Methods for Obtaining Progenitor Cells and Uses Thereof in the Treatment of Tissue or Organ Damage

Related Applications

[0001] This application claims priority to European Patent Application No. EP 08 300 109.9 filed on February 22, 2008 and entitled "Method for Obtaining Progenitor Cells". This European patent application is incorporated herein by reference in its entirety.

Field of the Invention

[0002] The invention relates to a method for obtaining progenitor cells by co-culturing adult human mesenchymal stem cells and adult fully differentiated cells. The invention also relates to the use of such progenitor cells for the treatment of pathologies, and/or for tissue reconstitution or regeneration.

Background of the Invention

[0003] For several years, technologies in the field of regenerative medicine have focussed on stem cells, as these cells have the capacity to differentiate into specialized cell types. Indeed, certain tissues or organs, such as heart tissue and neural tissue cannot regenerate alone or, at least, cannot regenerate efficiently, due to their very limited capacity of self-renewal. Regenerative medicine involves transplanting cells of interest with the goal of repairing and regenerating a target tissue and/or target organ.

[0004] Thus, heart failure (HF) is among the main causes of death in Western countries, affecting more than 6,5 million people in Europe and more than 5 million in the USA

(Tendera M, 2005). According to the World Health Organization, about 16.7 million people die globally each year from cardiovascular disease, accounting for 29% of all deaths in the world. As adult cardiomyocytes loose their proliferative potential, they fail to allow regeneration of myocardium damage occurring after myocardial infarction or other cardiac diseases, such as genetic disorders. Statistics show that about 22% of men and about 44% of women will develop heart failure within 6 years of a heart attack.

[0005] With regard to this serious public health problem, rebuilding the injured heart is a critical challenge. During the past decade, intensive efforts have been devoted to the development of methods for cardiac repair based on cell transplantation. Different cell types have been utilized in such methods, including skeletal myoblasts, neonatal myocytes,

hematopoietic stem cells and mesenchymal and embryonic stem cells. But each of these approaches exhibits limitations (e.g., extensive death of transplanted cells, lack of electrochemical junctions with host cardiomyocytes, risk of immune rejection, and teratoma formation) (Rosenthal N, 2003; Torella D et al, 2004; Leri A et al, 2004). Even if some of these strategies provide beneficial effects, these are mainly due to neoangiogenesis and arteriogenesis. However, none of these methods promote cardiac remuscularization, which is vital to improve heart function in the long term (Invernici G et al, 2008).

[0006] More recently, cardiac progenitors have been reported to generate new viable cardiomyocytes electrically integrated in the injured heart (Beltrami AP et al, 2003; Oh H et al, 2003; Messina E et al, 2004; Dawn B et al, 2005)

[0007] Nevertheless, clinical feasibility of this approach is compromised by the fact that cardiac progenitors are very difficult to isolate due to limited myocardium accessibility and progenitors rarity, and are also very difficult to expand in vitro in amounts sufficient to allow autologous transplantation.

[0008] Currently, there exists two main ways to obtain cardiac progenitors in large amounts. The first one consists in culturing adult stem cells with a chemical agent, such as polyethylene glycol (PEG). However, this leads to the formation of heterokaryons (formed by cell fusion), which cannot divide. Moreover, such heterokaryons cannot be used in therapy since they possess two nuclei (Takei et al, 2005). The second method consists in inducing cardiomyogenic differentiation of embryonic stem cells. But this method also exhibits drawbacks, since differentiation is never totally complete, which could lead to possible neoplasma and/or teratoma formation. In addition, there is still a risk of immune rejection. These drawbacks raise a major problem in the use of regenerative therapy based on embryonic stem cells (Behfar et al, 2007; Nussbaum et al, 2007). [0009] Studies and scientific reports on differentiation of mesenchymal stem cells into cardiomyocytes have never disclosed or suggested the possibility of obtaining cardiac progenitors by coculturing mesenchymal stem cells with adult cardiomyocytes. Indeed, only the differentiation to cardiomyocytes, i.e., to fully differentiated cells, has been described (Rangappa et al, 2003; Wang et al, 2005; Wang et al, 2006). On the contrary, other experiments suggest the impossibility of obtaining cardiomyocytes from mesenchymal stem cells by coculture with adult cardiomyocytes (Yoon et al, 2005).

[0010] Therefore, there still remains, in the art, an ongoing and undisputed need for a simple and effective method for obtaining progenitor cells in amounts that are useful in therapeutic applications, such as cardiac or neural progenitors, in high quantity and in a substantially pure form.

Summary of the Invention

[0011] The present invention encompasses the recognition by the applicants that, in contrast to what has been reported in the prior art, progenitor cells can be obtained by coculturing adult human stem cells with adult fully differentiated cells. In particular, the applicants have shown that coculturing adult human stem cells with adult cardiomyocytes in vitro provides cells which result from the reprogramming of cardiomyocytes by adult human stem cells and which exhibit morphologic and structural properties substantially similar neonatal or fetal cardiomyocytes that are able to proliferate. These reprogrammed cells can be obtained in large amounts in vitro, are non-immunogenic, and their administration in small animal models as well as large animal models have been demonstrated to promote heart remuscularization accompanied by improvement of contractile function.

[0012] Thus, in one aspect, the present invention provides a method for obtaining a population of progenitor cells which comprises a step of coculturing adult human mesenchymal stem cells and adult fully differentiated cells in an appropriate culture medium. The progenitor cells obtained by coculture may then be amplified. During the coculture, the adult human mesenchymal stem cells and adult fully differentiated cells are in physical contact. In the methods of present invention, the adult fully differentiated cells and the progenitor cells formed are of the same mammalian species.

[0013] In certain embodiments, the population of progenitor cells obtained is a substantially homogeneous population of progenitor cells. In other embodiments, the population of progenitor cells obtained is a heterogeneous population of progenitor cells. In particular, the population may comprise progenitor cells and adult human mesenchymal stem cells.

[0014] The adult human stem cells used in a method of the invention may be obtained from any appropriate human tissue. In certain embodiments, the adult human stem cells are derived from a human tissue selected from the group consisting of adipose tissue, skeletal muscle, bone marrow, dental pulp, blood, umbilical cord blood, cornea, retina, brain, liver,

skin, lining of the gastrointestinal tract, pancreas, and any combination thereof. In certain preferred embodiments, the adult human stem cells are derived from human adipose tissue and/or human skeletal muscle. The human tissue is preferably obtained from a healthy subject. In certain embodiments, the healthy donor is a healthy adult donor.

[0015] The adult fully differentiated cells used in a method of the invention may be any of a variety of appropriate adult fully differentiated cells. In certain embodiments, the adult fully differentiated cells are selected from the group consisting of adult cardiomyocytes, adult neurons, adult skeletal cells, and adult hepatocytes.

[0016] Progenitor cells that can be obtained according to a method of the present invention include, but are not limited to, cardiac progenitor cells, neural progenitor cells, adipogenic cells, chondrogenic cells, dermatogenic cells, hematopoietic cells, endothelial cells, myogenic cells, nephrogenic cells, urogenitogenic cells, osteogenic cells, stromal cells, pleurigenic cells, and splanchogenic cells. In certain preferred embodiments, progenitor cells obtained by a method of the invention are cardiac progenitor cells, neural progenitor cells, skeletal progenitor cells or liver progenitor cells.

[0017] Adult human stem cells to be used in the practice of the present invention may be obtained by any suitable method. However, in certain embodiments, adult human stem cells are obtained by a process comprising steps of: culturing cells isolated from a human tissue; and - selecting, from the cells isolated from the human tissue, a cell sub-population called

"CA" and exhibiting an adhesion rate lower than about 12 hours to obtain adult human stem cells.

[0018] In such a method, the human tissue from which cells are obtained may be any appropriate tissue. The human tissue is preferably obtained from a healthy subject. In certain embodiments, the healthy subject is a healthy adult subject.

[0019] The present invention also provides a method for obtaining adult human stem cells which comprises steps of: enzymatically disgesting a sample of human tissue to obtain a digested tissue sample; if adipocytes are present in the tissue sample, eliminating adipocytes from the digested tissue sample to obtain a cellular fraction essentially free of adipocytes; culturing, in vitro, cells from the cellular fraction essentially free of adipocytes; and

selecting, from the culture cells, a cell sub-population called "CA" and exhibiting an adhesion rate lower than about 12 hours to obtain adult human stem cells.

[0020] In such a method, the step of eliminating adipocytes may be carried out by centrifugation. Preferably, the cells from the cellular fraction are not filtered before the step of culturing, in order to avoid losing cells by filtration. Expansion of adult stem cells may be carried out in the presence or in the absence of a growth factor. In embodiments where a growth factor is used, the growth factor may be selected from the group consisting of bFGF, PDGF, EGF, NGF, SCF, and any combination thereof. Preferably, the growth factor is bFGF.

