7 (DSHB) and GFP (Abcam). Antibodies for Myosins isoforms have been described in (S. J. Mathew Dev 138, 371 (2011). Tissue sections were incubated overnight with primary antibodies. Secondary antibodies conjugated with AlexaFluor (Molecular probes) were used at 1:500. Imaging was performed on a Zeiss Axio observer and images processed with Adobe photoshop.

(47) Results

Example 1: Use of an Activator of the Wnt Signaling Pathway

(48) Production of Induced Paraxial Mesoderm Progenitor (iPAM) Cells In-Vitro

(49) We first aimed at identifying key molecular players promoting differentiation of the paraxial mesoderm lineage from ES cells. First, we investigated the time-course of paraxial mesoderm induction during mouse ES cell differentiation after formation of embryoid bodies, in DMEM based medium supplemented with 15% Fetal Bovine serum (FBS15%). Differentiation in paraxial mesoderm progenitor cells was characterized by activation of the Brachyury/T, Tbx6, and Msgn1 markers detected by PCR. Our data suggest that between day 1 and 4 of culture, some differentiated cells are in a presomitic mesoderm-like stage.

(50) The Msgn1RepV Reporter ES Line Characterization.

(51) In order to follow the differentiation of ES cells toward the first stage of paraxial mesoderm differentiation (ie presomitic fate), which represents the first step of skeletal muscle differentiation after acquisition of a mesodermal identity, we generated a transgenic mouse ES cell line harboring a fluorescent reporter specifically expressed in paraxial mesoderm progenitors. We used the promoter from the mouse Msgn1, a gene specific for the presomitic mesoderm, to drive the expression of Venus (a modified YFP). The transgenic Msgn1RepV (Mesogenin1 Reporter Venus) mouse ES cell line was subsequently validated using the tetraploid aggregation method to generate embryos entirely derived from the transgenic ES cells. As expected, transgenic mouse embryos exhibit fluorescently labeled paraxial mesoderm tissue, thus, validating the tissue specificity of Venus expression in the transgenic ES cell line.

(52) R-Spondins Identification.

(53) In order to optimize the differentiation conditions for paraxial mesoderm progenitors, we developed a manual screening assay, testing candidate growth factors and drugs interfering with various signaling pathways on ES cells. The Msgn1RepV reporter cells were plated at a defined density in 24-well plates coated with gelatin (0.1%). Two basal culture media were selected: a DMEM based medium containing 15% fetal bovine serum (FBS, high serum) and a defined serum-free medium containing 15% KSR (Invitrogen/Gibco). These basal media were supplemented with candidate factors on day 0 of differentiation. Control and experimental conditions were cultured in parallel. Cells were left to differentiate for three to four days with medium changed on day 2 or 3. Cell cultures were analyzed on day 3 and 4 of differentiation visually and by flow cytometry for YFP+ population quantification.

(54) After 4 days of differentiation, control differentiation in 15% FBS results in a low and variable induction of YFP+ cells (typically 1 to 15% of the culture), and differentiation in defined medium 15% KSR (Invitrogen) results in an even lower induction (typically 1%). Among the set of candidates tested, we identified the secreted R-spondin3 protein as being able to increase dramatically the induction of YFP+ cells. In our assay, R-spondin3 at 10 ng/mL is sufficient to increase significantly the induction of YFP+ cells both in FBS based medium and KSR based medium, up to 70% (FIGS. 1, 2 and 7). The R-spondin3 response saturated between 30 to 100 ng/mL. While at day 0, the YFP+ population is <1% of the cells, in R-spondin3 supplemented differentiation medium, YFP+ population can represent more than 50%, up to 70% of the cells at day4 (FIG. 2B). In human ES cells, induction of the paraxial mesoderm progenitor markers, Brachyury, PDGFRa, Tbx6 and Msgn1 is observed after 3 to 10 days of culture in 15% FBS containing medium when treating huES1 with R-spondin3 (FIG. 10).

