PDF(Pubmed)
Abstract：
Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC self-renewal and differentiation, but whether and how metabolic sensor O-GlcNAcylation, which can be modulated via an inhibition of its cycling enzymes O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT), contributes to hematopoiesis remains largely unknown. Herein, isogenic, single-cell clones of OGA-depleted (OGAi) and OGT-depleted (OGTi) human induced pluripotent stem cells (hiPSCs) were successfully generated from the master hiPSC line MUSIi012-A, which were reprogrammed from CD34+ hematopoietic stem/progenitor cells (HSPCs) containing epigenetic memory. The established OGAi and OGTi hiPSCs exhibiting an increase or decrease in cellular O-GlcNAcylation concomitant with their loss of OGA and OGT, respectively, appeared normal in phenotype and karyotype, and retained pluripotency, although they may favor differentiation toward certain germ lineages. Upon hematopoietic differentiation through mesoderm induction and endothelial-to-hematopoietic transition, we found that OGA inhibition accelerates hiPSC commitment toward HSPCs and that disruption of O-GlcNAc homeostasis affects their commitment toward erythroid lineage. The differentiated HSPCs from all groups were capable of giving rise to all hematopoietic progenitors, thus confirming their functional characteristics. Altogether, the established single-cell clones of OGTi and OGAi hiPSCs represent a valuable platform for further dissecting the roles of O-GlcNAcylation in blood cell development at various stages and lineages of blood cells. The incomplete knockout of OGA and OGT in these hiPSCs makes them susceptible to additional manipulation, i.e., by small molecules, allowing the molecular dynamics studies of O-GlcNAcylation.
摘要：
造血在整个生命中持续产生来自造血干细胞(HSC)的所有类型的血细胞。代谢状态是HSC自我更新和分化的已知调节因子，但是代谢传感器是否以及如何进行O-GlcNAcylation，可以通过抑制其循环酶O-GlcNAcase（OGA）和O-GlcNAc转移酶（OGT）来调节，对造血的贡献在很大程度上仍然未知。在这里，等基因,OGA耗尽（OGAi）和OGT耗尽（OGTi）人类诱导多能干细胞（hiPSCs）的单细胞克隆成功地从主hiPSC系MUSIi012-A，从包含表观遗传记忆的CD34造血干/祖细胞（HSPC）重新编程。已建立的OGAi和OGTihiPSC表现出细胞O-GlcNAcylation的增加或减少，伴随着OGA和OGT的丧失，分别,表型和核型正常，并保留了多能性，尽管它们可能倾向于向某些细菌谱系分化。在通过中胚层诱导和内皮到造血转变进行造血分化时，我们发现OGA抑制加速了hiPSC对HSPCs的承诺,而O-GlcNAc稳态的破坏会影响其对红系谱系的承诺.来自所有组的分化的HSPC能够产生所有造血祖细胞，从而证实了它们的功能特征。总之,已建立的OGTi和OGAihiPSC的单细胞克隆为进一步剖析O-GlcNAcylation在血细胞发育中不同阶段和谱系的作用提供了有价值的平台.这些hiPSC中OGA和OGT的不完全敲除使它们容易受到额外的操纵，即，通过小分子，允许O-GlcNAcylation的分子动力学研究。