[0021] In still another aspect, the invention provides a population of progenitor cells that is obtained by any of the methods of coculture disclosed herein. In certain embodiments, the population of progenitor cells is a substantially homogenous population of progenitor cells. In other embodiments, the population of progenitor cells obtained is a heterogeneous population of progenitor cells. In particular, the population may comprise progenitor cells and adult human mesenchymal stem cells. [0022] In certain embodiments, the population of progenitor cells is a population of cardiac progenitor cells that results from coculture of adult human stem cells and adult cardiomyocytes. These cardiac progenitor cells exhibit morphologic, structural and/or functional properties that are similar or substantially similar to cardiac progenitors found in fetal or adult cardiac tissue. [0023] In other embodiments, the population of progenitor cells is a population of neural progenitor cells that results from coculture of adult human stem cells and adult neurons. These neural progenitor cells exhibit morphologic, structural and/or functional properties that are similar or substantially similar to neural progenitors found in fetal or adult brain tissue.

[0024] In yet another aspect, the invention provides a pharmaceutical composition comprising a population of progenitor cells obtained by any of the methods disclosed herein, and optionally, a pharmaceutically acceptable carrier or excipient. A pharmaceutical composition according to the present invention may further comprise at least one biologically active substance or bioactive factor.

[0025] A population of progenitor cells obtained by a method of the present invention, or a pharmaceutical composition thereof, may be used in the treatment of pathologies, in particular pathologies associated with tissue or organ damage, injury, dysfunction,

degeneration or abnormality, for reconstruction or regeneration of the tissue or organ. In most cases, the progenitor cells are allogenic to the subject to be treated. In certain embodiments, a population of progenitor cells, or a pharmaceutical composition thereof, is used in the treatment of a cardiac pathology, for example, a cardiac pathology selected from the group consisting of heart failure, myocardial infarction, cardiac ischemia, and inherited genetic cardiomyopathies such as Duchenne muscular dystrophy and Emery Dreiffuss. In other embodiments, a population of progenitor cells, or a pharmaceutical composition thereof, is used in the treatment of a neuronal pathology, for example, a neurodegenerative disease selected from the group consisting of polyglutamine diseases, spinocerebelloar degeneration, amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease.

[0026] In a related aspect, the present invention provides a method for treating a subject suffering from a pathology associated with tissue or organ damage, which comprises a step of administering to the subject in need thereof an efficient amount of a population of progenitor cells obtained by any of the methods disclosed herein, or a pharmaceutical composition thereof. In most cases, the progenitor cells are allogenic to the subject to be treated. In certain preferred embodiments, the population of progenitor cells comprises cardiac progenitor cells and the pathology associated with tissue or organ damage is a cardiac pathology such as heart failure, myocardial infarction, cardiac ischemia, and inherited genetic cardiomyopathies such as Duchenne muscular dystrophy or Emery Dreiffuss. In other preferred embodiments, the population of progenitor cells comprises neural progenitor cells and the pathology associated with tissue or organ damage is a neuronal pathology such as a neurodegenerative disease, i.e., polyglutamine diseases, spinocerebelloar degeneration, amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease.

[0027] The population of progenitor cells used in the methods of treatment of the present invention may be substantially homogenous or heterogeneous. For example, the population may comprise progenitor cells and adult human mesenchymal stem cells.

[0028] Administration of a population of progenitor cells to a subject suffering from a pathology associated with tissue or organ damage may be performed by any suitable method. In certain preferred embodiments, the population of progenitor cells is administered locally {i.e., at or near the site of tissue damage or degeneration). In certain embodiments, a method of treatment of the invention further comprises a step of pharmacologically immunosuppressing the subject prior to administering the population of progenitor cells. However, in most methods of treatment of the present invention, it is not necessary to

pharmacologically immunosuppressing the subject prior to administration of the population of progenitor cells since the cells administered are non- immunogenic.

[0029] These and other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art having read the following detailed description of the preferred embodiments.

Brief Description of the Figures

[0030] Figure 1 is a set of fluorescence images of hybrid cells containing material of both human and murine origin after 12 hours and after 24 hours of coculture of murine adult cardiomyocytes and adult human mesenchymal stem cells. Prior to coculture, human stem cells and murine adult cardiomyocytes were labelled with QTracker 525 (green fluorescence, Invitrogen) and CM DiI (red fluorescence, Invitrogen), respectively.

[0031] Figure 2 is a set of fluorescence images illustrating the formation of micronuclei from the murine adult cardiomyocytes and human mesenchymal stem cells.

[0032] Figure 3 is a set of two images (screen captures of a videomicroscopy) showing the presence of nanotubes between cells in coculture (i.e., murine adult cardiomyocytes and human adult stem cells) allowing intercellular exchange of materials and/or information. The nanotubes are formed from cardiomyocytes to adult stem cells (hMADS) (Figure 3A), from adult stem cells to cardiomyocytes (see Figure 3B), as well as from adult stem cells to adult stem cells (Figure 3C). [0033] Figure 4 is a set of images showing the formation of colonies of cardiac progenitors (small rounded cells).

[0034] Figure 5 is a set of fluorescence images showing the expression of GATA-4 transcription factor, an early cardiomyogenic differentiation marker, and proliferation Ki-67 nuclear antigen marker in colony-derived cells at day 7 of coculture of human adult stem cells and murine cardiomyocytes.

[0035] Figure 6 is a set of fluorescence images showing cell cardiomyogenic markers at passage 0 and passage 4.

[0036] Figure 7 is a set of electronic microscopy images showing that murine cardiac progenitor cells obtained exhibit a morphology that is typical of cardiac progenitors including

a disorganized striation pattern, a characteristic distribution of mitochondria, and a lack of sarcomers.

[0037] Figure 8 is a set of fluorescence images showing that the GATA-4 positive cardiac progenitors obtained do not express nuclear markers of human origin (human lamin AJC), but in contrast express nuclear markers of murine origin (lamin AJC).

[0038] Figure 9 is a fluorescence image of a double FISH analysis using murine COT-I (green coloration) and human COT-I (red coloration) showing that nuclei of the GATA-4 positive cardiac progenitors obtained (stained by Hoescht 33342 - blue coloration) only contain genomic material of murine origin. [0039] Figure 10 is a set of two graphs showing the results of a quantitative RT-PCR analysis of the transcriptional activity of murine and human genes involved in cardiomyogenic differentiation and in cellular proliferation as a function of time: at the start of the coculture of human adult stem cells and murine adult cardiomyocytes (0), 3.5 hours after the start of the coculture (3.5h), and at day 1 (Jl), day 4 (J4), day 7 (J7) and day 15 (J15) after the beginning of the coculture.

[0040] Figure 11 is a set of four graphs showing a comparison of the results of a quantitative RT-PCR analysis of the transcriptional activity of murine and human genes in a coculture of human adult stem cells and murine adult cardiomyocytes, wherein the cells are in physical contact (on the left hand side) and wherein the adult stem cells and cardiomyocytes are isolated from each other using a Millicell™ (on the right hand side). Measurements were performed at the start of the coculture (0), and at 3.5 hours, 1 day (Jl), 4 days (J4), 7 days (J7)and 15 days (J 15) after the beginning of the coculture

[0041] Figure 12 is a graph presenting the results of an echocardiographic analysis of ischemic mice engrafted with control medium (HBSS), human adult stem cells alone (hMADS), and cardiac progenitors.

[0042] Figure 13 is a set of pictures showing the left infracted myocardium wall thickness in mice engrafted with control medium (HBSS), human adult stem cells alone (hMADS), and cardiac progenitors. The pictures were taken 20 days post-ischemia.

[0043] Figure 14 is a graph presenting the results of an echocardiographic analysis of mdx mice engrafted with control medium (HBSS), human adult stem cells alone (hMADS), cardiac progenitors resulting from the coculture of human adult mesenchymal stem cells and

adult cardiomyocytes from mdx mice with a genetic deficiency in dystrophin (Mdx), and cardiac progenitors resulting from the coculture of human adult mesenchymal stem cells and adult cardiomyocytes from wild- type mice (WT).

[0044] Figure 15 is a set of pictures illustrating the dystrophin expression into progenitor- treated mdx mice engrafted with cardiac progenitors resulting from the coculture of human adult mesenchymal stem cells and adult cardiomyocytes from wild type mice (coculture cardio WT) and with cardiac progenitors resulting from the coculture of human adult mesenchymal stem cells and adult cardiomyocytes from mdx mice (coculture cardio Mdx).

[0045] Figure 16 is a fluorescence image showing the expression of GATA-4 transcription factor, an early cardiomyogenic differentiation marker, and proliferation Ki-67 nuclear antigen marker in colony-derived cells at day 5 of coculture of human adult stem cells and porcine adult cardiomyocytes.

[0046] Figure 17 is a set of two immunohistochemistry pictures of neural progenitor cells showing the expression of β3 tubulin (in red) and of Ki-67 (in green).

