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*

Background: hPSCs cloning efficiency is still low. Results: Pioglitazone, a PPARγ agonist, along with Rho kinase inhibitor, Y-27632, increased cloning efficiency (2–3-fold versus Y-27632) through enhanced membrane localization of β-catenin and E-cadherin. Conclusion: Cloning efficiency in individualized hPSCs was enhanced synergistically by PPARγ activation and Rho kinase inhibition. Significance: This offers a new approach to hPSCs expansion for biomedical applications. 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.

signaling (9). Additionally, it was shown that PPAR␥ activation significantly reduced apoptosis of isolated rat cardiomyocytes that were subject to hypoxia/reoxygenation, at least in part by facilitation of Akt rephosphorylation (10). We reported that the PPAR␥ agonist enhanced the proliferation and survival rate of mouse embryonic stem cells (11). Therefore, we hypothesized that the PPAR␥ agonist, pioglitazone, might positively affect survival of dissociated single hPSCs and increase colony formation.
The ROCK inhibitor Y-27632 (Calbiochem, 688000) was added to the culture medium at a final concentration of 10 M (6). Pioglitazone (Cayman, 18570) and GW9662 (Sigma, M6191) were dissolved in dimethyl sulfoxide (DMSO). To find an effective dose of pioglitazone, we treated the cells with 2, 4, 8, and 16 M of ROCK inhibitor. Y-27632 (6) and GW9662 (11) were prepared at a final concentration of 10 M. All small molecules were added to the culture medium for the first 24 h after the cells were replated. Subsequently, the cell cultures were continued in the absence of small molecules. To induce differentiation, hPSCs were grown in suspension as embryoid bodies in hPSC medium without basic FGF and small molecules for 2 weeks.
The CHO-K1 cell line (Pasteur Institute, Tehran, Iran) was also used for transfection experiments. CHO cells were cultured and maintained as previously described (15).
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 10 3 hPSCs/well of a 6-well plate in hPSC medium. The cloning efficiency was calculated by ImageJ software version 1.4 (8).
Plasmids and Co-transfection-We used the following plasmids in this study: PPAR␥-EGFP expression plasmid (16), PDSred-N1 (Clontech), RhoA V14, and PIP5K1␣ (kindly provided by Dr. Nicolai E. Savaskan, Friedrich Alexander University of Erlangen-Nuremberg, Germany). Co-transfection of plasmids into CHO cells was performed using Lipofectamine LTX reagent (Invitrogen, 15338-100). The cell numbers and amount of plasmids for each transfection were determined based on the manufacturer's instructions. Two days post-transfection, we used the cells for further analyses.
Gene Expression Analysis-Total RNA was extracted using the RNeasy Kit (Qiagen, 74004), and cDNA was synthesized starting with 1 g of total RNA using reverse transcriptase and a hexamer primer (TaKaRa). Real-time (SYBR Green) PCR was performed in a thermal cycler Rotor gene 6000 (Corbett) according to the manufacturer's protocol (TaKaRa). The PCR mixture contained 10 l of Rotor-Gene SYBR Green PCR Master Mix (TaKaRa), 3 pmol of each primer, and 25 ng of cDNA for each reaction in a final volume of 20 l. All samples were assessed in relation to the levels of GAPDH expression as an internal control.
All measurements were performed in triplicate. Real-time specific primer pairs were designated by Beacon Designer software (version 7.2) as obtained from Metabion (Planegg/ Steinkirchen, Germany). The primer sequences are listed in Table 1. Real-time data were assessed and reported according to the ⌬⌬Ct method.
