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Generation of bladder urothelium from human pluripotent stem cells under chemically defined serum- and feeder-free system.

ABSTRACT: Human stem cells are promising sources for bladder regeneration. Among several possible sources, pluripotent stem cells are the most fascinating because they can differentiate into any cell type, and proliferate limitlessly in vitro. Here, we developed a protocol for differentiation of human pluripotent stem cells (hPSCs) into bladder urothelial cells (BUCs) under a chemically defined culture system. We first differentiated hPSCs into definitive endoderm (DE), and further specified DE cells into BUCs by treating retinoic acid under a keratinocyte-specific serum free medium. hPSC-derived DE cells showed significantly expressed DE-specific genes, but did not express mesodermal or ectodermal genes. After DE cells were specified into BUCs, they notably expressed urothelium-specific genes such as UPIb, UPII, UPIIIa, P63 and CK7. Immunocytochemistry showed that BUCs expressed UPII, CK8/18 and P63 as well as tight junction molecules, E-CADHERIN and ZO-1. Additionally, hPSCs-derived BUCs exhibited low permeability in a FITC-dextran permeability assay, indicating BUCs possessed the functional units of barrier on their surfaces. However, BUCs did not express the marker genes of other endodermal lineage cells (intestine and liver) as well as mesodermal or ectodermal lineage cells. In summary, we sequentially differentiated hPSCs into DE and BUCs in a serum- and feeder-free condition. Our differentiation protocol will be useful for producing cells for bladder regeneration and studying normal and pathological development of the human bladder urothelium in vitro.

SUBMITTER: Kang M

PROVIDER: S-EPMC4057664 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Generation of bladder urothelium from human pluripotent stem cells under chemically defined serum- and feeder-free system.

Kang Minyong M Kim Hyeon Hoe HH Han Yong-Mahn YM

International journal of molecular sciences 20140425 5

Human stem cells are promising sources for bladder regeneration. Among several possible sources, pluripotent stem cells are the most fascinating because they can differentiate into any cell type, and proliferate limitlessly in vitro. Here, we developed a protocol for differentiation of human pluripotent stem cells (hPSCs) into bladder urothelial cells (BUCs) under a chemically defined culture system. We first differentiated hPSCs into definitive endoderm (DE), and further specified DE cells into ...[more]

PMID: 24776760

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Project description:BackgroundThe successful establishment of human induced pluripotent stem cells (hiPSCs) has increased the possible applications of stem cell research in biology and medicine. In particular, hiPSCs are a promising source of cells for regenerative medicine and pharmacology. However, one of the major obstacles to such uses for hiPSCs is the risk of contamination from undefined pathogens in conventional culture conditions that use serum replacement and mouse embryonic fibroblasts as feeder cells.Methodology/principal findingsHere we report a simple method for generating or culturing hiPSCs under feeder- and serum-free defined culture conditions that we developed previously for human embryonic stem cells. The defined culture condition comprises a basal medium with a minimal number of defined components including five highly purified proteins and fibronectin as a substrate. First, hiPSCs, which were generated using Yamanaka's four factors and conventional undefined culture conditions, adapted to the defined culture conditions. These adapted cells retained the property of self renewal as evaluated morphologically, the expression of self-renewal marker proteins, standard growth rates, and pluripotency as evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo (teratoma formation in immunodeficient mice). Moreover, levels of nonhuman N-glycolylneuraminic acid (Neu5Gc), which is a xenoantigenic indicator of pathogen contamination in human iPS cell cultures, were markedly decreased in hiPSCs cultured under the defined conditions. Second, we successfully generated hiPSCs using adult dermal fibroblast under the defined culture conditions from the reprogramming step. For a long therm culture, the generated cells also had the property of self renewal and pluripotency, they carried a normal karyotype, and they were Neu5Gc negative.Conclusion/significanceThis study suggested that generation or adaption culturing under defined culture conditions can eliminate the risk posed by undefined pathogens. This success in generating hiPSCs using adult fibroblast would be beneficial for clinical application.

Dataset Information

Generation of bladder urothelium from human pluripotent stem cells under chemically defined serum- and feeder-free system.

Publications

Generation of bladder urothelium from human pluripotent stem cells under chemically defined serum- and feeder-free system.

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