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Differentiation Protocol-Dependent Variability in hiPSC-Derived Endothelial Progenitor Functionality

ABSTRACT: With multiple groups generating methods for differentiating induced pluripotent stem cells (hiPSCs) into endothelial cells/endothelial progenitors (ECs/EPs), there is a need to better understand the specific endothelial subtypes that different differentiation protocols produce. This is especially important for accurate tissue modeling as researchers continue to incorporate the endothelium into engineered tissues. We illustrated the heterogeneity of cells produced from different differentiation protocols by focusing on two selected protocols, one driving differentiation exclusively using small molecules (SM Protocol) and one driving differentiation primarily using growth factors (GF Protocol). We characterized the cells through a combination of vasculogenic computational analysis following encapsulation in 3D hydrogels, RNA sequencing, and measurement of soluble factor release. Vasculogenic computational analysis indicates that cells from the GF protocol formed more dense and interconnected vasculature compared to cells from the SM protocol. Likewise, RNA-seq analysis showed GF-derived cell enrichment in pathways involved in cell migration and angiogenesis. In addition, GF-derived cells favor differentiation to arterial endothelial cells as well as predisposition to undergoing a partial endothelial-to-mesenchymal transition, whereas SM-derived cells resemble immature progenitors based on higher proliferation rates as well as ECM remodeling. These trends also persisted following extended 3D culture. The results demonstrate that despite using the same starting hiPSC population and isolating EPs using the same surface marker, the two differentiation protocols yield highly distinct cell populations. This work highlights the importance of understanding the specific endothelial subtypes produced by different differentiation protocols for the creation of more accurate tissue models.

ORGANISM(S): Homo sapiens

PROVIDER: GSE278088 | GEO | 2024/09/26

REPOSITORIES: GEO

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Project description:Regular physical exercise evokes profound physiological responses which are strongly associated with many health benefits. However, the details of cellular networks and mechanisms underlying the adaptive responses to exercise remain to be elucidated. We have previously shown the usefulness of electrical pulse stimulation (EPS) to investigate metabolic effects of exercise in cultured human myotubes. The aim of the present study was to uncover networks of signaling pathways and regulatory molecules responsible for the metabolic effects of exercise in human skeletal muscle cells exposed to chronic EPS. Differentiated myotubes were subjected to two different EPS protocols (protocol 1; 2 ms, 10 V, 0.1 Hz for 24 h or protocol 2; 2 ms, 30 V, and 1 Hz for 48 h). Fuel handling was assessed using radiolabeled substrates. The transcriptome, cell proteome, and secreted proteins from EPS-treated and untreated myotubes were analyzed using a combination of high-throughput RNA sequencing, microarray, and high-resolution liquid chromatography mass spectrometry. Independent validation of selected putative myokines were measured using ELISA or multiplex assay. Oxidative metabolism was enhanced in human myotubes exposed to EPS protocol 1. A total of 81 differentially regulated proteins and 952 differentially expressed genes (DEGs) were observed in the cells after EPS protocol 1. Gene ontology (GO) analysis indicated that significantly regulated proteins and genes were enriched in biological processes related to glycolytic pathways, positive regulation of fatty acid oxidation, and oxidative phosphorylation, as well as muscle contraction, autophagy/mitophagy, and oxidative stress. Moreover, proteomic identification of secreted proteins revealed extracellular levels of 137 proteins were changed in myotubes subjected to EPS protocol 1. We also found some degree of overlap between the DEGs found in myotubes submitted to EPS protocol 1 and protocol 2. Among these DEGs was a myokine, leukemia inhibitory factor, which showed to enhance the glucose uptake in skeletal muscle cells, indicating autocrine mechanism to maintain metabolic homeostasis. Collectively, our data provides new insight into the transcriptome, proteome and secreted proteins alterations following in vitro exercise and is a valuable resource for understanding the molecular mechanisms and regulatory molecules mediating the beneficial metabolic effects of exercise.

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Differentiation Protocol-Dependent Variability in hiPSC-Derived Endothelial Progenitor Functionality

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