In this study, because excessive polycythemia is a predominant trait in some high-altitude dwellers (chronic mountain sickness [CMS] or Monges disease) but not others living at the same altitude in the Andes, we took advantage of this human experiment of nature and used a combination of induced pluripotent stem cell technology, genomics, and molecular biology in this unique population to understand the molecular basis for hypoxia-induced excessive polycythemia. is an essential downstream target of SENP1 and that the differential expression and response of GATA1 and Bcl-xL are a key mechanism underlying CMS pathology. Introduction Chronic mountain sickness (CMS) or Monges disease occurs in up to 20% of individuals residing at high altitude in various regions of the world (Len-Velarde et al., 2000; Meja et al., 2005; Wu, 2005; Jiang et al., 2014). Three large high-altitude populations (Andeans, Ethiopians, and Tibetans) have been extensively analyzed (Beall, 2000, 2006; Zhou et al., 2013; Udpa et al., 2014), and these have provided a unique opportunity to investigate the mechanisms of adaptation to high-altitude hypoxia and development because these PhiKan 083 human populations have been under selection pressure for centuries (Beall, 2000, 2006; Zhou et al., 2013; Udpa et al., 2014). For Tibetans, have seemingly been under positive selection as illustrated in multiple studies (Simonson et al., 2010; Xiang et al., 2013; Lorenzo et al., 2014; Luo et al., 2014). In the Andean populace, several studies, including our own, have pointed out that there are several candidate genes, such as (have been linked to adaptation (Alkorta-Aranburu et al., 2012; Scheinfeldt et al., 2012; Udpa et al., 2014; Gonzales and Chaupis, 2015). It is important to note that some of these DNA-selected regions and candidate genes, as in our previous studies (Zhou et al., 2013; Udpa et al., 2014), have been shown to be causally related to the phenotype of tolerance to high-altitude hypoxia. Furthermore, hypoxia-inducible factor (or gene polymorphisms and polycythemia (Meja et al., 2005). This suggested to us that there must be other possible mechanisms that play an important role in excessive erythropoiesis in high-altitude Andean polycythemia. One major reason for our desire for this extreme phenotype is that we hypothesize that this molecular mechanisms that are underlying this phenotype may train us about other related diseases at sea level or about protection of tissues when they are hypoxic or ischemic, as we have recently shown from studies at high altitude (Stobdan et al., 2015). Results Generation of human induced pluripotent stem cells (iPSCs) from CMS and non-CMS subjects followed by in vitro erythroid differentiation To understand the genetic basis of CMS, we acquired blood samples as well as skin biopsies from your same individuals (CMS and non-CMS) residing in Peru (4338 m; corresponding to 59% of O2 at sea level). We sequenced the whole genomes from 20 subjects (10 individuals with CMS and non-CMS) and reported on these in a previous study (Zhou et al., 2013). We have now reprogrammed fibroblasts and generated human iPSCs from five CMS and four non-CMS subjects (Table 1), as well as from three sea-level subjects used as controls. The iPSCs were characterized using DNA Rabbit polyclonal to FAT tumor suppressor homolog 4 fingerprinting, high-resolution karyotyping, and alkaline phosphatase staining, as well as assessing the expression of multilineage differentiation markers, as explained in detail in the Characterization of iPSCs section of Materials and methods as well as in our previous work (Zhao et al., 2015). DNA fingerprinting analysis confirmed that this iPSC lines were identical to parental fibroblast lines. The reprogramming of iPSCs was confirmed by staining for pluripotency markers and alkaline phosphatase and the ability to differentiate into three germ layers in vitro (Zhao et al., 2015). The expression of transgenes in the mRNA of iPSCs was low or undetectable, and stem array confirmed that this karyotypes of iPSC colonies were normal (Zhao et al., 2015). Table 1. Summary of non-CMS and CMS subjects from Cerro PhiKan 083 de Pasco used in the current study as well as their medical test scores We transformed iPSC lines into erythroid cells (refer to the Erythroid induction and differentiation section of Materials and methods) by adopting a previously published protocol (Kobari et al., 2012). We used sequential cytokines mixtures for induction and maturation of erythroid populace, as previously explained (Fig. 1 A; Kobari et al., 2012). A quantitative assessment was performed of surface PhiKan 083 markers such as CD34, CD45 (leukocyte common antigen), CD71 (transferrin receptor protein 1), CD36, and CD235a (glycophorin A). Fig. 1 B shows the CD profile under normoxic conditions, including the progressive increase of CD71 and its subsequent fall and the increase in CD235a with time. Sturgeon et al. (2014) have shown that primitive hematopoietic progenitors are KDR (kinase place domain receptor)+CD235a+, but we believe that CD235a represents erythroid lineage in our studies because their appearance is late in.