The Synergistic Enhancement of Cloning Efficiency in Individualized Human Pluripotent Stem Cells by Peroxisome Proliferative-activated Receptor-γ (PPARγ) Activation and Rho-associated Kinase (ROCK) Inhibition

Nasim-Sadat Kajabadi,Ali Ghoochani,Maryam Peymani,Kamran Ghaedi,Abbas Kiani-Esfahani,Motahareh-Sadat Hashemi,Mohammad Hossein Nasr-Esfahani,Hossein Baharvand

doi:10.1074/jbc.m114.624841

Abstract

Although human pluripotent stem cells (hPSCs) provide valuable sources for regenerative medicine, their applicability is dependent on obtaining both suitable up-scaled and cost effective cultures. The Rho-associated kinase (ROCK) inhibitor Y-27632 permits hPSC survival upon dissociation; however, cloning efficiency is often still low. Here we have shown that pioglitazone, a selective peroxisome proliferative-activated receptor-γ agonist, along with Y-27632 synergistically diminished dissociation-induced apoptosis and increased cloning efficiency (2-3-fold versus Y-27632) without affecting pluripotency of hPSCs. Pioglitazone exerted its positive effect by inhibition of glycogen synthase kinase (GSK3) activity and enhancement of membranous β-catenin and E-cadherin proteins. These effects were reversed by GW-9662, an irreversible peroxisome proliferative-activated receptor-γ antagonist. This novel setting provided a step toward hPSC manipulation and its biomedical applications.

Highlights

Elevated Colony Formation of Dissociated Single human pluripotent stem cells (hPSCs) in the Presence of Pioglitazone and Y-27632—We took into consideration our previous findings (11) of the positive effect of PPAR␥ activation on mouse embryonic stem cell proliferation to determine if a potent agonist of PPAR␥ could serve as a potential factor to improve hPSCs viability along with Y-27632 (ROCK inhibitor)
In two other hPSC lines we observed that single cells co-treated with Y-27632 plus pioglitazone had the best cloning efficiency, which was significantly reduced by the PPAR␥ antagonist, GW9662 (Fig. 1C, at least p Ͻ 0.05)
Proliferation, and apoptosis analyses showed no significant alteration in the evaluated parameters in dissociated single hPSCs after treatment of pioglitazone plus Y-27632 compared with Y-27632

Summary

Experimental Procedures

Cell Culture—We used hESC lines (RH5, RH6) (12) and the hiPSC line (hiPSC9) (13) in this study. hPSCs were expanded on Matrigel (Sigma E1270)-coated tissue culture dishes under feeder-free conditions in hPSC medium that included DMEM/ F-12 (Gibco, 21331-020) supplemented with 20% knock-out serum replacement (KOSR, Gibco, 10828-028), 2 mM L-glutamine (Gibco, 25030-024), 0.1 mM ␤-mercaptoethanol (Sigma, M7522), 1% nonessential amino acids (Gibco, 11140-035), 100 units/ml penicillin and 100 ␮g/ml streptomycin (Gibco, 15070063), insulin-transferrin-selenite (ITS, Gibco, 41400045), and 100 ng/ml basic fibroblast growth factor (bFGF, Royan Institute) (14). Colony Formation of Single Dissociated Single hPSCs—We evaluated the effect of PPAR␥ activation on cloning efficiency of single dissociated single hPSCs [(number of alkaline phosphatase-positive colonies/number of seeded cells) ϫ 100] by analyzing the numbers of feeder-independent colonies For this purpose single cells were plated into Matrigel-coated tissue culture dishes at a density of 60 ϫ 103 hPSCs/well of a 6-well plate in hPSC medium. Protein Preparation and Western Blot Analysis—Cells were lysed using TRI reagent (Sigma, 93289) according to the manufacturer’s protocol. After sonication and homogenization of the pellet by a tight glass homogenizer in homogenization buffer that contained protease inhibitor, the suspension was centrifuged at 3000 ϫ g for 15 min At this step the pellet included the nucleus and plasma membrane.

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