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
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04
  • 2024-05
  • 2024-06
  • 2024-07
  • 2024-08
  • 2024-09

    • The following are the supplementary data related

    2018-10-20

    The following are the supplementary data related to this article.
    Conflict of interest
    Introduction Mammalian eyes contain retinal stem cells (RSCs) that are capable of proliferating and differentiating into all of the cell types of the neural retina as well as the retinal pigmented epithelium (RPE) in vitro (Tropepe et al., 2000; Ahmad et al., 2000; Demontis et al., 2012; Fang et al., 2013; Ballios et al., 2012; Coles et al., 2004). These stem cells are located at the retinal periphery in the bi-layered ciliary epithelium (CE), which overlays the ciliary body muscles that control the lens. This CE contains an inner non-pigmented layer that is contiguous with the neural retina and a pigmented outer layer that is contiguous with RPE (Xu et al., 2002; Bharti et al., 2006); however, RSCs only reside within the pigmented CE layer. The ciliary marginal zone (CMZ) is a region in the peripheral retina of fish and meclofenamate that is capable of continuous retinal regeneration (Reh & Levine, 1998). The similar peripheral location of mammalian RSCs suggests there may be some homology in the RSC niche between these animal classes (Reh & Levine, 1998). The two CE layers can be distinguished by the expression of cadherins, in that the non-pigmented CE (and the neural retina) expresses N-Cad (Cdh2) and the pigmented CE (and the RPE) expresses P-Cad (Cdh3) (Xu et al., 2002; Nose & Takeichi, 1986; Inagaki et al., 2005). We have shown through FACS and single cell analyses that RSCs arise from a rare cell within the pigmented CE that expresses P-Cad but not N-Cad (Ballios et al., 2012). These rare prospectively isolated cells proliferate to form clonally derived spheres in vitro that are multipotential and self-renewing, arguing that they are stem cells and not cells derived via transdifferentiation as suggested by others (Cicero et al., 2009). We sought to distinguish whether P-Cad or N-Cad are simply indicative of the CE tissue of origin or whether they are functionally relevant to the maintenance or proliferation of RSCs in vivo and in vitro.
    Material and methods
    Results
    Discussion Adhesion molecules have been implicated in forming the stem cell niche in many systems (Raymond et al., 2009), including germ cells in Drosophila testis (Voog et al., 2008), murine mammary stem cells (Stingl et al., 2006) and murine neural stem cells in the brain (Shen et al., 2008). For example, in the fly testis if there is a loss of adhesion of the stem cells to the hub, then the stem cells will lose their ability to maintain their stemness (Raymond et al., 2009; Voog et al., 2008). In mammalian mammary cells if there is a loss of B1 Integrin, then the stem cells appear to lose proliferative ability (Taddei et al., 2008). In the present study, P-Cad was found to be restricted to the RPE and pigmented CE and N-Cad was restricted to the NR and non-pigmented CE, with no overlap of expression of P-Cad and N-Cad in single cells in vivo. We also demonstrated that P-Cad was not required in vivo to maintain the RSC niche, and that the CE appeared to be morphologically intact in P-Cad−/− mice. P-Cad is expressed in the cap and myoepithelial cells of the mammary gland; however, in the P-Cad−/− mouse these cells are not affected and maintain their structural integrity (Radice et al., 1997), suggesting that there may be compensatory mechanisms in place that prevent tissue from disorganizing in vivo with the loss of one of its main adhesion proteins. A similar compensatory adhesive mechanism may be at work to maintain the structure of pigmented CE and its RSCs in vivo. However, the CE from P-Cad−/− mice was easier to dissociate, which appears to have led to more of the RSCs and CE cells in general surviving the combination of enzymatic digestion and harsh mechanical dissociation into single cells. It is possible that we have been underestimating the total number of stem cells in the wild type CE due to cell death during the dissociation of the tissue. Indeed, the total number of clonal spheres obtained per retina was significantly greater in the P-Cad−/− mice compared with wild type controls, but the frequencies of clonal sphere formation were not different significantly. Thus, if there is a compensatory adhesive mechanism, it does not appear to completely rectify the loss of P-Cad mediated adhesion, as evinced by the greater ease of dissociation of pigmented CE cells in vitro. In addition, there appears to a low upregulation of NCad expression in a subset of the PCad−/− pigmented CE cells which may suggest that NCad may be compensating for the loss of PCad in these mice. However, blocking NCad in the PCad−/− cells has no affect which leads us to conclude that there may be other compensatory mechanisms in these mice.