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  • A variety of suitable murine xenograft models are currently

    2018-11-08

    A variety of suitable murine xenograft models are currently used in hematopoietic research, yet these animals require conditioning prior to experimentation. Given the increasingly restricted access to radiation sources, NBSGW mice are broadly enabling, allowing laboratories without access to radiation to accelerate human hematopoietic research. The NBSGW model and other homozygous mice possessing the Kit allele are significant steps toward providing a standardized framework and an improved understanding of human HSC biology.
    Experimental Procedures
    Author Contribution
    Acknowledgments
    Introduction During embryonic development, cell fate is determined by both intrinsic programs and external cell niche. The animal studies suggested that endothelial cell niche provides both supportive and inductive roles throughout pancreas development (Eberhard et al., 2010). Early studies showed that signals from endothelial cyp3a inhibitors are essential for the induction of pancreatic organogenesis (Lammert et al., 2001). Endothelial cells specifically promote early dorsal pancreas development by inducing Ptf1a+ pancreatic progenitors (PPs) by activating FGF10 signaling (Yoshitomi and Zaret, 2004; Jacquemin et al., 2006). Interestingly, some groups recently reported that the endothelial cell niche could restrain epithelium branching and endocrine development. One group shows that blood vessel ablation results in increased pancreatic organ size (Sand et al., 2011). Another group showed that elimination of endothelial cells increases the size of pancreatic buds (Magenheim et al., 2011). Similarly, another group showed that overexpressing vascular endothelial growth factor A increases embryonic endothelial cell populations and perturbs pancreatic endocrine differentiation (Cai et al., 2012). However, a complete understanding of the role of endothelial cells in human pancreatic development is still missing.
    Results and Discussion
    Experimental Procedures
    Author Contributions
    Acknowledgments
    Introduction Human embryonic stem cells (hESCs) provide an attractive alternative cell source for bone regeneration. Although recent reports demonstrated methods to generate osteogenic cells from hESCs (Arpornmaeklong et al., 2010, 2011; Mateizel et al., 2008), it is difficult to evaluate the osteogenic development of hESCs and to identify specific cell populations based on surface antigen phenotypes, especially when surface antigens are not cell lineage specific. Also, assessing the homogeneity of cells during differentiation can be challenging. Therefore, a more efficient monitoring system is desirable to evaluate the osteogenic development of hESCs. RUNX2 is known as a critical regulator (Komori, 2010; Maruyama et al., 2007; Sudhakar et al., 2001) during osteogenic development. RUNX2 plays an essential role upstream of osteoblastic differentiation in osteogenic specification (Hill et al., 2005), and it induces the expression of osteogenic extracellular matrix genes during osteoblast maturation, such as collagen-Iα, alkaline phosphatase (ALP), and osteocalcin (BGLAP) (Cohen, 2009; Dalle Carbonare et al., 2012; Lian et al., 2006). Therefore, RUNX2 initiates osteogenesis in a manner that is precisely controlled temporally and spatially, and loss of RUNX2 expression at this early stage impairs osteogenic differentiation in bone development (Otto et al., 1997; Cohen, 2013). To better define the kinetics of osteogenic development from hESCs and to more efficiently identify osteoprogenitor cells for clinical applications, we have generated a novel reporter system for RUNX2 expression by utilizing the RUNX2 P1 promoter driven expression of YFP. The P1 promoter directs expression of the longest RUNX2 isoform and is activated by developmental signaling pathways in mesenchymal progenitor cells (Zhang et al., 2009). We then stably introduced this RUNX2 P1 promoter-YFP cassette with a constitutively active luciferase (luc) gene for in vivo imaging into hESCs (H9 cell line). After verifying the reliability of this reporter system, we tracked the osteogenic differentiation using this RUNX2-YFP-integrated H9 hESCs, evaluated the efficiency of such osteogenic differentiation of hESCs, and tested the osteogenic phenotypes of these differentiated cells in vivo.