Developmental regulation of endothelial-to-hematopoietic transition from iPSCs [sci-RNA-seq]. Developmental regulation of endothelial-to-hematopoietic transition from iPSCs [sci-RNA-seq]

Hematopoietic stem cells (HSCs) arise in mid-gestation from a specialized hemogenic endothelium (HE). In this process, HE cells undergo a unique fate change termed endothelial-to-hematopoietic transition, or EHT. While induced pluripotent stem cells (iPSCs) give rise to HE with robust hemogenic potential, the generation of bona fide HSCs from iPSCs remains a challenge. Here, we map single cell dynamics of EHT during embryoid body differentiation from iPSCs and integrate it with human embryo datasets to identify key transcriptional differences between in vitro and in vivo cell states. We further map ligand-receptor interactions associated with differential expression of developmental programs in the iPSC system. We found that the expression of endothelial genes was incompletely repressed during iPSC EHT. Elevated FGF signaling by FGF23, an endothelial pathway ligand, was associated with differential gene expression between in vitro and in vivo EHT. Chemical inhibition of FGF signaling during EHT increased HSPC generation in the zebrafish system, while an FGF agonist had the opposite effect. Consistently, chemical inhibition of FGF signaling increased hemogenic output from iPSCs. In summary, we map the dynamics of EHT from iPSCs at single cell resolution and identify ligand-receptor interactions that can be modulated to improve iPSC differentiation protocols. We show, as proof of principle, that chemical inhibition of FGF signaling during EHT improves hematopoietic output in zebrafish and the iPSC system. Overall design: iPSCs were formed into embryoid bodies (day 0) and treated with 100ng/ml hSCF, 100ng/ml hFLT3, 50ng/ml hG-CSF, 25ng/ml IL-6 and 10ng/ml IL-3 starting on day 1 and throughout the rest of any given experiment. Unsorted cells from each time point (days 7, 8, 11, 18 and 21 of differentiation) were used for single-cell combinatorial indexing RNA-seq (sci-RNA-seq) samples. CD34+, CD43+ or CD45+ were sorted using FACS for the 10X 3' sc-RNA-seq day 8 sample.

造血干细胞（Hematopoietic stem cells, HSCs）于妊娠中期由特化的生血内皮细胞（hemogenic endothelium, HE）分化产生。在此过程中，HE细胞会经历一种独特的命运转变，即内皮向造血转化（endothelial-to-hematopoietic transition, EHT）。尽管诱导多能干细胞（induced pluripotent stem cells, iPSCs）可分化为具有强生血潜能的HE，但从iPSCs诱导生成真正的造血干细胞仍是一项挑战。
本研究对iPSCs经拟胚体（embryoid body）分化过程中的EHT单细胞动态变化进行了图谱绘制，并将其与人类胚胎数据集整合，以鉴定体外（in vitro）与体内（in vivo）细胞状态之间的关键转录差异。我们进一步绘制了iPSC系统中与发育程序差异表达相关的配体-受体相互作用图谱。
研究发现，在iPSC来源的EHT过程中，内皮细胞基因的表达并未被完全抑制。由内皮通路配体FGF23介导的增强型成纤维细胞生长因子（Fibroblast Growth Factor, FGF）信号通路，与体外和体内EHT之间的差异基因表达密切相关。在EHT阶段对FGF信号通路进行化学抑制，可提升斑马鱼系统中的造血干祖细胞（hematopoietic stem and progenitor cells, HSPC）生成效率，而FGF激动剂则会产生相反效果。与之一致的是，对FGF信号通路的化学抑制可提升iPSCs的生血产出。
综上，本研究以单细胞分辨率绘制了iPSCs来源的EHT动态变化图谱，并鉴定出可通过调控以优化iPSC分化方案的配体-受体相互作用。作为原理验证，我们证实了在EHT过程中对FGF信号通路进行化学抑制，可提升斑马鱼和iPSC系统的造血产出。
总体实验设计：将iPSCs制备为拟胚体（第0天），并于第1天起添加100ng/ml人干细胞因子（human stem cell factor, hSCF）、100ng/ml人FMS样酪氨酸激酶3配体（human FMS-like tyrosine kinase 3 ligand, hFLT3）、50ng/ml人粒细胞集落刺激因子（human granulocyte colony-stimulating factor, hG-CSF）、25ng/ml白细胞介素6（IL-6）及10ng/ml白细胞介素3（IL-3），直至实验结束。收集各时间点（分化第7、8、11、18和21天）的未分选细胞，用于制备单细胞组合索引RNA测序（single-cell combinatorial indexing RNA-seq, sci-RNA-seq）样本。针对分化第8天的样本，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分选出CD34+、CD43+或CD45+细胞，用于10X 3'端单细胞RNA测序（10X 3' sc-RNA-seq）。