Secretome Analysis Performed During in vitro Cardiac Differentiation: Discovering the Cardiac Microenvironment

Abstract

Human pluripotent stem cells are an important tool for the study of developmental processes, such as cardiomyogenic differentiation. Despite the advances made in this field, the molecular and cellular signals involved in the commitment of embryonic stem cells to the cardiac phenotype are still under investigation. Therefore, this study focuses on identifying the extracellular signals involved in in vitro cardiac differentiation of human embryonic stem cells. Using a three-dimensional cardiomyogenic differentiation protocol, the conditioned medium and the extracellular matrix (ECM) of embryoid body cultures were collected and characterized at four specific time points. Mass spectrometry (MS) and antibody array analysis of the secretome identified a number of secreted proteins related to signaling pathways, such as Wnt and TGFβ, as well as many ECM proteins. When comparing the proteins identified at selected time points, our data pointed out protein interactions and biological process related to cardiac differentiation. Interestingly, the great changes in secretome profile occurred during the cardiac progenitor specification. The secretome results were also compared with our previous RNAseq data, indicating that the secreted proteins undergo some level of gene regulation. During cardiac commitment it was observed an increase in complexity of the ECM, and some proteins as IGFBP7, FN1, HSPG2, as well as other members of the basal lamina could be highlighted. Thus, these findings contribute valuable information about essential microenvironmental signals working on cardiomyogenic differentiation that may be used in future strategies for cardiac differentiation, cardiomyocyte maturation, and in advances for future acellular therapies.

The secretome from embryonic stem cell cardiomyogenesis: Same signals, different cellular feedbacks

“…Previously, our group verified that many proteins secreted at the final stages of hESC cardiac differentiation (D8/D9 and D15) were related to the cardiovascular system development (Robert et al, 2020). However, here, we demonstrated that CMd8 and Several studies showed the beneficial effects of using cell secretome in in vivo and in vitro investigations, assaying different types of cells and disease models (Ellison-Hughes & Madeddu, 2017;Ferreira et al, 2018;Leitolis, Suss et al, 2019;Li et al, 2019;S.…”

“…The CM was obtained at four time points of cardiac differentiation: Days 1 (CMd1), 4 (CMd4), 8 (CMd8), and 15 (CMd15), as previously described (Robert et al, 2020). After collection, CM was subjected to two centrifugations to remove cell debris and large apoptotic bodies (Figure S1).…”

“…Advances in embryonic stem cell (ESC) research (or induced pluripotent stem cells [iPSCs]) have allowed investigations on cardiac development through the use of in vitro cardiomyogenic differentiation protocols (Leitolis, Robert et al, 2019; Mummery et al, 2012). Wolling and colleagues, as well as our own group (Robert, Pereira, Dallagiovanna, & Stimamiglio, 2020) characterized the secretome of human embryonic stem cells (hESCs) during in vitro cardiomyogenic differentiation. Both results identified a set of proteins with relevant importance to cardiac development.…”

“…Both results identified a set of proteins with relevant importance to cardiac development. Furthermore, considering the proteins modulated during in vitro cardiomyogenesis, interesting biological processes, such as the regulation of cell migration, proliferation, and cardiovascular and blood vessel development, were identified (Robert et al, 2020).…”

Reactivation of atrium genes is a primer for heart infarction or regeneration

“…We also found in the present unbiased bioinformatics approach that atrial cardiomyocytes displayed secretory phenotype. The heart is regarded as a endocrine organ thirty years ago, and secretome profiling also the hallmark during cardiomyogenesis 27 . Expression of Nppa was an early and specific marker for the differentiating working myocardium of the atria and ventricles of the developing heart 28 .…”