Subcellular Fractionation-The discontinuous sucrose gradient approach was used to isolate nuclear and plasma membrane fractions. At the initial step, we added a homogenization buffer (0.25 M sucrose, 10 mM HEPES, pH 7.5) that contained protease inhibitor mixture (Calbiochem, 539134) to freshly harvested cells. The cells were incubated on ice for 10 min. 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. Next, the suspension was centrifuged on a sucrose buffer gradient (buffer A (0.3 M sucrose, 50 mM Tris, pH 7.5, 1 mM MgCl 2 ) and buffer B (1.8 M sucrose, 50 mM Tris, pH 7.5, 1 mM MgCl 2 )) at 110,000 ϫ g for 90 min. Both buffers contained protease inhibitor mixture. Finally, the nucleus fraction (pellet of the previous step) was washed with buffer A at 15,000 ϫ g for 15 min.
Chromatin Immunoprecipitation (ChIP)-We used the Pierce™ Agarose ChIP kit (Life Technologies, Inc., 26156) according to the manufacturer's protocol to investigate PPAR␥ response element within ␤-catenin and E-cadherin promoters.
Co-immunoprecipitation-Pierce co-immunoprecipitation (Co-IP, Life Technologies, 26149) was performed to analyze ␤-catenin and E-cadherin interaction according to the manufacturer's instructions.
Alkaline Phosphatase and Immunofluorescence Staining-The colony formation assay was performed with an alkaline phosphatase kit (Sigma, 86R) according to the manufacturer's instructions.
For immunostaining, colonies were fixed with 4% paraformaldehyde (Sigma, P6148) for 30 min at 4°C followed by permeabilization with 0.4% Triton X-100 (Sigma, T8532) in PBS, blocked with secondary antibody-related host serum for 1 h, treated with the primary antibody for 1 h, and incubated with secondary antibody for 1 h. Primary antibodies used in this study were: anti-Oct4 (1:100, Santa Cruz Biotechnology, SC-5279), anti-Nanog Flow Cytometry Analysis of Cell Cycle, Proliferation, and Apoptosis-For cell-cycle analysis, hPSCs seeded for 24 h were fixed in 70% ethanol. After washing, the cells were suspended in PBS that included RNase A and propidium iodide (1 mg/ml) solution. For identifying and examining proliferating cells, we incubated the cycling cells with 5-bromo-2Ј-deoxyuridine (BrdU) for 1 h. After DNA denaturation, the cells were stained with monoclonal anti-BrdU (Sigma, B2531) as the primary antibody and IgM-FITC (Millipore, AP124F) as the secondary antibody. Apoptosis analysis was conducted at 24 h after cell seeding by using the following three protocols: Annexin V, terminal transferase dUTP nick end labeling (TUNEL) and caspase-3 activity. For annexin V analysis, cells were labeled with propidium iodide and Annexin V-FITC (IQ Products, IQP-120F) according to the manufacturer's protocol.
For the TUNEL assay, cells were stained to detect apoptotic nuclei by the DeadEnd Fluorometric TUNEL System (Promega, G3250) according to the manufacturer's instructions, then analyzed by flow cytometry. Improvement in cellular viability was further confirmed by the caspase-3/7 activation assay as a cellular marker of apoptosis using a commercially available kit (APT403, Millipore) according to the manufacturer's instructions. Cells were analyzed by FACS Caliber flow cytometer (BD Biosciences), and the data were processed according to the ModFit LT™ version 4.0 program.
Statistical Analysis-Data were expressed as the means Ϯ S.E. Statistical analysis of RT-qPCR and Western blotting with three independent cultures were performed by Graphpad prism software version 6 and Image J software, respectively. The results were subsequently compared using one-way analysis of variance followed by Tukey's post-hoc test or the t test when two independent groups were compared. The mean difference was significant at the p Ͻ 0.05 level.

