Atsushi Asakura Lab

Angiogenic Therapy for DMD
Sponsor: NIH, MDA

Project Description: DMD is an X-linked recessive genetic disease affecting one in 3,500 children caused by mutations in the gene coding for the protein dystrophin, which results in progressive muscle degeneration and vascular deficiency. For an effective form of therapy of DMD, both the muscle and the vasculature need to be addressed. To reveal the developmental relationship between muscular dystrophy and vasculature, mdx mice, an animal model for DMD, were crossed with Flt-1 gene knockout mice to create a model with increased vasculature. Flt-1 is a decoy receptor for vascular endothelial growth factor (VEGF), and therefore both homozygous (Flt-1-/-) and heterozygous (Flt-1+/-) Flt-1 gene knockout mice display increased endothelial cell proliferation and vascular density during development. We show the mdx:Flt-1+/- mice display improved muscle histology, and an increase in muscle blood flow and force production, compared to the mdx mice. Consequently, the mdx:utrophin-/-:Flt-1+/- mice display improved muscle histology and significantly higher survival rates compared to the mdx: utrophin-/- mice which show more severe muscle phenotypes than the mdx mice. Importantly, increased vascular niche in the mdx:Flt-1+/- mice results in increased satellite cell number, which may be responsible for the improved muscle phenotype seen in these mice (Verma et al., Hum. Mol. Genet., 2010). These data suggest that increasing the vasculature in neuromuscular diseases including DMD and ALS may ameliorate the histological and functional phenotypes associated with this disease (Ennen, et al., Skeletal Muscle, 2013).

Genetically Modified Muscle Stem Cell Therapy
Sponsor: NIH

Project Description: MyoD is a muscle-specific transcription factor that plays essential roles in muscle satellite cell differentiation and regeneration. We have demonstrated that myoblasts derived from MyoD-/- satellite cells engrafted with significantly higher efficacy compared to wild-type myoblasts after injection into regenerating muscle (Asakura et al., Proc. Natl. Acad. Sci. U S A, 2007) and infarcted heart (Nakamura et al., PLoS ONE, 2012). Importantly, in MyoD-/- myoblasts, anti-apoptotic genes such as Pax3, Bcl-2 and Bcl-xL were up-regulated through down-regulation of MyoD-target microRNAs, miR-1 and miR-206, while some pro-apoptotic genes such as GADD45 were down-regulated (Hirai et al., J. Cell Biol., 2010). Consistent with these expression profiles, MyoD-/- myoblasts were revealed to possess remarkable resistance to apoptosis and increased survival in vitro and in vivo. Therefore, MyoD is not only regulating terminal differentiation but also regulating apoptosis during myogenic differentiation. In addition, down-regulation of MyoD expression may be required for maintenance of self-renewing muscle stem cells. Our outcomes will reveal the developmental relationship between satellite cells as well as the molecular biology of cell differentiation, apoptosis and self-renewal processes. Furthermore, our data offer evidence for novel therapeutic stem cell transplantation, in which suppression of MyoD in myogenic progenitor cells would be beneficial to the therapy for DMD and ischemic heart diseases by providing a selective advantage for expansion of the stem cells.

Myoblast-derived induced Pluripotent Stem (iPS) cells
Sponsor: NIH

Project Description: Expression of the four transcription factors; Oct4, Sox2, cMyc and Klf4 has been shown to generate induced Pluripotent Stem (iPS) cells from many types of differentiated somatic cells. It remains unclear, however, whether committed skeletal muscle stem cells and progenitor cells have the potency to undergo reprogramming to develop iPS cells in line with previously reported cases. To test this, we have isolated satellite cells from adult mouse muscles. Upon being infected with retroviral vector expressing the four factors, these satellite cell-derived myoblasts gave rise to embryonic stem (ES) cell-like colonies. These cells expressed ES specific genes and were competent to differentiate into all three germ layers and germ cells, indicating the successful generation of muscle-derived iPS cells (Watanabe et al., Stem Cells, 2011). Therefore, it would be advantageous to utilize the skeletal muscle system as a well defined differentiation model to further elaborate on the effects of iPS cell reprogramming in somatic cells. Our hypothesis is that myoblast-derived iPS cells may maintain epigenetic memory of myogenic status, which might contribute to the higher myogenic differentiation potential. Therefore, the use of myoblast-derived iPS cells will be a potential cell therapy for DMD.