(55) Paraxial Mesoderm Progenitors Characterization.

(56) To confirm induction of a paraxial mesoderm progenitor cell fate upon differentiation of ES cells in vitro, we sorted the YFP+ cell population after four days of differentiation in presence of R-spondin3 (FIG. 3A) and analyzed the YFP+ versus YFP cells by qRT-PCR for the key paraxial mesoderm markers Msgn1 and Tbx6 (FIG. 3B). We confirmed that the YFP+ population strongly expresses the Msgn1 endogenous gene, as well as Tbx6, demonstrating that we are able to generate paraxial mesoderm progenitors (iPAM) in vitro.

(57) R-Spondin Family and Wnt Signaling.

(58) We next asked whether other members of the R-spondin family can induce paraxial mesoderm progenitor iPAM cells (Msgn1-YFP+ paraxial mesoderm progenitors). ES cells were cultured in medium containing recombinant R-spondin proteins (R-spondins 1-4) supplemented with 15% FBS and allowed to differentiate for 4 days (FIG. 4B). Two members of the family, R-spondin2 and R-spondin3, exhibit comparable activities and significantly increase the number of YFP+ cells. The activity of R-spondin family proteins has been associated with canonical Wnt/Beta catenin signaling (Kim et al., 2008; Nam et al., 2006) and more recently with Wnt/PCP signaling (Ohkawara et al., 2011). We analyzed the effect of the inhibition of canonical Wnt signaling on R-spondin dependent differentiation using the secreted Dkk1 inhibitor, (FIG. 4A). Supplementation of the medium with the extracellular Wnt antagonist Dkk1 results in a sharp decrease of YFP+ induction. Moreover, adding Dkk1 to FBS-containing medium blocks the effect of R-spondin3, suggesting that R-spondin3 effect is mediated by the Wnt canonical pathway. We also analyzed the expression of luciferase from a plasmid driven by a promoter responding to canonical Wnt signaling (BAT-luc) transfected in ES cells treated or not with R-spondin3, and with Dkk1 or with the compound LiCl which can activate the Wnt pathway (FIG. 4C). Luciferase was strongly activated by R-spondin3 treatment suggesting that it activates the canonical Wnt pathway in this context.

(59) To further test whether R-spondin3 effect is mediated by the Wnt canonical pathway, we tested the effect of CHIR99021, a well described GSK-3 inhibitor (Ring et al., 2003). FIG. 5 shows that after 4 days, CHIR99021 is as efficient as Rspo-3 in inducing YFP+ cells, suggesting that R-spondin3 effect is mediated by activation of the canonical Wnt pathway.

(60) Dimethyl sulfoxide (DMSO) has been shown to promote the differentiation of several cell types, notably mesoderm from the P19 Embryonic Carcinoma (EC) cell line (McBurney et al., 1982; Skerjanc, 1999). The exact mechanism of action of DMSO in cell culture is not known, and it has been hypothesized that DMSO modifies the plasma membrane properties, making the cells more responsive to extracellular signals present in the differentiation medium. Addition of 0.5% of DMSO to FBS-containing medium, results in an increase of YFP+ cells after 4 days in culture (FIGS. 5, 6 and 7), although this increase is modest compared to the increase due to the addition of R-spondin2 or R-spondin3, or both. Interestingly, the addition of R-spondins and DMSO synergizes to enhance paraxial mesoderm progenitors differentiation (FIGS. 5, 6 and 7). Optimal conditions for paraxial mesoderm differentiation were observed when both DMSO and R-spondin 2 and/or 3 were combined (FIGS. 5, 6 and 7). Importantly, this effect is also seen in a serum-free, defined KSR based medium (FIG. 7B).

(61) Paraxial Mesoderm Progenitors Differentiation Potential

(62) We next explored the differentiation potential of the iPAM cell population. In vivo, paraxial mesoderm progenitor cells are fated to become skeletal muscles, vertebral tissues (cartilage, bone), dorsal dermis, endothelium, and other tissues such as adipose tissues.

