Y a selective Rho associated kinase ROCK inhibitor is known

Y-27632, a selective Rho-associated kinase (ROCK) inhibitor, is known to facilitate hESC/hiPSC culture growth by diminishing dissociation-induced apoptosis (Ohgushi et al., 2010; Watanabe et al., 2007). We therefore compared doxycycline- and Y-27632-mediated hESC survival by the colony-forming assay using H9 glutathione reductase in Matrigel coating-mTeSR1 medium culture. Cell numbers increased more during 5 days of culture in the doxycycline-supplemented cultures than in either the untreated or Y-27632-treated cultures (Figure 2E). The number of colonies (colony-forming efficiency) was robustly greater in the cultures treated with doxycycline (81.9 ± 12.7 colonies/well) or Y-27632 (79.6 ± 11.7 colonies/well) than in the untreated control (20.3 ± 6.7 colonies/well); the doxycycline and Y-27632 effects were indistinguishable (Figure 2F). However, colony sizes (average numbers of cells assembled in a colony) in the doxycycline-treated group (136.2 ± 8.1 cells/colony) were significantly larger than those in the Y-27632-treated group (103.8 ± 10.5 cells/colony) and untreated control (80.9 ± 6.3 cells/colony) (Figure 2G). Cell colonies in Y-27632-treated cultures differed somewhat in shape as compared with those of the control cultures, with less compactly arranged cells that exhibited bipolar cellular processes (Figure 2H), probably because of altered Rho kinase activity, which is associated with changes in cell morphology (Harb et al., 2008) (the cell morphologies returned to normal within several hours after Y-27632 withdrawal). In contrast, cell and colony shapes in the doxycycline-treated cultures were indistinguishable from those of the untreated culture. Notably, the percent of AP+ undifferentiated hESC colonies in the total number of colonies was significantly higher in the doxycycline-treated cultures than in the untreated control and Y-27632-treated cultures (74.7% ± 1.6% in doxycycline-treated, 66.9% ± 1.8% in Y-27632-treated, 64.3% ± 3.3% in untreated cultures, Figure 2I). Expression of genes encoding human transcription factors generally appeared similar for doxycycline-treated and untreated hESCs in gene expression arrays, but expression levels of genes related to hESC pluripotency were greater in the doxycycline-treated cultures (Figure 2J). The increased pluripotency marker gene expression was confirmed using real-time PCR (data not shown). Doxycycline’s ability to maintain an undifferentiated state was also apparent in mouse ESCs (mESCs) that had been engineered to contain Oct3/4 promoter-driven GFP: doxycycline treatments increased the percentage of GFP+ cells (Figure S2). Taken together, these findings indicate that doxycycline supplementation generates greater yields of undifferentiated hESCs in culture, not only by promoting cell survival but also by maintaining self-renewal. Because of their vulnerability after cell dissociation, hESC/iPSCs are commonly maintained in clusters without dissociation on a feeder layer of mouse embryonic fibroblasts (MEFs). Thus, we examined whether doxycycline supplementation would confer a practical benefit: facilitating the expansion and maintenance of undifferentiated hESC/iPSCs in culture for long periods with multiple cell passages. The doxycycline-mediated hESC survival and self-renewal shown in colony-forming assays (Figure 2) were further confirmed using fluorescence-activated cell sorting (FACS) on hESC/iPSCs cultured in clusters (Figures 3A and 3B). Cells on apoptosis (Annexin V+/PI+) were greatly reduced by doxycycline treatment (Figure 3A), along with decrease of dead cell populations (Figure 3B). In addition, significantly more cell populations in hESC (H9) and hiPSC (Lenti-1) cultures were accumulated in S phase by doxycycline treatment (Figure 3B). When H9 hESCs were subcultured in clusters on MEF feeders, increases of >5-fold and >120-fold in cell number were attained over 56 days (8 passages) and 140 days of culture (20 passages), respectively, by simply adding doxycycline (Figures 3C and 3D), as compared with untreated cells, without altering the normal karyotype (Figure 3E). Immunocytochemical (Figure 3F) and FACS (Figure 3G) analyses showed that, in the multipassaged cultures with doxycycline supplementation, most cells expressed undifferentiated hESC markers. In addition, pluripotent potentials of the hESCs maintained with doxycycline supplementation were confirmed by in vitro differentiation into three germ layers (Figures S3A and S3B) and in vivo teratoma formation (Figure S3C).