红系发育是造血干细胞增殖分化最终形成成熟红细胞的精细调控过程。其中红细胞脱核是哺乳动物特有的生物学现象。虽然该现象早已被发现，但其机制尚不明确。这也是制约我们利用胚胎干或诱导多能干细胞体外生成功能性红细胞的一个瓶颈。本课题组红系发育各阶段转录组数据显示：PIM1表达水平在晚幼红细胞中显著上调，且是晚幼红细胞表达最高的蛋白激酶。前期我们对PIM1在红系发育中的作用进行了初步研究，发现敲低人CD34+细胞的PIM1不影响红细胞增殖、分化和凋亡，只特异性地影响脱核。此外比对哺乳和非哺乳动物PIM1蛋白序列，我们发现PIM1存在特异变化的氨基酸位点。由此我们推测其在哺乳动物红细胞脱核过程中发挥了重要的调控作用。本项目拟利用流式、细胞成像、磷酸化组学、动物模型等多手段深入探讨PIM1在脱核中的分子机制。项目的完成不仅有助于揭示红细胞脱核的分子调控机制，也有望为体外生成功能性红细胞提供了新的理论策略。

Erythropoiesis is a highly regulated multistage process by which hematopoietic stem cells proliferate, differentiate and eventually form mature erythrocytes. Among this step-by-step process, erythroblast enucleation is the most unique biological event in mammals. Although this distinctive feature has been discovered for more than a century, the molecular mechanisms remain largely unclear. This bottleneck also restricts the generation of functional erythrocytes using embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Our RNA-seq analysis of erythroid cells at each developmental stage revealed that serine/threonine kinase PIM1 was significantly up-regulated in both human and mouse orthochromatic erythroblasts and was the highest expressed protein kinase in this stage. To explore the role of PIM1 in human erythroblast enucleation, we knocked down PIM1 by shRNA in human CD34+ cells and found that PIM1 knockdown specifically resulted in remarkable decrease in enucleation with no effects on cell growth, differentiation and apoptosis. Moreover, comparison of PIM1 sequence revealed that specific amino acid sites existed between mammals and non-mammals. Base on these findings, we hypothesize that PIM1 plays important roles in mammalian erythroblast enucleation. In this application, we will explore the molecular mechanisms by which PIM1 regulate enucleation using state-of-the art approaches including flow cytometry, cell sorting, cell imaging, phosphoproteomics in conjunction with animal models. Successful accomplishment of proposed studies will not only uncover novel mechanisms of mammalian erythroblast enucleation, but may also help in developing strategy for generation of functional erythrocytes ex vivo.