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Another Step in Reprogramming Uncovered

"Gata4 Blocks Somatic Cell Reprogramming By Directly Repressing Nanog"

Slowly but surely, the processes which take place during induced pluripotent stem cell (iPSC) formation are being uncovered, allowing a deeper understanding of the process while allowing researchers to streamline techniques with which to make fully reprogrammed pluripotent cells. Gata4 is a transcription factor known to be essential for definitive endoderm formation and, within the inner cell mass, Gata4 and the pluripotency factor Nanog, exhibit a "salt and pepper" distribution of Nanog-positive and Gata-positive cells and these are not co-expressed in any cell throughout the E3.5 blastocyst (Chazaud et al), suggestive of reciprocal inhibition. Now researchers from the laboratory of Roque Bort at the Instituto de Investigación Sanitaria La Fe, Valencia, Spain have investigated the role of Gata4 in reprogramming. In their study published in Stem Cells, they report the finding that Gata4 expression inhibits the reprogramming process by inhibiting the transcription of Nanog by its interaction with a distal enhancer element (Serrano et al).

iPSCs Get the OK from The Immune System

"Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells"

The main advantage of induced pluripotent stem cells (iPSCs) over embryonic stem cells (ESCs) is their ability to provide a patient-specific source of cells for tissue replacement. However, it was feared that incomplete reprogramming, any mutations caused during the reprogramming process (Polo et al and Gore et al) or simply the reprogramming process itself, which remains relatively unknown, could cause iPSCs, and potentially their progeny, to cause an unwanted immune response. This fear was brought sharply into focus in 2011 with the finding that iPSC-, but not ESC-derived tumours, elicited a significant immune response (Zhao et al). However, controversy around these findings (Okita et al) led researchers from the group of Masumi Abe at the National Institute of Radiological Sciences, Chiba, Japan to re-examine this potential threat by focusing their study on differentiated cell types from mouse iPSCs and mouse ESCs, a more clinically relevant experiment. Encouragingly, they found no difference in the immunological response from pluripotent cells from either source and, moreover, they observed limited or no immune responses to the transplanted differentiated cells (Araki et al).

Can Peripheral Blood Provide an Optimal Source for iPSC Generation?

"A Practical and Efficient Cellular Substrate for the Generation of Induced Pluripotent Stem Cells from Adults: Blood-Derived Endothelial Progenitor Cells"

An ideal source of cells for the generation of human induced pluripotent stem cells (iPSCs) has been investigated thoroughly since the inception of this technology, but to this day no cell type has been deemed optimal over others; indeed fibroblasts are still often the cell of choice in reprogramming techniques, even though the reprogramming efficiency tends to be very low (Takahashi and Yamanaka and Takahashi et al.). However, the optimal parameters have been delineated; cells must be clearly defined, easily and reproducibly available from all patients from all disease states, expandable, storable, stable and capable of allowing for high efficiency reprogramming. Stem cells from the blood have been suggested as a potentially exciting alternative, although the availability of stem cells such as HSCs, which are perhaps more amenable to reprogramming, is limiting. This led to researchers from the groups of Amer Ahmed Rana, Ludovic Vallier and Nicholas W. Morrell from University of Cambridge, United Kingdom to investigate other cellular sources derived from peripheral blood. They have discovered that late-outgrowth endothelial progenitor cells (L-EPCs; also known as blood-outgrowth endothelial cells), which arise from the mononuclear cell fraction of peripheral blood (PBMNC) under endothelial-selective conditions (Lin et al. and Medina et al.), are a potentially attractive source of cells for generating iPSCs (Getia, Ormistonb and Rouhania et al.).

iPSCs – Differentiation and Purification aids Cartilage Production

"Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells"

