Freshly isolated muscle stem cells (MuSCs) exhibit robust regenerative capacity in vivo that is rapidly lost in culture. Using a bioengineered substrate to recapitulate key biophysical and biochemical niche features in conjunction with a novel highly automated single cell tracking algorithm, we show that substrate elasticity is a potent regulator of MuSC fate in culture. Unlike MuSCs on rigid plastic dishes (~106kPa), MuSCs cultured on soft hydrogel substrates that mimic the elasticity of muscle (12kPa) self-renew in vitro and contribute extensively to muscle regeneration when subsequently transplanted into mice and assayed histologically and quantitatively by non-invasive bioluminescence imaging. Our studies provide novel evidence that by recapitulating physiological tissue rigidity, propagation of adult muscle stem cells is possible, enabling future cell-based therapies for muscle wasting diseases.
Adult muscle satellite cells have a principal role in postnatal skeletal muscle growth and regeneration1. Satellite cells reside as quiescent cells underneath the basal lamina that surrounds muscle fibres2 and respond to damage by giving rise to transient amplifying cells (progenitors) and myoblasts that fuse with myofibres. Recent experiments showed that, in contrast to cultured myoblasts, satellite cells freshly isolated3–5 or satellite cells derived from the transplantation of one intact myofibre6 contribute robustly to muscle repair. However, because satellite cells are known to be heterogeneous4,6,7, clonal analysis is required to demonstrate stem cell function. Here we show that when a single luciferase-expressing muscle stem cell is transplanted into the muscle of mice it is capable of extensive proliferation, contributes to muscle fibres, and Pax7+luciferase+ mononucleated cells can be readily re-isolated, providing evidence of muscle stem cell self-renewal. In addition, we show using in vivo bioluminescence imaging that the dynamics of muscle stem cell behaviour during muscle repair can be followed in a manner not possible using traditional retrospective histological analyses. By imaging luciferase activity, real-time quantitative and kinetic analyses show that donor-derived muscle stem cells proliferate and engraft rapidly after injection until homeostasis is reached. On injury, donor-derived mononucleated cells generate massive waves of cell proliferation. Together, these results show that the progeny of a single luciferase-expressing muscle stem cell can both self-renew and differentiate after transplantation in mice, providing new evidence at the clonal level that self-renewal is an autonomous property of a single adult muscle stem cell.
Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2-3 weeks), the frequency is low (, 0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide the first evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.Reprogramming of somatic cell nuclei towards pluripotency has been achieved by nuclear transfer into enucleated oocytes 1,2 and the introduction of four defined factors to generate iPS cells [3][4][5][6] . These remarkable advances enable the generation of patient-specific cells for tissue replacement, modelling human diseases in tissue culture, and drug discovery. However, an understanding of the molecular mechanisms underlying nuclear reprogramming has been elusive, largely due to the technical challenges associated with nuclear transfer and the low efficiency of iPS cell generation.
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