Stem cells for retinal repair.

Casaroli-Marano RP, Zarbin MA (eds): Cell-Based Therapy for Retinal Degenerative Disease. Dev Ophthalmol. Basel, Karger, 2014, vol 53, pp 70–80 (DOI: 10.1159/000357328)

Stem Cells for Retinal Repair Jeffrey Stern · Sally Temple

Abstract

Stem Cells for Retinal Repair

Remarkable progress over the past decade has led to the first clinical studies of stem cell therapy for retinal disease. The unique access retina offers for implantation, monitoring, and ablation is well suited for stem cell trials, and retinal applications have now moved to the forefront of the field of regenerative medicine. Retinal progeny derived from either pluripotent stem cells or tissue-specific retinal and retinal pigment epithelium (RPE) stem cells have the capacity both to replace damaged retina and to provide trophic support that slows disease progression. In contrast, bone marrow and neural stem cells produce nonretinal progeny that provide trophic support but with limited integration and capacity to differentiate into retinal progeny that can replace damaged retinal tissue. Embryonic and induced pluripotent stem cells differentiated into neural retinal and RPE progeny provide an unlimited supply of human cells for transplantation and disease modeling but raise the risks of aberrant differentiation and over proliferation. Tissue-specific stem cells isolated from neural retina or RPE that are naturally committed to retinal fates have a restricted lineage potential that improves the margin of safety. This improved safety of retina and RPE stem cells is balanced, however, by a restricted proliferative potential, which limits the quantity of progeny produced. In this chapter, we review the types of stem cells under development for retinal therapy.

Progress to develop stem cell-based therapy for the retina and retinal pigment epithelium (RPE) has accelerated over the past few years. Key advances in stem cell science, combined with the unique factors that favor retina as a target for cellular therapy, have brought us to the point of clinical application [1]. Advantages of the RPE and outer retina for cellular therapy include surgical access, high-resolution, noninvasive measurement of anatomy and visual function, relatively simple connectivity, ability to ablate uncontrolled growth, a degree of immune privilege, and great unmet medical need for the treatment of blinding degenerative retinal diseases. Notably, pioneering transplantation of retina and RPE provides proof of principle that restoration of vision is possible in animal models and patients. These early transplantation experiments were limited by a lack of donor material, a challenge now addressed by stem cell technology, which provides ample source cells. This chapter surveys the types of stem cells and transplant strategies being used to replace damaged retina and to provide trophic support for retinal survival and function.

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Neural Stem Cell Institute, Rensselaer, N.Y., USA

Pluripotent stem cells produce progeny from all three germinal lines: endoderm, ectoderm, and mesoderm. Pluripotent stem cells isolated from the inner cell mass of an early blastocyst can be used to develop embryonic stem cell (ESC) lines [2, 3] that can be banked effectively and differentiated into a full array of progeny [4–7]. The advent of ESC culture has provided an ample supply of human cells and sparked rapid advances in related stem cell research. A second type of pluripotent stem cell, induced pluripotent stem cells (iPSCs), was more recently developed by reprogramming somatic cells such as fibroblasts. This transformative concept was recognized by the 2012 Nobel Prize in Medicine shared by John Gurdon and Shinya Yamanaka. Cellular reprogramming was demonstrated in seminal studies showing that an adult somatic cell nucleus transferred into a denucleated egg became ‘reprogrammed’ into a pluripotent state capable of generating an entire animal such as a frog or sheep [8, 9]. This process, also termed somatic cell nuclear transfer (SCNT), has been attempted using human somatic nuclei and donated oocytes in order to generate early embryos for production of patient-matched SCNT cell lines, envisioned as a valuable therapeutic cell source. However, it has proven to be very difficult technically, and the approach has been largely replaced by directed reprogramming of somatic cells into ESC-like cells by the insertion of key genes, thus producing iPSC lines. One combination of pluripotency genes used to create iPSC is OCT-4, Nanog, Sox2, and c-Myc; other combinations include Lin28 and microRNAs [10–13]. The iPSCs are pluripotent and closely matched to the donor in genetic makeup, which should in theory favor immune matching. Testing of the long-term safety of implanted iPSC-derived progeny is underway. These reprogrammed pluripotent cells are also valuable for in vitro disease modeling, including diseases with known genetic contributions.

