Quality Of Pluripotent Stem Cells Research Articles

Blastocyst complementation using Prdm14-deficient rats enables efficient germline transmission and generation of functional mouse spermatids in rats

Murine animal models from genetically modified pluripotent stem cells (PSCs) are essential for functional genomics and biomedical research, which require germline transmission for the establishment of colonies. However, the quality of PSCs, and donor-host cell competition in chimeras often present strong barriers for germline transmission. Here, we report efficient germline transmission of recalcitrant PSCs via blastocyst complementation, a method to compensate for missing tissues or organs in genetically modified animals via blastocyst injection of PSCs. We show that blastocysts from germline-deficient Prdm14 knockout rats provide a niche for the development of gametes originating entirely from the donor PSCs without any detriment to somatic development. We demonstrate the potential of this approach by creating PSC-derived Pax2/Pax8 double mutant anephric rats, and rescuing germline transmission of a PSC carrying a mouse artificial chromosome. Furthermore, we generate mouse PSC-derived functional spermatids in rats, which provides a proof-of-principle for the generation of xenogenic gametes in vivo. We believe this approach will become a useful system for generating PSC-derived germ cells in the future.

Academic application of Good Cell Culture Practice for induced pluripotent stem cells_suppl

Human induced pluripotent stem cells (hiPSC) are a promising tool for replacing animal-based experiments. To warrant data reproducibility, quality-controlled research material is recommended. While the need for global harmonization of quality standards for stem cell banking centers, commercial providers, pre-clinical and clinical use of cells is well doc­umented, there are no recommendations available for quality control of hiPSC in an academic research environment to date. To fill this gap, we here give an example of a quality-controlled, two-tiered banking process producing a fully characterized master cell bank (MCB) and partially characterized respective working cell banks (WCB). Characteri­zation includes the study of morphology, mycoplasma contamination, cell line identity, karyotype stability, cell antigen expression and viability, gene expression, pluripotency, and post-thaw recovery. Costs of these procedures are cal­culated. We present the results of the proposed testing panel of two hiPSC MCBs and show that both fulfil the defined specifications regarding the above-mentioned characterization assays during and upon banking. In conclusion, we propose a panel of eight assays, which are practical and useful for an academic research laboratory working with hiPSCs. Meeting these proposed specifications ensures the quality of pluripotent stem cells throughout diverse experi­ments at moderate costs.

Academic application of Good Cell Culture Practice for induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSC) are a promising tool for replacing animal-based experiments. To warrant data reproducibility, quality-controlled research material is recommended. While the need for global harmonization of quality standards for stem cell banking centers, commercial providers, pre-clinical and clinical use of cells is well doc­umented, there are no recommendations available for quality control of hiPSC in an academic research environment to date. To fill this gap, we here give an example of a quality-controlled, two-tiered banking process producing a fully characterized master cell bank (MCB) and partially characterized respective working cell banks (WCB). Characteri­zation includes the study of morphology, mycoplasma contamination, cell line identity, karyotype stability, cell antigen expression and viability, gene expression, pluripotency, and post-thaw recovery. Costs of these procedures are cal­culated. We present the results of the proposed testing panel of two hiPSC MCBs and show that both fulfil the defined specifications regarding the above-mentioned characterization assays during and upon banking. In conclusion, we propose a panel of eight assays, which are practical and useful for an academic research laboratory working with hiPSCs. Meeting these proposed specifications ensures the quality of pluripotent stem cells throughout diverse experi­ments at moderate costs.

Application for Lifestyle disease by iPS cells technologies

Currently it is less advanced to understand the pathology of lifestyle disease by using iPS cells because there is partly less direct connection between life style disease and iPS cells. So much more scientists focus on regenerative medicine such as beta cells therapy using iPS cells technologies. It will be indeed a powerful tool to generate beta cells from iPS cells as even in type2 diabetes patients, hyposecretion of insulin from beta cells in pancreas is one of causes. Another reason is complexity of the pathology of life style disease. There are a lot of reasons to cause lifestyle disease. Lifestyle diseases include cancer, chronic liver disease, Type 2 diabetes, heart disease, metabolic syndrome, chronic renal failure, stroke, and obesity. Since obesity is one of major causes of lifestyle diseases, we want to focus on adipogenesis from iPS cells in this review. We analysed and established the differentiation protocol into adipocytes from mouse ES cells and human iPS cells. The other point in this review is the starting pluripotent cells for differentiation. Quality of pluripotent stem cells are one of most critical factors to succeed in getting well-differentiated cells. Recently, we have developed new naive human pluripotent stem cells (PSC),"Reset cells". Naive PSC have more similar to human epibast cells than conventional human PSC. They will be more ideal cells for differentiation because of their hypomethylated status and earlier stage of development.

3,2/-Dihydroxyflavone-Treated Pluripotent Stem Cells Show Enhanced Proliferation, Pluripotency Marker Expression, and Neuroprotective Properties

Efficient maintenance of the undifferentiated status of embryonic stem cells (ESCs) may be important for preparation of high-quality cell sources that can be successfully used for stem cell research and therapy. Here we tried to identify a compound that can enhance the quality of pluripotent stem cells. Treatment of ESCs and induced pluripotent stem cells (iPSCs) with 3,2'-dihydroxyflavone (3,2'-DHF) led to increases in cell growth, colony formation, and cell proliferation. Treatment with 3,2'-DHF resulted in high expression of pluripotency markers (OCT4, SOX2, and NANOG) and significant activation (STAT3 and AKT) or suppression (GSK3β and ERK) of self-renewal-related kinases. 3,2'-DHF-treated high-quality pluripotent stem cells also showed enhanced differentiation potential. In particular, treatment of iPSCs with 3,2'-DHF led to elevated expression of ectodermal differentiation markers and improved differentiation into fully matured neurons. Next, we investigated the in vivo effect of 3,2'-DHF-pretreated iPSCs (3,2'-DHF iPSCs) in a peripheral nerve injury model and found that transplantation of 3,2'-DHF iPSCs resulted in more efficient axonal regeneration and functional recovery than in controls. Upon histopathological and gene expression analyses, we found that transplantation of 3,2'-DHF iPSCs stimulated expression of cytokines, such as TNF-α, in the early phase of injury and successfully reduced convalescence time of the injured peripheral nerve, showing an effective neuroprotective property. Taken together, our data suggest that 3,2'-DHF can be used for more efficient maintenance of pluripotent stem cells as well as for further applications in stem cell research and therapy.

Advances in reprogramming to pluripotency.

Pluripotent stem cells (PSCs) derived from somatic cells represent a powerful experimental tool for investigating the molecular mechanisms underlying the disease phenotype; with prospects to advance medical therapies. They also have significant potential as a renewable source of autologous cells for cellular therapy. Various approaches for PSC derivation from somatic cells have been reported in the literature. The method used for reprogramming is particularly relevant as it may affect the characteristics and quality of PSCs. This review will present an overview of the basic strategies and methods for reprogramming to pluripotency. These strategies will be briefly discussed in the context of how the mechanism of reprogramming could influence PSC characteristics with respect to safety and quality. Aspects of the reprogramming approach that can influence PSC properties, such as culture conditions and donor cell source, are also discussed.

244. TaqMan® Assays for Assessment of Genome, Epigenome and Quality of Pluripotent Stem Cells

244. TaqMan® Assays for Assessment of Genome, Epigenome and Quality of Pluripotent Stem Cells