A comparison of epithelial and neural properties in progenitor cells derived from the adult human ciliary body and brain☆
Introduction
During embryogenesis, the multipotent neuroectoderm of the optic cup develops into the central retina and a peripheral region called the ciliary margin (CM) (Klassen et al., 2004a). While the central retina becomes an integrated part of the central nervous system (CNS), the CM gives rise to two non-neural structures: the double-layered epithelium of the ciliary body (CB) and the distal iris (Perron and Harris, 2000, Perron et al., 1998). However, the CM contains multipotential retinal progenitor cells even at a late stage of development, and in certain lower vertebrates it is a source of progenitor cells for retinal regeneration throughout life (Wetts et al., 1989).
In adult humans, the retina is considered to have limited regenerative potential, and severe injuries will lead to permanent damage. Interestingly, current studies have revealed that the CB (Coles et al., 2004, Tropepe et al., 2000, Xu et al., 2007), in addition to the retina itself (Carter et al., 2007, Klassen et al., 2004b, Lawrence et al., 2007, Mayer et al., 2005), even in adulthood harbors cells with the characteristics of neural stem cells (NSCs); (1) they are undifferentiated and have the capability of cell division and self-renewal (2) they are multipotent, i.e. they have the ability to differentiate into both neurons and glia found in the retina (Boulton and Albon, 2004, Gage, 2000).
In vitro, NSCs grow in suspension as characteristic spherical aggregates (neurospheres) containing a mixture of multipotent neural stem cells normally present in very small numbers as well as progenitor cells that are more restricted in their proliferative and phenotypic potential (Arsenijevic et al., 2001, Kukekov et al., 1999). Previous studies of CB spheres from the adult eye have mainly focused on the differentiation of cells along various retinal phenotypic pathways after attached colony formation or after grating in vivo (Coles et al., 2004, Tropepe et al., 2000). Only recently, studies have focused on the characterization of the sphere composition during in vitro cultivation (Kohno et al., 2006, Xu et al., 2007). As CB spheres are isolated from a non-neural epithelium of neuroepithelial origin, it is vital to map their regulation of epithelial and neural properties to fully utilize them as a clinical source of retinal progenitor cells in the future.
In the present paper, we have for the first time combined immunohistochemistry, molecular biology and electron microscopy to compare CB spheres isolated from the adult human eye with neurospheres derived from the adult human subventricular zone (SVZ), which is considered the largest and best characterized stem cell niche in the CNS (Gage, 2002, Liu et al., 2003). We have also compared the two sphere-forming cell populations from adult rats. Our data indicate that adult human ciliary body spheres contain epithelial-like cells with decreased expression of neural stem cells' markers compared to CNS neurospheres.
Section snippets
Material and methods
The research was conducted in accordance with the Declaration of Helsinki and all tissue harvesting was approved by the Norwegian National Committee for Medical Research Ethics. All animal experiments were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Eyes were enucleated from cadavers (n = 8) as previously described (Slettedal et al., 2007). The CB epithelium was carefully dissected under an operating microscope and placed in Leibowitz-15
Sphere-forming capacity
Isolated cells from the CB divided to form spheres with a uniform, well-defined spherical contour containing a variable amount of pigmentation (Figs. 1A–B and 3A), whereas SVZ spheres did not contain pigment (Fig. 1D). The measured growth rate of individual spheres ten days after the first passage (P1 + 10D) showed that human CB spheres had a diameter of 134 ± 50 μm (n = 45), whereas human SVZ spheres only measured 69 ± 24 μm (n = 45, p < 0.01) (Fig. 1C). In contrast, there was no statistical
Discussion
Adult retinal stem/progenitor cells may open a door of opportunity for autotransplantation, where cells can be isolated and expanded in culture outside of their natural niche before transplantation back into the diseased eye (Djojosubroto and Arsenijevic, 2008). Further, these progenitors may be manipulated before transplantation, for instance by induced differentiation towards a photoreceptor fate (Coles et al., 2004, Klassen et al., 2004c), by overexpression of disease modulating substances
Acknowledgements
The authors would like to thank Eli Gulliksen and Kristiane Haug (Center for Eye Research), Elin Kampenhaug and Birthe Mikkelsen (Vilhelm Magnus Laboratory), and Steinar Stølen and Yiqing Cai (Department of Oral Biology, Faculty of Dentistry, University of Oslo) for their excellent technical assistance and support. The authors thank Dr. Wayne Murrell for critical reading and helpful suggestions on this manuscript. Excellent working conditions at Institute for Surgical Research were provided by
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2015, Progress in Retinal and Eye ResearchCitation Excerpt :While initially identified in rodents, these cells have subsequently been isolated from rabbit (Inoue et al., 2005), pig (Gu et al., 2007; Macneil et al., 2007), monkey (Martinez-Navarrete et al., 2008) and human (Bhatia et al., 2009; Coles et al., 2004; Jasty et al., 2012; Martinez-Navarrete et al., 2008; Mayer et al., 2005; Moe et al., 2009; Xu et al., 2007a) eyes, suggesting an evolutionary conservation of this population in the adult vertebrate eye. Demonstrating a cardinal feature of stem cells, the resulting neurospheres can be dissociated and grown after several passages, though many reports have suggested that the growth of these cells as free-forming spheres become less vigorous after multiple passages, especially when compared to brain-derived neural stem cells (Moe et al., 2009; Xu et al., 2007a; Yanagi et al., 2006). However, these cells have a broader expansion potential when cultured as monolayers, maintaining a progenitor phenotype (Coles et al., 2004; Gualdoni et al., 2010; Macneil et al., 2007; Xu et al., 2007a).
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Grant information: Supported by the Norwegian Association of the Blind and Partially Sighted, the Blindmission IL, the Research Council of Norway, Ullevål University Hospital and Rikshospitalet University Hospital.