The availability of human pluripotent ESC and iPSC lines that can be expanded and differentiated to produce quantities of retinal and RPE progeny has sparked activity to transplant these progeny for cell replacement of retinal disease. An important step toward clinical applications has been expansion of human pluripotent stem cells under xeno-free conditions without animal contamination [14, 15], including retinal and RPE cells [16, 17]. Rapid progress is being made to improve the efficient expansion and differentiation of pluripotent stem cells into pure progeny populations of the desired type for transplantation. For example, our laboratory has shown that providing pluripotent cells with a stable growth factor environment increases pluripotency markers, minimizes spontaneous differentiation, and increases cell number, while improving differentiation efficiency [18]. Neural Retinal Fate To effect retinal production, hESCs are first directed into an anterior neural fate and then into the neural retinal fate. Neural differentiation was initially promoted by factors such as retinoic acid or undefined stromal or astrocyte feeders [19, 20] but has been made highly efficient by the use of small molecules, such as the dual Smad inhibition protocol that generates highly pure Pax6+ anterior neural cells [21]. Early stem cell transplant studies in mouse indicated a cautionary note: when mouse ESCs were differentiated into neural progeny and then transplanted into the subretinal space of wildtype or rhodopsin-knockout mice, tumors were observed after 2 months in half the recipients [22]. However, subsequent studies using mouse and human cells indicate that transplantation of pluripotent stem cell-derived progeny into animal models can result in benefits such as rescue of outer retina function without tumor formation [23–25]. Trophic factor support provided by the transplanted cells has a significant role in rescue of retinal function, while integration of ESC-

Stem Cells for Retina Casaroli-Marano RP, Zarbin MA (eds): Cell-Based Therapy for Retinal Degenerative Disease. Dev Ophthalmol. Basel, Karger, 2014, vol 53, pp 70–80 (DOI: 10.1159/000357328)

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Pluripotent Stem Cells

Retinal Pigment Epithelium Fate In comparison to neural retinal cells, production of highly differentiated RPE is more readily accomplished, and RPE transplantation is leading the way toward the clinical application of pluripotent stem cells. The hESCs show spontaneous, albeit slow and inefficient, production of RPE progeny [38], but this process can be enhanced by the addition of appropriate factors that first promote neural fate (as described above), followed by factors that promote RPE differentiation in vivo, such as activin A [39]. The resulting cultures are

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still impure, but isolated islands of RPE differentiation can be enriched by manual selection, and other selection approaches to improve efficiency are being developed [40, 41] including the addition of a myosin inhibitor [42] or a combination of retinal inducing factors including IGF1, Noggin, Dkk1, bFGF, and others added at specific times [43]. Even with manual selection, remarkably pure differentiated monolayers are formed that exhibit salient features of RPE, including expression of markers such as RPE65, bestrophin, MITF, and ZO-1, phagocytosis of photoreceptor outer segments, production of polarized cobblestone monolayers with apical microvilli and basal nuclei, electrophysiological properties such as appropriate transepithelial resistance, polarized secretion of growth factors, and pigment processing [44]. Physiological and molecular characterization reveals several similarities and differences between hESC- or iPSC-derived RPE and native RPE [44] that should be considered in transplantation strategies. The transcription profile of hESC-derived RPE more closely resembles that of native fetal than native adult RPE [45]. Such differences might affect the ability of the cells to integrate and survive after transplantation, which, in turn, may depend on stage of maturity of the donor cells. To test the efficacy of RPE transplantation, a widely used model is the Royal College of Surgeons (RCS) rat, which has a MERTK gene defect that limits phagocytosis of outer segments [46] resulting in a retinal degeneration phenotype. Proof of principle demonstrations that subretinal transplantation of RPE reverses RCS rat retinal degeneration have long been established [47]. The hESC-derived RPE can phagocyte outer segments in the RCS rat and improve vision [24, 48, 41]. Transplantation of iPSC-derived RPE into rodent models restores visual function [49–51] even after xenografted cells are lost, raising the possibility that a secondary protective host cellular response contributes to photoreceptor cell rescue [40]. This result is consistent with previously

