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Title:
MESODERMAL PROGENITOR CELLS
Document Type and Number:
WIPO Patent Application WO/2015/172207
Kind Code:
A1
Abstract:
The present invention relates to methods for culturing and generation of mesodermal progenitor cells, more particularly methods to produce mesodermal progenitor cells, starting from pluripotent stem cells. The invention relates to methods for isolation of such cells by selecting them on the basis of the expression of marker genes such as CD44, CD140a and CD140b. The invention is also directed to the cells produced by the methods of the invention. The cells are useful, among other things, for treatment of disorders, diseases or injuries, such as conditions leading to deterioration of mesodermal derivative tissues (e.g. congenital myopathies, heart diseases and skeletal muscle diseases).

Inventors:
SAMPAOLESI Maurillo ('s Hertogenwijngaard 41, Leuven, B-3000, BE)
QUATTROCELLI Mattia (Bloemenberggang 15, Leuven, B-3000, BE)
Application Number:
BE2015/000021
Publication Date:
November 19, 2015
Filing Date:
May 11, 2015
Export Citation:
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Assignee:
KATHOLIEKE UNIVERSITEIT LEUVEN KU LEUVEN RESEARCH & DEVELOPMENT (Waaistraat 6 - box 5105, Leuven, B-3000, BE)
International Classes:
C12N5/0775; A61K35/28; A61K35/44; A61P9/00; A61P21/00
Domestic Patent References:
WO2013005053A22013-01-10
Foreign References:
US20090317909A12009-12-24
US20110306131A12011-12-15
Other References:
DIMARIO J X: "Myogenesis: Methods and Protocols", 2012, METHODS IN MOLECULAR BIOLOGY, ISBN: 978-1-61779-342-4, article QUATTROCELLI M ET AL: "Mouse and human mesoangioblasts: isolation and characterization from adult skeletal muscles", pages: 65 - 76, XP008177297, 798
QUATTROCELLI M ET AL: "Cell therapy strategies and improvements for muscular dystrophy", CELL DEATH AND DIFFERENTIATION, vol. 17, no. 8, August 2010 (2010-08-01), pages 1222 - 1229, XP055211410, ISSN: 1350-9047, DOI: 10.1038/cdd.2009.160
SAMPAOLESI M ET AL: "Cell therapy of primary myopathies.", ARCHIVES ITALIENNES DE BIOLOGIE, vol. 143, no. 3-4, September 2005 (2005-09-01), pages 235 - 242, XP055209453, ISSN: 0003-9829
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Claims:
CLAIMS

1. An in vitro method to produce mesodermal progenitor cells from pluripotent stem cells, the method comprising:

a) culturing pluripotent stem cells in a mesoderm inducing medium; and b) isolate mesodermal progenitor cells by selecting the differentiated cells of step (a) that express all three factors CD44, CD140a and CD140b.

2. The method of claim 1 , wherein step (a) further comprises a step wherein the pluripotent stem cells are cultured in a low- or non-attachment vessel.

3. The method according to claim 1 or 2, wherein the isolation in step (b) is performed by any method which can select for cells that express said factors such as fluorescence- activated cell sorting (FACS), transgenic cell tracing and immunolabeling-based isolation.

4. The method according to any of claims 1 to 3, wherein the mesoderm inducing medium of step (a) comprises at least one factor selected from the group consisting of holo- transferrin, thyoglycerol and ascorbic acid.

5. The method according to any of claims 1 to 4, wherein step (a) further comprises:

(i) an embryoid body (EB) formation step wherein the pluripotent stem cells are cultured in low- or non-attachment vessels, which allow them to form embryoid bodies;

(ii) an embryoid body maturation step wherein the EBs of step (i) are further committed to the mesodermal phenotype by culturing them in medium comprising at least one factor selected from the group consisting of holo- transferrin, thyoglycerol and ascorbic acid; and

(iii) a cell spreading step wherein the matured EBs of step (ii) are further cultured in vessels for adherent culture.

6. The method of claim 5, wherein the cells of step (iii) express the factors Brachyury, MixM , Meoxl , Mespl , Pax3, Pax7, Desmin, Gata4, Tbx5, and Flkl

7. The method of any of claims 1 to 6, wherein the pluripotent stem cells are human cells.

8. The method of any of claims 1 to 7, wherein the pluripotent stem cells are iPSCs.

9. The method of claim 8, wherein the iPSCs are mesoangioblast-derived iPSCs.

10. The method of any of the previous claims, wherein the serum concentration in step a, (i), (ii), and (iii) is about 0 % to about 25%, and preferentially about 20% in step (i) and preferentially about 15%) in step (ii) and (iii).

1 1. The method of claim 4 or 5, wherein the concentration of holo-transferrin is about 1 μ9/ιηΙ to about 500 μ9/ηιΙ, and preferentially about 250 μ9/ιηΙ; the concentration of thyoglycerol is about 1 mM to 10 mM, and preferentially about 5mM; and the concentration of ascorbic acid is about 1 μg/rr)\ to 200 μ9/ιηΙ, and preferentially about 60 μg/ml.

12. The method of any of claims 2 to 1 1 , wherein the low- or non-attachment vessels are non-culture-treated plastic dishes.

13. The method of any of claims 5 to 12, wherein said vessels for adherent culture are collagen-coated cell-culture-treated plastic vessels.

14. The method of any of the previous claims, wherein the cells are cultured in step (a) for about 0 days to about 14 days.

15. The method of any of the previous claims, wherein the cells are cultured in one or more steps for at least 1 day, and preferentially for about 3 days.

16. The method of any of the previous claims, wherein the cells are cultured in each step for at least 1 day.

17. The method of any of the previous claims, wherein the cells are cultured in each step for about 3 days.

18. The method of any of the previous claims, wherein the cells are cultured in step (a) for about 9 days.

19. The mesodermal progenitor cells produced by the method of any of the previous claims and characterized in that said cells express the factors CD44, CD140a, CD140b, Brachyury, Mixl1 , Meoxl , Mespl , Pax3, Pax7, Desmin, Gata4, Tbx5, and Flk1.

20. A pharmaceutical composition comprising the mesodermal progenitor cells of claim 19 and one or more pharmaceutically acceptable carriers.

21. The pharmaceutical composition of claim 20, which comprises a therapeutically effective amount of said mesodermal progenitor cells.

22. The mesodermal progenitor cells of claim 19 or the pharmaceutical composition of claim 20 or 21 for use in medical therapy.

23. The mesodermal progenitor cells of claim 19 or the pharmaceutical composition of claim 20 or 21 , for administration to a subject in need thereof.

24. The mesodermal progenitor cells of claim 19 or the pharmaceutical composition of claim 20 or 21 for use in a medical therapy for the treatment of a myopathic patient.

25. The mesodermal progenitor cells of claim 22 or 23, wherein the medical therapy or the administration is for a patient or subject that has: a congenital myopathy; a heart disease; a skeletal muscle disease; and/or any condition leading to deterioration of mesodermal derivative tissues.

26. The mesodermal progenitor cells of any of claims 22 to 25, wherein the cells are autologous.

27. The mesodermal progenitor cells of any of claims 22 to 25, wherein the cells are allogeneic.

28. The mesodermal progenitor cells of any of claims 23 to 27, wherein the subject or patient is a mammal, preferentially a human.

29. A method of repairing or treating a damaged tissue in a patient, comprising administering to the patient the mesodermal progenitor cells of claim 19 or the pharmaceutical composition of claim 20 or 21 , wherein said cells or pharmaceutical composition comprise a therapeutically effective amount of said cells.

30. The method of claim 29, wherein the patient is a mammal, preferentially a human.

31. The method of claim 29 or 30 wherein said patient has: a congenital myopathy; a heart disease; a skeletal muscle disease; and/or any condition leading to deterioration of mesodermal derivative tissues.

Description:
MESODERMAL PROGENITOR CELLS

FIELD OF THE INVENTION

The present invention relates to methods for culturing and generation of mesodermal progenitor cells, more particularly methods to produce mesodermal progenitor cells, starting from pluripotent stem cells. The invention relates to methods for isolation of such cells by selecting them on the basis of the expression of marker genes such as CD44, CD140a and CD140b. The invention is also directed to the cells produced by the methods of the invention. The cells are useful, among other things, for treatment of disorders, diseases or injuries, such as conditions leading to deterioration of mesodermal derivative tissues (e.g. congenital myopathies, heart diseases and skeletal muscle diseases). The invention is also directed to cell banks that can be used to provide cells for administration to a subject, the banks comprising cells having a desired potency for achieving the effects as described in this invention. The invention is also directed to compositions comprising cells of specific potency for achieving these effects, such as in pharmaceutical compositions. The cells can be characterized by one or more of the following: extended replication in culture and expression of markers of extended replication, such as telomerase; expression of markers of mesodermal lineages and broad differentiation potential, more precisely differentiation potential to certain mesodermal lineages; absence of tumorigenic or transformed phenotypes; and a normal karyotype.

BACKGROUND OF THE INVENTION

Muscular dystrophies (MDs) are congenital myopathies, featuring irreversible degeneration of the cardiac and skeletal muscles. No curative treatments are still available for MD patients. Stem cell-based medicine is a promising tool for MD treatment as it aims at providing novel functional fibers to the degenerating muscles. However, the stem cell-based treatments currently under pre-clinical or clinical trials aim at the regeneration of one muscle type, namely either the skeletal muscle or the heart. Induced pluripotent stem cells (iPSCs) present a unique and unprecedented potential for further development of stem cell strategies. In fact, iPSCs are reprogrammed from somatic cells via transient overexpression of pluripotency factors or via specific chemical cues. Once stabilized in culture, iPSCs can virtually give rise to all tissues of the body and still match donor's genotype. Also, iPSCs can be coaxed towards the mesodermal lineage, for example in the presence of HGF and fibronectin matrix (US2011/0306131). Interestingly, iPSCs can present "epigenetic memory", namely a l commitment bias, inherited from the source cells, towards a determined lineage. However, combining the differentiation efficiency and safety of iPSC derivatives is still troublesome, given the high cellular heterogeneity and the difficult removal of uncommitted cells. Therefore, iPSCs could pave the way to novel complex therapeutical approaches for systemic myopathies, yet a combined iPSC-based approach for both cardiac and skeletal muscle regeneration in vivo is still lacking.

In this perspective, several scientific/methodological/clinical questions still remain open:

• lack of a unique cell pool to regenerate both cardiac and skeletal muscles in vivo;

• lack of a unique cell pool to regenerate both muscle types via systemic delivery;

• lack of a system to generate said unique cell pool with isolation/expansion properties compatible with the clinical needs;

• lack of a system to generate this unique cell pool in autologous setting with the recipient;

• lack of a system to generate this unique cell pool without genomic engineering and/or specific matrices.

There is therefore a clear need in the field for a novel cellular system with more differentiation and expansion potential and which reveals a more pure cell pool with broader applications. The existing methods for inducing/generating mesodermal progenitor cells are still lacking the properties as mentioned hereabove, e.g. the method of Rudy-Reil (US2011/0306131) describes the induction of mesoderm derived cells from pluripotent stem cells, that comprises a differentiation step with a mesoderm associated factor, but that method lacks a purification/selection step such that the generated cells are still highly diverse and not useful for further (medical) applications.

The present invention provides more pure mesoderm progenitor cells, which are suitable for many applications, including medical applications such as applications for disorders or injuries wherein cardiac and skeletal muscles need to be repaired. Therefore, the present invention provides a novel method for obtaining transgene-free, expandable, injectable mesodermal progenitor cells from pluripotent cells; such pluripotent cells can be of mammalian origin, comprising murine, canine and human. Moreover, the present invention provides mesodermal progenitor cells, which can be used for differentiation applications for mesoderm derivative tissues, comprising cardiac and skeletal muscles. Thus the present invention is the first invention specifically combining pluripotent cells with a novel isolation method, without requiring genetic engineering, genomic manipulation or supportive matrices, for yielding a cell product suitable for regeneration of mesoderm derivative tissues such as both cardiac and skeletal muscles, in the context of diseases such as muscular dystrophies for which there is still a huge need in the field.

SUMMARY OF THE INVENTION

The present invention provides mesodermal progenitor cells, more specifically mesodermal progenitor cells derived from pluripotent stem cells, using a unique method comprising a unique selection/isolation step.

In a first aspect, the present invention provides for a method to produce purified mesodermal progenitor cells, such method comprising a purification/selection step wherein the mesodermal cells are selected by their expression pattern, comprising at least one, preferably at least two and more preferably all three markers from the group: CD44, CD140a and CD140b. Said selection/isolation step can be a sequential isolation process wherein the cells are first selected for one marker (said marker being selected from CD44, CD140a and CD140b) followed by a second round wherein the first selected cells are further selected by selecting for a second marker from said group of markers and potentially further selected for the third marker of said group of markers. Alternatively said selection/isolation step can be performed by selection for expression of 2 genes or 3 genes of the group consisting of CD44, CD140a and CD140b.

