Definitive endoderm formation is followed by the establishme

Definitive endoderm formation is followed by the establishment of the primitive gut tube that is patterned into the hindgut, midgut and foregut, from which the endoderm organs are specified (Zorn and Wells, 2009). To follow the initial stages of this process we purified the endoderm progenitor Sephin1 and analyzed their differentiation course in three different time points (Fig. 3A). By isolating these cells we significantly reduced the influence of other cells on their differentiation. Unbiased functional annotation of the endoderm derivatives gene expression demonstrated that the endoderm progenitor cells could differentiation into both foregut (liver (Corrected P-value- 2.2E−9), bile (Corrected P-value- 4.3E−2)) and hindgut (colon (Corrected P-value- 4.3E−2) tissues. Up regulation of both liver and bile genes suggest that the differentiation occurred through hepatoblasts - progenitor cells that can differentiate into both hepatocytes and cholangiocytes (bile duct epithelial cells) (Zaret and Grompe, 2008). The expression onset of endodermal genes in our system resembled their expression timing in normal endoderm development demonstrating the system ability to recapitulate aspects of normal endoderm development. Accordingly, FOXA2, HHEX and HNF1B that are expressed during early endoderm formation were the first endodermal genes to be expressed in our system. In contrast, ALB, HNF4A and TTR that are expressed later on during endoderm specification, were expressed only after further differentiation of the endoderm progenitor cells (Table 1, Fig. 3C). Interestingly, some hepatic genes were unexpectedly down regulated upon Mid-CXCR4+ differentiation. This down regulation could occur due to the isolation of the endoderm cells from other cells, which may have prevented a cross talk between different cell types that may be necessary for their differentiation and maintenance. The interaction between cardiac mesoderm and foregut endoderm, which leads to hepatic differentiation through FGF2 signaling was demonstrated in several models. It was also demonstrated that when HESCs form teratomas, the hepatic like cells develop next to cardiac mesoderm like cells (Lavon et al., 2004). Congruently, in our system we demonstrated, both in expression level and in single cell microscopy level, the importance of FGF2 in endoderm derivative maintenance. FGF2 had an opposite effect on CXCR4- cells, demonstrating context depended nature of FGF signaling. Finally, we present the major steps of endoderm differentiation that can be recognized in our model (Fig. 5). We demonstrate that as the cells proceed through differentiation the differences between them and the cell of origin are more pronounced (Fig. 5).
Materials and methods
Acknowledgments We thank Dr. Danny Kitsberg for kindly providing the primers for the real time SYBR green reactions. We also thank Nadav Sharon and Dr. Yoav Mayshar for critically reading the manuscript. N.B. is the Herbert Cohn Chair in Cancer Research. This research was partially supported by funds from the ISF-Morasha Foundation (grant no. 1801/10) to N.B. We gratefully acknowledge support for this project provided by a grant from the Legacy Heritage Fund of New York to N.B.
Introduction It is generally believed that root formation results from the interactions between Hertwig\'s epithelial root sheath (HERS) and adjacent undifferentiated mesenchymal cells. Stem cells from the apical papilla (SCAPs) represent a population of early mesenchymal stem/progenitor cells residing in the root apex of immature permanent teeth. These postnatal stem cells can generate the calcium nodules in the osteo/odontogenic medium and Oil red O-positive lipid clusters following the adipogenic induction in vitro(Sonoyama et al., 2006). Besides, they can bring about the formation of bone-like tissues and dentin-like tissues in vivo (Abe et al., 2008; Sonoyama et al., 2006; Sonoyama et al., 2008). In physiological conditions, SCAPs contribute to the formation of developing radicular pulp as well as odontoblasts that are responsible for the root dentinogenesis, and they also play a paramount role in the pulp healing and regeneration (Huang et al., 2008; Sonoyama et al., 2008). Recent clinical reports indicate that SCAPs are important to the apexogenesis of developing roots and continuous root maturation in teenagers suffering from the endodontic diseases (Banchs and Trope, 2004; Chueh and Huang, 2006). Moreover, SCAPs have been used as a competent candidate for dental tissue regeneration. Dentin-pulp-like tissues in the empty root canal space and bioengineered roots which can support a porcelain crown have been respectively generated by utilizing SCAPs recombined with biological scaffolds in vivo (Huang et al., 2009; Huang et al., 2010; Sonoyama et al., 2006).