Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • The current study demonstrates that extracellular ATP releas

    2018-10-20

    The current study demonstrates that extracellular ATP, released during passaging, binds to P2X7 allowing calcium influx which in turn correlated with initiation of dynamic blebbing through activation of the Rho pathway. The interplay between Rac and Rho determined whether Octreotide acetate underwent dynamic blebbing or attached. We showed that activation of Rac reduced dynamic blebbing and that activated-Rac was less abundant in dynamically blebbing cells than in attached hESC. Moreover, inhibition of Rac by NSC23766 extended dynamic blebbing. Rac and Rho regulate motility in many cells in which Rac signaling antagonizes Rho directly at the GTPase level (Parri & Chiarugi, 2010). For example, the balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH 3T3 fibroblasts (Sander et al., 1999). The above results identify several targets that could be used to prevent initiation of dynamic blebbing (Fig. 7). One strategy would be to decrease the release of ATP during passaging by using gentle methods that minimize cell death or by using reagents, such as apyrase, that hydrolyze extracellular ATP. Another potential target would be calcium which influxes upon activation of the P2X7 receptor. Inclusion of a chelating agent helped reduce dynamic blebbing, but may also interfere with cell attachment. Rac activation could be used to prevent dynamic blebbing and may be a possible target for improving attachment and plating of single hESC in the future. The best method that we found for inhibiting initiation of dynamic blebbing was inclusion of the P2X7 inhibitor (KN62) in culture medium during passaging. This inhibitor is more specific than the ROCK inhibitors, is relatively inexpensive, and does not stress or alter cell morphology, as seen with ROCK inhibitors. In embryos, dynamic blebbing is a driving force that enables cell migration (Blaser et al., 2006; Trinkaus, 1973; Wourms, 1972; Trinkaus, 1996). While a blebbing cell in suspension does not have progressive motility, in a three-dimensional matrix, blebbing cells can gain space and would be able to escape environmental danger or migrate in an embryo (Fackler & Grosse, 2008). hESC bleb more vigorously than other cells types (Weng, 2016), indicating that hESC have a cytoskeleton that is poised for hyperactivation. Cultured hESC resemble cells of the epiblast (Ohgushi et al., 2010; Ohgushi & Sasai, 2011), which undergo massive movements during gastrulation. The dynamic blebbing observed in vitro may occur because hESC (epiblast-like) would normally gastrulate in an embryo and migrate first as sheets of epiblast and then as mesenchymal cells following extrusion from the primitive steak. While this point would require future investigation, the data in this study may help understand the massive cell movements characteristic of gastrulating embryos. In contrast to the hESC used in our study, mouse embryonic stem cells (mESC), which resemble the inner cell mass from which they were derived (Ohgushi et al., 2010), show relatively little dynamic blebbing in vitro during passaging and attach rapidly to their substrate (Weng, 2016). Recently, new methods for deriving hESC have been successful in obtaining “naïve” states of pluripotent hESC, which like mESC, resemble cells of the inner cell mass, not the epiblast (Ohgushi et al., 2010). It is likely that naïve hESC lines would be less prone to dynamic blebbing and may offer an additional alternative to avoid dynamic blebbing during passaging of pluripotent cells. The following are the supplementary data related to this article.
    Authors contributions
    Conflicts of interest
    Funding NW was supported by a NSF IGERT fellowship in Video Bioinformatics (grant # DGE 093667 awarded to Dr. Bir Bhanu). Video imaging was done in the UCR Stem Cell Core, which was supported by grant # CL1-00508 from the California Institute for Regenerative Medicine to PT. Portions of the work were funded by grant # 22RT-0127 from the Tobacco-Related Disease Research Program of California to PT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation Octreotide acetate of the manuscript.