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Failure of Y-27632 to improve the culture of adult human adipose-derived stem cells
No full textNuno Jorge Lamas,1–3 Sofia C Serra,1,2 António J Salgado,1,2 Nuno Sousa1,2 1Life and Health Sciences Research Institute (ICVS), School of Health Sciences (ECS), University of Minho, Braga, Portugal; 2ICVS/3B’s-PT Government Associate Laboratory, Braga/Guimarães, Portugal; 3Clinical Pathology Department, Centro Hospitalar do Alto Ave (CHAA), EPE, Guimarães, Portugal Abstract: Y-27632 is a well-known inhibitor of the Rho-associated coiled kinase (ROCK) and has been shown to significantly improve the culture of a variety of multipotent stem cell types. However, the effects of Y-27632 on the expansion of adult human adipose-derived stem cell (hADSC) cultures remain to be established. Here, we aimed to characterize the effects of Y-27632 on the culture of hADSCs. Adult hADSCs were isolated from subjects submitted to elective plastic surgery procedures and cultivated in vitro under optimized conditions. Our results show that the continuous supplementation of hADSC cultures with Y-27632 led to decreased numbers of cells and decreased global metabolic viability of hADSC cultures when compared with control conditions. This effect appeared to be dependent on the continuous presence of the drug and was shown to be concentration-dependent and significant for 10 µM and 20 µM of Y-27632. Moreover, the Y-27632-induced decrease in hADSC numbers was not linked to a block in global cell proliferation, as cell numbers consistently increased from the moment of plating until passaging. In addition, Y-27632 was not able to increase the number of hADSCs present in culture 24 hours after passaging. Taken together, our results suggest that, in contrast to other stem cell types, Y-27632 supplementation is not a suitable strategy to enhance hADSC culture expansion. Keywords: human mesenchymal stem cells, human multipotent stromal cells (hMSCs), ROCK inhibitor, MTS assa
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Y-27632 mediated signal transduction during differentiation.
No full text<p>Total mRNA collected from all groups was converted to cDNA and probed to assay levels of β-catenin (A). β-catenin levels increased under all differentiation induction conditions, however, levels decreased significantly when Y-27632 was added during mesodermal and ectodermal differentiation. Immunofluorescent detection of β-catenin in undifferentiated cells (B) and cells treated with RA (C) RA plus Y-27632 (D) or RA with Y-27632 pre-treatment (E). To assay the activity of the TGF-β pathway, cell lysates were probed with a phospo-SMAD2/3 antibody (F). Results demonstrate that a significant decrease in SMAD activation occurred when Y-27632 was present or after pre-treatment and removal of the inhibitor. cDNA from RA induced cells was assayed for Ezrin, Radixin and Moesin expression levels (G). A pan-ERM antibody was used to stain undifferentiated cells (H) and those exposed to RA (I) RA plus Y-27632 (J) or RA with Y-27632 pre-treatment (K). Model representing ROCK interaction with multiple pathways influencing the ‘fate’ of unspecified stem cells (L). Scale bar equals 50 µm, green staining represents the primary antibody, and blue staining is the nuclear dye TOTO-3. * Significance accepted at p<0.05.</p
Y-27632 rescues neuritogenesis.
No full text<p><i>A-D</i>, The <i>Vil2<sup>+/+</sup></i> and <i>Vil2<sup>kd/kd</sup></i> neurons treated with DMSO (<i>A</i>,<i>C</i>) or 40 µM Y-27632 (24 h, <i>B</i>,<i>D</i>) were fixed at 2 DIV and stained with an anti-neuronal class III β-tubulin antibody (green) and rhodamine phalloidin (red). Scale bars, 50 µm. <i>E-G</i>, The number (<i>E</i>) and length (<i>F</i>) of neurites, and length of axon (<i>G</i>) were quantified in the <i>Vil2<sup>+/+</sup></i> and <i>Vil2<sup>kd/kd</sup></i> neurons treated with DMSO (white columns, n = 30) or 40 µM Y-27632 (black columns, n = 30). Three independent experiments were performed. <i>*p</i><0.05, <i>**p</i><0.01, <i>***p</i><0.001 (DMSO-treated vs. Y-27632-treated), <i>###p</i><0.001 (DMSO-treated <i>Vil2<sup>+/+</sup></i> vs. DMSO-treated <i>Vil2<sup>kd/kd</sup></i>), Student's t test. Data represent mean ± SE.</p
Y-27632 enhances the differentiation of P19 cells into ectoderm.