One embodiment of the present invention comprises the selection for CD44 expressing cells, another embodiment comprises the selection of CD140a expressing cells and yet another embodiment comprises the selection of CD140b expressing cells. In other embodiments of the present invention, said selection step comprises an isolation of CD44 and CD140a expressing cells, another embodiment comprises the isolation of CD44 and CD140b expressing cells, and yet another embodiment comprises the isolation of CD140a and CD140b expressing cells. In yet another preferred embodiment of the present invention the selection/isolation step is a selection step wherein CD140a, CD140b, and CD44 expressing cells, thus cells which all individually express all said 3 markers, are isolated.

The present invention relates to a method which comprises a first step (a) wherein pluripotent stem cells are differentiated to the mesodermal lineage; and a second step wherein said mesodermal cells of step (a) are further purified or selected by selecting for those cells which express at least one, at least 2 or preferably all 3 markers selected from the group: CD44, CD140a and CD140b. Said first step (a) can be any known mesodermal differentiation/induction step that is known in the art. Said second isolation step can be performed with any known method or process known in the art for selecting cells based on their expression of certain marker genes, especially those technologies who can select cells based on their expression pattern of genes on their surfaces, such as but not limited to fluorescence-activated cell sorting (FACS), immune-labeled based isolation and cell tracing based methods (e.g. transgenic cell tracing methods).

The first step of the present invention can be performed by a mesoderm inducing step which comprises a procedure wherein said pluripotent stem cells are cultured in a low-attachment vessel or a non-attachment vessel. In a more specific embodiment of the present invention, said first step comprises mesoderm induction process which further comprises the following steps: (i) an embryoid body (EB) formation step wherein the pluripotent stem cells are cultured in low- or non-attachment vessels; (ii) an embryoid body maturation step wherein the EBs of step (i) are further committed to the mesodermal phenotype by culturing them in medium comprising at least one factor selected from the group consisting of holo-transferrin, thyoglycerol and ascorbic acid; and (iii) a cell spreading step wherein the matured EBs of step (ii) are further cultured in vessels for adherent culture. Said step wherein the pluripotent stem cells are cultured in low- or non-attachment vessels allow the pluripotent stem cells to form EBs.

In another embodiment of the present invention, said first step comprises a culturing/differentiation step wherein said pluripotent stem cells are cultured in a medium comprising at least one mesoderm inducing factor. In a more specific embodiment of the present invention, said factor is at least one factor, at least 2 factors or three factors from the group consisting of holo-transferrin, thyoglycerol and ascorbic acid. In another yet more specific embodiment of the present invention said factors are used in the following concentration: holo-transferrin is preferentially used in the concentration range from about 1 μg/ml to about 500 μg/ml, and preferentially the concentration of holo-transferrin is about 250 μg/ml; thyoglycerol is preferentially used in the concentration range from about 1 mM to about 10 mM, and preferentially the concentration of thyoglycerol is about 5mM; and ascorbic acid is preferentially used in the concentration range from about 1 μg/ml to about 200 μg/ml, and preferentially the concentration of ascorbic acid is about 60 μg/ml.

In specific embodiments of the present invention, the cells at the end of step (a) or at the end of step (iii) express at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or all 10 markers selected from the group consisting of Brachyury, MixH , Meoxl , Mespl , Pax3, Pax7, Desmin, Gata4, Tbx5, and Flk1. In a more specific embodiment of the present invention, said Brachyury- and/or MixH- and/or Meoxl - and/or Mespl - and/or Pax3- and/or Pax7- and/or Desmin- and/or Gata4- and/or Tbx5- and/or Flk1 -expressing cells further express at least one, two or three markers from the list consisting of CD44, CD140a and CD140b. The method of the present invention concerns any type of pluripotent stem cells as starting material, such as iPSCs, which can be of any origin, but more preferably of human origin. In a more specific embodiment said IPSCs are mesoangioblast-derived iPSCs.

The method of the present invention comprises all kinds of known mesodermal differentiation procedures and protocols which are used to differentiate said pluripotent stem cells towards the mesodermal lineage, thereby using all known culturing conditions, and all kinds of differentiation factors known to the skilled person. Said differentiation step can be performed in the absence of serum as well as in the presence of serum. In more specific embodiments said serum concentration can range from 0 to 25%, or from about 5 % to about 25 %, or from about 15% to about 20%. In certain embodiments of the present invention, said serum concentration can vary during the differentiation process in certain steps of the whole differentiation procedure. In a certain embodiment of the present invention, said EB formation step (i) is performed in about 15 % serum and said embryoid body maturation step (ii) and cell spreading step (iii) are performed in about 25% serum.

In certain specific embodiments of the present invention, said low- or non-attachment vessels are non-culture-treated plastic dishes. Such dishes/vessels are well known to the skilled person and are used to allow stem cells to form aggregates and to form EBs from such stem cells.

In other specific embodiments of the present invention, said vessels for adherent culture are well known to the skilled person and can be described as cell-culture-treated plastic vessels coated with any adherence factor that increases adhesion and preferentially also promotes commitment of the cells, examples of said adherence factor include, but are not limited to collagen, gelatin and fibronectin. In specific embodiments of the present invention, said adherence factor is collagen.

In certain embodiments of the present invention, the cells are cultured for about 1 to about 14 days in said first differentiation step (a). In a more specific embodiment of the present invention, the cells are cultured for about 6 to about 12 days, more specifically about 9, 10 or 11 days in said first differentiation step (a). In even more specific embodiments of the present invention, said differentiation step (a) comprises a first EB formation step (i) wherein the cells are cultured for at least 1 day in low- or non-adherent vessels, more specifically for about 3 or 4 days. In other specific embodiments of the present invention, said differentiation step (a) further comprises an EB maturation step wherein the cells or EBs are cultured for at least 1 day in low- or non-adherent vessels in mesoderm-induction medium comprising at least one mesoderm induction factor, more specifically for about 3 or 4 days in said vessels and said medium. In other specific embodiments of the present invention, said differentiation step (a) further comprises a cell spreading step wherein the cells or EBs are further cultured in vessels for adherent culture for at least 1 day, more specifically for about 3 or 4 days. In yet other specific embodiments of the present invention, said EB maturation step (ii) and said cell spreading step (iii) are performed in the same medium; and in certain embodiments thereof the vessels are changed between step (ii) and (iii), more specifically the low- or non-adherent vessel of step (ii) are changed to vessels for adherent culture in step (iii), meaning that the cells/EBs of step (ii) are replated in step (iii).

Another aspect of the present invention relates to the cells produced by the methods of the present invention. Said cells, mesodermal progenitor cells are characterized in that they all express at least one, at least two, and preferably at least 3 marker genes selected from the group consisting of CD44, CD140a, and CD140b. In specific embodiments of the present invention said cells all individually express the 2 or more preferably all 3 markers from the group CD44, CD140a, and CD140b. In more specific embodiments, said CD44- and/or CD140a- and/or CD140b-expressing cells additionally express at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or all 10 markers from the group consisting of Brachyury, MixH , Meoxl , Mespl , Pax3, Pax7, Desmin, Gata4, Tbx5, and Flkl In a more specific embodiment of the present invention, said mesodermal progenitor cells express CD44, CD140a, CD140b, and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or all 10 markers from the group consisting of Brachyury, MixH , Meoxl , Mespl , Pax3, Pax7, Desmin, Gata4, Tbx5, and Flkl In an even more specific embodiment of the present invention, said mesodermal progenitor cells express CD44, CD140a, CD140b, Brachyury, MixM , Meoxl , Mespl , Pax3, Pax7, Desmin, Gata4, Tbx5, and Flkl (Figure 5).

Further, the cells may not be tumorigenic, such as not producing teratomas. If cells are transformed or tumorigenic, and it is desirable to use them for infusion, such cells may be disabled so they cannot form tumors in vivo, as by treatment that prevents cell proliferation into tumors. Such treatments are well known in the art. In view of the property of the cells to achieve the desired effects, cell banks can be established containing cells that are selected for having a desired potency to achieve any of the desired effects. The bank can provide a source for making a pharmaceutical composition to administer to a subject. Cells can be used directly from the bank or expanded prior to use. Especially in the case that the cells are subjected to further expansion, after expansion it is desirable to validate that the cells still have the desired potency. Banks allow the "off the shelf use of cells that are allogeneic to the subject. Accordingly, the invention also is directed to diagnostic procedures conducted prior to administering the cells to a subject. The procedures include assessing the potency of the cells to achieve the effects described in this application. The cells may be taken from a cell bank and used directly or expanded prior to administration. In either case, the cells could be assessed for the desired potency and/or their expression of the relevant/desired markers. Especially in the case that the cells are subjected to further expansion, after expansion it is desirable to validate that the cells still have the desired potency. Or the cells can be derived from the subject and expanded prior to administration. In this case, as well, the cells could be assessed for the desired potency prior to administration back to the subject (autologous).

Accordingly, the invention also is directed to diagnostic procedures conducted prior to administering the cells to a subject, the pre-diagnostic procedures including assessing the potency of the cells to achieve one or more of the desired effects. The cells may be taken from a cell bank and used directly or expanded prior to administration. In either case, the cells would be assessed for the desired potency. Or the cells can be derived from the subject and expanded prior to administration. In this case, as well, the cells would be assessed for the desired potency prior to administration.

Although the cells selected for the effects are necessarily assayed during the selection procedure, it may be preferable and prudent to again assay the cells prior to administration to a subject for treatment to confirm that the cells still achieve the effects at desired levels. This is particularly preferable where the cells have been stored for any length of time, such as in a cell bank, where cells are most likely frozen during storage.

With respect to methods of treatment with cells that achieve the desired effects, between the original isolation of the cells and the administration to a subject, there may be multiple (i.e., sequential) assays for the effects. This is to confirm that the cells can still achieve the effects, at desired levels, after manipulations that occur within this time frame. For example, an assay may be performed after each expansion of the cells. If cells are stored in a cell bank, they may be assayed after being released from storage. If they are frozen, they may be assayed after thawing. If the cells from a cell bank are expanded, they may be assayed after expansion. Preferably, a portion of the final cell product (that is physically administered to the subject) may be assayed.

The invention further includes post-treatment diagnostic assays, following administration of the cells, to assess efficacy.

The invention is also directed to a method for establishing the dosage of such cells by assessing the potency of the cells to achieve one or more of the desired effects. In this case, the potency would be determined and the dosage adjusted accordingly.

In this case, one would monitor efficacy, by methods including one or more of the assays described in this invention, to establish and maintain a proper dosage regimen.

The invention is also directed to compositions comprising a population of the cells having a desired potency to achieve the desired effects. Such populations may be found as pharmaceutical compositions suitable for administration to a subject and/or in cell banks from which cells can be used directly for administration to a subject or expanded and/or further differentiated, e.g. to a certain mesodermal (terminally differentiated) lineage, prior to administration. In one embodiment, the cells have enhanced (increased) potency compared to the previous (parent) cell population. Parent cells are as defined herein. Enhancement can be by selection of natural expressors or by external factors acting on the cells.

Accordingly, any of the indicators described herein may be monitored during treatment with the methods and cells according to the current invention.

For all these treatments, one would administer the cells that achieve the effects described in this application. Such cells could have been assessed for the potency and selected for desired potency.

It is understood, however, that for treatment of any of the conditions or diseases of the present invention, it may be expedient to use such cells; that is, one that has been assessed for achieving the desired effects and selected for a desired level of efficacy prior to administration for treatment of the condition.

The cells may be prepared by the isolation and culture conditions as described in the present invention.

Another aspect of the present invention relates to a pharmaceutical composition, comprising the mesodermal progenitor cells of the present invention and one or more pharmaceutically acceptable carriers. In another embodiment of the present invention said pharmaceutical composition comprises a therapeutically effective amount of the mesodermal progenitor cells of the present invention. In a more specific embodiment, said pharmaceutical composition comprises at least 10000 mesodermal progenitor cells of the present invention. In certain embodiments of the present invention, any suitable number of cells may be administerd to a subject or patient. For example, at least, or about, 0.5 x 10 6 , 1.5 x 10 6 , 4.0 x 10 6 or 5.0 x 10 6 cells per kg of patient may be administered. For example, at least, or about, 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 cells may be administered. As a guide, the number of cells of the invention to be administered may be from 10 5 to 10 9 , preferably from about 10 6 to about 10 8 . Typically, up to 2 x 10 8 cells are administered to each patient. Any of the specific numbers discussed above with reference to the populations of the invention may be administered. In such cases where cells are administered or present, culture medium may be present to facilitate the survival of the cells. In some cases the cells of the invention may be provided in frozen aliquots and substances such as DMSO may be present to facilitate survival during freezing. Such frozen cells will typically be thawed and then placed in a buffer or medium either for maintenance or for administration.

Another aspect of the present invention relates to the use of the cells or the pharmaceutical composition of the present invention in medical therapy. In certain embodiments of the present invention, said medical therapy is to threat patients or subjects that are in need thereof. Said Patients can be any patient can be any patient that is injured or has a disease or disorder comprising congenital myopathies, heart disease, skeletal muscle disease and/or any condition or injury that leads to a deterioration of mesodermal derivative tissues. In a specific embodiment of the present invention said patient is a myopathic patient.

