Graduate Student Haihe Laboratory of Cell Ecosystem, Tianjin, China (People's Republic)
Abstract: The transplantable therapeutic properties of HSCs and the differentiation potential of mature blood cells have attracted many hematological researchers for years. iPSC cells have made it possible to generate HSCs in vitro. The visualization of hematopoietic stem cells can certainly provide a solution to accurately grasp the time window of their emergence, track their dynamics and development, optimize the hematopoietic stem cell niche, and establish an effective expansion model in vitro. By combining traditional knowledge with bioinformatics and deep machine learning, we have selected highly probable long-term and short-term HSCs fate-determining transcription factors, HOXA9, HLF, TCF16, CD34, and CD82 etc, to be integrated with fluorescent proteins through CRISPR/Cas9 gene editing. Subsequently, we established a new ex vivo differentiation and developmental system by optimizing the induction process and growth factors as well as small molecules to mimic the three-dimensional embryonic development in vivo, that effectively enhances the endothelial-to-hematopoietic transition. hematopoietic transition using an embryoid body (EB) model. In developmental tracing ex vivo of HSC, DsRed+ cells began to appear in the EB organoids from day 10 onwards, followed by outgrowth and shedding, which was showed at day 13, DsRed-dim positive hematopoietic progenitor subpopulation, CD34+CD43+CD45+ HSC cells, with strong multicellular type colony forming capability. The floating cells on day 16, on the other hand, formed more homogeneous erythroid colonies. The HSC visualization iPSC line established in this study provides a powerful tool for ex vivo tracking and expansion of HSC and also enables further in vitro simulation of the optimal spatial location of the HSC niche with EBs.
