JP2014040485A - Skeletal muscle augmentation utilizing muscle-derived progenitor compositions, and treatments thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide skeletal muscle augmentation utilizing muscle-derived progenitor compositions, and treatments thereof.SOLUTION: An object of the present invention is to provide novel muscle-derived progenitor cells (MDCs) and MDC compositions exhibiting long-term survival following transplantation. The MDCs of this invention and compositions containing the MDCs comprise early progenitor muscle cells, i.e., muscle-derived stem cells that express progenitor cell markers, including, but not limited to, desmin, M-cadherin, MyoD, myogenin, CD34, and Bcl-2. In addition, these early progenitor muscle cells express the Flk-1, Sca-1, MNF, and c-met cell markers, but do not express the CD45 or c-Kit cell markers.

Description

  The present invention relates to muscle-derived progenitor cells (MDC) and MDC compositions and their use in strengthening body tissues, particularly skeletal muscle. In particular, the present invention relates to muscle-derived progenitor cells that exhibit long-term survival after introduction into skeletal muscle, methods of isolating MDC, and methods of using MDC-containing compositions for strengthening human or animal skeletal muscle. About. The present invention also relates to a novel use of muscle-derived progenitor cells for the treatment of cosmetic or functional conditions such as, but not limited to, skeletal muscle weakness, muscular dystrophy, muscular atrophy, spasticity, myoclonus and muscle pain. . The present invention also relates to a novel use of MDC for increasing skeletal muscle mass in exercisers or other organisms that require above average skeletal muscle mass.

  Myoblasts, or myofiber precursors, are mononuclear myocytes that fuse to form postmitotic multinucleated myotubes that allow long-term expression and delivery of bioactive proteins (Non-patent document 1; Non-patent document 2; Non-patent document 3; Non-patent document 4; Non-patent document 5).

  Cultured myoblasts contain a subpopulation of cells that exhibit some of the self-renewal characteristics of stem cells (Non-patent Document 6). Such cells cannot fuse to form myotubes and do not divide unless separated and cultured (A. Barofio et al., Non-Patent Document 6). Myoblast transplantation studies (see below) show that most transplanted cells die rapidly, but some survive and mediate the formation of new muscle (7). This small number of cells exhibit distinguishable behavior, such as slow growth in tissue culture and rapid growth after transplantation, indicating the possibility that these cells are myoblast stem cells ( JR Beuchamp et al., Non-Patent Document 7).

  Myoblasts have been used as a vehicle for gene therapy in the treatment of various muscle and non-muscle related disorders. For example, transplantation of genetically modified or unmodified myoblasts has been used for the treatment of Duchenne muscular dystrophy (Non-patent document 8; Non-patent document 9; Non-patent document 10; Non-patent document). 11; Non-patent document 12; Non-patent document 13). Furthermore, myoblasts are proinsulin for the treatment of type 1 diabetes (non-patent document 14), factor IX for the treatment of type B hemophilia (non-patent document 15; non-patent document 16; non-patent document). Reference 17; Non-patent document 18), Adenosine deaminase for treatment of adenosine deaminase deficiency syndrome (Non-patent document 19), Erythropoietin for treatment of chronic anemia (Non-patent document 20; Non-patent document 21), and growth retardation Have been genetically engineered to produce human growth hormone (22).

  Myoblasts are those that are damaged or diseased of muscle tissue, as disclosed in US Pat. It is also used for treatment. Furthermore, myoblast transplantation has also been used to repair myocardial dysfunction (Non-Patent Document 23; Non-Patent Document 24; Non-Patent Document 25).

  Despite the above, the main myoblast-inducing therapy is associated with low cell survival after transplantation due to migration and / or phagocytosis. In order to circumvent this problem, Atala et al., US Pat. No. 6,057,028, incorporated herein by reference, discloses the use of myoblasts suspended in a liquid polymer such as alginate. The polymer solution acts as a matrix that prevents myoblasts from migrating and / or phagocytosing after injection. However, polymer solutions present problems similar to the biopolymers described above. Furthermore, the Atala patent is limited to the use of myoblasts in muscle tissue only, excluding other tissues.

  Thus, there is a need for other different tissue-enhancing substances that last for a long time, are compatible with a wide range of host tissues, and minimize inflammation, scar formation, and / or tonicity of the tissue surrounding the transplant site. . Therefore, the muscle-derived progenitor cell (MDC) -containing composition of the present invention is provided as an improved novel substance for strengthening skeletal muscle. Furthermore, methods for producing muscle-derived progenitor cell compositions that exhibit long-term viability after transplantation and various cosmetic and / or functional defects such as, but not limited to, skeletal muscle weakening, muscular dystrophy, muscular atrophy Methods for utilizing MDC and compositions containing MDC to treat spasticity, myoclonus and myalgia are provided. Similarly, methods are provided for using MDC and compositions containing MDC for increased skeletal muscle mass in exercisers or other organisms that require above-average skeletal muscle mass.

  It is clear that previous attempts to use myoblasts for strengthening non-muscle tissue have failed (Patent Document 4 to Atala). Thus, the findings disclosed herein show that muscle-derived progenitor cells of the present invention can be successfully transplanted into non-muscle tissue, such as skeletal muscle tissue, and exhibit long-term survival. It was not done. As a result, MDC and compositions containing MDC can be used as a general strengthening substance for skeletal muscle production. Furthermore, since the muscle-derived progenitor cells and compositions of the present invention can be derived from autologous sources, immunological complications in the host, such as reabsorption of reinforcing substances, and inflammation of the tissue surrounding the transplant site and / or Or the risk of scar formation is reduced.

  Mesenchymal stem cells are present in various connective tissues of the body including muscle, skeletal muscle, cartilage and the like (Non-patent document 26; Non-patent document 27), but the term mesenchyme is not derived from muscle but in bone marrow of skeletal muscle. It has been used historically to refer to a class of stem cells purified from. Thus, mesenchymal stem cells can be distinguished from the muscle-derived precursor cells of the present invention. Furthermore, mesenchymal cells do not express the CD34 cell marker expressed by the muscle-derived progenitor cells described herein (Non-patent Document 28).

  Descriptions herein of inconveniences and problems associated with known compositions and methods are not intended to limit the scope of the embodiments described herein. Indeed, certain embodiments may encompass one or more known compositions, compounds, or methods without the disadvantages or problems so described.

