Multipotent stem cells isolated from the adult mouse retina are capable of producing functional photoreceptor cells

npg Retinal stem cells produce functional photoreceptors 788 ORIGINAL ARTICLE Cell Research (2013) 23:788-802. © 2013 IBCB, SIBS, CAS All rights reserved 1001-0602/13 $ 32.00 www.nature.com/cr Multipotent stem cells isolated from the adult mouse retina are capable of producing functional photoreceptor cells Tianqing Li1, *, Michelle Lewallen1, *, Shuyi Chen1, *, Wei Yu1, Nian Zhang1, Ting Xie1, 2 1 Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA; 2Department of Anatomy and Cell Biology, University of Kansas School of Medicine, 3901 Rainbow Blvd, Kansas City, KS 66160, USA Various stem cell types have been tested for their potential application in treating photoreceptor degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Only embryonic stem cells (ESCs) have so far been shown to generate functional photoreceptor cells restoring light response of photoreceptordeficient mice, but there is still some concern of tumor formation. In this study, we have successfully cultured Nestin+Sox2+Pax6+ multipotent retinal stem cells (RSCs) from the adult mouse retina, which are capable of producing functional photoreceptor cells that restore the light response of photoreceptor-deficient rd1 mutant mice following transplantation. After they have been expanded for over 35 passages in the presence of FGF and EGF, the cultured RSCs still maintain stable proliferation and differentiation potential. Under proper differentiation conditions, they can differentiate into all the major retinal cell types found in the adult retina. More importantly, they can efficiently differentiate into photoreceptor cells under optimized differentiation conditions. Following transplantation into the subretinal space of slowly degenerating rd7 mutant eyes, RSC-derived photoreceptor cells integrate into the retina, morphologically resembling endogenous photoreceptors and forming synapases with resident retinal neurons. When transplanted into eyes of photoreceptor-deficient rd1 mutant mice, a RP model, RSC-derived photoreceptors can partially restore light response, indicating that those RSC-derived photoreceptors are functional. Finally, there is no evidence for tumor formation in the photoreceptor-transplanted eyes. Therefore, this study has demonstrated that RSCs isolated from the adult retina have the potential of producing functional photoreceptor cells that can potentially restore lost vision caused by loss of photoreceptor cells in RP and AMD. Keywords: retinal stem cells; photoreceptor cells Cell Research (2013) 23:788-802. doi:10.1038/cr.2013.48; published online 9 April 2013 Introduction Dysfunction and death of retinal neurons are among the most common health problems for aged people. Each day, millions of people live in darkness caused by retinal degenerative diseases, such as glaucoma, retinitis pigmentosa (RP), and age-related macular degeneration (AMD). Current therapies for these diseases are largely preventative and treatments capable of curing these diseases by restoring lost visual function remain to be developed. With the aging population rapidly growing, the *These three authors contributed equally to this work. Correspondence: Ting Xie E-mail: Received 5 December 2012; revised 17 January 2013; accepted 28 February 2013; published online 9 April 2013 need to develop effective methods to recover vision for retinal degeneration disease patients is more urgent than ever. Among methods currently undergoing development, stem cell-based cell replacement therapy represents one of the most promising. In 2006, Maclaren et al. [1] showed that immature post-mitotic photoreceptor precursors, when transplanted into the subretinal space, could integrate into the outer photoreceptor cell layer, differentiate into rod photoreceptors, and establish synaptic connections with interneurons in the inner nuclear layer. Later, the same group further showed that the transplanted cells are able to form visual circuitry all the way to the visual cortex and restore vision of rod photoreceptorimpaired mice [2]. These transplantation experiments proved the principle that cell transplantation therapy is an effective method for treating blindness. Now, the biggest challenge facing the medical community for the use Cell Research | Vol 23 No 6 | June 2013 npg Tianqing Li et al . npg 789 of cell-replacement therapy to treat retinal degenerative diseases is to find appropriate cellular sources for transplantation. Photoreceptor precursors isolated from the developing retina have given the best vision recovery results [2], however, the source for such cells is extremely limited and their clinical use raises ethical concerns. Embryonic stem cells (ES cells) and induced pluripotent stem (iPS) cells represent two of the most attractive subjects in regenerative medicine research, due to their unlimited proliferation ability and tremendous differentiation potential. It has been shown that ES cells can be induced to differentiate to retinal progenitor cells [3, 4], rod and cone photoreceptor cells, retinal pigmented epithelial (RPE) cells [5], and can even generate a three-dimensional optic cup [6, 7] in culture dishes. These exciting results have inspired several groups of researchers to test the idea of using ES cell- or iPS cell-derived retinal cells to treat diseases of retinal degeneration. So far, the results are promising: ES/iPS cell-derived retinal cells are able to integrate into retina tissues, express markers for retinal neurons, and even rescue the light response of photoreceptor-impaired animals [8-10]. While the use of iPS cells circumvents the ethical drawbacks that accompany the use of ES cells, the oncogenic properties of both ES and iPS cells are currently an unavoidable safety issue when using them for clinical therapy. Indeed, transplantation experiments using ES cell-derived retinal cells have shown teratoma formation in transplanted eyes due to contamination of undifferentiated ES cells [10, 11]. When compared with ES cells, tissue-specific adult stem cells are more efficient at generating the cells of the tissues from which they originate. This combined with the reduced concern for tumor formation makes them another attractive renewable cellular source for cell replacement therapy. Hence, the presence and location of stem cells in the adult retina is under intense investigation. In 2000, Tropepe et al. [12] reported isolation of adult retinal stem cells from pigmented ciliary body epithelium, which could form neurospheres and could differentiate into retina-specific cell types, including rod photoreceptors. Following this report, different groups have isolated similar cell populations from rat, pig, and human ciliary body [13-15]. Recently, the retinal stem cell properties of ciliary body-derived cells have been challenged by several reports, and their usage for retinal cell replacement therapy is being scru (...truncated)