(63) Thus, we performed sequential differentiation protocols, aiming at first generating iPAM cells, and then differentiating them further in 15% FBS medium or by applying various described differentiation protocols (see below), in particular Myogenic and Chondrogenic media.

(64) For example, between day0 to day4, ES cells were exposed to optimized differentiation conditions (ie. R-spondin3 10 ng/mL, DMSO 0.5%, in 15% FBS basal medium). On day 4, culture medium was changed and cells were exposed to specific differentiation media until day 18, with medium replacement every 3 days. At day 18, cell cultures were fixed and analyzed by tissue specific histochemical staining or immunofluorescence (FIG. 8). Under optimized differentiation conditions, cell cultures were positive for Cartilage (Alcian blue positive nodules), Muscles (Desmin positive fibers) and Endothelium (CD31/PECAM1). Alternatively, after 4 days in differentiation conditions (ie. Rspo3 10 ng/mL, DMSO 0.5%, in FBS15% basal medium), cells were switched to FBS 15% or FBS 1% or FBS1% plus Sonic Hedgehog (Shh) and Retinoic acid (F1ShhRA) or plus Shh, Noggin and LiCl (F1SNLi). Cells were harvested the next day and analyzed by qRT-PCR for the dermal marker Dermo1 and the muscle marker Myf5 (FIG. 9). Significant activation of these markers was observed indicating differentiation of the iPAM cells toward the dermal and muscle lineages respectively.

(65) Myogenic Protocol:

(66) Alternatively, induced paraxial mesoderm progenitors (iPAM) cells can be differentiated in two-dimensional culture into muscle cells using SFO3 medium complemented with BMP4, ActivinA and IGF-1 for 3 days, followed by 3 days of SFO3 medium complemented with LiCl and Shh.

(67) Induced paraxial mesoderm progenitors (iPAM) cells can be cultured in a hanging drop for 3 days at 800 cells/20 uL in differentiation medium, composed of DMEM supplemented with 10% fetal calf serum (FCS), 5% horse serum (Sigma), 0.1 mM 2-mercaptoethanol, 0.1 mM nonessential aminoacids, and 50 ug/ml penicillin/streptomycin. After 3 days, the medium is changed and cell aggregates are transferred on a low attachment plate. At day 6, cells are plated and cultured in differentiation medium on plates coated with Matrigel (BD Bioscience, Bedford, Mass., USA). Myogenic differentiation is achieved by withdrawal of FBS from confluent cells and addition of 10 ug/ml insulin, 5 ug/ml transferrin, and 2% horse serum.

(68) Induced paraxial mesoderm progenitors (iPAM) cells can also be cultured for 3 weeks in Skeletal Muscle Cell Medium (Lonza, Chemicon) complemented with EGF, insulin, Fetuin, dexamethasone, and bFGF (100 ng/mL).

(69) Osteogenic Protocol:

(70) For skeletal lineages, Induced paraxial mesoderm progenitors (iPAM) cells are exposed to 200 ng/ml human or mouse recombinant BMP4 or a combination of 1 uM retinoic acid and 10 mM Lithium Chloride. Alternatively, cells are plated on gelatin-coated plates at a density of 1-310^3 per well (24-well plate) and cultured for 28 days in bone differentiation medium (DMEM, 10% FBS, 2 mM 1-Glutamine, 1 Penicillin/streptomycin (P/S), 0.1 M dexamethasone, 50 M ascorbic acid 2-phosphate, 10 mM -glycerophosphate, 10 ng/mL BMP4) in order to observe cells expressing bone specific markers or secreting alcian blue positive extracellular matrix. Differentiated skeletal cell lineages are identified using specific stainings for extracellular matrix components of bone and cartilage including alcian blue or alizarin red, as well as by immunofluorescence using chondrocyte- and/or osteocyte specific antibodies.

