Generation and clonal isolation of retinal stem cells from human embryonic stem cells

Abstract

Retinal stem cells (RSCs) are present within the pigmented ciliary epithelium (CE) of the adult human eye and produce progeny that differentiate in vitro into all neural retinal subtypes and retinal pigmented epithelium (RPE). We hypothesized that a RSC population, similar to the adult CE-derived RSC, is contained within pigmented colonies that arise in long-term cultures of hESCs suggested to recapitulate retinal development in vitro. Single pigmented hESC-derived cells were isolated and plated in serum-free media containing growth factors and, after 2 weeks, clonal sphere colonies containing both pigmented and nonpigmented cells were observed. These colonies expressed the early retinal transcription factors Rx, Chx10 and Pax6, and could be dissociated and replated as single cells to form secondary clonal colonies. When allowed to differentiate, expression of markers for both RPE and neurons was observed. Rhodopsin expression was detected after explant coculture and transplantation into the developing mouse eye as well as following treatment with soluble factors in vitro. We show that RSCs emerge in an in vitro model of retinal development and are a potential source of human photoreceptors for use in transplantation.

A new immunodeficient pigmented retinal degenerate rat strain to study transplantation of human cells without immunosuppression

“…Transplantation of fetal retinal sheets together with and without its RPE has resulted in visual improvements in animal models (review [17–19]) and in human patients [20]. Several laboratories are working on transplantation of human stem cell-derived retinal progenitors or RPE cells with the goal of restoring vision using mostly immunosuppressed animal models [22–25, 27, 35, 41–43]. However, immunosuppression is very labor-intensive and can have negative effects on the health of the recipient, especially with long-term use; e.g., the most commonly used immunosuppressant cyclosporin A can have nephrotoxic effects [44, 45].…”

Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue

“…5a). We manually dissected nonpigmented Rx::Venus-strong tissues in the tapered NR margin (CM-like tissues, hereafter) from hESCderived retina (day 60-85), dissociated them, and then subjected them to retinosphere culture (1,000 cells per well) 17,52,53 . For comparison, we dissected nonmarginal NR (central NR) tissues from the same aggregates.…”

“…Namely, the stemlike cells reported by Clarke et al 53 were derived from a rare population within hESC-derived pigmented cells (stem cells from pigmented ciliary epithelial cells are also described in ref. 61).…”

Cell replacement and visual restoration by retinal sheet transplants

“…Therefore, another cell source needs to be found. Retinal and photoreceptor progenitor cells have been derived from human embryonic stem cells (hESCs) (Lamba et al, 2006; Meyer et al, 2009; Yue et al, 2010; Meyer et al, 2011; Hambright et al, 2012; Clarke et al 2012) and from induced pluripotent stem cells (iPSCs) (Lamba et al, 2010; Parameswaran et al, 2010; Meyer et al, 2011; Tucker et al, 2011). Lamba et al (Lamba et al, 2009) transplanted hESC-derived retinal progenitors, differentiated according to a previously published protocol to newborn and adult wild-type mice and to adult transgenic CRX −/− mice, a retinal degeneration model.…”