Label-free imaging flow cytometry for analysis and sorting of enzymatically dissociated tissues.

Biomedical research relies on identification and isolation of specific cell types using molecular biomarkers and sorting methods such as fluorescence or magnetic activated cell sorting. Labelling processes potentially alter the cells' properties and should be avoided, especially when purifying cells for clinical applications. A promising alternative is the label-free identification of cells based on physical properties. Sorting real-time deformability cytometry (soRT-DC) is a microfluidic technique for label-free analysis and sorting of single cells. In soRT-FDC, bright-field images of cells are analyzed by a deep neural net (DNN) to obtain a sorting decision, but sorting was so far only demonstrated for blood cells which show clear morphological differences and are naturally in suspension. Most cells, however, grow in tissues, requiring dissociation before cell sorting which is associated with challenges including changes in morphology, or presence of aggregates. Here, we introduce methods to improve robustness of analysis and sorting of single cells from nervous tissue and provide DNNs which can distinguish visually similar cells. We employ the DNN for image-based sorting to enrich photoreceptor cells from dissociated retina for transplantation into the mouse eye.

Photoreceptor precursor cell integration into rodent retina after treatment with novel glycopeptide PKX-001.

Cell replacement therapy is emerging as an important approach in novel treatments for neurodegenerative diseases. Many problems remain, in particular improvements are needed in the survival of transplanted cells and increasing functional integration into host tissue. These problems arise because of immune rejection, suboptimal precursor cell type, trauma during cell transplantation, and toxic compounds released by dying tissues and nutritional deficiencies. We recently developed an ex vivo system to facilitate identification of factors contributing to the death of transplanted neuronal (photoreceptor) and showed 2.8-fold improvement in transplant cell survival after pretreatment with a novel glycopeptide (PKX-001). In this study, we extended these studies to look at cell survival, maturation, and functional integration in an in vivo rat model of rhodopsin-mutant retinitis pigmentosa causing blindness. We found that only when human photoreceptor precursor cells were preincubated with PKX-001 prior to transplantation, did the cells integrate and mature into cone photoreceptors expressing S-opsin or L/M opsin. In addition, ribbon synapses were observed in the transplanted cells suggesting they were making synaptic connections with the host tissue. Furthermore, optokinetic tracking and electroretinography responses in vivo were significantly improved compared to cell transplants without PKX-001 pre-treatment. These data demonstrate that PKX-001 promotes significant long-term stem cell survival in vivo, providing a platform for further investigation towards the clinical application to repair damaged or diseased retina.

Restoration of visual function by transplantation of optogenetically engineered photoreceptors.

A major challenge in the treatment of retinal degenerative diseases, with the transplantation of replacement photoreceptors, is the difficulty in inducing the grafted cells to grow and maintain light sensitive outer segments in the host retina, which depends on proper interaction with the underlying retinal pigment epithelium (RPE). Here, for an RPE-independent treatment approach, we introduce a hyperpolarizing microbial opsin into photoreceptor precursors from newborn mice, and transplant them into blind mice lacking the photoreceptor layer. These optogenetically-transformed photoreceptors are light responsive and their transplantation leads to the recovery of visual function, as shown by ganglion cell recordings and behavioral tests. Subsequently, we generate cone photoreceptors from human induced pluripotent stem cells, expressing the chloride pump Jaws. After transplantation into blind mice, we observe light-driven responses at the photoreceptor and ganglion cell levels. These results demonstrate that structural and functional retinal repair is possible by combining stem cell therapy and optogenetics.

Magnetic Iron Oxide Nanoparticle Labeling of Photoreceptor Precursors for Magnetic Resonance Imaging.

IMPACT STATEMENT: This study describes the methods and results of superparamagnetic iron oxide nanoparticle (SPION) labeling and magnetic resonance imaging (MRI) tracking of human embryonic stem cell-derived photoreceptor precursors transplanted into the subretinal space of Royal College of Surgeons rats. SPION labeling and MRI tracking provide information about the biodistribution of transplanted photoreceptor precursors, which is necessary for improving the functional benefits of cell therapy for degenerative retinal diseases.

Gold nanoparticles for multimodal high-resolution imaging of transplanted cells for retinal replacement therapy.

