Vascular cells differentiated from peripheral blood mononuclear cell- versus urine cell-derived induced pluripotent stem cells: A comparative analysis

Abstract

Abstract Background: The use of peripheral blood mononuclear cells (PBMCs) and urine-derived epithelial cells for reprogramming towards induced pluripotent stem cells (iPSCs) has shown to be highly effective. Due to their easy accessibility, these cell sources hold promising potential for non-invasive and repetitive isolation from patients. This study aims to conduct a comparative analysis of the phenotype, differentiation efficacy, and functional properties of iPSCs derived from PBMCs and urine towards endothelial cells (EC) and vascular smooth muscle cells (VSMC). Methods: PBMC-derived iPSCs and urine-derived iPSCs were differentiated to ECs via embryoid body formation, followed by an in-vitro monolayer culture. SMCs were generated through a defined monolayer culture. The expression profiles of iPSCs, iPSC-derived ECs, and iPSC-derived VSMCs were assessed through various techniques such as immunofluorescence, RT-qPCR, western blot, and flow cytometry analysis. Functionality of ECs was evaluated with a tube formation assay, while the functional properties of VSMCs were assessed by measuring the contractile response to carbachol. Results: Both PBMC-derived and urine-derived iPSCs were successfully and efficiently differentiated into functional ECs and VSMCs. The efficacy of EC differentiation did not differ significantly between the two cell types, with both yielding approximately 45% mature ECs. The derived ECs displayed morphological and functional characteristics consistent with native ECs, including marker expression and tube formation. However, pluripotency marker SOX2 continued to be upregulated, while OCT4, KLF4, c-Myc, and SSEA-4 were downregulated. Functional assessment via tube formation assays showed no significant difference in the amount of newly formed tubes and branches between the two cell types. VSMC differentiation resulted in 96% and 94% α-SMA positive cells for PBMC-derived and urine-derived iPSCs, respectively. VSMCs of both origins exhibited a spindle-shaped, contractile morphology and expressed α-SMA, calponin, and transgelin consistent with native VSMCs. The generated VSMC lines from both cell sources demonstrated adequate contractility in response to carbachol. Conclusions: This study demonstrates a comparative analysis of functional ECs and VSMCs generated from PBMC-derived and urine-derived iPSCs. Comparison of morphology, expression profile, and functionality of vascular cells generated from both cell sources did not reveal significant differences.