Stem Cells and Muscular Dystrophy Rita Perlingeiro, PhD Lillehei Heart Institute Dept. of Medicine University of Minnesota [email protected] BioC/Phsl/BMEn 5444 April 24 , 2014 The Dystrophin-Associated Protein Complex Pathophysiology of DMD Mutation within the Dystrophin gene Absence of dystrophin Muscle membrane injury Cascade of events leading to muscle fiber injury and degradation degeneration regeneration Fibrosis, contractures, and weakness Filareto, A. Animal Models for Muscular Dystrophy Gene therapy Cell therapy Cell/Gene therapy From Allamand & Campbell, Hum. Mol. Gen., 2000 OUTLINE Myoblasts and clinical trials Muscle stem cells: satellite cells* Bone marrow and peripheral blood Mesoangioblasts/pericytes Pluripotent ES/iPS cells Muscle Regeneration in Adult Mouse CTX Day 2 CTX Day 7 CTX Day 15 Abmayr S M , Development 2012 ADULT MYOGENESIS Satellite Cell Pax7 Myoblasts Multinucleated myotube Myf5, Mrf4, MyoD MyoD, Mrf4, Myogenin Muscle fiber DIFFERENTIATION MCK Fusion - Limited migration - Poor survival Skeletal muscle hierarchy Satellite cells Pax7 (Pax3) Myoblasts Multinucleated myotube Myf5, Mrf4, MyoD MyoD, Mrf4, Myogenin Muscle fiber MCK - Myoblasts: post-mitotic - Stem/satellite cells: self-renew, proliferate, and give rise to myoblasts Ideal cell population for therapeutic application Satellite cells reside beneath the basal lamina of the muscle fiber N‐CAM Laminin Renault et al., Exp. Gerontol. 2002 Satellite Cells Pax7: green Laminin: red Nuclei: blue Halevy et al, Dev. Dyn., 2004 Experimental evidence of their regenerative potential: Transplantation of single intact myofibers into injured muscle results not only in efficient muscle regeneration but also expansion of the satellite cell pool. Single myofiber Resulting engraftment Collins C.A. et al, Cell, 2005 What is the bottleneck? Hunting for another source of cells able to give rise to functional myoblasts/myotubes and satellite cells Bone Marrow (hematopoietic and stromal cells) Bone Marrow Cells and Muscle regeneration • Mouse BM cells differentiate into muscle (Ferrari et al, Science, 1998) (Bittner et al, Anat Embryol., 1999) (Gussoni et al, Nature, 1999) (Fukada et al, J. Cell Science, 2002) Cardiotoxin mdx mdx mdx/cardiotoxin • Persistence of donor nuclei in a DMD patient receiving BMT (Gussoni et al, J Clin Invest, 2002) • Human circulating AC133+ cells restore dystrophin expression in mdx mice (Torrente et al, J Clin Invest, 2002) • Human adipose tissue induces dystrophin expression in the mdx mouse (Rodriguez et al, J Exp Med, 2005) FUSION, not regeneration Mesenchymal Stem Cells (bone marrow, fat tissues) Osteoblasts Adipocytes Chondrocytes 5-azacytidine Petri dish Myocytes Not efficient – probably fusion in vivo (mouse): make muscle? Can we make mesenchymal stem cells more prone to make skeletal muscle? Primary MSCs MSC line (B9) PAX3 IRES GFP IRES GFP Fat Bone Cartilage Muscle ? Oil Red O Staining Alizarin Red S Staining Alcian Blue Staining Gang E.J. Exp. Cell Res., 2009 2- Mesoangioblasts (Pericytes) - Cells of adult origin with myogenic potential - Great in vitro expansion potential: feasibility for transplantation - No need for genetic modification (except correction if using autologous cells Science. 