Aim: Longitudinal tracking of transplanted cells in clinical and experimental setups is crucial for evaluating the efficiency of retinal cell replacement therapies. Materials & methods: Gold nanoparticle-labeled photoreceptor precursors were transplanted in the vitreous and subretinal space of rats and were longitudinally tracked for over a month using optical coherence tomography, computed tomography and fluorescence fundus imaging. Results: This multimodal imaging approach enabled high-resolution long-term tracking and estimation of cell survival in the retina and vitreous, while displaying no toxic effects on the cells or the retina. Conclusion: These observations highlight the applicability of using gold nanoparticle for retinal cell tracking in existing experimental settings and its translational potential for providing more efficient retinal cell therapy in humans.

Transplantation of photoreceptors into the degenerative retina: Current state and future perspectives.

The mammalian retina displays no intrinsic regenerative capacities, therefore retinal degenerative diseases such as age-related macular degeneration (AMD) or retinitis pigmentosa (RP) result in a permanent loss of the light-sensing photoreceptor cells. The degeneration of photoreceptors leads to vision impairment and, in later stages, complete blindness. Several therapeutic strategies have been developed to slow down or prevent further retinal degeneration, however a definitive cure i.e. replacement of the lost photoreceptors, has not yet been established. Cell-based treatment approaches, by means of photoreceptor transplantation, have been studied in pre-clinical animal models over the last three decades. The introduction of pluripotent stem cell-derived retinal organoids represents, in principle, an unlimited source for the generation of transplantable human photoreceptors. However, safety, immunological and reproducibility-related issues regarding the use of such cells still need to be solved. Moreover, the recent finding of cytoplasmic material transfer between donor and host photoreceptors demands reinterpretation of several former transplantation studies. At the same time, material transfer between healthy donor and dysfunctional patient photoreceptors also offers a potential alternative strategy for therapeutic intervention. In this review we discuss the history and current state of photoreceptor transplantation, the techniques used to assess rescue of visual function, the prerequisites for effective transplantation as well as the main roadblocks, including safety and immune response to the graft, that need to be overcome for successful clinical translation of photoreceptor transplantation approaches.

Transplantation of rat embryonic stem cell-derived retinal cells restores visual function in the Royal College of Surgeons rats.

AIM OF STUDY: To evaluate the feasibility of transplantation of embryonic stem cell (ESC)-derived retinal cells in the treatment of retinal degeneration. MATERIALS AND METHODS: Rat ESCs were isolated and induced into retinal progenitor cells (RPCs) in vitro, which were subsequently induced into retinal pigment epithelium cells (RPEs) and photoreceptors (PRCs). All cells were identified by Western blot detection of their specific markers. RPEs and PRCs were, respectively, injected into the retina of Royal College of Surgeons (RCSs) rats. Control group was injected with PBS. Post-transplantation visual function was determined by electroretinography (ERG). The histology of the whole eye was compared by H&E staining. RESULTS: RPEs and PRCs were successfully derived from rat ESCs through the two-step differentiation as indicated by the presence of ESC- (Oct-3/4, Nanog, TRA-1-60 and TRA-1-81), RPC- (Rx, Mitf, Pax6 and Chx10), RPE- (RPE65 and keratin) and PRC-specific markers (blue opsin, red/green opsin, recoverin and rhodopsin) in Western blot. The amplitude of ERG a- and b-wave in RPE- and PRC-transplanted groups at week 2 and 10 after transplantation was markedly higher compared with PBS controls. Retinal injury and vascular stress response was not detected in any of the RCS rats after transplantation. CONCLUSION: The developed stepwise protocol can derive retinal cells from ESCs. Transplantation of these retinal cells can restore visual function of RCS rats. Our study provides evidence for potential clinical application of ESC-based cell therapy for retinal degeneration.

Müller Glia Reactivity and Development of Gliosis in Response to Pathological Conditions.

Within the mammalian retina, both Müller glia and astrocytes display reactivity in response to many forms of retinal injury and disease in a process termed gliosis. Reactive gliosis is a complex process that is considered to represent a cellular response to protect the retina from further damage and to promote its repair following pathological insult. It includes morphological, biochemical and physiological changes, which may vary depending on the type and degree of the initial injury. Not only does gliosis have numerous triggers, but also there is a great degree of heterogeneity in the glial response, creating multiple levels of complexity. For these reasons, understanding the process of glial scar formation and how this process differs in different pathological conditions and finding strategies to circumvent these barriers represent major challenges to the advancement of many ocular therapies.

Pluripotent Stem Cells for Retinal Tissue Engineering: Current Status and Future Prospects.

