The NIH SCU has routinely grown hECs on the NIH registry since 2003, and the Unit has provided evidence that hESCs can be grown for long periods without genetic change. From this work, the SCU generated an mRNA database of information that includes both undifferentiated and differentiated samples. With help from the NINDS bioinformatics team, they have designed a public webpage in which this database of information may be easily interrogated and charts plotted. This provides an excellent reference source to which new hESC and iPSC lines can be compared. During the past year, the NIH SCU has also moved into the generation and characterization of induced pluripotent stem cells (iPSCs). Initially, they were involved with characterization of iPSCs derived by reprogramming fibroblasts using four reprogramming factors (Sox2, Oct4, c-Myc and Klf4) in individual retroviral vectors (NIH-i1-i10, the McKay lab), and compared them to hES cells. Their analyses indicate that there is strong similarity between iPSCs and hESs, and that any apparent variation is due to normal variation in the human population. In addition, the NIH SCU developed a new series of iPSCs (SCU-i1-10) from human bone marrow stromal cells and skin fibroblasts using a polycistronic lentiviral vector with the same four factors. All of these lines were initially propagated as colonies of mouse embryonic fibroblasts (MEFs), some were then transitioned as colonies onto MEF-free plates coated with Matrigel, and then finally into Matrigel coated plates as a novel monolayer culture generated by single cell suspensions. Of note, work has also focused on the role of stress reduction improving the growth of hESCs and iPSCs. By FACS, all SCU lines were negative for SSEA-1 and positive for SSEA-4, Tra-1-60 and Tra-1-81. By immunohistochemistry and qRT-PCR, POU5F1 and NANOG were at levels comparable to hESCs (WA01 and UC06). Current studies are in progress to determine if SCU-derived iPSCs are pluripotent based on differentiation into cell types representative of all three germ layers in vitro, and the ability to form teratomas in vivo in comparison to WA01. The SCU is also evaluating commercially available reagents needed for reprogramming, and subsequent growth in order to provide the best advice possible to the NIH community. The Unit has trained a number of investigators in the growth and maintenance of pluripotent stem cells, and have been cited for their contributions (Kumari D and Usdin K. (2010) The distribution of repressive histone modifications on silenced FMR1 alleles provides clues to the mechanism of gene silencing in fragile X syndrome. Human Molecular Genetics. 19(23):463442;Kuznetsov SA, Cherman N, Robey PG. (2011) In vivo bone formation by progeny of human embryonic stem cells.Stem Cells and Development. 20(2):269-87).
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