(71) Induced paraxial mesoderm progenitors (iPAM) cells can also be differentiated into the bone lineage using the following differentiation medium composed of DMEM, 10% FBS, 2 mM L-Glutamine, 1P/S, 0.1 mM Dexamethasone, 50 mM ascorbic acid 2-phosphate, 10 mM beta-glycerophosphate, and 10 ng/mL BMP4, and vitamin D3 for 20 days, with medium changed every 3 days. Bone formation can be confirmed by staining the differentiating culture with Alizarin red, well known in the art that results to stain differentiated bone in red. Extracellular accumulation of calcium can also be visualized by von Kossa staining. Alternatively, differentiating cells can be lysed and assayed for ALP activity using BBTP reagent. Alternatively, differentiating cells can be analyzed for osteoblast lineage markers expression, for example Osterix (Osx) and Cbfa1/Runx2, alkaline phosphatase, collagen type I, osteocalcin, and osteopontin.

(72) Chondrogenic Protocol:

(73) For chondrogenic cell differentiation, induced paraxial mesoderm progenitors (iPAM) cells are plated at a density of 810^4 per well (24-well plate) and cultured for 30 minutes in a 37 C incubator in cartilage differentiation medium (MEM, 10% FBS, 2 mM 1-Glutamine, 1P/S, 0.1 M Dexamethasone, 170 M ascorbic acid 2-phosphate). Next, an equal amount of cartilage cell differentiation medium with 10 ng/mL TGF beta3 is added to the well. After one week, the medium is replaced with cartilage differentiation medium supplemented with 10 ng/mL Bmp2. After 21 days cartilaginous nodules secreting extracellular matrix can be observed. Induced paraxial mesoderm progenitors (iPAM) cells can also be differentiated into cartilage cells using a differentiation medium based on alphaMEM, 10% FBS, 2 mM L-Glutamine, 1P/S, 0.1 mM Dexamethasone, and 170 mM ascorbic acid 2-phosphate or DMEM supplemented with 0.1 mM dexamethasone, 0.17 mM ascorbic acid, 1.0 mM sodium pyruvate, 0.35 mM L-proline, 1% insulin-transferrin sodium, 1.25 mg/ml bovine serum albumin, 5.33 ug/ml linoleic acid, and 0.01 ug/ml transforming growth factor-beta), as well as TGFbeta3 or BMP2. Cells are cultured for several weeks, with medium changed every 3 days. Differentiation can also be performed at high density on 3D scaffold such as Alginate beads in a DMEM based medium containing 10% FBS and antibiotic supplemented with 100 ng/ml recombinant human Bone Morphogenic Protein-2 (BMP-2) and 50 mg ascorbic acid. Cartilage formation can be confirmed by Alcian Blue staining of the differentiating culture, well known in the art that results in the staining of Muco-glycoproteins in blue. Alternatively, a safranin O staining can be performed.

(74) Dermal Fibroblast Protocol:

(75) Induced paraxial mesoderm progenitors (iPAM) cells can be differentiated into dermal fibroblasts by culturing them on a scaffold of collagen in medium containing a fibroblast growth factor such as bFGF (basic Fibroblast Growth Factor) or a member of the Wnt family of growth factors.

(76) Next, to confirm that R-spondin also induces paraxial mesoderm progenitors (iPAM) cells from human ES cells differentiation, HUES1 cells were plated as single cells and differentiated in 15% FBS containing medium with or without Rspo3. qRT-PCR time course analysis for paraxial mesoderm progenitor markers expression was performed (FIG. 10). Strong activation of Msgn1 and Tbx6 during hES cells differentiation in presence of R-spondin3, demonstrate that iPAM cells can be differentiated from hES cells.

