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Pluripotent Stem Cells

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.

Forthcoming Article in Stem Cells - Human Embryonic Stem Cells Suffer from Centrosomal Amplification

By Stuart P. Atkinson

Current protocols for the differentiation of embryonic stem cells (ESC) to clinically relevant cell types are woefully inefficient with many millions or tens of millions of ESC used for each differentiation, only to yield small proportions of the desired cell type. This entails large scale culture and amplification of ESC, often over a large period of time. Unfortunately, long term culture of ESCs is also known to lead to karyotypic instability perhaps rendering these cell types at best less functional and at worst potentially tumorigenic. Therefore, a deeper understanding of the processes which lead to karyotypic instability in cultured ESCs is of major interest, and indeed could allow for formulation of strategies to mediate the repression or inhibition of this instability. One potential mechanism fueling chromosome karyotypic instability is the amplification of centrosomes which can perturb chromosome segregation in mitosis. In the upcoming edition of Stem Cells, researchers from the lab of Aleš Hampl at the Masaryk University, Brno, undertake an analysis of centrosome amplification in hESC. In this work they uncover a link between centrosome amplification and karyotypic instability and suggest the implementation of new culture techniques to inhibit such instability (Holubcová et al).

Phosphorylation of Sox2 Cooperates in Reprogramming to Pluripotent Stem Cells

From the December Issue of Stem Cells

By Stuart P Atkinson

Modifications of histone proteins have been well studied in ESCs and iPSCs and we are beginning to understand the importance that these modifications have in relation to chromatin structure and gene regulation and indeed the enzymes which mediate these modifications. However, research into modifications of non-histone proteins in pluripotent cells has perhaps lagged behind. Studies in 2009 Saxe et al, Swaney et al and Van Hoof et al) have provided an excellent insight into the global picture of protein phosphorylation in ECs and ESCs, which included the modification of both OCT4 and SOX2, and recently Nanog has been shown to be regulated by phosphorylation Moretto-Zita et al). Now, in the December issue of Stem Cells, the lab of Zigang Dong from The Hormel Institute at the University of Minnesota provides data from an in depth analysis of the specific role of Sox2 phosphorylation in mouse ESC Jeong et al), supporting a potential role in pluripotency and reprogramming.

Reprogramming Somatic Cells – Down to One Gene!

From Cell Stem Cell

A new study from the lab of Sheng Ding (The Scripps Research Institute), published recently in Cell Stem Cell, reports the reprogramming of human primary somatic cells using only OCT4 gene transduction and a combination of small molecules. Kim et alhad previously shown than one factor reprogramming is possible, but in human foetal neural stem cells, a cell source perhaps not readily available. iPSCs were generated at an efficiency of roughly 1 colony in 250,000 target cells over an 8 week period utilising neonatal human epidermal keratinocytes (NHEKs). The group also went on to generate iPSCs from human umbilical vein endothelial cells (HUVECs) and amniotic fluid-derived cells (AFDCs). Through a screening process, a novel small molecule activator of 3-phosphoinositide-dependent kinase-1 (PDK1), PS48, was also discovered to be useful in the reprogramming process. It was hypothesised that this small molecule may facilitate a metabolic conversion from mitochondrial oxidation to glycolysis during the reprogramming process, or in other words, metabolically reprogram the cells. The authors note that “differential use of glycolytic metabolism over mitochondrial oxidation by pluripotent cells would favour pluripotency by promoting proliferation/cell cycle transitions with less oxidative stress.”

iPSCs Provide HIV “Killer” Cells

by Stuart P. Atkinson

From the lab of Dan Kaufman at the Department of Medicine and Stem Cell Institute, University of Minnesota comes a report on the potential use of patient specific human induced pluripotent stem cells (hiPSCs) in the battle against HIV-1 infection. Kaufman’s lab have previously shown that embryonic stem cell (ESC) derived Natural Killer (NK) cells, a key component of the innate immune system, can potentially kill tumour cells in vivo and dramatically reduce tumour size in vitro Woll et aland Woll et al). Several studies have also shown that NK cells can provide protection against HIV-1 replication in which sufferers show decreased NK cell function. In their most recent study published in the Journal of Virology, Kaufman’s lab now demonstrate that NK cells derived from human ESC (hESC) and also hiPSCs have potent anti-HIV-1 activity Ni et al).