No full text<p>P19 cell aggregates were treated with RA+LIF to induced neural-ectoderm differentiation. Total mRNA was collected, converted to cDNA and used in a PCR to examine GFAP expression, a marker of ectodermal derived neural cell types (A). GFAP levels increased with RA+LIF treatment in the presence or absence of Y-27632. Increased GFAP expression was also detected in the DMSO treated groups supplemented with Y-27632. Immunofluorescent detection of GFAP in undifferentiated (B) RA+LIF (C) RA+LIF and Y-27632 (D) or RA+LIF with Y-27632 pre-treated cells (E). qPCR with NF-68 primers showed an increase in expression in cells exposed to Y-27632 during differentiation (F). Immunofluorescent detection of NF-68 in undifferentiated cells or those exposed to the following: (G) RA+LIF (H) RA+LIF and Y-27632 (I) or RA+LIF with Y-27632 pre-treatment (J). FACS analysis demonstrates increased numbers of GFAP- (trend) and NF-68-positive cells with Y-27632 treatment during RA+LIF induced differentiation (K). Scale bar equals 50 µm, green staining represents the primary antibody, and blue staining is the nuclear dye TOTO-3. * Significance accepted at p<0.05.</p
Reversibility of the effect of Y-27632 on cell morphology.
No full text<p>Images of Y-27632-treated HeLa JW cells were taken prior to addition of the drug, after 30 min incubation with 10 µM Y-27632, and 2 h after the drug was washed off. The effect of Y-27632 on cell morphology was reversible, as cells regained normal morphology within 2 hours of reagent withdrawal.</p
Dynamics of FA plaque proteins following Y-27632 treatment.
No full text<p>(<b>A</b>) Normalized averaged FRAP curves (n=20-30) of FA plaque proteins in untreated and Y-27632 treated cells. For some proteins, no major changes were observed in the FRAP curves in untreated vs. treated cells (talin, FAK, VASP, ILK), whereas other proteins displayed higher (vinculin, kindlin-2) or lower (paxillin, zyxin) FRAP rates following Y-27632 treatment. (<b>B</b>) Kinetic parameters of FA proteins as derived from FRAP measurements within the first 9 min of Y-27632 treatment. The half-time to full recovery (T<sub>1/2</sub>) and mobile fraction (R<sub>f</sub>) values of the FRAP curves prior to and following Y-27632 treatment were not indicative of the overall FA disassembly state. However, the differences between the k<sub>on</sub> and k<sub>off</sub> values derived from fitting the FRAP data to the full diffusion-exchange model represent the disassembly rate of FAs for each protein, following Y-27632 treatment. (<b>C</b>) Theoretical FA disassembly curves calculated from the differences in the expected disassembly rates of tested proteins from the FRAP data. (<b>D</b>) Actual disassembly curves measured directly from time-lapse movies, following treatment of HeLa JW cells with Y-27632. Note the remarkable similarity between the calculated and measured values.</p
Y-27632 promoted the proliferation of human limbal epithelial cells.
No full text<p>Phase contrast photographs of human limbal epithelial cells expanded on plastic dishes with or without adding various concentrations of Y-27632 in culture medium (A). Magnification: 200×. Cells with prominent nuclei and high nucleus/cytoplasm ratio as indicated by arrows were noted in dishes with Y-27632. There was a positive dose-dependent correlation between the concentrations of Y-27632 and proliferation of expanded limbal epithelial cells (B). Statistical analyses were performed using a paired test, and <i>*</i>p<0.05 was considered significant.</p
Though dissociation-induced PTEN activation is suppressed by Y-27632, it does not contribute to Y-27632 induced increase of the cloning efficiency of prostate stem/progenitor cells.
No full text<p>(A) Western blot analyses of protein expression in prostate colony cells grown with and without 10 µM Y-27632 and in dissociated primary prostate colony cells cultured in suspension for 4 hours with and without 10 µM Y-27632. (B) Bar graph shows the sphere-forming units of WT and PTEN KO prostate cells in the presence and absence of Y-27632. Error bars represent means and STD from 3 wells. * <i>P</i><0.05, ** <i>P</i><0.005.</p
ROCK inhibitor Y-27632 on the gene expression of porcine trophoblast cells.
No full text<p>(A-B) The real-time PCR analyses of gene expression changes of pTR cells cultured with Y-27632. Most trophoblast genes analyzed are up-regulated in Y-27632 treated cells (pIVFTR-7 A) and pPATR-5 B)). ROCK1 and ROCK2 are down-regulated. (C) Western blot detected proteins changes pTR cells cultured with Y-27632. CDX2 was remarkably increased in Y-27632 treated pIVFTR-7 and pPATR-5 cells. pTR cells cultured without Y-27632 in the same condition are used as control. β-ACTIN serves as an endogenous control. The * indicates P < 0.05 and the **indicates P < 0.001.</p
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