The retina is a very fine and layered neural tissue, which vitally depends on the preservation of cells, structure, connectivity and vasculature to maintain vision. There is an urgent need to find technical and biological solutions to major challenges associated with functional replacement of retinal cells. The major unmet challenges include generating sufficient numbers of specific cell types, achieving functional integration of transplanted cells, especially photoreceptors, and surgical delivery of retinal cells or tissue without triggering immune responses, inflammation and/or remodeling. The advances of regenerative medicine enabled generation of three-dimensional tissues (organoids), partially recreating the anatomical structure, biological complexity and physiology of several tissues, which are important targets for stem cell replacement therapies. Derivation of retinal tissue in a dish creates new opportunities for cell replacement therapies of blindness and addresses the need to preserve retinal architecture to restore vision. Retinal cell therapies aimed at preserving and improving vision have achieved many improvements in the past ten years. Retinal organoid technologies provide a number of solutions to technical and biological challenges associated with functional replacement of retinal cells to achieve long-term vision restoration. Our review summarizes the progress in cell therapies of retina, with focus on human pluripotent stem cell-derived retinal tissue, and critically evaluates the potential of retinal organoid approaches to solve a major unmet clinical need-retinal repair and vision restoration in conditions caused by retinal degeneration and traumatic ocular injuries. We also analyze obstacles in commercialization of retinal organoid technology for clinical application.

Cellular regeneration strategies for macular degeneration: past, present and future.

Despite considerable effort and significant therapeutic advances, age-related macular degeneration (AMD) remains the commonest cause of blindness in the developed world. Progressive late-stage AMD with outer retinal degeneration currently has no proven treatment. There has been significant interest in the possibility that cellular treatments may slow or reverse visual loss in AMD. A number of modes of action have been suggested, including cell replacement and rescue, as well as immune modulation to delay the neurodegenerative process. Their appeal in this enigmatic disease relate to their generic, non-pathway-specific effects. The outer retina in particular has been at the forefront of developments in cellular regenerative therapies being surgically accessible, easily observable, as well as having a relatively simple architecture. Both the retinal pigment epithelium (RPE) and photoreceptors have been considered for replacement therapies as both sheets and cell suspensions. Studies using autologous RPE, and to a lesser extent, foetal retina, have shown proof of principle. A wide variety of cell sources have been proposed with pluripotent stem cell-derived cells currently holding the centre stage. Recent early-phase trials using these cells for RPE replacement have met safety endpoints and hinted at possible efficacy. Animal studies have confirmed the promise that photoreceptor replacement, even in a completely degenerated outer retina may restore some vision. Many challenges, however, remain, not least of which include avoiding immune rejection, ensuring long-term cellular survival and maximising effect. This review provides an overview of progress made, ongoing studies and challenges ahead.

Organoid technology for retinal repair.

A major cause for vision impairment and blindness in industrialized countries is the loss of the light-sensing retinal tissue in the eye. Photoreceptor damage is one of the main characteristics found in retinal degeneration diseases, such as Retinitis Pigmentosa or age-related macular degeneration. The lack of effective therapies to stop photoreceptor loss together with the absence of significant intrinsic regeneration in the human retina converts such degenerative diseases into permanent conditions that are currently irreversible. Cell replacement by means of photoreceptor transplantation has been proposed as a potential approach to tackle cell loss in the retina. Since the first attempt of photoreceptor transplantation in humans, about twenty years ago, several research groups have focused in the development and improvement of technologies necessary to bring cell transplantation for retinal degeneration diseases to reality. Progress in recent years in the generation of human tissue derived from pluripotent stem cells (PSCs) has significantly improved our tools to study human development and disease in the dish. Particularly the availability of 3D culture systems for the generation of PSC-derived organoids, including the human retina, has dramatically increased access to human material for basic and medical research. In this review, we focus on important milestones towards the generation of transplantable photoreceptor precursors from PSC-derived retinal organoids and discuss recent pre-clinical transplantation studies using organoid-derived photoreceptors in context to related in vivo work using primary photoreceptors as donor material. Additionally, we summarize remaining challenges for developing photoreceptor transplantation towards clinical application.

[Photoreceptor Transplantation into the Degenerative Retina]. / Photorezeptortransplantation in die degenerative Netzhaut.