In certain embodiments of the present invention, the cells for use in the treatment of a patient are autologous. In other embodiments, the cells for use in the treatment of a patient are allogeneic. In certain embodiments of the present invention, said allogeneic or autologous cells are used to repair or threat specific tissues in a patient; said tissue can be a damaged tissue, eg. a certain tissue that is damaged due to an injury or disease; in certain more specific embodiments of the present invention, said tissue need to be treated with a therapeutically effective amount of the cells of the present invention.

In one embodiment of the present invention, the subject to be treated is a patient, more preferably a vertebrate such as a mammal even more preferably a human. Mammals include, but are not limited to humans, dogs, cats, horses, cows and pigs.

In some more specific embodiments of the present invention, the mesodermal progenitor cells of the present invention are used for medical treatment, more specifically in combination with at least one pharmaceutically acceptable carrier. In other embodiments of the present invention, the mesodermal progenitor cells of the present invention are further cultured and/or differentiated to specific lineages, including terminally differentiated lineages, such as cardiomyocytes, skeletal muscle cells, osteocytes, adipocytes, and smooth muscle cells. In other specific embodiments, said further cultured and/or differentiated cells are used for medical treatment or therapies, preferably for treatment of tissues in a patient that comprise cells of the same lineage. Said patient preferentially is treated with cells that are generated and differentiated from its own progenitor cells or its own pluripotent cells, by methods of the present invention, thus using an autologous setting or treatment. In another embodiment, said patient is treated with cells that are generated and differentiated from progenitor cells or pluripotent cells from another subject or person, by methods of the present invention, thus using an allogeneic setting or treatment. In certain embodiments of the present invention, such allogeneic treatments are accompanied by an immunosuppressive treatment of said patient, for example by co-administration of immuno-suppressive compounds, before and/or simultaneous with and/or after the cellular treatment as contemplated in the present invention.

Numbered statements of this invention are:

1. An in vitro method to produce mesodermal progenitor cells from pluripotent stem cells, the method comprising:

a) culturing pluripotent stem cells in a mesoderm inducing medium; and b) isolate mesodermal progenitor cells by selecting the differentiated cells of step (a) that express all three factors CD44, CD140a and CD140b. The method of statement 1 , wherein step (a) further comprises a step wherein the pluripotent stem cells are cultured in a low- or non-attachment vessel. The method according to statement 1 or 2, wherein the isolation in step (b) is performed by any method which can select for cells that express said factors such as fluorescence-activated cell sorting (FACS), transgenic cell tracing and immunolabeling-based isolation. The method according to any of statements 1 to 3, wherein the mesoderm inducing medium of step (a) comprises at least one factor selected from the group consisting of holo-transferrin, thyoglycerol and ascorbic acid. The method according to any of statements 1 to 4, wherein step (a) further comprises:

(i) an embryoid body (EB) formation step wherein the pluripotent stem cells are cultured in low- or non-attachment vessels, which allow them to form embryoid bodies;

(ii) an embryoid body maturation step wherein the EBs of step (i) are further committed to the mesodermal phenotype by culturing them in medium comprising at least one factor selected from the group consisting of holo- transferrin, thyoglycerol and ascorbic acid; and

(iii) a cell spreading step wherein the matured EBs of step (ii) are further cultured in vessels for adherent culture. The method of statement 5, wherein the cells of step (iii) express the factors Brachyury, MixH , Meoxl , Mespl , Pax3, Pax7, Desmin, Gata4, Tbx5, and Flkl The method of any of statements 1 to 6, wherein the pluripotent stem cells are human cells. The method of any of statements 1 to 7, wherein the pluripotent stem cells are iPSCs. The method of statement 8, wherein the iPSCs are mesoangioblast-derived iPSCs. The method of any of the previous statements, wherein the serum concentration in step a, (i), (ii), and (iii) is about 0 % to about 25%, and preferentially about 20% in step (i) and preferentially about 15% in step (ii) and (iii). The method of statement 4 or 5, wherein the concentration of holo-transferrin is about 1 μ9/ιηΙ to about 500 μg/ml, and preferentially about 250 μg/ml; the concentration of thyoglycerol is about 1 mM to 10 mM, and preferentially about 5mM; and the concentration of ascorbic acid is about 1 μg/ml to 200 μ9/ιηΙ, and preferentially about 60 μg/ml. The method of any of statements 2 to 11 , wherein the low- or non-attachment vessels are non-culture-treated plastic dishes. The method of any of statements 5 to 12, wherein said vessels for adherent culture are collagen-coated cell-culture-treated plastic vessels. The method of any of the previous statements, wherein the cells are cultured in step (a) for about 0 days to about 14 days. The method of any of the previous statements, wherein the cells are cultured in one or more steps for at least 1 day, and preferentially for about 3 days. The method of any of the previous statements, wherein the cells are cultured in each step for at least 1 day. The method of any of the previous statements, wherein the cells are cultured in each step for about 3 days. The method of any of the previous statements, wherein the cells are cultured in step (a) for about 9 days. The mesodermal progenitor cells produced by the method of any of the previous statements and characterized in that said cells express the factors CD44, CD 140a, CD140b, Brachyury, Mixl1 , Meoxl , Mespl , Pax3, Pax7, Desmin, Gata4, Tbx5, and Flkl A pharmaceutical composition comprising the mesodermal progenitor cells of statement 19 and one or more pharmaceutically acceptable carriers. The pharmaceutical composition of statement 20, which comprises a therapeutically effective amount of said mesodermal progenitor cells. The mesodermal progenitor cells of statement 19 or the pharmaceutical composition of statement 20 or 21 for use in medical therapy. The mesodermal progenitor cells of statement 19 or the pharmaceutical composition of statement 20 or 21 , for administration to a subject in need thereof. The mesodermal progenitor cells of statement 19 or the pharmaceutical composition of statement 20 or 21 for use in a medical therapy for the treatment of a myopathic patient. The mesodermal progenitor cells of statement 22 or 23, wherein the medical therapy or the administration is for a patient or subject that has: a congenital myopathy; a heart disease; a skeletal muscle disease; and/or any condition leading to deterioration of mesodermal derivative tissues.

The mesodermal progenitor cells of any of statements 22 to 25, wherein the cells are autologous. The mesodermal progenitor cells of any of statements 22 to 25, wherein the cells are allogeneic. The mesodermal progenitor cells of any of statements 23 to 27, wherein the subject or patient is a mammal, preferentially a human. A method of repairing or treating a damaged tissue in a patient, comprising administering to the patient the mesodermal progenitor cells of statement 19 or the pharmaceutical composition of statement 20 or 21 , wherein said cells or pharmaceutical composition comprise a therapeutically effective amount of said cells. 30. The method of statement 29, wherein the patient is a mammal, preferentially a human.

31. The method of statement 29 or 30 wherein said patient has: a congenital myopathy; a heart disease; a skeletal muscle disease; and/or any condition leading to deterioration of mesodermal derivative tissues.

DETAILED DESCRIPTION OF THE INVENTION

Brief description of the figures of the invention

Figure 1. Schematic representation of possible application of the invention in autologous setting. Schematic representation of the generation of mesoderm progenitor cells according to certain embodiments of the present invention and application in an autologous setting applied to a human myopathic patient.

Figure 2. Experimental data suffragating the potential of the invention, when generated from murine cells. Representative brightfield pictures related to the different stages of MiP isolation, when generated from murine pluripotent cells (A). Once isolated and traced with a fluorescent reporter, murine MiPs are able to engraft in both cardiac and skeletal muscles of dystrophic cardiomyopathic mice (Sgcb-null) under immunosuppressive regimen, as shown by immunostaining (B, arrows point at engrafted tissue patches, indicated by a dashed line). Sgcb-null mice are a relevant genetic model of sarcoglycanopathy LGMD-2E and a relevant pathological/transplantation model for the congenital myopathies featuring chronic degeneration of both cardiac and skeletal muscles. Scale bars, 100μηι.

Figure 3. Experimental data suffragating the potential of the invention, when generated from canine cells. Representative brightfield pictures related to the different stages of MiP isolation, when generated from canine pluripotent cells (A). Once isolated and traced with a fluorescent reporter, canine MiPs are able to engraft in both cardiac and skeletal muscles of immunodeficient mice (Rag2-null; j -null), after transient occlusion of the coronary artery (ischemia/reperfusion-induced cardiac damage) and cardiotoxin-induced skeletal muscle wastage, as shown by immunostaining (B, arrows point at engrafted tissue patches, indicated by a dashed line). Said murine model is a relevant pathological/transplantation model for acute and/or ischemic conditions of the cardiac and skeletal muscles. Scale bars, 100μΐη.

Figure 4. Experimental data suffragating the potential of the invention, when generated from human cells. Representative brightfield pictures related to the different stages of MiP isolation, when generated from human pluripotent cells (A). Once isolated and traced with a fluorescent reporter, human MiPs are able to participate in both cardiac and skeletal myocytes during in vitro differentiation, as shown by immunostaining on co-cultures with neonatal cardiomyocytes (left) or fetal myoblasts (right) (B, arrows point at chimeric cardiomyocytes or myotubes). Scale bars, 100μΐη.

Figure 5. Experimental data suffragating the expression of mentioned markers in isolated MiPs. The chart depicts average fold change enrichment in the expression levels of reported markers (indicated on the x axis). Y axis, fold change values as compared to iPSCs. *, P<0.05 MiPs vs iPSCs (n=3), t-test. The average data are related to human cells, but similar results are obtainable with murine or canine cells. The individual values may vary according to species of origin, or to the intrinsic characteristics of the MiP preparation.

Description DEFINITIONS

"A" or "an" means one or more than one. Where the plural form is used herein, it generally includes the singular.

"Comprising" means, without other limitation, including the referent, necessarily, without any qualification or exclusion on what else may be included. For example, "a composition comprising x and y" encompasses any composition that contains x and y, no matter what other components may be present in the composition. Likewise, "a method comprising the step of x" encompasses any method in which x is carried out, whether x is the only step in the method or it is only one of the steps, no matter how many other steps there may be and no matter how simple or complex x is in comparison to them. "Comprised of and similar phrases using words of the root "comprise" are used herein as synonyms of "comprising" and have the same meaning.

"Effective amount" generally means an amount which provides the desired local or systemic effect. For example, an effective amount is an amount sufficient to effectuate a beneficial or desired clinical result. The effective amounts can be provided all at once in a single administration or in fractional amounts that provide the effective amount in several administrations. The precise determination of what would be considered an effective amount may be based on factors individual to each subject, including their size, age, injury, and/or disease or injury being treated, and amount of time since the injury occurred or the disease began. One skilled in the art will be able to determine the effective amount for a given subject based on these considerations which are routine in the art. As used herein, "effective dose" means the same as "effective amount."

The term "therapeutically effective amount" refers to the amount of an agent or cells determined to produce any therapeutic response in a mammal. For example, effective anti-inflammatory therapeutic agents may prolong the survivability of the patient, and/or inhibit overt clinical symptoms. Treatments that are therapeutically effective within the meaning of the term as used herein, include treatments that improve a subject's quality of life even if they do not improve the disease outcome per se. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art. Thus, to "treat" means to deliver such an amount. Thus, treating can prevent or ameliorate any pathological symptoms of the diseases or injuries described in the present invention.

"Treat," "treating" or "treatment" are used broadly in relation to the invention and each such term encompasses, among others, preventing, ameliorating, inhibiting, or curing a deficiency, dysfunction, disease, or other deleterious process, including those that interfere with and/or result from a therapy or injury.

"Pharmaceutically acceptable carrier" is any pharmaceutically-acceptable medium for the cells used in the present invention. Such a medium may retain isotonicity, cell metabolism, pH, and the like. It is compatible with administration to a subject in vivo, and can be used, therefore, for cell delivery and treatment.

The term "potency" refers to the ability of the cells to achieve the various effects described in this invention. Accordingly, potency refers to the effect at various levels, including, but not limited to, reducing symptoms of the diseases/injuries described in the present invention, ability to differentiate in terminally differentiated (mesodermal) cells such as cardiomyocytes, skeletal muscle cells, osteocytes, adipocytes, and smooth muscle cells, ability to re-express missing therapeutic genes once terminally differentiated or fused with host cells or tissues.

"Subject" or "patient" means a vertebrate, such as a mammal, such as a human. Mammals include, but are not limited to, humans, dogs, cats, horses, cows and pigs. "Stem cell" means a cell that can undergo self-renewal (i.e., progeny with the same differentiation potential) and also produce progeny cells that are more restricted in differentiation potential. A stem cell encompasses an undifferentiated cell capable of (1 ) long term self-renewal, or the ability to generate at least one identical copy of the original cell, (2) differentiation at the single cell level into multiple, and in some instances only one, specialized cell type and/or (3) of in vivo functional regeneration of tissues. Stem cells may further be subclassified according to their developmental potential as totipotent, pluripotent, multipotent and oligo/unipotent. Within the context of the invention, a stem cell would also encompass a more differentiated cell that has de-differentiated, for example, by nuclear transfer, by fusion with a more primitive stem cell, by introduction of specific transcription factors, or by culture under specific conditions. See, for example, Wilmut et al., Nature, 385:810-813 (1997); Ying et al., Nature, 416:545-548 (2002); Guan et al., Nature, 440:1 199-1203 (2006); Takahashi et al., Cell, 126:663-676 (2006); Okita et al., Nature, 448:313-317 (2007); and Takahashi et al., Cell, 131 :861 -872 (2007).