US Pat. No. 5,130,141 US Pat. No. 5,538,722 US Pat. No. 6,866,842 US Pat. No. 5,667,778

T.A. A. Partridge and K.M. E. Davies, 1995, Brit. Med. Bulletin 51: 123-137 J. et al. Dhawan et al., 1992, Science 254: 1509-12. A. D. Grinnell, 1994, Myology 2nd edition G. Engel and C.I. F. Armstrong, McGraw-Hill, Inc. , 303-304 S. Jiao and J.H. A. Wolff, 1992, Brain Research 575: 143-7 H. Vandenburg, 1996, Human Gene Therapy 7: 2195-2200. A. Barofio et al., 1996, Differentiation 60: 47-57 J. et al. R. Beuchamp et al., 1999, J. MoI. Cell Biol. 144: 1113-1122 E. Gussoni et al., 1992, Nature, 356: 435-8. J. et al. Hard et al., 1992, Muscle & Nerve, 15: 550-60. G. Karpati et al., 1993, Ann. Neurol. , 34: 8-17 J. et al. P. Tremblay et al., 1993, Cell Transplantation, 2: 99-112. P. A. Moisset et al., 1998, Biochem. Biophys. Res. Commun. 247: 94-9 P. A. Moisset et al., 1998, Gene Ther. 5: 1340-46 L. Gros et al., 1999, Hum. Gen. Ther. 10: 1207-17 M.M. Roman et al., 1992, Somat. Cell. Mol. Genet. 18: 247-58 S. N. Yao et al., 1994, Gen. Ther. 1: 99-107 J. et al. M.M. Wang et al., 1997, Blood 90: 1075-82. G. Hortelano et al., 1999, Hum. Gene Ther. 10: 1281-8 C. M.M. Lynch et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 1138-42. E. Regulier et al., 1998, Gene Ther. 5: 1014-22 B. Dalle et al., 1999, Gene Ther. 6: 157-61 K. Anwer et al., 1998, Hum. Gen. Ther. 9: 659-70 C. E. Murry et al., 1996, J. MoI. Clin. Invest. 98: 2512-23 B. Z. Atkins et al., 1999, Ann. Thorac. Surg. 67: 124-129 B. Z. Atkins et al., 1999, J. MoI. Heart Lung Transplant. 18: 1173-80 H. E. Young et al., 1993, In Vitro Cell Dev. Biol. 29A: 723-736 H. E. Young et al., 1995, Dev. Dynam. 202: 137-144 M.M. F. Pittenger et al., 1999, Science 284: 143-147.

(Summary of Invention)
An object of the present invention is to provide novel muscle-derived progenitor cells (MDC) and MDC compositions that exhibit long-term survival after transplantation. MDCs and compositions containing MDCs of the present invention include early progenitor muscle cells, ie muscles that express progenitor cell markers including, but not limited to, desmin, M-cadherin, MyoD, myogenin, CD34, and Bcl-2 Contains induced stem cells. Furthermore, these early progenitor muscle cells express Flk-1, Sca-1, MNF, and c-met cell markers, but not CD45 or c-Kit cell markers.

  Another object of the present invention is to provide a method for isolating and enriching muscle-derived progenitor cells from a starting muscle cell population. This method results in enrichment of MDC with long-term viability after implantation or introduction into a soft tissue site. The MDC population according to the present invention is particularly enriched for cells that express progenitor cell markers including, but not limited to, desmin, M-cadherin, MyoD, myogenin, CD34, and Bcl-2. This MDC population also expresses Flk-1, Sca-1, MNF, and c-met cell markers, but does not express CD45 or c-Kit cell markers.

  Yet another object of the present invention is to provide a method of using MDC and a composition comprising MDC for strengthening muscle tissue, including skeletal muscle, which does not require a polymeric carrier or special culture medium for transplantation. That is. Such methods include, for example, administration of the MDC composition by direct injection into or on the surface of the tissue or by introduction into the skeletal muscle by systemic distribution of the composition.

  Yet another object of the present invention is to provide a method for strengthening skeletal muscle after injury, wound, surgery, trauma, non-trauma, or other treatments that result in fissures, openings, depressions, wounds, etc. It is.

  Another object of the present invention is to provide MDC and compositions comprising MDC that are modified through the use of chemicals, growth media, and / or genetic engineering. Such MDCs and compositions thereof include chemically or genetically modified cells useful for the production and delivery of biological compounds and the treatment of various diseases, conditions, injuries, or illnesses.

  Another object of the present invention is to provide modified MDCs and compositions comprising MDC through the use of chemicals, growth media, and / or genetic engineering. Such MDCs and compositions thereof include chemically or genetically modified cells useful for the manufacture and delivery of biological compounds and the treatment of various diseases, conditions, injuries, or illnesses.

  Yet another object of the present invention is to provide a pharmaceutical composition comprising MDC and a composition comprising MDC. The pharmaceutical composition comprises isolated MDC. This MDC may then be propagated by cell culture after isolation. In one aspect of this embodiment, these MDCs are frozen prior to delivery to a subject in need of a pharmaceutical composition.

  The present invention also provides a method of performing MDC isolation using the composition and a single plating procedure. MDCs are isolated from skeletal muscle biopsies. In one embodiment, skeletal muscle obtained from a biopsy may be stored for 1-6 days. In one aspect of this embodiment, biopsy-derived skeletal muscle is stored at 4 ° C. Cells are crushed and digested with collagenase, dispase, another enzyme or a combination of enzymes. After washing the enzyme from the cells, the cells are cultured in a flask in a culture medium for about 30 to about 120 minutes. During this period, “rapidly adhering cells” adhere to the wall of the flask or vessel, while “slowly adhering cells” or MDCs remain in suspension. “Slowly adherent cells” are transferred to a second flask or vessel where they are cultured for 1-3 days. During this second period, “slowly adherent cells” or MDCs adhere to the wall of the second flask or vessel.

  In another embodiment of the invention, these MDCs are grown to any number of cells. In a preferred aspect of this embodiment, the cells are grown in a fresh culture medium for about 10-20 days. More preferably, the cells are grown for 17 days.

  MDCs, whether grown or not, may be preserved for transport or stored for a period prior to use. In one embodiment, the MDC is frozen. Preferably, the MDC is frozen at -20 to -90 ° C. More preferably, the MDC is frozen at about −80 ° C. This frozen MDC is used as a pharmaceutical composition.

  MDC may be used to treat many skeletal muscle degenerative pathological conditions, either frozen, preserved as a pharmaceutical composition, or used as is. This condition includes, but is not limited to, skeletal muscle weakness, muscular dystrophy, muscular atrophy, spasticity, myoclonus and muscle pain. Increased skeletal muscle mass in athletes or other organisms that require skeletal muscle mass above average MDC, either frozen, preserved as a pharmaceutical composition, or used as is May be used for.

