STEM CELL BIOLOGY

Stem Cell Biology

Stem Cell Biology

Introduction

Mesoangioblasts (MABs) are a subset of muscle-derived pericytes able to restore dystrophic phenotype in mice and dogs. However, their lifespan is limited and they undergo senescence after 25-30 population doublings. Recently, induced pluripotent stem cells (iPSCs) generated from reprogrammed fibroblasts have been demonstrated to have in vitro and in vivo myogenic potential when sorted for the SM/C-2.6 antigen. Furthermore, chimeric mice from mdx-iPSCs (DYS-HAC) cells showed tissue-specific expression of dystrophin. Nevertheless, myogenic differentiation protocols and the potential of iPSCs generated from different cell sources still present unanswered questions (Darabi, 2008).

Here we show that iPSCs generated from prospectively sorted MABs (MAB-iPSCs) are pluripotent as fibroblast-derived iPSCs (f-iPSCs). However, both teratoma formation and genetic cell manipulation assays identify a durable epigenetic memory in MAB-iPSCs, resulting in stronger myogenic commitment. Striated muscle tissue accounts for up to 70% of MAB-iPSC teratomas. Moreover, transfection with Pax3 and Pax7 induces a more robust myogenic differentiation in MAB-iPSCs than in f-iPSCs. A larger amount of CD56+ progenitors can be sorted from the MAB-iPSCs differentiating pool and, after transplantation into asg-KO mice, can efficiently participate to skeletal muscle regeneration and restore asg expression. Our data strongly suggest that iPSCs are a heterogeneous population and, when generated from myogenic adult stem cells, they exhibit a stronger commitment, paving the way for creating custom-made cell protocols for muscular dystrophies.

Discussion and Analysis

Muscular dystrophies are a set of genetically inherited diseases involving muscle wastage and motility impairment 1. Although steroid administration and positive pressure ventilation can alleviate pain, a therapy for long-term muscle repair is still missing. Duchenne muscular dystrophy (DMD) and sarcoglycanopathies represent the most severe and common forms. Sarcoglycanopathies, such as recessive forms of limb-girdle muscular dystrophy (LGMD), are characterized by the absence of sarcoglycan proteins, resulting in a dysfunction of the dystrophin-sarcoglycan complex (DSC) 2. DSC impairment leads to progressive detachment of sarcomeric units from the sarcolemmal membrane and chronic muscle inflammation. a-Sarcoglycan-KO (asg-KO) mice show severe impairment of skeletal fibre architecture, kyphosis and progressive loss of motility 3. Thus, asg-KO mice represent a valuable model to study the severe muscle damage that is common in human dystrophies.

Several strategies are currently being investigated to repair muscle architecture in dystrophic muscles. Beside gene delivery and drug administration, cell therapy has become a promising perspective in the last decade. Cell therapy for muscular dystrophy is based on in vivo injection of committed myogenic stem cells, in order to promote regeneration and restore muscle architecture. Transplantation of donor-derived or autologous genetically corrected cells aims to restore the expression of the missing protein in newly generated muscle fibres.

MABs are multipotent stem cells, but still their proliferation and potency rates decline with age. Therefore, we decided to reprogramme MABs towards a pluripotent state with indefinite proliferation capability. Sorted AP+ MABs were reprogrammed by retroviral-mediated over-expression of Oct4, Sox2, Klf4 and cMyc. After 6-10 days on a feeder layer of inactivated murine embryonic fibroblasts (iMEFs), and in the presence of LIF, ESC-like colonies appeared and ...