Project description:Gene editing using engineered nucleases frequently produces unintended genetic lesions in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogenous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201+CD150+CD48-c-Kit+Sca-1+Lin-(KSL) population of pre-cultured HSCs. This dataset compares the gene expression in three different populations: (1) CD201+CD150+CD48-KSL (2) CD201+CD150+CD48+KSL and (3) CD201-KSL cells.
Project description:Precise study of HSCs during regeneration has been impeded by the rarity of the HSC population and depletion of phenotypic HSCs early following genotoxic stresses, such as total body irradiation (TBI). We isolated bone marrow (BM) ckit+sca-1+lin- (KSL) cells, which are enriched for HSCs, from adult C57Bl6 mice before and at several time points following TBI, as a means to map the dynamic molecular response of HSC regeneration. We isolated BM KSL cells and myeloid progenitor cells (c-kit+sca-1-lin- cells) at day +14 after irradiation and compared the gene expression profile of regenerating HSCs versus steady state HSCs (non-irradiated) and committed progenitor cells.
Project description:To understand the underlying mechanism by which the Hif1a gene is required by hematopoietic stem cells (HSCs), we performed a comparative DNA microarray analysis using total RNA isolated from wild type Lin-Sca-1+c-Kit+ cells and Hif1a-/- Lin-Sca-1+c-Kit+ cells. The result was validated by quantitative real-time PCR analysis of wild type Lin-Sca-1+c-Kit+ and Hif1a-/- Lin-Sca-1+c-Kit+ cells.