Results
Elevated Colony Formation of Dissociated Single 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). Pioglitazone, a highly specific PPAR␥ agonist, was added to the culture medium at various concentrations (2-16 M) along with Y-27632 (Fig. 1, A and  B) for the first 24 h after plating of dissociated single hPSCs. Subsequently the cells were cultured for 7 days. Only pioglitazone did not result in alkaline phosphatase-positive colonies in the same manner as DMSO alone (data not shown). According to the results, the 8 M concentration of pioglitazone was the most efficient in terms of colony formation as detected by the numbers of alkaline phosphatase (AP)-positive colonies (Fig. 1, A and B). Therefore, this concentration was used for additional experimentation. To confirm that PPAR␥ activation led to enhanced cloning efficiency, we treated the cells simultaneously with Y-27632 and a PPAR␥ antagonist (GW9662). In this condition, a drop in colony formation was observed (p Ͻ 0.05, Fig. 1C). Of note, 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). Therefore, treatment by Y-27632 plus pioglitazone on dissociated single hPSCs had a synergistic effect on colony formation compared with Y-27632 alone. Furthermore, due to universality of the PPAR␥ effect on cloning efficiency, we continued the other experiments with a hESC cell line, RH5.
Pioglitazone Did Not Modulate hPSCs Apoptosis and Proliferation-We sought to determine if the increase in cloning efficiency with Y-27632 plus pioglitazone could be attributed to a decreased rate of apoptosis or enhancement of proliferation. We used three distinct assays (annexin V, TUNEL, and caspase-3 activation) to evaluate apoptosis. The results showed a clearly evident significant decrease in the rate of apoptotic cells after Y-27632 treatment compared with untreated cells (at least p Ͻ 0.01; Fig. 2, A-C). However, co-treatment of the cell with Y-27632 and pioglitazone or GW9662 did not significantly change the rate of apoptotic cells.
To validate if co-treatment of Y-27632 and pioglitazone increased the proliferation rate, we repeated the previous experiments and assessed the numbers of cells that were in S phase. Flow cytometry data showed no significant difference in S phase cell numbers co-treated with Y-27632 and pioglitazone compared with only Y-27632 (Fig. 2D, p Ͼ 0.05). The BrdU proliferation assay confirmed the same trend in proliferation rate (Fig. 2E).
Therefore, cell preservation under this circumstance was possibly not due to reduced apoptosis or an increased proliferation rate. We proposed that adhesion alteration resulted in colony formation enhancement.
Down-regulation of PPAR␥, ␤-Catenin, and E-cadherin Proteins in Dissociated Single hPSCs-Cytoskeletal components play a major role in cell-ECM/cell interactions which result in increasing viability. During cell dissociation cytoskeletal phosphorylation leads to dissociation induced apoptosis due to disruption of cytoskeletal components (4,17). In this experiment we measured the transcript and protein levels of ␤-catenin and E-cadherin as cell-ECM/cell components and PPAR␥ in dissociated single hPSCs after 4 h. We detected no significant changes in mRNA expression in dissociated single cells and colonies (Fig. 3A). Surprisingly, dissociation of hPSCs resulted in down-regulation of the protein contents of E-cadherin, ␤-catenin, and PPAR␥ (Fig. 3, B and C). This was also demonstrated by immunostaining (Fig. 3D). Therefore, it seems that PPAR␥ was involved in repair of cell-ECM/cell interaction disruption.
Augmentative Role of Pioglitazone in Colony Formation through ␤-Catenin and E-cadherin Escalation-The role of E-cadherin and its associated molecule ␤-catenin in cell-cell interaction is critical for the survival and differentiation of hPSCs (18). Therefore, changes in E-cadherin and ␤-catenin expression under pioglitazone treatment have been determined by protein level analysis and co-immunoprecipitation   OCTOBER 23, 2015 • VOLUME 290 • NUMBER 43 at 4 h post-treatment of dissociated single hPSCs. Co-treatment of Y-27632 and pioglitazone up-regulated E-cadherin and ␤-catenin proteins compared with Y-27632-treated cells (Fig. 4, A and B). Interestingly, the PPAR␥ antagonist (GW-9962) reversed the conditions generated by pioglitazone.

Pioglitazone Augmented Cloning Efficiency of hPSCs
It has been reported that ␤-catenin performance in hPSCs is dependent on its subcellular localization (19). Additionally, colony formation is a consequence of ␤-catenin localization in the membrane, whereas nuclear localization of ␤-catenin results in nuclear gene expression (20,21). Therefore, we conducted immunofluorescence staining to study ␤-catenin subcellular localization after treatment of hPSCs with pioglitazone. Immunostaining data showed membrane localization of ␤-catenin in hPSCs (Fig. 4C).