Additional objects and advantages gained by the present invention will become apparent from the detailed description and examples set forth below.
For example, the present invention provides the following items.
(Item 1)
A method for strengthening skeletal muscle in a mammal in need of skeletal muscle strengthening, comprising:
a) isolating MDC;
b) growing the isolated MDC in culture for 10-20 days; and
c) administering a therapeutically effective portion of the expanded MDC to mammalian skeletal muscle in need of skeletal muscle strengthening;
A method of strengthening skeletal muscle in a mammal in need thereof.
(Item 2)
The method of item 1, wherein the mammal in need of strengthening skeletal muscle is afflicted with a pathological condition of the muscle.
(Item 3)
Item 2. The method according to Item 1, wherein the mammal is a human.
(Item 4)
(A) suspending human skeletal muscle cells in the first cell culture vessel for 30 to 120 minutes;
(B) transferring the medium from the first cell culture container to the second cell culture container;
(C) attaching the remaining cells in the medium to the wall of the second cell culture vessel; and
(D) isolating cells from the wall of the second cell culture vessel, wherein the isolated cells are MDCs;
The method according to Item 1, wherein the MDC is isolated by the method of isolating the MDC by comprising.
(Item 5)
(A) plating a suspension of skeletal muscle cells derived from skeletal muscle tissue in a first container to which fibroblasts of the skeletal muscle cell suspension adhere;
(B) re-plating non-adherent cells from step (a) into a second container, wherein the re-plating step is after about 15 to about 20% of the cells have adhered to the first container And
(C) repeating step (b) at least once;
The method according to Item 1, wherein the MDC is isolated by the method of isolating the MDC by comprising.
(Item 6)
The method of item 1, wherein the MDC is administered by injecting the MDC into mammalian skeletal muscle.
(Item 7)
The method of item 1, wherein the mammalian muscle is defective.
(Item 8)
The method of item 1, wherein MDC is administered by applying MDC to said defect.
(Item 9)
9. A method according to item 8, wherein the MDC is applied via injection.
(Item 10)
A method for strengthening skeletal muscle in a mammal in need of skeletal muscle strengthening, comprising:
a) isolating MDC;
b) freezing the isolated MDC to a temperature of about -25 ° C to -90 ° C;
c) thawing frozen isolated MDC; and
d) administering the thawed MDC to mammalian skeletal muscle in need of skeletal muscle strengthening;
A method of strengthening skeletal muscle in a mammal in need thereof.
(Item 11)
11. The method of item 10, wherein the mammal in need of skeletal muscle is afflicted with a muscular pathological condition.
(Item 12)
Item 11. The method according to Item 10, wherein the mammal is a human.
(Item 13)
(A) suspending human skeletal muscle cells in the first cell culture vessel for 30 to 120 minutes;
(B) transferring the medium from the first cell culture container to the second cell culture container;
(C) attaching the remaining cells in the medium to the wall of the second cell culture vessel; and
(D) isolating cells from the wall of the second cell culture vessel, wherein the isolated cells are MDCs;
11. The method according to item 10, wherein the MDC is isolated by the method of isolating the MDC.
(Item 14)
(A) plating a suspension of skeletal muscle cells derived from skeletal muscle tissue in a first container to which fibroblasts of the skeletal muscle cell suspension adhere;
(B) re-plating non-adherent cells from step (a) into a second container, wherein the re-plating step is after about 15 to about 20% of the cells have adhered to the first container And
(C) repeating step (b) at least once;
11. The method according to item 10, wherein the MDC is isolated by the method of isolating the MDC.
(Item 15)
11. The method of item 10, wherein the MDC is administered by injecting the MDC into mammalian skeletal muscle.
(Item 16)
Item 11. The method according to Item 10, wherein the mammalian muscle is defective.
(Item 17)
12. The method of item 10, wherein the MDC is administered by applying MDC to the defect.
(Item 18)
18. A method according to item 17, wherein the MDC is applied via injection.

  The patent or patent application file contains at least one photocopy made in color. Copies of this patent or patent application in color photocopies will be provided by the US Patent Office upon application and payment of the necessary fee.

The accompanying drawings are presented to further explain the present invention and to facilitate its understanding through clarification of various aspects thereof.
FIGS. 1A and 1B show human MDC (hMDC) injected into mouse muscle with human dystrophin and mouse Y chromosome stained to show chimeragenicity in fused human and mouse muscle cells. FIG. 2 is a bar graph showing the percentage of dystrophin positive fibers in mice injected with hMDC.

(Detailed description of the invention)
The present invention provides a method of using human MDC and such cells to form skeletal muscle tissue to repair damaged skeletal muscle or to increase skeletal muscle volume and / or strength above wild-type levels. provide. The present invention further provides methods of treating skeletal muscle disorders, including but not limited to skeletal muscle weakness, muscular dystrophy, muscular atrophy, spasticity, myoclonus and muscle pain. By isolating human muscle-derived cells (MDC) from adult tissue, increased skeletal muscle density and skeletal muscle volume can be achieved in a human subject administered these cells.

Muscle-derived cells and compositions The present invention comprises early progenitor cells (also referred to herein as muscle-derived progenitor cells or muscle-derived stem cells) that exhibit long-term survival after transplantation into body tissue, preferably skeletal muscle. Provide MDC. To obtain the MDCs of the present invention, muscle explants, preferably skeletal muscle, are obtained from animal donors, preferably mammals including humans. This extracorporeal tissue functions as a structural and functional syncytium that encompasses the “residue” of muscle progenitor cells (TA Partridge et al., 1978, Nature 73: 306-8; BH Lipton et al., 1979). , Science 205: 12924).

  Cells isolated from primary muscle tissue contain a mixture of fibroblasts, myoblasts, adipocytes, hematopoietic progenitor cells and muscle derived progenitor cells. Progenitor cells of the muscle-derived population exhibit the differential adhesion characteristics of primary muscle cells on collagen-coated tissue flasks as described in US Pat. No. 6,866,842 to Chancellor et al., Which is incorporated herein by reference. Can be enriched while using. Cells with slow adhesion are morphologically round and express high levels of desmin and have the ability to fuse and differentiate into multinucleated myotubes (Chancellor et al., US Pat. No. 6,866,662). 842). These subpopulations of cells have been engineered in vitro by expressing high levels of alkaline phosphatase, parathyroid hormone-dependent 3 ′, 5′-cAMP, and other markers of the osteogenic and myogenic lineage in vitro. It has been shown to respond to muscle morphogenic protein 2 (rhBMP-2) (US Pat. No. 6,866,842 to Chancellor et al .; T. Katagiri et al., 1994, J. Cell Biol., 127: 1755-1766). ).