Detailed Description of the Invention

[0047] The present inventors have demonstrated that progenitor cells can be obtained by coculturing adult mesenchymal human stem cells with adult fully differentiated cells. In certain embodiments, adult fully differentiated cells are cells which do not divide or divide poorly in vivo, or for which progenitor cells do not exist or are extremely rare. In other embodiments, adult fully differentiated cells are cells for which progenitors do exist in vivo but have genetic defect. In such embodiments, progenitors generated by a method according to the invention will have their genetic defect corrected by coculture of the differentiated cells with adult stem cells, hence their interest in therapy. In yet other embodiments, adult fully differentiated cells are cells which have a genetic defect and for which progenitors do not exist in vivo or are rare.

[0048] As intended herein, the term "coculturing" refers to a process in which at least two different types of cells are cultured together in an appropriate culture medium. In the methods of the present invention, human adult stem cells and fully differentiated cells are cocultured.

[0049] As used herein, the term "appropriate culture medium" refers to a culture medium that contains nutrients necessary to support the growth and/or survival of the cocultured cells and of the population of progenitor cells obtained from the coculture, but that does not contain

any chemical reagent generally used for the cell fusion of stem cells with somatic cells, such as polyethylene glycol (PEG). An appropriate culture medium may or may not further comprise growth factors. By way of examples, growth factors of interest may be bFGF (also known as FGF-2), BMP2, IGFl, TNF-α, TGFβ-1, BMP-2, BMP-4, Activin-A, FGF-2, FGF- 4, IL-6, IGF-I, IGF-2, VEGF-A, EGF, and any combination of these or other growth factors. For example, an appropriate culture medium may further comprise a cocktail of growth factors such as cocktails used for the differentiation of human or canine mesenchymal cells (Bartunek et ah, 2007, which is incorporated herein by reference in its entirety) or for the differentiation of murine embryonic stem (ES) cells in cardiomyocytes (Behfar A et ah, 2007, which is incorporated herein by reference in its entirety).

[0050] Thus, an appropriate culture medium according to the invention may consist in a minimal medium in which cells can grow, such as for example Dulbecco modified Eagle's minimal essential medium (DMEM) supplemented or not with decomplemented fetal calf serum (FCS). Adult human mesenchymal stem cells and adult fully differentiated cells may be plated in coated (e.g., gelatine-coated) or uncoated plates.

[0051] As used herein, the term "adult human mesenchymal stem cells" generally refers to undifferentiated cells found in a differentiated (specialized) tissue and that are capable of making identical copies of themselves (self-renewal) for the lifetime of the organism. Adult human mesenchymal stem cells that can be used in the context of the present invention thus include any suitable adult human stem cells (i.e., cells with an ability for self-renewal) derived from any suitable tissue using any appropriate isolation method. For example, adult human mesenchymal stem cells that can be used in the methods of the present invention include cells previously described in international patent application PCTYFR2003/002439 (the content of which is incorporated herein by reference in its entirety) and in Rodriguez et ah, 2005. These cells are called human Multipotent Adipose tissue Derived Stem cells (or hMADS). Other adult human mesenchymal stem cells that can be used in the methods of the present invention are cells derived from adipose tissue and skeletal muscle of an adult person and obtained using a method disclosed in international patent application PCT/FR2003/002439 or a variation of this- that method developed by the present inventors and described herein.

[0052] Such mesenchymal stem cells exhibit a very important ability for self-renewal and, in particular, are capable of sustained self-renewal during at least 130 doublings of the population. Other mesenchymal stem cells also exhibiting an important capacity for self-

renewal and that may be used in the practice of the methods of the present invention include, but are not limited to, adult multilineage inducible (MIAMI) cells (D'Ippolito G et al, 2004), MAPC (also known as MPC) (Reyes M et al, 2002), cord blood derived stem cells (Kogler G et al, 2004), and mesoangioblasts (Sampaolesi M et al, 2006; Dellavalle A et al, 2007). In particular, umbilical cord blood stem cells are easy to expand in vitro, are multipotent, have been reported to be non- immunogenic (Wang et al, 2009; Ji et al, 2008) and have been shown to fail to elicit an immune response in allogeneic hosts after engraftment into the diseased heart (Henning et al, 2007). Furthermore, umbilical cord blood banks (e.g., Etablissement Francais du Sang, in France) provide secure and easily available sources of such cells for transplantation.

[0053] As used herein, the term "adult fully differentiated cells" refers to cells specialized for a particular function (e.g., adult cardiomyocytes, adult neurons) and that do not have the ability to generate other kinds of cells. It must be further noted that certain tissues or organs cannot, or at least cannot efficiently, regenerate. Thus, as already mentioned above, in certain embodiments of the present invention, adult fully differentiated cells of interest are cells for which no endogenous progenitors exist in vivo or at least are extremely rare. In other embodiments of the present invention, adult fully differentiated cells are cells for which endogenous progenitors do exist in vivo but have a genetic defect. In such embodiments, coculture of adult fully differentiated cells with adult stem cells according to the present invention will lead to progenitor cells in which the genetic defect would have been corrected. In certain embodiments, adult fully differentiated cells are cells that combine rarity of progenitor cells in vivo and presence of a genetic defect.

[0054] In the context of the present invention, adult fully differentiated cells may be from any appropriate mammal origin (e.g., mouse, rat, rabbit, pig, dog or human cells). For example, adult cardiomyocytes that can be used according to the invention may be mouse, rat, rabbit, pig, dog or human adult cardiomyocytes.

[0055] The terms "progenitor cells" and "precursor cells" are used herein interchangeably. They refer to cells that occur in fetal or adult tissue and are partially specialized. These cells divide and give rise to differentiated cells. Progenitor or precursor cells belong to a transitory amplifying population of cells derived from stem cells. Compared to stem cells, they have a limited capacity for self-renewal and differentiation. Such capacity for self-renewal (or proliferation) is demonstrated by the expression of proliferation markers such as, for example, Ki-67 nuclear antigen. Moreover, since progenitor cells are committed to a particular

differentiation process, progenitor cells also express specific markers. The terms "progenitor cells" and "precursor cells" also encompass cells obtained by the methods disclosed herein and which result from the reprogramming of adult fully differentiated nuclei by adult human stem cells. These cells are "progenitor-like cells" in that they exhibit morphologic, structural and functional properties that are similar or substantially similar to partially specialized cells found in fetal or adult tissue.

[0056] As used herein, the term "marker" refers to a protein, glycoprotein or other molecule expressed on the surface of a cell or into a cell, and which can be used to help identify the cell (e.g., identify the type of cell). A marker can generally be detected by conventional methods. Specific non-limiting examples of methods that can be used for the detection of a cell surface marker are immunohistochemistry, fluorescence activated cell sorting (FACS), and enzymatic analysis.

[0057] It should also be highlighted that progenitor cells obtained according to a method of the present invention are uninucleated. Advantageously, such progenitor cells can easily divide in vivo and also retain the non- immunogenic properties previously described for the mesenchymal stem cells (Le Blanc K., 2007; Rodriguez et al., 2005).

[0058] In certain preferred embodiments of the present invention, progenitor cells are cardiac progenitors or neural progenitors.

[0059] The terms "cardiac progenitors" and "cardiomyogenic progenitors" are used herein interchangeably, and refer to progenitor cells that can divide and give rise to cardiac cells.

The terms also encompass progenitor cells that are obtained by a method disclosed herein

(i.e., by coculturing human adult stem cells and adult cardiomyocytes) and that can divide and give rise to cardiac cells. These progenitor cells exhibit morphologic, structural and functional properties that are similar or substantially similar to cardiac progenitors found in fetal or adult heart tissue. Thus, preferably, cardiac progenitors of the invention express both specific cardiac (or cardiomyogenic) markers, such as GATA-4, desmin and sarcomeric alpha

(α) actinin, and proliferation markers such as Ki-67 nuclear antigen. More preferably, cardiac progenitors of the invention express cardiac markers at levels generally found in cardiac progenitors that occur in the heart and not at levels generally found in cardiomyocytes. Moreover, cardiac progenitors that occur in the heart have specific morphologic characteristics; they resemble neonatal or fetal cardiomyocytes that are able to proliferate.

Thus, preferably, cardiac progenitors of the present invention display a disorganized striation pattern and a characteristic distribution of mitochondria, but lack of sarcomere.

[0060] The terms "neural progenitors" and "neural precursors" are used herein interchangeably, and refer to progenitor cells that can divide and give rise to neural cells. The terms also encompass progenitor cells that are obtained by a method disclosed herein (i.e., by coculturing human adult stem cells and adult neurons) and that can divide and give rise to neural cells. These progenitor cells exhibit morphologic, structural and functional properties that are similar or substantially similar to neural progenitors found in the brain. Thus, preferably, neural precursors of the present invention express both specific neuronal markers, such as class III β-tubulin (p ₃ -tubulin), and proliferation markers, such as Ki-67 nuclear antigen. More preferably, neural progenitors of the invention express neuronal markers at levels generally found in neural progenitors that occur in the brain and not at levels generally found in neurons.