Next, we sought to determine whether plasma membrane localization of ␤-catenin increased upon pioglitazone treatment. Thus, Western blotting of plasma membrane and nuclear fractions at 4 h post-treatment of dissociated single hPSCs was performed. The subcellular plasma membrane fraction of ␤-catenin increased significantly in Y-27632 plus pioglitazone; however, the nuclear fraction showed no significant change (Fig. 4D). c-Myc and Tau proteins were used as positive controls for nuclear and plasma membrane proteins, respectively (Fig. 4D). We performed co-immunopre-cipitation for ␤-catenin and subsequent Western blotting for E-cadherin to show if pioglitazone could also influence the interactions of E-cadherin and ␤-catenin. The result revealed that pioglitazone synergistically affected the assembly of E-cadherin and ␤-catenin (Fig. 4E).
Furthermore, to evaluate whether Wnt signaling was involved in membrane-tethered ␤-catenin, we analyzed hPSC extracts for phospho-GSK3 (p-GSK3) by using a phospho-specific antibody that reacted with phosphorylated GSK3␤,-ser9 (P-GSK3,-Ser-9). The activity of GSK3 is inhibited via phosphorylation of Ser-9 (22). We observed that the level of P-GSK3,-Ser-9 increased upon pioglitazone co-treatment with Y-27632 (Fig. 5A). As already depicted, there was an increased accumulation of ␤-catenin and E-cadherin proteins in the Y-27632 plus pioglitazone cell extracts (Fig. 4, A and B). However, ␤-catenin and E-cadherin transcripts were not significantly induced (Fig. 5B), which suggested that both alterations occurred at on the protein level. For additional confirmation, we examined the recruitment existence of PPAR␥ on E-cadherin and ␤-catenin promoters. Chromatin was isolated from hPSCs maintained in hPSC medium that contained Y-27632 and/or Y-27632 plus pioglitazone using the PPAR␥ antibody. ChIP analysis indicated that recruitment of PPAR␥ on E-cadherin and ␤-catenin promoters in Y-27632 plus pioglitazonetreated cells was similar to Y-27632-treated cells (Fig. 5C). Collectively, these data show that pioglitazone induces accumulation of membrane-tethered ␤-catenin and the E-cadherin protein complex.
PPAR␥ Expression Regulated by the Rho/ROCK Signaling Pathway during hPSCs Dissociation-To determine whether modulation of PPAR␥ expression after hPSC dissociation (Fig.  3) resulted from Rho/ROCK activation during dissociation, we treated dissociated single hPSCs with Y-27632 as an inhibitor of the ROCK signaling pathway and assessed expression levels of PPAR␥, ␤-catenin, and E-cadherin. There was a significant increase in expressions of E-cadherin and ␤-catenin in Y-27632-treated cells within 4 h after treatment, whereas increased PPAR␥ expression occurred within the second hour after treatment of dissociated single hPSCs (Fig. 6A). These findings suggested prior up-regulation of PPAR␥ compared with ␤-catenin and E-cadherin transcripts in Y-27632-treated cells.
Next, we sought to determine whether the Rho/ROCK pathway directly affected PPAR␥ expression. We chose two factors from the beginning and end of this pathway, RhoA and PIP5K, respectively. These factors were separately co-transfected with a PPAR␥ expression plasmid under the regulation of a CMV promoter in a CHO cell line. Co-transfection results showed a considerable decrease in PPAR␥ protein expression that was affected by PIP5K as one of the final factors of the Rho/ROCK pathway (Fig. 6, B and C). According to the data the Rho/ROCK signaling pathway exerted its regulatory role on PPAR␥ by a direct inhibitory effect on its expression. There is no significant difference between DMSO and GW9662 treatments. C, immunostaining showed localization of ␤-catenin in the cell membrane at day 3. Scale bar: 100 m. D, Western blotting for plasma membrane (PM) and nuclear (N) fractions 4 h post-treatment of dissociated single hPSCs. We observed enhancement of ␤-catenin in the plasma membrane fragment. c-Myc and Tau proteins were used as positive controls for nuclear and plasma membrane proteins, respectively. E, co-immunoprecipitation (IP) for ␤-catenin and subsequent Western blotting (WB) for E-cadherin. Pioglitazone synergistically regulated the assembly of E-cadherin and ␤-catenin. ***, p Ͻ 0.001.