  In one embodiment of the present invention, slow adherent cells (MDC) may be differentiated from rapidly adherent cells by using a pre-plating operation. In accordance with the present invention, populations of fast-adhering MDC (PP1-4) and slow-adhesion round MDC (PP6) are isolated and enriched from skeletal muscle explants using various immunohistochemical methods. The presence of pluripotent cells among slowly adherent cells was examined by testing for expression (Example 1 of US Pat. No. 6,866,842 to Chancellor et al.). As shown in Table 1 of Example 1 herein, PP6 cells expressed myogenic markers such as desmin, MyoD, and myogenin. PP6 cells also expressed two genes, c-met and MNF, which are expressed at an early stage of muscle formation (JB Miller et al., 1999, Curr. Top. Dev. Biol. 43: 191 to 219; see Table 3). In PP6, the percentage of cells that expressed the satellite cell-specific marker M-cadherin was lower (A. Irinchev et al., 1994, Development Dynamics 199: 326-337), but at an early stage of muscle formation. The percentage of cells expressing Bcl-2, a marker limited to that cell, was higher (JA Dominov et al., 1998, J. Cell Biol. 142: 537-544). PP6 cells also express human hematopoietic progenitor cells, as well as CD34, a marker identified in stromal cell precursors in skeletal muscle bone marrow (RG Andrews et al., 1986, Blood 67: 842-845; C. I. Civin et al., 1984, J. Immunol. 133: 157-165; L. Fina et al., 1990, Blood 75: 2417-2426; P. J. Simons et al., 1991, Blood 78: 2848-2853; ). PP6 cells also expressed Flk-1, a mouse homologue of the human KDR gene recently identified as a marker for hematopoietic cells with stem cell-like characteristics (BL Ziegler et al., 1999, Science 285: 1553). 1558; see Table 3). Similarly, PP6 cells expressed Sca-1, a marker present in hematopoietic cells with stem cell-like characteristics (M. van de Rijn et al., 1989, Proc. Natl. Acad. Sci. USA 86: 4634- 8; M. Osawa et al., 1996, J. Immunol. 156: 3207-14; However, PP6 cells did not express CD45 or c-Kit hematopoietic stem cell markers (LK Ashman, 1999, Int. J. Biochem. Cell. Biol. 31: 1037-51; GA Koretzky, 1993. , FASEB J. 7: 420-426; see Table 3).

  In one embodiment of the present invention, a PP6 population of muscle-derived progenitor cells having the characteristics described herein is provided. These muscle-derived progenitor cells express desmin, CD34, and Bcl-2 cell markers. According to the present invention, PP6 cells are isolated by the technique described herein (Example 1) to obtain a population of muscle-derived progenitor cells that have long-term viability after transplantation. The PP6 muscle-derived progenitor cell population includes a large percentage of cells that express progenitor cell markers including, but not limited to, desmin, CD34, and Bcl-2. Furthermore, PP6 cells express Flk-1 and Sca-1 cell markers, but do not express CD45 or c-Kit markers. Preferably, more than 95% of PP6 cells express desmin, Sca-1 and Flk-1 markers, but do not express CD45 or c-Kit markers. PP6 cells are preferably used within about 1 day or about 24 hours after the last plating.

  In a preferred embodiment, fast adherent cells and slow adherent cells (MDC) are separated from each other using a single plating technique. One such technique is shown in Example 2. First, cells are prepared from a biopsy sample of skeletal muscle. The biopsy sample may contain about 100 mg of cells. Biopsy samples in the size range of about 50 mg to about 500 mg are used according to both the pre-plating method and the single plating method of the present invention. In addition, 50, 100, 110, 120, 130, 140, 150, 200, 250, 300, 400 and 500 mg biopsy samples are used according to both the pre-plating method and the single plating method of the present invention.

  In a preferred embodiment of the invention, the tissue from the biopsy sample is then stored for 1-7 days. This storage is performed at a temperature of about room temperature to about 4 ° C. Stress is applied to the skeletal muscle tissue biopsied during this waiting period. Although this stress is not necessary for MDC isolation using a single plating technique, the use of a waiting period generally results in higher yields of MDC.

  According to a preferred embodiment, the tissue from the biopsy sample is crushed and centrifuged. The pellet is resuspended and digested with digestive enzymes. Enzymes that can be used include, but are not limited to, collagenase, dispase, or combinations of these enzymes. After digestion, the enzyme is washed away from the cells. Cells are transferred to flasks in a culture medium for the isolation of rapidly adherent cells. Many culture media may be used. Particularly preferred culture media include those designed for the culture of endothelial cells including Cambrex endothelial growth medium. This medium may be supplemented with other components including fetal bovine serum, IGF-1, bFGF, VEGF, EGF, hydrocortisone, heparin, and / or ascorbic acid. Other media that can be used in the single plating technique include InCell M310F media. This medium may be replenished as described above or used unsupplemented.

  The process for isolation of fast adherent cells may require culturing in the flask for about 30 to about 120 minutes. Rapid adherent cells adhere to the flask within 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 minutes. After the cells adhere, the slowly adherent cells are separated from the rapidly adherent cells by removing the culture medium from the flask to which the rapidly adherent cells are attached.

  The culture medium removed from the flask is then transferred to the second flask. Cells are centrifuged and resuspended in culture medium before being transferred to a second flask. Cells are cultured in this second flask for 1-3 days. Preferably, the cells are cultured for 2 days. During this period, slowly adherent cells (MDC) adhere to the flask. After the MDC has adhered, the culture medium is removed and new culture medium is added to promote MDC growth. The number of MDCs may be increased by culturing for about 10 to about 20 days. The number of MDCs may be increased by culturing for 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days. Preferably, the MDC is subjected to a 17 day growth culture.

  As an alternative to the pre-plating method and the single plating method, the MDC of the present invention can be obtained by fluorescence activated cell sorting (FACS) using an antibody labeled against one or more cell surface markers expressed by the MDC. Can be isolated (C. Webster et al., 1988, Exp. Cell. Res. 174: 252-65; JR Blanton et al., 1999, Muscle Nerve 22: 43-50). For example, FACS analysis has been described herein to select populations of PP6-like cells that exhibit long-term viability when introduced into host tissue. This can be done using labeled antibodies that specifically bind to other cell surface markers. Also encompassed by the present invention is the use of one or more fluorescent detection labels, such as fluorescein or rhodamine, for antibody detection of different cell marker proteins.

  Any MDC isolation method described above or known in the art may be used to preserve MDCs to be transported or not used for a period of time using any method known in the art. For example, isolated MDC may be frozen at a temperature in the range of about −25 to about −90 ° C. Preferably, the MDC is frozen at about −80 ° C. on dry ice for delayed use or transportation. Freezing may be performed in any cryopreservation medium known in the art.

Muscle-Induced Cell-Based Treatment In one embodiment of the present invention, MDC is isolated from a skeletal muscle source and introduced or transplanted into the muscle or non-muscle soft tissue of interest, or into skeletal muscle. Conveniently, the MDCs of the present invention are isolated and enriched to contain a large number of progenitor cells that exhibit long-term survival after transplantation. Furthermore, the muscle-derived progenitor cells of the present invention express a number of characteristic cell markers such as desmin, CD34, and Bcl-2. Furthermore, the muscle-derived progenitor cells of the present invention express Sca-1 and Flk-1 cell markers but do not express CD45 or c-Kit cell markers (see Example 1).

