原始人,先前已显示新生儿和成人红系细胞具有活跃的磷酸戊糖途径(PPP),为各种过程提供动力。然而,目前尚不清楚PPP是否在来自血源性内皮(HE)的红细胞祖细胞出现过程中发挥作用。在这项研究中,我们探讨了PPP及其在发育性红细胞生成中的遗传调控。我们诱导造血分化的人诱导多能干细胞(hiPSCs)获得HE细胞。这些细胞用携带针对FOXO1的shRNA的慢病毒载体处理,或用针对PPP的抑制剂处理,NRF2或AKT。红系分化,通过流式细胞术评估增殖和频率。通过qPCR或通过分析可用的RNAseq数据来评估基因表达。我们发现PPP对于HE细胞的红系分化是必不可少的,并且它部分地促进了核苷酸的生物合成。此外,我们发现NRF2和AKT是必不可少的,虽然FOXO1是有害的,用于HE衍生的红细胞分化。相比之下,阻断FOXO1表达不影响脐血HSPCs的红系分化。机械上,HE细胞中的FOXO1抑制导致PPP的非氧化分支增加。在发育性红细胞生成期间,FOXO1的逐渐减少激活了PPP并促进了核苷酸生物合成和细胞增殖。
Primitive, neonatal and adult erythroid cells have been previously shown to have an active pentose phosphate pathway (PPP) that fuels various processes. However, it is unclear whether the PPP plays a role during the emergence of erythroid progenitors from hemogenic endothelium (HE). In this study, we explored PPP and its genetic regulation in developmental erythropoiesis. We induced hematopoietic differentiation of human induced pluripotent stem cells (hiPSCs) to obtain HE cells. These cells were treated with lentiviral vectors harboring shRNAs against FOXO1, or with inhibitors against the PPP, NRF2 or AKT. Erythroid differentiation, proliferation and frequency were evaluated by flow cytometry. Gene expression was assessed by qPCR or by analysis of available RNAseq data. We found that PPP is indispensable for the erythroid differentiation of HE cells and it partially fuels nucleotide biosynthesis. Moreover, we showed that NRF2 and AKT are essential, while FOXO1 is detrimental, for HE-derived erythroid differentiation. In contrast, blocking FOXO1 expression did not affect erythroid differentiation of cord-blood HSPCs. Mechanistically, FOXO1 inhibition in HE cells led to an increase in the non-oxidative branch of the PPP. During developmental erythropoiesis, the gradual decrease in FOXO1 activates the PPP and fuels nucleotide biosynthesis and cell proliferation.