221 DERIVATION OF INSULIN-PRODUCING CELLS FROM HUMAN DERMAL FIBROBLASTS AT INTERMEDIATE PHASE OF REPROGRAMMING

Abstract

Process of somatic cell reprogramming into induced pluripotent stem (iPS) cells can broadly be divided into an early stochastic phase and a later, hierarchical and more deterministic phase of gene activation. Between the stochastic onset and the deterministic progression while approaching pluripotency, cells upon reprogramming undergo a transient activation or repression of developmental regulators. In these intermediate cells yet acquiring pluripotency, many somatic gene expressions are lost, while various lineage-specific factors are activated. In the present study, we produced pancreatic β cells by exposing human dermal fibroblasts (HDF) at the intermediate phase of reprogramming to pancreas-specific culture conditions. To obtain the intermediate cells, reprogramming factor Oct4, Sox2, Klf4, and c-Myc were transduced into HDF using retroviruses. By Day 9 after transduction, the expression of fibroblast-associated Thy1 was lost, while the early reprogramming marker CD49d was activated, suggesting that the cells are at the early stage of intermediate phase. These cells were capable of differentiating into 3 germ layers when cultured in tri-differentiation medium (including 100 ng mL^–1 Activin A, 0.1 ng mL^–1 Wnt3 for endodermal differentiation; 20 ng mL^–1 BMP4, 20 ng mL^–1 Activin A for mesodermal differentiation; 10 μM SB431542, 600 ng mL^–1 Dorsomorphin, and 35 ng mL^–1 Noggin for ectodermal differentiation) and expressing lineage-specific markers Sox17 (endoderm), Brachyury (mesoderm), and Otx2 (ectoderm). Further, the endodermal cells derived from HDF at intermediate phase of reprogramming were cultured in pancreas-induction medium (KO DMEM with 1% insulin-transferrin-selenium (ITS), 10 ng mL^–1 BMP4, 10 mM nicotinamide, 10 μM forskolin, 10 μM dexamethasone, and 50 ng mL^–1 exendin-4). After 5 days in culture, cells expressed pancreatic progenitor marker Pdx1, as examined by immunostaining and fluorescence-activated cell sorting (FACS) analysis. After culture for additional 10 days, the cells formed spherical clusters and expressed pancreatic islet-specific genes including insulin, somatostatin, glucagon, Nkx6.1, and MafA, as determined by RT-PCR. Moreover, the production of insulin and C-peptide from the derived β cells was confirmed by immunocytochemistry and FACS analysis. In response to glucose stimulation, these cells secreted insulin and C-peptide, as measured by ELISA. The present study demonstrates that fully differentiated functional cells could be converted from somatic cells at intermediate phase of reprogramming by exposing cells to specific culture conditions. The results shown in this study may provide a faster and potentially safer way to produce functional pancreatic cells without isolation and differentiation of iPS cells.