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Urothelial differentiation of human amniotic fluid stem cells by urothelium specific conditioned medium.

ABSTRACT: Human amniotic fluid stem cells (HAFSCs) have a high proliferative capacity and a good differentiation potential, and may thus be suitable for regenerative medicine. To date, urothelial differentiation mechanisms of HAFSCs are poorly understood. We have investigated the urothelial differentiation potential of HAFSCs so that they can be therapeutically applied to cure defective diseases of bladder. To induce the stem cell differentiation, HAFSCs were cultured in a bladder cancer-derived conditioned medium. After 2 weeks of culture, HAFSCs began to express the urothelial lineage-specific markers (UPII, CK8 and FGF10). Meanwhile, pluripotency markers (Oct-4, Sox-2 and Nanog) were downregulated at both RNA and protein levels in the differentiated HAFSCs. Immunocytochemistry data revealed that differentiated HAFSCs expressed urothelial markers of UPII and CK8. We have screened the receptor tyrosine kinase arrays with the differentiated HAFSCs. The screening showed that MuSK, Tie-1 and EphA4 receptor tyrosine kinases were upregulated, whereas EphA7 and FGF R1 kinases were downregulated in HAFSCs. The data suggest that HAFSCs can be an important urothelium cell source, which can be used for urinary tract engineering.

SUBMITTER: Kang HH

PROVIDER: S-EPMC3959875 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Urothelial differentiation of human amniotic fluid stem cells by urothelium specific conditioned medium.

Kang Henry H HH Kang Julia J JJ Kang Hyung-Gyoo HG Chung Seyung S SS

Cell biology international 20140113 4

Human amniotic fluid stem cells (HAFSCs) have a high proliferative capacity and a good differentiation potential, and may thus be suitable for regenerative medicine. To date, urothelial differentiation mechanisms of HAFSCs are poorly understood. We have investigated the urothelial differentiation potential of HAFSCs so that they can be therapeutically applied to cure defective diseases of bladder. To induce the stem cell differentiation, HAFSCs were cultured in a bladder cancer-derived condition ...[more]

PMID: 24375948

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Project description:BackgroundObesity is a metabolic disorder syndrome characterized by excessive fat accumulation that is related to many diseases. Human amniotic mesenchymal stem cells (hAMSCs) have a great potential for cell-based therapy due to their characteristics such as pluripotency, low immunogenicity, no tumorigenicity, potent paracrine effects, and no ethical concern. Recently, we observed that both hAMSCs and their conditioned medium (hAMSCs-CM) efficiently repaired skin injury, inhibited hepatocellular carcinoma, and alleviated high-fat diet (HFD)-induced diabetes. However, the effects and the underlying mechanisms of hAMSCs-CM on high-fat diet (HFD)-induced obesity were not explored.MethodsThe characteristics of hAMSCs were confirmed by flow cytometry, RT-PCR, and immunofluorescence. Obese mice were induced by administrating HFD for 15 weeks and simultaneously, the mice were intraperitoneally injected with hAMSCs-CM weekly to evaluate the effects of hAMSCs-CM on HFD-induced obesity. GTT and ITT assays were used to assess the effects of hAMSCs-CM on HFD-induced glucose tolerance and insulin resistance. The lipid accumulation and adipocytes hypertrophy in mouse adipose tissues were determined by histological staining, in which the alterations of blood lipid, liver, and kidney function were also examined. The role of hAMSCs-CM in energy homeostasis was monitored by examining the oxygen consumption (VO2), carbon dioxide production (VCO2), and food and water intake in mice. Furthermore, the expressions of the genes related to glucose metabolism, fatty acid β oxidation, thermogenesis, adipogenesis, and inflammation were determined by western blot analysis, RT-PCR, and immunofluorescence staining. The roles of hAMSCs-CM in adipogenesis and M1/M2 macrophage polarization were investigated with 3T3-L1 preadipocytes or RAW264.7 cells in vitro.ResultshAMSCs-CM significantly restrained HFD-induced obesity in mice by inhibiting adipogenesis and lipogenesis, promoting energy expenditure, and reducing inflammation. The underlying mechanisms of the anti-obesity of hAMSCs-CM might be involved in inhibiting PPARγ and C/EBPα-mediated lipid synthesis and adipogenesis, promoting GLUT4-mediated glucose metabolism, elevating UCP1/PPARα/PGC1α-regulated energy expenditure, and enhancing STAT3-ARG1-mediated M2-type macrophage polarization.ConclusionOur studies demonstrated that hAMSCs significantly alleviated HFD-induced obesity through their paracrine effects. Obviously, our results open up an attractive therapeutic modality for the prevention and treatment of obesity and other metabolic disorders clinically. The cytokines, exosomes, or micro-vesicles secreted from hAMSCs significantly inhibited HFD-induced obesity in mice by inhibiting lipid production and adipogenesis, promoting energy consumption, and reducing inflammation.

Project description:Human amniotic fluid stem cells (AFSC) are an exciting and very promising source of stem cells for therapeutic applications. In this study we investigated the effects of short-term treatments of small molecules to improve stem cell properties and differentiation capability. For this purpose, we used epigenetically active compounds, such as histone deacetylase inhibitors Trichostatin A (TSA) and sodium butyrate (NaBut), as well as multifunctional molecules of natural origin, such as retinoic acid (RA) and vitamin C (vitC). We observed that combinations of these compounds triggered upregulation of genes involved in pluripotency (KLF4, OCT4, NOTCH1, SOX2, NANOG, LIN28a, CMYC), but expression changes of these proteins were mild with only significant downregulation of Notch1. Also, some alterations in cell surface marker expression was established by flow cytometry with the most explicit changes in the expression of CD105 and CD117. Analysis of cellular energetics performed using Seahorse analyzer and assessment of gene expression related to cell metabolism and respiration (NRF1, HIF1α, PPARGC1A, ERRα, PKM, PDK1, LDHA, NFKB1, NFKB2, RELA, RELB, REL) revealed that small molecule treatments stimulate AFSCs toward a more energetically active phenotype. To induce cells to differentiate toward neurogenic lineage several different protocols including commercial supplements N2 and B27 together with RA were used and compared to the same differentiation protocols with the addition of a pre-induction step consisting of a combination of small molecules (vitC, TSA and RA). During differentiation the expression of several neural marker genes was analyzed (Nestin, MAP2, TUBB3, ALDH1L1, GFAP, CACNA1D, KCNJ12, KCNJ2, KCNH2) and the beneficial effect of small molecule treatment on differentiation potential was observed with upregulated gene expression. Differentiation was also confirmed by staining TUBB3, NCAM1, and Vimentin and assessed by secretion of BDNF. The results of this study provide valuable insights for the potential use of short-term small molecule treatments to improve stem cell characteristics and boost differentiation potential of AFSCs.

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Urothelial differentiation of human amniotic fluid stem cells by urothelium specific conditioned medium.

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Urothelial differentiation of human amniotic fluid stem cells by urothelium specific conditioned medium.

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