During the last fiscal year, the NIH SCCF has made progress in a number of areas as highlighted below. We have assisted our collaborators by providing material and cell lines, resulting in the publication of several manuscripts. We continued to mentor and teach standard and feeder-free, pluripotent stem cell culture, provided assistance and advice on the generation of iPSCs from collaborators samples, as well as assistance and advice on differentiation strategies as requested. This included a collaboration with Dr. Alan Koretsky at NINDS, in which induced pluripotent stem cell lines were generated from embryonic fibroblasts of a transgenic mouse expressing GFP under the control of microglia-specific CX3CR1 promoter. In terms of bringing pluripotent stem cells to the clinic, we have provided advice on the establishment of assays and GMP protocols related to Dr. Kapil Bhartis (NEI) clinical initiative regarding iPSC derivation and differentiation into retinal pigmented epithelial cells. Along with members of the Cell Processing Section, we have established methods for reprogramming of rare CD34+ cells in peripheral blood, an easily obtainable source of cells from even severely compromised patients. In order to generate a cellular model for Hutchinson-Gilford Progeria Syndrome (HGPS), human iPSC lines were generated by targeting safe-harbor sites, CLYBL, on chromosome 13 and by random integration. The cell lines express either normal or mutant LMNA gene (progerin) in a drug-inducible manner as confirmed by RFP expression. Cells overexpressing progerin demonstrated aberrations in nuclear morphology, mitosis and differentiation. This work was presented at the ISSCR annual meeting in 2016. To study human aging from a different angle to the Progeria work, another cellular aging model is being generated by knocking out the WRN gene in human iPSC lines. Mutations in both alleles of WRN gene cause Werner Syndrome (WS), also known as adult progeria, which is an autosomal recessive syndrome characterized by the appearance of premature aging. The gene has been successfully disrupted in iPSCs, and the structural determination of targeted genomic region in each iPSC clone is in progress. In collaboration with Dr. Richard Leapman's laboratory at the NIBIB, we contributed to understanding the biological implications of some fundamental physiological processes in pluripotent stem cells. Our novel findings, published in 2015, regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. In collaboration with Dr. Ettore Appella's lab at the NCI, we are investigating the role of p53 in the regulation of self-renewal and differentiation of murine neural precursors or neural stem cells. These data should provide critical insights into genomic-stress management in hPSC culture and differentiation. As always, we update the SCU website with protocols and information as it becomes available to aid other researchers in their studies.
Showing the most recent 10 out of 25 publications
