Abstract 17442: Retinoic Acid Protocol Combined With Glucose Starvation and Matrigel Plating Enhances Commitment of Human IPSC-derived Cardiomyocytes Into Atrial-like Phenotype
Introduction: Current cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hIPSC) mainly recapitulate immature ventricular-like phenotypes. However, this approach has not yet been used to generate patient-specific IPSC-derived atrial CMs. Here, we explore the role of retinoic acid (RA) combined with newer enrichment and monolayer-based cardiac differentiation methods (Matrigel mattress and small molecule-based differentiation) to direct hIPSC-CMs into an atrial-like phenotype.
Hypothesis: We hypothesize that a combined approach of RA with glucose starvation (gluc-starv) and Matrigel mattress method increases hIPSC-derived CMs commitment into a more atrial fate.
Methods: hIPSCs (Stanford IPSC Biobank) were plated at low density in human recombinant vitronectin (hVTN) and cultured until 90% confluence previous to cardiac differentiation. After small molecule treatment, cells were exposed to either RA or dimethyl-sulfoxide (DMSO, used to dissolve RA) and enriched by gluc-starv . Cells were replated in either Matrigel mattress, Matrigel coated plates or hVTN at day 15 after differentiation. RT-PCR, beating frequency (BF) and spontaneous calcium release events (sCRE) were recorded for all groups studied.
Results: Differentiated hIPSC-CMs showed upregulation of cardiac markers (NKX2.5, TNNT2 and GJA5). HIPSC-CMs also showed expression of Ca2+ handling genes (NCX1, RYR2 and ATP2A2) and ionic channels (SCN5A, KCNQ1 and KCNJ3). RA treated hIPSC-CMs showed enhanced expression of atrial specific markers (MLC2a, NPPA, COUPTFI and HEY1) and a higher BF vs. DMSO treated hIPSC-CMs (56.3±4.0 vs 30.4±1.7 beats/min., n=5, p<0.001). RA treated hIPSC-CM also have a size comparable to human atrial CMs. sCRE in CM exposed to RA+gluc-starv and plated in Matrigel mattress showed smaller slope (59.3±2.2 vs 104.1±7.9 F/Fo*s-1, n=5, p<0.001), shorter duration (131.2±2.1 vs 201.6±12.9 ms, n=5, p<0.001), shorter cycle length (193.4±5.1 vs 236.7±20.6 ms, n=5, p<0.001), and a smaller time to peak (20.3±0.5 vs 33.1±2.0 ms, n=5, p<0.001) than DMSO treated hIPSC-CM.
Conclusions: A combined approach of RA+gluc-starv. and Matrigel mattress plating enhances the commitment of CMs-derived from hIPSCs into a more mature atrial-like phenotype.
Author Disclosures: M. Argenziano: None. E. Savio Galimberti: None. D. Darbar: None.
- © 2016 by American Heart Association, Inc.