Cell replacement with stem cell-derived retinal ganglion cells from different protocols

Ziming Luo ,Kun-Che Chang

Abstract Glaucoma,characterized by a degenerative loss of retinal ganglion cells,is the second leading cause of blindness worldwide.There is currently no cure for vision loss in glaucoma because retinal ganglion cells do not regenerate and are not replaced after injury.Human stem cell-derived retinal ganglion cell transplant is a potential therapeutic strategy for retinal ganglion cell degenerative diseases.In this review,we first discuss a 2D protocol for retinal ganglion cell differentiation from human stem cell culture,including a rapid protocol that can generate retinal ganglion cells in less than two weeks and focus on their transplantation outcomes.Next,we discuss using 3D retinal organoids for retinal ganglion cell transplantation,comparing cell suspensions and clusters.This review provides insight into current knowledge on human stem cell-derived retinal ganglion cell differentiation and transplantation,with an impact on the field of regenerative medicine and especially retinal ganglion cell degenerative diseases such as glaucoma and other optic neuropathies.

Key Words: cell clumps;cell suspension;cell transplantation;differentiation;direct-induced protocol;glaucoma;optic neuropathy;regenerative medicine;retinal ganglion cell;retinal organoids;stem cells

Introduction

Glaucoma and other optic neuropathies cause blindness by damaging retinal ganglion cells (RGCs).As part of the central nervous system,RGCs do not regenerate.Thus,cell replacement strategies have emerged as an attractive therapeutic solution to restore vision (Singh et al.,2020;Zhang et al.,2021b).Transplantation of primary RGCs in animal models has been successfully demonstrated (Hertz et al.,2014;Venugopalan et al.,2016;Wareham et al.,2020),but the supply of primary cells is limited.Thus,there is an unmet need to develop a stem cell-based source not only to protect endogenous neurons (Wu et al.,2018;Lim et al.,2022) but also to scale-up donor RGCs for cell replacement therapy.

While transplanting human stem cell-derived RGCs is a feasible approach for functional restoration (Do et al.,2021;Zhang et al.,2021b) and several encouraging studies have advanced RGC replacement strategies (Rabesandratana et al.,2020;Wu et al.,2021;Croteau et al.,2022),low donor RGC survival and integration into retinas remain major barriers due to dramatic cell death and lack of cell-cell interactions after transplantation,respectively.This review provides an overview of cell replacement studies involving stem cell-derived RGCs generated from different protocols and their transplant outcomes.We begin by discussing RGC transplants from direct induction,which is directly differentiating RGCs or retinal progenitor cells from stem cells attached to a cell culture dish.Next,we discuss RGC transplant from retinal organoids,which unlike direct-induced RGCs are neurons in tissue architectures,which may influence cell replacement outcomes.Further,we discuss transplant results by donor organoid RGCs implanted in either tissue pieces or cell suspensions (Figure 1andTable 1).

Table 1｜Stem cell-derived RGC transplantation

Search Strategy

We searched the PubMed database and screened relevant literature published from September 2006 to January 2023.A combination of the following terms was used to maximize search specificity and sensitivity: “stem cell transplantation,” “retinal ganglion cell,” “glaucoma,” “optic neuropathy,” and “organoids.” Results were further screened by title and abstract.Studies exploring cell transplantation using cell suspensions and cell clusters were included.

Direct-Induced Retinal Ganglion Cells and Transplant

There is a long history of directly inducing neuronal differentiation from stem cells,with different pluripotency levels generating different types of retinal neurons.Many direct-differentiation protocols have been well-summarized in previous reviews (Ji and Tang,2019;Yuan et al.,2021),so here we limit discussion to studies in the past 5 years.

In 2020,we used a robust chemically defined protocol to differentiate human embryonic stem cells into RGC-like neurons (Zhang et al.,2020).Calcium imaging showed that the generated RGCs had an immature gamma-aminobutyric acid response,similar to the immature primary rodent RGCs.After intravitreal injection into adult rats,these human RGClike cells successfully migrated into the ganglion cell layer in 1 week.We later significantly shortened the differentiation protocol by overexpressing NGN2 and supplying an RGC-conditioning medium with glial cell line-derived neurotrophic factor,which induced RGC-like cells in less than 2 weeks (compared to one month previously) (Luo et al.,2022).We also confirmed the single-cell transcriptome profile of the induced neurons,and analysis with human fetal retinas showed that RGC markers were co-expressed in several clusters.Although cell survival was not high after transplant,the study showed a promising neuroprotective effect on host RGCs,saving them from apoptosis after optic nerve crush.

