In summary the cell density

In summary, the cell density of PDX1+ posterior foregut vorapaxar positively correlates with the induction of PDX1+NKX6.1+ pancreatic bud cells in hESC/iPSC differentiation cultures. Although the molecules that regulate the differentiation step remain unknown, the signals elicited by high cell density are crucial for the pancreatic bud induction, and synergistically work with soluble factors. To our knowledge, this is the first study clearly demonstrating that the signals derived from spatial and morphological contexts are associated with the differentiation of hESCs/iPSCs into pancreatic lineages. Further studies to elucidate the molecular nature of the signals based on our findings may contribute to the development of hESC/iPSC-based cell therapy for diabetes. The following are the supplementary data related to this article.
Acknowledgments This work was supported in part by funding from the Takeda Science Foundation to T.T., the Suzuken Memorial Foundation to T.T. and the Life Science Foundation of Japan to K.O., by the Japan Society for the Promotion of Science (JSPS) through its ‘Funding Programme for World-Leading Innovative R&D on Science and Technology (FIRST Programme)’ to K.O. and Grant-in-Aid for Young Scientists (B) to T.T. and by the Japan Science and Technology Agency (JST) through its research grant “Core Center for iPS Cell Research and Projects for Technological Development, Research Center Network for Realization of Regenerative Medicine” to Y.K. and K.O. T.T. wrote the first draft of the manuscript. T.T., S.M., Y.K. and K.O. contributed to manuscript edits and revisions. T.T., S.M., Y.K., M.F., Y.H., Y.K. and K.O. designed, directed, and interpreted the experiments. T.T., S.M., H.T. and T.S. performed the experiments. K.O. supervised the study. The authors thank Kanae Mitsunaga for her technical assistance in the flow cytometry analysis and Yasuhiro Nakano for providing valuable advice on this research.
Introduction Pediatric myelodysplastic syndrome (MDS) represents a range of disorders characterized by dysplastic morphology comprising in total less than 5% of pediatric hematological malignancies (Hasle et al., 2004). The spectrum of MDS ranges from refractory cytopenia of childhood (RCC) to advanced MDS with excess of blasts (RAEB) with increasing risk of leukemic transformation (Hasle et al., 2003). Survival has increased from 30 to 60% since hematopoietic stem cell transplantation (HSCT) is applied (Strahm et al., 2011; Sasaki et al., 2001; Woods et al., 2002). The pathophysiology of MDS is not fully elucidated. However, genetic predisposition, acquired cytogenetic abnormalities and abnormal immune responses have been linked to MDS (Strahm et al., 2011; Hasle and Niemeyer, 2011). These aspects do not explain the entire range of disease in pediatric or adult MDS. Recently, it has been suggested in adult MDS that impaired interaction between hematopoietic precursor cells and their bone-marrow microenvironment might contribute to the disease (Zhang et al., 2012). In children, no conclusive data is yet available. Mesenchymal stromal cells (MSCs) have been identified as supporting cells of hematopoietic stem cells (HSC) in vivo and in vitro (Morikawa et al., 2009; Mendez-Ferrer et al., 2010; Sugiyama et al., 2006) and linked to disease, as aberrant MSC function was shown to contribute to the pathophysiology of malignant disorders in murine models (Raaijmakers et al., 2010; Schepers et al., 2013). Characteristics of MSCs from adult MDS patients have been extensively studied focusing on cytogenetic and molecular abnormalities (Blau et al., 2011; Lopez-Villar et al., 2009; Flores-Figueroa et al., 2008) as well as gene and protein expressions (Flores-Figueroa et al., 2008; Marcondes et al., 2008; Santamaria et al., 2012). In addition, abnormal immunomodulation (Marcondes et al., 2008; Wang et al., 2013; Zhao et al., 2012a) as well as decreased hematopoietic support (Zhao et al., 2012a; Ferrer et al., 2013) by MSCs has been reported in MDS. However, these data remain conflicting with other studies reporting no abnormalities in stromal function (Flores-Figueroa et al., 2008; Klaus et al., 2010; Alvi et al., 2001). Differences in results may be explained not only by a variety of MSC expansion protocols and experimental set-up, but also by the heterogeneity of the disease (Aizawa et al., 1999). Studies reporting on (cyto)genetics and function of MDS-MSC have been summarized in Supplementary Tables S1 and S2.