Direct Induction of hemogenic endothelial progenitors from hPSCs by defined factors revealed by single-cell transcriptome analysis

Transcription factors play critical roles in stem cell maintenance and differentiation. Using single cell RNA sequencing, we investigated transcription factors expressed in endothelial progenitors differentiated from human pluripotent stem cells (hPSCs) and identified upregulated differential expression of SOXF subgroup members SOX7, SOX17, and SOX18. To test whether overexpression of these factors increases differentiation efficiency, we established inducible hPSC lines and found only SOX17 improved differentiation of CD34+VEC+ cells. Temporal expression analysis of SOX17 and VEC revealed that SOX17 was turned on immediately before VEC, indicating SOX17 may be a causative factor in determining hemogenic endothelial differentiation. Upon Cas13d mediated repression of SOX17, differentiation was significantly abrogated. We found SOX17 forward programming is sufficient to generate more than 50% CD34+VEC+CD73- cells. Further differentiation of SOX17 forward programmed cells generated hematopoietic progenitors that emerged via an endothelial to hematopoietic transition and significantly upregulated definitive hematopoietic transcriptional programs. Our analyses reveal an uncharacterized function of SOX17 in directing hPSCs differentiation towards hematopoietic lineages. Overall design: One sample of hPSCs differentiated to day 5 endothelial progenitors was collected to sequence 2000-3000 cells via scRNAseq

转录因子 (Transcription Factors) 在干细胞维持与分化过程中发挥关键调控作用。本研究采用单细胞RNA测序 (Single-Cell RNA Sequencing, scRNA-seq) 技术，对人多能干细胞 (Human Pluripotent Stem Cells, hPSCs) 分化得到的内皮祖细胞中表达的转录因子进行分析，鉴定出SOXF亚家族成员SOX7、SOX17与SOX18存在上调差异表达。
为验证这些转录因子过表达是否可提升分化效率，我们构建了诱导型人多能干细胞系，实验结果显示仅SOX17能够改善CD34+VEC+细胞的分化效率。
通过对SOX17与VEC进行时序表达分析，我们发现SOX17的激活早于VEC，提示SOX17可能是调控造血内皮分化的关键因子。
经Cas13d介导的SOX17基因沉默后，细胞分化过程显著被抑制。
我们发现，仅通过SOX17正向编程即可生成占比超过50%的CD34+VEC+CD73-细胞。
对经SOX17正向编程的细胞进行后续分化，可获得经由内皮向造血转化 (Endothelial-to-Hematopoietic Transition, EHT) 过程产生的造血祖细胞，且该类细胞显著上调了定型造血转录程序。
本研究揭示了SOX17此前未被阐明的功能：可引导人多能干细胞向造血谱系分化。
总体实验设计：收集1份由人多能干细胞分化至第5天的内皮祖细胞样本，通过单细胞RNA测序对2000~3000个细胞进行测序。