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 human induced pluripotent stem cells (hiPSCs) from collaborators samples, as well as assistance and advice on differentiation strategies as requested. This included a collaboration with Dr. Keith Killian (NCI), in which cortical spheroids (mini-brains) were generated from human embryonic stem cell (hESC) lines for analysis of methylation patterns. We have also been evaluating nave pluripotent culture protocols, which could be important in studying diseases related to the X chromosome, and developing simple assays to assess the naivety of the culture. In terms of bringing pluripotent stem cells to the clinic, we have provided advice on assays and culture of iPSCs related to Dr. Kapil Bhartis (NEI) clinical initiative regarding iPSC derivation and differentiation into retinal pigmented epithelial cells. To understand the pathogenesis of Werner Syndrome, a rare, autosomal recessive progeroid syndrome, as well as the progression of normal aging, one or both WRN alleles in human iPSC lines have been disrupted and a fluorescent protein gene inserted, using CRISPR-Cas9 technology, to monitor activity of endogenous WRN. The effect of single or double disruption of the WRN locus on cell survival and function is being analyzed in these iPSCS as well as various cell types differentiated from them. In collaboration with Dr. Yihong Ye (NIDDK), the control mechanism of protein misfolding in normal and pathological conditions is being investigated. To do this, two genes of interest have been knocked out in hiPSCs using CRISPR-Cas9 technology and the resulting cell lines are being differentiated into neural precursor cells. These cell lines will be a good model system to study the folding of proteins such as alpha synuclein and the transmission of misfolded proteins between neurons and glial cells. Deficiency in GLI-similar 3 (GLIS3) gene causes many pathological conditions including neonatal diabetes and congenital glaucoma. In collaboration with Dr. Anton Jetten (NIEHS), GLIS3 gene has been knocked out in hESCs using CRISPR/Cas9 technology and the resulting cells are being differentiated into multiple cell types to study its role in various biological contexts. In collaboration with Dr. Ettore Appella lab (NCI), we are investigating the role of p53 in the regulation of self-renewal and differentiation of neural precursors or neural stem cells. Our data also provide critical insights into genomic-stress management in human pluripotent stem cell (hPSC) culture and differentiation. This work has led to a manuscript in preparation (Gete Y et al. manuscript in preparation). We are also assisting Dr. Melvin L. DePamphilis (NICHD), in the study of the role of geminin, (GMNN), in controlling mouse and human pluripotent stem cell growth and differentiation. Finally, in collaboration with Dr. Taiping Chen (MD Anderson) and Dr. Alexander Meissner (Harvard University) we are examining cellular stress and genomic instability in nave and primed human pluripotent stem cells. 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