2003 Jul 25;301(5632):487-92 Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Sampaolesi M, Torrente Y, Innocenzi A, Tonlorenzi R, D'Antona G, Pellegrino MA, Barresi R, Bresolin N, De Angelis MG, Campbell KP, Bottinelli R, Cossu G. Dog mesoangioblasts Ucal Treated SCID/mdx mice Dystrophin/human AC lamin AC Treated dogs Vrillie Variable degree of improvement Valgus Vaccin Alessandro Magli Clinical trials are ongoing but there is a challenge: “senescence” Mesoangioblasts from iPS cells Tedesco et al , Science Translational Medicine, 2012 - Cells of adult origin with myogenic potential ?? - Great in vitro expansion potential: feasibility for Transplantation ?? - No need for genetic modification (except correction if using autologous cells) ?? From pluripotent stem cells to skeletal muscle Pax3/Pax7 enables the generation of mouse ES-derived skeletal myogenic progenitor cells that are endowed with in vivo regenerative potential Myf5 MyoD MHC Proliferation Pax7 Differentiation in vitro GFP in vivo Pax7 Dystrophin GFP Merge Merge Darabi R. et al, 2008 Nat. Med. Stem Cells, 2011 Filareto et al, 2012 J. Stem Cell Res. Ther. Magli et al, 2012 Stem Cells Dystrophic iPS cells: ex vivo genetic correction Filareto et al, Nat. Comm., 2013 In vivo regenerative potential of μUTRN-corrected iPS-derived myogenic precursors * (18.8) (0) * PBS-Control Contractile properties of TA muscles of transplanted dKO mice Filareto et al, Nat. Comm., 2013 UTRN+ and α1-SYN+ myofibers in TA muscles of dKO mice after IV cell transplantation Filareto et al, Nat. Comm., 2013 Expansion Potential of iPax7 Human ES/iPS Cells Purification of Pax7+ (GFP+) cells EB s iPax7-H9 ES cells EB-derived monolayer 14% GFP Pax7 Induction + dox Cell count 10 10 10 10 Expansion of Pax7+ progenitors 8 7 H9 IPRN 14.57 IPRN 13.13 6 5 0 4 8 12 16 Days Cell Stem Cell, 2012 Myogenic differentiation potential of human ES/iPS-derived cells. Differentiation Proliferation Myogenin MHC Pax7 Myogenin MHC iPax7-iPS-2 iPax7-iPS-1 iPax7-H9 Pax7 Cell Stem Cell, 2012 Transplantation of human ES/iPS-derived myogenic progenitors into NSG-mdx4cv mice Human Lamin AC Human Dystrophin Merge PBS-control H9 IPRN 13.13 IPRN 14.57 Cell Stem Cell, 2012 Total Number of Human Dystrophin Positive Fibers/TA Section Keys: NSG gram 100 60 H9 NSG mdx iPS1 iPS2 iPS1: IPRN 13.13 iPS2: IPRN 14.57 40 PBS Cell 20 50 0 H9 iPS1 0 iPS2 2 6 10 s 14 F0 + * ** 135 * 50 25 0 NSG NSG -mdx H9 1 iPS1 +++ ** * * 45 0 iPS2 NSG NSG -mdx H91 iPS1 iPS2 Weight 7.5 90 mg +++ 2.5 0 NSG NSG- mdx 90 CSA 5 PBS Cell 16 Specific force (sF0) KN/m2 gram 75 mm2 Average Number Improved strengthen in TA muscles of transplanted NSG-mdx4cv mice 60 +++ 30 NSG NSG -mdx H9 1 iPS1 iPS2 0 NSG NSG -mdx H91 iPS1 iPS2 Cell Stem Cell, 2012 HSKM Average Number HDF Total Number of Human Dystrophin Positive Fibers/TA Section 39 26 13 0 HDF 1 HSKM Specific force (sF0) 125 100 KN/m2 Human Dystrophin / DAPI What happens when we transplant fibroblasts or myoblasts into NSG-mdx4cv mice? 75 50 25 0 HDF 1 HSKM Cell Stem Cell, 2012 Satellite cell engraftment by human ES/iPS-derived myogenic progenitors Pax7 Pax7 / hLamin AC / DAPI Pax7 hLamin AC Pax7 hLamn AC/DAPI Laminin Laminin Merge Merge H9 IPRN 13.13 IPRN 14.57 Cell Stem Cell, 2012 Safety and long-term engraftment of human ES/iPS-derived myogenic progenitors: 11-month cohort Human Specific Dystrophin / DAPI Control H9 iPS1 iPS2 Human Dystrophin / Human Lamin AC / DAPI H9 iPS1 iPS2 Cell Stem Cell, 2012 Summary Rinaldi and Perlingeiro, Transl Res, 2014
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