Example 2: Use of an Activator of the Wnt Signaling Pathway and an Inhibitor of BMP Signaling Pathway

(77) Characterization of the Msgn1-YFP+ Cell Population

(78) Using R-spondin proteins and DMSO, we are able to produce in a single step 70% of Msgn1-YFP+ cells (FIG. 2B). To directly compare the transcriptome of Msgn1-YFP+ to their in-vivo counterpart (ie. presomitic mesoderm, PSM), we used microarrays to generate a global transcriptome profiling of consecutive mouse fragments representing progressively more mature stages of differentiation (data not shown). The PSM fragments spanned from the tail bud level to the somitic region where the myogenic program is first activated. Based on this microarray series, we were able to define sets of signature genes defining the progressive maturation stage of cells from the tail bud (epiblast) to the presomitic mesoderm and somitic stages. In parallel, we differentiated ES cells in presence of R-spondin3 and DMSO (RD), sorted the Msgn1-YFP+ cell population and generated microarrays of this population at day3 and day4 of differentiation respectively. Global transcriptomes and signature genes sets were compared between the in vivo PSM and in vitro differentiated Msgn1-YFP+ cells (data not shown).

(79) We confirm that Msgn1-YFP+ cells express a number of key paraxial mesoderm markers already validated by Q-PCR such as Msgn1 and Tbx6 (FIG. 3B). We noticed that, in contrast to the native PSM cells, the Msgn1-YFP+ population also activates cardiac and angiogenic markers, which are a signature of more ventral/lateral mesoderm derivatives. Also, we found that TGF/BMP signaling tends to be up-regulated in Msgn1-YFP+. Unexpectedly, we found that the Msgn1-YFP+ cell population expresses BMP4 at a significant level whereas BMP4 is detected only at very low level in vivo in the PSM. This was problematic because BMP4 has been shown to prevent cells to acquire a paraxial mesoderm fate at the expenses of lateral plate fates (Pourqui O et al, 1996, Tonegawa A et al, 1997).

(80) Noggin to Counteract BMP4 Signaling

(81) To counteract this BMP4 activity, we tested the effect of addition of recombinant Noggin (Nog) protein known to inhibit BMP4, to the differentiation medium. We found that addition of Noggin from day 0 or day 1 of differentiation, Msgn1-YFP+ cells effectively repress BMP4 expression compared to cells cultured in differentiation medium lacking Noggin. We further defined both the optimal concentration and timing of Noggin addition. Noggin does not change the total number of Msgn1-YFP+ induced Paraxial Mesoderm progenitor (iPAM) cells (efficiency of production) or the total cell number in culture but rather changes the maturation of the Msgn1-YFP+ (efficiency of maturation). To better characterize the impact of Noggin on Msgn1-YFP+ cells, we performed microarrays on the Msgn1-YFP+ induced paraxial mesoderm progenitors (iPAM) population at day 3 and 4 of differentiation (FIG. 11, compare conditions RDN and RD).

(82) We show that adding Noggin to the medium represses BMP4 expression in the Msgn1-YFP+ cells, leading to the upregulation of several PSM specific markers including Tbx6, Pcdh8, Pax3, Foxc1, Raldh2 and Ripply2 (FIG. 12B and data not shown).

(83) BMP Inhibitors and BMP Signaling

(84) We detected strong upregulation of the BMP inhibitors Follistatin (Fst) and Cerberus (Cer1) in the mouse PSM microarray series and other BMP inhibitors such as Chordin, Noggin, and Gremlin1 are known to be expressed by the adjacent tissues during development [McMahon J A et al, 1998; Stafford D A et al, D2011 and Scott I C et al, 2000]. This suggests that in vivo the PSM requires BMP inhibition to mature properly, and that in vitro BMP inhibition is also required to the proper maturation of iPAM cells.