[0061] One skilled in the art will understand that the type of progenitor cells obtained using a method of the present invention will depend on the type of adult fully differentiated cells used in the coculture. Thus, for example, the use of adult cardiomyocytes as fully differentiated cells in a coculture according to the invention will yield cardiac progenitors. Similarly, the use of adult neurons as fully differentiated cells in a coculture according to the invention will yield neuron progenitors. [0062] A method according to the present invention provides a population of progenitor cells. In certain embodiments, the method provides a substantially homogeneous population of progenitor cells. The term "substantially homogeneous population", as used herein, refers to a population of cells wherein the majority (e.g., at least about 80%, preferably at least about 90%, more preferably at least about 95%) of the total number of cells have the specific characteristics of the progenitor cells of interest. In other embodiments, the method provides a heterogeneous population of progenitor cells. The term "heterogeneous population", as used herein, refers to a population of cells comprising the progenitor cells of interest and at least one additional (different) type of cells. For example, a heterogeneous population of progenitor cells may comprise progenitor cells of interest and adult human mesenchymal stem cells. Generally, a heterogeneous population of progenitor cells will comprise at least about 40%, preferably at least about 50%, more preferably at least about 60% of progenitor cells.

I - Progenitor Cells Cardiac Progenitors

[0063] In an attempt to overcome the problems described in the state of the art that are associated with isolation and in vitro expansion of cardiac progenitors, the present inventors have developed a simple method for obtaining cardiac progenitors. This method involves a step comprising coculturing adult human stem cells and adult cardiomyocytes. The inventors have found that this coculture step during which adult human stem cells and adult cardiomyocytes are in physical contact is crucial to the reprogramming of adult fully differentiated nuclei by stem cells leading to the formation of cardiomyogenic progenitors. Cardiac progenitors resulting from a method of the present invention are able to proliferate in vitro for long periods of time (for example, more than about 3 months) with no apparent changes in the phenotype.

[0064] The inventive method provides a valuable alternative to obtain an infinite number of cardiac progenitor cells that can be used as pharmacological and/or therapeutic tools. [0065] Indeed, a method according to the present invention allows for the formation of repairing cells with higher survival ability than stem cells, following engraftment to the damaged heart. For example, administration of cardiac progenitors, obtained according to the present invention, to mouse myocardial infarction models efficiently improves ventricular function by myocardium muscle regeneration, as demonstrated by the applicants and illustrated herein in the Examples section. Similar results were obtained in the pig, a relevant large animal model closely reflecting human cardiac pathology (see Examples section).

[0066] In addition, the present applicants have also observed similar beneficial effects in dystrophin-deficient mdx mice, a genetic cardiomyophathy model (see Examples section). In this context, cardiac progenitors were found to contribute to the correction of genetic defects by inducing dystrophin expression that consequently gives rise to partial cardiac restoration. This means that in a clinical setting, it would not be necessary to perform gene therapy which would require introduction of a gene in progenitor cells of interest.

[0067] Advantageously, cardiac progenitors obtained according to a method of the present invention share a common immune privilege behavior with human adult mesenchymal stem cells, which are used in their formation. Indeed, both in rodents and in large animals, the cardiac progenitors failed to elicit an immune response in the host engrafted heart (see

Examples section). This opens up the possibility of treating cardiac dysfunction in an allotransplantation context, bypassing immune rejection risk.

Neural Progenitors

[0068] The present inventors have also shown that the method described herein for obtaining progenitor cells may also be used to obtain neural progenitors. In this context, adult human mesenchymal stem cells are cocultured with adult neurons in an appropriate culture medium. During coculture, the human adult mesenchymal stem cells and the adult neurons are in physical contact. Using this method, neural progenitor cells can be generated in an amount that is suitable for use in therapeutic applications. [0069] Thus, neural progenitor cells obtained by a method of the present invention may be useful in cell transplantation protocols for the healing, repair and/or regeneration of damaged, injured, degenerated or abnormal (e.g., exhibiting a genetic defect) tissues of the central nervous system (brain and/or spinal cord).

Other Progenitor Cells [0070] As mentioned above, adult fully differentiated cells of interest are cells for which no endogenous progenitors exist in vivo or at least are rare or difficult to isolate and/or to expand in vitro. Alternatively, adult fully differentiated cells of interest may be cells for which endogenous progenitors exist in vivo and are capable of self-renewal, but have a genetic defect. [0071] Thus, a method according to the present invention may also be used to obtain any other type of progenitor cells (i.e., other than cardiac and neural progenitors described above). Exemplary progenitors that can be obtained using a method of the invention include, but are not limited to, progenitors from skeletal muscle, liver, pancreas, and the like. Other examples include adipogenic cells, chondrogenic cells, dermatogenic cells, hematopoietic cells, endothelial cells, myogenic cells, nephrogenic cells, urogenitogenic cells, osteogenic cells, stromal cells, pleurigenic cells, splanchogenic cells, and the like.

II - Methods for the Generation of Progenitor Cells

[0072] The method disclosed herein for generating progenitor cells comprises a step of coculturing adult human mesenchymal stem cells and adult fully differentiated cells in an appropriate culture medium. In a particular embodiment, the method comprises steps of:

a) providing adult mesenchymal stem cells and adult fully differentiated cells, and b) coculturing said cells in an appropriate culture medium.

In and during the coculture, the adult mesenchymal stem cells and adult fully differentiated cells are in physical contact.

Providing Adult Human Mesenchymal Stem Cells and Adult Differentiated Cells

[0073] In this particular context, the term "providing" herein refers to a process in which cells are isolated and provided in a state suitable for in vitro culture. As used herein, the term "isolated" refers to a cell which has been separated from at least some components of its natural environment. This term includes gross physical separation of the cell from natural environment {e.g., removal from the donor). Preferably, "isolated" includes alteration of the cell's relationship with the neighboring cells with which it is in direct contact, for example, by dissociation.

[0074] Within the context of the invention, human stem cells are preferably adult human mesenchymal stem cells, which may be derived from a large variety of tissues. In certain embodiments, human mesenchymal stem cells are derived from adipose tissue. In other embodiments, human mesenchymal stem cells are derived from skeletal muscle. In yet other embodiments, human mesenchymal stem cells are derived from adipose tissue and skeletal tissue. In still other embodiments, human mesenchymal stem cells are derived from bone marrow, dental pulp, blood, and umbilical cord blood. [0075] In most of the present document, the method for obtaining progenitor cells is described as involving the coculture of adult human mesenchymal stem cells and adult fully differentiated cells in an appropriate culture medium. However, it is to be understood that the present invention encompasses methods for obtaining progenitor cells wherein adult human stem cells (i.e., adult human non- mesenchymal stem cells) are used in place of adult human mesenchymal stem cells. Examples of tissues from which adult human (non-mesenchymal) stem cells may be obtained include, but are not limited to, tissues of endodermal origin such as the liver and pancreas, and tissues of ectodermal origin such as the cornea and/or the retina of the eye, the brain and the skin.

[0076] Similarly, it is to be understood that the present invention encompasses methods for obtaining progenitor cells wherein adult non-human mammalian stem cells are used in place of adult human stem cells. Adult non-human mammalian stem cells that can be used in the practice of the present invention include any adult stem cells of a non-human mammalian

origin, such as, for example, of mouse, rat, dog, cat, pig, guinea pig, hamster, or monkey origin and the like.

[0077] As already mentioned above, adult fully differentiated cells may be differentiated cells derived from any tissue or organ sample (e.g., brain, liver, lung, heart, kidney, skin, muscle, bone, bone marrow, or blood). Suitable cell types include, but are not limited to, basal cells, epithelial cells, platelets, lymphocytes, T-cells, B-cells, natural killer cells, reticulocytes, granulocytes, monocytes, mast cells, neurocytes, neuroblasts, cytomegalic cells, dendritic cells, macrophages, blastomeres, endothelial cells, interstitial cells, Kupffer cells, Langerhans cells, littoral ceils, tissue cells such as muscle cells and adipose cells, and the like. In certain preferred embodiments, differentiated cells are selected from the group consisting of adult cardiomyocytes, adult neurons, and any other cells for which no endogenous progenitors exist in vivo, or at least are extremely rare. Alternatively or additionally, differentiated cells are any cells for which endogenous progenitors exist in vivo but have a genetic defect. [0078] In the context of the present invention, a cell is "derived from" a subject or a sample (e.g., a biological sample) if the cell is obtained (e.g., isolated, extracted, or purified) from the subject or sample. A cell derived from an organ, tissue, cell line, etc. may be modified in vitro after it is obtained. Such a modified cell is still considered to be derived from the original source. [0079] Thus, adult human mesenchymal stem cells and adult fully differentiated cells may be independently isolated from any suitable tissue sample. The term "tissue sample", as used herein, refers to any sample of tissue harvested from a suitable mammal, as already mentioned above. In the context of the present invention, tissue samples are preferably obtained from healthy donors. The donor may be of any age. However, in certain preferred embodiments, the donor is a healthy adult.

