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Pluripotent Stem Cells

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New organs to help us Liver longer?

“Vascularized and functional human liver from an iPSC-derived organ bud transplant”  

The ex vivo manufacture of organs destined for use as transplants has gained much momentum following a recent paper in Nature, in which scientists from multiple centres in Japan have reported the generation of three-dimensional (3D) vascularised and functional human liver tissue from human induced pluripotent stem cells (hiPSCs).   Led by Professor Hideki Taniguchi at Yokohama City University, the team demonstrated that hiPSCs can produce liver (hepatic) cells which can self-organise into 3D cell clusters that, when transplanted into mice, are able to connect with host vessels and integrate within the host system, maturing into cells that are very similar to that of adult human liver tissue in their structure and function. 

Nanog Expression in ESCs Re-evaluated

Nanog is oft the first transcription factor to come to mind when the word pluripotency is mentioned, and several studies have suggested that its expression ranges from high to low in ESCs grown in classic feeder-based systems with serum + LIF (Young) with a fraction of cells also observed to fluctuate from a Nanog low to Nanog high state (Chambers et al).   This metastability was proposed to be a characteristic of the pluripotent state (also observed for Stella (Hayashi et al)and Rex1 (Toyooka et al)), although when ESCs are reverted to their “naïve” ground state under a 2 inhibitor based media formulation (2i) without feeders, pluripotency is maintained even though the ESCs are fully homogenous (Wray et al).   Under 2i conditions, it has been proposed that Nanog is expressed mono-allelically, but bi-allelically under feeder-based growth conditions (Miyanari and Torres-Padilla), and additional research has found that Nanog activity is autorepressive and may regulate allelic switching (Fidalgo et al and Navarro et al), altogether leaving unclear mechanistic insights and functional relevance.   Now two studies have been published in Cell Stem Cell (one from the research groups of Konstantinos Anastassiadis and Timm Schroeder and the other from the group of Rudolf Jaenisch) which combine single cell ESC analysis linked with allele-specific reporter systems to show that Nanog is bi-allelically expressed independently of culture conditions, and its expression is very similar to that of several other homogenously expressed pluripotency markers (Filipczyk et al and Faddah et al).

Chemically Induced iPSCs

The generation of induced pluripotent stem cells (iPSCs) generally relies on the ectopic expression of pluripotency associated transcription factors. However, some pioneering studies have shown that some of these factors can be replaced by small molecule drugs, which are non-immunogenic, cost-effective, and easily synthesized, preserved, and standardised (Zhu et al). Now for the first time, in a study published in Science, researchers from the laboratory of Hongkui Deng have identified a small molecule “cocktail” which can reprogram mouse somatic cells to pluripotency without the need for exogenous gene expression (Hou et al). These chemically induced pluripotent stem cells (CiPSCs) resemble mouse embryonic stem cells (ESCs) and could have great potential for clinical applications if it is possible to recapitulate this for the generation of human iPSCs.

Reprogramming by Attenuating Differentiation

“Induction of Pluripotency in Mouse Somatic Cells with Lineage Specifiers”

OCT4 is generally understood as being a hugely important transcription factor associated with pluripotent stem cells and its expression in somatic cells can allow for the derivation of induced pluripotent stem cells (iPSCs) (Li et al and Zhu et al). Upon screening for factors which may substitute for Oct4, to further the understanding of the reprogramming process and the pluripotent state, the groups of Chao Tang and Hongkui Deng at the Peking University, Beijing, China have somewhat unexpectedly identified eight lineage specifiers as Oct4 substitutes. They show that these function by inhibiting differentiation towards specific lineages, which is usually triggered by other reprogramming factors (SOX2, KLF4, and c-MYC), and in doing so facilitating the induction of pluripotency (Shu, Wu, Wu, Li et al).

Removing the Risk from ESC-derived cells

“Immunologic and chemical targeting of the tight-junction protein Claudin-6 eliminates tumorigenic human pluripotent stem cells”

The problem of potentially tumorigenic cells contained within differentiated cultures of human pluripotent stem cells is one of the major obstacles for the transition of stem cell therapy from the laboratory to the clinic. Cell surface markers have been used to attempt the selective removal of pluripotent cells with some success (Choo et al, Fong et al and Tang et al), and this approach has now been further assessed through expression microarrays by researchers from the laboratory of Nissim Benvenisty at the Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel. This work reports that the tight junction protein Claudin-6 (CLDN-6) is highly specific for pluripotent cells and the use of a cytotoxin-conjugated antibody efficiently kills undifferentiated cells (Ben-David et al).

Muse-ing on an Alternative to iPSCs and ESCs

“Awakened by Cellular Stress: Isolation and Characterization of a Novel Population of Pluripotent Stem Cells Derived from Human Adipose Tissue”

Using adult stem cells for regenerative purposes faces many problems; one of which is low cell survival and engraftment, due in part to the cellular stresses generated during the process. Challenging both haematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) with moderate hypoxic conditions before transplantation has been shown to increase expansion, survival, and self-renewal while maintaining the capability for multi-lineage differentiation (Chacko et al, Eliasson et al and Grayson et al) and is a possible solution to this problem (Also see Is hypoxic the new normoxic?). However, another potentially more advantageous adult stem cell has recently been reported; the so-called Multilineage Differentiating Stress-Enduring (Muse) Cell, isolated from mesenchymal tissues under conditions of cellular stress. While being mesenchymal in origin, Muse cells display pluripotent characteristics (Kuroda et al), and have been identified as a primary source of induced pluripotent stem cells (iPSCs) generated from fibroblasts (Wakao et al) but, interestingly and seemingly paradoxically, do not produce teratomas in vivo (Kuroda et al and Wakao et al). Now, researchers from the group of Gregorio Chazenbalk at the David Geffen School of Medicine at University of Los Angeles, USA have described the purification of Muse cells from human adipose tissue under severe cellular stress conditions. The authors describe the protocol for their derivation and show them to show multiple pluripotent characteristics including multilineage differentiation (Heneidi et al).

