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Secretome analysis during human induced pluripotent stem cell-derived cardiomyocyte differentiation

ABSTRACT: Proteomic analysis of conditioned media during cardiomyocyte differentiation from induced pluripotent stem cells.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yuki Saito

PROVIDER: PXD074366 | JPOST Repository | Thu Aug 06 00:00:00 BST 2026

REPOSITORIES: jPOST

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Project description:Cardiovascular diseases are a major cause of life-threatening burden around the world. The heart has a very low regeneration capacity and donor organs for transplantation are scarce. Therefore regeneration of lost myocardium with stem cell-derived cardiomyocytes (CMs) provides an attractive strategy for heart repair. Human pluripotent stem cells (hPSCs) can be efficiently differentiated in vitro into CMs but the molecular mechanisms behind this process are still not fully understood. In particular identification of secreted autocrine and/or paracrine factors that function as important extrinsic signals remained elusive because the mass spectrometry (MS)-based identification of secreted proteins from cell culture supernatants is impeded by high levels of albumin present in common differentiation media. Thus we established an albumin-free cardiomyogenic differentiation medium and performed secretomics at seven different time points during in vitro differentiation. This analysis led to the identification of 4832 proteins with 1802 being significantly altered during differentiation and 431 of these were annotated as secreted according to gene ontology. Bioinformatics revealed enrichment of extrinsic Wnt pathway-related proteins 3 days upon induction of differentiation and of extracellular matrix proteins in the resulting CMs. Numerous extrinsic components of Wnt, Activin A, Nodal, TGFβ, BMP or FGF signaling pathways were quantitatively assessed during differentiation. Notably, the abundance of pathway agonists was generally lower compared to the respective antagonists but their curves of progression over timer were rather similar. We hypothesize that Activin A, Nodal and TGFβ signaling are turned down shortly upon initiation of cardiac differentiation whereas BMP signaling is switched on. Wnt and FGF signaling peaks between d0 and d3 of differentiation and interestingly, Activin A and TGFβ signaling seem to be reactivated at the cardiac progenitor stages and/or in early CMs.

Project description:The identified stromal factors SDF1alpha, sFRP1 and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells. Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons are potentially useful for treating Parkinson’s disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by co-culture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factor(s) produced by stromal cells that constitute SDIA is unknown. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons in the human embryonal carcinoma stem cell line, NTera2. Here we further examined the effects of PA6-conditioned medium and found that it can induce DA neuronal differentiation in both the NTera2 cell line and the hESC line, I6. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with proteomic analysis of PA6-conditioned medium. Four candidate factors (hepatocyte growth factor (HGF), stromal cell-derived factor-1 alpha (SDF1alpha), secreted frizzled-related protein 1 (sFRP1) and vascular endothelial growth factor D (VEGFD)) were identified and immunoaffinity capillary electrophoresis (ICE) was used to establish the protein concentration of these factors in conditioned medium. Upon addition of SDF1alpha, sFRP1, and VEGFD, we observed an increase in the number of tyrosine hydroxylase- and TuJ1- positive cells in both the NTera2 and I6 cell lines. These results indicate that SDF1alpha, sFRP1 and VEGF-D are major components of SDIA, and suggest the potential use of these defined factors to elicit dopaminergic differentiation of pluripotent stem cells as a therapeutic intervention in PD. Mouse embryonic fibroblasts were grown in DMEM medium supplemented with 10% FBS; this conditioned media was used as the control. PA6, mouse stromal cells, were grown in MEM-alpha supplemented with 10% FBS; this conditioned media is known to induce differentiation in hES cells.

			Action	DRS
	E01_hiPSC-CM_Day10.raw	Raw
	E01_hiPSC-CM_Day12.raw	Raw
	E01_hiPSC-CM_Day14.raw	Raw
	E01_hiPSC-CM_Day16.raw	Raw
	E01_hiPSC-CM_Day17.raw	Raw

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Secretome analysis during human induced pluripotent stem cell-derived cardiomyocyte differentiation

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