br Methods and materials br Results br Discussion Existing m

Methods and materials
Results
Discussion Existing methods for hPSC differentiation to endothelial progenitors require the addition of growth factors and/or xenogenic components, limiting their application for large-scale production and therapeutic applications (Bautch, 2011; Wilson et al., 2014). Here, we report a defined, albumin-free, non-xenogenic differentiation system for directing hPSCs to endothelial progenitors. We showed that a completely defined medium, DMEM supplemented with 100μg/mL ascorbic acid, is sufficient to efficiently generate CD34+ CD31+ endothelial progenitors from hPSCs following Gsk-3β inhibition. These hPSC-derived endothelial progenitors are multipotent and can be further directed into smooth muscle Manumycin A or endothelial cells upon subsequent culture in appropriate inductive media. CD31+/VE-cadherin+ endothelial cells differentiated under serum-free conditions exhibited uptake of acetylated low-density lipoprotein (Ac-LDL) and formed tube-like structures when cultured on Matrigel in the presence of VEGF. However, long-term expansion of these cells required serum-containing medium. Albumin has been reported to increase growth rate and overall cell health (Ashman et al., 2005; Zoellner et al., 1996). Here, however, we demonstrate that albumin is dispensable in endothelial progenitor differentiation. In spite of the greater simplicity of this new albumin free-medium, it supported endothelial progenitor induction of hPSCs comparably to LaSR basal medium. This simplified medium offers several advantages in both research and clinical applications of hPSC-derived endothelial progenitors. First, it eliminates batch-to-batch variability of albumin, likely increasing reproducibility of differentiation processes. Second, it provides a simpler chemical background for examining and screening factors regulating gene expression, differentiation, and proliferation. For example, albumin can bind and sequester lipids, proteins and small molecules (Garcia-Gonzalo and Izpisúa Belmonte, 2008). Third, it can reduce the risk of potential pathogen contamination and cell immunogenicity, facilitating therapeutic applications of hPSC-derived endothelial progenitor cells. Finally, this new system can significantly reduce reagent cost and simplify quality control for endothelial progenitor cell differentiation.
Conclusions
Introduction Homeostasis in the intestinal epithelium requires the concerted action of multiple stem and progenitor populations. Some of these populations are highly proliferative, while others are typically quiescent (Barker, 2014). Highly-proliferative, or fast-cycling, stem cells are present throughout the intestine and are responsible for the daily maintenance of the epithelium, and therefore represent the “work-horse” for intestinal homeostasis (Barker et al., 2007). Slowly-cycling, or quiescent, stem cells are less prevalent and have been shown to be able to replace fast-cycling stem cells after injury, and therefore are considered clonogenic reserve cells that can maintain intestinal homeostasis (Buczacki et al., 2013; Tian et al., 2011). While the precise relationships between the different types of stem and progenitor cells remain to be determined, it is clear that altering their growth properties can lead to pathology, for example cancer (Barker et al., 2009). We demonstrated previously that activation of the K-Ras oncoprotein promotes expansion and hyper-proliferation within the intestinal transit amplifying cells (TACs), a population that is normally highly proliferative (Haigis et al., 2008). Similarly, oncogenic K-Ras has been shown to accelerate the rate of cell division in Lgr5+ stem cells in the small intestine (Snippert et al., 2014). Here, we have studied if/how mutant K-Ras can affect the proliferative and self-renewal properties of intestinal stem cells that are normally quiescent.