Cell replacement therapy is a promising treatment for irreversible retinal cell death in diverse diseases such as Stargardt's disease, age-related macular degeneration, and retinitis pigmentosa. The final impact of all retinal dystrophies is the loss of photoreceptors; hence, there is a pressing need for research into replacement. Seminal work has shown that a simple three-dimensional culture system enables differentiation of human pluripotent stem cells to retinal organoids containing large numbers of photoreceptors developing alongside retinal neurons and Müller glia cells in a laminated structure that resembles the native retina. Despite these promising developments, current protocols show different efficiencies across pluripotent stem cells and result in retinal organoids with a mixture of photoreceptor cells at varying maturation states, along with nonphotoreceptor cell types. In this study, we investigated the impact of stage-specific addition of retinoic acid (RA), 9-cis-retinal, 11-cis-retinal, levodopa (l-DOPA), triiodothyronine (T3), and γ-secretase inhibitor ((2S)-N-[(3,5-Difluorophenyl)acetyl]-l-alanyl-2-phenyl]glycine1,1-dimethylethyl ester2L [DAPT]) in the generation of cone and rod photoreceptors. Our results indicate that addition of RA + T3 during days 90 to 120 of differentiation enhanced the generation of rod and S-cone photoreceptor formation, while the combined addition of DAPT from days 28 to 42 with RA during days 30 to 120 of differentiation led to enhanced generation of L/M-cones at the expense of rods. l-DOPA when added together with RA during days 90 to 120 of differentiation also promoted the emergence of S-cones at the expense of rod photoreceptors. Collectively, these data represent an advance in our ability to direct generation of rod and cone photoreceptors in vitro.

Induced pluripotent stem cell (iPSC)-derived retinal organoids provide a platform to study human retinogenesis, disease modeling, and compound screening. Although retinal organoids may represent tissue structures with greater physiological relevance to the in vivo human retina, their generation is not without limitations. Various protocols have been developed to enable development of organoids with all major retinal cell types; however, variability across iPSC lines is often reported. Modulating signaling pathways important for eye formation, such as those involving bone morphogenetic protein 4 (BMP4) and insulin-like growth factor 1 (IGF1), is a common approach used for the generation of retinal tissue in vitro. We used three human iPSC lines to generate retinal organoids by activating either BMP4 or IGF1 signaling and assessed differentiation efficiency by monitoring morphological changes, gene and protein expression, and function. Our results showed that the ability of iPSC to give rise to retinal organoids in response to IGF1 and BMP4 activation was line- and method-dependent. This demonstrates that careful consideration is needed when choosing a differentiation approach, which would also depend on overall project aims.

Nerve growth factor (NGF) has demonstrated great benefit in the treatment of neurotrophic corneal ulcers. There is evidence for multiple modes of action in promoting corneal healing, but only indirect evidence exists for NGF's effects on limbal stem cells (LSCs). Understanding the role of NGF in LSC biology will improve our understanding of paracrine regulation of the limbal niche and the design of stem cell-based therapies for conditions such as LSC deficiency. In this article, we studied the regulation of NGF signaling components during LSC differentiation and the role of NGF in LSC proliferation and maintenance of the stem cell phenotype. LSC differentiation was induced by prolonged (40 day) culture which resulted in a significant increase in cell size, decrease in colony-forming efficiency and expression of putative LSC markers. A protein microarray measuring expression of 248 signaling proteins indicated the low affinity NGF receptor p75NTR to be the most downregulated protein upon differentiation. Further confirmation by Western blotting and real-time quantitative polymerase chain reaction indicated that NGF and p75NTR are expressed in early LSC cultures and downregulated upon differentiation. LSC cultures grown in the presence of anti-NGF antibody showed decreased colony-forming efficiency, DNA replication and expression of putative LSC markers ABCG2 and C/EBPδ. Supplementation of LSC culture medium with NGF extended the life span of LSC cultures in vitro and increased the expression of putative LSC markers ΔNp63α and ABCG2. Taken together, our data indicate that NGF signaling is a key promoter of LSC proliferation, colony-forming efficiency, and a maintainer of the LSC phenotype. Stem Cells 2019;37:139–149