Vision impairment and blindness due to photoreceptor loss represents one of the major causes for disability in industrialized societies. Whereas rod photoreceptors allow vision under dim light conditions, cone photoreceptors provide high-acuity vision in daylight conditions and color detection. Several therapeutic strategies are currently developed to repair vision loss, including cell-based interventions. Within the last decade, major progress regarding the replacement of photoreceptors by transplantation has been made in pre-clinical animal models. This includes defining the necessary conditions, like the optimal ontogenetic stage of transplantable donor photoreceptors, cell-specific enrichment procedures and robust transplantation technologies. Moreover, first studies provided evidence for functional improvements by photoreceptor transplantation in mouse models of retinal dysfunction. Furthermore, advances in cell culture technology were made by introducing methods to generate photoreceptor-containing retinal organoids, derived from pluripotent stem cells, that provide theoretically unlimited sources for the production of photoreceptor transplants. Interestingly, the recently identified transfer of cytoplasmic material between donor and host photoreceptors might represent an additional treatment option for cell transplantation approaches. Within this review, we focus on the main developments within the photoreceptor transplantation field and discuss important achievements, challenges and hurdles to develop photoreceptor transplantation towards clinical applications.

Structural divergence of essential triad ribbon synapse proteins among placental mammals - Implications for preclinical trials in photoreceptor transplantation therapy.

As photoreceptor transplantation rapidly moves closer to the clinic, verifying graft efficacy in animal models may have unforeseen xenogeneic barriers. Although photoreceptor transplants have most convincingly exhibited functional synaptogenesis in conspecific studies, such evidence (while ruling out false-positives due to: viral graft labeling, fusion/cytosolic transfer, or neuroprotection) has not yet been shown for discordant xenografts. From this, a fundamental question should be raised: is useful xenosynaptogenesis likely between human photoreceptors and mouse retina? The triad ribbon synapse (TRS) that would normally form is unique and contains trans-synaptic proteins essential to its formation and function. Thus, could interspecific structural divergence be present that may inhibit this trans-synaptic bridge in discordant xenografts? In an effort to address this question computationally, we compared eight recently confirmed (including subcellular location) TRS specific (or predominantly expressed at the TRS) proteins among placental mammals (1-to-1 orthologs) using HyPhy selection analysis (a predictive measure of structural divergence) and by using Phyre2 tertiary structural modeling. Here, selection analysis revealed strong positive (diversifying) selection acting on a particularly important TRS protein: pikachurin. This positive selection was localized to its second Laminin-G (LG)-like domain and on its N-terminal domain - a putative region of trans-synaptic interaction. Localization of structural divergence to the N-terminus of each putative post-translational cleavage (PTC) product may suggest neofunctionalization from ancestral uncleaved pikachurin - this would be consistent with a recent counter-paradigm report of pikachurin cleavage predominating at the TRS. From this, we suggest a dual role after cleavage where the N-terminal fragment can still mediate the trans-synaptic bridge, while the C-terminal fragment may act as a diffusible trophic or "homing" factor for bipolar cell dendrite migration. Tertiary structural models mirrored the conformational divergence predicted by selection analysis. With human and mouse pikachurin (as well as other TRS proteins) likely to diverge considerably in structure among placental mammals - alongside known inter-mammalian variation in TRS phenotype and protein repertoire, high levels of diversifying selection acting on genes involving sensation, considerable timespans allowing for genetic drift that can create xenogeneic epistasis, and uncertainty surrounding the extent of xenosynaptogenesis in PPC transplant studies to date - use of distantly related hosts to test human photoreceptor graft therapeutic efficacy should be considered with caution.

A Reinterpretation of Cell Transplantation: GFP Transfer From Donor to Host Photoreceptors.

The utilization of fluorescent reporter transgenes to discriminate donor versus host cells has been a mainstay of photoreceptor transplantation research, the assumption being that the presence of reporter+ cells in outer nuclear layer (ONL) of transplant recipients represents the integration of donor photoreceptors. We previously reported that GFP+ cells in the ONL of cone-GFP transplanted retinas exhibited rod-like characteristics, raising the possibility that GFP signal in recipient tissue may not be a consequence of donor cell integration. To investigate the basis for this mismatch, we performed a series of transplantations using multiple transgenic donor and recipient models, and assessed cell identity using nuclear architecture, immunocytochemistry, and DNA prelabeling. Our results indicate that GFP+ cells in the ONL fail to exhibit hallmark elements of donor cells, including nuclear hetero/euchromatin architecture. Furthermore, GFP signal does not appear to be a consequence of classic donor/host cell fusion or transfating post-transplant, but is most likely due to material exchange between donor and host photoreceptors. This transfer can be mediated by rods and cones, is bidirectional between donor and host cells, requires viable photoreceptors, occurs preferentially at sites of outer limiting membrane disruption and can be detected in second-order retinal neurons and Müller glia. Collectively, these data warrant re-evaluation of the use of lineage tracing fluorescent reporters in transplantation studies involving the retina and other CNS tissues. Furthermore, the reinterpretation of previous functional rescue data, based on material exchange, rather than cell integration, may offer a novel approach to vision rescue. Stem Cells 2017;35:932-939.