Mitochondrial iPSC Models Developed

“Induced Pluripotent Stem Cells with a Mitochondrial DNA Deletion” and ““Disease-Causing Mitochondrial Heteroplasmy Segregated Within Induced Pluripotent Stem Cell Clones Derived from a Patient with MELAS”

Mitochondrial defects are associated with common diseases as well as rare genetic disorders which result when abnormal mitochondria accumulate and manifest as an unhealthy mixture of mutant mitochondrial DNA (mtDNA) which affects cellular function (Stewart et al, Taylor and Turnbull, and Wallace). The heterogeneity of mutant vs. wild type mtDNA can impact on disease severity as can the nature of the mutation, the interaction between nuclear and mitochondrial genomes, and the metabolic threshold tolerated by specific tissues. Strategies to eliminate diseased mitochondria have recently been devised including the modulation of mitochondrial heteroplasmy through stem cell manipulation (Craven et al and Tachibana et al). Two studies, published in Stem Cells, now report on the derivation of induced pluripotent stem cells (iPSCs) with mitochondrial gene mutations for use as model systems. The first, by Cherry and Gagne et al from the groups of George Q. Daley and Suneet Agarwal, report on their studies into Pearson marrow pancreas syndrome (PS), a rare congenital disorder caused by large deletions in mtDNA in which the cause of the associated life-threatening bone marrow failure is unknown (Pearson et al and Rotig et al). After derivation and cultivation of iPSCs, the group were able to isolate cells with different levels of mtDNA heteroplasmy and found that defects in growth and mitochondrial function were linked to the levels of mutant mtDNA, while haematopoietic differentiation revealed a tissue-specific phenotype characteristic of PS. Similarly, Folmes, Martinez-Fernandez and Perales-Clemente et al from the group of Timothy J. Nelson generated iPSCs from a patient with MELAS (lactic acidosis and stroke-like episodes) syndrome in which an mtDNA mutation is implicated. They also found a link between mtDNA mutation load and phenotype in progenitor cells and lineage-restricted progeny as well as cell fate regulation.

iPSCs to Neurons: Now Quick and Easy?

“Rapid Generation of Functional Dopaminergic Neurons From Human Induced Pluripotent Stem Cells Through a Single-Step Procedure Using Cell Lineage Transcription Factors”

From Stem Cells Translational Medicine

The degeneration of dopamine (DA) neurons in the substantia nigra leads to Parkinson’s disease (PD), the second most common neurodegenerative disease (Lees et al). The molecular mechanisms behind the disease are slowly being uncovered, but these efforts were not helped by the relative lack of a modelable cell type. The discovery of induced pluripotent stem cell (iPSC) technology has allowed the generation of PD-specific pluripotent cells to allow the study of development down the neuronal lineage (Cooper et al, Devine et al and Nguyen et al) in an effort to mimic early human development. However, known protocols are often laborious and time-expensive, a problem which led researchers from the laboratory of Vania Broccoli at the Stem Cells and Neurogenesis Unit, Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy to study and produce a quicker, more efficacious means of producing DA neurons. They now report, in Stem Cells Translational Medicine, that the forced expression of three specific transcription factors allows for the conversion of iPSCs into functional DA neurons at a high efficiency and in a short time period, and additionally apply this protocol successfully to PD-specific iPSCs (Theka et al).

Reprogramming Super Molecule Found?

“Nicotinamide Overcomes Pluripotency Deficits and Reprogramming Barriers”

From Stem Cells

Current technologies for the efficient large scale production of human induced pluripotent stem cells (iPSCs) are developing daily and are moving steadily towards clinical application, although challenges still exist.  Yee Sook Cho from the Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea, in a study published in Stem Cells, now report on their investigation of the role of metabolic networks in the reprogramming process.  Specifically, they have studied the role of Nicotinamide Adenine Dinucleotide, or NAD+, a cofactor for enzymes that participate in a variety of cellular responses.  NAD+ is synthesised either through the conversion of tryptophan or its assembly from nicotinamide (NAM), nicotinic acid (NA) or nicotinamide riboside through the salvage pathway (Belenky et al and Bogan and Brenner). Analysis of these pathways now suggests that NAD+ content is crucial for pluripotency, by enhancing metabolism and increasing resistance to cellular stress (Son and Son et al).

Factor Release from ECM Promotes Self-Renewal

“Matrix Remodeling Maintains Embryonic Stem Cell Self-Renewal by Activating Stat3”

From Stem Cells

Previous studies from the laboratory of Joel Voldman at the Massachusetts Institute of Technology, USA have shown that extracellular matrix (ECM) remodelling is necessary for mouse embryonic stem cell (mESC) self-renewal (Przybyla and Voldman).   The ECM is known to act as a reservoir for growth factors/cytokines (Hynes) with remodelling allowing the release of these factors to act in a paracrine or autocrine manner (Taipale and Keski-Oja).   Now, in new research from the laboratory of Joel Voldman, published in Stem Cells, how the feeder layer of mouse embryonic fibroblasts (MEFs) used in classical mESC cultivation affects remodelling of the ECM and self-renewal is studied, finding a key role the activation of JAK and Stat3 signalling (Przybyla et al).

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