Monsalve Anuntxi, Canals Isaac, Oburoglu Leal
Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden.
Neurology, Lund Stem Cell Center, Lund University, Lund, Sweden.
Front Cell Dev Biol. 2022 Oct 12;10:1039636. doi: 10.3389/fcell.2022.1039636. eCollection 2022.
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.
原始、新生儿和成人红系细胞此前已被证明具有活跃的磷酸戊糖途径（PPP），该途径为各种过程提供能量。然而，尚不清楚PPP在造血内皮（HE）来源的红系祖细胞出现过程中是否发挥作用。在本研究中，我们探索了发育性红细胞生成过程中的PPP及其基因调控。我们诱导人诱导多能干细胞（hiPSC）进行造血分化以获得HE细胞。这些细胞用携带针对FOXO1的短发夹RNA（shRNA）的慢病毒载体处理，或用针对PPP、NRF2或AKT的抑制剂处理。通过流式细胞术评估红系分化、增殖和频率。通过qPCR或分析可用的RNA测序数据评估基因表达。我们发现PPP对于HE细胞的红系分化是不可或缺的，并且它部分为核苷酸生物合成提供能量。此外，我们表明NRF2和AKT对于HE来源的红系分化至关重要，而FOXO1则具有不利影响。相反，阻断FOXO1表达并不影响脐血造血干细胞（HSPC）的红系分化。从机制上讲，抑制HE细胞中的FOXO1会导致PPP非氧化分支增加。在发育性红细胞生成过程中，FOXO1的逐渐减少会激活PPP并为核苷酸生物合成和细胞增殖提供能量。