Selected Publications

J. P. Ennen, M. Verma, A. Asakura. (2013) Vascular-targeted therapies for Duchenne muscular dystrophy. Skeletal Muscle. 3(1):9.

N. Motohashi, Y. Asakura, A. Asakura. (2013) Isolation, culture and transplantation of muscle satellite cells. J Vis Exp. In press

S. Biressi, A. Asakura. (2012) Satellite Cells and the Universe of Adult Muscle Stem Cells. J. Stem Cell Res. & Ther. S11-001.

A. Asakura. (2012) Skeletal Muscle-derived Hematopoietic Stem Cells: Muscular Dystrophy Therapy by Bone Marrow Transplantation. J. Stem Cell Res. & Ther. S11-005.

N. Motohashi, A. Asakura. (2012) Molecular Regulation of Muscle Satellite Cell Self-Renewal. J. Stem Cell Res. & Ther. S11-e002.

Y. Nakamura, Y. Asakura, B. A. Piras, H. Hirai, C. T. Tastad, M. Verma, A. J. Christ, J. Zhang, T. Yamazaki, M. Yoshiyama, A. Asakura. (2012) Increased angiogenesis and improved left ventricular function after transplantation of myoblasts lacking the MyoD gene into infarcted myocardium. PLoS ONE. 7(7):e41736.

J. L. Mull, A. Asakura. (2012) A New Look at an Immortal DNA Hypothesis for Stem Cell Self-Renewal. J. Stem Cell Res. Ther. 2:e105.

S. Watanabe, H. Hirai, Y. Asakura, C. Tastad, M. Verma, C. Keller, J. R. Dutton, A. Asakura. (2011) MyoD gene suppression by Oct4 is required for reprogramming in myoblasts to produce induced pluripotent stem cells. Stem Cells. 29:505-516.

M. Verma, A. Asakura. (2011) Efficient single muscle fiber isolation from alcohol fixed adult muscle following beta-galactosidase staining for satellite cell detection. J. Histochem. Cytochem. 59:60-67.

H. Hirai, M. Verma, S. Watanabe, C. Tastad, Y. Asakura, A. Asakura. (2010) MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3. J. Cell. Biol. 191:347-365.

M. Verma, Y. Asakura, H. Hirai, S. Watanabe, C. Tastad, G.-H. Fong, M. Ema, J. A. Call, D. A. Lowe, A. Asakura. (2010) Flt-1 haploinsufficiency ameliorates muscular dystrophy phenotype by developmentally increased vasculature in mdx mice. Hum. Mol. Genet. 19:4145-4159.

H. Hirai, L. Romanova, S. Kellner, M. Verma, S. Rayner, A. Asakura*, N. Kikyo*. (2010) Post-mitotic role of nucleostemin as a promoter of skeletal muscle cell differentiation. Biochem. Biophys. Res. Commun. 39:299-304. (*double corresponding authors)

A. Asakura, (2008) Stem Cells for Muscle Regeneration. Stem Cell Biology. World Scientific Press. 145-175

A. Asakura. (2008) Vascular endothelial growth factor (VEGF) gene regulation by HEXIM1 in heart. Circ. Res. 102:398-400.

A. Asakura, H. Hirai, B. Kablar, S. Morita, J. Ishibashi, B. A. Piras, A. J. Christ, M. Verma, K. A. Vineretsky, M. A. Rudnicki. (2007) Increased survival of muscle stem cells lacking the MyoD gene after transplantation into regenerating skeletal muscle. Proc. Natl. Acad. Sci. U S A 104:16552-16557.

A. Asakura. (2007) Hematopoietic potential cells in skeletal muscle. Cell Rec. 17:836-838.

G. Grenier, A. Scim�, F. Le Grand, A. Asakura, C. Perez-Iratxeta, M. A. Andrade-Navarro, P. A. Labosky, M. A. Rudnicki. (2007) Resident Endothelial Precursors in Muscle, Adipose and Dermis Contribute to Post-Natal Vasculogenesis. Stem Cells. 25:3101-3110.

Y. Nakamura, X. Wang, C. Xu, A. Asakura, M. Yoshiyama, A. H. From, J. Zhang. (2007) Xenotransplantation of Long Term Cultured Swine Bone Marrow-Derived Mesenchymal Stem Cells. Stem Cells. 25:612-20.

