Don Rix Protein Identification Facility, Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada.
Mol Cell Proteomics. 2012 Dec;11(12):1924-36. doi: 10.1074/mcp.M112.020057. Epub 2012 Sep 27.
The cellular microenvironment comprises soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to regulate cellular behavior. Pluripotent stem cells utilize interactions between support cells and soluble factors in the microenvironment to assist in the maintenance of self-renewal and the process of differentiation. However, the ECM also plays a significant role in shaping the behavior of human pluripotent stem cells, including embryonic stem cells (hESCs) and induced pluripotent stem cells. Moreover, it has recently been observed that deposited factors in a hESC-conditioned matrix have the potential to contribute to the reprogramming of metastatic melanoma cells. Therefore, the ECM component of the pluripotent stem cell microenvironment necessitates further analysis. In this study we first compared the self-renewal and differentiation properties of hESCs grown on Matrigel™ pre-conditioned by hESCs to those on unconditioned Matrigel™. We determined that culture on conditioned Matrigel™ prevents differentiation when supportive growth factors are removed from the culture medium. To investigate and identify factors potentially responsible for this beneficial effect, we performed a defined SILAC MS-based proteomics screen of hESC-conditioned Matrigel™. From this proteomics screen, we identified over 80 extracellular proteins in matrix conditioned by hESCs and induced pluripotent stem cells. These included matrix-associated factors that participate in key stem cell pluripotency regulatory pathways, such as Nodal/Activin and canonical Wnt signaling. This work represents the first investigation of stem-cell-derived matrices from human pluripotent stem cells using a defined SILAC MS-based proteomics approach.
细胞微环境由可溶性因子、支持细胞和细胞外基质（ECM）的组成部分组成，它们共同调节细胞行为。多能干细胞利用微环境中支持细胞和可溶性因子之间的相互作用来协助维持自我更新和分化过程。然而，细胞外基质在塑造人类多能干细胞（包括胚胎干细胞（hESCs）和诱导多能干细胞）的行为方面也起着重要作用。此外，最近观察到 hESC 条件培养基中的沉积因子有可能有助于转移性黑色素瘤细胞的重编程。因此，需要进一步分析多能干细胞微环境中的细胞外基质成分。在这项研究中，我们首先比较了在 hESC 预条件化的 Matrigel™上生长的 hESC 的自我更新和分化特性与未条件化的 Matrigel™上生长的 hESC 的特性。我们确定，当培养基中去除支持性生长因子时，在条件化 Matrigel™上培养可防止分化。为了研究和鉴定可能对此有益效果负责的因素，我们对 hESC 条件化 Matrigel™进行了基于 SILAC MS 的定义蛋白组学筛选。从这个蛋白质组学筛选中，我们鉴定了 hESC 和诱导多能干细胞条件化 Matrigel™中超过 80 种细胞外蛋白。这些包括参与关键干细胞多能性调节途径的基质相关因子，如 Nodal/Activin 和经典 Wnt 信号通路。这项工作代表了使用基于 SILAC MS 的定义蛋白组学方法首次对人类多能干细胞衍生的基质进行的研究。