Cartilage tissue engineering hopes to provide replacement tissues, but many problems have arisen during progress in this area. A good source of cells is lacking; as autologous chondrocytes, although useful, require surgical procedures which can lead to problems at the donor site (Lee et al.), while adult stem cell sources for chondrogenesis, such as mesenchymal stem cells (MSCs) or adipose-derived stem cells (ASCs) are lacking in number (Pittenger et al.), chondrogenic potential (Diekman et al.), or proliferative/differentiation potential due to age of donor (Dexheimer et al.) or disease (Murphy et al.). Another possible source of cells for cartilage replacement is patient specific induced pluripotent stem cells (iPSC s); with perhaps the lack of a sufficiently efficient differentiation protocol being the only barrier to their use (Yoshida and Yamanaka). This led Farshid Guilak and researchers from Duke University, Durham, USA addressed this problem in mouse iPSCs through the pre-differentiation of iPSCs to the chondrogenic lineage, purification, followed by creation of tissue-engineered cartilage constructs (Diekman et al.).

UPS and downs of Pluripotency Control

"Regulation of Pluripotency and Cellular Reprogramming by the Ubiquitin-Proteasome System"

The regulation of pluripotency has been assessed on many different levels; transcription factor networks, epigenetic regulation and miRNA analysis. However, the role of posttranslational modifications in embryonic stem cell (ESC) pluripotency and differentiation has not been fully assessed although studies have suggested a link transcription-independent regulation of proteins and ESC fate (>Lu et al.). Additionally, previous studies have shown strong links between proteasome function and the regulation of gene expression and cell cycle in ESCs (Szutorisz et al. and Atkinson and Colin et al.). With this knowledge, researchers from the group of Iannis Aifantis at the Howard Hughes Medical Institute and Department of Pathology, New York University School of Medicine, USA to study the role of the ubiquitin-proteasome system (UPS) in protein turnover in ESCs. Through shotgun proteomics, the ubiquitinated protein landscape during of ESC and induced pluripotent stem cells and during ESC differentiation has been uncovered. Additionally the deubiquitinating enzyme Psmd14 and the E3 ligase Fbxw7 have been identified as important to ESC pluripotency and cellular reprogramming (Buckley, Aranda-Orgilles, Strikoudis et al.).

Moving Reproductive Medicine from Monkey to Human

"Towards germline gene therapy of inherited mitochondrial diseases"

Human disease caused by mutations in mitochondrial DNA (mtDNA) is thought to affect between 1000 and 4000 children born in the US every year (Haas et al. and Schaefer et al.) although some estimates have this at a much higher level (Elliott et al.). While there are no cures for mitochondrial disorders and treatments aim to alleviate symptoms and delay the progression of the given disease, studies have begun try to address this problem. Pronuclear transfer between abnormally fertilized human zygotes results in minimal carry-over of mtDNA and leads to blastocyst stage formation in vitro (Craven et al). Meanwhile, studies in monkeys (Tachibana et al. 2009) have suggested that the removal of mtDNA from a patient's oocytes and the replacement with healthy mitochondria from another donor through spindle transfer (ST; also called spindle–chromosomal complex transfer) (Tachibana et al. 2010) is feasible, highly effective and can allow for the birth of healthy offspring. In a follow up study from their previous work in monkeys, researchers from the laboratory of Shoukhrat Mitalipov at the Oregon Health & Science University, USA have now studied this concept in healthy donated human oocytes (Tachibana et al. 2011). They have been able to show the successful fertilisation of ST oocytes, blastocyst formation and the derivation of embryonic stem cells with normal euploid karyotypes which contained exclusively donor mtDNA.

Functional Oocyte formation from Pluripotent Cells

Recent studies have begun to describe means of directing the differentiation of male and female germ cells from embryonic stem cells (ESCs) towards sperm and oocytes. The group of Mitinori Saitou at Kyoto University, Japan have previously described the production of primordial germ cell–like cells (PGCLCs) from male mouse ESCs and induced pluripotent stem cells (iPSCs) with which allow for spermatogenesis and offspring production (Hayashi et al, 2011), while another group has shown the potential for sperm production from human pluripotent stem cells (Easley et al). Meanwhile, the existence of adult female germline progenitors has been postulated (White et al, Zou et al 2009) but remains controversial (Zhang H, Zheng W, et al) and the production of functional oocytes from PGCLCs from female pluripotent stem cells has not yet been described. Herein, Hayashi et al, 2012 describe a culture system to develop PGCLCs into oocytes which after maturation can contribute to fertile offspring.