Dedifferentiation may also be caused by the administration of certain compounds or exposure to a physical environment in vitro or in vivo that would cause the dedifferentiation. Stem cells also may be derived from abnormal tissue, such as a teratocarcinoma and some other sources such as embryoid bodies (although these can be considered embryonic stem cells in that they are derived from embryonic tissue, although not directly from the inner cell mass). Stem cells may also be produced by introducing genes associated with stem cell function into a non-stem cell, such as an induced pluripotent stem cell.

"Self-renewal" refers to the ability to produce replicate daughter (stem) cells having differentiation potential that is identical to those from which they arose. A similar term used in this context is "proliferation."

Multipotent," with respect to (stem) cells, refers to their ability to transdifferentiate into derivatives of more than one primitive germ layer (i.e., endoderm, mesoderm and ectoderm) upon differentiation, such as all three, including their ability to give rise to more than one tissue derivative inside the same germ layer.

A totipotent stem cell is a cell that can give rise to a new individual if provided with appropriate maternal support. After sperm fertilizes the egg, a zygote is formed which then has the potential to develop into a complete embryo. The fertilized egg (and its immediate progeny cells during the blastula stage) is a totipotent stem cell, which has the potential to create any type of cell necessary for embryonic development, included extraembryonic membranes and tissues required for mammalian development, the embryo itself, and all postembryonic tissues and organs. Approximately four days after fertilization, compaction and blastocyst formation occurs; within a blastocyst, the inner cell mass contains cells which are pluripotent as they can give rise to all cell types of the embryo proper, including somatic and germ cells. These cells are no longer totipotent as they cannot generate the trophectoderm. From this inner cell mass, embryonic stem (ES) cells can be generated. Pluripotency of ES cells is defined based on the following criteria: they generate embryoid bodies in vitro, can generate teratomas in vivo and when injected in the blastocyst, can contribute to all somatic and germline cell types. These features are maintained by a set of transcription factors and genes that have recently been defined, including Oct3a/4, Nanog, Sox2, Essrb, Dppa4, Tcl1 , and Tbx3. Embryonic development and the subsequent adult life are viewed as a continuum of decreasing potencies.

During gastrulation and subsequent developmental steps, pluripotent cells further specialize into multipotent stem cells, also termed adult stem cells. Adult stem cells are termed multipotent as they are committed to differentiate into the multiple cell types of a single tissue/lineage. For example, the hematopoietic stem cell (HSC) - the best studied multipotent stem cell - undergoes self-renewing cell divisions and, if transplanted at the single cell level can give rise to all the lineages of the blood system and functionally repopulates the hematopoietic system of a myeloablated animal or human. More recently, tissue-specific stem cells have been isolated from many organs, including among others the central nervous system (CNS), epidermis, intestine, liver, lung, retina, skeletal muscle and heart. Like HSCs, most of these organ-derived stem cells fulfil all the basic stem cell criteria: they are undifferentiated but tissue committed stem cells, they self-renew - albeit for many of these adult stem cells at a very slow rate, and generate within the tissue of origin differentiated progeny. Adult stem cells usually reside in a quiescent state under tissue homeostatic conditions and become activated upon injury or specific environmental cues. Usually, once isolated and expanded in vitro, adult stem cells tend to undergo senescence and potency limitation at high passage number.

Other cells termed unipotent stem cells also fulfill all criteria for stem cells except that they are able to contribute only to one mature cell type. In the category of unipotent, lineage-committed stem cells we can include, for example, corneal epithelial cells or endothelial progenitor cells, muscle (myo)satellite cells and spermatogonia! stem cells. Aside of unipotent spermatogonia! stem cells, the isolation of new populations of adult spermatogonia! stem cells from neonatal and adult testis have recently been reported. Such testis derived stem cells appear to be truly pluripotent, as they generate teratomas and contribute to somatic and germline lineages when injected in the blastocyst. Pluripotent stem cells

"EC cells" were discovered from analysis of a type of cancer called a teratocarcinoma. In 1964, researchers noted that a single cell in teratocarcinomas could be isolated and remain undifferentiated in culture. This type of stem cell became known as an embryonic carcinoma cell (EC cell).

"Embryonic Stem Cells (ESC)" are well known in the art and have been prepared from many different mammalian species for many years. Embryonic stem cells are stem cells derived from the inner cell mass of an early stage embryo known as a blastocyst. They are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm. These include each of the more than 220 cell types in the adult body. The ES cells can become any tissue in the body, excluding a placenta. Only the morula's cells are totipotent, able to become all tissues and a placenta. Some cells similar to ESCs may be produced by nuclear transfer of a somatic cell nucleus into an enucleated fertilized egg.

"Induced pluripotent stem cells (IPSC or IPS cells)" are somatic cells that have been reprogrammed, for example, by introducing exogenous genes that confer on the somatic cell a less differentiated phenotype. These IPS cells can then be induced to differentiate into tissue derivatives of the three germ layers. IPS cells have been derived using modifications of an approach originally discovered in 2006 (Yamanaka, S. et al., Cell Stem Cell, 1 :39-49 (2007)). For example, in one instance, to create IPS cells, scientists started with skin cells that were then modified by a standard laboratory technique using retroviruses to insert genes into the cellular DNA. In one instance, the inserted genes were Oct4, Sox2, Lif4, and c-myc, known to act together as natural regulators to keep cells in an embryonic stem cell-like state. These cells have been described in the literature. See, for example, Wernig et al., PNAS, 105:5856-5861 (2008); Jaenisch et al., Cell, 132:567-582 (2008); Hanna et al., Cell, 133:250- 264 (2008); and Brambrink et al., Cell Stem Cell, 2: 151-159 (2008). These references are incorporated by reference for teaching IPSCs and methods for producing them. It is also possible that such cells can be created by specific culture conditions (exposure to specific agents).

The term "isolated" refers to a cell or cells which are not associated with one or more cells or one or more cellular components that are associated with the cell or cells in vivo. An "enriched population" means a relative increase in numbers of a desired cell relative to one or more other cell types in vivo or in primary culture.

However, as used herein, the term "isolated" does not indicate the presence of only the cells of the invention. Rather, the term "isolated" indicates that the cells of the invention are removed from their (natural tissue) environment/mix of cells and are present at a higher concentration as compared to the normal (tissue) environment. Accordingly, an "isolated" cell population may further include cell types in addition to the cells of the invention and may include additional tissue components. This also can be expressed in terms of cell doublings, for example. A cell may have undergone 10, 20, 30, 40 or more doublings in vitro or ex vivo so that it is enriched compared to its original numbers, in its original mix of cells, in vivo , or in its original (tissue) environment (e.g., bone marrow, peripheral blood, placenta, umbilical cord, umbilical cord blood, adipose tissue, etc.).

Isolation of cells includes positive and/or negative selection of cells, wherein said selection could be used to isolate cells via a combination of cell-specific markers. Both positive and negative selection techniques are available to those of skill in the art, and numerous monoclonal and polyclonal antibodies suitable for positive/negative selection purposes are also available in the art (see, for example, Leukocyte Typing V, Schlossman, et al., Eds. (1995) Oxford University Press) and are commercially available from a number of sources.

Techniques for mammalian cell separation from a mixture of cell populations have also been described by Schwartz, et al., in U. S. Patent No. 5,759,793 (magnetic separation), Basch et al., 1983 (immunoaffinity chromatography), and Wysocki and Sato, 1978 (fluorescence- activated cell sorting (FACS)).

A "cell bank" is industry nomenclature for cells that have been grown and stored for future use. Cells may be stored in aliquots. They can be used directly out of storage or may be expanded after storage. This is a convenience so that there are "off the shelf cells available for administration. The cells may already be stored in a pharmaceutically-acceptable excipient so they may be directly administered or they may be mixed with an appropriate excipient when they are released from storage. Cells may be frozen or otherwise stored in a form to preserve viability. In one embodiment of the invention, cell banks are created in which the cells have been selected for enhanced potency to achieve the effects described in this invention. Following release from storage, and prior to administration to the subject, it may be preferable to again assay the cells for potency. This can be done using any of the assays, direct or indirect, described in this application or otherwise known in the art. Then cells having the desired potency can then be administered to the subject for treatment. Following release from storage, and prior to administration to the subject, it may be necessary to further differentiate the cells further, eg to the desired terminally differentiated cells, and assay said terminally differentiated cells for their potency, before the administration toi the subject for treatment Banks can be made using cells derived from the individual to be treated (from their pre-natal tissues such as placenta, umbilical cord blood, or umbilical cord matrix or expanded from the individual at any time after birth, such as by using IPSC technology). Or banks can contain cells for allogeneic uses.

"Progenitor cells" are cells produced during differentiation of a stem cell that have some, but not all, of the characteristics of their terminally-differentiated progeny. Defined progenitor cells, such as "mesodermal progenitor cells" and "cardiac progenitor cells" are committed to a lineage, but not to a specific or terminally differentiated cell type. A progenitor cell can form a progeny cell that is more highly differentiated than the progenitor cell.

"Selecting" a cell with a desired level of potency can mean identifying (as by assay), isolating, and expanding a cell. This could create a population that has a higher potency than the parent cell population from which the cell was isolated. The "parent" cell population refers to the parent cells from which the selected cells divided. "Parent" refers to an actual P1 → F1 relationship (i.e., a progeny cell). So if cell X is isolated from a mixed population of cells X and Y, in which X is an expressor and Y is not, one would not classify a mere isolate of X as having enhanced expression. But, if a progeny cell of X is a higher expressor, one would classify the progeny cell as having enhanced expression.

To select a cell that achieves the desired effect would include both an assay to determine if the cells achieve the desired effect and would also include obtaining those cells. The cell may naturally achieve the desired effect in that the effect is not achieved by an exogenous transgene/DNA. But an effective cell may be improved by being incubated with or exposed to an agent that increases the effect. The cell population from which the effective cell is selected may not be known to have the potency prior to conducting the assay. The cell may not be known to achieve the desired effect prior to conducting the assay. As an effect could depend on gene expression and/or secretion, one could also select on the basis of one or more of the genes that cause the effect.

Selection could be from cells in a tissue. For example, in this case, cells would be isolated from a desired tissue, expanded in culture, selected for achieving the desired effect, and the selected cells further expanded.

Selection could also be from cells ex vivo, such as cells in culture. In this case, one or more of the cells in culture would be assayed for achieving the desired effect and the cells obtained that achieve the desired effect could be further expanded.

Cells could also be selected for enhanced ability to achieve the desired effect. In this case, the cell population from which the enhanced cell is obtained already has the desired effect. Enhanced effect means a higher average amount per cell than in the parent population. The parent population from which the enhanced cell is selected may be substantially homogeneous (the same cell type). One way to obtain such an enhanced cell from this population is to create single cells or cell pools and assay those cells or cell pools to obtain clones that naturally have the enhanced (greater) effect (as opposed to treating the cells with a modulator that induces or increases the effect) and then expanding those cells that are naturally enhanced.

However, cells may be treated with one or more agents that will induce or increase the effect. Thus, substantially homogeneous populations may be treated to enhance the effect.

If the population is not substantially homogeneous, then, it is preferable that the parental cell population to be treated contains at least 100 of the desired cell type in which enhanced effect is sought, more preferably at least 1 ,000 of the cells, and still more preferably, at least 10,000 of the cells. Following treatment, this sub-population can be recovered from the heterogeneous population by known cell selection techniques and further expanded if desired.

Thus, desired levels of effect may be those that are higher than the levels in a given preceding population. For example, cells that are put into primary culture from a tissue and expanded and isolated by culture conditions that are not specifically designed to produce the effect may provide a parent population. Such a parent population can be treated to enhance the average effect per cell or screened for a cell or cells within the population that express greater degrees of effect without deliberate treatment. Such cells can be expanded then to provide a population with a higher (desired) expression.

"Validate" means to confirm. In the context of the invention, one confirms that a cell is an expressor with a desired potency. This is so that one can then use that cell (in treatment, banking, drug screening, etc.) with a reasonable expectation of efficacy. Accordingly, to validate means to confirm that the cells, having been originally found to have/established as having the desired activity, in fact, retain that activity. Thus, validation is a verification event in a two-event process involving the original determination and the follow-up determination. The second event is referred to herein as "validation."

