Sarah Decembrini

Adult ciliary epithelial cells, previously identified as retinal stem cells with potential for retinal repair, fail to differentiate into new rod photoreceptors.

The ciliary margin in lower vertebrates is a site of continual retinal neurogenesis and a stem cell niche. By contrast, the human eye ceases retinal neuron production before birth and loss of photoreceptors during life is permanent and a major cause of blindness. The discovery of a proliferative cell population in the ciliary epithelium (CE) of the adult mammalian eye, designated retinal stem cells, raised the possibility that these cells could help to restore sight by replacing lost photoreceptors. We previously demonstrated the feasibility of photoreceptor transplantation using cells from the developing retina. CE cells could provide a renewable source of photoreceptors for transplantation. Several laboratories reported that these cells generate new photoreceptors, whereas a recent report questioned the existence of retinal stem cells. We used Nrl.gfp transgenic mice that express green fluorescent protein in rod photoreceptors to assess definitively the ability of CE cells to generate new photoreceptors. We report that CE cells expanded in monolayer cultures, lose pigmentation, and express a subset of eye field and retinal progenitor cell markers. Simultaneously, they continue to express some markers characteristic of differentiated CE and typically lack a neuronal morphology. Previously reported photoreceptor differentiation conditions used for CE cells, as well as conditions used to differentiate embryonic retinal progenitor cells (RPCs) and embryonic stem cell‐derived RPCs, do not effectively activate the Nrl‐regulated photoreceptor differentiation program. Therefore, we conclude that CE cells lack potential for photoreceptor differentiation and would require reprogramming to be useful as a source of new photoreceptors. STEM Cells 2010;28:1048–1059

Derivation of traceable and transplantable photoreceptors from mouse embryonic stem cells.

Summary Retinal degenerative diseases resulting in the loss of photoreceptors are one of the major causes of blindness. Photoreceptor replacement therapy is a promising treatment because the transplantation of retina-derived photoreceptors can be applied now to different murine retinopathies to restore visual function. To have an unlimited source of photoreceptors, we derived a transgenic embryonic stem cell (ESC) line in which the Crx-GFP transgene is expressed in photoreceptors and assessed the capacity of a 3D culture protocol to produce integration-competent photoreceptors. This culture system allows the production of a large number of photoreceptors recapitulating the in vivo development. After transplantation, integrated cells showed the typical morphology of mature rods bearing external segments and ribbon synapses. We conclude that a 3D protocol coupled with ESCs provides a safe and renewable source of photoreceptors displaying a development and transplantation competence comparable to photoreceptors from age-matched retinas.

Comparative Analysis of the Retinal Potential of Embryonic Stem Cells and Amniotic Fluid-Derived Stem Cells

Photoreceptors have recently been generated from mouse and human embryonic stem cells (ESCs), although ethics concerns impede their utilization for cell replacement therapy for retinal disease. Extra-embryonic tissues have received attention as alternative therapeutic sources of stem cells. Human and mouse amniotic fluid-derived stem cells (AFCs) have been reported to be multipotent and express embryonic and adult stem cell markers. Here, in vitro conditions that generate retinal cells from ESCs were used to analyze and compare the retinal potential of murine AFCs and ESCs. We show that AFCs express pluripotency markers (Nanog, Sox2, and Oct3/4) as well as retinal transcription factor genes (Et, Lhx2, Tll1, Six6, Otx2, Pax6, and Fgf15). AFCs from amniotic fluid of Fgf15.gfp, Nrl.gfp, and Crx.gfp embryos cultured in retinal proliferation and differentiation conditions failed to switch on these retinal transgenes. AFCs cultured in retinal-promoting conditions, effective on ESCs, showed reduced expression of retinal markers. Retinal co-cultures activated retinal genes in ESCs but not in AFCs, and migration assays in retinal explants showed limited migration of AFCs compared with ESCs. Unlike ESCs, AFCs do not express the early embryonic ectodermal gene Utf1 and Western analysis of AFCs identified only the B isoform of Oct3/4, rather than the isoform A present in ESCs. We conclude that AFCs have restricted potential and differ considerably from ESCs and retinal progenitor cells. Reprogramming to induce pluripotency or new differentiation protocols will be required to confer retinal potential to AFCs as expression of a subset of pluripotency and retinal markers is not sufficient.

Differentiation and Transplantation of Embryonic Stem Cell-Derived Cone Photoreceptors into a Mouse Model of End-Stage Retinal Degeneration

Summary The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs). Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor β2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1−/− mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.

