Insulin-like growth factor binding protein 4 (IGFBP4) has been reported to play critical role in cardiomyocytes differentiation of embryonic stem cells (ESCs). But whether it promotes cardiomyocytes induction of iPSCs is unclear. In the present study, we aim to explore the role of IGFBP4 in the cardiogenesis of mouse iPSCs. We observed that IGFBP4 treatment at late stage during differentiation process of mouse iPSCs greatly enhanced the beating frequency of embryoid bodies (EBs). The expressions of Nkx2.5 (cardiac-specific transcription factor), ?-MHC, ?-actinin, and Troponin I (cardiac-specific protein) were significantly enhanced by IGFBP4 treatment. Immunostaining analysis showed that ?-MHC, TNNT2 and connexin 43, typical cardiac markers, were obviously expressed in isolated cardiomyocytes from iPSCs with or without IGFBP4 treatment. Further study revealed that IGFBP4 had little effect on the apoptosis of EBs, but it significantly promoted the proliferation of cardiomyocytes from iPSCs characterized by higher ratio EdU positive cells in differentiated cardiomyocytes. We next observed that IGFBP4 inhibited ?-catenin expression in cytosol of EBs at late stage during differentiation of iPSCs. Knockdown of ?-catenin using a siRNA technique promoted the proliferation of differentiated cardiomyocytes and enhanced cardiomyocytes induction of iPSCs, suggesting that the effect of IGFBP4 on cardiomyocytes differentiation of iPSCs has relationship with ?-catenin signaling pathway. In conclusion, IGFBP4 promotes cardiogenesis of iPSCs by enhancing the proliferation of differentiated cardiomyocytes through inhibiting ?-catenin signaling.
Low efficiency of cardiomyocyte differentiation from induced pluripotent stem cells (iPSCs) hinders the clinical application of iPSC technology for cardiac repair strategy. Recently, we screened out nucleosome assembly protein 1-like 1 (Nap1l1), which was downregulated during the differentiation of P19CL6 cells into cardiomyocytes. Here, we attempted to study the role of Nap1l1 in cardiomyogenesis of iPSC. Nap1l1 was downregulated during the differentiation of iPSC. Knockdown of Nap1l1 dramatically enhanced the differentiation of iPSC into functional cardiomyocytes while overexpression of Nap1l1 sharply lowered the differentiation. Moreover, although Nap1l1-knockdown had little effect on endoderm differentiation, the Nap1l1 modulation significantly accelerated mesoderm development. Re-expressing Nap1l1 in Nap1l1-knockdown-iPSC rescued the effects of Nap1l1. Inducibly overexpressing Nap1l1 at early stage of differentiation greatly inhibited mesoderm induction and cardiogenesis of iPSC. However, mesoderm stem cells (Flk-1-positive cells) originated from Nap1l1-knockdown- or -overexpression-iPSC showed no difference in further cardiomyocyte differentiation compared with that of control-iPSC. Further study revealed that Nap1l1-overexpression increased ?-secretase activity and the expression of Notch intracellular domain (NICD) and downstream genes during the differentiation of iPSC. ?-Secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycinet-butyl ester) greatly suppressed the production of NICD and abolished the inhibitory effects of Nap1l1-overexpression on mesoderm induction and cardiogenesis. These findings demonstrate that downregulation of Nap1l1 significantly enhances mesodermal induction and subsequent cardiogenesis of murine iPSC via inhibition of ?-secretase-regulated Notch signaling, which would facilitate the application of iPSC in heart diseases.
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