Recently, we used a functional proteomic analysis to screen 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 the cardiogenesis of mouse iPSCs. We observed Nap1l1 was downregulated during the differentiation of iPSCs. Knockdown of Nap1l1 dramatically enhanced the differentiation of iPSCs to cardiomyocytes characterized by the increased number of beating embryonic bodies (EBs), the larger alpha-myosin heavy chain (α-MHC)-stained area and the upregulation of cardiac transcription factors (Nkx2.5, GATA4, Mef2c, Tbx5). The effects were sharply inhibited by Nap1l1 overexpression in iPSCs. Cardiomyocytes derived from Nap1l1-knockdown-iPSCs exhibited proper cell biological characteristics. Further study revealed that Nap1l1 knockdown in iPSCs promoted mesoderm (Flk-1, Brachyury and Mesp1) development, but Nap1l1 overexpression inhibited the effect. To explore whether Nap1l1 knockdown in iPSCs enhances cardiomyocytes differentiation by mesoderm induction. Mesoderm cells (Flk-1 positive cells) from iPSCs development were sorted by fluorescent-assisted cell sorting (FACS) and recultured to induce cardiomyocytes differentiation. The result revealed that the same number of Flk-1(mesodermal marker) positive cells from Nap1l1 knockdown, Nap1l1 overexpression or their control iPSCs didn’t show obvious difference in cardiomyocyte differentiation. Loss of Notch signaling in ES cells has been reported to favor commitment to a mesoderm and to induce cardiogenesis. The present study revealed that NICD and downstream genes (Hes1, Hes5, Hey1 and Hey 2) were positively regulated by Nap1l1 expression during differentiation of iPSCs. Notch signaling inhibitor greatly rescued the inhibitory effects of Nap1l1 overexpression on mesoderm induction and cardiogenesis. These findings demonstrate that downregulation of Nap1l1 significantly enhances mesodermal induction and subsequently promotes cardiogenesis from mouse iPSCs via regulating Notch signaling, which will facilitate application of iPSCs for heart diseases.
Nucleosome Assembly Protein 1-Like 1 (Nap1l1) Regulates the Proliferation of Murine Induced Pluripotent Stem Cells