Pluripotent Stem Cells

From PNAS
By Stuart P. Atkinson

Mouse embryonic stem cells (mESCs) have a remarkably short G1 and G2 phase, which in somatic cells is determined by Cdk activity alongside other cell-cycle related proteins which fluctuate during the cell cycle due to APC/C mediated degradation. APC/C is a large multi-subunit E3 ubiquitin ligase, which can be activated by Cdc20 and Cdh1 interaction at the end of mitosis and inactivated by Emi1 (or Fbxo5) and degradation of Cdh1 just before S-phase. Inhibition of Cdk activity in G1 phase allows the replication factors Cdt1 and Cdc6 to recruit Mcm proteins onto chromatin, form pre-replicative complexes (pre-RCs) and license DNA for replication. Previous studies found that in mESCs APC/C substrates were constant and Cdk activity high throughout the mESC cell cycle (White et al, Fujii-Yamamoto et al and Yang et al). However, the careful re-appraisal of protein levels and activity in mESC by researchers from the laboratory of Marc W. Kirschner have uncovered oscillations in APC/C substrate levels and Cdk activity which, alongside other key findings, promote the abbreviated cell cycle of mESCs (Ballabeni et al).

From Genes and Development
By Stuart P. Atkinson

Although induced stem cell technology promises a source of patient-specific cells for cell replacement in diseased or dysfunctional tissues, multiple studies have shown that activation of cellular senescence pathways is a barrier to the reprogramming process and some studies have shown that cells differentiated from iPSCs show reduced functionality and early senescence (Feng et al and Liu et al). This suggests that currently utilised iPSC-derivation protocols may not achieve complete reprogramming of somatic cells to produce stem cells which share characteristics of youthful embryonic stem cells (ESCs). Further, this may have additional implications regarding the age of the cell-donor. Now, a study from the group ofJean-Marc Lemaitre at the Institute of Functional Genomics, Montpellier, France, published in Genes and Development has shown the ability of an optimized 6-factor reprogramming protocol to efficiently reverse these aging characteristics and allowing the derivation of fully pluripotent iPSCs with key characteristics of young cells (Lapasset et al).

From Stem Cells
By Stuart P. Atkinson

Current knowledge of the mechanisms of neural induction in mammals is not clear, but data from other model systems has allowed some details to be uncovered. Examinations on mammalian neural induction are mostly performed using in vitro models of neuroepithelial differentiation from embryonic stem cells (ESCs) and confirm the requirement of BMP inhibition and FGF activation in neural differentiation of mouse ESCs. Research from the group whose research is presented herein has found that phospho-SMAD1 was not altered in human ESCs undergoing neural differentiation in the presence of FGF signaling inhibitors, suggesting that FGFs regulate hESC neural differentiation independent of BMP/SMAD signaling (LaVaute et al). This led the group of Su-Chun Zhang, University of Wisconsin, Wisconsin ,USA to study in detail how FGF signalling regulates neuroectoderm specification of hESCs (Yoo et al) using a previously described efficient differentiation protocol for neuroepithelial  (Pankratz et al).

From Stem Cells
By Stuart P. Atkinson

In mouse, Pten plays a negative role in the self-renewal of embryonic and adult cell types (Stiles et al, Korkaya et al and Groszer et al) through negative regulation of PI3K/Akt signalling and while studies have shown the importance of PI3K/Akt signalling in embryonic stem cells (ESCs) (Ding et al and Storm et al), the role of PTEN in human ESCs (hESCs) is relatively unknown.

From Stem Cells
By Stuart P. Atkinson

The ability to monitor the homogeneity of pluripotent cell cultures and their differentiated derivatives is potentially useful if such cells are to be made applicable to cellular therapy. Another use for such monitoring systems is in the generation of pure induced pluripotent stem cultures (iPSCs) containing only fully reprogrammed pluripotent cell types. At present, identification of “true” iPSCs relies on monitoring morphological criteria and is time consuming, even for well-trained operators. A potentially effective system to allow the purification of fully pluripotent cells has been posited by the group of Vania Broccoli at the San Raffaele Scientific Institute, Milan, Italy, which takes advantage of the current knowledge of microRNAs (miRNAs) and their role in pluripotency and differentiation. Building on previous work (Brown et al and Kelly et al), the group describe a reporter system based on Let7a and mir-292 miRNA-mediated regulation for selecting and maintaining the pluripotent state of human iPSCs (hiPSCs) (Di Stefano et al).

