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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

The Rho Kinase Pathway Regulates Mouse Adult Neural Precursor Cell Migration

Article Focus for this Month’s Edition of Stem Cells

Paper commentary by Carla B. Mellough

The subventricular zone (SVZ) is a multicellular structure that lines the lateral walls of the lateral ventricles of the brain. SVZ ependymal cells face the ventricular lumen and are involved in the production and circulation of cerebrospinal fluid. Further, the SVZ is an established site of adult neurogenesis, boasting the largest population of proliferative cells in the brain of mature rodents, monkeys and humans. The neural precursor cells (NPCs) of the SVZ produce neuroblasts which migrate to the olfactory bulb via the rostral migratory stream (RMS). These ordinarily act to replenish olfactory neurons, however following central nervous system (CNS) damage they become capable of migrating towards ectopic sites of injury. The mobilisation and guidance of NPCs towards a distinct neural destination involves numerous external signals which must be integrated and translated by neuroblasts to produce an appropriate response, allowing specific and directed migration. The Rho-GTPase family of molecules and their related regulatory members such as the Rac and PIk3 proteins have previously been demonstrated to influence cell migration by regulating the translation of external signals into cytoskeletal reorganisation, yet their role in the migration of neuroblasts through the adult RMS had not yet been established. In the February edition of Stem Cells, new results by Leong et al. from Ann Turnley’s laboratory at the Centre for Neuroscience at The University of Melbourne, begin to reveal the role of the Rho-GTPase pathway in the migration of adult mouse SVZ NPCs.

The Rho Kinase Pathway Regulates Mouse Adult Neural Precursor Cell Migration

Article Focus for this Month’s Edition of Stem Cells

Paper commentary by Carla B. Mellough

The subventricular zone (SVZ) is a multicellular structure that lines the lateral walls of the lateral ventricles of the brain. SVZ ependymal cells face the ventricular lumen and are involved in the production and circulation of cerebrospinal fluid. Further, the SVZ is an established site of adult neurogenesis, boasting the largest population of proliferative cells in the brain of mature rodents, monkeys and humans. The neural precursor cells (NPCs) of the SVZ produce neuroblasts which migrate to the olfactory bulb via the rostral migratory stream (RMS). These ordinarily act to replenish olfactory neurons, however following central nervous system (CNS) damage they become capable of migrating towards ectopic sites of injury. The mobilisation and guidance of NPCs towards a distinct neural destination involves numerous external signals which must be integrated and translated by neuroblasts to produce an appropriate response, allowing specific and directed migration. The Rho-GTPase family of molecules and their related regulatory members such as the Rac and PIk3 proteins have previously been demonstrated to influence cell migration by regulating the translation of external signals into cytoskeletal reorganisation, yet their role in the migration of neuroblasts through the adult RMS had not yet been established. In the February edition of Stem Cells, new results by Leong et al. from Ann Turnley’s laboratory at the Centre for Neuroscience at The University of Melbourne, begin to reveal the role of the Rho-GTPase pathway in the migration of adult mouse SVZ NPCs.

PRC2 Complexes with JARID2, and esPRC2p48 in ES Cells to Modulate ES Cell Pluripotency and Somatic Cell Reprogramming

From the February 2011 Issue of Stem Cells

Paper commentary by Stuart Atkinson

Recently, the Polycomb repressor complex PRC2 and its known constituents (notably Jarid2 and Pcl2 (or Mtf2)) have received an appraisal of their roles in the pluripotent nature and differentiation of embryonic stem cells (ESC) (Reviewed in Margueron and Reinberg). PRC2 is known to have methyltransferase activity targeted towards lysine 27 of histone H3 (K27 H3) catalysed by Ezh2 which initiates epigenetic silencing of genes, maintained by the subsequent binding of the methylated K27 H3 by the CBX subunits in the PRC1 multi-subunit complex. This mechanism of regulation is vitally important in the silencing of non-specific lineage associated gene expression in development, and as demonstrated recently, in ESCs. A new study (Zhang et al.) from the laboratories of Tim M. Townes and Hengbin Wang at the Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham in the February edition of Stem Cells, reports similar findings to previous studies (see further reading below) by identifying Jarid2 and Mtf2 (Pcl2) as members of the PRC2 complex in mouse ESCs (mESCs). However they also go on to describe a novel mESC specific subunit, esPRC2p48 (Hypothetical protein E130012A19Rik) which is demonstrated to be as critical to proper PRC2 function as the other subunits.

PRC2 Complexes with JARID2, and esPRC2p48 in ES Cells to Modulate ES Cell Pluripotency and Somatic Cell Reprogramming

From the February 2011 Issue of Stem Cells

Paper commentary by Stuart Atkinson

Recently, the Polycomb repressor complex PRC2 and its known constituents (notably Jarid2 and Pcl2 (or Mtf2)) have received an appraisal of their roles in the pluripotent nature and differentiation of embryonic stem cells (ESC) (Reviewed in Margueron and Reinberg). PRC2 is known to have methyltransferase activity targeted towards lysine 27 of histone H3 (K27 H3) catalysed by Ezh2 which initiates epigenetic silencing of genes, maintained by the subsequent binding of the methylated K27 H3 by the CBX subunits in the PRC1 multi-subunit complex. This mechanism of regulation is vitally important in the silencing of non-specific lineage associated gene expression in development, and as demonstrated recently, in ESCs. A new study (Zhang et al.) from the laboratories of Tim M. Townes and Hengbin Wang at the Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham in the February edition of Stem Cells, reports similar findings to previous studies (see further reading below) by identifying Jarid2 and Mtf2 (Pcl2) as members of the PRC2 complex in mouse ESCs (mESCs). However they also go on to describe a novel mESC specific subunit, esPRC2p48 (Hypothetical protein E130012A19Rik) which is demonstrated to be as critical to proper PRC2 function as the other subunits.

Osteoblasts Derived from Induced Pluripotent Stem Cells form Calcified Structures in Scaffolds both in vitro and in vitro

From the February 2011 Issue of Stem Cells

Paper commentary by Stuart Atkinson

The correct and efficient differentiation of pluripotent cell types to clinically relevant cell types is a major common goal in stem cell biology. Recent advances in induced pluripotent stem cell (iPSC) technologies have put the prize of patient-specific stem cells for autologous cellular therapy firmly within biomedical sciences’ grasp. The regeneration of the musculoskeletal system is one such system in which iPSC technology could have a great impact. Current strategies involve bone autografts and autologous transplantation of mesenchymal stem cells (MSCs) from the bone marrow, but both have severe limitations. Bone autografts are invasive and have a high morbidity rate (Arrington et al.) and while autologous MSCs exhibit great potential for musculoskeletal regeneration, their proliferative potential decreases greatly with age, perhaps limiting this type of therapy to younger patients (Stenderup et al.). The potential of iPSCs to be differentiated to MSCs has been demonstrated previously (Lian et al.), and indeed these iPSC-derived MSCs were shown to contribute to tissue regeneration more than bone marrow-derived MSCs in a rodent model of hind limb ischemia. Moreover, the MSCs generated in this particular study were able to proliferate for 120 population doublings with no observable karyotypic abnormalities, overall making iPSC-derived MSCs a very attractive proposition. Now,Bilousova et al. from the laboratory of Susan M. Majka at the University of Colorado, Denver in the forthcoming edition of Stem Cells extend these previous studies to further evaluate the potential of iPSCs for use in the regeneration of the musculoskeletal system.

Osteoblasts Derived from Induced Pluripotent Stem Cells form Calcified Structures in Scaffolds both in vitro and in vitro

From the February 2011 Issue of Stem Cells

Paper commentary by Stuart Atkinson

The correct and efficient differentiation of pluripotent cell types to clinically relevant cell types is a major common goal in stem cell biology. Recent advances in induced pluripotent stem cell (iPSC) technologies have put the prize of patient-specific stem cells for autologous cellular therapy firmly within biomedical sciences’ grasp. The regeneration of the musculoskeletal system is one such system in which iPSC technology could have a great impact. Current strategies involve bone autografts and autologous transplantation of mesenchymal stem cells (MSCs) from the bone marrow, but both have severe limitations. Bone autografts are invasive and have a high morbidity rate (Arrington et al.) and while autologous MSCs exhibit great potential for musculoskeletal regeneration, their proliferative potential decreases greatly with age, perhaps limiting this type of therapy to younger patients (Stenderup et al.). The potential of iPSCs to be differentiated to MSCs has been demonstrated previously (Lian et al.), and indeed these iPSC-derived MSCs were shown to contribute to tissue regeneration more than bone marrow-derived MSCs in a rodent model of hind limb ischemia. Moreover, the MSCs generated in this particular study were able to proliferate for 120 population doublings with no observable karyotypic abnormalities, overall making iPSC-derived MSCs a very attractive proposition. Now,Bilousova et al. from the laboratory of Susan M. Majka at the University of Colorado, Denver in the forthcoming edition of Stem Cells extend these previous studies to further evaluate the potential of iPSCs for use in the regeneration of the musculoskeletal system.

2010 Winner - Cinzia Rota

Law, Ethics, Religion, and Clinical Translation in the 21st Century  – A Conversation with Cinzia Rota

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Another Blow to the iPSC Field?

From Nature

A recent report in Nature (Lister et al.) has suggested that human induced pluripotent stem cells (iPSC), the great hope for personalised cellular therapy, are not as similar to embryonic stem cells (ESCs) as hoped, and that this may affect their use as a replacement for ESC in disease modelling and cellular therapy.

Centromeric and telomeric regions in iPSCs were found to maintain a DNA methylation state similar to that of their cell of origin, and were further linked to changes in methylation of histone H3 and transcriptional activity. Some regions of difference were shared between the multiple iPSC lines studied, suggesting that certain loci may be “inherently susceptible to aberrant methylation” while other regions of difference were iPSC specific, also suggesting “a stochastic element to reprogramming”. It was also observed that these differences are transmitted through differentiation of iPSC towards a trophoblastic lineage.

Another Blow to the iPSC Field?

From Nature

A recent report in Nature (Lister et al.) has suggested that human induced pluripotent stem cells (iPSC), the great hope for personalised cellular therapy, are not as similar to embryonic stem cells (ESCs) as hoped, and that this may affect their use as a replacement for ESC in disease modelling and cellular therapy.

Centromeric and telomeric regions in iPSCs were found to maintain a DNA methylation state similar to that of their cell of origin, and were further linked to changes in methylation of histone H3 and transcriptional activity. Some regions of difference were shared between the multiple iPSC lines studied, suggesting that certain loci may be “inherently susceptible to aberrant methylation” while other regions of difference were iPSC specific, also suggesting “a stochastic element to reprogramming”. It was also observed that these differences are transmitted through differentiation of iPSC towards a trophoblastic lineage.

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