Another group recently used a two-step differentiation technique (Chavali et al.,2020) to induce RGCs from human stem cells and transplanted the cells into mouse retinas (Vrathasha et al.,2022).Cells survived up to five months after transplant,much longer than in previous studies with direct-induced donor RGCs.Moreover,these donor neurons exhibited action potentialsin vivo.Using surviving donor cell number per transplanted cell number as an indicator,transplantation efficiency was on average 0.134%,similar to 0.1% in previous studies (Venugopalan et al.,2016;Oswald et al.,2021).Although the encouraging results of cell transplantation in either rodents or pigs were reported,studies of cell transplantation into non-human primates are still needed to advance the field.

Further,there are other hurdles in the translational setting,as most directinduction protocols involve genetic manipulation and/or multiple smallmolecule cocktails,which limit clinical application.Hence,retinal organoids,which were developed partially by internal drive,could be a more feasible source of donor cells.

Retinal Organoids and Transplant

Cell suspensions as donor material

Since stem cell-derived retinal organoids were first developed in 2012 (Nakano et al.,2012),they have become a promising source of donor cells for RGC transplants.Differentiation of retinal organoids recapitulates the development of fetal retinas,in which RGCs first develop at 6-8 weeks in humans (Luo et al.,2019).During retinal organoid formation,retinal progenitors self-organize into 3D tissue architectures,protruding from the neurosphere or culture dish to form a cup-like structure.Due to the 3D structure of retinal organoids,many studies refer to harvesting RGCs from retinal organoids as a 3D protocol.Compared to chemically defined RGC-like cells in 2D protocols,RGCs from 3D protocols follow natural retinal development and show similar transcriptional profiles as human fetal RGCs (Sridhar et al.,2020;Finkbeiner et al.,2022).Therefore,increasing studies have recently used organoid-derived RGCs for cell replacement.

Interestingly,a few years before the organoid technique was established,a study reported transplant with embryonic stem cell-derived eye-like structures (Aoki et al.,2008).In this study,mouse embryonic stem cells differentiated and formed multi-layered cell masses with surrounding pigmented cells,which the authors referred to as eye-like structures (Aoki et al.,2006).For the cell transplant study,dissociating the structures into cell suspensions and injecting them in wild-type or N-methyl-D-aspartate (NMDA)-treated mice showed that donor cells could integrate into the host retinas of both wild-type and NMDA-treated mice (60% of wild-type eyes and >90% of NMDA-treated eyes had integration 10 days after transplant) (Aoki et al.,2008).However,only donor cells in NMDA-treated models expressed RGC markers,which the authors interpreted as resulting from increased niches to receive grafted cells after injury.Although it did not refer to the cells as organoids,this study provided the first proof-of-concept cell replacement using donor cells from stem cell-derived 3D tissues.At this stage,3D structure and retinal cell type composition are not well-defined,so the study did not clearly identify cell type before transplant.Since the establishment of the retinal organoid differentiation system,RGC development in retinal organoids has been well-studied.Initially,a group transplanted cells that dissociated from embryoid bodies,which is the first structure formed during organoid differentiation (Zhou et al.,2021).Neural progenitors form spontaneously in the embryoid bodies (Brickman and Serup,2017).They demonstrated that donor cells further differentiation into RGCin vivo,expressing specific markers with axon-like structures.Next,researchers collected donor cells by enzymatically dissociating organoids,generating mostly retinal progenitor cells.One study showed that five weeks after injection into NMDA-treated mice,0.5% of donor progenitors integrated into host retinas and differentiated into RGC-like cellsin vivo(Wang et al.,2019).

