MC1568 cost Density dependent Notch signaling rapidly induce

Density-dependent Notch signaling rapidly induced a vascular progenitor cell state characterised by expression of EC and vSMC markers. Since VEGFR1 is not endothelial-specific, despite being a major EC product (Supplementary Fig. S5), several other endothelial markers were utilized including VEGF-A, VE-cadherin, PECAM-1, and vWF. We found that VEGF-A was strongly induced by Notch, which in turn stimulated commitment of progenitor HBMSCs along the EC lineage. Interestingly, exposure to exogenous VEGF-A at standard density over 24h was not sufficient to induce HBMSCs to express EC markers (Supplementary Fig. S4). Therefore, VEGF-A is not sufficient to initiate HBMSC differentiation to ECs, although it supports differentiation in sustained high density cultures. In vivo studies have documented how VEGF can disrupt vSMC function, ablating pericyte coverage and causing vessel destabilisation (Greenberg et al., 2008). Thus, the Notch-induced VEGF-A may have inhibited HBMSC differentiation to vSMCs while promoting HBMSC differentiation to ECs, and tubule formation. Our experiments indicate that low density culture suppresses the potential of HBMSCs to differentiate along vascular lineages. Therefore, a double-labeling immunofluorescence approach was used to address whether our HBMSC populations might be bipotential with the ability to differentiate along either SMC or EC lineages. Many MC1568 cost were found to express both vSMC and EC markers, so it was clear that both lineages can be induced in the same cells over an initial 24h after plating at high density. However, there may be some heterogeneous potential within our HBMSC populations. HBMSCs are known to have therapeutic potential for tissue remodeling and repair. Being multipotent, they can not only differentiate along a range of lineages, they can also enhance tissue repair by secreting anti-inflammatory factors (Lee et al., 2009b). Their culture conditions can affect their phenotypic status; for example, culture in 3D spheres was recently shown to enhance the expression of anti-inflammatory molecules (Bartosh et al., 2010). In this study, we have demonstrated that it is possible to control their vascular differentiation in vitro, through regulating plating density and cell–cell contact. High density triggers EC differentiation by rapid induction of Notch signaling which in turn up-regulates VEGF-A, a growth factor that consolidates EC differentiation. These high density in vitro conditions may mirror extensive cell contact areas in EC monolayers in vivo. Our study, which has defined mechanisms of vascular differentiation of HBMSCs, provides new opportunities to regulate HBMSCs in therapeutic neovascularisation.
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Materials and methods
Acknowledgments
Introduction The isolation of high quality hepatocytes from organs and tissues for toxicological studies and clinical therapies remains a challenge and alternative cellular resources are urgently needed. Various protocols for directed hepatic differentiation of stem cells have been developed as alternatives for large scale hepatocyte production. Embryonic and, more recently, induced pluripotent stem (iPS) cells where shown to substantially expand and differentiate into hepatocyte-like cells in culture [1–3]. Their maintenance, growth and differentiation using cytokine and growth factor supplemented differentiation media, however, are time consuming and expensive. Furthermore, pluripotent stem cells, for now, are not considered for cell therapy applications due to risks of teratoma formation. Adult extrahepatic tissues and normal postnatal liver provide an alternative potential source for the generation of hepatocytes for in vitro studies and cell therapy. Evidence has been provided that adult stem cells from various extrahepatic sources could occasionally overcome lineage borders and differentiate into hepatic lineage cells, upon coordinated in vitro stimulation [4,5]. The number of stem/progenitor cells, that can be isolated from normal adult liver, is generally low and not sufficient for most of the in vitro and in vivo applications.