The present invention is mainly described by the use of pluripotent stem cells as starting material for the methods to generate the mesodermal progenitor cells and their further use. It is however contemplated by the present invention that other types of stem cells can be used in the present invention, including their use as starting material to generate the mesodermal progenitor cells of the present invention, eg. by using the methods and step(s) as described in the present invention. Thus the present invention relates to a method, more specifically an in vitro method, to produce mesodermal progenitor cells from stem cells, the method comprising the isolation of mesodermal progenitor cells from said stem cells, which optionally are further cultured and/or differentiated to a mesodermal lineage or phenotype, by selecting said cells that express one or more factors, more specifically two or more factors and even more specifically all three factors, selected from the group consisting of CD44, CD140a and CD140b. Thus the present invention also relates to a method, more specifically an in vitro method, to produce mesodermal progenitor cells from stem cells, the method comprising: (a) culturing said stem cells in a mesoderm inducing medium; and (b) isolate mesodermal progenitor cells by selecting the differentiated cells of step (a) that express one or more factors, more specifically two or more factors and even more specifically all three factors, selected from the group consisting of CD44, CD140a and CD140b.

Stem Cells

The present invention can be practiced, preferably, using stem cells of vertebrate species, such as humans, non-human primates, domestic animals, livestock, and other non-human mammals. These include, but are not limited to, those cells described below.

Embryonic Stem Cells

The most well studied stem cell is the embryonic stem cell (ESC) as it has unlimited self- renewal and multipotent differentiation potential. These cells are derived from the inner cell mass of the blastocyst or can be derived from the primordial germ cells of a post-implantation embryo (embryonal germ cells or EG cells). ES and EG cells have been derived, first from mouse, and later, from many different animals, and more recently, also from non-human primates and humans. When introduced into mouse blastocysts or blastocysts of other animals, ESCs can contribute to all tissues of the animal. ES and EG cells can be identified by positive staining with antibodies against SSEA1 (mouse) and SSEA4 (human). See, for example, U.S. Patent Nos. 5,453,357; 5,656,479; 5,670,372; 5,843,780; 5,874,301 ; 5,914,268; 6, 1 10,739 6,190,910; 6,200,806; 6,432,71 1 ; 6,436,701 , 6,500,668; 6,703,279; 6,875,607; 7,029,913; 7,1 12,437; 7,145,057; 7,153,684; and 7,294,508, each of which is incorporated by reference for teaching embryonic stem cells and methods of making and expanding them. Accordingly, ESCs and methods for isolating and expanding them are well- known in the art.

A number of transcription factors and exogenous cytokines have been identified that influence the potency status of embryonic stem cells in vivo. The first transcription factor to be described that is involved in stem cell pluripotency is Oct4. Oct4 belongs to the POU (Pit-Oct-Unc) family of transcription factors and is a DNA binding protein that is able to activate the transcription of genes, containing an octameric sequence called "the octamer motif within the promoter or enhancer region. Oct4 is expressed at the moment of the cleavage stage of the fertilized zygote until the egg cylinder is formed. The function of Oct3/4 is to repress differentiation inducing genes (i.e., FoxaD3, hCG) and to activate genes promoting pluripotency (FGF4, Utf1 , Rex1 ). Sox2, a member of the high mobility group (HMG) box transcription factors, cooperates with Oct4 to activate transcription of genes expressed in the inner cell mass. It is essential that Oct3/4 expression in embryonic stem cells is maintained between certain levels. Overexpression or downregulation of >50% of Oct4 expression level will alter embryonic stem cell fate, with the formation of primitive endoderm/mesoderm or trophectoderm, respectively. In vivo, Oct4 deficient embryos develop to the blastocyst stage, but the inner cell mass cells are not pluripotent. Instead they differentiate along the extraembryonic trophoblast lineage. Sall4, a mammalian Spalt transcription factor, is an upstream regulator of Oct4, and is therefore important to maintain appropriate levels of Oct4 during early phases of embryology. When Sall4 levels fall below a certain threshold, trophectodermal cells will expand ectopically into the inner cell mass. Another transcription factor required for pluripotency is Nanog, named after a Celtic tribe "Tir Nan Og": the land of the ever young. In vivo, Nanog is expressed from the stage of the compacted morula, is subsequently defined to the inner cell mass and is downregulated by the implantation stage. Downregulation of Nanog may be important to avoid an uncontrolled expansion of pluripotent cells and to allow multilineage differentiation during gastrulation. Nanog null embryos, isolated at day 5.5, consist of a disorganized blastocyst, mainly containing extraembryonic endoderm and no discernable epiblast.

Non-Embryonic Stem Cells

Stem cells have been identified in most tissues.

A stem cell that has been studied extensively in the art is the mesenchymal stem cell (MSC). MSCs are derived from the embryonal mesoderm and can be isolated from many sources, including adult bone marrow, peripheral blood, fat, placenta, and umbilical blood, among others. MSCs can differentiate into many mesodermal tissues, including muscle, bone, cartilage, fat, and tendon. There is considerable literature on these cells. See, for example, U.S. Patent Nos. 5,486,389; 5,827,735; 5,81 1 ,094; 5,736,396; 5,837,539; 5,837,670; and 5,827,740. See also Pittenger, M. et al, Science, 284: 143-147 (1999).

Another example of an adult stem cell is constituted by the mesoangioblasts (MABs) which have been isolated from skeletal and cardiac muscles, typically by organ culture and isolation of AP+ cells. MABs resemble MSCs on certain aspects, except that MABs are more myogenically committed, are systemically injectable and have been shown to regenerate murine and canine dystrophic muscles. Moreover, a clinical trial on the skeletal muscles of DMD (Duchenne Muscular Dystrophy) patients is actually on going (EudraCT no. 201 1- 000176-33). A method of isolation has been described in U.S. 2009/317909. See also Sampaolesi et al, /vafure,_444(71 19):574-9 (2006).

One of the best characterized adult stem cells is the hematopoietic stem cell (HSC). HSCs are mesoderm-derived cells that can be purified using cell surface markers and functional characteristics. They have been isolated from bone marrow, peripheral blood, cord blood, fetal liver, and yolk sac. They initiate hematopoiesis and generate multiple hematopoietic lineages. When transplanted into lethally-irradiated animals, they can repopulate the erythroid neutrophil-macrophage, megakaryocyte, and lymphoid hematopoietic cell pool. They can also be induced to undergo some self-renewal cell division. See, for example, U.S. Patent Nos. 5,635,387; 5,460,964; 5,677, 136; 5,750,397; 5,681 ,599; and 5,716,827. U.S. Patent No. 5, 192,553 reports methods for isolating human neonatal or fetal hematopoietic stem or progenitor cells. U.S. Patent No. 5,716,827 reports human hematopoietic cells that are Thy- 1 + progenitors, and appropriate growth media to regenerate them in vitro. U.S. Patent No. 5,635,387 reports a method and device for culturing human hematopoietic cells and their precursors. U.S. Patent No. 6,015,554 describes a method of reconstituting human lymphoid and dendritic cells. Accordingly, HSCs and methods for isolating and expanding them are well-known in the art.

Other stem cells that are known in the art include gastrointestinal stem cells, epidermal stem cells, and hepatic stem cells, which have also been termed "oval cells" (Potten, C, et al., Trans R Soc Lond B Biol Sci, 353:821-830 (1998), Watt, F., Trans R Soc Lond B Biol Sci, 353:831 (1997); Alison et al., Hepatology, 29:678-683 (1998).

Other non-embryonic cells reported to be capable of differentiating into cell types of more than one embryonic germ layer include, but are not limited to, cells from umbilical cord blood (see U.S. Publication No. 2002/0164794), placenta (see U.S. Publication No. 2003/0181269, umbilical cord matrix (Mitchell, K.E. et al., Stem Cells, 21 :50-60 (2003)), small embryonic-like stem cells (Kucia, M. et al., J Physiol Pharmacol, 57 Suppl 5:5-18 (2006)), amniotic fluid stem cells (Atala, A., J Tissue Regen Med, 1 :83-96 (2007)), skin-derived precursors (Toma et al., Nat Cell Biol, 3:778-784 (2001)), and bone marrow (see U.S. Publication Nos. 2003/0059414 and 2006/0147246), each of which is incorporated by reference for teaching these cells.

Strategies of Reprogramming Somatic Cells

Several different strategies such as nuclear transplantation, cellular fusion, and culture induced reprogramming have been employed to induce the conversion of differentiated cells into an embryonic state. Nuclear transfer involves the injection of a somatic nucleus into an enucleated oocyte, which, upon transfer into a surrogate mother, can give rise to a clone ("reproductive cloning"), or, upon explantation in culture, can give rise to genetically matched embryonic stem (ES) cells ("somatic cell nuclear transfer," SCNT). Cell fusion of somatic cells with ES cells results in the generation of hybrids that show all features of pluripotent ES cells. Explantation of somatic cells in culture selects for immortal cell lines that may be pluripotent or multipotent. At present, spermatogonia! stem cells are the only source of pluripotent cells that can be derived from postnatal animals. Transduction of somatic cells with defined factors can initiate reprogramming to a pluripotent state. These experimental approaches have been extensively reviewed (Hochedlinger and Jaenisch, Nature, 441 : 1061-1067 (2006) and Yamanaka, S., Cell Stem Cell, 1 :39-49 (2007)).

Nuclear Transfer

Nuclear transplantation (NT), also referred to as somatic cell nuclear transfer (SCNT), denotes the introduction of a nucleus from a donor somatic cell into an enucleated oocyte to generate a cloned animal such as Dolly the sheep (Wilmut et al., Nature, 385:810-813 (1997). The generation of live animals by NT demonstrated that the epigenetic state of somatic cells, including that of terminally differentiated cells, while stable, is not irreversible fixed but can be reprogrammed to an embryonic state that is capable of directing development of a new organism. In addition to providing an exciting experimental approach for elucidating the basic epigenetic mechanisms involved in embryonic development and disease, nuclear cloning technology is of potential interest for patient-specific transplantation medicine.

Fusion of Somatic Cells and Embryonic Stem Cells

Epigenetic reprogramming of somatic nuclei to an undifferentiated state has been demonstrated in murine hybrids produced by fusion of embryonic cells with somatic cells. Hybrids between various somatic cells and embryonic carcinoma cells (Solter, D., Nat Rev Genet, 7:319-327 (2006), embryonic germ (EG), or ES cells (Zwaka and Thomson, Development, 132:227-233 (2005)) share many features with the parental embryonic cells, indicating that the pluripotent phenotype is dominant in such fusion products. As with mouse (Tada et al., CurrBiol, 1 1 :1553-1558 (2001)), human ES cells have the potential to reprogram somatic nuclei after fusion (Cowan et al., Science, 309:1369-1373(2005)); Yu et al., Science, 318:1917-1920 (2006)). Activation of silent pluripotency markers such as Oct4 or reactivation of the inactive somatic X chromosome provided molecular evidence for reprogramming of the somatic genome in the hybrid cells. It has been suggested that DNA replication is essential for the activation of pluripotency markers, which is first observed 2 days after fusion (Do and Scholer, Stem Cells, 22:941 -949 (2004)), and that forced overexpression of Nanog in ES cells promotes pluripotency when fused with neural stem cells (Silva et al., Nature, 441 :997-1001

(2006) ).

Culture- Induced Reprogramming

Pluripotent cells have been derived from embryonic sources such as blastomeres and the inner cell mass (ICM) of the blastocyst (ES cells), the epiblast (EpiSC cells), primordial germ cells (EG cells), and postnatal spermatogonial stem cells ("maGSCsm" "ES-like" cells). The following pluripotent cells, along with their donor cell/tissue is as follows: parthogenetic ES cells are derived from murine oocytes (Narasimha et al., Curr Biol, 7:881 -884 (1997)); embryonic stem cells have been derived from blastomeres (Wakayama et al., Stem Cells, 25:986-993 (2007)); inner cell mass cells (Eggan et al., Nature, 428:44-49 (2004)); embryonic germ and embryonal carcinoma cells have been derived from primordial germ cells (Matsui et al., Cell, 70:841-847 (1992)); GMCS, maSSC, and MASC have been derived from spermatogonial stem cells (Guan et al., Nature, 440:1 199-1203 (2006); Kanatsu-Shinohara et al., Cell, 1 19:1001-1012 (2004); and Seandel et al., Nature, 449:346-350 (2007)); EpiSC cells are derived from epiblasts (Brons et al., Nature, 448:191 -195 (2007); Tesar et al., Nature, 448:196-199(2007)); parthogenetic ES cells have been derived from human oocytes (Cibelli et al., Science, 295L819 (2002); Revazova et al., Cloning Stem Cells, 9:432-449 (2007)); human ES cells have been derived from human blastocysts (Thomson et al., Science, 282: 1 145-1 147 (1998)); MAPC (an acronym for multipotent adult progenitor cell) have been derived from bone marrow (Jiang et al., Nature, 418:41 -49 (2002); Phinney and Prockop, Stem Cells, 25:2896-2902 (2007)); cord blood cells derived from cord blood (van de Ven et al., Exp Hematol, 35:1753-1765 (2007)); neurosphere derived cells derived from neural cell (Clarke et al., Science, 288: 1660-1663 (2000)). Donor cells from the germ cell lineage such as PGCs or spermatogonial stem cells are known to be unipotent in vivo, but it has been shown that pluripotent ES-like cells (Kanatsu-Shinohara et al., Cell, 1 19:1001 -1012 (2004) or maGSCs (Guan et al., Nature, 440: 1 199-1203 (2006), can be isolated after prolonged in vitro culture. While most of these pluripotent cell types were capable of in vitro differentiation and teratoma formation, only ES, EG, EC, and the spermatogonial stem cell-derived maGCSs or ES-like cells were pluripotent by more stringent criteria, as they were able to form postnatal chimeras and contribute to the germline. Recently, multipotent adult spermatogonial stem cells (MASCs) were derived from testicular spermatogonial stem cells of adult mice, and these cells had an expression profile different from that of ES cells (Seandel et al., Nature, 449:346-350

(2007) ) but similar to EpiSC cells, which were derived from the epiblast of postimplantation mouse embryos (Brons et al., Nature, 448: 191-195 (2007); Tesar et al., Nature, 448:196-199 (2007)).

