Abstract
Cardiomyocyte regeneration is limited in adults. The adipose tissue-derived stromal vascular fraction (Ad-SVF) contains pluripotent stem cells that rarely transdifferentiate into spontaneously beating cardiomyocyte-like cells (beating CMs). However, the characteristics of beating CMs and the factors that regulate the differentiation of Ad-SVF toward the cardiac lineage are unknown. We developed a simple culture protocol under which the adult murine inguinal Ad-SVF reproducibly transdifferentiates into beating CMs without induction. The beating CMs showed the striated ventricular phenotype of cardiomyocytes and synchronised oscillation of the intracellular calcium concentration among cells on day 28 of Ad-SVF primary culture. We also identified beating CM-fated progenitors (CFPs) and performed single-cell transcriptome analysis of these CFPs. Among 491 transcription factors that were differentially expressed (≥ 1.75-fold) in CFPs and the beating CMs, myocyte-specific enhancer 2c (Mef2c) was key. Transduction of Ad-SVF cells with Mef2c using a lentiviral vector yielded CFPs and beating CMs with ~ tenfold higher cardiac troponin T expression, which was abolished by silencing of Mef2c. Thus, we identified the master gene required for transdifferentiation of Ad-SVF into beating CMs. These findings will facilitate the development of novel cardiac regeneration therapies based on gene-modified, cardiac lineage-directed Ad-SVF cells.
Highlights
The morbidity burden of heart failure is growing as a result of the worldwide increase in the incidence of ischemic heart disease
The mean number of beating SVF cell colonies on day 28 was 9.2 ± 0.7 per 100 mm dish, accounting for ~ 0.15% of all cells, whereas no beating cells were observed among passage 3 adipose tissue-derived stem cells (ASCs) (Supplementary Figure S1)
The action potential of a beating SVF cell on day 28 of primary culture was characterised by an initial rapid depolarisation phase that merged with a terminal repolarisation phase with a small plateau phase (Fig. 1n)
Summary
The morbidity burden of heart failure is growing as a result of the worldwide increase in the incidence of ischemic heart disease. Stem cell therapy has emerged as an adjuvant treatment for heart failure. As clinical trials have shown, somatic stem cells, including bone-marrow-derived stromal cells (BMSCs) and adipose tissue-derived stromal vascular fraction (Ad-SVF), have advantages in terms of safety and availability over iPS c ells[2,15], but they rarely differentiate into spontaneously beating cardiomyocytes. (e,f) Phase-contrast (e) and corresponding calcium ion fluorescence image of SVF beating clusters loaded with the calcium indicator fluo-8/AM (f) on day 14. Phase-contrast image of a cluster of beating SVF cells (e) and the corresponding calcium ion fluorescence image (i). (m) A representative calcium ion transient trace, represented as ΔF/F0, for a single beating SVF cell. (n) A representative action potential of a spontaneously beating SVF that was recorded on day 28 of primary culture (m) A representative calcium ion transient trace, represented as ΔF/F0, for a single beating SVF cell. (n) A representative action potential of a spontaneously beating SVF that was recorded on day 28 of primary culture
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