We have performed extensive characterization of the 21 Bush-era human embryonic stem cells and deposited the data with NCBI GEO for public access. We have created a user-friendly gene expression search engine which allows a casual user to interrogate the data for their particular gene of interest. In our mission to facilitate pluripotent stem cell research, we have performed in-depth characterization of a control induced pluripotent stem cell (iPSC) line, BC1. This includes FACS analysis and immunocytochemistry as well as gene expression microarray analysis. In collaboration with the NIH-CRM, we generated iPSCs derived from neuronal precursors differentiated from H1 ES cell lines and compared their gene expression and methylation profiles to the parental line. This provides a direct comparison of ESCs to iPSCs as all lines have the same genome. Analysis shows no global difference although there may be more subtle effects to be determined. In collaboration with NIH-CRM, 5 transgenic hESC lines, which express traceable markers from cell type-specific promoters, have been generated using a Zinc Finger nuclease-assisted gene-targeting method to integrate the transgenes into AAVS1, one of the known safe harbor sites in the human genome. Each transgene contains a gene for ZS green and a drug-selection marker. Correct transgene integration as well as normal karyotype has been confirmed for each line and expression of ZS green from appropriate cell types has been confirmed in 2 lines. All lines will be deposited with WiCell shortly to facilitate distribution to the community. We have extended our studies on the novel non-colony type (NCM) monolayer method for pluripotent cell culture using different small molecules and alternative substrates. We have demonstrated improved efficiency of transfection or transduction of plasmid DNAs, lentiviral particles, and short oligonucleotide-based microRNAs using this method. We will continue to improve the method for application to high thoughput and scalability for drug screening and therapeutic use. In addition, to generate homogeneous populations of specific cell types efficiently and reproducibly, directed differentiation has been attempted starting from NCM culture. These differentiation strategies do not include the formation of embryoid bodies, which are a major source of heterogeneity in many differentiation protocols. Several of the traceable transgenic hESC lines described above have been successfully differentiated into neural precursor cells (endoderm), beating cardiomyocytes (mesoderm), and SOX17-positive cells (endoderm) with relatively high efficiency and homogeneity. Further optimization and refinement of the differentiation protocols will be pursued. In terms of bringing pluripotent stem cells to the clinic, we have been evaluating novel xeno-free substrates, media and small molecule inhibitors as well as non-integrating methods of reprogramming. These methods include Sendai virus and microRNA boosted mRNA- based reprogramming. We will also be evaluating episomal plasmid-based reprogramming strategies in the future and testing novel strategies to reprogram blood cells. Finally, we have been involved in mentoring and teaching standard and feeder-free, pluripotent stem cell culture, assisting and advising on the generation of iPSCs from patient samples as well as assisting and advising on differentiation strategies as requested. We update the SCU website with protocols and information as it becomes available to aid other researchers in their studies.
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