J. Ishibashi, R. L. Perry, A. Asakura, M. A. Rudnicki. (2005) MyoD induces myogenic differentiation through cooperation of its NH2- and COOH-terminal regions. J. Cell Biol. 171:471-482.

J. Lee, A. Asakura, J. Zhang. (2005) Experimental cell transplantation for myocardial repair. Handbook of Cardiac Anatomy, Physiology and Devices. Humana Press. 427-438.

C. J. Storbeck, K. Daniel, Y. H. Zhang, J. Lunde, A. Scime, A. Asakura, B. Jasmin, R. G. Korneluk, L. A. Sabourin. (2004) Ste20-like kinase SLK displays myofiber type specificity and is involved in C2C12 myoblast differentiation. Muscle Nerve 29:553-64.

M. Komaki, A. Asakura, M. A. Rudnicki, S. Cheifetz, J. Sodek. (2004) MyoD enhances BMP-induced osteogenic differentiation of myogenic cell cultures. J. Cell Sci. 117:1457-1468.

A. Asakura, M. A. Rudnicki. (2003) Rhabdomyosarcomagenesis-Novel pathway found. Cancer Cell 4: 421-422.

A. Asakura. (2003) Stem cells in adult skeletal muscle. Trends Cardiovasc. Med. 13:123-128.

A. Asakura, P. Seale, A. Girgis-Gabardo, M. A. Rudnicki. (2002) Myogenic specification of side population cells in skeletal muscle. J. Cell Biol. 159:123-134.

A. Asakura, M. A. Rudnicki. (2002) Side population cells from diverse adult tissues are capable of in vitro hematopoietic differentiation. Exp. Hematol. 30:1339-1345.

A. Asakura, M. A. Rudnicki. (2002) Cellular and Molecular Mechanisms Regulating Skeletal Muscle development. Mouse Development: Patterning, Morphogenesis, and Organogenesis. Academic Press. 253-278.

P. Seale, A. Asakura, M. A. Rudnicki. (2001) The potential of Muscle Stem Cells. Dev. Cell 1: 333-342.

A. Asakura, M. Komaki, M. A. Rudnicki. (2001) Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation. Differentiation 68:245-253.

J. M. Olson*, A. Asakura*, L. Snider, R. Hawkes, A. Strand, S. J. Tapscott, (2001) NeuroD2 is necessary for normal development and survival of central nervous system neurons. Dev. Biol. 238:174-187. (*contributed equally).

A. Asakura, S. J. Tapscott. (1998) Apoptosis of epaxial myotome in Danforth's short-tail (Sd) mice in somites that form following notochord degeneration. Dev. Biol., 203:276-289.

A. Asakura, G. E. Lyons, S. J. Tapscott. (1995) The regulation of myoD gene expression: Conserved elements mediated expression in embryonic axial muscle. Dev. Biol. 171:386-398.

A. Asakura, A. Fujisawa-Sehara, T. Komiya, Y. Nabeshima, Y.-I. Nabeshima. (1993) MyoD and myogenin act on the chicken myosin light chain 1 gene as distinct transcriptional factors. Mol. Cell. Biol. 13:7153-7162.

Principal Investigator

• Atsushi Asakura, PhD

Postdoctoral Fellows

• Norio Motohashi, PhD
• Yuko Motohashi, PhD
• Tomohide Takaya, PhD

Graduate Students

• Mayank Verma
• Nandkishore R. Belur

Collaborators

• Charles P. Emerson Jr. (University of Massachusetts)
• David J. Goldhamer (University of Connecticut)
• Shihuan Kuang (Purdue University)
• Charles Keller (Oregon Health and Science University)
• LaDora V. Thompson (University of Minnesota)
• Michael Kyba (University of Minnesota)
• Dawn A. Lowe (University of Minnesota)
• James R. Dutton (University of Minnesota)

Undergraduate Student and Volunteer Positions

• Undergraduate Student and Volunteer Positions are currently available in the area of muscle stem cells and muscular dystrophy biology to understand molecular mechanisms of muscle regeneration and muscular dystrophy diseases.

Contact Information

Atsushi Asakura, PhD
Associate Professor

Phone: 612-624-7108
Fax: 612-624-2436
Email: asakura@umn.edu
Address: Stem Cell Institute, University of Minnesota Medical School, MTRF 4-220, 2001 6th St SE, Minneapolis, MN 55455