Cone Genesis Tracing by the Chrnb4-EGFP Mouse Line: Evidences of Cellular Material Fusion after Cone Precursor Transplantation

The cone function is essential to mediate high visual acuity, color vision, and daylight vision. Inherited cone dystrophies and age-related macular degeneration affect a substantial percentage of the world population. To identify and isolate the most competent cells for transplantation and integration into the retina, cone tracing during development would be an important added value. To that aim, the Chrnb4-EGFP mouse line was characterized throughout retinogenesis. It revealed a sub-population of early retinal progenitors expressing the reporter gene that is progressively restricted to mature cones during retina development. The presence of the native CHRNB4 protein was confirmed in EGFP-positive cells, and it presents a similar pattern in the human retina. Sub-retinal transplantations of distinct subpopulations of Chrnb4-EGFP-expressing cells revealed the embryonic day 15.5 high-EGFP population the most efficient cells to interact with host retinas to provoke the appearance of EGFP-positive cones in the photoreceptor layer. Importantly, transplantations into the DsRed retinas revealed material exchanges between donor and host retinas, as >80% of transplanted EGFP-positive cones also were DsRed positive. Whether this cell material fusion is of significant therapeutic advantage requires further thorough investigations. The Chrnb4-EGFP mouse line definitely opens new research perspectives in cone genesis and retina repair.

Mutations in CEP78 Cause Cone-Rod Dystrophy and Hearing Loss Associated with Primary-Cilia Defects

Cone-rod degeneration (CRD) belongs to the disease spectrum of retinal degenerations, a group of hereditary disorders characterized by an extreme clinical and genetic heterogeneity. It mainly differentiates from other retinal dystrophies, and in particular from the more frequent disease retinitis pigmentosa, because cone photoreceptors degenerate at a higher rate than rod photoreceptors, causing severe deficiency of central vision. After exome analysis of a cohort of individuals with CRD, we identified biallelic mutations in the orphan gene CEP78 in three subjects from two families: one from Greece and another from Sweden. The Greek subject, from the island of Crete, was homozygous for the c.499+1G>T (IVS3+1G>T) mutation in intron 3. The Swedish subjects, two siblings, were compound heterozygotes for the nearby mutation c.499+5G>A (IVS3+5G>A) and for the frameshift-causing variant c.633delC (p.Trp212Glyfs(∗)18). In addition to CRD, these three individuals had hearing loss or hearing deficit. Immunostaining highlighted the presence of CEP78 in the inner segments of retinal photoreceptors, predominantly of cones, and at the base of the primary cilium of fibroblasts. Interaction studies also showed that CEP78 binds to FAM161A, another ciliary protein associated with retinal degeneration. Finally, analysis of skin fibroblasts derived from affected individuals revealed abnormal ciliary morphology, as compared to that of control cells. Altogether, our data strongly suggest that mutations in CEP78 cause a previously undescribed clinical entity of a ciliary nature characterized by blindness and deafness but clearly distinct from Usher syndrome, a condition for which visual impairment is due to retinitis pigmentosa.

Isolation and Culture of Adult Ciliary Epithelial Cells, Previously Identified as Retinal Stem Cells, and Retinal Progenitor Cells

The protocols described in this unit provide detailed information on how to isolate and expand, in culture, ciliary epithelial cells (CECs), previously identified as retinal stem cells, from the adult mouse eye, and embryonic retinal progenitor cells (RPCs) from the developing retina. CECs are initially cultured in floating conditions as neurospheres and then expanded in monolayer cultures. RPCs are cultured in floating conditions. Detailed protocols for retinal differentiation, as well as exogenous gene expression using lentivirus are also described.

Intracameral Chemotherapy for Globe Salvage in Retinoblastoma with Secondary Anterior Chamber Invasion

