Here we show that cholesterol metabolites

Here, we show that cholesterol metabolites play an important role in cardiac differentiation in mouse embryonic stem cells (mESC). Gcrt receptor expression in the early mouse embryo and its exclusive localization to cell nuclei within the visceral endoderm then prompted us to further investigate a possible role for this class of cholesterol-derived steroid hormones. As the visceral endoderm is an important signaling center for the formation of anterior structures including head and heart (Arai et al., 1997; Perea-Gomez et al., 2002), we studied Gcrt function in differentiating mESC as an in vitro model of the developing mouse embryo. We found that Gcrt controls expression of Cerberus-1 (Cer1), a secreted inhibitor of Nodal and BMP signaling, with an essential role in cardiac differentiation in AMG-517 and ESC (Cai et al., 2013; Foley et al., 2007). These studies suggest that cholesterol metabolites such as the Gcrts may function much earlier in embryonic development than previously anticipated and furthermore indicate that Gcrts regulate the heart-inducing properties of the anterior visceral endoderm.
Results and discussion
Materials and methods
Author contributions
Acknowledgments The authors would like to thank Drs. Fred Levine and Marcia Dawson for helpful discussions, Yoav Altman for FACS and Caroline Kemp for assistance in graphic design. This research was supported by grants from the NIH (R37HL059502, R01HL108176 to MM; and P30 CA030199 and P30 AR061303 to SBMRI Functional Genomics and Cytometry); the California Institute for Regenerative Medicine (CIRM) (RC1-000132 to MM); the Mayo Clinic Center for Regenerative Medicine (to AMF and AT) and the Fondation Leducq (to AT and MM). In addition, we are grateful for a Doctoral Fellowship from the Science and Technology Foundation of the Portuguese Ministry of Science Technology and Higher Education (to JCT) and a Howard Hughes Med-into-Grad Program fellowship (to JCT), a CIRM (TG2-01162) postdoctoral fellowship (to EW) and American Heart Association Postdoctoral fellowships (to EW and AMF).
Introduction Human pluripotent stem cells (hPSCs) are increasingly used in vascular research, including disease modeling, drug screening, and development of regenerative therapies (Ashton et al., 2011; Bautch, 2011; Kinney et al., 2014; Kusuma et al., 2014; Murry and Keller, 2008; Segers and Lee, 2008). Recently, dramatic improvements in the efficiency of directed differentiation protocols to produce endothelial cells have been reported by stage-specific modulation of pathways including TGFβ superfamily (James et al., 2010; Rufaihah et al., 2011; Wang et al., 2007), VEGF (vascular endothelial growth factor) (Goldman et al., 2009; James et al., 2010; Rufaihah et al., 2011; Wang et al., 2007), and Notch signaling (Marcelo et al., 2013; Sahara et al., 2014). However, most of these approaches require animal cells, fetal bovine serum, or cytokines and growth factors, limiting their applications for large-scale endothelial cell production for research or therapeutic applications. Recently, we reported a rapid and robust endothelial progenitor differentiation protocol under serum-free conditions, which only employs a Gsk-3β inhibitor in LaSR basal medium (Advanced DMEM/F12, 2.5mM GlutaMAX and 60μg/mL ascorbic acid) (Lian et al., 2014). The presence of bovine serum albumin (BSA) in this medium increases the cost, adds xenogenic components, and heightens lot-to-lot variability. Toward developing a defined, xeno-free endothelial progenitor differentiation platform, we screened several commercially available basal media, supplemented with insulin and ascorbic acid, for the ability to generate CD34+ CD31+ cells after treatment with 5μM CHIR99021. We found that DMEM supplemented with 100μg/mL ascorbic acid generated 20–30% CD34+ CD31+ endothelial progenitors that were enriched to >95% CD34+ progenitors via MACS. This minimal, defined differentiation platform should facilitate generation of proliferative endothelial progenitor cells from hPSCs for both research and clinical applications.