From Stem Cells
By Stuart P. Atkinson

Induced pluripotent stem cell (iPSC) technology not only has the capability to produce patient-specific but also disease-specific pluripotent cells. In monogenic diseases, there lies the potential for gene modification allowing differentiation of therapeutically relevant “corrected” cells, as has been demonstrated in mouse iPSCs where a sickle cell anemia mutation was targeted and corrected through the use of homologous recombination and an exogenous DNA donor template (Hanna et al). However, moving this technology into human cells is technically difficult due to the reported low efficiency of gene targeting and the requirement for large donor templates. However, the use of engineered zinc finger nucleases (ZFNs), customizable restriction enzymes which can be targeted to a desired DNA element (Kim et al) may allow higher gene targeting efficiency. The groups of Marius Wernig and J. Keith Joung from the Harvard Medical School, Boston, Massachusetts, USA have investigated the ability to efficiently correct a genetic mutation in human iPSCs (Sebastiano et al) and have shown the applicability of ZFN technology to human iPSCs containing a sickle cell anemia mutation, demonstrating a promising means of genetic correction.

From Cell
By Stuart P. Atkinson

Although multiple studies over the last 5-10 years have begun to unravel the complex network which underlies the pluripotent state of embryonic stem cells (ESCs), bringing together transcription factors, small RNA species, chromatin and DNA methylation events, the specific molecular determinants of the pluripotent state are yet to be fully appreciated. However, two recent papers in Cell now reveal two previously unappreciated pluripotency-associated elements: the role of a DNA damage complex in transcriptional control and transcription factor splicing.

From the October Edition of Stem Cells
By Stuart P. Atkinson

A brief report in the October edition of Stem Cells from the laboratory of Steven L. Stice at the University of Georgia, Athens, Georgia, USA describes the ability of pig induced pluripotent stem cells (piPSCs) to be transmitted through the germline and studies the effect of piPSC incorporation on offspring development and tumorigenicity, the first experiment of its kind outside the mouse model (West et al, 2011). Using pig as a model for such studies holds great interest due to the relative similarity of pigs to humans when compared to the more widely used mouse model.

From Nature
By Stuart P. Atkinson

When induced pluripotent stem cell (iPSC) technology broke onto the scene in 2007, the attainment of a practical and ethical source of patient specific pluripotent stem cells seemed to be within our grasp. The use of somatic cell nuclear transfer (SCNT) for the derivation of human embryonic stem cells (hESCs) was all but relegated to the past, along with its ethical and technical difficulties. Further, as noted in an editorial in Nature, previous fraudulent claims in this field had added a degree of unwarranted scepticism to research undertaken in this field. However, iPSC derivation and its detailed analysis has shown that iPSC may have only a limited use in a therapeutic context due to differences between blastocyst-derived stem cells at the level of gene expression, epigenetic patterning, differentiation potential and genomic integrity. It is suggested that these differences may lead to dysfunctionality and/or tumorigenicity and have led some groups to return to SCNT-mediated techniques. The group of Dieter Egli from the The New York Stem Cell Foundation Laboratory, New York initiated a rigorous study designed to understand the obstacles to blastocyst development in SCNT in order that we can use this process to generate bona fide patient-specific hESCs. In their paper, published in Nature (Noggle et al), the group found that transfer of the somatic genome into an enucleated oocyte failed to develop and this was associated with an apparent arrest of developmentally-associated gene expression. They found however, that if the oocyte genome is left in place upon somatic genome transfer, the embryo can develop to the blastocyst stage and hESCs can be derived.

From the July Edition of Stem Cells
By Stuart P. Atkinson

The risk of immune rejection and tumorigenesis of cells derived from pluripotent stem cells are both major concerns for regenerative medicine. The generation of patient specific induced pluripotent stem cells (iPSCs) promises a way round the problem of immunotolerance in cell/tissue replacement therapy, but iPSC could also be useful in the generation of a human leukocyte antigen (HLA)-haplotyped bank of pluripotent stem cell lines which could be used for widespread clinical use. Mauritanian cynomolgus macaques (CMs) exhibit limited major histocompatibility complex (MHC) diversity and may be useful in modelling human diseases, including neurodegeneration and transplantation research in non human primates (NHP). For this reason, the group of Ole Isacson at the Harvard Medical School/McLean Hospital, Massachusetts, USA attempted to produce MHC-matched iPSCs which could be of great value for immunological research and preclinical validation of the use of iPSCs. Their findings are presented in the July edition of Stem Cells (Deleidi et al).