Novel synthetic small molecules dramatically increase hESC survival after single cell dissociation by enhancing cell-ECM mediated integrin activity. (A) Chemical structures of Thiazovivin/Tzv and Pyrintegrin/Ptn as indicated. (B) ALP staining of hESC colonies that had grown from dissociated single cells seeded in low density for four days and treated as indicated. Bar, 20 μm. (C) Ratio of ALP positive colonies vs. total initially seeded cells (n = 2). (D) Immunostaining of hESCs long-term maintained in chemically defined media containing Ptn or Tzv as indicated. Bar, 50 μm. (E) Representative phase contrast images of hESCs 12 h after seeding on the different matrices and treated with the indicated compounds. Bar, 20 μm. If not specified, all the above hESCs were grown in the chemically defined medium and feeder-free condition on Matrigel coated plates. All error bars indicate ± SEM.

Tzv stabilizes E-cadherin after cell dissociation to protect hESCs from death under ECM-free conditions. (A) Phase contrast images of hESCs grown on noncoated plates treated with the indicated molecules. Bar, 50 μm. (B) Western blot analysis of full-length E-cadherin in hESCs before and after trypsin. (C) A time-course Western blot analysis of full-length E-cadherin expression in hESCs after trypsin dissociation and treatment with DMSO, Tzv, or Ptn for indicated time. (D) Flow cytometry analysis of E-cadherin surface level in hESCs after trypsin treatment in the presence of Tzv. DMSO was used as a control. (E) E-cadherin endocytosis analysis in the absence or presence of Tzv (n = 2). (F) Cell survival analysis of hESCs grown on BSA or different concentrations of E-cad-Fc chimera-coated plates for five days (n = 2). All error bars indicate ± SEM.

Tzv is a novel ROCK inhibitor and Rho-ROCK axis regulates cell-ECM and cell-cell adhesion (A) In vitro Rho-kinase assay treated with indicated compounds (n = 3). (B) Staining of F-actin in hESCs 6 h after seeding on Matrigel-coated plates in the absence or presence of Y-27632, a selective ROCK inhibitor. Bar, 10 μm. (C). Flow cytometry analysis of E-cadherin surface level in hESCs 6 h after trypsin treatment in the presence of indicated compounds. DMSO was used as a control. (D) Phase contrast images of hESCs grown on noncoated plates treated with the indicated molecules. Bar, 100 μm. All error bars indicate ± SEM.

Cell-cell interaction regulates cell-ECM interaction and contributes to survival and self-renewal of hESCs. (A) Western blot showing the active Rho level in hESCs before or after trypsin. (B) Rho-kinase assay showing the ROCK activity in hESCs before or after trypsin treatment (n = 3). (C) Western blot showing the active Rho level in hESCs 30 min after replating at the two different densities on the Matrigel-coated plates. (D) Rho-kinase assay showing the ROCK activity in hESCs 30 min after replating at the two different densities on Matrigel-coated plates (n = 3). (E) Western blot showing the active Rho level in hESCs 30 min after replating on BSA or different concentrations of E-cad-Fc chimera-coated plates. (F) Cell attachment on Matrigel-coated plates for different densities of hESCs (n = 2). LC, low-density culture (1.5 × 10⁴ cells per well of 6-well plate). HC, higher-density culture (15 × 10⁴ cells per well of 6-well plate). All graphs show mean ± SEM.

hESCs maintained in mESCs culture condition have better survival and are dependent on different cell adhesion signaling for survival and self-renewal. (A) A time-course Western blot analysis of full-length E-cadherin expression in mESCs after replating for the indicated time. (B) Phase contrast images of the conventional hESCs and converted hESCs 24 h after replating on noncoated plates. Bar, 100 μm. (C) Immunostaining of converted hESCs with typical pluripotency markers. Bar, 50 μm. (D) Phase contrast images of the conventional hESCs and converted hESCs. Bar, 50 μm. (E) Fluorescent images of Oct4-GFP hESCs and converted Oct4-GFP hESCs two days after replating on the Matrigel-coated plates in the presence of control IgG, integrin β1-blocking antibodies, and E-cadherin blocking antibodies (5–10 μg/mL). Representative pictures are shown from three independent experiments. (F) Cell-cell adhesion and cell-ECM adhesion regulate each other through Rho-ROCK signaling. Two cell adhesion systems cooperate with growth factor signaling to control survival, self-renewal of hESCs. (G) Pluripotent stem cell states may exhibit a broad range, and distinct cell states within this dynamic range could be created by different inputs from the two cell adhesion signaling pathway. All graphs show mean ± SEM.