PUMA在人外周血源iPS细胞分化为血小板中的作用研究

Allogenic platelet transfusions protect patients from bleeding episodes and also make aggressive medical procedures such as those involving marrow transplants requiring chemotherapy and/or radiotherapy possible. The patients who suffer from bleeding duo to thrombopenia or platelet dysfunction are fully dependent upon the supply of platelets which can sometimes be in short supply due to high demands coupled with an extremely short expiration date for platelet products of only 5 days. One approach that is under investigation to overcome platelet shortages, especially for patients with antibodies or a rare HLA, is to use the extraordinary capabilities of stem cells to proliferate and differentiate to produce clinical scale quantities of functional platelets in bioreactors. To generate the functional platelets from human induced pluripotent stem cells (hiPSCs), the key point is the safety of iPSCs and to enhance the efficiency of reprogramming and differentiation. Apoptosis has been recognized as the central mechanism of platelet production from megakaryocytes (MKs). Bcl-XL and Bax have an important effect on MKs maturation and platelet production. p53-upregulated mediator of apoptosis (PUMA) is a BH-3-only pro-apoptotic gene in the Bcl-XL family. The PUMA protein interacts with Bcl-XL and promotes mitochondrial translocation and multimerization of Bax. We try to establish a PUMA knockdown iPSCs from human peripheral blood using Episomal vectors. According to a feeder-free adherent monolayer differentiation culture protocol in serum-free medium with sequential addition of growth factors, we plan to investigate the effect of PUMA on hiPSCs differentiation to MKs and platelets. We plan to add PUMA inhibitor to culture medium during normal hiPSCs differentiation to appreciate the regulatory mechanisms of PUMA at each phase of a developmental process，such as iPSCs differentiation to MKs, MKs maturation and platelet production, which can accomplish clinic scale platelet production in the end.

输注血小板是大出血、移植、放化疗后的重要保障，是预防血小板数量或功能异常出血的有效方法。由于血源紧张、保存期短，血小板始终库存不足。诱导多能干细胞（iPSCs）体外分化生产血小板为临床供应带来了新希望，尤其对HLA配型困难及有血小板抗体的患者。iPSCs的安全性和诱导分化效率是生产临床级血小板的关键。研究发现Bcl-XL和Bax在调控巨核细胞成熟和血小板产生中起重要作用。凋亡蛋白PUMA不仅直接结合Bcl-XL促进线粒体易位和Bax多聚化，还调控iPSCs基因组稳定性和重编程效率。本课题拟构建PUMA表达下调人外周血源iPSCs，通过无血清无支持细胞单层贴壁培养方法体外生产血小板，解析PUMA基因在iPSCs分化产生血小板中的作用及机制；通过小分子抑制剂研究PUMA在iPSCs诱导分化为巨核细胞、巨核细胞成熟及释放血小板的不同阶段的作用，为人外周血源iPSCs生产血小板的临床应用提供依据。

本课题已按计划执行，并按时完成。项目共发表SCI论文4篇，中文期刊6篇，获得专利6项。课题研究目标圆满完成，主要研究结果如下：1）应用CRISPR-cas9技术建立了PUMA 敲除的 iPS细胞；2）PUMA 敲除的 iPS细胞，无外源基因插入，表达干细胞基因，具有三胚层分化能力；3）PUMA敲除的iPS细胞往造血细胞分化能力显著高于对照组，生成CD34+CD45+造血干祖细胞的数量是对照iPS细胞的20倍（p<0.05）；4 ）PUMA敲除的iPS细胞生成GEMM-CFU、巨核细胞和血小板的数量显著高于对照iPS细胞（p<0.05）。本项目建立了人外周血源 iPS细胞无血清、无支持细胞、单层贴壁培养诱导分化血小板方法，证实PUMA在iPS细胞分化产生血小板中发挥重要的作用，敲除PUMA有利于iPS细胞诱导分化为巨核细胞和血小板。