[0080] Methods of harvesting samples from tissues and organs are known in the art and can be used in the practice of the present invention. Preferably, methods of harvesting are not excessively destructive for the tissue being harvested. Thus, for example, human tissue samples are preferably not obtained by liposuction. [0081] Isolation of cells of interest from a tissue sample preferably occurs in an aseptic environment. In embodiments where the tissue sample is solid or semi-solid, blood and debris are removed from the tissue sample prior to isolation of the cells. For example, the

tissue sample may be washed with a buffer solution (e.g., buffered saline) optionally comprising antimytotic and/or antibiotic agents.

[0082] In certain embodiments, the different cell types present in the tissue sample are fractionated into subpopulations from which the cells of interest can be isolated. This may be accomplished using techniques for cell separation including, but not limited to, mechanical treatment (e.g., mincing or shear forces) and/or enzymatic digestions (e.g., using one or more proteolytic enzymes or combination of proteolytic enzymes such as neutral proteases, metalloproteases, serine proteases, deoxyribonucleases, for example, collagenase, trypsin, chymotrypsin, thermolysin, dispase, elastase, hyaluronidase, pepsin, and the like to dissociate the tissue sample into its component cells, followed by cloning and selection of specific cell types.

[0083] Any suitable clonal selection and cell separation techniques may be used in the practice of the present invention. Suitable methods of cell selection and/or separation include, but are not limited to, selection based on morphologic and/or biochemical markers, selective growth of desired cells (positive selection), selective destruction of unwanted cells (negative selection), separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal elutriation, and the like.

[0084] In certain embodiments, adult human mesenchymal stem cells are isolated and obtained as described in international patent application PCT/FR2003/002439

(WO/2004/013275) with the difference that any suitable tissue sample may be used (including those described above) and that the donor may be an adult donor and not just a child of less than 10 years of age. Other differences include the fact that the stem cells are not necessarily quiescent or do not necessarily have the ability to become quiescent (in contrast to the method disclosed in PCT/FR2003/002439).

[0085] In certain embodiments, adult human mesenchymal stem cells may be obtained using a method comprising one or more of the following steps: a) enzymatic digestion of a tissue sample (e.g., in the presence of collagenase for about 10 minutes or less) to obtain a cellular fraction; b) in vitro culture of the cellular fraction; and

c) selection, from the cellular fraction, of a cell sub-population of cells called "CA" and exhibiting an adhesion rate lower than about 12 hours to obtain adult human mesenchymal stem cells.

If the tissue sample is an adipose tissue, the method further comprises, prior to step (b), a step of elimination of adipocytes from the digested tissue sample obtained in step (a) (e.g., by filtration), which leads to a cellular fraction essentially free of adipocytes.

[0086] Before coculture, adult human mesenchymal stem cells may be cultured according to standard cell culture techniques. For example, cells are often grown in a suitable vessel in a sterile environment at 37 ⁰ C in an incubator containing a humidified 95% air - 5% CO ₂ atmosphere. Vessels may contain stirred or stationary cultures. Cell culture techniques are well known in the art and established protocols are available for the culture of diverse cell types (see, for example, R.I. Freshney, "Culture of Animal Cells: A Manual of Basic Technique", 2 ^nd Edition, 1987, Alan R. Liss, Inc.).

[0087] If desired, cell viability can be determined, prior to coculture, for example, using standard techniques including histology, quantitative assessment with radioisotopes, visual observation using a light or scanning electron microscope or a fluorescent microscope. Alternatively, cell viability may be assessed by Fluorescence- Activated Cell Sorting (FACS).

[0088] If desired, adult human mesenchymal stem cells and/or adult fully differentiated cells, either freshly isolated from a tissue sample or following expansion in culture, can be independently cryopreserved for future use in a coculture according to the present invention. In such a case, the cells are preferably cryopreserved under such conditions that most of the cells are viable upon recovery (i.e., thawing). Preferably, more than about 50%, 75%, 80%, or 85% of the cryopreserved cells are viable after recovery. More preferably, more than about 90% of the cryopreserved cells are viable after recovery. Even more preferably, more than about 95% or about 99% of the cryopreserved cells are viable after recovery. Preferably, the cryopreservation conditions are such that viable cells have identical morphologic and functional characteristics as the cells prior to cryopreservation.

[0089] Methods for the cryopreservation of different types of cells are known in the art. Any suitable method of cryopreservation may be used in the practice of the present invention. Typically, the cryopreservation medium contains dimethyl sulfoxide (DMSO). The cryopreservation medium may further comprise cryopreservation agents such as, methylcellulose. Once frozen, the adult mesenchymal stem cells and the adult fully

differentiated cells may be independently stored indefinitely under liquid nitrogen until needed, as long as care is taken to prevent the possibility of accidental thawing or warming of the frozen cells at any time during their storage period.

[0090] When the cells are to be used in a method of the present invention, they can be thawed under controlled conditions, for example by transferring the vial(s) containing frozen cells to a water bath set at 37 ⁰ C. The thawed contents of the vial(s) may then be rapidly transferred under sterile conditions to a culture vessel containing an appropriate medium. The thawed cells can then be tested for viability, growth properties, etc,

Coculturinε of Adult Human Mesenchymal Stem Cells and Adult Differentiated Cells [0091] Coculture of adult human mesenchymal stem cells and adult fully differentiated cells may be carried out using any suitable method. The adult human mesenchymal stem cells and adult fully differentiated cells are cocultured under conditions where they are in physical contact. The applicants have shown that physical contact is required for the reprogramming of adult differentiated nuclei to take place leading to the formation of progenitor cells (see Examples section). As used herein, the term "physical contact" has its general meaning. For example, cells are in physical contact with each other when they are in a conformation or arrangement that allows for intercellular exchange of materials and/or information to take place without the involvement of a soluble factor. Such conformations or arrangements include, but are not limited to, configurations comprising junction gaps, intercellular nanotubes, interactions between membrane receptors and membrane ligands, and the like.

[0092] In certain embodiments, the adult human mesenchymal stem cells and adult fully differentiated cells are first put in suspension together in an appropriate culture medium before being plated, which leads to an aggregation of the two different kinds of cells. In other embodiments, the adult human mesenchymal stem cells are plated in an appropriate culture medium in order to obtain a cell lawn of mesenchymal stem cells, and then the adult fully differentiated cells are added onto the plate of mesenchymal stem cells.

[0093] In certain particular embodiments, the cells are cocultured in a culture medium that does not comprise any growth factor. Indeed, as shown by the inventors (see Examples section), a method according to the invention may be successfully carried out in the absence of any growth factors.

[0094] In other particular embodiments, adult human mesenchymal stem cells are plated on coated plates, e.g., gelatine-coated plates.

[0095] Adult human mesenchymal stem cells and adult fully differentiated cells may be cocultured for any efficient amount of time, i.e., any amount of time that is necessary to allow the formation of progenitor cells. One skilled in the art will know how to determine such an amount of time. In certain embodiments, adult human mesenchymal stem cells and adult fully differentiated cells are cocultured for at least about 7 days and preferably for at least about 15 days in an appropriate culture medium, as described herein.

[0096] Moreover, adult human stem cells and adult fully differentiated cells may be cocultured in any efficient ratio, i.e., in any ratio that leads to the formation of progenitor cells. One skilled in the art will know how to determine such a ratio, and will also know how to identify optimal ratio conditions for the efficient formation of progenitor cells. For example, in certain embodiments, adult human mesenchymal stem cells and adult fully differentiated cells may be cocultured in a ratio of about 1:1.

[0097] Advantageously, from 2 days after initiation of the coculture of mesenchymal stem cells and adult fully differentiated cells, colonies of small rounded cells appear in the plates, leading to the formation of colony-derived cells, which are the progenitor cells of interest. Progenitor cells thus formed may then be recovered and amplified.

[0098] Therefore, in a particular embodiment, a method according to the present invention further comprises a step of amplifying (or expanding) the progenitor cells obtained. More specifically, this step of amplifying may consist in dissociating cells, for example, using trypsin and then replating the dissociated cells. It should be further noted that the step of amplifying can be repeated several times.

[0099] Thus, a method according to the present invention constitutes a quick and easy way to obtain an unlimited number of progenitor cells that can be used in therapeutic applications. In addition, a method according to the present invention generally provides a substantially homogeneous or substantially pure population of progenitor cells, thus avoiding an additional step of sorting cells prior to their use in therapy (for example in allotransplantation).

II - Progenitor Cells

[0100] In another aspect, the present invention relates to a population of progenitor cells, obtainable according to a method of the invention or an obvious variation thereof. In certain

embodiments, the population of progenitor cells is substantially homogenous. In other embodiments, the population of progenitor cells is heterogeneous.

[0101] The Applicants have demonstrated that formation of progenitor cells according to the invention results from nuclear reprogramming of adult fully differentiated cells by human adult stem cells. Thus, the progenitor cells obtained are of the same mammalian species as the adult fully differentiated cells used in the preparation method. In other words, using human adult stem cells and murine adult fully differentiated cells according to the invention will yield murine progenitor cells; while using human adult stem cells and porcine adult fully differentiated cells will yield porcine progenitor cells.

