冷冻保存过程中细胞骨架对人多能干细胞命运的调控机制研究及应用

Human pluripotent stem cells (hPSCs) include human embryonic stem cells and human induced pluripotent stem cells. Due to the unlimited capacity of self-renew and the ability of differentiation into all somatic cells of human body, hPSCs have become precious cell sources for regenerative medicine and drug screening. hPSCs are very fragile and sensitive to the stresses caused by dissociation, osmosis and freezing during cryopreservation and easily damaged. The low recovery rate of hPSCs after cryopreservation has limited its application in regenerative medicine. In this project, we will focus on the effects of cytoskeletal changes on the cellular morphology of hPSCs and the roles of cytoskeleton in adhesion, movement, cell death, cell self-renew and differentiation through investigating the changes of cytoskeletal components, such as microtubule, microfilament and other cytoskeleton-related proteins, to reveal the regulation mechanism of cytoskeleton in cell fate during cryopreservation. With understanding the regulation mechanism, we aim to design a novel strategy for rapid recovery after cryopreservation to elevate the cell recovery rate and improve functional recovery of hPSCs. The implementation of this project will not only provide a method for long term storage of hPSCs but also lay the foundation for its clinical applications.

人多能干细胞包括人胚胎干细胞和人诱导多能干细胞，由于具备无限增殖和多元分化能力而成为细胞治疗和药物筛选等的首选细胞源。人多能干细胞非常脆弱，对冷冻保存过程中产生的解离、渗透和冷冻应力非常敏感，易形成冷冻损伤，并最终导致人多能干细胞冷冻后的低复苏率，严重抑制其应用。本项目以冷冻过程中细胞骨架的变化对人多能细胞的生物功能的影响为研究对象，考察细胞骨架中的微管、微丝和肌动蛋白等在不同冷冻条件下的形态和数量变化，确定细胞骨架变化对细胞形态的影响，探讨冷冻过程中细胞骨架变化对细胞黏附、运动、细胞死亡、自我更新和分化等的作用，揭示细胞骨架在冷冻过程中对人多能干细胞命运的调控机制，设计人多能干细胞冷冻后快速恢复策略，促进细胞的生物功能恢复，提高冷冻复苏率，为解决人多能干细胞在临床应用中面临的规模化长期稳定存储问题奠定理论基础。

本项目以人多能干细胞为研究对象，发现解离过程和冷冻过程导致细胞复苏后E-cad下调，coflin和Filamin 1上调，而Vimentin先上调而后维持稳定，并进而导致细胞的大量死亡。研究结果表明当细胞解离和冷冻复苏后，细胞凋亡过程通过下列途径：PKA蛋白激活，PP1磷酸酶活性增加，进一步导致 Cofilin 去磷酸化激活，去磷酸化激活的Cofilin和Bax相互作用并转位到线粒体；同时p53在Ser15发生磷酸化,与PKA互作，进而在胞内与线粒体蛋白发生互作；由此促进线粒体Cyt c的释放，继而发生Caspase依赖的凋亡。由此在解离和冷冻复苏液中引入H89、Pft或CytoD抑制剂将不同程度的抑制上述线粒体凋亡途径组分的表达，进而抑制细胞凋亡，提升细胞活率。本项目的研究结果为解决人多能干细胞在临床应用中面临的规模化长期稳定存储问题奠定理论基础。