Mass Spectrometry–based Proteomic Analysis of the Matrix Microenvironment in Pluripotent Stem Cell Culture

Abstract

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 MSbased 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 matrixassociated 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. Molecular & Cellular Proteomics

Extracellular Matrices Decellularized from Embryonic Stem Cells Maintained Their Structure and Signaling Specificity

“…6,10 The results of the present study demonstrated the organization-and lineage-specific characteristics of ESC-derived ECMs and their differential signaling capacity in regulating ESC fate.…”

“…26 Parallel to the organizational dependence, the characteristics of PSC-derived ECMs are also influenced by lineage specifications. 6,23,29 For example, cerberus, a small antagonist of bone morphogenetic protein (BMP), was detected in the secretome and ECMs of ESCs undergoing cardiac differentiation, but not neural differentiation. 6,28 Finally, the ECMs derived at the early or late stage of EBs exhibited different signaling capacities, suggesting the influence of the developmental stage on ECM characteristics.…”

“…6,23,29 For example, cerberus, a small antagonist of bone morphogenetic protein (BMP), was detected in the secretome and ECMs of ESCs undergoing cardiac differentiation, but not neural differentiation. 6,28 Finally, the ECMs derived at the early or late stage of EBs exhibited different signaling capacities, suggesting the influence of the developmental stage on ECM characteristics. 10,11,28 The goal of the present study is to evaluate the influence of ESC organizations (i.e., undifferentiated monolayers, undifferentiated 3D aggregates, and differentiated EBs) and lineage commitment on the characteristics of the decellularized ECMs.…”

“…20,21 Compared to the ECMs derived from adult stem cells or somatic tissues, the decellularized matrices from PSCs may have a broader spectrum of signaling capacity owing to their embryonic origin, serving as novel biomaterials for in vivo cell and/or matrix delivery as well as in vitro cell expansion and differentiation. 6,10 The PSC-derived ECMs may have the reduced risk of tumor formation compared to the live cells, significantly improving their prospects in clinical applications.…”

“…4,5 In vitro cultured PSCs produce endogenous ECM proteins (i.e., fibronectin [FN], basement membrane proteins) that regulate PSC fate through cell adhesion and/or binding with autocrine factors (i.e., leukemia inhibitory factor [LIF], Wnt, and Activin). [6][7][8][9] Thus, the derivation of ECMs from PSC cultures while preserving their distinct signaling capacities will greatly enhance their potential in cell delivery and tissue repair. 10,11 Stem cell-derived ECMs have been used to support in vitro cell expansion and differentiation as well as in vivo tissue regeneration.…”