[0102] In certain preferred embodiments, the population of progenitor cells is a population of cardiac progenitors or a population of neural progenitors. In other embodiments, the population of progenitor cells is a population of skeletal muscle progenitor cells or liver progenitor cells. In still other embodiments, the population of progenitor cells is a population of adipogenic cells, chondrogenic cells, dermatogenic cells, hematopoietic cells, endothelial cells, myogenic cells, nephrogenic cells, urogenitogenic cells, osteogenic cells, stromal cells, pleurigenic cells, pancreagenic cells, or splanchogenic cells. In yet other embodiments, the population of progenitor cells is population of progenitor cells devoid of genetic defects.

[0103] As already mentioned above, the present applicants have demonstrated that a population of cardiac progenitors obtained according to the invention is efficient at improving remuscularization of damaged myocardium. This remuscularization results in a significant and efficient reconstitution and/or regeneration, which has previously never been observed when other populations of cells (i.e., differentiated cells or stem cells) have been used. As already mentioned, according to the prior art, injections of such other populations of cells only led to a slight improvement of angiogenesis limiting cardiomyocyte loss after infraction but without being able to regenerate new heart tissue.

[0104] Thus, the inventors have demonstrated the potential role of progenitor cells in cell therapy, as illustrated, for example, in the case of cardiac progenitors.

[0105] Furthermore, use of a population of progenitor cells according to the invention will avoid possible neoplasma and/or teratoma formation, and will eliminate, or at least significantly reduce, the risk of immune rejection in a grafted patient, in contrast to previously injected populations of cells in the prior art.

HI - Pharmaceutical Compositions and Use Thereof

[0106] A further aspect of the invention relates to a pharmaceutical composition comprising a population of progenitor cells, preferably a substantially homogenous population of progenitor cells, and optionally a pharmaceutically acceptable carrier or excipient. In certain embodiments, a pharmaceutical composition may further comprise at least one biologically active substance or bioactive factor.

[0107] As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the progenitor cells, and which is not excessively toxic to the host at the concentrations at which it is administered. Examples of suitable pharmaceutically acceptable carriers or excipients include, but are not limited to, water, salt solution (e.g., Ringer's solution), alcohols, oils, gelatins, carbohydrates (e.g., lactose, amylase or starch), fatty acid esters, hydroxymethylcellulose, and polyvinyl pyroline. Pharmaceutical compositions may be formulated as liquids, semi-liquids (e.g., gels) or solids (e.g., matrix, lattices, scaffolds, and the like). If desired, the pharmaceutical composition may be sterilized.

[0108] As used herein, the term "biologically active substance or bioactive factor" refers to any molecule or compound whose presence in a pharmaceutical composition of the invention is beneficial to the subject receiving the composition. As will be acknowledged by one skilled in the art, biologically active substances or bioactive factors suitable for use in the practice of the present invention may be found in a wide variety of families of bioactive molecules and compounds. For example, a biologically active substance or bioactive factor useful in the context of the present invention may be selected from anti- inflammatory agents, anti-apoptotic agents, immunosuppressive or immunomodulatory agents, antioxidants, growth factors, and drugs. [0109] In certain preferred embodiments, the pharmaceutical composition comprises a population of cardiac progenitors, preferably a substantially homogenous population of cardiac progenitors. In other preferred embodiments, the pharmaceutical composition comprises a population of neural progenitors, preferably a substantially homogenous population of neural progenitors. [0110] Another aspect of the invention pertains to such pharmaceutical compositions for treating pathologies, and/or for tissue reconstitution or regeneration.

[0111] A related aspect of the invention concerns a method for treating a subject suffering from a pathology associated with tissue or organ damage, said method comprising a step of administering to the subject an efficient amount of a population of progenitor cells as described herein, or a pharmaceutical composition thereof.

[0112] In the context of the invention, the term "treating" or "treatment", as used herein, refers to a method that is aimed at delaying or preventing the onset of a pathology, at reversing, alleviating, inhibiting, slowing down or stopping the progression, aggravation or deterioration of the symptoms of the pathology, at bringing about ameliorations of the symptoms of the pathology, and/or at curing the pathology.

[0113] As used herein, the term "subject" refers to a mammal, preferably a human being, that can suffer from a pathology associated with tissue or organ damage, but may or may not have the pathology. The term "subject" does not denote a particular age, and thus encompasses adults, children, and newborns.

[0114] As used herein, the term "efficient amount" refers to any amount of a population of progenitor cells (or a pharmaceutical composition thereof) that is sufficient to achieve the intended purpose.

[0115] As used herein, the term "pathology" refers to any disease or condition, in particular to any disease or condition associated with tissue or organ damage. The term "pathology associated with tissue or organ damage" refers to any disease or clinical condition characterized by tissue or organ damage, injury, dysfunction, defect, or abnormality. Thus, the term encompasses, for example, injuries, degenerative diseases and genetic diseases. Such pathologies may affect any tissues or organs.

[0116] In certain embodiments, pathologies of interest (i.e., pathologies that can be treated with the progenitor cells or pharmaceutical compositions thereof) are cardiac or neuronal pathologies. Examples of cardiac degenerative diseases include, but are not limited to, heart failure, myocardial infarction, cardiac ischemia, myocarditis, arythmia, and the like. Examples of neuronal pathologies include, but are not limited to, neurodegenerative diseases such as polyglutamine diseases, spinocerebellar degeneration, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, and the like.

[0117] In other embodiments, pathologies of interest are genetic diseases. Examples of genetic diseases include, but are not limited to, inhereited genetic cardiomyocytes such as

Duchenne muscular dystrophy, and Emery Dreiffuss dilated cardiomyopathy, mental retardation caused by genetic abnormality such as fragile X chromosome and other inborn errors of metabolism such as phenylketonura gene defect, and the like.

[0118] In most methods of treatment of the present invention, the progenitor cells are allogenic to the subject being treated. As used herein, the term "allogenic" has its art understood meaning. More specifically, the term "allogenic", when used herein in relation to the progenitor cells, means (1) that neither the adult human mesenchymal stem cells nor the adult fully differentiated cells used in the preparation of the progenitor cells were obtained from the subject to be treated, and (2) that the adult fully differentiated cells were obtained from a donor of the same species as the subject to be treated.

[0119] A population of progenitor cells (or a pharmaceutical composition thereof) according to the present invention may be administered to a subject using any suitable method. Generally, the method of administration will be selected based on the site of tissue damage to be treated. Suitable methods of administration include, but are not limited to, parenteral methods such as intravenous, intra-arterial, intracardial (e.g., epicardial, intramyocardial), and percutaneous administration. For example, progenitor cells may be surgically implanted, injected, or delivered using a catheter or syringe. They may be delivered at or near the site of tissue damage or degeneration (e.g., to the deficient heart of a subject). [0120] Patients may receive a single administration of a population of progenitor cells. Alternatively, they may receive at least two administrations of progenitor cells.

[0121] Progenitor cells of the invention may be implanted in a subject alone or in combination with other cells, and/or in combination with other biologically active factors or reagents, drugs. As will be appreciated by those skilled in the art, these other cells, biologically active factors, reagents, and drugs may be administered simultaneously (i.e., substantially at the same time) or sequentially with (e.g., prior to and/or following administration of) the cells of the invention. Alternatively, progenitor cells of the invention may be seeded and grown on a scaffold or any other three-dimensional tissue engineered construct support, either alone or in combinations with other cells, and /or in combination with biologically active factors or reagents. The scaffold or construct, which may be configured to replace an entire organ or a portion of an organ, can then be implanted into a subject.

[0122] In certain embodiments, a treatment according to the present invention further comprises pharmacologically immunosuppressing the subject prior to initiating the cell-based treatment. Methods for the systemic or local immunosuppression of a subject are well known in the art. However, in most cases, a treatment according to the present invention will not require to pharmacologically immunosuppress the subject prior to administration of the population of progenitor cells since, as already mentioned above, these cells are non- immunogenic.

[0123] In fact, the non-immunogenicity of the cells disclosed herein is an important aspect of the present invention. It provides the possibility of easily treating a large range of patients, irrespective of their genotype. This is of crucial interest in the treatment of patients with severe diagnosis in cases where time is of the essence, and for whom harvest of their own stem cells is precluded. The non-immunogenicity of the progenitor cells of the present invention is also of crucial interest in the treatment of patients with genetic defects. Indeed, since these cells can contribute to gene defect correction, gene therapy (which otherwise would have required introduction of a gene in defective progenitor cells of the patient) becomes unnecessary. Thus, the methods of treatment provided herein overcome the economic, ethic and therapeutic limitations generally associated with stem cell-based therapies.

[0124] Administration regimens (including the optimal time of administration, e.g. following a heart attack) and effective dosages to be used in the methods of treatment of the present invention can be readily determined by good medical practice based on the nature of the pathology of the subject, and will depend on a number of factors including, but not limited to, the extent of the symptoms of the pathology and extent of damage or degeneration of the tissue or organ of interest, and characteristics of the subject (e.g., age, body weight, gender, general health, and the like).

