Attenuation of extrinsic signaling reveals the importance of matrix remodeling on maintenance of embryonic stem cell self-renewal

Abstract

The role of extrinsic factors in maintaining self-renewal of embryonic stem cells (ESCs) has been extensively studied since the cells' isolation, but the necessity for cell-secreted factors in self-renewal has remained undefined to date. Although it is generally accepted that addition of leukemia inhibitory factor (LIF) together with either serum or bone morphogenetic protein 4 (BMP4) is sufficient to maintain mouse ESCs (mESCs) in a selfrenewing state, this does not preclude the possibility that autocrine factors are also required. Here we make use of a microfluidic perfusion device that is able to globally diminish diffusible autocrine signaling by applying continuous media flow to deplete cellsecreted factors. We demonstrate mESC culture for several days under continuous microfluidic perfusion and show that cell-secreted factors are removed and can be recovered downstream. We find that perturbing cell-secreted signaling causes mESCs to exit their stable self-renewing state in defined conditions that normally support self-renewal and to exhibit properties characteristic of epiblast cells. This state change is not due to the presence of the known autocrine differentiation inducer fibroblast growth factor 4, but, remarkably, it can be prevented by global remodeling of the extracellular matrix (ECM). We also find that cell-secreted matrix remodeling proteins are removed under perfusion and that inhibition of extracellular matrix remodeling causes mESCs to differentiate. Taken together, our data indicate that LIF and BMP4 are not sufficient to maintain self-renewal and that cell-secreted factors are necessary to continuously remodel the ECM and thereby prevent differentiation, revealing a previously undescribed level of mESC regulation through the use of microfluidic perfusion technology. I t has long been known that cell-secreted signals are required for cellular processes such as growth, survival, differentiation, metastasis, and apoptosis (1-5). However, the precise contributions of autocrine and/or paracrine signals to a particular process are often difficult to determine. When the cell-secreted factors and/or receptors are known, one can use chemical or genetic inhibition of target molecules, derivation of knockout cell lines, or overexpression of candidate molecules and receptors to study autocrine/paracrine processes. However, when the cell-secreted factors are unknown, one is typically limited to varying cell density and looking for density-dependent phenotypes. Because autocrine loops can be self-sufficient even at clonal density (6), these methods are incomplete.Pluripotent stem cells isolated from the developing blastocyst are well-suited for the study of cell-secreted signaling, because extrinsic signals generated by the embryo are essential for proper development (7,8), and autocrine and paracrine signals are likewise important in stem cell self-renewal (9), growth (3), and differentiation (1, 10). Mouse embryonic stem cells (mESCs) are pluripotent cells derived from the inner cell mass of preimplanta...

LIF supports primitive endoderm expansion during pre-implantation development

“…Perhaps one mechanism by which LIF supports the expansion of the ICM is through the production of basement membrane required for its survival. It has been shown in ESCs that basement membrane is important to maintain ESCs in an undifferentiated state (Przybyla and Voldman, 2012). Additionally, the only way in which individual Epi cells can be shown to generate ESC lines in the presence of a pharmacological block to PrE specification (inhibition of MEK) is by the provision of specific ECM components (Boroviak et al, 2014).…”

Mesenchymal stem cell mechanobiology and emerging experimental platforms

“…MSC heterogeneity is efficiently studied using microfluidic platforms that provide controllable medium perfusion [152][153][154] for long-term culture of individually captured cells [155], high-density arrayed cell cultures (figure 7b) [156] and arrayed cell niches [157,158]. These and similar platforms enabled on-chip gene profiling [156,159,160] and highthroughput single cell bioinformatics [161].…”

Differential Expression of Extracellular Matrix and Growth Factors by Embryoid Bodies in Hydrodynamic and Static Cultures

“…[10][11][12][13] The use of continuous microfluidic perfusion devices, for example, can alter extracellular matrix (ECM) remodeling processes and cause mouse ESCs to spontaneously differentiate. 14 Furthermore, the expression profile of extracellular molecules has been shown to be dynamically modulated during ESC differentiation in a rotary orbital culture system. 15 Altogether, previous studies suggest that ECM and growth factor (GF) molecules comprising the microenvironment play a key role in directing stem cell fate, but the differences in endogenous matrix production and expression of relevant molecules elicited by simple changes in hydrodynamics in differentiating PSCs have yet to be elucidated.…”