Bone morphogenetic protein-4 promotes induction of cardiomyocytes from human embryonic stem cells in serum-based embryoid body development

Abstract

Cardiomyocytes derived from human embryonic stem (ES) cells are a potential source for cell-based therapy for heart diseases. We studied the effect of bone morphogenetic protein (BMP)-4 in the presence of fetal bovine serum (FBS) on cardiac induction from human H1 ES cells during embryoid body (EB) development. Suspension culture for 4 days with 20% FBS produced the best results for the differentiation of early mesoderm and cardiomyocytes. The addition of Noggin reduced the incidence of beating EBs from 23.6% to 5.3%, which indicated the involvement of BMP signaling in the spontaneous cardiac differentiation. In this condition, treatment with 12.5–25 ng/ml BMP-4 during the 4-day suspension optimally promoted the cardiomyocyte differentiation. The incidence of beating EBs at 25 ng/ml BMP-4 reached 95.8% on day 6 of expansion and then plateaued until day 20. In real-time PCR analysis, the cardiac development-related genes MESP1 and Nkx2.5 were upregulated in the EB outgrowths by 25 ng/ml BMP-4. The activation of BMP signaling in EBs was confirmed by the increase in the phosphorylation of Smad1/5/8 and by the nuclear localization of phospho-Smad1/5/8 and Smad4. The addition of 150 ng/ml Noggin considerably decreased the incidence of beating EBs and Nkx2.5 expression, and Noggin alone increased Nestin expression and neural differentiation in EB outgrowths. The cardiomyocytes induced by 25 ng/ml BMP-4 showed proper cell biological characteristics and a course of differentiation as judged from isoproterenol administration, gene expression, protein assay, immunoreactivity, and subcellular structures. No remarkable change in the extent of apoptosis and proliferation in the cardiomyocytes was observed by BMP-4 treatment. These findings showed that BMP-4 in combination with FBS at the appropriate time and concentrations significantly promotes cardiomyocyte induction from human ES cells.

Mesoderm is committed to hemato-endothelial and cardiac lineages in human embryoid bodies by sequential exposure to cytokines

“…Brachyury is also expressed at the cardiac precursor stage [33]. Previous work showed that BMP4 at 25 ng/ml concentration in combination with FBS at the appropriate time significantly promotes cardiomyocyte induction from human ES cells [34], consistent with the data generated in our study. We also analyzed the expression of Flk1 in all groups and observed that the levels of Flk1 in bFGF-induced cells were significantly increased at day 7 of differentiation and then sharply decreased thereafter.…”

Transcriptional Networks of Embryonic Stem Cell-Derived Cardiomyogenesis

“…Laflamme et al (2007) demonstrate that Bmp4 treatment increased in vitro cardiomyogenesis using human embryonic stem cells and Paige et al (2010) has elegantly shown a balanced interplay between activin A/Bmp4 and Wnt/-catenin is needed to efficiently induce mesodermal lineage formation and subsequent cardiomyocyte development in human embryonic stem cells. Similar findings are also observed using mouse embryonic stem cells (Taha et al, 2006(Taha et al, , 2007Taha & Valojerdi, 2008;Takei et al, 2009;Verma & Lenka, 2010). In addition to the role of Bmp signaling, several studies have reported the pivotal role of Fgf signaling controlling mouse embryonic stem cell-derived cardiomyogenesis (Dell´Era et al, 2003;Ronca et al 2009).…”

The primary cilium coordinates early cardiogenesis and hedgehog signaling in cardiomyocyte differentiation

“…Wnt and PDGF signaling are regulated by the cilium in a series of other cell types involved in growth control, migration and differentiation (reviewed by Christensen et al, 2008;Gerdes and Katsanis, 2008). BMP promotes the induction of cardiomyocytes from the mouse stem cell line P19.SI (Angello et al, 2007) and human embryonic stem cells (Takei et al, 2009). Based on their findings on Pkd2 -/-mouse embryos, Slough and colleagues (Slough et al, 2008) also hypothesized that primary cilia in the heart and/or vasculature can function as mechanosensors to Immunofluorescence microscopy analysis of acetylated tubulin (tb) on primary cilia in the heart of E11.5 WT and Ift88-null mice (tb, red; DAPI, blue).…”