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Reprogramming of Human Urine Stem Cells into Cardiomyocytes via a Small Molecule Cocktail in Xeno-free conditions

ABSTRACT: Reprogramming autologous somatic cells into alternative lineages holds significant promise as a crucial cell source for regenerative therapy. Among various sources, human urine stem cells (hUSCs) have emerged as a highly promising candidate for reprogramming due to their ease of collection and high transdifferentiation potential. In this study, we present the first evidence for cardiac regeneration by directly converting hUSCs into functional cardiomyocytes under xeno-free conditions using a cocktail of 15 small molecules. This innovative protocol ensures a safe, animal-free reprogramming environment, enhancing its clinical applicability. The resulting chemically induced human cardiomyocyte-like cells (hCiCMs) exhibit several key characteristics of native cardiomyocytes, including the expression of cardiomyocyte-specific markers, display sarcomeric organization, and exhibit typical cardiac calcium flux and electrophysiological properties. Notably, transplantation of hCiCMs into the infarcted hearts of mice led to substantial improvements in cardiac function, characterized by enhanced contractility and reduced infarct size. Our findings validate the efficacy of the xeno-free differentiation protocol and highlight the potential of hUSC-derived hCiCMs as a viable and effective source for regenerative therapies in myocardial infarction. The use of hUSCs provides a readily accessible, autologous cell source, thereby paving the way for personalized and safer regenerative treatments for cardiac diseases.

ORGANISM(S): Homo sapiens

PROVIDER: GSE272220 | GEO | 2025/07/01

REPOSITORIES: GEO

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Project description:Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persists for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods. Mouse embryonic fibroblasts were treated with different combinations of transcription factors to drive transdifferentiation to induced cardiomyocytes (iCMs). Putative iCMs were enriched by zeocin selection. Zeocin resistance was conferred to iCMs via the TroponinT-GCaMP5-Zeo lentiviral reporter.

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Reprogramming of Human Urine Stem Cells into Cardiomyocytes via a Small Molecule Cocktail in Xeno-free conditions

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