  The MDCs and compositions comprising MDCs of the present invention can be used to repair, treat, or ameliorate various cosmetic or functional conditions (eg, defects) through skeletal muscle strengthening. In particular, such compositions can be used to treat skeletal muscle disorders. Multiple and consecutive MDC administrations are also encompassed by the present invention.

For MDC-based therapy, skeletal muscle explants are preferably obtained from autologous or heterologous human or animal sources. Autologous animal sources or human sources are more preferred. The MDC composition is then prepared and isolated as described herein. In order to introduce or transplant MDC and / or a composition comprising MDC according to the present invention into a human or animal recipient, a suspension of mononuclear muscle cells is prepared. Such suspensions contain the concentrate of muscle-derived progenitor cells of the present invention in a physiologically acceptable carrier, excipient, or diluent. For example, a suspension of MDC for administration to a subject may contain 10 ⁸ to 10 ⁹ cells / ml in a sterile solution of complete medium modified to contain the subject's serum instead of fetal bovine serum. Can be included. Alternatively, the MDC suspension can be a serum-free sterile solution, such as a cryopreservation solution (Celox Laboratories, St. Paul, Minn.). The MDC suspension can then be introduced into one or more sites of the donor tissue, for example via injection.

  The described cells are administered as a pharmaceutically or physiologically acceptable preparation or composition containing a physiologically acceptable carrier, excipient, or diluent and are intended for inclusion in humans or non-human animals. It can be administered to the tissue of the recipient organism. MDC-containing compositions can be prepared by resuspending the cells in a suitable liquid or solution, such as sterile saline or other physiologically acceptable aqueous liquid for injection. One skilled in the art can usually determine the amount of ingredients used in such compositions.

  MDC or a composition thereof can be administered by adding an MDC suspension onto an absorbable or adhesive substance, such as a collagen sponge matrix, and inserting the MDC-containing substance in or on the site of interest. Alternatively, MDC can be administered by parenteral route injection, including subcutaneous, intravenous, intramuscular, and intrasternal. Other modes of administration include, but are not limited to, intranasal, intrathecal, intradermal, transdermal, enteral, and sublingual. In one embodiment of the invention, the administration of MDC can be mediated by endoscopic surgery.

  For injection administration, the compositions can be present as sterile solutions or suspensions, or can be resuspended in a pharmaceutically and physiologically acceptable aqueous or oily vehicle that contains preservatives, stabilizers And materials to make the solution or suspension isotonic with the recipient's body fluid (ie blood). Non-limiting examples of excipients suitable for use include water, phosphate buffered saline pH 7.4, 0.15 M aqueous sodium chloride solution, dextrose, glycerol, dilute ethanol and the like and mixtures thereof. Exemplary stabilizers are polyethylene glycols, proteins, sugars, amino acids, inorganic acids, and organic acids, which may be used alone or as a mixture. The amounts used as well as the route of administration are individually determined and correspond to the amounts used in similar types of applications or indications known to those skilled in the art.

  In order to optimize the outcome of transplantation, the closest possible immunological agreement between the donor and the recipient is desirable. If autologous sources are not available, the closest match that can be used can be determined by analyzing donor and recipient class I and class II histocompatibility antigens. This minimizes or eliminates immune rejection and reduces the need for treatment with immunosuppressive or immunomodulating agents. If desired, treatment with immunosuppressive or immunomodulating agents can be initiated before, during, and / or after the transplantation procedure. For example, cyclosporin A or other immunosuppressive agent can be administered to the graft recipient. Immunological tolerance may also be induced prior to transplantation by alternative methods known in the art (DJ Watt et al., 1984, Clin. Exp. Immunol. 55: 419; D. Faustman et al., 1991). , Science 252: 1701).

Consistent with the present invention, MDC can be administered to body tissues including skeletal muscle. The number of cells in the MDC suspension and the mode of administration may vary depending on the site and condition to be treated. About 1.0 × 10 ⁵ to about 1 × 10 ⁸ MDCs may be administered according to the present invention. As a non-limiting example, according to the present invention, about 0.5-2.0 × 10 ⁶ MDC is administered through a collagen sponge matrix for the treatment of about 5 mm area of the cranial defect (Example 3 reference).

  For strengthening skeletal muscle or treating skeletal muscle disorders, MDC may be prepared as described above and administered for additional skeletal muscle strength, for example by administration via injection on, inside, or around the skeletal muscle. And / or volume can be obtained. The number of MDCs introduced is adjusted as needed to provide varying amounts of skeletal muscle density and / or skeletal muscle volume, or includes the use of MDCs of the invention in augmenting skeletal muscle volume. To do. Skeletal muscle disorders include, but are not limited to, skeletal muscle weakness, muscular dystrophy, muscle atrophy, spasticity, myoclonus and muscle pain. The present invention also relates to a novel use of MDC for increasing skeletal muscle mass in exercisers or other organisms that require skeletal muscle mass above average.

Genetically engineered muscle-derived cells In another embodiment of the present invention, the MDC of the present invention contains a nucleic acid sequence encoding one or more active biological molecules, and the biological molecules such as proteins, polypeptides May be genetically engineered to express peptides, hormones, metabolites, drugs, enzymes, and the like. Such MDCs may be histocompatible (autologous) or non-histocompatible (allogeneic) to recipients, including humans. The cells are used for various treatments, such as, but not limited to, skeletal muscle weakness, muscular dystrophy, muscular atrophy, spasticity, myoclonus and myalgia and long-term treatment for pathologic conditions. Can function as a local delivery system.

  Preferred in the present invention are autologous muscle-derived progenitor cells that are not recognized as foreign by the recipient. In this regard, the MDC used for cell-mediated gene transfer or delivery is desirably consistent with respect to the major histocompatibility locus (MHC or HLA in humans). Such MHC or HLA matching cells are autologous. Alternatively, the cells may be from an individual having the same or similar MHC or HLA antigen profile. The patient may also be tolerated against allogeneic MHC antigens. The present invention also encompasses the use of cells lacking MHC class I and / or II as described in US Pat. No. 5,538,772, incorporated herein by reference.

  MDC may be genetically engineered by various molecular techniques and methods known to those skilled in the art, such as transfection, infection, or transduction. As used herein, transduction generally refers to a cell that has been genetically engineered to contain a foreign or heterologous gene through the introduction of a viral or non-viral vector into the cell. . Transfection refers to cells that have been genetically engineered to contain foreign genes that are more commonly carried in plasmids or non-viral vectors. MDC has been transfected or transduced with a different vector and can therefore serve as a gene delivery vehicle to transfer the expression product into muscle.