Functional 3D Tissue Formation from ESCs

While the overexpression of specific transcription factors in embryonic stem cells (ESCs) can allowed the production of specific cell types such as haematopoietic stem/progenitor cells, adipocytes and neural progenitors, our ability to form functional artificial organs from these cells is still limited (Eiraku et al, Eiraku and Sasai, and Suga et al). Now, in a study published in Nature, researchers from the group of Sabine Costagliola at the Institute of Interdisciplinary Research in Molecular Human Biology (IRIBHM), Université Libre de Bruxelles, Belgium have shown that transient overexpression of the transcription factors Nkx2-1 and Pax8 in mouse ESCs directs them to differentiate into thyroid follicular cells (TFCs) that can organize into three-dimensional (3D) follicular structures and mimic thyroid function in vivo after grafting into athyroid mice (Antonica et al).

Understanding Heterogeneity in Stem Cells

Original article from STEM CELLS

“Cellular Heterogeneity During Embryonic Stem Cell Differentiation to Epiblast Stem Cells Is Revealed by the ShcD/RaLP Adaptor Protein”

The Shc (Src homolog and collagen homolog) family of adaptor proteins is a crucial linker of a wide range of receptors to their downstream effectors (Pelicci et al). Four Shc proteins (ShcA isoforms p52 and p46, p66ShcA and ShcC) had previously been identified in mammals and implicated in distinct signaling pathways, but now a 4th family member and 5th protein, ShcD/RaLP, has been identified. Studies have found ShcD to be expressed in the neuromuscular junction, mediating muscle-specific kinase signaling (Jones et al), in melanocytes, implicated in mitogen-activated protein kinase (MAPK) signalling (Fagiani et al), and in the brain (Jones et al), and has been reported to be involved in the metastatic progression of melanoma. Now, in a report in Stem Cells, researchers from the laboratory of Luisa Lanfrancone at the Department of Experimental Oncology, European Institute of Oncology, Milan, Italy have found ShcD to be an important modulator in the switch of key pathways involved in determining EpiSC identity; ShcD is transiently upregulated during early differentiation of ESCs, corresponding to the ESC to epiblast stem cells (EpiSCs) transition, while loss of ShcD perturbs the commitment process (Turco et al). Additionally, loss of ShcD in ESCs increased the heterogeneity of cells during both differentiation and EpiSC determination as measured through the loss of Oct4 and gain of Cdx2 in distinct subpopulations of cells.

Inducing Apoptosis to Increase Safety of Pluripotent Cell Derivatives

Original article from STEM CELLS Translational Medicine

“Apoptotic Susceptibility to DNA Damage of Pluripotent Stem Cells Facilitates Pharmacologic Purging of Teratoma Risk”

The potential for dysregulated cell growth and teratoma formation after the transplantation of embryonic stem cell (ESC)- or induced pluripotent stem cell (iPSC)-derived cells is an important consideration for their therapeutic translation. One potential solution to this problem is the separation or deletion of pluripotent cells from differentiated cell-cultures using techniques which leave the required differentiated cells unscathed and functional (Tang et al), such as the use of suicide genes (Goldring et al and Cheng et al) or apoptotic induction (Knoepfler and Bieberich et al). One proposed mechanism which can be targeted pharmacologically in ESCs/iPSCs is their propensity to rapidly and efficiently undergo apoptosis in response to DNA-damage in order to prevent accumulated mutational load (see paper for extended references), rather than utilising the cell cycle arrest and repair mechanism observed in differentiated cells. With this in mind, researchers from the laboratory of Timothy J. Nelson at the Mayo Clinic, Minnesota, USA, aimed to construct a strategy of selectively ridding cultures of pluripotent stem cells in response to DNA damage-inducing agents (Smith et al), and have now demonstrated in a report in Stem Cells Translational Medicine that etoposide treated cultures become depleted of pluripotent and potentially teratogenic cells while leaving differentiated progenitors unharmed and functional.

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