Reprogramming by Defined Transcription Factors

Takahashi and Yamanaka have reported reprogramming somatic cells back to an ES-like state (Takahashi and Yamanaka, Cell, 126:663-676 (2006)). They successfully reprogrammed mouse embryonic fibroblasts (MEFs) and adult fibroblasts to pluripotent ES- like cells after viral-mediated transduction of the four transcription factors Oct4, Sox2, c-myc, and Klf4 followed by selection for activation of the Oct4 target gene Fbx15. Cells that had activated Fbx15 were coined iPS (induced pluripotent stem) cells and were shown to be pluripotent by their ability to form teratomas, although they were unable to generate live chimeras. This pluripotent state was dependent on the continuous viral expression of the transduced Oct4 and Sox2 genes, whereas the endogenous Oct4 and Nanog genes were either not expressed or were expressed at a lower level than in ES cells, and their respective promoters were found to be largely methylated. This is consistent with the conclusion that the Fbx15-iPS cells did not correspond to ES cells but may have represented an incomplete state of reprogramming. While genetic experiments had established that Oct4 and Sox2 are essential for pluripotency (Chambers and Smith, Oncogene, 23:7150-7160 (2004); Ivanona et al., Nature, 442:5330538 (2006); Masui et al., Nat Cell Biol, 9:625-635 (2007)), the role of the two oncogenes c-myc and Klf4 in reprogramming is less clear. Some of these oncogenes may, in fact, be dispensable for reprogramming, as both mouse and human iPS cells have been obtained in the absence of c-myc transduction, although with low efficacy (Nakagawa et al., Nat Biotechnol, 26: 191 -106 (2008); Werning et al., Nature, 448:318-324 (2008); Yu et al., Science, 318: 1917-1920 (2007)).

Cell Culture

In general, cells useful for the invention can be maintained and expanded in culture medium that is available and well-known in the art. Also contemplated is supplementation of cell culture medium with mammalian sera. Additional supplements can also be used advantageously to supply the cells with the necessary trace elements for optimal growth and expansion. Hormones can also be advantageously used in cell culture. Lipids and lipid carriers can also be used to supplement cell culture media, depending on the type of cell and the fate of the differentiated cell. Also contemplated is the use of feeder cell layers.

Cells in culture can be maintained either in suspension or attached to a solid support, such as extracellular matrix components. Stem cells often require additional factors that encourage their attachment to a solid support, such as type I and type II collagen, chondroitin sulfate, fibronectin, "superfibronectin" and fibronectin-like polymers, gelatin, poly-D and poly-L-lysine, thrombospondin and vitronectin. One embodiment of the present invention utilizes collagen. See, for example, Ohashi et al., Nature Medicine, 13:880-885 (2007); Matsumoto et al., J Bioscience and Bioengineering, 105:350-354 (2008); Kirouac et al., Cell Stem Cell, 3:369-381 (2008); Chua et al., Biomaterials, 26:2537-2547 (2005); Drobinskaya et al., Stem Cells, 26:2245-2256 (2008); Dvir-Ginzberg et al., FASEB J, 22:1440-1449 (2008); Turner et al., J Biomed Mater Res Part B: AppI Biomater, 82B: 156-168 (2007); and Miyazawa et al., Journal of Gastroenterology and Hepatology, 22:1959-1964 (2007).

Cells may also be grown in "3D" (aggregated) cultures. An example is PCT/US2009/31528.

Once established in culture, cells can be used fresh or frozen and stored as frozen stocks, using, for example, DMEM with 40% FCS and 10% DMSO. Other methods for preparing frozen stocks for cultured cells are also available to those of skill in the art.

Pharmaceutical Formulations

In certain embodiments, the cell populations are present within a composition adapted for and suitable for delivery, i.e., physiologically compatible. In some embodiments the purity of the cells (or conditioned medium) for administration to a subject is about 100% (substantially homogeneous). In other embodiments it is 95% to 100%. In some embodiments it is 85% to 95%. Particularly, in the case of admixtures with other cells, the percentage can be about 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%- 50%, 60%-70%, 70%-80%, 80%-90%, or 90%-95%. Or isolation/purity can be expressed in terms of cell doublings where the cells have undergone, for example, 10-20, 20-30, 30-40, 40- 50 or more cell doublings.

The choice of formulation for administering the cells for a given application will depend on a variety of factors. Prominent among these will be the species of subject, the nature of the condition/disease or injury being treated, its state and distribution in the subject, the nature of other therapies and agents that are being administered, the optimum route for administration, survivability via the route, the dosing regimen, and other factors that will be apparent to those skilled in the art. For instance, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form.

Final formulations of the aqueous suspension of cells/medium will typically involve adjusting the ionic strength of the suspension to isotonicity (i.e., about 0.1 to 0.2) and to physiological pH (i.e., about pH 6.8 to 7.5). The final formulation will also typically contain a fluid lubricant. In some embodiments, cells/medium are formulated in a unit dosage injectable form, such as a solution, suspension, or emulsion. Pharmaceutical formulations suitable for injection of cells/medium typically are sterile aqueous solutions and dispersions. Carriers for injectable formulations can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.

The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and/or carrier in compositions to be administered in methods of the invention. Typically, any additives (in addition to the cells) are present in an amount of 0.001 to 50 wt % in solution, such as in phosphate buffered saline. The active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, most preferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and most preferably about 0.05 to about 5 wt %. In some embodiments cells are encapsulated for administration, particularly where encapsulation enhances the effectiveness of the therapy, or provides advantages in handling and/or shelf life. Cells may be encapsulated by membranes, as well as capsules, prior to implantation. It is contemplated that any of the many methods of cell encapsulation available may be employed.

A wide variety of materials may be used in various embodiments for microencapsulation of cells. Such materials include, for example, polymer capsules, alginate-poly-L-lysine-alginate microcapsules, barium poly-L-lysine alginate capsules, barium alginate capsules, polyacrylonitrile/polyvinylchloride (PAN/PVC) hollow fibers, and polyethersulfone (PES) hollow fibers.

Techniques for microencapsulation of cells that may be used for administration of cells are known to those of skill in the art and are described, for example, in Chang, P., et al., 1999; Matthew, H.W., et al., 1991 ; Yanagi, K., et al., 1989; Cai Z.H., et al., 1988; Chang, T.M., 1992 and in U.S. Patent No. 5,639,275 (which, for example, describes a biocompatible capsule for long-term maintenance of cells that stably express biologically active molecules. Additional methods of encapsulation are in European Patent Publication No. 301 ,777 and U.S. Pat. Nos. 4,353,888; 4,744,933; 4,749,620; 4,814,274; 5,084,350; 5,089,272; 5,578,442; 5,639,275; and 5,676,943. All of the foregoing are incorporated herein by reference in parts pertinent to encapsulation of cells.

Certain embodiments incorporate cells into a polymer, such as a biopolymer or synthetic polymer. Examples of biopolymers include, but are not limited to, fibronectin, fibrin, fibrinogen, thrombin, collagen, and proteoglycans. Other factors, such as the cytokines discussed above, can also be incorporated into the polymer. In other embodiments of the invention, cells may be incorporated in the interstices of a three-dimensional gel. A large polymer or gel, typically, will be surgically implanted. A polymer or gel that can be formulated in small enough particles or fibers can be administered by other common, more convenient, non-surgical routes.

The dosage of the cells will vary within wide limits and will be fitted to the individual requirements in each particular case. In general, in the case of parenteral administration, it is customary to administer from about 0.01 to about 20 million cells/kg of recipient body weight. The number of cells will vary depending on the weight and condition of the recipient, the number or frequency of administrations, and other variables known to those of skill in the art. The cells can be administered by a route that is suitable for the tissue or organ. For example, they can be administered systemically, i.e., parenterally, by intravenous administration, or can be targeted to a particular tissue or organ; they can be administrated via subcutaneous administration or by administration into specific desired tissues.

The cells can be suspended in an appropriate excipient in a concentration from about 0.01 to about 5x10 6 cells/ml. Suitable excipients for injection solutions are those that are biologically and physiologically compatible with the cells and with the recipient, such as buffered saline solution or other suitable excipients. The composition for administration can be formulated, produced, and stored according to standard methods complying with proper sterility and stability.

Dosing

Doses for humans or other mammals can be determined without undue experimentation by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art. The dose of cells/medium appropriate to be used in accordance with various embodiments of the invention will depend on numerous factors. The parameters that will determine optimal doses to be administered for primary and adjunctive therapy generally will include some or all of the following: the disease being treated and its stage; the species of the subject, their health, gender, age, weight, and metabolic rate; the subject's immunocompetence; other therapies being administered; and expected potential complications from the subject's history or genotype. The parameters may also include: whether the cells are syngeneic, autologous, allogeneic, or xenogeneic; their potency (specific activity); the site and/or distribution that must be targeted for the cells/medium to be effective; and such characteristics of the site such as accessibility to cells/medium and/or engraftment of cells. Additional parameters include co-administration with other factors (such as growth factors and cytokines). The optimal dose in a given situation also will take into consideration the way in which the cells/medium are formulated, the way they are administered, and the degree to which the cells/medium will be localized at the target sites following administration.

The optimal dose of cells could be identified by the skilled person using the parameters as described hereabove. For fairly pure preparations of cells, optimal doses in various embodiments may range from 10 4 to 10 8 cells/kg of recipient mass per administration. In some embodiments the optimal dose per administration will be between 10 5 to 10 7 cells/kg. In other embodiments the optimal dose per administration will be 5x10 5 to 5x10 6 cells/kg.

In various embodiments, cells/medium may be administered in an initial dose, and thereafter maintained by further administration. Cells/medium may be administered by one method initially, and thereafter administered by the same method or one or more different methods. The levels can be maintained by the ongoing administration of the cells/medium. Various embodiments administer the cells/medium either initially or to maintain their level in the subject or both by intravenous injection. In a variety of embodiments, other forms of administration are used, dependent upon the patient's condition and other factors, discussed elsewhere herein.

Cells/medium may be administered in many frequencies over a wide range of times. Generally lengths of treatment will be proportional to the length of the disease process, the effectiveness of the therapies being applied, and the condition and response of the subject being treated.

"Co-administer" means to administer in conjunction with one another, together, coordinately, including simultaneous or sequential administration of two or more agents (including cells, medium, etc.).

Uses.

Administering the cells is useful to reduce any of the overt symptoms of any of the diseases/conditions/injuries as described in this invention, such as (congenital) myopathies, heart diseases, and any condition leading to deterioration of mesodermal derivative tissues. This may be based on underlying effects of the cells, such as, their capacity to further differentiation and/or homing to the desired tissue/cell lineage.

In addition, other uses are provided by knowledge of the biological mechanisms described in this invention. One of these includes drug discovery. This aspect involves screening one or more compounds for the ability to affect the cell's ability to achieve any of the effects described in this invention. Accordingly, the assay may be designed to be conducted in vivo or in vitro. Assays could assess the effect at any desired level, e.g., differentiation capacity, homing capacity, cell survival, etc.

In a specific embodiment, the cells are screened for an agent that enhances the cells' ability to prevent or reduce the events associated with a certain condition/disease or injury as described in this invention. Assessment could be in vivo, in appropriate animal models.

The assays for potency as described in this invention can include in vitro assays that measure the effects of the cells, including assays on differentiation, general viability, proliferation, and lifespan. It may also involve analysis of the function of those cells, including, appropriate or inappropriate gene expression.

Gene expression can be assessed by directly assaying protein or RNA. This can be done through any of the well-known techniques available in the art, such as by FACS and other antibody-based detection methods and PCR and other hybridization-based detection methods. Indirect assays may also be used for expression, such as the effect of gene expression.

Assays for expression/secretion (in case factors are secreted from the cells) include, but are not limited to, ELISA, Luminex. qRT-PCR, anti-factor western blots, and factor immunohistochemistry on tissue samples or cells.

A further use for the invention is the establishment of cell banks to provide cells for clinical administration. Generally, a fundamental part of this procedure is to provide cells that have a desired potency for administration in various therapeutic clinical settings.

In a specific embodiment of the invention, the cells are selected for having a desired potency for engraftment into a diseased/damaged tissue, particularly a tissue of the mesodermal lineage. In specific embodiments of this invention said diseased/damaged tissue, include at least one, or two different mesodermal tissues (or different terminally differentiated cell lineages).