Stern · Temple Casaroli-Marano RP, Zarbin MA (eds): Cell-Based Therapy for Retinal Degenerative Disease. Dev Ophthalmol. Basel, Karger, 2014, vol 53, pp 70–80 (DOI: 10.1159/000357328)


NSCs into host retina occurs, but to a limited extent [25]. Neural differentiation can be extended to induce progeny displaying a variety of retinal progenitor cell (RPC) markers, shown for mouse ESCs [26] and hESCs in which Wnt inhibition via Dkk treatment and IGF1 promote the retinal fate [27]. The resulting cells exhibit markers of all the major cell types, and current efforts are aimed at increasing purity and authentic differentiation of specific retinal cell types, and, in particular, of photoreceptor cells [28]. Promisingly, transplantation of hESC-derived photoreceptors into Crxdeficient mice restores some visual function [29], and one hope is that these efforts can be improved with enhanced differentiation protocols [30]. Neural retinal cells can also be derived from iPSCs [28, 31]. A culture protocol to differentiate iPSC-derived RPCs into photoreceptor cell precursors has been developed, and these progeny integrate into mouse retina and differentiate into both rod and cone photoreceptor cells [32]. In addition to producing purified neural retinal populations, the remarkable ability of these multipotent progenitors to self-assemble into structures reminiscent of the eye cup with appropriate neural retina and RPE layers both enables studies of how these tissues are formed and provides the opportunity for multi-dimensional transplants and for enriched production of different layers [33–37].

entiation state of implanted cells. The matrix may minimize inappropriate differentiation, subretinal rosette formation, or other forms of unwanted cell growth including proliferative vitreoretinopathy. In addition, a synthetic substrate may improve results by replacing disease-damaged Bruch’s membrane. Although retina has immune privilege [58] and hESCs are naïve, with reduced immunogenicity [59], these conditions may not be sufficient to prevent cell rejection. After transplantation of allogeneic hESC-derived cells into retina, immune suppression is needed to prevent acute rejection [60]. Surviving transplanted RPE cells were not reported for the patient in the ongoing ACT study who discontinued immunosuppressive medications [56]. Immunosuppressive medications are not always well tolerated by elderly patients who make up a large proportion of those with targeted retinal diseases [61]. The immune response to retinal cells derived from human pluripotent sources transplanted into specific locations within the eye and in different disease states is an important unknown requiring further studies to optimize clinical success. A potential solution to the problem of immune rejection was raised with the advent of iPSC-derived RPE, which have the patient’s somatic cell genetic and immune profile [11, 62]. Although the genetic composition of iPSCs is derived from the donor, some immune rejection of iPSCs has been reported, in part arising from the inducing factors themselves [63]. However, in contrast to undifferentiated iPSCs, specialized progeny differentiated from iPSCs do not appear to be rejected after transplantation [64]. Other challenges raised by the use of iPSCs are inappropriate differentiation and overgrowth [65]. When compared to hESCs, iPSCs have been shown to generate more tumors after transplantation in vivo, be more virulent in homologous than xenografted hosts, and be more prone to produce inappropriate types of progeny, e.g. nonneural cells after placement in the CNS [66, 67]. The


