Abstract 17415: Generation of Cardiac/Skeletal Myogenic Progenitors From Human Induced Pluripotent Stem Cells Using a Small Molecule

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Abstract

Background: The aim was to evaluate the conversion of human induced pluripotent stem cells (hiPSCs) into cardiac (CMPs) or skeletal myogenic progenitors (SMPs) with a single small molecule for cell therapy.

Methods and Results: hiPSCs (purchased as cell line) were cultured as a monolayer and treated with a small molecule, Givinostat (GIV, a histone deacetylase inhibitor, 10-200nM) for 7 days. Higher cell viability (CCK8 Assay) and lower cytotoxicity (LDH Assay) was observed with concentration of 150nM GIV. GIV had dual effect by generating both CMPs and SMPs after 7 days. In addition to GIV, mTeSRTM1 serum-free medium was used with Rho-associated kinase (ROCK) inhibitors (5μM Thiazovivin or 1μM Y27632) for SMPs specification; and mTeSRTM1 serum-free medium was used with ALK4/5/7 (TGFβ type-I receptor) inhibitors (2μM SB431542 or 1μM A83-01) for CMPs specification; and was assessed after 4-8 days. GIV increased upregulation of skeletal myogenic genes (Real Time PCR-analysis) at first 4 days and then was further increased at a higher level after 8 days. Muscle genes included Meox1 (21.4-fold), Meox2 (3.9-fold), Tcf15 (3.5-fold), Pax3 (28-fold), Pax7 (3.2-fold), MyoD1 (1.7-fold), dystrophin (3.8-fold), myogenin (6-fold), Myh2 (5.6-fold), Myh6 (3.4-fold), Tbx1 (2.6-fold), Mesp1 (2.4-fold), desmin (1.9-fold) and β-catenin (1.6-fold) versus control. PCR results were confirmed with Western blots densitometry including Pax3 (7-fold), Pax7 (2.5-fold), Myf5 (8-fold), MyoD1 (2-fold), dystrophin (6-fold) and desmin (5-fold) versus control (P<0.05). Morphologically, the myotube or myocyte-like shape of differentiating cells were observed microscopically. GIV increased upregulation of cardiac myogenic genes after 4 days with a higher level after 8 days. Cardiac genes included Pitx2 (56-fold), ISl1 (6.9-fold), Nkx2.5 (48-fold), Hand1 (14.1-fold), GATA4 (5-fold), Tbx5 (3.8-fold), TnnT2 (5.7-fold), Myl7 (16.1-fold), MLC2v (8.9-fold), Myf2c (1.4-fold), Cdh4 (3.1-fold) and Lhx2 (2.7-fold) versus control.

Conclusions: Pretreatment of hiPSCs with GIV represents a viable strategy for producing both cardiac/skeletal myogenic progenitors in vitro for cell therapies against myocardial infarction and Duchenne muscular dystrophy.

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