Pioglitazone and ROCK Inhibitor Y-27632 Did Not Affect hPSC Pluripotency-We assessed hPSC colony growth to determine the presence of a possible effect of pioglitazone on their morphological quality. Colonies grown for 31 passages in vitro retained predominantly undifferentiated morphological features such as well defined borders and small cells with a high nucleus:cytoplasm ratio (Fig. 7A). They expressed standard undifferentiating markers (ALP, Oct4, SSEA3, SSEA4, TRA-1-60, and TRA-1-81; Fig. 7A). The effect of pioglitazone on the undifferentiated hPSC state was assessed by analyzing the expression levels of stemness factors (NANOG, OCT4, and SOX2; Fig. 7B) by RT-qPCR at passage-31 (Fig. 7C). Pioglitazone enhanced the expression level of the stemness factor NANOG (Fig. 6B) in the undifferentiated state. Additionally, the differentiation potential of hPSCs was evaluated by spontaneous differentiation and the expression of PAX6, Nestin, and SOX1 at the RNA level (Fig. 7C) and PAX6 (ectodermal), SOX7 (endodermal), and EOMES (mesodermal) at the protein level (Fig. 7D). Collectively, the data showed that co-treatment of pioglitazone with Y-27632 did not negatively affect hPSC self-renewal.

Discussion
To our knowledge this is the first report where co-implementation of pioglitazone as a highly selective PPAR␥ agonist, with a ROCK inhibitor, Y27632, has increased survival and cloning efficiency (2-3-fold versus Y27632 alone) of individualized hPSCs under feeder-free culture conditions. However, pioglitazone alone did not enhance cloning efficiency.
We conducted a search for a possible mechanism for the positive effect of pioglitazone. Our cell cycle, 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. We demonstrated that the addition of the ROCK inhibitor, Y-27632, to Matrigel as an ECM for expansion of hPSCs increased cloning efficiency compared with its presence solely in culture medium through up-regulation of adhesion integrins (8). Therefore, we proposed that alteration in cell adhesion cytoskeletal elements resulted in colony formation enhancement (Fig. 8). It was demonstrated that activation of the Rho/ROCK pathway after the loss of E-cadherin-dependent intercellular adhesion played a pivotal role in the apoptosis of dissociated single hPSCs (4). Inappropriate destabilization of ␤-catenin in induction of apoptosis has been shown in tumor cells (23). We observed that the FIGURE 5. Induction of ␤-catenin by pioglitazone. A, Western blot analysis for phospho-GSK3 (P-GSK3,-ser9) in dissociated single hPSCs. Data quantification showed enhanced P-GSK3 in cell extracts of Y-27632 plus pioglitazone (Pio). ***, p Ͻ 0.001. B, RT-qPCR analysis for ␤-catenin and E-cadherin transcripts were not significantly induced in Y-27632 plus pioglitazone. C, ChIP for the existence of ␤-cateninand E-cadherin-associated PPAR␥ binding sites was performed using related antibodies. There was no significant difference (NS) between both groups. There were no detected response elements for PPAR␥ on E-cadherin and ␤-catenin promoters. At the fourth hour, expressions of PPAR␥, E-cadherin, and ␤-catenin increased significantly. B and C, we sought to determine if the Rho/ROCK pathway directly affected PPAR␥ expression. RhoA and PIP5K were separately co-transfected with a PPAR␥ expression plasmid under the regulation of a CMV promoter in a CHO cell line. Analysis of Western blots showed PPAR␥ protein expression was influenced by PIP5K. *, p Ͻ 0.05. Therefore, the Rho/ROCK signaling pathway exerted its regulatory role on PPAR␥ by a direct inhibitory effect on its expression. protein levels of E-cadherin, ␤-catenin, and PPAR␥ down-regulated in individualized hPSCs. In contrast these protein levels up-regulated after application of PPAR␥ activation and ROCK inhibition. Immunostaining, co-immunoprecipitation of E-cadherin and ␤-catenin and plasma membrane, and nuclear fractionation of the cells showed more plasma membrane localization of ␤-catenin and its direct interaction with E-cadherin after pioglitazone treatment. This result was consistent with a previous study which reported that ligand-activated PPAR␥ directly interacted with ␤-catenin and resulted in retaining this component in the cytosol (24). The up-regulation of ␤-catenin in individualized hPSCs occurred through GSK3 inactivation by ligand-activated PPAR␥. This escalation of membranous ␤-catenin along with E-cadherin led to intensified colony formation in dissociated single hPSCs by pioglitazone and inhibition of ␤-catenin-mediated transcriptional pathways involved in promoting cell proliferation.