“linc”ing Non-Coding RNAs with Pluripotency and Reprogramming

By Stuart P. Atkinson

Little by little, the processes behind cellular reprogramming of lineage-committed cells to induced pluripotent stem cells (iPSCs) are being discovered and more detailed comparative studies between iPSC and embryonic stem cells (ESCs) are being undertaken. The similarity of these two cell types is generally viewed as being essential if hiPSCs are to be taken towards the clinic and while this has led to detailed analyses of many cellular attributes, such as mRNA expression, miRNA expression and chromatin structure, other “avenues” remain relatively unstudied.

Memory in Induced Pluripotent Stem Cells: Reprogrammed Human Retinal Pigmented Epithelial Cells Show Tendency for Spontaneous Redifferentiation

From this month’s edition of Stem Cells

By Carla Mellough

Elucidation of the best methods for somatic cell reprogramming which fully harness the stem cell-like capacity of induced pluripotent stem cells (iPSCs) is key prior to the utilisation of iPSC for regenerative medicine purposes. The translation of iPSC therapy from the bench to the clinic has so far been slowed by limitations in the efficiency and safety of reprogramming protocols, alongside recent reports which indicate that although reprogrammed cells exhibit numerous properties of human embryonic stem cells (hESC), not all iPSC are fully reprogrammed and significant differences still remain, with many studies now linking the profile of iPSC to their cell of origin. Adding to this body of evidence, results published in the November issue of Stem Cells by Hu et al. From the University of California Santa Barbara, assess the retention of epigenetic imprints in iPSC generated from retinal pigmented epithelial (RPE) cells and whether these iPSC have a propensity to redifferentiate back into RPE cells – their cell of origin.

Reprogramming the methods that induce pluripotency

By Carla Mellough

The clinical applicability of induced pluripotent stem cells (iPSC) has been limited by the inefficiency of reprogramming methods, modification of the genome using most reprogramming protocols, and the lack of failsafe differentiation protocols to then generate functional differentiated phenotypes from iPSCs to provide patient-specific cell types for autologous transplantation. Various non-integrating and thus safer methods of reprogramming have been achieved but these too come with their own limitations. For example, repeat administration of transient vectors demonstrate low reprogramming efficiency, it is challenging to generate and purify the quantities of recombinant proteins required in protein based strategies and methods based on DNA transfection come with the risk of genetic recombination or insertional mutagenesis. To this end, a recent paper in Cell Stem Cell from the laboratory of Derrick Rossi at Harvard Medical School describes how they achieve reprogramming of multiple human somatic cell types using a simple non-integrating method which achieves highly efficient reprogramming to levels much higher than what has previously been achieved.

Forthcoming Stem Cells Publication – Novel Hyperactive Transposons for Genetic Modification of Induced Pluripotent and Adult Stem Cells: A Non-viral Paradigm for Coaxed Differentiation

By Stuart P Atkinson

Non-viral genetic modification of cells is a relatively inefficient process, be it modifying adult cells or in the generation of induced pluripotent stems cells (iPSC). It has been suggested that transposon-based methods of gene transfer may overcome this limitation, but this technique has been plagued by inefficiency problems. However recent work has utilised in vitro molecular evolution to generate a transposon system derived from the Sleeping Beauty (SB) transposase which shows vastly enhanced transposition capabilities, allowing a significant enhancement of stable gene transfer efficiencies.

Make sure you take your vitamin C!

We all know that vitamin C is important for keeping us healthy through its many activities including its capacity as an antioxidant and its involvement as an enzyme co-factor in collagen synthesis. Indeed this second property is the basis of the most obvious consequence of vitamin c deficiency that was the demise of many an eighteenth century mariner known as Scurvy which messes up the integrity of mucous membranes because of the lack of normal collagen. The problem is easily solved by administering citrus fruit juices, a practice initiated by the British Royal Navy in 1795. As with sailors, so it is with cells. Ascorbic acid is now an important medium supplement used in serum free embryonic stem cell culture which moreover has the ability to enhance the generation of induced pluripotent stem cells. This interesting property was discovered by the research group of Duanqing Pei in Guangzhou, China but their approach to the utility of ascorbate was a little different to that of Chung etal in their article published in this month’s edition of Stem Cells. Pei and co-authors inferred that the factors that induce cells to become pluripotent were causing the cells to make the free radicals known as reactive oxygen species. Now, pluripotent cells have effective mechanisms for getting rid of these particularly nasty by-products of oxygen metabolism, but the fibroblasts used for iPSC generation do not, so Pei’s tem added vitamin C during the process in the hope that its antioxidant properties would promote survival of greater numbers of cells undergoing reprogramming. This seemed to be a success but the data described in Chung’s manuscript suggest another mechanism to account for ascorbic acid’s ability to improve iPSC generation.

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