Seon Pyo Hong 1, Sukhyun Song 2, Sung Woo Cho 3, Seungjoo Lee 3, Bong Ihn Koh 3, Hosung Bae 1 2, Kyun Hoo Kim 2 3, Jin-Sung Park 2 3, Hyo-Sang Do 4, Ilkyun Im 4, Hye Jin Heo 5, Tae Hee Ko 5, Jae-Hyeong Park 5, Jae Boum Youm 5, Seong-Jin Kim 6, Injune Kim 1 3, Jin Han 5, Yong-Mahn Han 4, Gou Young Koh 1 2 3
Abstract
Isolating actively proliferating cardioblasts is the first crucial step for cardiac regeneration through cell implantation. However, the origin and identity of putative cardioblasts are still unclear. Here, we uncover a novel class of cardiac lineage cells, PDGFRα+Flk1- cardioblasts (PCBs), from mouse and human pluripotent stem cells induced using CsAYTE, a combination of the small molecules Cyclosporin A, the rho-associated coiled-coil kinase inhibitor Y27632, the antioxidant Trolox, and the ALK5 inhibitor EW7197. This novel population of actively proliferating cells is cardiac lineage-committed but in a morphologically and functionally immature state compared to mature cardiomyocytes. Most important, most of CsAYTE-induced PCBs spontaneously differentiated into functional αMHC+ cardiomyocytes (M+CMs) and could be a potential cellular resource for cardiac regeneration.
(A) Protocol to generate PCBs from Flk1+ MPC by CsAYTE stimulation and subsequent analyses. (B) Phase-contrast images showing differentiating Flk1+ MPCs at day 6.0 incubated with control vehicle (Control), CsA, and CsAYTE. Scale bars, 100 μm. (C–K) Representative FACS analyses, quantifications, and images of PDGFRα+Flk1− PCBs, PDGFRα+ Nkx2.5+ cells, and PDGFRα+ cTnT+ cells differentiated from Flk1+ MPCs at day 6.0 incubated with Control, CsA, and CsAYTE (Scale bars, 100 μm). Each group, n = 3–6. p < 0.05 and *p < 0.01 versus Con; #p < 0.05 and ##p < 0.01 versus CsA. (L) Protocol for analyses of PCB-derived cardiomyocyte differentiation in a feeder-free culture. (M and N) Representative FACS analyses and percentages of PCBs-derived cTnT+ and αMHC-GFP+ cardiomyocytes grown in feeder-free culture. Each group, n = 4–5.
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