(85) We screened a set of BMP inhibitors, including recombinant proteins Noggin (Nog), Chordin (Chd), Chordin-like 1 (Chdl1), Follistatin (Fst), Follistatin-like 1 (Fstl1), Dan family protein including Cerberus 1 (Cerberus) and Gremlin 1 (Grem1); at varying concentration range (10-200 ng/mL), and various time-window (day 0-4, day 1-4) and analyzed the impact on BMP4 and the PSM marker Tbx6 expression. Additionally, we tested the chemical compound Dorsomorphin (Compound C), a specific BMP type I receptor inhibitor, at various concentration (0.1-1 M) and various time-window (day 0-4, day 1-4). Addition of the BMP inhibitors does not affect the number of induced paraxial mesoderm progenitors (iPAM) cells or the total cell number in culture (data not shown). Among the set of candidates tested, we identified in particular Noggin (Nog), Follistatin (Fst) and Dorsomorphin as promoting induced paraxial mesoderm progenitors (iPAM) maturation by counteracting BMP4 and activating Msgn1 and Tbx6 expression (data not shown).

(86) To monitor the PSM identity of Msgn1RepV cells differentiated in vitro, we purified them by FACS after both 3 and 4 days of differentiation in the presence of R-spondin3 and Noggin. qRT-PCR analysis confirmed that the sorted population strongly expresses Msgn1 and Tbx6, as expected for PSM cells (FIG. 3B). For a more systematic analysis, gene signatures were generated for both time-points as described above. Comparison of Day3 differentiated ES gene signature to that of the PSM transcriptional domains revealed that the differentiated ES cells expressed a large number of genes of the posterior PSM including T, Rspo3 and Fgf8 (FIG. 12A-B). Thus the cells expressing the Msgn1 reporter bear a close molecular resemblance to their counterparts in vivo.

(87) We then asked whether the maturation process could be pursued further in vitro. Strikingly, Day4 but not Day3 differentiated ES cells were found to activate genes specific for the anterior PSM such as Mesp2, Ripply2 or Foxc1, indicating that these cells have acquired an anterior PSM identity (FIG. 12B). In particular, these Day4 cells significantly upregulated the Pax3 gene. Pax3 regulates the differentiation of the myogenic lineage and, recently, overexpression of Pax3 in ES cells was shown to induce muscle [Darabi, R et al., 2008]. This suggested that, over time, R-spondin3 and Noggin treatment can transform undifferentiated ES cells into bona fide Pax3 positive muscle progenitors without genetic manipulation.

(88) In conclusion, these results demonstrate the potential use and the synergistic effect of an activator of the Wnt signaling pathway like R-spondin3 and of an inhibitor of the Bone Morphogenetic Protein (BMP) signaling pathway like Noggin to obtain induced paraxial mesoderm progenitor (iPAM) cells.

Example 3: Generation of Myogenic Lineage In Vivo

(89) To further drive the terminal differentiation of these precursors, we next took an in vivo approach. Pax3-positive cells obtained after 4 days of differentiation in medium containing R-spondin3 and Noggin were transduced with a GFP-expressing lentivirus to permanently label them. They were then injected into the Tibialis anterior muscle of adult Rag2/:c/ immunocompromised mice that had been injured by intramuscular injection of the snake venom cardiotoxin [Gayraud-Morel B. et al., 2012]. Examination of the transplanted muscles after 1 month showed that the grafted GFP-expressing cells reconstituted large areas filled with muscle fibers expressing laminin and dystrophin (n=3; FIG. 13A). The fibers expressed high levels of the differentiation marker Myogenin, had a small diameter and were not aligned as in adult muscle suggesting that they might correspond to embryonic primary fibers (FIG. 13B) [Gayraud-Morel B. et al., 2009]. This was confirmed by showing that these fibers expressed embryonic and perinatal isoforms of myosin heavy chain (FIG. 13C). Remarkably, a significant population of cells expressing the satellite cell-specific marker Pax7 was observed in the engrafted regenerating area suggesting that the differentiated ES cells were also able to produce a progenitor pool of muscle cells (FIG. 13B). Thus, when transplanted in vivo in injured muscles, Pax3-positive cells derived in vitro are able to continue their differentiation toward the myogenic lineage.

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