[0125] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

Examples

Materials and Methods for Experiments Related to Cardiac Progenitor Cells and their Uses in Mice:

[0126] Stem cells isolation and expansion: The stem cells used in the examples reported herein were isolated from human adipose tissue using a variation of a method that the applicants have previously developed (Rodriguez et al, 2005). The applicants have initially used adipose tissue from young children to avoid potential aging effects resulting in a loss of functionality of stem cells, as was previously reported (Geiger et al, 2002; Edelberg et al, 2002). They have now successfully isolated similar stem cells from adipose tissue and from skeletal muscle obtained from adult patients. In particular, the inventors have demonstrated that multipotent stem cells can be extracted (1) from other tissues and organs than adipose tissue and (2) irrespective of the sample donor age.

[0127] Adult cardiomyocytes isolation: Adult cardiomyocytes used in coculture, were isolated from the heart of 8-weeks-old WT mice C57BL6 or 8-weeks-old mdx mice sharing genetic deficiency in dystrophin. Adult cardiomyocytes were extracted according to a method routinely used in the laboratory of the applicants and which generally yields approximately 1 million rod-shaped myocytes per heart.

[0128] Coculture of human adult stem cells with murine adult cardiomyocytes and expansion of cardiomyogenic progenitors: Human adult stem cells were co-cultured with freshly isolated murine adult cardiomyocytes at a 1:1 ratio, onto uncoated tissue culture plates (VWR). Cocultures were maintained in DMEM (Invitrogen) supplemented or not with heat- inactivated fetal bovine serum (2 to 10%) (D. Dutschers) and antibiotics (100 U/ml of penicillin and 100 μg/ml of streptomycin (Invitrogen). After 7 to 15 days, adherent cocultured cells were dissociated (0.25% trypsin EDTA, Invitrogen) and replated at 1000- 3000 cells/cm". This dissociation step can be repeated several times in order to allow expansion of cardiac progenitors. Cardiomyogenic progenitor cell formation was confirmed by immunohistochemistry using several antibodies directed against cardiac markers (GATA-4 (R§D), desmin (Abeam) and sarcomeric α actinin (Sigma)) and proliferation markers such as Ki67 nuclear antigen (Abeam). [0129] Cardiac progenitors engraftment in different heart disease mouse models: It is important to note that all engraftment experiments were performed on male mice to avoid an oestrogen cardioprotective effect. In order to assess immune privilege behavior of

transplanted cells, all the animals used in this study were immunocompetent. Heart repairing potential of progenitor cells was investigated in both cardiac ischemia wild type mice and gene deficient mdx mice suffering from cardiomyopathy due to the absence of dystrophin.

[0130] WT mice experimental infarction: 8 weeks-old C57/BL6 male mice were submitted to experimental ischemia produced by ligation of the left coronary artery for approximatively 90 minutes followed by reperfusion. This partial occlusion led to the death of more than 80% of the myocardium. Echocardiography was used to confirm successful generation of heart infarction 5 days after surgery (decrease of left ventricular fractional shortening to 70% prior surgery to 40% after injury). Progenitor cells obtained by stem cells/WT cardiomyocytes coculturing were loaded with Cell Tracker CM-DiI (Invitrogen)

(red color) prior to be microinjected in the damaged myocardium (150 000 to 300 000 cells).

[0131] Mdx mice experimental infarction: Mdx mice exhibit cardiac dysfunction due to the absence of cardiac dystrophin. Ligation of the left coronary artery for a time in the range of 30 minutes to 90 minutes results in massive mortality (up to 90%) of this type of animals. For this reason, animals were just submitted to thoratocomy followed by progenitor cell transplantation. Prior to engraftment, cells were labelled with CM-DiI and then were microinjected in the left myocardium in a range of 150 000 to 300 000 cells. These mice were engrafted with progenitor cells resulting from two different cocultures: a first coculture of human adult mesenchymal stem cells and adult cardiomyocytes from wild-type mice and a second coculture of human adult mesenchymal stem cells and adult cardiomyocytes from mdx mice.

[0132] Cardiac function analysis: Cardiac function recovery was assessed by echocardiography performed 5, 20 and 40 days post-engraftment. An echocardiography was also performed 1 day before surgery for control and comparison purposes. [0133] Cardiac histological analysis: At 20 or 40 days after transplantation, mice were killed and the hearts were perfused with 4% PFA and were snap frozen in liquid nitrogen.

[0134] Cryostat serial sections of 10 μm were made in order to assess: (i) migration of Dil-labelled progenitor cells after engraftment;

(ii) extent of fibrosis taking place after ischemia in dead myocardium area (picrosirius staining);

(iii) inflammatory response (immunohistochemical staining to detect macrophages (CD45 and CDl Ib (Abeam)) and T lymphocytes (CD3 (Sigma));

(iv) cardiac remuscularization (immunohistochemistry with several cardiac makers (GATA-4 (R§d)desmin (Abeam), sarcomeric α actinin (Sigma)) and proliferation Ki 67 marker (Abeam)); and

(v) extent of gene defect correction process in dystrophin deficient mdx mice (antibody used for dystrophin detection was purchased from Abeam (rabbit polyclonal to dystrophin)).

Materials and Methods for Experiments Related to Cardiac Progenitor Cells and their Uses in Pigs:

[0135] Stem cells isolation and expansion: The stem cells used in the examples reported herein were isolated from human adipose tissue using a variation of a method that the applicants have previously developed (Rodriguez et al, 2005), as described above.

[0136] Adult cardiomyocytes isolation: Adult cardiomyocytes used in coculture, were isolated from the heart of farm pigs (weight 30-35 kg) according to a method previously described (Sipido and Stankovicova, 1998). [0137] Coculture of human adult stem cells with porcine adult cardiomyocytes and expansion of cardiomyogenic progenitors: The methods of coculture and expansion were similar to those used for the mouse cardiac progenitors. The porcine cardiac progenitor cells obtained had a "progenitor-like" phenotype expressing both early cardiac GATA-4 and proliferation Ki67 markers, and thus were easily expandable in vitro. [0138] Cardiac progenitor engraftment in a heart disease pig model: This study is ongoing. 10 pigs are currently being treated. Myocardial infarction model was performed in farm pigs (weight 30-35 kg) under anesthesia, by occlusion of the proximal left circumflex with a balloon angioplasty, as previously described (Simon and Maibach, 2000; Garot et al, 2003). This method leads to significant and reproducible infarct areas. Cell injections were realized five days after myocardial infarction, after left thoracotomy and under anesthesia. Each treatment consists in 20 to 30 injections of 20 to 30 millions of reprogrammed cells in the border area of ischemic myocardium.

[0139] Cardiac function analysis: As mentioned above, this study is in progress.

Ventricular function recovery was evaluated by serial echocardiography and magnetic resonance imaging (MRI) for each pig. The regional shortening of the necrotic myocardium and LV (left ventricle) myocardial velocities and strain (by Doppler Tissue Imaging and

Spicke Tracking) were measured by transthoracic echocardiography at the time of cell injection (baseline), 1 month and 2 months after cell injection, and by transepicardic echocardiography (direct heart contact after left thoracotomy to enhance ultrasound signal) at the time of cell injection and 2 months after cell injection. Infarct size and left ventricular regional contraction were also evaluated 2 months after cell injection by MRI which provides a very sensitive method for the evaluation of scar size.

[0140] Cardiac histological analysis: As mentioned above, this study is underway. Infarct size was measured from pictures of cross sectional hearts up to 2 months after cell injection. Standard histological examination and immunochemistry were performed on serial sections of the engrafted myocardium to assess survival and phenotype of labelled transplanted cells, myocardial recovery and degree of vascularisation.

Materials and Methods for Experiments Related to Neural Progenitor Cells:

[0141] Isolation of adult neurons: Adult neurons used for coculture were isolated from cerebellum and dorsal root ganglia of 8-weeks-WT mice C57BL6. Neurons isolation from cerebellum was performed according to the method previously described by Ahlemeyer B and Baumgart-Vogt E (2005) and those from dorsal root ganglia according to slightly modified protocols reported by Delree et al. (1989).

[0142] Coculture of human adult stem with murine adult neurons and expansion of neuronal progenitors. As described for the coculture with adult cardiomyocytes above, human adult stem cells were cocultured with freshly isolated murine neurons at a 1:1 ratio (the ratio was found not to be critical for the success of neuronal progenitor formation). The mixed cells were plated onto uncoated tissue culture plates (vWR), suggesting that extracellular matrix coating is not required. Cocultures were maintained in stem cells growth medium, i.e., DMEM supplemented with heat inactivated fetal bovine serum (2 to 10%) (D. Dutschers) and antibiotics (100U/ml penicillin and lOOμg/ml of streptomycin (Invitrogen). After 7 to 15 days, adherent cocultured cells were dissociated (0,25% trypsin EDTA; Invitrogen) and replated at 1000-3000 cells/cm ² . This dissociation step can be repeated several times. Neuronal progenitor cell formation was confirmed by immunohistochemistry using several antibodies directed against neuron markers such as βlll tubulin (R§D) and proliferation markers such as Ki67 nuclear antigen (Abeam).