In a banking procedure, the cells (or compositions) would be assayed for the ability to achieve any of the above effects. Then, cells would be selected that have a desired potency for any of the above effects, and these cells would form the basis for creating a cell bank.

It is also contemplated that potency can be increased by treatment with an exogenous compound, such as a compound discovered through screening the cells with large combinatorial libraries. These compound libraries may be libraries of agents that include, but are not limited to, small organic molecules, antisense nucleic acids, siRNA DNA aptamers, peptides, antibodies, non-antibody proteins, cytokines, chemokines, and chemo-attractants. For example, cells may be exposed to such agents at any time during the growth and manufacturing procedure. The only requirement is that there be sufficient numbers for the desired assay to be conducted to assess whether or not the agent increases potency. Such an agent, found during the general drug discovery process described above, could more advantageously be applied during the last passage prior to banking.

Another use is a diagnostic assay for efficacy and beneficial clinical effect following administration of the cells. Depending on the indication, there may be biomarkers available to assess. The dosage of the cells can be adjusted during the treatment according to the effect.

In a specific embodiment, the diagnostic assay involves assessing the engraftment capacity into a diseased/damaged tissue, particularly a tissue of the mesodermal lineage.

A further use is to assess the efficacy of the cell to achieve any of the above results as a pre- treatment diagnostic that precedes administering the cells to a subject. Moreover, dosage can depend upon the potency of the cells that are being administered. Accordingly, a pre- treatment diagnostic assay for potency can be useful to determine the dose of the cells initially administered to the patient and, possibly, further administered during treatment based on the real-time assessment of clinical effect.

In a specific embodiment, the pre-treatment diagnostic procedure involves assessing the potency of the cells to differentiate and/or engraft into a mesodermal lineage/tissue type in an in vitro or in vivo (animal model) setting, eg. By analysing the expression of certain markers more precisely those markers associated with said mesodermal lineage/tissue type.

It is also to be understood that the cells of the invention can be used not only for purposes of treatment, but also research purposes, both in vivo and in vitro to understand the mechanism involved normally and in disease models. In one embodiment, assays, in vivo or in vitro, can be done in the presence of agents known to be involved in the biological process. The effect of those agents can then be assessed. These types of assays could also be used to screen for agents that have an effect on the events that are promoted by the cells of the invention. Accordingly, in one embodiment, one could screen for agents in the disease model that reverse the negative effects and/or promote positive effects. Conversely, one could screen for agents that have negative effects in a non-disease model. Compositions

The invention is also directed to cell populations with specific potencies for achieving any of the effects described herein. As described above, these populations are established by selecting for cells that have desired potency. These populations are used to make other compositions, for example, a cell bank comprising populations with specific desired potencies and pharmaceutical compositions containing a cell population with a specific desired potency. In a specific embodiment, the cells have a desired potency to differentiate and/or engraft into a mesodermal lineage/tissue type.

Uses of cells of the invention for treatment of diseases

The diseases or disorders that can be treated by the methods of the present invention using the cells or compositions of the present invention include:

"Myopathies" are muscular diseases, in which the dysfunction of the muscle fibers or of the muscle cells results in variable degrees of muscle weakness. Myopathies can be either systemic, either limited to specific parts of the body. Myopathies include but are not limited to: muscle cramps, stiffness, spasms, myositis, drug-induced myopathies, and rhabomyolisis.

- "Congenital myopathies" are genetically inherited myopathies, in which the muscular disease generally originates from a genetic mutation. Congenital myopathies result in either stable, either degenerative muscle dysfunction with systemic or local character. Congenital myopathies include but are not limited to: muscular dystrophies (such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD)), myotonia, nemaline myopathy, multicore myopathy, myotubular myopathy, inflammatory myopathy, mitochondrial myopathy, and glycogen/lipid storage diseases.

- "Heart diseases" are acute or chronic dysfunctional conditions of the cardiac muscle, generally leading to myocardial dysfunction, dyspnea, peripheral edema, and heart failure. Heart diseases can be either acquired, either congenital. Heart diseases include but are not limited to: dilated cardiomyopathy, cardiac hypertrophy, restricted cardiomyopathy, arrhythmias, ischemia, mitochondrial cardiomyopathy, peripartum cardiomyopathy, peri- /myo-/endo-/pan-carditis.

- "Conditions leading to deterioration of mesodermal derivative tissues" include but are not limited to: medical conditions affecting the bone, the cartilage, the smooth muscles, the adipose tissue, the vessels, and other medical conditions affecting the heart and/or the skeletal muscles, which were not specifically defined among "myopathies", "congenital myopathies", and "heart diseases", such as other forms of acute or chronic muscle fiber or cardiomyocyte wastage conditions.

We can further describe our invention that comprises bipotential progenitor cells for regeneration of heart and skeletal muscle in vivo. We have identified a novel system to combine iPSC technology and epigenetic memory to produce autologous, expandable, injectable bipotential progenitors with efficient rates of migration, engraftment and regeneration in both muscle types.

Our invention embeds the generation and any possible manipulation of mesodermal iPSC- derived progenitors (MiPs) from pluripotent stem cells, preferrably - but not exclusively - from mesoangioblast-derived iPSCs (MAB-iPSCs), in - among other species - mice, dogs and humans.

This invention comprises as starting cells pluripotent stem cells, preferrably - but not exclusively - MAB-iPSCs, that are reprogrammed from somatic cells, preferrably - but not exclusively - MABs isolated from the skeletal muscle of the future or HLA-matched recipient according to published protocols, known to the skilled person.

In a specific embodiment of the present invention, the starting cells are pluripotent stem cells; in a more specific embodiment said pluripotent stam cells are IPS cells; in an even more specific embodiment, the starting cells are mesoangioblast-derived iPSCs (MAB-iPSCs).

The starting pluripotent stem cells, such as IPS cells, can be produced by - among others - retroviral, lentiviral and/or non-integrative systems carrying any efficient combination of reprogramming factors. Said starting pluripotent stem cells, can be isolated by any technique of cell isolation, including - among others - manual picking, clonal dilution and/or cell sorting. Said starting pluripotent stem cells, can be cultured and expanded according to any technique of pluripotent stem cell culture in undefined, defined and/or xeno-free conditions. The starting pluripotent stem cells, can be traced for any purpose with any method of cell tracing in vitro as well as in vivo. The starting pluripotent stem cells, can be object of any integrative or non- integrative methods of - among others - genome editing and/or genetic, epigenetic, transcriptional, protein and signaling manipulation.

The starting pluripotent stem cells, can then be further differentiated following a specific three- step differentiation protocol, involving the following steps, i) initial commitment, ii) mesodermal commitment and iii) mesodermal maturation. The initial commitment step (i) involves embryoid body formation in defined media formulation, including - among other factors - serum, L- glutamine, non-essential aminoacids, sodium pyruvate and 2-mercaptoethanol, and in specific vessels, including - among others - non-tissue-grade vessels, under controlled environmental conditions, including - among others - stable parameters of temperature, pH, carbon dioxide and oxygen. The mesodermal commitment step (ii) involves embryoid body maturation in defined media formulation, including - among other factors - serum, L-glutamine, serum, ascorbic acid, thioglycerol and transferrine, and in specific vessels, including - among others

- non-tissue-grade vessels, under controlled environmental conditions, including - among others - stable parameters of temperature, pH, carbon dioxide and oxygen. The mesodermal maturation step (iii) involves further skew of cells towards the mesodermal commitment in defined media formulation, including - among other factors - serum, L-glutamine, serum, ascorbic acid, thioglycerol and transferrine, and in specific vessels, including - among others

- tissue-grade vessels coated with preferrably - but not exclusively - collagen fibers, under controlled environmental conditions, including - among others - stable parameters of temperature, pH, carbon dioxide and oxygen. The duration of each step is, in certain embodiments of the present invention, extendable from 0 to 6 days. The commitment of differentiating cells during the three stages can be confirmed by expression levels of genes, such as - among others - Brachyury, MixH , Meoxl , Mespl , Pax3, Pax7, Desmin, Gata4, Tbx5 and Flk1 , according to any method of gene expression evaluation.

After differentiation, said MiPs can be further isolated for positiveness to CD140a (Pdgfr- alpha), CD140b (Pdgfr-beta) and CD44, in any combination and/or order of single/double/triple-antigen isolation, and according to any method of antigen-specific and/or antigen-triggered isolation and/or antigen-related transgenic/tracing systems. Said double- antigen isolation can be CD44 and CD140a; CD44 and CD140b; or CD140a and CD140b. Thus said selection step (b) in the current invention can be any combination of these 3 markers, including selecting for CD44; for CD140a, for CD140b, and/or any combination thereof, including CD44 and CD140a; CD44 and CD140b; or CD140a and CD140b; and CD44, CD140a and CD140b. MiPs can be cultured and expanded in defined media formulation, including - among other factors - serum, L-glutamine, non-essential aminoacids, 2-mercaptoethanol, ascorbic acid, thioglycerol and transferrine, and in specific vessels, including - among others - tissue-grade vessels coated with preferrably - but not exclusively

- collagen fibers, under controlled environmental conditions, including - among others - stable parameters of temperature, pH, carbon dioxide and oxygen. MiPs can be expanded for any number of passages from passage 0 onwards, till senescence arises. MiPs can be characterized by expression of genes, such as - among others - Brachyury, MixH , Meoxl , Mespl , Pax3, Pax7, Desmin, Gata4, Tbx5, and Flk1 , according to any method of gene expression evaluation. In addition, MiPs can be traced for any purpose with any method of cell tracing in vitro as well as in vivo. MiPs can be object of any integrative or non-integrative methods of - among others - genome editing and/or genetic, epigenetic, transcriptional, protein and signaling manipulation.

MiPs can present the ability of differentiating in vitro into preferrably - but not exclusively - (i) skeletal muscle fibers/skeletal myocytes/skeletal myocyte-like cells; and/or (ii) cardiomyocytes/cardiomyocyte-like cells; and/or (iii) osteocytes/osteocyte-like cells; and/or (iv) adipocytes/adipocyte-like cells; and/or (v) smooth muscle cells/smooth muscle-like cells and into any possible combination thereof. Differentiation towards skeletal muscle fibers/skeletal myocytes/skeletal myocyte-like cells (i) can be attained preferrably - but not exclusively - by co-differentiation with any source and any type of differentiating skeletal myoblasts at any relative cell concentration in defined media formulation, including - among other factors - serum, L-glutamine, non-essential aminoacids, sodium pyruvate and 2- mercaptoethanol, and in specific vessels, including - among others - tissue-grade vessels coated with preferrably - but not exclusively - collagen fibers, under controlled environmental conditions, including - among others - stable parameters of temperature, pH, carbon dioxide and oxygen. Differentiation time can expand from 0 days onwards, preferably said differentiation time is between 4 and 7 days, more preferably is 7 days. Differentiation status can be checked by any dedicated method known to the skilled person and differentiation rate can range from 0 to 100%, preferably said differentiation rate is close to 100% or is between 90 and 100%, more preferably is 100%. Differentiation towards cardiomyocytes/cardiomyocyte-like cells (ii) can be attained preferrably - but not exclusively - by low-confluence differentiation in any cell concentration in defined media formulation, including - among other factors - serum, L-glutamine, non-essential aminoacids, sodium pyruvate and 2-mercaptoethanol, and in specific vessels, including - among others - tissue- grade vessels coated with preferrably - but not exclusively - gelatin, under controlled environmental conditions, including - among others - stable parameters of temperature, pH, carbon dioxide and oxygen. Differentiation time can expand from 0 days onwards, preferably said differentiation time is between 3 and 5 days, more preferably is 5 days. Differentiation status can be checked by any dedicated method known to the skilled person and differentiation rate can range from 0 to 100%, preferably said differentiation rate is close to 100% or is between 90 and 100%, more preferably is 100%. Differentiation towards osteocytes/osteocyte-like cells (iii) can be attained preferrably - but not exclusively - by high- confluence differentiation in any cell concentration in defined media formulation, including - among other factors - serum, L-glutamine, dexamethasone, ascorbic acid and glycerol-2- phopshate, and in specific vessels, including - among others - tissue-grade vessels coated with preferrably - but not exclusively - vitronectin, under controlled environmental conditions, including - among others - stable parameters of temperature, pH, carbon dioxide and oxygen. Differentiation time can expand from 0 days onwards, preferably said differentiation time is between 20 and 30 days, more preferably is 28 days. Differentiation status can be checked by any dedicated method known to the skilled person and differentiation rate can range from 0 to 100%, preferably said differentiation rate is close to 100% or is between 90 and 100%, more preferably is 100%. Differentiation towards adipocytes/adipocyte-like cells (iv) can be attained preferrably - but not exclusively - by high-confluence differentiation in any cell concentration in defined media formulation, including - among other factors - serum, L- glutamine, dexamethasone, IBMX, insulin and indomethacin, and in specific vessels, including - among others - tissue-grade vessels coated with preferrably - but not exclusively - gelatin, under controlled environmental conditions, including - among others - stable parameters of temperature, pH, carbon dioxide and oxygen. Differentiation time can expand from 0 days onwards, preferably said differentiation time is between 20 and 30 days, more preferably is 28 days. Differentiation status can be checked by any dedicated method known to the skilled person and differentiation rate can range from 0 to 100%, preferably said differentiation rate is close to 100% or is between 90 and 100%, more preferably is 100%. Differentiation towards smooth muscle cells/smooth muscle-like cells (v) can be attained preferrably - but not exclusively - by low-confluence differentiation in any cell concentration in defined media formulation, including - among other factors - serum, L-glutamine and TGF-beta, and in specific vessels, including - among others - tissue-grade vessels coated with preferrably - but not exclusively - collagen fibers, under controlled environmental conditions, including - among others - stable parameters of temperature, pH, carbon dioxide and oxygen. Differentiation time can expand from 0 days onwards, preferably said differentiation time is between 3 and 10 days, more preferably is 10 days. Differentiation status can be checked by any dedicated method known to the skilled person and differentiation rate can range from 0 to 100%, preferably said differentiation rate is close to 100% or is between 90 and 100%, more preferably is 100%.