The mechanism of pioglitazone and Y-27632 action in enhancing E-cadherin is an intriguing question that awaits future investigation. A recent report has suggested that ␤-catenin transcriptional activity through modulation of Tcf3 activity plays a role in preventing exit from the pluripo-tent state (25). In contrast, it has been shown that transcriptional activity of ␤-catenin is negligible during self-renewal, which is due to the tight association of ␤-catenin with plasma membrane, where it is in a complex with E-cadherin (26). On the other hand, E-cadherin also can recruit ␤-catenin to the cell membrane and prevent its nuclear localization and transactivation (27). The positive role of Wnt pathway activation by GSK3 inhibition in maintenance of the undifferentiated hPSCs has been presented previously (28), although this is controversial (29).
Of interest, we observed that the expression of PPAR␥ transcripts increased after ROCK inhibition. On the other hand, overexpression of Rho/ROCK pathway components significantly decreased the amount of PPAR␥ protein. In the case of RhoA, there were no significant changes in the PPAR␥ level due to the absence of ROCK (a mediating factor in this pathway) to transfer this signaling. However, the level of PPAR␥ decreased significantly with PIP5K transfection. The inhibitory role of PPAR␥ agonists on the Rho/ROCK pathway in cultured rat aortic smooth muscle cells has previously been demonstrated (30). It was reported that Rho/ROCK activation inhibited expression of PPAR␥, which thereby caused reduced adipogen- RT-qPCR analysis for stemness genes (B) in undifferentiated state is shown. Shown are RT-qPCR (C) and Western blot (D) analyses after spontaneous differentiation by embryoid body formation in hPSC medium without basic FGF for 2 weeks. *, p Ͻ 0.05. esis (31) and increased cell proliferation in pulmonary artery smooth muscle cells of sheep (32). Those two pathways likely regulate each other in hPSCs.
Co-treatment of pioglitazone and Y-27632 also markedly increased the cloning efficiency of hPSCs without affecting their pluripotency. However, we observed up-regulation of NANOG that could be related to interaction with ␤-catenin. It was demonstrated that increased ␤-catenin or the addition of Wnt3A to the culture medium promoted pluripotency and led to NANOG expression (33).
Taken together, the addition of the PPAR␥ agonist, pioglitazone, and Y-27632 to culture medium synergistically increased cloning efficiency of both individualized hESCs and hiPSCs compared with Y-27632 alone in feeder-free culture conditions upon passaging. This might be related to adhesion through enhanced up-regulation and accumulation of membranous ␤-catenin and its interaction with E-cadherin as well as augmentation of signal transduction from the ECM, external environment, and the cell membrane into the cytoplasm, which resulted in changes to cellular dynamics and further downstream targets that regulated gene expression. These results provided a more favorable condition toward hPSCs manipulation and their biomedical applications.