Another study found that dissociated organoid cells expressed more RGC markers after seeding onto an adhesive substrate to allow axonal outgrowth,indicating further RGC differentiation and maturation (Rabesandratana et al.,2020).THY1 selection to harvest RGCs for intravitreal injection into mouse eyes showed that,despite no neurite outgrowth on the host retinas,there was robust cell survival,with RGC marker expression up to 4 weeks after transplant.Unfortunately,although theirin vitroresult showed RGC maturation after re-adhesion,the researchers did not compare different transplant outcomes of cells with versus without re-adhesion.

To further understand how the microenvironment affects donor cell development,a recent study recollected surviving mouse donor cells from mouse host retinas and performed RNA sequencing (Oswald et al.,2021).Similar intravitreal transplants after the dissociation of donor cells from mouse retinal organoids and selection by THY1 showed that cells survived up to 12 months after intravitreal injection.The impressive long-term survival might be due to mouse-to-mouse transplant (mouse retinal organoids injected into mouse retinas),unlike previous studies that transplanted donor cells originating from human stem cells.RNA sequencing revealed upregulation of pathways mediating axonal outgrowth,extension,and guidance,consistent with morphological observations that transplanted cells form axonal processes along the retinal surface and enter the optic nerve head.

Many rodent studies have successfully performed RGC transplantation by injecting cells into the vitreous cavity (Parameswaran et al.,2010).But,there are hurdles when translating these studies into eyes with large vitreous cavities.Unlike in rodent models,cell suspensions barely touch host retinas after injection into large eyes and tend to aggregate in the host vitreous (Becker et al.,2016).Although subretinal injection can deliver RGCs to a specific target (Sun et al.,2015),potential risks such as retinal detachment and subsequent neuronal injury should be considered.

Thus,having condensed donor cells attach to the ganglion cell layer without retinal invasion is an ultimate goal.One group working on a feline model prepared cellular scaffolds of compressed collagen to deliver donor cells.Compared with cell suspension transplant,donor cells did not aggregate and successfully migrated from the scaffold onto the inner retinal surface (Becker et al.,2016).Another group used a biodegradable tissue-engineered scaffold to overcome this issue (Li et al.,2017).Cells dissociated from day-55 retinal organoids were seeded onto a poly(lactic-co-glycolic) acid scaffold and cultured for another few days,and RGCs on scaffolds demonstrated dendrite architectures and long axons.Two weeks after transplant,the scaffold and cellular fluorescence were detected in both rabbit and monkey eyes.Although the study did not show robust evidence of cell survival because fluorescence can be detected from living or dead cells,this is the first study of dissociated organoid transplant into monkey models,indicating cell delivery by scaffolds could provide sufficient cell-host contact and facilitate cell survival.In addition,the delivery of a cell scaffold and its attachment to the host retina are relevant for clinical applications.Hence,cell scaffold transplantation requires multiple techniques applied in combination with stem cell and biomaterial transplantation.Additional studies are needed to advance this field,including determining the best biodegradable material and the optimal size and characteristics of the transplanted scaffold.

Cell clusters as donor material

Compared to induction in 2D cultures,retinal neurons in retinal organoids have the advantage of developing in well-organized tissues,sharing similar lamination with the retina.Unlike in cell suspension,retinal neurons in organoid tissues are supported by an extracellular matrix.A study has demonstrated that grafting donor cells in tissue-like cerebral organoids with 3D cytoarchitecture exhibit enhanced survival and robust vascularization from the host brain compared to transplants of dissociated neural progenitor cells (Daviaud et al.,2018).Hence,it is interesting to ask whether transplant leads to better outcomes when donor cells are engrafted as clusters of cells or pieces of tissue.

A recent study transplanted organoid fragments into mouse eyes (Wu et al.,2021).Retinal organoid fragments containing RGCs were prepared and placed near the retinal surface by air injection to ensure sufficient donorhost contact.Transplanted cells survived for 12 weeks and extended neurites into the inner plexiform layer of host retinas.Although the researchers did not perform cell suspension transplant as a comparison,survival duration and neurite extension were better than many cell suspension transplants described in previous experiments.