In certain embodiments of the present invention, said MiPs have the ability of differentiating in vivo preferrably- but not exclusively - into skeletal muscle fibers (i) and/or cardiomyocytes (ii) via any mechanism or technique of - among others - cell transplantation, cell injection, cell fusion, in vivo cell differentiation, cell-engineered scaffolds and/or any combination thereof. In vivo MiP differentiation can be achieved in xeno- or non-xenografts and/or in heterologous or autologous setting. In vivo MiP differentiation relies preferrably - but not exclusively - on injection into systemic circulation, such as - among others - injection into arterial or venous vessels. In vivo MiP differentiation can rely also on any other method of local or parenteral application, such as - among others - intramuscular, intramyocardial, intraperitoneal and/or subcutaneous injection, all well-known techniques to the skilled person. In vivo transplantation and/or injection and/or application of MiPs can be attained with any method of cell transplantation and/or injection and/or application as known to the person skilled in the art. In certain embodiments of the present invention, said in vivo MiP differentiation can be foreseen preferrably- but not exclusively - for regeneration of any type of cardiac and/or skeletal muscles, including any combination thereof. In vivo MiP differentiation time can expand from 0 to 1000 days, preferably from 10 to 100 days, more preferably from 20 to 90 days. Differentiation status can be checked by any dedicated method known to the skilled person and differentiation rate can range from 0 to 100%. In vivo MiP differentiation may result preferrably- but not exclusively - in variable levels of engraftment into host muscles and MiP- driven expression of - among other genes - markers of myogenesis and/or markers of mature muscle and/or genes whose correct expression is absent in congenital myopathies as known by the skilled person. Finally, in vivo MiP differentiation can be potentially applied to any subject/organism of any species, including - among others - Mus musculus, Canis familiaris and Homo sapiens, with a certain health/disease/injury status, in order to treat certain diseases, including - among others - congenital myopathies involving - among other symptoms - severe degeneration of cardiac and/or skeletal muscles including any combination of degeneration of cardiac an skeletal muscles.

The present invention is additionally described by way of the following illustrative, non-limiting Examples that provide a better understanding of the present invention and of its many advantages.

EXAMPLES

EXAMPLE 1 : Mesodermal progenitor cells: generation, isolation and in vitro differentiation

Murine fibroblast-derived (f-) and mesoangioblast-derived (MAB-) iPSCs were cultured on a feeder layer of mytomycin-treated primary murine embryonic fibroblasts in iPSC medium (high-glucose DMEM, supplemented with 20% KnockOut serum, 1 % sodium pyruvate, 1 % Pen/Strep, 1 % L-Glutamine, 1 % non-essential aminoacids (NEAA), 0.2% 2-mercaptoethanol, and 100U/ml LIF; all reagents from Thermo Fisher Scientific, LIF from Millipore) at 5%0 2 /5%C0 2 /37°C. Once the feeder layer removed by brief pre-plating on gelatin-coated plastic vessels (NUNC), GFP + iPSCs were then induced to form embryoid bodies (EBs) in suspension culture in non-culture-treated plastic dishes (10 5 cells in 8ml/10cm dish; Becton Dickinson) in DMEM20% medium (high-glucose DMEM, supplemented with 20% FBS, 1 % sodium pyruvate, 1% Pen/Strep, 1 % L-Glutamine, 1 % non-essential aminoacids (NEAA), 0.2% 2-mercaptoethanol) for 72hours at 5%0 2 /5%C0 2 /37 0 C. Subsequently, EBs were collected, centrifuged, disaggregated by thorough pipetting and resuspended as single cells and seeded for suspension culture in an equal number of non-culture-treated dishes as previous step, in EB medium (IMDM supplemented with 15% FBS, 1 % Pen/Strep, 1 % L- Glutamine (all from Thermo Fisher Scientific), 5mM thyoglycerol, 60μg/ml ascorbic acid, 250μg/ml holo-transferrin (all from Sigma-Aldrich)) for 72hours at 5%0 2 /5%00 2 /37°0. Then EBs were gently collected avoiding disruption, and plated on an equal number of collagen- coated culture-treated plastic dishes (NUNC) in EB medium for 72hours at 5%0 2 /5%C0 2 /37°C. At this stage, cells were gently detached by means of TrypLE (Thermo Fisher Scientific) and FACS-sorted at FACSAria III (Becton Dickinson) using APC-conjugated anti-CD140a antibody (eBioscience). Both CD140a + and CD140a " fractions were collected for further positive and negative selections respectively, and seeded in DMEM20% medium on collagen-coated 3.5cm dishes and incubated at 5%0 2 /5%C0 2 /37°C. Upon 90% confluence, in analogous conditions to the first sorting step, cells were further purified using APC- conjugated CD140b antibody and, as last step, CD44 antibody (eBioscience). After the three sorting steps (Figures 1-2), CD140+/CD140b+/CD44+ cells (murine MiPs) were cultured on collagen-coated culture-treated plastic vessels (NUNC) in DMEM20% medium at 5%0 2 /5%C0 2 /37°C. Cardiomyogenic differentiation of murine MiPs was performed in co- culture with neonatal rat cardiomyocytes or in spontaneous differentiation. Neonatal rat cardiomyocytes were harvested from newborn rat pups before 24hours post-birth by digesting the minced hearts in ADS buffer (tissue-culture water supplemented with 0.0085% NaCI, 0.0005% KCI, 0.00015% monohydrated NaH 2 P0 4 , 0.00125% monohydrated MgS0 , 0.00125% glucose, 0.00595% Hepes; all reagents from Sigma-Aldrich) supplemented with 0.4% collagenasell and 0.6% pancreatin (Sigma-Aldrich) in 3 rounds of 20min shaking in 37°C-warmed water bath, and further purified using a gradient of Percoll (0.458g/ml- 0.720g/ml; GE Healthcare). MiPs were seeded in co-culture with rat neonatal cardiomyocytes with 1 :10 MiP/cardiomyocytes ratio in cardiomyocyte maintenance medium (high-glucose DMEM supplemented with 18% Medium 199, 5% horse serum, 5% FBS, 1 % Pen/Strep, 1% L-Glutamine; all reagents from Thermo Fisher Scientific) on gelatin-coated vessels (NUNC) for 72hours at 5%0 2 /5%C0 2 /37°C. Cardiomyogenic spontaneous differentiation was performed seeding murine MiPs at low density (5000cells/cm 2 ) on collagen-coated vessels (NUNC) in RPMI20%10% medium (RPMI supplemented with 20% FBS, 10% horse serum, 1% Pen/Strep, and 1% L-Glutamine) for 72hours at 5%0 2 /5%C0 2 /37°C, then further matured in DMEM2% medium (high-glucose DMEM supplemented with 2% horse serum, 1% Pen/Strep, and 1% L-Glutamine) for 24-48hours at 5%0 2 /5%C0 2 /37°C. Skeletal myogenic differentiation of murine MiPs was performed in co-culture with C2C12 fetal myoblasts or through pulse of Pax3-Pax7 overexpression. Murine MiPs were seeded in co-culture with C2C12 fetal myoblasts with 1 :10 MiP/myoblast ratio in DMEM20% medium on collagen- coated vessels for 24hours, then differentiated in DMEM2% medium for 96-120hours at 5%0 2 /5%C02/37°C. Murine MiPs were pulsed with Pax3-Pax7 overexpression through transfection in proliferative conditions with two pSPORT6.1 plasmids bearing Pax3 and Pax7 cDNA sequences, and with Lipofectamine (Thermo Fisher Scientific) according to manufacturer's instructions, then differentiated in DMEM2% medium for 10 days. Osteogenic and adipogenic differentiation of murine MiPs were performed by means of osteogenic and adipogenic kits (Millipore) according to manufacturer's instructions. Smooth muscle differentiation was performed on MiPs seeded at low density on collagen-coated vessels in DMEM2% medium supplemented with 50ng/ml TGF i (Peprotech).

Canine and human f- and MAB-iPSCs were cultured on a feeder layer of mytomycin- treated primary murine embryonic fibroblasts in hiPSC medium (DMEM-F12 supplemented with Ham's nutrient mix, 20% KnockOut serum, 1 % Pen/Strep, 1 % L-Glutamine, 1 % nonessential aminoacids (NEAA), 0.2% 2-mercaptoethanol, and 5ng/ml bFGF (Peprotech)). Differentiation of canine and human f- and MAB-iPSCs was performed in analogous conditions to murine iPSCs, except for the first step of EB formation in hiPSC medium and for the duration of 96hours for each of the three steps. Canine and human MiPs were FACS-sorted in analogous conditions to murine MiPs, using APC-conjugated anti-CD140a, antiCD140b and anti-CD44 antibodies (BioSS) (Figures 3-4). Moreover, culture of canine and human MiPs and differentiation towards the different mesodermal lineages (cardiomyogenic, skeletal myogenic, osteogenic, adipogenic, and smooth muscle) was performed following the procedures optimized for murine MiPs. Thus for human MiPs we show here that, once isolated and traced with a fluorescent reporter, human MiPs are able to participate in both cardiac and skeletal myogenesis after in vitro co-culture with neonatal cardiomyocytes and fetal myoblasts respectively, as shown by immunostaining.

EXAMPLE 2: Mesodermal progenitor cells: In vivo injections

Sgcb-null mice are a relevant genetic model of sarcoglycanopathy LGMD-2E and a relevant pathological/transplantation model for the congenital myopathies featuring chronic degeneration of both cardiac and skeletal muscles. Dystrophic cardiomyopathic Sgcb-null mice were divided in randomized groups at 3 months post-birth (n=8, sham; n=8, f-MiPs; n=8, MAB-MiPs) and injected with GFP + murine MiPs. All mice groups were kept under cyclosporine-based immunosuppressive treatment throughout the whole experiment. MiPs were injected in parallel in the left ventricle myocardium and in both femoral arteries under isofluorane anesthesia into each animal (5x10 5 cells/1 ΟμΙ in the myocardium; 5x10 5 cells/1 ΟΟμΙ per femoral artery). Sham controls received equal treatment and amounts of cell- free saline solution. Engraftment, regeneration and functional outcome were investigated at 4 and 8 weeks post-injection (Figure 2). Here we show that, once isolated and traced with a fluorescent reporter, murine MiPs are able to engraft in both cardiac and skeletal muscles of dystrophic cardiomyopathic mice (Sgcb-null) under immunosuppressive regimen, as shown by immunostaining.

Immunodeficient Rag2-null; ^c-null mice, after transient occlusion of the coronary artery (ischemia/reperfusion-induced cardiac damage) and cardiotoxin-induced skeletal muscle wastage, constitute a relevant pathological/transplantation model for acute and/or ischemic conditions of the cardiac and skeletal muscles, being said transplantation model suitable for testing non-murine cells .Immunodeficient Rag2-null/yc-null mice were divided in randomized groups at 2 months post-birth (n=7, sham; n=7, f-MiPs; n=7, MAB-MiPs) and injected with canine MiPs. At 10 days before cardiac injection, hindlimb skeletal muscles of all mice groups were locally injected with cardiotoxin (50 l/muscle, 10μΜ solution). At 7 days before cardiac injection, mice were injected with cells or sham in both femoral arteries (5x10 5 cells/1 ΟΟμΙ per femoral artery) under isofluorane anesthesia. Cardiac injection was performed directly in the left ventricle myocardium (5x10 5 cells/1 ΟμΙ) at 1 hour post-coronary artery ligation. Engraftment, regeneration and functional outcome were investigated at 4 weeks post-injection (Figure 3). Here we show that, once isolated and traced with a fluorescent reporter, canine MiPs are able to engraft in both cardiac and skeletal muscles of immunodeficient mice (in said Rag2-null; ?c-null model). Thus for murine and canine MiPs, we show here that, once isolated and traced with a fluorescent reporter, murine and canine MiPs are able to engraft and regenerate both cardiac and skeletal muscles, in animal models of chronic or acute dual myogenic impairment, as shown by immunostaining.