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reported evidence that RPE transplants rescue photoreceptors in the RCS rat both through replacement of lost cells as well as through production of diffusible trophic factors [51]. The extent to which pluripotent stem cell progeny mediate rescue by incorporation into the host retina or via trophic factor release is uncertain. Rescue of retinal degeneration models by nonintegrating cellular therapy to provide neurotrophic factors is known [52]. Viral transfection of an RPE cell line to increase secretion of ciliary neurotrophic factor (CNTF) has led to a clinical trial using encapsulated cells with initially promising results for retinitis pigmentosa (RP) patients [53, 54]. This approach could be broadened to include pluripotent stem cell-derived progeny virally transformed to provide neurotrophic factors such as CNTF, brain-derived growth factor (BDNF), glial-derived growth factor, rod-derived cone survival factor(s), and others known to rescue retina in models of RP delivered by cell transplantation. Anatomic and functional restoration in the RCS model after subretinal transplantation of ESC-derived RPE has allowed rapid progression to clinical studies of hESC-derived RPE (hESCRPE) replacement therapy. The first trials, sponsored by Advanced Cell Technology Inc. (ACT), targeted Stargardt disease and age-related macular degeneration (AMD) with an initial dose of 50,000 hESC-RPE cells injected into a surgically created submacular retinal bleb (ClinicalTrials. gov NCT01469832 and NCT01344993). Initial reports of 2 patients describe good safety and preliminary positive efficacy, particularly in the patient with Stargardt disease [56] and the ACT study continues to enroll patients, utilizing escalating doses of subretinal hESC-RPE suspensions. In contrast to ACT’s use of a cellular suspension, studies by Pfizer Inc., the London Project, the California Institute for Regenerative Medicine, and others [55, 57] use hESC- or iPSCRPE monolayers on an implantable matrix to improve epithelial polarity and stabilize the differ-

Retinal Pigment Epithelial Stem Cells

During normal embryogenesis, the RPE is one of the first cell types to fully differentiate. Terminal differentiation begins early, around 4–6 weeks of gestation in humans. The resulting differentiated RPE cells then normally remain mitotically inactive from their birth in the early embryo throughout life. Our laboratory has discovered a subpopulation of quiescent adult RPE cells that can be activated to self-renew when cultured under appropriate proliferative conditions. In addition to

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self-renewal, this specialized RPE stem cell (RPESC) differentiates into a variety of progeny including RPE and mesenchymal lineages when cultured under specific differentiation conditions [71, 72]. The RPESC is a multi-, not pluri-, potent stem cell capable of limited self-renewal and producing limited ectodermal and mesenchymal lineages. This adult stem cell, specialized to produce RPE, readily produces pure populations of RPE progeny (RPESC-RPE) with physiologic and morphologic properties closely resembling those of native RPE. RPESC have less proliferative capacity than pluripotent stem cells, producing a few billion cells per donor, rather than unlimited progeny. This reduced proliferative capacity is accompanied by reduced potential for tumor formation. RPESCs do not display uncontrolled growth under conditions where pluripotent stem cells cause teratomas [71]. Although tumor formation has not been observed in hESC-derived RPE trials, the possibilities of aberrant differentiation or overgrowth remain a concern, which makes the restricted lineage potential and proliferative capacity of RPESC-RPE attractive. This improved safety profile arising from reduced proliferative capacity is balanced by practical considerations in providing source tissue for large clinical populations. If 100,000 cells are needed for each patient, the few billion cells produced by an RPESC provide RPE progeny sufficient for hundreds of recipients, in contrast to the unlimited resource of pluripotent stem cells. As discussed above, immune compatibility is an important consideration for disease targets such as AMD that include older recipient populations who are relatively intolerant of immunosuppressive regimes. Immune-matched source tissue, as is standard for solid organ and bone marrow transplantation, may also improve the immune compatibility of transplanted retinal tissue. Immune-specified RPESCs can be obtained from eye banks, which provide a ready source of donor eyes from which RPESCs are harvested.



iPSC cultures acquire small genetic mutations during or after reprogramming [68], raising concerns that these could lead to growth or differentiation instability, and yet these complications are difficult to discern prior to the use of the line. Although several strategies are being researched to address features of iPSCs related to oncogenic foci, such as limited induction to multi-, rather than pluripotent iPSC-like specialized stem cells [69], additional work is needed to demonstrate whether these approaches increase retina and/or RPE transplant safety. Approvals for a clinical trial of iPSC-derived RPE led by Masayo Takahashi announced in early 2013 by the Riken Center in Japan is planned to evaluate the use of patientmatched iPSC-RPE for individualized medicine [70]. The ongoing debate as to whether hESCs and hiPSCs are distinguishable, and whether hiPSCs are inherently less safe, remains unresolved. When small numbers of lines are tested, differences between hESCs and iPSCs have been noted, but when large samples are compared, distinguishing features are difficult to find [62]. It is becoming clear that both hESCs and hiPSCs are highly variable, in terms of gene expression profiles, DNA methylation patterns, and differentiation propensities. Ultimately, extensive safety data on individual hESC or iPSC lines are needed for wide clinical use of ESCs and iPSCs.

Neural Retina Stem and Progenitor Cells

Retinal stem cells (RSCs) were first isolated from the ciliary epithelium at the marginal periphery of the retina [73–75]. Questions about the ability of these stem cells to self-renew [76] have been answered [77]. RSCs and RPCs have also been isolated from the neural retina [78, 79]. In nonmammalian species, RSCs have been documented to arise from Müller glial cells. For example, in teleosts, Müller glia have the capacity to regenerate damaged retina in situ [80], shown unequivocally by lineage tracing experiments in transgenic fish in which bright light damage induced Müller glia to divide and replace rods and cones [81]. Müller glia at the periphery of the chick retina also can be activated to proliferate and reexpress progenitor markers [82]. There is accumulating evidence that Müller glia can be activated to divide and show plasticity in adult mammals, for example, in mice after NMDA-induced damage [83] or α-aminoadipate binding [84] or by sonic hedgehog, Wnt, or Notch pathway activation [85, 86]. RSCs and RPCs expanded through multiple passages can be differentiated into the major neu-

ral retinal cell types [74, 75], although differentiation into photoreceptor cells is limited [76, 87– 89]. When transplanted, RSCs integrate into host retina and rescue vision in animal models of injured retina [90, 91]. RPCs transplanted into the subretinal space of young mice survive, migrate, integrate, and differentiate into appropriate cell types including photoreceptor cells [75]. In healthy adult recipients, however, transplanted RSCs preferentially express ganglion cell or glial markers rather than the photoreceptor cell markers desirable for retinal degenerative diseases [92]. Young RSCs may integrate more extensively than RSCs from older donors, and injured host retina also promotes integration [93–95]. Transplantation of RSCs isolated at the developmental age that normally generates photoreceptor cells improved photoreceptor cell differentiation within grafts in adult retina [96]. Viral transformation of host retina to overexpress neurotrophic CNTF improves transplanted photoreceptor progenitor cell integration [97]. RPCs isolated from human fetal retina have been expanded through multiple passages to produce quantities of progeny sufficient for transplantation [98–100]. Remaining challenges for clinical application of RSC- and RPC-based replacement therapy include obtaining sufficient fetal donors, directing appropriate differentiation, and controlling immune reactivity. In addition to use for replacement therapy, RPC transplantation may provide trophic factors that support the outer retina including rod photoreceptor cell factors that promote cone survival, which, in RP mouse models, are lost [101, 102]. The use of intravitreal RPCs to release cone survival factors has been proposed as a possible therapy for RP. Neural Stem and Progenitor Cells

Neural stem cells (NSCs) first isolated from the fetal murine nervous system [103] are prevalent during development and are retained through-


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We envision multiple RPESC banks to provide clinical quantities of immune-matched source RPE for replacement therapy. The RPESC offers the further possibility of autologous transplantation if this proves necessary to achieve immune compatibility. RPESC-based autologous RPE transplantation would require two vitrectomies, one to harvest and the other to replace RPE, and consideration of increased surgical risk needs to be balanced by the risks associated with immune rejection. The recently discovered RPESC is a promising new stem cell source for replacement therapy and delivery of trophic factors. Furthermore, it is possible that in the future this cell could be activated in situ to replace lost cells in the patient’s eye to treat AMD and forms of RP without surgical transplantation.