  Although viral vectors are preferred, one of skill in the art will know how to engineer cells to contain nucleic acid sequences encoding the desired protein or polypeptide, cytokine, etc., such as US Pat. No. 5,538,722. Methods such as fusion, transfection, lipofection mediated by the use of liposomes, electroporation, precipitation with DEAE-dextran or calcium phosphate, particle collisions with nucleic acid coated particles (eg gold particles) (biolistics) ), It should be understood that it may be performed by microinjection or the like.

  Vectors for introducing heterologous (ie, exogenous) nucleic acid (DNA or RNA) into muscle cells for the expression of biologically active products are well known in the art. Such vectors carry a promoter sequence, preferably a promoter that is cell specific and is located upstream of the sequence to be expressed. The vector may also optionally contain one or more expressible marker genes for good transfection and expression as an indicator of expression of the nucleic acid sequence contained within the vector.

Representative examples of vehicles or vector constructs for transfection or infection of muscle-derived cells of the invention include replication deficient viral vectors, DNA viruses or RNA virus (retroviral) vectors such as, but not limited to, adenovirus, herpes simplex virus and Includes adeno-associated viral vectors. Adeno-associated viral vectors are single stranded and allow efficient delivery of multiple copies of nucleic acids to the cell nucleus. Preferred is an adenovirus vector. Vectors are usually substantially free of any prokaryotic DNA and may contain many different types of functional nucleic acid sequences. Examples of such functional sequences are polynucleotides such as DNA or RNA, sequences including transcriptional and translational initiation and termination regulatory sequences, such as promoters (eg strong promoters, inducible promoters) and enhancers that are active in muscle cells. Is included. As part of the functional sequence, an open reading frame (polynucleotide sequence) encoding the protein of interest is also included, and a flanking sequence may also be included for site-directed integration. In some situations, the 5′-flanking sequence is a property of the transcription initiation region to allow homologous recombination, for example to provide inducible or non-inducible transcription to increase or decrease the level of transcription. Can be changed.

  In general, a nucleic acid sequence that is desired to be expressed by a muscle-derived progenitor cell is, for example, a structural gene, or gene of a gene that is heterologous to the muscle-derived progenitor cell and encodes a desired protein or polypeptide product. Functional fragment, segment or part. The encoded and expressed product may be retained within the cell, ie, in the cytoplasm, nucleus, or organelle, or may be secreted by the cell. For secretion, the natural signal sequence present in the structural gene may be retained, or a signal sequence not naturally present in the structural gene may be used. If the polypeptide or peptide is a fragment of a larger protein, a signal sequence may be provided such that the desired protein has the native sequence upon processing at the secretion and processing site. Examples of genes of interest for use in accordance with the present invention include genes encoding cell growth factors, cell differentiation factors, cell signaling factors and programmed cell death factors. Specific examples include, but are not limited to, genes encoding BMP-2 (rhBMP-2), IL-1Ra, Factor IX, and connexin 43.

  As described above, a marker may be present for selection of cells containing the vector construct. The marker may be an inducible or non-inducible gene and generally allows positive selection under induction or without induction, respectively. Examples of commonly used marker genes include neomycin, dihydrofolate reductase, glutamine synthetase and the like.

  The vectors used generally include an origin of replication commonly used by those skilled in the art and other genes necessary for replication in the host cell. As an example, a replication system that includes an origin of replication and any protein associated with replication encoded by a particular virus may be included as part of the construct. The replication system must be chosen so that the gene encoding the product required for replication does not ultimately transform muscle-derived cells. Such replication systems are described in, for example, G. Acsadi et al., 1994, Hum. Mol. Genet3: represented by replication deficient adenovirus constructed as described by 579-584, also Epstein-Barr virus. Examples of replication defective vectors, particularly retroviral vectors that are replication defective, are described in Price et al., 1987, Proc. Natl. Acad. Sci. USA, 84: 156, and Sanes et al., 1986, EMBO J. et al. , 5: 3133. It should be understood that the final gene construct may contain one or more genes of interest, for example genes encoding biologically active metabolic molecules. Furthermore, cDNA, synthetically produced DNA or chromosomal DNA may be used using methods and protocols known and practiced by those skilled in the art.

  If desired, the cells may be genetically engineered prior to in vivo injection of the cells by using infectious replication deficient viral vectors. In this regard, the vector may be introduced into retroviral producer cells for amphotropic packaging. The natural growth of muscle-derived progenitor cells into adjacent regions eliminates the need for multiple injections within or at the site of interest.

  In another aspect, the present invention involves the use of early progenitor muscle cells that have been virally transduced with an MDC, eg, an adenoviral vector engineered to contain a heterologous gene encoding a desired gene product. Through ex vivo gene delivery to cells and tissues of recipient mammalian hosts, including humans. Such ex vivo procedures offer the advantage of efficient viral gene transfer over direct gene transfer procedures. The ex vivo procedure uses muscle-derived progenitor cells derived from isolated cells of muscle tissue. A muscle biopsy sample that functions as a source of muscle-derived progenitor cells can be obtained from the site of injury or from another area that may be more easily obtained from a clinician.

  According to the present invention, clonal isolates can be obtained from various procedures known in the art, such as muscle-derived progenitor cells (ie PP6 using a single plating procedure) using limiting dilution plating in tissue culture medium. Cell or “slowly adherent” cells). Clonal isolates contain genetically identical cells originating from a single solitary cell. Furthermore, clonal isolates can be derived using FACS analysis as described above, then achieving single cells per well by limiting dilution and establishing clonally isolated cell lines. An example of a clonal isolate derived from a PP6 cell population is mc13 as described in Example 1. Preferably, MDC clonal isolates are utilized in the methods of the invention, as well as for genetic manipulation for the expression of one or more biologically active molecules, or in gene replacement therapy.

  MDC is first infected with a heterologous gene encoding a desired gene product into an engineered viral vector containing at least one, and a physiologically acceptable carrier or excipient such as saline or phosphate buffered saline. Suspend in water and then administer to the appropriate site in the host. Consistent with the present invention, MDC can be administered to body tissues including skeletal muscle as described above. The desired gene product is expressed by the injected cells, thereby introducing the gene product into the host. Thereby, the introduced and expressed gene product is expressed over a long period of time by the MDC of the present invention that survives in the host for a long time and can be used to treat, repair or ameliorate injury, dysfunction or disease. .