Results of Experiments with Cardiac Progenitor Cells and Their Uses in Mice Preparation of hybrid cells containing material of both human and murine origin:

[0143] Cytoplasmic exchanges: Prior to coculture, human stem cells and murine adult cardiomyocytes were labelled with Qtracker 525 (green fluorescence color, Invitrogen) and CM-DiI (red fluorescent color, Invitrogen), respectively. Twelve hours after coculture, a large number of hybrid cells were formed that contain cytoplasmic material of both human and murine origin as demonstrated on Figure 1 by the presence of both green fluorescence and red fluorescence in the hybrid cells.

[0144] Nuclear exchanges (nuclear fusion and reprogramming): Cytoplasmic events were accompanied by nuclear exchanges between both types of cells. This is strongly suggested by the presence of aberrant nuclei morphology observable shortly (24-36 hours) after initiation of the coculture. In addition, at the same time, micronuclei formation from the two types of cells was detected, as shown on Figure 2. This event seems to play an important role in nuclear reprogramming leading to the formation of cardiac progenitors. [0145] Intercellular Communications: Using videomicroscopy, the applicants have observed the presence of nanotubes between murine adult cardiomycytes and human adult stem cells allowing exchange of cytoplasmic and mitochondrial material (see Figure 3). These nanotubes are formed from cardiomyocytes to adult stem cells (see Figure 3A), from adult stem cells to cardiomyocytes (see Figure 3B), as well as from adult stem cells to adult stem cells (see Figure 3C). The formation of other types of conformations allowing exchange of material, including gap junctions, has also been observed between murine adult cardiomycytes and human adult stem cells (data not shown).

Characterization of hybrid cells in terms of cardiomyogenic, proliferative and nuclear markers: [0146] Colony Formation of Cardiac GATA-4 positive Progenitors: From 2 days after initiation of coculture, the applicants observed the formation of colonies of growing small rounded cells (see Figure 4). Colony-derived cells progressively migrated and changed morphology, becoming more elongated (see Figure 4, last picture of the set). Colony-derived cells expressed GATA-4 transcription factor, an early cardiomyogenic differentiation marker and proliferation Ki-67 nuclear antigen marker indicating that these cells are cycling (see Figure 5). These unique features are those of cardiac progenitor cells.

[0147] Maintenance of Cardiomyogenic Marker Expression along Serial Trypsinization-dependant Passages: In addition to GAT A-4, progenitor cells express other cardiomyogenic markers, for example, desmin and sarcomeric α actinin. However, desmin and sarcomeric α-actinin, which are cardiomyogenic markers of late differentiation, are expressed only weakly in the progenitor cells compared to cardiomyocytes. Such commited cells can successfully be expanded as revealed by the persistence of both cardiomyogenic and proliferation markers along culture passages (see Figure 6).

[0148] Morphological Properties of the Cardiac G AT A-4 positive Progenitors:

Analysis of the cardiac GAT A-4 positive progenitor cells obtained by electronic microscopy showed that these cells display a disorganized striation pattern, a characteristic distribution of mitochondria (asymmetric concentration of mitochondria localized in the non-striated zone), and a lack of sarcomere (see Figure 7). This morphology is typical of neonatal or fetal cardiomyocytes that are able to proliferate in vitro.

[0149] G AT A-4 Nuclei Characterization: The present applicants have hypothesized that cell fusion and nuclear reprogramming mechanisms were involved in the process of double positive Ki-67/GATA-4 cells formation. This hypothesis was reinforced by the fact that these cells never expressed nuclear markers of human origin (human lamin A/C). But in contrast, they expressed nuclear markers of murine origin (lamin A/C). These results, which are presented on Figure 8, indicate that cardiomyogenic progenitor cells did not result from human adult stem cell transdifferentiation but rather from stem cell-mediated nuclear reprogramming processes of somatic nuclei towards progenitor cells. This was confirmed by a double FISH analysis, which showed that the nuclei of cardiac GAT A-4 positive progenitors only contained genomic material of murine origin (see Figure 9).

[0150] The Applicants have also performed a quantitative RT-PCR analysis of the transcriptional activity of human and murine genes involved in cardiomyogenic differentiation and in cellular proliferation as a function of time during the coculture (see

Figure 10). As far as murine genes are concerned, this analysis showed that from the time cells are combined for coculture (0) to day 15 (J 15) after the beginning of the coculture, one observes: (i) a large increase (at least a 7 fold increase) in the transcriptional expression of early cardiomyogenic differentiation genes such as GATA-4; (ii) a lack of activation of late cardiomyogenic differentiation genes such as desmin, and (iii) a strong increase (higher than a

15 fold increase) in murine messengers such as Ki67. As far as human genes are concerned

and during the same time range, one observes: (i) a lack of genes involved in cardiomyogenesis such as GATA-4 and Desmin; and (ii) a temporary increase in the expression of Ki67 followed by a tremendous decrease (more than a 30 fold-decrease at J 15).

[0151] These results suggest that human adult stem cells are temporarily activated 24 hours after the beginning of the coculture, i.e., before the formation of colonies of progenitor cells (which is detectable 2 days after the beginning of the coculture), and then enter quiescence.

[0152] Physical Contact between Cells during Coculture: The applicants have shown that physical contact between murine adult cardiomycytes and human adult stem cells was required for reprogramming of adult cardiomyocytes by adult human stem cells to take place. A quantitative RT-PCR analysis of the transcriptional activity of human and murine genes similar to that described above was performed for a coculture in which human adult stem cells and murine cardiomyocytes were isolated from each other using a Millicell™ with a biopore membrane. In this setting no formation of colonies of progenitor cells was observed. As shown in Figure 11, absence of reprogramming has been confirmed by RT-PCR, indicating lack of induction of murine cardiac and proliferative genes.

Cardiac Progenitor Benefits after Engraftment into Diseased Heart (Infarctus and Genetic Cardiomyopathy):

[0153] Progenitor Engraftment into Experimental Ischemia WT Mice: Cardiac transplantation of a low number of progenitor cells (approximately 100 000 to 300 000 cells) into WT mice submitted to experimental ischemia (ligation of the left coronary artery for 90 minutes followed by reperfusion) improved cardiac function as assessed by echocardiography. In contrast, transplantation of adult stem cells alone had no significant beneficial effect (see Figure 12). [0154] Histological analysis of the infarcted myocardium revealed that injected cells: are able to survive and migrate throughout the ischemic area; are not immunologically rejected (lack of T lymphocyte and macrophage infiltration); and efficiently contribute to cardiac remuscularization (detection of GATA positive cells in infarcted area) leading to fibrosis reduction (see Figure 13). [0155] In particular, the significant difference of damaged myocardium between control mice and progenitor-treated mice should be highlighted.

[0156] Progenitor Engraftment into mdx Mice, an Experimental Model of Genetic Cardiomyopathy: Mdx mice were engrafted with either progenitor cells resulting from the coculture of human adult mesenchymal stem cells and adult cardiomyocytes from wild-type mice or with progenitor cells results from the coculture of human adult mesenchymal stem cells and adult cardiomyocytes from mdx mice. Engraftment of cardiac progenitor cells into genetically dystrophin deficient mdx mice was also found to efficiently improve cardiac function as assessed by echocardiography (see Figure 14). The inventors also detected, by immunohistochemistry, a large number of dystrophin positive cardiomyocytes, demonstrating that progenitor cells contribute to gene defect correction (see Figure 15). In addition, no inflammatory response (no detection of T lymphocytes and macrophages) was observed after engraftment, suggesting that progenitor cells are non-immunogenic.

Results of Experiments with Cardiac Progenitor Cells and Their Uses in Pigs

[0157] Porcine Cardiac Progenitor Cells: Colony-derived cells resulting from coculture of human adult mesnchymal stem cells and porcine adult cardiomyocytes expressed GATA-4 transcription factor, an early cardiomyogenic differentiation marker and proliferation Ki-67 nuclear antigen marker indicating that these cells are cycling (see Figure 16). These unique features are those of cardiac progenitor cells.

[0158] Progenitor Engraftment into Pigs: The preliminary results obtained indicate that the population of cardiac progenitor cells resulting from coculture of human adult mesenchymal stem cells and porcine adult cardiomyocytes failed to elicit an immune response in pig engrafted hearts. Lack of T-lymphocyte infiltration was assessed by CD3 immunohistochemistry. The preliminary results also show that injected cells are disseminated in perinfarct area from 10 days to 2 months after transplantation, suggesting that reprogrammed cells are (1) alive, (2) are not rejected, and (3) home to the site of injury.

Results of Experiments with Neuronal Progenitor Cells

[0159] Formation of Neuronal βlll tubulin Positive Progenitor Cells: From 3-5 days after initiation of the coculture as described above, neuronal progenitor cells were detected. These cells were found to express both neuronal markers such as βlll tubulin and proliferation markers such as Ki-67 (see Figure 17).

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