Cardiac Progenitors Induced from Human Induced Pluripotent Stem Cells with Cardiogenic Small Molecule Effectively Regenerate Infarcted Hearts and Attenuate Fibrosis

Abstract

Cardiac progenitor cells (CPCs) being multipotent offer a promising source for cardiac repair due to their ability to proliferate and multiply into cardiac lineage cells. Here, we explored a novel strategy for human CPCs generation from human induced pluripotent stem cells (hiPSCs) using a cardiogenic small molecule, isoxazole (ISX-9) and their ability to grow in the scar tissue for functional improvement in the infarcted myocardium. CPCs were induced from hiPSCs with ISX-9. CPCs were characterized by immunocytochemistry and RT-PCR. The CPC survival and differentiation in the infarcted hearts were determined by in vivo transplantation in immunodeficient mice following left anterior descending artery ligation and their effects were determined on fibrosis and functional improvement. ISX-9 simultaneously induced expression of cardiac transcription factors, NK2 homeobox 5, islet-1, GATA binding protein 4, myocyte enhancer factor-2 in hiPSCs within 3 days of treatment and successfully differentiated into three cardiac lineages in vitro. Messenger RNA and microRNA-sequencing results showed that ISX-9 targeted multiple cardiac differentiation, proliferation signaling pathways and upregulated myogenesis and cardiac hypertrophy related-microRNA. ISX-9 activated multiple pathways including transforming growth factor β induced epithelial-mesenchymal transition signaling, canonical, and non-canonical Wnt signaling at different stages of cardiac differentiation. CPCs transplantation promoted myoangiogenesis, attenuated fibrosis, and led to functional improvement in treated mice.

miRNAs in Extracellular Vesicles from iPS-Derived Cardiac Progenitor Cells Effectively Reduce Fibrosis and Promote Angiogenesis in Infarcted Heart

“…Human iPSC cell line ACS-1021 (ATCC, USA) and CPCs induced by ISX-9 were cultured as described [15]. In some cases, EBs and commercial human CPCs were also cultured.…”

“…Animal experiments were carried out both at the University of Illinois at Chicago and Augusta University according to experimental protocols approved by the University of Illinois at Chicago and Augusta University Animal Care and Use Committee, and the methods were performed in accordance with the guide for the Care and Use of Laboratory Animals by the Institute of Animal Resources. MI model was generated as previously described [15]. Briefly, MI was induced in 8-9-week-old NOD/SCID mice (The Jackson Laboratory) or C57/B6 mice which were anaesthetized with 2% isoflurane (isoflurane USP, Henry Schein), intubated, and ventilated.…”

“…Echocardiography was performed in mice anesthetized mildly with inhaled isoflurane (0.5%) using a Philips iE33 ultrasound machine, equipped with a L15-7io probe as described previously [15]. Hearts were imaged in 2D in the parasternal long-axis and/or parasternal short-axis views at the level of the highest LV diameter.…”

“…Multiple miRNAs in EV act as mediators of cell-cell communication within the cardiovascular system [2] and can be transferred into recipient cells to regulate gene expression, thus leading to cardioprotection [11, 13, 14]. We reported that a small molecule, ISX-9, could render CPCs (CPC ISX-9 ) highly resistant to oxidative stress, thus permitting better survival and engraftment in the infarcted myocardium [15]. Development of CPC ISX-9 may represent a significant advance in the cardiac stem cell field, as ISX-9 treatment circumvents the need to genetically reprogram the cells in order to enhance their function.…”

Cardiac Progenitor Cells from Stem Cells: Learning from Genetics and Biomaterials

“…Furthermore, the study used co-culture with END2 cells to induce cardiac differentiation in iPSCs, which prevents reproducibility of the protocol due to the presence of END2-derived growth factors at unknown concentrations [135]. Another study also demonstrated that treating human iPSCs with the cardiogenic small molecule isoxazole (ISX-9) for 7 days stimulated the expression of CPC markers [136]. These CPCs expressed NKX2.5, GATA4, ISL1, and MEF2C and were able to generate cardiomyocytes, smooth muscle cells and endothelial cells under basal differentiation conditions.…”