In an animal model study of myoblast-mediated gene therapy, transplantation of 10 ⁶ myoblasts per 100 mg muscle was required for partial correction of muscle enzyme deficiency (JE Morgan et al., 1988, J Neuro.Sci.86: 137; see TA Partridge et al., 1989, Nature 337: 176). By analogy from this data, approximately 10 ¹² MDC suspended in a physiologically compatible medium can be transplanted into muscle tissue for gene therapy for a 70 kg human. This number of MDCs of the present invention can be produced from a single 100 mg skeletal muscle biopsy sample from a human source (see below). For the treatment of specific injury sites, injection of genetically engineered MDC into a given tissue or site of injury is a therapeutically effective amount of cells in solution or suspension, preferably physiologically acceptable. About 10 ⁵ to 10 ⁶ cells per cm ^{3 of} tissue to be treated.

Example 1. Enrichment, isolation and analysis of MDC by pre-plating method MDC was prepared as described (U.S. Pat. No. 6,866,842 to Chancellor et al.). Muscle explants come from many sources: 3 week old mdx (dystrophic) mice (C57BL / 10ScSn mdx / mdx, Jackson Laboratories), 4-6 week old normal female SD (Sprague Dawley) rats, or Obtained from the hind limb of SCID (severe combined immunodeficiency) mice. Muscle tissue from each of the animal sources was incised to remove any bone and ground into a slurry. The slurry was then digested at 37 ° C. by continuous incubation for 1 hour with 0.2% type XI collagenase, dispase (II grade, 240 units) and 0.1% trypsin. The resulting cell suspension was passed through 18, 20, and 22 gauge needles and centrifuged at 3000 rpm for 5 minutes. Cells were then suspended in growth medium (DMEM supplemented with 10% fetal bovine serum, 10% horse serum, 0.5% chicken embryo extract, and 2% penicillin / streptomycin). The cells were then pre-plated in collagen coated flasks (Chancellor et al., US Pat. No. 6,866,842). After about 1 hour, the supernatant was removed from the flask and re-plated into a new collagen-coated flask. The cells that adhered rapidly within this 1 hour incubation were mostly fibroblasts (above Z. Qu et al .; Chancellor et al. US Pat. No. 6,866,842). The supernatant was removed and re-plated after 30-40% of the cells had adhered to each flask. After about 5 to 6 successive platings, the culture is enriched in small round cells, namely those designated PP6 cells, which are isolated from the starting cell population and used for subsequent studies. did. Adherent cells isolated in early plating were pooled and designated PP1-4 cells.

  The populations of mdxPP1-4, mdxPP6, normal PP6, and fibroblasts were examined by immunohistochemical analysis for expression of cell markers. The results of this analysis are shown in Table 1.

ｍｄｘＰＰ１−４、ｍｄｘＰＰ６、正常ＰＰ６、および線維芽細胞は予備プレーティング技術により誘導し、免疫組織化学的分析により検査した。「−」は細胞の２％未満が発現を示したことを示し、「（−）」、「−／＋」は細胞の５〜５０％が発現を示したことを示し、「＋／−」は細胞の約４０〜８０％が発現を示したことを示し、「＋」は細胞の９５％超が発現を示したことを示し、「ｎｏｒ」は正常細胞を示し、「ｎａ」は免疫組織化学的データが得られなかったことを示す。

mdxPP1-4, mdxPP6, normal PP6, and fibroblasts were induced by pre-plating technique and examined by immunohistochemical analysis. “−” Indicates that less than 2% of the cells showed expression, “(−)”, “− / +” indicates that 5-50% of the cells showed expression, and “+/−” Indicates that approximately 40-80% of the cells showed expression, “+” indicates that more than 95% of the cells showed expression, “nor” indicates normal cells, and “na” indicates immune tissue Indicates that no chemical data was available.

  It should be understood that both mdx and normal mice showed all identical distributions of cell markers tested in this assay. That is, the presence of the mdx mutation does not affect cell marker expression in the isolated PP6 muscle cell derived population.

  Growth medium containing MDC in DMEM (Dulbecco's modified Eagle medium) and 10% FBS (fetal bovine serum), 10% HS (horse serum), 0.5% chicken embryo extract, and 1% penicillin / streptomycin), or , Were grown in a fusion medium containing DMEM supplemented with 2% fetal bovine serum and 1% antibiotic solution. All media materials were purchased from Gibco Laboratories (Grand Island, NY).

Example 2 Enrichment, isolation and analysis of MDCs by a single plating method Populations of fast-adhering and slow-adhering MDCs were isolated from skeletal muscle of mammalian subjects. The subject may be a human, rat, dog or other mammal. The biopsy sample size ranged from 42 to 247 mg.

Skeletal muscle biopsy tissue is immediately placed in cold medium (HYPOTHERMOSOL® (BioLife) supplemented with gentamicin sulfate (100 ng / mL, Roche)) and stored at 4 ° C. After 3-7 days, the biopsy tissue is removed from storage and production begins. If there is connective or non-muscle tissue, it is excised from the biopsy sample. Weigh the remaining muscle tissue used for isolation. The tissue is ground in Hank's Balanced Salt Solution (HBSS), transferred to a triangular test tube, and centrifuged (2500 × g, 5 minutes). The pellet is then resuspended in digestive enzyme solution (Liberase Blendzyme 4 (0.4-1.0 U / mL, Roche)). 2 mL of digestive enzyme solution is used per 100 mg of biopsy tissue and incubated on a rotating plate at 37 ° C. for 30 minutes. The sample is then centrifuged (2500 × g, 5 minutes). The pellet is resuspended in culture medium and passed through a 70 μm cell strainer. The culture medium used for the procedure described in this example was added to the Cambrex endothelial growth medium EGM-2 basal medium with the following components: (i.) 10% (v / v) fetal bovine serum, and (ii) .) Cambrex EGM-2 SingleQuot kit with insulin growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), hydrocortisone, Heparin and those containing ascorbic acid were supplemented. The filtered cell solution was then transferred to a T25 culture flask and incubated at 37 ° C. for 30-120 minutes under 5% CO ₂ . The cells attached to the flask are “rapidly attached cells”.

After incubation, the cell culture supernatant is removed from the T25 flask and placed in a 15 mL triangular test tube. Rinse the T25 culture flask with 2 mL of warmed culture medium and transfer to the 15 mL triangular test tube described above. Centrifuge 15 mL triangular test tube (2500 × g, 5 min). The pellet is resuspended in culture medium and transferred to a new T25 culture flask. The flask is incubated for about 2 days at 37 ° C. under 5% CO ₂ (cells attached to the flask are “slowly adherent cells”). After incubation, the cell culture supernatant is aspirated and fresh culture medium is added to the flask. The flask is then returned to the incubator and allowed to grow. Standard culture passages are performed from here to maintain cell confluency in the culture flask below 50%. Adherent cells are detached from the flasks during passage using trypsin EDTA (0.25%, Invitrogen). It takes an average of 17 days (typically from the date of start of production) for typical growth of “slowly adherent cells” to achieve an average total viable cell number of 37 million cells.

  After the desired cell number is achieved, the cells are harvested from the flask using trypsin EDTA and centrifuged (2500 × g, 5 minutes). The pellet is resuspended in BSS-P solution (HBSS supplemented with human serum albumin (2% v / v, Sera Care Life)) and counted. The cell solution is then centrifuged again (2500 × g, 5 minutes) and the desired temperature using cryopreservation medium (CryoStor (Biolife) supplemented with human serum albumin (2% v / v, Sera Care Life Sciences)). Resuspend to cell concentration and fill into appropriate vials for cryopreservation. The cryovial is placed in a freezing container and placed in a -80 ° C freezer. Cells are administered by thawing a frozen cell suspension with an equal volume of saline at room temperature and injected directly (no other manipulations). The lineage characteristics of the slowly adherent cell population are myogenic (87.4% CD56 +, 89.2% desmin +), endothelium (0.0% CD31 +), hematopoietic system (0.3% CD45 +), and fibroblasts ( 6.8% CD90 + / CD56-).

After dissociation of skeletal muscle biopsy tissue, two fractions of cells were collected based on rapid or slow attachment to the culture flask. Cells were then grown in culture with growth medium and then frozen (3 × 10 ⁵ cells in ¹⁵ μL) in a 1.5 mL Eppendorf tube cryopreservation medium. As a control group, 15 μL of cryopreservation medium alone was placed in a test tube. These tubes were stored at −80 ° C. until the time of injection. Immediately prior to injection, the tubes were removed from storage, thawed at room temperature, and resuspended in 15 μl of 0.9% sodium chloride solution. The resulting 30 μL solution was then aspirated into a 0.5 cc insulin syringe with a 30 gauge needle. The investigator who performed the surgery and injection was blinded regarding the contents of the test tube.

Cell number and viability were counted using a Guava flow cytometer and Viacount assay kit (Guava). CD56 was measured by flow cytometry (Guava) using PE-conjugated anti-CD56 antibody (1:50, BD Pharmingen) and PE-conjugated isotype control monoclonal antibody (1:50, BD Pharmingen). Desmin was measured by flow cytometry (Guava) against paraformaldehyde-fixed cells (BD Pharmingen) using monoclonal desmin antibody (1: 100, Dako) and isotype control monoclonal antibody (1: 200, BD Pharmingen). Fluorescence labeling was performed using a Cy3-conjugated anti-mouse IgG antibody (1: 250, Sigma). Between steps, cells were washed with permeabilization buffer (BD Pharmingen). For creatine kinase (CK) assay, 1 × 10 ⁵ cells were plated per well in a 12-well plate in differentiation induction medium. After 4-6 days, cells were harvested by trypsinization and centrifuged to pellet. Cell lysis supernatant is CK
Assayed for CK activity using the Liqui-UV kit (Stanbio).

Example 3 Strengthening skeletal muscle using MDC By testing a population of human muscle-derived cells (hMDC) isolated from human muscle biopsy samples using pre-plating techniques, hMDC is similar to its murine counterpart. It has sex characteristics and reproduction characteristics.

Method Pre-plating technique: This technique is disclosed throughout the application, especially in Example 1 above.

Isolation and cell culture: Candidate populations were obtained using pre-plating techniques. These cells are grown in EGM ^™ -2 medium (Cambrex) at a density of 600 cells / cm ² and every 72-96 hours until confluence is reached under standard conditions (5.0% CO ₂ , 37 ° C.). Passed on.

  Flow cytometry: hMDC was analyzed for the presence of cell surface clusters of differentiation markers CD34, CD56, CD144, and CD146.

  Immunochemistry: hMDC were stained with desmin, myosin heavy chain, and dystrophin.

  Bioinformatic live cell imaging: Cells were grown in a cell culture system capable of capturing dynamic images. Images were taken at 10 minute intervals. We measured many parameters, such as doubling rate, growth rate, total cell number, degree of extension, area, and circumference, for various populations at early and late passages. We have identified both molecular and behavioral features that are thought to predict regeneration efficiency in vivo by comparing human MDC phenotype profiles between various pre-plating fractions.

  In vivo regeneration: We transplanted an early passage human population into the gastrocnemius muscle of mdx / SCID mice. The inventors collected muscles 2 weeks after transplantation. Muscles were frozen and cut into 10 μm sections. For immunohistochemical analysis, we used mouse anti-human dystrophin or anti-mouse dystrophin (Novocastra, DYS3 / 2, 1:50), biotinylated goat anti-mouse secondary Ab (Vector, 1: 500) and Streptavidin-Cy3 (Sigma, 1: 500) was used. We have used human nuclear antigen antibodies to label human nuclei in skeletal muscle sections.

Results The inventors examined three preliminary plating fractions: preliminary plating 1 and 2 (pp1-2), preliminary plating 2-3 (pp2-3) and preliminary plating 5-6). All populations were negative for CD34 and CD144 and positive for CD56 and CD146. A decrease in CD56 and CD146 was observed over time. Immunostaining revealed the ability of the cells to fuse into multinucleated myotubes and the expression of desmin, myosin, and dystrophin, revealing its myogenic potential.

  With time-lapse imaging, great variability was observed in parameters such as cell division time, population doubling time, cell motility behavior, and morphological parameters. The inventors observed a morphological medium-specific change. Cells grown in EGM2 showed a decrease in growth rate as the cells proliferated. According to the present inventors' analysis so far, no such behavioral difference regarding the pre-plating fraction has been observed.

  We injected several pre-plating populations: pp2 (n = 11 muscle), pp4 (n = 19), pp6 (n = 20). Cross-sex transplantation of hMDC into host mdx-scid muscle resulted in fusion from donor cells to host skeletal muscle fibers and subsequent delivery and expression of dystrophin in the host. Chimerism was measured by the use of human specific antibodies and donor specific Y chromosome (FIG. 1). The number of dystrophin-positive fibers regenerated was significantly higher in transplants using the pp6 fraction compared to the pp2 fraction (P = 0.037, two-sided two test, FIG. 2). However, there was no significant difference in the level of regeneration between pp2 and pp4 transplants. All groups had higher dystrophin levels than PBS sham controls (FIG. 2).

  This study shows that hMDC is similarly obtained from pp6, which appears to be different from pp2 obtained from human muscle biopsy samples. In the transplantation test, the number of dystrophin-positive regenerated muscle fibers was lower than that observed with mouse MDC, but the present inventors have observed human dystrophin expression. Initial results indicate that culture in SkGM results in higher hMDC proliferation than cultures in either EGM-2 or DMEM. Ongoing imaging studies will determine whether this increased growth is associated with higher therapeutic efficacy and different cell phenotypes.

Claims (1)

Invention described in this specification.

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