人体细胞重编程过程中细胞“自塑”微环境的解析

Human induced pluripotent stem cells (hiPSC) are embryonic-like stem cells obtained by the process of cell reprogramming. Since hiPSCs can be derived from easily accessible somatic cells, they represent a very interesting source for in vitro studies of human development, and for patient-specific and disorder-specific studies and applications. However, the reprogramming process is currently very difficult to control because it is not well understood, especially at its intermediate stages. Exploring the molecular events triggering the full reprogramming of human fibroblasts is complicated by the fact that the process is very inefficient and cells undergoing full reprogramming to hiPSC are only a very minor fraction of all the cells in culture. Currently genetically unmodified human fibroblasts can be reprogrammed with a maximal efficiency of ~2%. .Our preliminary data show that down-scaling human fibroblast reprogramming to micro-liter scale strongly promotes the process, with ~120 hiPSC colonies produced from 100 cells seeded. The soluble microfluidic culture environment differs from that in conventional culture wells because of the very small medium volume per cell. Thus, any exchange of molecules between cells and soluble environment strongly affects medium composition. Our preliminary data support the accumulation of cell-secreted factors as the basis of the high-efficiency quasi-deterministic reprogramming we obtained..In this project, we will study this self-regulated high-efficiency reprogramming environment to understand which endogenous signals are driving the process. We will identify the cell-secreted molecular players that critically accumulate in the culture medium along the process and are responsible for such a great promotion of reprogramming efficiency at micro-scale. Bioinformatics analysis and literature mining will shed light on the mechanistic progression of cell reprogramming, also at the less understood intermediate stages..This project has a sound scientific interest in dissecting the endogenously-shaped cell microenvironment during reprogramming. Moreover, it will make an important step forward to the controlled production of high-quality hiPSC, and to all the downstream hiPSC-based applications.

人诱导多能干细胞（hiPSC）是将人已分化的体细胞经重编程而得到的类似胚胎干细胞一种细胞类型。人诱导多能干细胞在科学研究及临床中具有巨大的应用前景。然而，由于我们对重编程的理解还不够透彻，目前还很难做到对重编程的有效控制。.我们前期的实验数据显示，将重编程的规模缩小至微升级别，能极大的提高重编程效率，每一百个种植细胞能够产生出约120个干细胞克隆。因为在微流控培养环境中，每个细胞能够分到的培养基体积非常少，细胞和环境中的任何分子交换都会极大地影响培养基中的成分。所以我们认为微流控培养环境与传统培养环境有很大区别。我们猜想：细胞分泌因子的积累能极大的提高细胞重编程的效率。之前的实验数据支持我们的猜想。.在这个项目中，我们将聚焦于这种自我调节的高效重编程过程，分析其独特的细胞培养微环境，找到影响重编程过程的重要分子，并分析其时序性。借助于生物信息学分析和文献挖掘技术，阐明细胞重编程的过程机制。

人诱导多能干细胞（hiPSC）是将人已分化的体细胞经重编程而得到的类似胚胎干细胞一种细胞类型。人诱导多能干细胞在科学研究及临床中具有巨大的应用前景。然而，由于我们对重编程的理解还不够透彻，目前还很难做到对重编程的有效控制。.我们前期的实验数据显示，将重编程的规模缩小至微升级别，能极大的提高重编程效率。.该项目旨在研究微流体内人体细胞重编程过程中的微环境。由于细胞分泌因子在小体积培养环境中积累，我们可以研究微流体中的细胞微环境。.我们建立了一种通过质谱分析法，对微流体中的细胞分泌蛋白进行蛋白质组学分析的强大而灵敏的实验方案。我们专注于两种允许在不同条件之间进行相对定量的蛋白质标记方法：通过细胞培养中的氨基酸进行稳定同位素标记（SILAC）和串联质谱标签（TMT）。.此外，通过使用低蛋白的成分确定培养基，使得重编程方案本身更加符合蛋白质组学的要求。.最后，我们研究了高效重编程过程中，内源性细胞外微环境的蛋白质组学组成，并致力于验证该过程中涉及的调控机制。.我们的结果展示了，在微流体重编程过程中，不同的细胞亚群是如何出现，以及是如何从这些互相影响的亚群中获得高效率的。我们的研究结果概述了内源性重编程微环境发展、形成的方式。此外，还概述了小鼠模型中观察到的一些发现，这些发现目前尚无法通过进行体内的人体测试而检验。因此，我们的重编程微流体模型对于人类体内重编程的应用具有广阔的前景。