Table 1. Comparison of stem cell sources for retinal repair

Stem cell type

Rescue vision

Immune compatible

Differentiation stability

Expansion capacity

Source

ESC-RPE iPSC-RPE RPESC ESC-NSC NSC RSC/RPC BMSC UCSC

yes yes unknown yes yes yes yes yes

no improved for self yes for self no no no yes for self no

improving improving, less yes improving yes yes yes yes

extensive extensive moderate extensive moderate moderate moderate moderate

embryo donor self, donor or cadaver self, donor or cadaver embryo donor fetal donor fetal donor self, donor or cadaver umbilical cord

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Mesenchymal Stem Cells

Transplantation of mesenchymal stem cells (MSCs) of mesodermal origin such as bone marrow stem cells (BMSCs) or umbilical cord blood stem cells (UCSCs) is being developed as a means of trophic support and to promote retinal revascularization. Restoration of inner retinal vasculature by BMSCs presents a rational approach to treat vascular disorders such as diabetic retinopathy [114]. Improved circulation after injection of BMSCs into the eye enhances survival of outer retinal neurons, accompanied by visual improvement, in both ischemic and nonischemic mouse models of retinal degeneration [115, 116]. Neurotrophic effects due to improved circulation after MSCs transplantation is a promising new strategy [117, 118]. Preservation of photoreceptor cells has been observed even after intravenous, rather than intraocular, administration of autologous BMSCs raising the possibility of minimally invasive cellular therapy for retinal disease [119]. It is important to distinguish these conceptually sound allogenic or autologous MSCs studies, which produce reliable controlled data, from those profit-driven, uncontrolled and as yet unproved MSC therapies that are widely marketed to attract patients with untreatable blinding conditions. In addition to improving retinal circulation, limited integration of transplanted MSCs into the



out life in restricted regions of the forebrain [104]. When transplanted into mouse retina, NSCs show limited integration and limited expression of retinal cell phenotypes [105]. Like RPCs, host age and health influence NSCs transplant fate [79, 105–108]. When transplanted into adult monkey, NSCs display minimal migration or integration, forming a monolayer of stable NSCs [109]. Transplantation of NSCs derived from committed central nervous system tissue rescues photoreceptor cells in animal models presumably by release of growth factors and/or metabolic processing of phototransduction byproducts [52, 110]. In order to enhance trophic effects, NSCs have been virally transduced to release GDNF [111], a factor that supports retinal function in a mouse model of RP [112]. It is known that transplantation of Schwann cells, which secrete GDNF, brain-derived growth factor, and CNTF, rescues rod photoreceptor cells [113], and native or virally transformed NSCs provide a promising stem cell-based source tissue for neurotrophic rescue of retinal degenerations. Primary NSCs from donor brains can also be expanded extensively. A clinical study (sponsored by Stem Cells Inc.), transplanting primary NSCs to support photoreceptor cells as therapy for AMD, has been approved by the FDA (Clinicaltials.gov No. NCT01632527).

neural retina seems to be accompanied by acquisition of neural retina-like features, which has led to the suggestion that some replacement of outer retinal cells may occur in BMSCs-mediated rescue of vision [120–122]. Others, however, report that MSC-mediated rescue of the photoreceptor cell layer is entirely due to improved circulation and secretion of growth factors essential for photoreceptor cell survival without significant cell replacement [123, 124]. Although controversy about the ability of BMSCs to form and replace brain tissues remains active, BMSC transplantation clearly prevents vision loss in animal models and may benefit retinal patients with AMD, RP, or diabetic retinopathy. Clinical development of MSC therapy is ongoing at Janssen Biotech Inc. (formerly Centocor Biotech). In phase I, immune rejection was limited, and complications related to surgical technique were addressed [125]. A phase II study is ongoing for subretinal injection of UCSC doses ranging from 47,500 to 470,000 cells (Clinicaltrials.gov No. NCT00458575).

Conclusions

The retina is ideally suited for stem cell-based replacement therapy, and several types of stem cells are currently under development for this clinical application (table 1). As with other paradigm shifts in medicine, such as the advent of antibiotics, the early stages of stem cell-based replacement therapy are evolving rapidly. Pursuit of multiple cell sources and approaches is important in order to identify those that favor specific circumstances, thus providing a range of therapeutic options for retinal patients. Studies to identify specific stem cells and transplant strategies with potential benefit for patients struggling with retinal disease are being pursued on many fronts with expectations that breakthrough therapies for otherwise untreatable blinding conditions are near.

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Jeffrey Stern, PhD, MD Neural Stem Cell Institute One Discovery Drive Rensselaer, NY 12144 (USA) E-Mail [email protected]

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