Transplanted photoreceptor precursors transfer proteins to host photoreceptors by a mechanism of cytoplasmic fusion.

Photoreceptor transplantation is a potential future treatment for blindness caused by retinal degeneration. Photoreceptor transplantation restores visual responses in end-stage retinal degeneration, but has also been assessed in non-degenerate retinas. In the latter scenario, subretinal transplantation places donor cells beneath an intact host outer nuclear layer (ONL) containing host photoreceptors. Here we show that host cells are labelled with the donor marker through cytoplasmic transfer-94±4.1% of apparently well-integrated donor cells containing both donor and host markers. We detect the occurrence of Cre-Lox recombination between donor and host photoreceptors, and we confirm the findings through FISH analysis of X and Y chromosomes in sex-discordant transplants. We do not find evidence of nuclear fusion of donor and host cells. The artefactual appearance of integrated donor cells in host retinas following transplantation is most commonly due to material transfer from donor cells. Understanding this novel mechanism may provide alternate therapeutic strategies at earlier stages of retinal degeneration.

Donor and host photoreceptors engage in material transfer following transplantation of post-mitotic photoreceptor precursors.

Photoreceptor replacement by transplantation is proposed as a treatment for blindness. Transplantation of healthy photoreceptor precursor cells into diseased murine eyes leads to the presence of functional photoreceptors within host retinae that express an array of donor-specific proteins. The resulting improvement in visual function was understood to be due to donor cells integrating within host retinae. Here, however, we show that while integration occurs the majority of donor-reporter-labelled cells in the host arises as a result of material transfer between donor and host photoreceptors. Material transfer does not involve permanent donor-host nuclear or cell-cell fusion, or the uptake of free protein or nucleic acid from the extracellular environment. Instead, RNA and/or protein are exchanged between donor and host cells in vivo. These data require a re-evaluation of the mechanisms underlying rescue by photoreceptor transplantation and raise the possibility of material transfer as a strategy for the treatment of retinal disorders.

Retinal transplantation of photoreceptors results in donor-host cytoplasmic exchange.

Pre-clinical studies provided evidence for successful photoreceptor cell replacement therapy. Migration and integration of donor photoreceptors into the retina has been proposed as the underlying mechanism for restored visual function. Here we reveal that donor photoreceptors do not structurally integrate into the retinal tissue but instead reside between the photoreceptor layer and the retinal pigment epithelium, the so-called sub-retinal space, and exchange intracellular material with host photoreceptors. By combining single-cell analysis, Cre/lox technology and independent labelling of the cytoplasm and nucleus, we reliably track allogeneic transplants demonstrating cellular content transfer between graft and host photoreceptors without nuclear translocation. Our results contradict the common view that transplanted photoreceptors migrate and integrate into the photoreceptor layer of recipients and therefore imply a re-interpretation of previous photoreceptor transplantation studies. Furthermore, the observed interaction of donor with host photoreceptors may represent an unexpected mechanism for the treatment of blinding diseases in future cell therapy approaches.

Function of human pluripotent stem cell-derived photoreceptor progenitors in blind mice.

Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement may hold the potential for repair in a completely degenerate retina by reinstating light sensitive cells to form connections that relay information to downstream retinal layers. This study assessed the therapeutic potential of photoreceptor progenitors derived from human embryonic and induced pluripotent stem cells (ESCs and iPSCs) using a protocol that is suitable for future clinical trials. ESCs and iPSCs were cultured in four specific stages under defined conditions, resulting in generation of a near-homogeneous population of photoreceptor-like progenitors. Following transplantation into mice with end-stage retinal degeneration, these cells differentiated into photoreceptors and formed a cell layer connected with host retinal neurons. Visual function was partially restored in treated animals, as evidenced by two visual behavioral tests. Furthermore, the magnitude of functional improvement was positively correlated with the number of engrafted cells. Similar efficacy was observed using either ESCs or iPSCs as source material. These data validate the potential of human pluripotent stem cells for photoreceptor replacement therapies aimed at photoreceptor regeneration in retinal disease.