Dataset Information

RNAseq time course for mature hiPS-CMs induced to proliferate with 4 cell cycle factors

ABSTRACT: The regenerative capacity of the heart after myocardial infarction (MI) is limited. Our previous study showed that ectopic introduction of Cdk1/CyclinB1 and Cdk4/CyclinD1 complexes (4F) promotes cardiomyocyte proliferation in 15-20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after MI. Here, we aim to identify the necessary reprogramming stages during the forced cardiomyocyte proliferation with 4F on a single cell basis. Temporal bulk RNAseq of mature hiPS-CMs treated with either LacZ or 4F adenoviruses revealed full cell cycle reprogramming in cardiomyocytes after 48 h post-infection with 4F, which was associated with sarcomere disassembly and metabolic reprogramming.

ORGANISM(S): Homo sapiens

PROVIDER: GSE174309 | GEO | 2022/04/05

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

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.

Dataset Information

RNAseq time course for mature hiPS-CMs induced to proliferate with 4 cell cycle factors

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure