Dataset Information

Improved Survival and Initiation of Differentiation of Human Induced Pluripotent Stem Cells to Hepatocyte-Like Cells upon Culture in William's E Medium followed by Hepatocyte Differentiation Inducer Treatment.

ABSTRACT: Hepatocyte differentiation inducer (HDI) lacks both glucose and arginine, but is supplemented with galactose and ornithine, and is added together with other reagents such as apoptosis inhibitor and oncostatin M. Although human induced pluripotent stem (iPS) cells initiate hepatocyte differentiation, most die within 7 days. In this study, we investigated both HDI and conventional media for their potential to improve cell survival.201B7 iPS cells were cultured in conventional media. This consisted of three cycles of 5-day culture in William's E (WE) medium, followed by a 2-day culture in HDI.Expression levels of ?-feto protein (AFP) were higher in cells cultured in WE and in Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham (DF12). 201B7 cells expressed the highest AFP and albumin (ALB) when cultured in HDI for 2 days following 7-day culture in WE. After three cycles of 5-day culture in WE followed by 2 days in HDI, 201B7 cells expressed AFP and ALB 54 ± 2.3 (average ± standard deviation) and 73 ± 15.1 times higher, respectively, than those cultured in ReproFF (feeder-free condition).201B7 cells survived culture in WE for 7 days followed HDI for 2 days. After three cycles of culture under these conditions, hepatocyte differentiation was enhanced, as evidenced by increased AFP and ALB expression.

SUBMITTER: Tomizawa M

PROVIDER: S-EPMC4830564 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

Improved Survival and Initiation of Differentiation of Human Induced Pluripotent Stem Cells to Hepatocyte-Like Cells upon Culture in William's E Medium followed by Hepatocyte Differentiation Inducer Treatment.

Tomizawa Minoru M Shinozaki Fuminobu F Motoyoshi Yasufumi Y Sugiyama Takao T Yamamoto Shigenori S Ishige Naoki N

PloS one 20160413 4

<h4>Background</h4>Hepatocyte differentiation inducer (HDI) lacks both glucose and arginine, but is supplemented with galactose and ornithine, and is added together with other reagents such as apoptosis inhibitor and oncostatin M. Although human induced pluripotent stem (iPS) cells initiate hepatocyte differentiation, most die within 7 days. In this study, we investigated both HDI and conventional media for their potential to improve cell survival.<h4>Materials and methods</h4>201B7 iPS cells we ...[more]

PMID: 27073925

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Project description:Understanding essential signaling network requirements and making appropriate adjustments in culture conditions are crucial if porcine pluripotent stem cells (PSC) are to achieve their full potential. Here, we first used two protein factors (LIF and FGF2) and kinase inhibitor combinations in attempts to convert primed type lentiviral-reprogrammed porcine induced PSC (Lv-piPSC) into naïve-like state and developed a medium called FL6i. In addition to FGF2 and LIF, this medium contained inhibitors of MAPK14, MAPK8, TGFB1, MAP2K1, GSK3A and BMP. Crucially, the usual TGFB1 and BMP4 protein components of many stem cell media were replaced in FL6i with inhibitors of TGFB1 and BMP. With this medium, Lv-piPSC were readily transformed from their original primed state into cells that formed colonies with typical features of naïve-state stem cells. The FL6i medium also assisted generation of naïve-type piPSC lines from porcine embryonic fibroblasts with non-integrating episomal plasmids (Epi-piPSC). These lines, despite retaining variable amounts of vector DNA, expressed higher endogenous pPOU5F1 and pSOX2 than Lv-piPSC. They have been cultured without obvious morphological change for >45 passages and retained pluripotent phenotypes in terms of upregulation of genes associated with pluripotency, low expression of genes linked to emergence of somatic cell lineages, and ability to generate well differentiated teratomas in immune-compromised mice. FL6i conditions, therefore, appear to support elevated pluripotent phenotypes. However, FL6i was less able to support the generation of embryonic stem cells from porcine blastocysts. Although colonies with dome-shaped morphologies were evident and the cells had some gene expression features linked to pluripotency, the phenotypes were ultimately not stable. Pathway analysis derived from RNAseq data performed on the various cell lines generated in this study suggest the benefits of employing the FL6i medium on porcine cells reside in its ability to minimize TGFB1 and BMP signaling, which would otherwise de-stabilize the stem cell state.

Dataset Information

Improved Survival and Initiation of Differentiation of Human Induced Pluripotent Stem Cells to Hepatocyte-Like Cells upon Culture in William's E Medium followed by Hepatocyte Differentiation Inducer Treatment.

Publications

Improved Survival and Initiation of Differentiation of Human Induced Pluripotent Stem Cells to Hepatocyte-Like Cells upon Culture in William's E Medium followed by Hepatocyte Differentiation Inducer Treatment.

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