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Multiple roles for Oct4 in induced pluripotency from MEFs and mouse myoblasts

From the March 2011 Issue of Stem Cells

Paper Commentary by Carla Mellough

Original reports describing the generation of induced pluripotent stem cells (iPSCs) set the foundations of the reprogramming process as the exogenous expression of four transcription factors Oct4, Sox2, Klf4, c-Myc and/or Lin28 and Nanog. Subsequent work has shown that with the use of small molecules direct reprogramming can also be achieved by overexpression of a subset of these factors, in some cases with exogenous Oct4 only. Oct4 expression seems to be integral to the reprogramming process and two articles in the March 2011 issue of Stem Cells now reveal additional roles for Oct4. The first article, by Yuan et al. from Sheng Ding’s laboratory at the Scripps Research Institute in California, reports a new small molecule which can facilitate reprogramming. The authors screened 100 small molecules under TGF-β receptor inhibition in mouse embryonic fibroblasts (MEFs) that had been retrovirally transduced with Oct4 and their results show that AMI-5, an inhibitor of protein arginine methyltransferase (PRMT) activity, could greatly facilitate the reprogramming process.

Multiple roles for Oct4 in induced pluripotency from MEFs and mouse myoblasts

From the March 2011 Issue of Stem Cells

Paper Commentary by Carla Mellough

Original reports describing the generation of induced pluripotent stem cells (iPSCs) set the foundations of the reprogramming process as the exogenous expression of four transcription factors Oct4, Sox2, Klf4, c-Myc and/or Lin28 and Nanog. Subsequent work has shown that with the use of small molecules direct reprogramming can also be achieved by overexpression of a subset of these factors, in some cases with exogenous Oct4 only. Oct4 expression seems to be integral to the reprogramming process and two articles in the March 2011 issue of Stem Cells now reveal additional roles for Oct4. The first article, by Yuan et al. from Sheng Ding’s laboratory at the Scripps Research Institute in California, reports a new small molecule which can facilitate reprogramming. The authors screened 100 small molecules under TGF-β receptor inhibition in mouse embryonic fibroblasts (MEFs) that had been retrovirally transduced with Oct4 and their results show that AMI-5, an inhibitor of protein arginine methyltransferase (PRMT) activity, could greatly facilitate the reprogramming process.

Mitochondrial Function Controls Proliferation and Early Differentiation Potential of Embryonic Stem Cells

From the March 2011 Issue of Stem Cells

Paper Commentary by Carla Mellough

Mitochondrial function is understood to play a key role in the ageing process and mitochondrial dysfunction underlies the pathophysiology of various diseases. Whilst much attention has focused on the role of the genetic and epigenetic state on cell function and differentiation in stem cells, little work thus far has addressed the contribution of cell metabolism in stem cell function and activity. New results reported in the March edition of Stem Cells by Mandal et al. from the University of California and Indian Institute of Science Education and Research, now begin to reveal the relationship between the mitochondria and stem cell proliferation, differentiation and tumorigenesis.

Mitochondrial Function Controls Proliferation and Early Differentiation Potential of Embryonic Stem Cells

From the March 2011 Issue of Stem Cells

Paper Commentary by Carla Mellough

Mitochondrial function is understood to play a key role in the ageing process and mitochondrial dysfunction underlies the pathophysiology of various diseases. Whilst much attention has focused on the role of the genetic and epigenetic state on cell function and differentiation in stem cells, little work thus far has addressed the contribution of cell metabolism in stem cell function and activity. New results reported in the March edition of Stem Cells by Mandal et al. from the University of California and Indian Institute of Science Education and Research, now begin to reveal the relationship between the mitochondria and stem cell proliferation, differentiation and tumorigenesis.

Stem Cells Translational Medicine

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Anthony J. Atala, MD, Editor

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STEM CELLS TRANSLATIONAL MEDICINE is dedicated to significantly advancing the clinical utilization of stem cell molecular and cellular biology. By bridging stem cell research and clinical trials, SCTM will help move applications of these critical investigations closer to accepted best practices.

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The potential of stem cells therapies and regenerative medicine is both provocative and powerful, offering the distinct possibility of eventually repairing or replacing tissues damaged from disease, including certain cancers.

By helping speed expertly executed translations of emerging lab discoveries into legitimate clinical trials and bedside application, STEM CELLS TRANSLATIONAL MEDICINE ultimately will improve patient outcomes.

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HIV Therapeutics– Gene Editing Shows Promise in Clinic

From Nature News

The Stem Cell Portal has recently reported on new studies which demonstrate a potential cure for HIV/AIDs based around the CCR5 receptor (Stem Cell Cures for HIV?). It is known that HIV can infect CD4+ T cells through the CCR5 cell surface receptor and the subsequent destruction of the immune system is driven by this loss of normal CD4+ T cells. It has been discovered that a homozygous deletion in the CCR5 allele entails a resistance to HIV infection, while heterozygous patients show slower disease progression. Now Nature News reports on results from a phase I safety trial presented at the Conference on Retroviruses and Opportunistic Infections by Sangamo BioSciences of a zinc finger nuclease (ZFN) which targets and artificially disrupts the CCR5 gene.

