This is similar to our findings with C2C12 cells on fibronectin

over time. Mean SEM relative expression level reported as fold difference from CD133+ starting population shown on Y-axis. Days after initiating PS formation shown along X-axis. Proliferative and maturation phases shown at bottom. A, Upregulated genes are: marker of proliferation Ki-67, Neuronal differentiation 1, Neurogenin 3, v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A, v-maf avian musculoaponeurotic fibrosarcoma oncogene family, protein B, paired box 4, paired box 6. B, Down regulated genes are: SRY -box 9, pancreatic and duodenal homeobox 1, ISL LIM homeobox 1, NK6 homeobox 1, GLIS family zinc finger 3, motor neuron and pancreas homeobox 1, Hairy enhancer of split 1. C, Isolation and endocrine PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19754931 differentiation of NGN3+ cells. After death, pancreas is removed and transported to an islet isolation facility where a biopsy is taken for histology. The pancreas is then digested and separated into islets and Dihydroartemisinin price exocrine tissue. Exocrine culture is initiated on day 2 post mortem. On day 6 post mortem, tissue is digested to single cells, which are labeled and sorted for expression of CD133. CD133+ cells are plated on pancosphere day 0. Samples are collected on pancosphere days 4, 6, 9, 13, 15, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19756449 19 and 21. Cell proliferation marker KI67 expression peaks at pancosphere day 9. On pancosphere day 19 IGF II is withdrawn and cells begin final maturation. Days between each step are indicated below arrows. Exocrine tissue culture and phases of pancosphere differentiation are shown above the timeline. doi:10.1371/journal.pone.0133862.g006 cells. Both cell population express hepatocyte nuclear factor 1, ONECUT1 and 2, PDX1, NKX6.1, SRY box 9, NGN3 and GLIS3, but low levels or no PAX6, ISL1, MAFB or NEUROD1. Although murine E12.5 cells express PAX4 and NKX2.2, failure to detect expression of these genes in NGN3+ cells prior to differentiation is consistent with their absence in human 4752 day post conception NGN3+ cells, a population which otherwise corresponds to murine E12.5 endocrine progenitors. In addition to an endocrine progenitor phenotype, NGN3+ cells undergo a pattern of gene expression that resembles fetal endocrine development. Although the efficiency of in vitro endocrine differentiation in PS is low, it results in cells expressing MAFA and MAFB mRNA and a protein expression phenotype that resembles beta cells. These mRNA and protein expression results strongly suggest NGN3+ cells from the adult human pancreas are capable of acquiring an endocrine cell fate. The role of NGN3 in this process may be to integrate proendocrine signals then activate target genes such as NEUROD1 to initiate cell cycle exit and endocrine fate commitment. Although murine in vivo reprogramming, exogenous NGN3 expression in human exocrine cultures and this work demonstrate exocrine cells have the capacity to acquire an endocrine cell fate after expression of NGN3, it does not necessarily imply this process occurs under normal circumstances in vivo. Studies demonstrating beta cell replacement in adult mice requires pre-existing beta cells and expression of NGN3 following injury is insufficient to drive differentiation to an endocrine cell fate suggest beta cell replication may be the dominant or exclusive method to maintain beta cell mass. Nevertheless, reprogramming demonstrates even subtle and transient changes to exocrine cell gene expression or cytokine milieu can establish a permissive environment for conversion to an endocrine cell fate. Given th