Organoid cluster transplants have also been performed on large animal models (Singh et al.,2019;Luo et al.,2021).However,the biggest issue of transplantation in large animals is how to ensure reliable donor-host contact with larger vitreal cavities.In one study,human stem cell-derived retinal organoids on days 60-70,when RGCs are developing,were delivered to the feline subretinal space (Singh et al.,2019).In the presence of an immunosuppressant,the graft survived up to one month after transplant and formed synaptic connections with the host ganglion cell layer.However,only human cytoplasm was stained to identify donor cells and their axons,which did not fully exclude the possibility of cytoplasmic transfusion.Donor survival and integration could be false positive results since host feline cells could have received human cytoplasmic material and thus stained as human donor cells.Further,although transplanting into the narrow subretinal space allowed reliable and long-term contact between the graft tissue and donor retina,donor RGCs had a long distance to migrate to their target layer by traveling across dense plexiform layers,which further decreases the chance for donor RGCs to survive and properly integrate.

Another study transplanted organoids to the inner retina surface of monkeys (Luo et al.,2021).Organoids were seeded on a poly(lactic-co-glycolic) acid scaffold,allowing RGCs in the organoids to migrate and extend their neurites.The cellular scaffold was then fixed onto the retinal surface following vitrectomy.Donor cells survived up to one year,and robust neurite outgrowth was noted along host neural fibers,reaching the optic nerve head.This study was conducted by the same group that seeded cell suspensions on a poly(lactic-co-glycolic) acid scaffold for transplant in 2017 (Li et al.,2017).However,the better outcome in 2021 may not be attributed exclusively to different forms of donor cells—the authors also optimized the surgical procedure (Luo et al.,2018) for better donor-host contact,which thereby may have contributed to the remarkable long-term survival.

Although several studies have been performed on organoid tissue/cluster transplant in rodent or large animal models,none have included a control group to compare with cell suspension.Hence,it is currently premature to draw conclusions about the more appropriate form of donor cells for transplant.In addition,several previous studies show that the extracellular matrix plays an essential role in neural development and axon extension,both in 2D-and 3D-culture systems (Fligor et al.,2018;Perepelkina et al.,2019).Therefore,we expect cell clusters to survive better after transplant due to their undisrupted cell-cell interactions supported by the extracellular matrix.Nonetheless,additional research is needed to identify the best donor cell format,which could contribute to efficient clinical translation.

Other donor cell-host factors

Studying donor cells on their own may not be sufficient to optimize transplant.For example,the development of high-throughput sequencing techniques has enabled further understanding of how similarin vitro-generated RGCs are to “real” human fetal or adult RGCs.However,similarity does not necessarily translate to effective cell replacement therapy.Other factors such as the capacity for successful synaptic connectivity between the donor cells and the host are also key to restoring visual function and thus demand further study.To that end,cell penetration into the ganglion cell layer to form synaptic integration with the visual circuit remains a challenge because of the existence of an internal limiting membrane (Zhang et al.,2021c).An exciting study showed that digestion of the internal limiting membrane by pronase E overcomes this barrier in anex vivomodel (Zhang et al.,2021c).However,more studies are needed to determine the optimal dose of pronase E forin vivoapplication to move this therapeutic strategy to pre-clinical study.

Microglia are the main immune cells in the central nervous system.These cells are considered harmful when they are activated,as microglia secrete cytotoxic cytokines in many disorders such as Alzheimer’s disease (Huang et al.,2022;Braatz et al.,2023) and Parkinson’s disease (Yu et al.,2023).Although the eye is an immune-privileged area and an increased number of microglia have not been observed in the mouse retina after human-induced pluripotent stem cell-derived RGC transplantation (Vrathasha et al.,2022),it remains controversial whether activated microglia infiltrate the retina after transplantation and consequently reduce donor RGC survival.

Both donor cell survival and integration are key concerns for successful stem cell transplantation,and long-term survival of transplanted RGCs remains an important barrier to overcome.Besides immune cell responses,there may also be a lack of growth factors to support donor cell survival.Cell transplant into an injury model is the ultimate goal for RGC replacement therapy,but whether a retina with RGC loss is a better host than a healthy eye for donor RGC survival remains debatable (Aoki et al.,2006;Rabesandratana et al.,2020;Luo et al.,2022).In addition,the investigation of donor cell survival between models of acute (e.g.,optic nerve crush) and chronic (e.g.,intraocular pressure elevation) RGC degeneration is another important area of work for clinical translation.