HIV Therapeutics– Gene Editing Shows Promise in Clinic

From Nature News

The Stem Cell Portal has recently reported on new studies which demonstrate a potential cure for HIV/AIDs based around the CCR5 receptor (Stem Cell Cures for HIV?). It is known that HIV can infect CD4+ T cells through the CCR5 cell surface receptor and the subsequent destruction of the immune system is driven by this loss of normal CD4+ T cells. It has been discovered that a homozygous deletion in the CCR5 allele entails a resistance to HIV infection, while heterozygous patients show slower disease progression. Now Nature News reports on results from a phase I safety trial presented at the Conference on Retroviruses and Opportunistic Infections by Sangamo BioSciences of a zinc finger nuclease (ZFN) which targets and artificially disrupts the CCR5 gene.

Engineered colour coded lentiviral vectors to visualise iPSC generation

From the laboratory of Axel Schambach in Germany comes a report describing a system which can be utilized to study the subtle dynamics of early reprogramming. Published in Molecular Therapy, Warlich et al. demonstrate the use of specially engineered colour-coded lentiviral vectors containing reprogramming factors (Oct4, Klf4, Sox2 and c-Myc) under the control of a retroviral promoter which induce rapid high level expression of reprogramming factors followed by rapid silencing. Using mouse embryonic fibroblasts containing an Oct4-EGFP reporter, the authors were able to observe cell-intrinsic stochastic processes following the induction of pluripotency, enabling the visualisation of the conversion of fibroblasts to induced pluripotent stem cells (iPSC). In their system, the expression of red fluorescent protein in transduced cells indicates reprogramming factor expression whilst green fluorescence indicates the emergence of iPSC. Using fluorescence microscopy, long term single cell tracking and live cell imaging, they demonstrate that vector silencing occurs prior to or at the onset of the expression of the pluripotency marker Oct4. They reveal that stochastic epigenetic changes are required for reprogramming and that early reprogrammed colonies can emerge as a genetic mosaic formed on the basis of epigenetic variability, particularly under conditions that increase reprogramming efficiency (e.g. the addition of valproic acid during induction). They observed heterogeneity of EGFP expression in iPSC colonies and report that not all cells within an iPSC colony expressed EGFP - some cells within the colony maintained the expression of exogenous reprogramming factors and failed to reprogramme. The observed heterogeneity within clonal colonies of genetically identical cells undergoing reprogramming in this study is particularly interesting and supports Yamanaka’s hypothesis of a stochastic model for induced pluripotency in somatic cells. Studies such as this help to discern the mechanisms underlying and variables guiding the reprogramming process by gene transfer of reprogramming transcription factors into somatic cells.

Engineered colour coded lentiviral vectors to visualise iPSC generation

From the laboratory of Axel Schambach in Germany comes a report describing a system which can be utilized to study the subtle dynamics of early reprogramming. Published in Molecular Therapy, Warlich et al. demonstrate the use of specially engineered colour-coded lentiviral vectors containing reprogramming factors (Oct4, Klf4, Sox2 and c-Myc) under the control of a retroviral promoter which induce rapid high level expression of reprogramming factors followed by rapid silencing. Using mouse embryonic fibroblasts containing an Oct4-EGFP reporter, the authors were able to observe cell-intrinsic stochastic processes following the induction of pluripotency, enabling the visualisation of the conversion of fibroblasts to induced pluripotent stem cells (iPSC). In their system, the expression of red fluorescent protein in transduced cells indicates reprogramming factor expression whilst green fluorescence indicates the emergence of iPSC. Using fluorescence microscopy, long term single cell tracking and live cell imaging, they demonstrate that vector silencing occurs prior to or at the onset of the expression of the pluripotency marker Oct4. They reveal that stochastic epigenetic changes are required for reprogramming and that early reprogrammed colonies can emerge as a genetic mosaic formed on the basis of epigenetic variability, particularly under conditions that increase reprogramming efficiency (e.g. the addition of valproic acid during induction). They observed heterogeneity of EGFP expression in iPSC colonies and report that not all cells within an iPSC colony expressed EGFP - some cells within the colony maintained the expression of exogenous reprogramming factors and failed to reprogramme. The observed heterogeneity within clonal colonies of genetically identical cells undergoing reprogramming in this study is particularly interesting and supports Yamanaka’s hypothesis of a stochastic model for induced pluripotency in somatic cells. Studies such as this help to discern the mechanisms underlying and variables guiding the reprogramming process by gene transfer of reprogramming transcription factors into somatic cells.

Use of poly(β-amino esters) as a non-viral means to induce pluripotency

From the Journal of Biological Chemistry

Since the generation of the first induced pluripotent stem cells (iPSC) from somatic cells was reported in 2006, various alternative ways of achieving pluripotency have been attempted in order to improve the safety and efficiency of the reprogramming process and of the resultant iPSC. The production of the viral particles commonly used to express the reprogramming factors is a time and labour intensive process and the risk of insertional mutagenesis is of significant clinical concern. From the Center of Regenerative Medicine of Barcelona now comes a report by Montserrat et al.1 which describes that human fibroblasts can successfully be reprogrammed using poly(β-amino esters) as the transfection reagent, avoiding the use of viral vectors entirely. The authors report that using serial transfection with poly(β-amino esters), which are biodegradable polymers that are easy to synthesise and have low toxicity, they can successfully transfect human fibroblasts with a CAG driven vector expressing reprogramming factors (Oct4, Sox2, Klf4 and c-Myc tagged with a GFP reporter gene) as a single polycistronic plasmid, to generate iPSC in 20-28 days. Moreover, they do this with higher efficiency than commercially available transfection reagents, an important consideration given the usually low transfection efficiency observed in human cells. Although this method does not remove the need for transgenes, alternative methods to transfect cells to achieve induced pluripotency, coupled with new advances in transgene free methods (perhaps for example with the use of microRNAs), will avoid potential complications associ

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