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- Title
Pioglitazone, A Synthetic Agonist for PPARγ, and b-FGF Enhanced Dissociated Human Embryonic Stem Cells Self-Renewal.
- Authors
Ghaedi, K.; Ghoochani, A.; Peymani, M.; Kajabadi, E.; Nasr Esfahani, M. H.; Baharvand, H.
- Abstract
Objective: Human embryonic stem cells (hESCs) are pluripotent cells with ability to differentiate to three germ layers. However, dissimilar to mouse embryonic stem cells (mESCs), hESCs are vulnerable to be cultured in dissociated state due to induced apoptosis that has been reflected as an important obstacle to extend genetically manipulation of them. This phenomenon is arisen by activation of Rho\Rock signaling pathway is adversely affecting dissociated hESCs proliferation and survival through E-Cadherin dependent cell-cell adhesion disruption. E-cadherin is one of cytoskeleton elements that will be decreased after Rho/Rock pathway activation. Thus attenuation of Rho/Rock pathway may inhibit apoptosis of dissociated hESCs. One of the proposed components, which have been shown to be potential attenuator of this signaling pathway, is Pioglitzone. Pioglitzone, a specific of agonist of proxisome proliferative activated receptor γ (PPARγ), decreases the MYPT phosphorylation level a key mediator of Rho/Rock signaling pathway. Proxisome proliferative activated receptor γ (PPARγ) is a member of the PPAR superfamily ligand dependent nuclear receptors (α, β/δ,γ) that has numerous roles in variety of cells. PPARγ could be activated by both natural and synthetic ligands. The aim of this study is implementation of Rock inhibitor (Y-27632) with Pioglitzone to increase hESCs colonies formation rate through induction of E-Cadherin and β-Catenin expression independent of cells survival. Furthremore, we have evaluated the effects of bFGF supplementation on essential transcriptional factors involving in pluripotency of dissociated hESCs and combinational effects of Pioglitazone and bFGF on proliferation state of dissociated hESCs have been examined. Materials and Methods: To study effects of Pioglitazone on β-Catenin and E-Cadherin along with Rock inhibitor (Y-27632), hESCs were treated with Pioglitazone and Y-27632. Four and 24 hours and 1 week post treating, cells were harvested and quantitative real time PCR was applied to demonstrate effects of using Pioglitazone and Y-27632 on E-cadherin and β-catenin expression. To analyze colony formation rate and cells survival, alkaline phosphatase (AP) and annexinV/ PI tests were performed respectively. Furthermore, hESCs were grown in serum-free N2B27-based media and treated with Pioglitazone in presence or absence of bFGF for 4 days and expression level of pluripotent markers was assessed by real time PCR. Localization and intensity of these markers were also carried out by immunostaining. Results: Data showed that treating with Rock inhibitor along with Pioglitzone increased hESCs colonies formation rate significantly without influencing cell survival as compared with Rock inhibitor implementation. Moreover, treatment of dissociated hESCs with Y-27632 and Pioglitazone increased E-cadherin and β -catenin expression. Co-treating with bFGF and Pioglitazone increased Nanog expression, whereas did not any significantly effect on OCT4 or SOX2 expression as compared with bFGF supplementation. Moreover, pioglitazone decreased Pax6 and Sox1 as neural precursor cell markers. Conclusion: The present study suggests that implementation of Rock inhibitor (Y-27632) with Pioglitazone increases hESCs colonies efficiency formation rate through an induction of E-Cadherin and β-catenin expression in an independent pathway of cells survival. FGF/ERK signaling plays a positively acting role in hESCs by supporting Nanog expression. Thus, Pioglitazone could positively effect on NANOG, downstream of FGF2 and decrease PAX6 and SOX1 neural precursor genes, which lead to improvement in self-renewal of hESCs.
- Subjects
PIOGLITAZONE; FIBROBLAST growth factors; EMBRYONIC stem cells
- Publication
Cell Journal (Yakhteh), 2013, Vol 15, Issue Sup 1, p22
- ISSN
2228-5806
- Publication type
Abstract