(535d) Systems-Level and Subcellular Resolution Dynamic Analysis of Trophectodermal Differentiation In Human Embryonic Stem Cells
AIChE Annual Meeting
Wednesday, October 19, 2011 - 1:30pm to 1:50pm
The pluripotency of human embryonic stem cells (hESCs) is regulated by a complex network of interacting pathways. Here we present a systems-level quantitative analysis of the molecular mechanisms regulating differentiation of hESCs along the trophectodermal lineage. The subcellular localization of proteins within a pathway, and their relative abundance govern the influence of a specific pathway on hESC fate. We developed a discontinuous sucrose gradient based protocol for simultaneous isolation of the nuclear, membrane and cytosolic subcellular fractions from undifferentiated and differentiating hESCs and conducted proteomic analysis on each of these three fractions. Further, we also used the method of spectral counting to estimate the relative abundance of all proteins identified in the cytosolic fraction. The integration of protein identifications with subcellular localization data and estimates of protein abundance allowed us to uncover several potential regulatory mechanisms pertaining to both hESC pluripotency and trophectodermal differentiation.
The hESC microenvironment plays a significant role in the regulation of signaling pathways in hESCs. The complex microenvironment of hESCs is determined in part by endogenous factors that are secreted by hESCs. However, experimental characterization of the hESC secretome is challenging. Since secreted proteins are synthesized in the endoplasmic reticulum (ER), we quantitatively characterized the microenvironment of hESCs as well as cells undergoing differentiation through proteomic analysis of the ER fractions. Interestingly, the list of secreted proteins identified using this approach is consistent with a previous proteomic analysis of hESC conditioned medium.
Taken together along with real-time PCR data, our proteomic analysis provides a quantitative understanding of the molecular processes regulating trophectodermal differentiation in hESCs.