One of the main challenges in limbal stem cell (LSC) biology and transplantation is the lack of definitive cell surface markers which can be used to identify and enrich viable LSCs. In this study, expression of 361 cell surface proteins was assessed in ex vivo expanded limbal epithelial cells. One marker, CD200 was selected for further characterization based on expression in a small subset of limbal epithelial cells (2.25 ± 0.69%) and reduced expression through consecutive passaging and calcium induced differentiation. CD200 was localized to a small population of cells at the basal layer of the human and mouse limbal epithelium. CD200⁺cells were slow cycling and contained the majority of side population (SP) and all the holoclone forming progenitors. CD200⁺ cells displayed higher expression of LSCs markers including PAX6, WNT7A, CDH3, CK14, CK15, and ABCB5 and lower expression of Ki67 when compared to CD200⁻. Downregulation of CD200 abrogated the ability of limbal epithelial cells to form holoclones, suggesting an important function for CD200 in the maintenance and/or self‐renewal of LSCs. A second marker, CD109 which was expressed in 56.29 ± 13.96% of limbal epithelial cells, was also found to co‐localize with ΔNp63 in both human and mouse cornea, albeit more abundantly than CD200. CD109 expression decreased slowly through calcium induced cell differentiation and CD109⁺ cells were characterized by higher expression of Ki67, when compared to CD109⁻ subpopulation. Together our data suggest that CD200 expression marks a quiescent population of LSCs with holoclone forming potential, while CD109 expression is associated with a proliferative progenitor phenotype. Stem Cells 2018

Stem cell-derived retinal organoids offer the opportunity to cure retinal degeneration of wide-ranging etiology either through the study of in vitro models or the generation of tissue for transplantation. However, despite much work in animals and several human pilot studies, satisfactory therapies have not been developed. Two major challenges for retinal regenerative medicine are (a) physical cell-cell interactions, which are critical to graft function, are not formed and (b) the host environment does not provide suitable queues for development. Several strategies offer to improve the delivery, integration, maturation, and functionality of cell transplantation. These include minimally invasive delivery, biocompatible material vehicles, retinal cell sheets, and optogenetics. Optimizing several variables in animal models is practically difficult, limited by anatomical and disease pathology which is often different to humans, and faces regulatory and ethical challenges. High-throughput methods are needed to experimentally optimize these variables. Retinal organoids will be important to the success of these models. In their current state, they do not incorporate a representative retinal pigment epithelium (RPE)-photoreceptor interface nor vascular elements, which influence the neural retina phenotype directly and are known to be dysfunctional in common retinal diseases such as age-related macular degeneration. Advanced coculture techniques, which emulate the RPE-photoreceptor and RPE-Bruch’s-choriocapillaris interactions, can incorporate disease-specific, human retinal organoids and overcome these drawbacks. Herein, we review retinal coculture models of the neural retina, RPE, and choriocapillaris. We delineate the scientific need for such systems in the study of retinal organogenesis, disease modeling, and the optimization of regenerative cell therapies for retinal degeneration. Significance statement The light-sensitive neural retina is nourished by the retinal pigment epithelium (RPE), while the choriocapillaris, a dense capillary network, supplies oxygen and metabolites. Coculture of these tissues is therefore required to understand normal retinal development and disease. Transplanted retinal precursors fail to fully integrate within host tissues and form the normal RPE-photoreceptor and RPE-choriocapillaris interactions which sustain vision. Coculture techniques will enable in vitro optimization of regenerative cell therapies for degenerative retinal diseases, forming a step to successful in vivo transplant experiments. Furthermore, coculture of neural retina, RPE, and choriocapillaris will facilitate the development of transplantable multitissue sheets.

Limbal stem cell (LSC) deficiency causes progressive loss of vision but may be treated by transplant of autologous LSCs. Cryopreservation has the potential to indefinitely extend the lifespan of LSCs allowing re-transplant in case of graft failure. In this study, we aimed to identify the optimal cryoprotectant and cryoprotectant concentration for LSC cultures. Suspension cultures derived from cadaveric corneoscleral rims were cooled to 4 °C with Me2SO, propylene glycol or ethylene glycol at a concentration of 5%, 10% or 15%. Cell tolerance was measured in terms of membrane integrity, colony-forming efficiency and alamarBlue® reduction. Increasing cryoprotectant concentration above 5% reduced membrane integrity, metabolism and colony-forming efficiency. Cryoprotectant choice did not significantly influence these characteristics. Cells demonstrating Side Population were maintained after cryopreservation with 5% propylene glycol in vapour phase liquid nitrogen for 1 week, indicating that cryopreservation of LSCs with relatively low cryoprotectant concentration (5%) has promise in low-temperature eye banking.