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Transient inactivation of Rb and ARF yields regenerative cells from postmitotic mammalian muscle. Pajcini KV, Corbel SY, Sage J, Pomerantz JH, Blau HM. Cell Stem Cell. 2010 Aug 6;7(2):198-213.

Pubmed (Abstract) Stanford Medical School Wall Street Journal Nature comment New-York Times More than 240 years ago it was discovered that salamanders have the capacity to regrow entire limbs following amputation. Centuries later, we remain mystified as to how and why certain vertebrates, such as teleost fish and urodele amphibians, are capable of such extensive regeneration, while rodents and humans have near to negligible capacity to regenerate tissues post-natally. A crucial event in urodele muscle regeneration is the formation of the blastema where dedifferentiation of muscular, cartilage, and nerve tissues occurs. Although the factors responsible for initiation of regeneration in newts and salamanders remain poorly understood, several downstream regulatory steps have been elucidated. One of the early molecular events following the formation of the blastema is the inactivation of the retinoblastoma (Rb) protein by phosphorylation, which allows post-mitotic nuclei of skeletal myotubes to re-enter the cell cycle and eventually initiate mitosis. However the role of Rb in mammalian muscle differentiation remains controversial. Given the importance of this question to determine if mature mammalian myocytes can re-enter the cell cycle, we conducted experiments to eliminate Rb by transiently knocking down its expression in postmitotic, multinucleated skeletal myotubes. The loss of Rb by delivering Rb-specific siRNA duplexes to C2C12 myotubes, yielded BrdU labeled indicating that myonuclei in mature are capable of entering S-phase and synthesizing new DNA. By contrast with C2C12 myotubes, when Rb-expression was removed in primary multinucleated myotubes the myonuclei rarely incorporated BrdU. We postulated that primary myotubes contained a second mechanism for inhibiting S-phase re-entry that was absent in the cell line. We used siRNAs that would target both INK4a family members, p16 and p19. Subsequent experiments showed that transient loss of both INK4a gene products combined with either transient (siRNA-mediated) or permanent (tamoxifencre-mediated) loss of Rb led to BrdU labeling in approximately 50% of the myotube nuclei. Our data showed that mature mammalian myotubes lacking Rb and p19ARF are capable of cell-cycle re-entry, structural dedifferentiation and upregulation of cytokinetic components important for cell division, all of which are features shared by regenerating muscle in urodele amphibians and provide evidence that crucial and temporally regulated dedifferentiation steps can be induced that are indicative of a mammalian regenerative process.

Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture Gilbert PM, Havenstrite KL, Magnusson KE, Sacco A, Leonardi NA, Kraft P, Nguyen NK, Thrun S, Lutolf MP, Blau HM

Pubmed (Abstract) Stanford Medical School Skeletal muscle wasting is a common clinical feature of a large number of pathologies that negatively impacts patient quality of life. Currently, there are no effective strategies to combat this condition, although cell transplantation approaches or therapeutics aimed at `rejuvenating' aged, injured or diseased skeletal muscle tissue hold promise. The ultimate goal of these studies was to identify culture conditions that permit expansion of this currently limited muscle stem cell source (MuSCs) in culture. We utilized a novel bioengineered hydrogel culture platform and automated cell tracking software developed by our laboratory in conjunction with in vivo functional assays. Using this approach, we demonstrated that recapitulating the stiffness of muscle tissue in culture could substantially increase MuSC survival, prevent their differentiation and promote self-renewal in culture for the first time. Intriguingly, during muscle wasting disease there is a progressive `stiffening' of the tissue, which we predict lends to pathogenesis. These observations constitute the first evidence that a biophysical niche property (substrate elasticity) can regulate adult stem cell self-renewal in culture. This advance now allows our laboratory and other researchers to study molecular mechanisms regulating MuSCs in highly simplified culture `microevironments' in which complexity can be gradually added to the system. The long term culture of MuSCs is a critical advance to the eventual use of human MuSCs in cell based therapeutic approaches to combat muscle wasting. Notably, soft hydrogel cultured MuSCs are still not as effective as freshly isolated MuSCs to regenerate damaged tissue.

Reprogramming towards pluripotency requires AID-dependent DNA demethylation Bhutani N, Brady JJ, Damian M, Sacco A, Corbel SY, Blau HM. Nature. 2010 Feb 25;463(7284):1042-7.

Pubmed (Abstract) Stanford Medical School The generation of pluripotent cells from a patient's own somatic cells is the holy grail of regenerative medicine. A variety of techniques have been used to attempt nuclear `reprogramming' including transfer of somatic nuclei into oocytes (SCNT) which led to cloning of the sheep `Dolly' and fusion of somatic cells with embryonic stem cells (ES) leading to cellular hybrids with stem cell characteristics. A recent breakthrough was the demonstration by Yamanaka that the introduction of only four molecular factors (Oct 4, Sox2, Klf4 and c-myc) into skin fibroblasts could generate pluripotent cells indistinguishable from ES cells in global gene expression, methylation patterns, and their ability to generate all of the germ layers. However, due to the asynchronous onset, low efficiency, and slow rate of reprogramming, it has not been possible to study the mechanistic changes that drive the human somatic cells towards the stem cell fate using any of the above techniques. In addition, during reprogramming the individual cell population is very heterogeneous and asynchronous precluding the characterization of the underlying molecular events. To overcome these limitations, we have developed a cell fusion based heterokaryon system in which reprogramming is initiated rapidly (24 h), is efficient (>60%) and synchronous (immediately upon fusion), making it an ideal system to study the earliest events of reprogramming and identify novel regulators, ultimately leading to more efficient reprogramming. Specifically, a mouse embryonic stem (ES) cell is fused to a human fibroblast to form an interspecies heterokaryon. Because there is no cell division in the culture conditions used, there is no nuclear fusion, no chromosome rearrangement and no chromosome loss, and these stable multinucleate heterokaryons can be maintained in culture for several days. By skewing the ratio of the input cells so that ES cells outnumber fibroblasts, reprogramming toward a pluripotent state is favored, since gene dosage (protein factor ratio) determines the outcome with respect to nuclear reprogramming. Reprogramming is initiated immediately upon addition of the fusion agent, PEG, and is synchronously induced. Detection of expression of human Oct4 and Nanog transcription is evident as early as 1 day postfusion and rapid DNA demethylation is observed at the promoters of human Oct 4 and Nanog as early as day 1-2 post fusion, possibly stabilizing expression of the newly activated genes. To investigate the role of key regulators that could impede or prevent reprogramming, we use multiple siRNAs to induce transient downregulation of a gene of interest in heterokaryons. One of the candidate molecules that we focused on was the Cytidine deaminase, AID or AICDA, that is known to be involved in genetic recombination in B cells. We showed that loss of function of AID results in a delay in nuclear reprogramming, both with respect to pluripotency gene transcription and demethylation of their promoters. These data demonstrated for the first time that the enzyme AID is a critical component of the demethylation process in mammalian pluripotent cells.

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