The aged suffer from progressive muscle weakness and regenerative failure. We demonstrate that muscle regeneration is impaired with aging due in part to a cell-autonomous functional decline in skeletal muscle stem cells (MuSCs). Two-thirds of aged MuSCs are intrinsically defective relative to young MuSCs, with reduced capacity to repair myofibers and repopulate the stem cell reservoir in vivo following transplantation due to a higher incidence of cells that express senescence markers and that have elevated p38α/β MAPK activity. We show that these limitations cannot be overcome by transplantation into the microenvironment of young recipient muscles. In contrast, subjecting the aged MuSC population to transient inhibition of p38α/β in conjunction with culture on soft hydrogel substrates rapidly expands the residual functional aged MuSC population, rejuvenating its potential for regeneration, serial transplantation, and strengthening damaged muscles of aged mice. These findings reveal a synergy between biophysical and biochemical cues that provides a paradigm for a localized autologous muscle stem cell therapy in aged individuals.

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.

Transplanted bone marrow derived cells (BMDCs) have been reported to fuse with cells of diverse tissues [1][2][3][4][5][6][7][8][9][10][11][12][13] , but the extremely low frequency of fusion has led to the view that such events are biologically insignificant. Nonetheless, in mice with a lethal recessive liver disease (tyrosinaemia), transplantation of wild type BMDCs restored liver function by cell fusion and rescued the mice from death 3, 9 , indicating that cell fusion can have beneficial effects. Here we report that chronic inflammation resulting from severe dermatitis or autoimmune encephalitis leads to robust fusion of BMDCs with Purkinje neurons and formation of hundreds of binucleate heterokaryons, a 10-100 fold higher frequency than previously reported 8,10,11,14 . Single haematopoietic stem cell transplants showed that the fusogenic cell is in the haematopoietic lineage and parabiosis experiments revealed that fusion can occur without irradiation. Species-mismatched bone marrow transplants resulted in activation of dormant rat Purkinje neuron-specific genes in BMDC nuclei post-fusion with mouse Purkinje neurons consistent with nuclear reprogramming. The precise neurological role of these heterokaryons awaits elucidation, but their frequency in brain after inflammation is clearly much higher than previously appreciated.Although fusion of like cells has long been known to accompany the normal development of a number of tissues such as skeletal muscle, bone and the placenta 15-17 , recent evidence from numerous laboratories indicates that bone marrow derived cells (BMDC) can fuse with