Production of endothelial progenitor cells from skin fibroblasts by direct reprogramming for clinical usages

Abstract

Endothelial progenitor cells (EPCs) play an important role in angiogenesis. However, they exist in limited numbers in the human body. This study was aimed to produce EPCs, for autologous transplantation, using direct reprogramming of skin fibroblasts under GMP-compliant conditions. Fibroblasts were collected and cultured from the skin in DMEM/F12 medium supplemented with 5% activated platelet-rich plasma and 1% antibiotic-antimycotic solution. They were then transfected with mRNA ETV2 and incubated in culture medium under hypoxia (5% oxygen) for 14 d. Phenotype analysis of transfected cells confirmed that single-factor ETV2 transfection successfully reprogrammed dermal fibroblasts into functional EPCs. Our results showed that ETV2 mRNA combined with hypoxia can give rise to functional EPCs. The cells exhibited functional phenotypes similar to endothelial cells derived from umbilical cord vein; they expressed CD31 and VEGFR2, and formed capillary-like structures in vitro. Moreover, these EPCs could significantly improve hindlimb ischemia in mouse models. Although the direct conversion efficacy was low (3.12 ± 0.98%), altogether our study demonstrates that functional EPCs can be produced from fibroblasts and can be used in clinical applications.

Direct cell-fate conversion of somatic cells: Toward regenerative medicine and industries

“…Since higher animals, including humans, do not have reverse transcriptase, there are no risks of integration of the transduced mRNAs into their genome. Many studies using mRNA transduction have been reported, e.g., hepatocytes, 59),115) cardiomyocytes, 116) neuronal cells, 40) endothelial progenitor cells, 117) and neural stem/ precursor cells. 34),118) Nevertheless, several difficulties of this method, such as stability and transfection efficiency, remain.…”

Vascular Tissue Engineering: Advanced Techniques and Gene Editing in Stem Cells for Graft Generation

“…reprogrammed into functional EPCs by transfecting with ETV2-encoding modRNA in combination with hypoxia treatment. 126 In addition, induced differentiation of hESCderived Isl + cells into ECs has also been achieved by transfecting the modRNA encoding VEGFA. 127 hPSCs were also differentiated into typical endothelial phenotype by delivery of modRNA encoding ETV2.…”

“…123 It requires multiple transfections in cell fate conversion. 123,126 The high cost of modRNAs and their sensitivity to heat and nuclease limit their application in tissue engineering. 123 CRISPR/Cas9-mediated gene editing.…”

Concise Review: Application of Chemically Modified mRNA in Cell Fate Conversion and Tissue Engineering

“…Very recently, 7.5% 5‐iodo‐cytidine and 35% 5‐iodo‐uridine modification of BMP2 mRNA has been used for bone regeneration . Nonetheless, differentiation to endothelial progenitor cells using ETV2 cmRNA harboring no modified nucleotides has also been reported .…”

“…cmRNAs have been used for differentiation and trans‐differentiation into a variety of cell types including neurons , β‐cells , endothelial progenitors , cardiovascular cells , and myogenic cells . However, the majority of studies using cmRNA for cell differentiation and tissue engineering have been performed on bone, and to a lesser extent, on heart regeneration.…”