The presence of anterior chamber seeding (ACS), whether primary (1%) or secondary, is commonly treated by enucleation. Considering the new frontiers of conservative management of very advanced retinoblastoma, the reported incidence of secondary ACS leading to conservative failure has progressed from 6% after chemoreduction to 67% after intra-arterial chemotherapy (IAC). Anterior chamber seeding is considered an intractable form of retinoblastoma regardless of the treatment used because of the hypoxic radio-resistant nature of the seeds and inability to reach tumoricidal concentrations in the aqueous using known routes of chemotherapy administration. Successful attempts to treat ACS were reported only recently and concerned patients with primary ACS in diffuse anterior retinoblastoma of retinal origin by intracameral chemotherapy (ICC) and of extra-retinal origin by brachytherapy. In this retrospective review approved by the ethical committee of Vaud, Switzerland (authorization no. 2016-00149), 12 consecutive patients with ACS treated with ICC were identified between November 2011 and October 2016. One case was excluded with primary ACS class III (pseudo-hypopion) resulting from anterior diffuse retinoblastoma published previously with an event-free follow-up of 5.5 years. The study population consisted of 11 eyes with secondary ACS from 11 heavily pretreated patients for whom advised enucleation was refused by the parents, including 10 patients with relapse referred from other centers. There were 4 eyes with unilateral and 7 eyes with bilateral retinoblastoma, of which 4 were remaining eyes after previous enucleation of the fellow eyes. The demographic characteristics and the treatment received before ICC, as well as ACS classification, relapses, and concomitant ultrasound biomicroscopy features, are shown in Table S1 (available at www.aaojournal.org). Anterior chamber seeding was classified as class I in 5 eyes, class II in 5 eyes, and class III in 1 eye (Fig 1AeF). According to the integrity of anterior hyaloid, 2 distinct mechanisms of tumor progression into the anterior segment could be distinguished: (1) transhyaloid infiltration into the posterior chamber secondary to vitreous seeds attached to the anterior hyaloid in 3 eyes (no peripheral primary or secondary retinal tumors) or to the anterior progression of a peripheral parietal tumor overlying the pars plana in 4 eyes (Fig 1G, I); and (2) invasion via a disrupted anterior hyaloid of iatrogenic origin, that is, pars plana vitrectomy (Fig 1K, L), and clear corneal intracapsular lensectomy in 2 eyes each. Anterior chamber seeding was isolated (9 eyes) or associated with an anterior uveal infiltration (2 eyes) as assessed by ultrasound biomicroscopy. Intracameral chemotherapy with melphalan was administered (concentration of 15 or 20 mg/ml, the lower concentration used initially and the higher concentration used in case of ACS relapse/ persistence) as previously described. All but 3 eyes (vitrectomized with silicone oil or postintracapsular lensectomy, see Table S1, available at www.aaojournal.org) received concomitant intravitreal chemotherapy at least once via the pars plana with 20 to 30 mg of melphalan at a concentration of 200 mg/ml to treat the underlying vitreous disease or, if absent, to prevent crosscontamination between the aqueous and the vitreous. Tumor control was monitored clinically by slit-lamp examination as well as in the iterative aqueous taps by cytopathology and by cell culture when available. Intracameral chemotherapy was continued as long as the cytopathology or cell culture remained positive and at least 1 additional injection was given after a negative result. Complete aqueous seeding regression was initially obtained in all eyes after a mean of 0.7 months (range, 0.2e1.3) as assessed clinically and by negative cytopathology and cell culture, after a mean number of 4.3 injections (range, 2e7) with a mean dose of 6.3 mg (range, 2.9e15) per injection. Higher doses corresponded to a greater quantity of aqueous humor withdrawn in cases with disrupted hyaloid (mean 0.46 ml compared with 0.24 ml with the hyaloid intact), indirectly confirming disruption of the hyaloid. Anterior chamber seeding class I required notably less mean 3.2 injections compared with 5.2 injections in classes II and III. Treatment during or after ICC, ICC-related complications, globe preservation, functional results, and histopathology of the enucleated eyes are summarized in Table S2 (available at www.aaojournal.org).

An Advocacy for the Use of 3D Stem Cell Culture Systems for theDevelopment of Regenerative Medicine: An Emphasis on PhotoreceptorGeneration

The availability of stem cells is of great promise to study early developmental stages and to generate adequate cells for cell transfer therapies. Although many researchers using stem cells were successful in dissecting intrinsic and extrinsic mechanisms and in generating specific cell phenotypes, few of the stem cells or the differentiated cells show the capacity to repair a tissue. Advances in cell and stem cell cultivation during the last years made tremendous progress in the generation of bona fide differentiated cells able to integrate into a tissue after transplantation, opening new perspectives for developmental biology studies and for regenerative medicine. In this review, we focus on the main works attempting to create in vitro conditions mimicking the natural environment of CNS structures such as the neural tube and its development in different brain region areas including the optic cup. The use of protocols growing cells in 3D organoids is a key strategy to produce cells resembling endogenous ones. An emphasis on the generation of retina tissue and photoreceptor cells is provided to highlight the promising developments in this field. Other examples are presented and discussed, such as the formation of cortical tissue, the epithelial gut or the kidney organoids. The generation of differentiated tissues and well-defined cell phenotypes from embryonic stem (ES) cells or induced pluripotent cells (iPSCs) opens several new strategies in the field of biology and regenerative medicine. A 3D organ/tissue development in vitro derived from human cells brings a unique tool to study human cell biology and pathophysiology of an organ or a specific cell population. The perspective of tissue repair is discussed as well as the necessity of cell banking to accelerate the progress of this promising field.