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Direct reprogramming of adult adipose-derived regenerative cells toward cardiomyocytes using six transcriptional factors II

ABSTRACT: Direct reprogramming of adult adipose-derived regenerative cells toward cardiomyocytes using six transcriptional factors II

PROVIDER: PRJNA844031 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR19451758_1.fastq.gz	Fastqsanger.gz
	SRR19451758_2.fastq.gz	Fastqsanger.gz
	SRR19451759_1.fastq.gz	Fastqsanger.gz
	SRR19451759_2.fastq.gz	Fastqsanger.gz
	SRR19451760_1.fastq.gz	Fastqsanger.gz

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Project description:Human embryonic and induced pluripotent stem cells (hESCs/iPSCs) are promising cell sources for cardiac regenerative medicine. To realize hESC/iPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes should be required. Though marker gene transduction or fluorescent-based purification methods were reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purposes but have not been established yet. In this study, we used microarrays to detail the global gene program during cardiac differentiation and to identify cardiac-specific cell surface markers. hiPSCs (201B6) were differentiated toward cardiomyocytes using a modified-directed differentiation protocol (high density culture in RPMI+B27-insulin, sequential administration of Activin A 100ng/mL 1 day, BMP4 10ng/mL+bFGF 10ng/mL 4 days, and Dkk1 100ng/mL 2 days). Beating clusters were first observed at day 8-9 and spread by day 11 after Activin A administration. Cardiac troponin-T (cTnT)-positive cells appeared at day 7-8 after induction and were observed in 30-70% of cells at day 11. qPCR and genome-wide analysis reflected differentiation processes from the undifferentiated state to cardiomyocytes. Rapid downregulation of pluripotent stem cell markers such as NANOG and POU5F1 was observed within 2 days of differentiation. Early and cardiac mesodermal genes (T, MESP1, KDR, ISL1) were expressed during day 2-5, and cardiac genes (NKX2-5, MYH6, MYH7, MYL2, and MYL7) were expressed after day 7. We identified VCAM1 as a cardiac-specific cell surface marker by microarray and flow cytometry. Human induced pluripotent stem cells (iPSCs; 201B6) were differentiated toward cardiomyocytes (RPMI+B27 medium supplemented d0-1 Activin A, d1-5 BMP4+bFGF, d5-7 Dkk1). RNA was extracted from cells at day 0, day 2, day 5, day 7, day 9, and day 11. Cardiomyocytes appeared after day 7 and reached about 50% of total cells at day 11.

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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Direct reprogramming of adult adipose-derived regenerative cells toward cardiomyocytes using six transcriptional factors II

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