Pluripotent Stem Cells

From the April 2011 Issue of Stem Cells

Paper Commentary by Stuart P. Atkinson

The gastrula organizer, a structure first described in the amphibian by Spemann and Mangold in 1923, contains a population of organizer cells which signal the allocation of anterior versus posterior structures and axis formation in the developing vertebrate embryo. It is known that the TGFb and WNT pathways are involved in the induction of amphibian organizer cells (Crease et al) which are characterised by the presence of the Goosecoid gene. However, due to obvious ethical concerns, intimate studies of organizer cells in human have not yet been established. Now, for the first time, a study from the laboratory of Nissim Benvenisty at The Hebrew University of Jerusalem, published in the April edition of Stem Cells, demonstrates that differentiating human embryonic stem cells (hESC) can be used to recapitulate the characteristics and functions of gastrula organizer cells (Sharon et al.).

Review by Stuart P. Atkinson

Recent studies in embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) biology have turned from comparative studies at the RNA and chromatin level to focus on what’s happening at the DNA level (See iPSC don´t Forget their Origins and Another Blow to the iPSC Field?). Genomic integrity is of course vital for the future use of such pluripotent cells in the clinic; changes at the genomic level can, at best, lead to failure of transplanted cell function, and in the worst case potentially lead to tumorigenesis. This review hopes to condense some of the recent high impact papers which have studied genomic alteration in iPSC in great detail.

From the March 2011 Issue of Stem Cells

Paper Commentary by Stuart P. Atkinson

Induced pluripotent stem cells (iPSC) have the potential to provide a patient specific source of cells that can be used in cellular therapy. However doubts about their similarity to embryonic stem cells (ESCs) have arisen, both at the gene expression and epigenetic level, and also with their initial differentiation capabilities and the capacity of the differentiated cells to function properly. So what other potential sources do we have? Currently derived human ESC lines (hESC) may not have the genetic diversity required, whilst derivation of additional hESC lines has associated ethical (and funding) problems. Meanwhile, hESC-derivation from somatic cell nuclear transfer (SCNT) has yet to yield results and does not look likely to in the short term. For many, iPSCs still represent the future of stem cell research and stem cell derivation. However, another source of pluripotent cells which are often overlooked may be available, in the form of parthenogenetic stem cells. Parthenogenetic ESCs are derived from activated oocytes at the metaphase II stage and could provide a patient specific source of ESCs for the donor female, and perhaps relatives of the said donor (Hikichi et al. and Kim et al.). However donated oocytes at this stage of development are generally used in assisted reproduction and are therefore of scarce availability, a problem also associated with SCNT. However, Josef Fulka Jr’s group at the Institute of Animal Science, Pratelstvi, Prague have now demonstrated a new method of creating parthenogenetic ESCs from metaphase I oocytes, which are often discarded during the course of IVF, by using Butyrolactone I (BL1). This study (Fulka et al.) is presented in the March Edition of 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.

From the March 2011 Issue of Stem Cells

Paper Commentary by Stuart P. Atkinson

The RNA binding protein LIN28 (or LIN28A) is highly expressed in human embryonic stem cells (hESCs) and is often used in the generation of induced pluripotent stem cells (iPSCs) (Yu et al.). It is known to play a role in inhibiting the maturation and promoting the degradation of the Let7 family of microRNAs which are known to promote the expression of genes involved in differentiation. However multiple Let7 independent roles for LIN28 have also been observed, and have prompted a study from the laboratory of Yingqun Huang at the Yale Stem Cell Center presented in the March edition of Stem Cells.

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.

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.

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

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.

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.