The maturation stage of donor cells,either 2D-or 3D-derived,may also prove critical.Terminal and fully mature neurons likely have limited ability to migrate and integrate into a new circuit (Moore et al.,2009;Oswald et al.,2021).Neuronal cell transplantation studies exhibit that neural precursor donor cells survive and integrate well in spinal cord injury models (Kitagawa et al.,2022;Zheng et al.,2022),suggesting that immature RGCs are better donor cells than mature RGCs for transplantation.However,one study transplanting dissociated mouse stem cell derived-organoids into mouse retinas showed that younger (day 16) donor cells yielded poor outcomes (less than 10% success rate) compared to older (day 21) donor cells (Oswald et al.,2021).Hence,additional studies are needed to identify the most suitable developmental stage of stem cell-derived RGCs as donor cells.In addition to cell replacement,stem cells also play a role in neuroprotection (Zhang et al.,2021a).Most studies to date of directly induced RGC transplants report similarly low survival efficiency,probably due to unsatisfactory donor cell conditions such as lack of supportive factors or toxic host microenvironments including intrinsic immune responses.However,some studies have observed profound neuroprotective effects on host RGCs,regardless of the types of transplanted donor cells,including undifferentiated stem cells (Wu et al.,2018),neural progenitor cells (Rettinger et al.,2021),retinal neurons (Luo et al.,2022),or even non-retinal neurons (Wu et al.,2010).These protective effects are thought to be due to trophic factors secreted by the donor cells to support endogenous neuronal survival.These findings suggest that cell transplant might be more efficient to provide continuous secretion of trophic factors than direct injection of such factors.Identifying the supportive trophic factors and overexpressing them in donor cells before transplantation could amplify the neuroprotective effects on endogenous RGC survival after injury.Our previous study showed that stem cell-derived RGC transplantation also protects endogenous RGCs from death after injury (Luo et al.,2022).Co-transplantation of human stem cells with mouse RGCs into the mouse eyes can promote mouse RGC survivalin vivo(Wu et al.,2018).Further,mesenchymal stem cell transplantation significantly improves RGC survival and restores visual function in a rodent model of anterior ischemic optic neuropathy (Wen et al.,2021).Thus,stem cell transplantation could be a potential therapeutic strategy for RGC restoration by both neuroprotection and neuroregeneration (Gokoffski et al.,2020).

Conclusion and Future Prospects

Here we reviewed data on 2D-and 3D-derived RGC differentiation and cell survival after retinal transplantation.A 2D direct-induced protocol significantly shortens the time for RGC differentiation,while 3D retinal organoids provide a platform to simultaneously study multiple retinal neurons (e.g.,RGCs,photoreceptors).Although many studies have compared the transcriptional similarities of 2D-and 3D-derived RGCs versus primary human fetal RGCs,it remains unknown which better mimics primary RGCs and which better survives after transplant.Hence,further studies are needed to identify the most suitable donor cells,for example,from which differentiation system,at which stage,as well as in what form (e.g.,cell suspension,tissue cluster,cellular scaffold).Furthermore,an in-depth understanding of factors that contribute to better survival/integration after transplant will also be critical to further fine-tune donor cells.Finally,characterizing donor cell-host interactions to optimize transplant for visual functional restoration will be important for future work to achieve translation into therapies for glaucoma and other optic neuropathies.

Acknowledgments:We thank Dr.Jeffrey L.Goldberg at Stanford University for his critical revision of this manuscript.

Author contributions:Conceptualization,literature review: ZL,KCC;writingoriginal draft preparation: ZL;writing-review and editing: KCC.Both authors approved the final version of the manuscript.

Conflicts of interest:The authors declare no conflicts of interest.

Data availability statement:The data are available from the corresponding author on reasonable request.

Open access statement:This is an open access journal,and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License,which allows others to remix,tweak,and build upon the work non-commercially,as long as appropriate credit is given and the new creations are licensed under the identical terms.

Open peer reviewer:Tom Johnson,Johns Hopkins University,USA.

Additional file:Open peer review report 1.