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DMSO efficiently down regulates pluripotency genes in human embryonic stem cells during definitive endoderm derivation and increases the proficiency of hepatic differentiation.

ABSTRACT: BACKGROUND: Definitive endoderm (DE) is one of the three germ layers which during in vivo vertebrate development gives rise to a variety of organs including liver, lungs, thyroid and pancreas; consequently efficient in vitro initiation of stem cell differentiation to DE cells is a prerequisite for successful cellular specification to subsequent DE-derived cell types [1, 2]. In this study we present a novel approach to rapidly and efficiently down regulate pluripotency genes during initiation of differentiation to DE cells by addition of dimethyl sulfoxide (DMSO) to Activin A-based culture medium and report its effects on the downstream differentiation to hepatocyte-like cells. MATERIALS AND METHODS: Human embryonic stem cells (hESC) were differentiated to DE using standard methods in medium supplemented with 100ng/ml of Activin A and compared to cultures where DE specification was additionally enhanced with different concentrations of DMSO. DE cells were subsequently primed to generate hepatic-like cells to investigate whether the addition of DMSO during formation of DE improved subsequent expression of hepatic markers. A combination of flow cytometry, real-time quantitative reverse PCR and immunofluorescence was applied throughout the differentiation process to monitor expression of pluripotency (POUF5/OCT4 & NANOG), definitive endoderm (SOX17, CXCR4 & GATA4) and hepatic (AFP & ALB) genes to generate differentiation stage-specific signatures. RESULTS: Addition of DMSO to the Activin A-based medium during DE specification resulted in rapid down regulation of the pluripotency genes OCT4 and NANOG, accompanied by an increase expression of the DE genes SOX17, CXCR4 and GATA4. Importantly, the expression level of ALB in DMSO-treated cells was also higher than in cells which were differentiated to the DE stage via standard Activin A treatment.

SUBMITTER: Czysz K

PROVIDER: S-EPMC4320104 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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DMSO efficiently down regulates pluripotency genes in human embryonic stem cells during definitive endoderm derivation and increases the proficiency of hepatic differentiation.

Czysz Katherine K Minger Stephen S Thomas Nick N

PloS one 20150206 2

<h4>Background</h4>Definitive endoderm (DE) is one of the three germ layers which during in vivo vertebrate development gives rise to a variety of organs including liver, lungs, thyroid and pancreas; consequently efficient in vitro initiation of stem cell differentiation to DE cells is a prerequisite for successful cellular specification to subsequent DE-derived cell types [1, 2]. In this study we present a novel approach to rapidly and efficiently down regulate pluripotency genes during initiat ...[more]

PMID: 25659159

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Project description:ObjectiveIn vitro production of a definitive endoderm (DE) is an important issue in stem cell-related differentiation studies and it can assist with the production of more efficient endoderm derivatives for therapeutic applications. Despite tremendous progress in DE differentiation of human embryonic stem cells (hESCs), researchers have yet to discover universal, efficient and cost-effective protocols.Materials and methodsIn this experimental study, we have treated hESCs with 200 nM of Stauprimide (Spd) for one day followed by activin A (50 ng/ml; A50) for the next three days (Spd-A50). In the positive control group, hESCs were treated with Wnt3a (25 ng/ml) and activin A (100 ng/ml) for the first day followed by activin A for the next three days (100 ng/ml; W/A100-A100).ResultsGene expression analysis showed up regulation of DE-specific marker genes (SOX17, FOXA2 and CXCR4) comparable to that observed in the positive control group. Expression of the other lineage specific markers did not significantly change (p<0.05). We also obtained the same gene expression results using another hESC line. The use of higher concentrations of Spd (400 and 800 nM) in the Spd-A50 protocol caused an increase in the expression SOX17 as well as a dramatic increase in mortality rate of the hESCs. A lower concentration of activin A (25 ng/ml) was not able to up regulate the DE-specific marker genes. Then, A50 was replaced by inducers of definitive endoderm; IDE1/2 (IDE1 and IDE2), two previously reported small molecule (SM) inducers of DE, in our protocol (Spd-IDE1/2). This replacement resulted in the up regulation of visceral endoderm (VE) marker (SOX7) but not DE-specific markers. Therefore, while the Spd-A50 protocol led to DE production, we have shown that IDE1/2 could not fully replace activin A in DE induction of hESCs.ConclusionThese findings can assist with the design of more efficient chemically-defined protocols for DE induction of hESCs and lead to a better understanding of the different signaling networks that are involved in DE differentiation of hESCs.

Project description:We investigated the effects of hypoxia on spontaneous (SP)- and activin A (AA)-induced definitive endoderm (DE) differentiation of mouse embryonic stem cells (mESCs) and their subsequent differentiation into distal pulmonary epithelial cells. SP differentiation for 6 days of mESCs toward endoderm at hypoxia of 1% O2, but not at 3% or 21% (normoxia), increased the expression of Sox17 and Foxa2 by 31- and 63-fold above maintenance culture, respectively. Treatment of mESCs with 20 ng/mL AA for 6 days under hypoxia further increased the expression of DE marker genes Sox17, Foxa2, and Cxcr4 by 501-, 1,483-, and 126-fold above maintenance cultures, respectively. Transient exposure to hypoxia, as short as 24 h, was sufficient to enhance AA-induced endoderm formation. The involvement of hypoxia-inducible factor (HIF)-1α and reactive oxygen species (ROS) in the AA-induced endoderm enrichment was assessed using HIF-1α(-/-) mESCs and the ROS scavenger N-acetylcysteine (NAC). Under SP conditions, HIF-1α(-/-) mESCs failed to increase the expression of endodermal marker genes but rather shifted toward ectoderm. Hypoxia induced only a marginal potentiation of AA-induced endoderm differentiation in HIF-1α(-/-) mESCs. Treatment of mESCs with AA and NAC led to a dose-dependent decrease in Sox17 and Foxa2 expression. In addition, the duration of exposure to hypoxia in the course of a recently reported lung differentiation protocol resulted in differentially enhanced expression of distal lung epithelial cell marker genes aquaporin 5 (Aqp5), surfactant protein C (Sftpc), and secretoglobin 1a1 (Scgb1a1) for alveolar epithelium type I, type II, and club cells, respectively. Our study is the first to show the effects of in vitro hypoxia on efficient formation of DE and lung lineages. We suggest that the extent of hypoxia and careful timing may be important components of in vitro differentiation bioprocesses for the differential generation of distal lung epithelial cells from pluripotent progenitors.

Dataset Information

DMSO efficiently down regulates pluripotency genes in human embryonic stem cells during definitive endoderm derivation and increases the proficiency of hepatic differentiation.

Publications

DMSO efficiently down regulates pluripotency genes in human embryonic stem cells during definitive endoderm derivation and increases the proficiency of hepatic differentiation.

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