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A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog.

ABSTRACT: The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However, the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end, we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming. We show that one of these molecules functions in reprogramming by inhibiting Tgf-beta signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog.

SUBMITTER: Ichida JK

PROVIDER: S-EPMC3335195 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog.

Ichida Justin K JK Blanchard Joel J Lam Kelvin K Son Esther Y EY Chung Julia E JE Egli Dieter D Loh Kyle M KM Carter Ava C AC Di Giorgio Francesco P FP Koszka Kathryn K Huangfu Danwei D Akutsu Hidenori H Liu David R DR Rubin Lee L LL Eggan Kevin K

Cell stem cell 20091008 5

The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However, the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this ...[more]

PMID: 19818703

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Project description:BackgroundClinically, for stem cell-based therapy (SCBT), autologous stem cells are considered better than allogenic stem cells because of little immune rejection and no risk of communicable disease infection. However, severe maxillofacial bone defects restoration needs sufficient autologous stem cells, and this remains a challenge worldwide. Human gingival mesenchymal stem cells (hGMSCs) derived from clinically discarded, easily obtainable, and self-healing autologous gingival tissues, have higher proliferation rate compared with autologous bone marrow mesenchymal stem cells (hBMSCs). But for clinical bone regeneration purpose, GMSCs have inferior osteogenic differentiation capability. In this study, a TGF-β signaling inhibitor SB431542 was used to enhance GMSCs osteogenesis in vitro and to repair minipig severe maxillofacial bone defects.MethodshGMSCs were isolated and cultured from clinically discarded gingival tissues. The effects of SB431542 on proliferation, apoptosis, and osteogenic differentiation of hGMSCs were analyzed in vitro, and then, SB431542-treated hGMSCs composited with Bio-Oss® were transplanted into immunocompromised mice subcutaneously to explore osteogenic differentiation in vivo. After that, SB431542-treated autologous pig GMSCs (pGMSCs) composited with Bio-Oss® were transplanted into circular confined defects (5 mm × 12 mm) in minipigs maxillary to investigate severe bone defect regeneration. Minipigs were sacrificed at 2 months and nude mice at 8 weeks to retrieve specimens for histological or micro-CT or CBCT analysis. Effects of SB431542 on TGF-β and BMP signaling in hGMSCs were investigated by Western Blot or qRT-PCR.ResultsOne micromolar of SB431542 treatment induced a robust osteogenesis of hGMSCs in vitro, without adverse effect on apoptosis and growth. In vivo, 1 μM SB431542 treatment also enabled striking osteogenesis of hGMSCs subcutaneously in nude mice and advanced new bone formation of pGMSCs in minipig maxillary bone defect model. In addition, SB431542-treated hGMSCs markedly increased bone-related proteins expression, and BMP2 and BMP4 gene expression. Conversely, SMAD3 protein-dependent TGF-β signal pathway phosphorylation was decreased.ConclusionsOur study show that osteogenic differentiation of GMSCs treated with TGF-β signaling inhibitor SB431542 was increased, and SB431542-treated autologous pig GMSCs could successfully repair minipig severe maxillofacial bone defects. This preclinical study brings about a promising large bone regeneration therapeutic potential of autologous GMSCs induced by SB431542 in clinic settings.

Project description:Liver cancer is the fifth leading cause of cancer death worldwide, but early diagnosis and treatment of liver cancer remains a clinical challenge. How to screen and diagnose liver cancer early and prolong the survival rate is still the focus of researchers. Cell experiments were used to detect the effect of WZ35 on the colony formation ability and proliferation activity of hepatoma cells, nude mouse experiment to observe the in vivo anticancer activity and toxic side effects of WZ35; metabolomics analysis, glucose metabolism experiment and Seahorse analysis of liver cancer cells treated with WZ35; cell experiments combined with bioinformatics analysis to explore the mechanism of WZ35-mediated metabolic reprogramming to exert anticancer activity; tissue microarray and case analysis to evaluate the clinical significance of biomarkers for early diagnosis, treatment and prognosis evaluation of liver cancer. WZ35 inhibited the proliferation activity of various cell lines of liver cancer, and showed good therapeutic effect in nude mice model of hepatocellular carcinoma without obvious toxic and side effects; WZ35 inhibited the absorption of glucose in hepatoma cells, and the drug effect glycolysis, phosphorylation and purine metabolism are relatively seriously damaged; WZ35 mainly inhibits YAP from entering the nucleus as a transcription factor activator by activating oxidative stress in liver cancer cells, reducing the transcription of GLUT1, and finally reducing its GLUT1. Tissue microarray and case analysis showed that GLUT1 and YAP were highly expressed and correlated in liver cancer patients, and were associated with poor patient prognosis. The GLUT1-YAP risk model had a high score in predicting prognosis. The study confirms that WZ35 is a small molecule glycolysis inhibitor, and through its properties, it mediates metabolic reprogramming dominated by impaired glycolysis, oxidative phosphorylation and purine metabolism to inhibit the proliferation activity of liver cancer cells. Our findings present novel insights into the pathology of liver cancer and potential targets for new therapeutic strategies. GLUT1-YAP has important reference significance for predicting the stages of disease progression in liver cancer patients and have the potential to serve as novel biomarkers for the diagnosis and treatment of liver cancer.

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A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog.

Publications

A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog.

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