Transplanted human photoreceptors transfer cytoplasmic material but not to the recipient mouse retina.

BACKGROUND: The discovery of material transfer between transplanted and host mouse photoreceptors has expanded the possibilities for utilizing transplanted photoreceptors as potential vehicles for delivering therapeutic cargo. However, previous research has not directly explored the capacity for human photoreceptors to engage in material transfer, as human photoreceptor transplantation has primarily been investigated in rodent models of late-stage retinal disease, which lack host photoreceptors. METHODS: In this study, we transplanted human stem-cell derived photoreceptors purified from human retinal organoids at different ontological ages (weeks 10, 14, or 20) into mouse models with intact photoreceptors and assessed transfer of human proteins and organelles to mouse photoreceptors. RESULTS: Unexpectedly, regardless of donor age or mouse recipient background, human photoreceptors did not transfer material in the mouse retina, though a rare subset of donor cells (< 5%) integrated into the mouse photoreceptor cell layer. To investigate the possibility that a species barrier impeded transfer, we used a flow cytometric assay to examine material transfer in vitro. Interestingly, dissociated human photoreceptors transferred fluorescent protein with each other in vitro, yet no transfer was detected in co-cultures of human and mouse photoreceptors, suggesting that material transfer is species specific. CONCLUSIONS: While xenograft models are not a tractable system to study material transfer of human photoreceptors, these findings demonstrate that human retinal organoid-derived photoreceptors are competent donors for material transfer and thus may be useful to treat retinal degenerative disease.

Progenitor division and cell autonomous neurosecretion are required for rod photoreceptor sublaminar positioning.

Migration is essential for the laminar stratification and connectivity of neurons in the central nervous system. In the retina, photoreceptors (PRs) migrate to positions according to birthdate, with early-born cells localizing to the basal-most side of the outer nuclear layer. It was proposed that apical progenitor mitoses physically drive these basal translocations non-cell autonomously, but direct evidence is lacking, and whether other mechanisms participate is unknown. Here, combining loss- or gain-of-function assays to manipulate cell cycle regulators (Sonic hedgehog, Cdkn1a/p21) with an in vivo lentiviral labelling strategy, we demonstrate that progenitor division is one of two forces driving basal translocation of rod soma. Indeed, replacing Shh activity rescues abnormal rod translocation in retinal explants. Unexpectedly, we show that rod differentiation also promotes rod soma translocation. While outer segment function or formation is dispensable, Crx and SNARE-dependent synaptic function are essential. Thus, both non-cell and cell autonomous mechanisms underpin PR soma sublaminar positioning in the mammalian retina.

Hydrogel assisted photoreceptor delivery inhibits material transfer.

Cell therapy holds tremendous promise for vision restoration; yet donor cell survival and integration continue to limit efficacy of these strategies. Transplanted photoreceptors, which mediate light sensitivity in the retina, transfer cytoplasmic components to host photoreceptors instead of integrating into the tissue. Donor cell material transfer could, therefore, function as a protein augmentation strategy to restore photoreceptor function. Biomaterials, such as hyaluronan-based hydrogels, can support donor cell survival but have not been evaluated for effects on material transfer. With increased survival, we hypothesized that we would achieve greater material transfer; however, the opposite occurred. Photoreceptors delivered to the subretinal space in mice in a hyaluronan and methylcellulose (HAMC) hydrogel showed reduced material transfer. We examined mitochondria transfer in vitro and cytosolic protein transfer in vivo and demonstrate that HAMC significantly reduced transfer in both contexts, which we ascribe to reduced cell-cell contact. Nanotube-like donor cell protrusions were significantly reduced in the hydrogel-transplanted photoreceptors compared to the saline control group, which suggests that HAMC limits the contact required to the host retina for transfer. Thus, HAMC can be used to manipulate the behaviour of transplanted donor cells in cell therapy strategies.

Photoreceptor nanotubes mediate the in vivo exchange of intracellular material.

Emerging evidence suggests that intracellular molecules and organelles transfer between cells during embryonic development, tissue homeostasis and disease. We and others recently showed that transplanted and host photoreceptors engage in bidirectional transfer of intracellular material in the recipient retina, a process termed material transfer (MT). We used cell transplantation, advanced tissue imaging approaches, genetic and pharmacologic interventions and primary cell culture to characterize and elucidate the mechanism of MT. We show that MT correlates with donor cell persistence and the accumulation of donor-derived proteins, mitochondria and transcripts in acceptor cells in vivo. MT requires cell contact in vitro and is associated with the formation of stable microtubule-containing protrusions, termed photoreceptor nanotubes (Ph NTs), that connect donor and host cells in vivo and in vitro. Ph NTs mediate GFP transfer between connected cells in vitro. Furthermore, interfering with Ph NT outgrowth by targeting Rho GTPase-dependent actin remodelling inhibits MT in vivo. Collectively, our observations provide evidence for horizontal exchange of intracellular material via nanotube-like connections between neurons in vivo.

InVision: An optimized tissue clearing approach for three-dimensional imaging and analysis of intact rodent eyes.

The mouse eye is used to model central nervous system development, pathology, angiogenesis, tumorigenesis, and regenerative therapies. To facilitate the analysis of these processes, we developed an optimized tissue clearing and depigmentation protocol, termed InVision, that permits whole-eye fluorescent marker tissue imaging. We validated this method for the analysis of normal and degenerative retinal architecture, transgenic fluorescent reporter expression, immunostaining and three-dimensional volumetric (3DV) analysis of retinoblastoma and angiogenesis. We also used this method to characterize material transfer (MT), a recently described phenomenon of horizontal protein exchange that occurs between transplanted and recipient photoreceptors. 3D spatial distribution analysis of MT in transplanted retinas suggests that MT of cytoplasmic GFP between photoreceptors is mediated by short-range, proximity-dependent cellular interactions. The InVision protocol will allow investigators working across multiple cell biological disciplines to generate novel insights into the local cellular networks involved in cell biological processes in the eye.

Modeling of Photoreceptor Donor-Host Interaction Following Transplantation Reveals a Role for Crx, Müller Glia, and Rho/ROCK Signaling in Neurite Outgrowth.

The goal of photoreceptor transplantation is to establish functional synaptic connectivity between donor cells and second-order neurons in the host retina. There is, however, limited evidence of donor-host photoreceptor connectivity post-transplant. In this report, we investigated the effect of the host retinal environment on donor photoreceptor neurite outgrowth in vivo and identified a neurite outgrowth-promoting effect of host Crx(-/-) retinas following transplantation of purified photoreceptors expressing green fluorescent protein (GFP). To investigate the noncell autonomous factors that influence donor cell neurite outgrowth in vitro, we established a donor-host coculture system using postnatal retinal aggregates. Retinal cell aggregation is sensitive to several factors, including plate coating substrate, cell density, and the presence of Müller glia. Donor photoreceptors exhibit motility in aggregate cultures and can engraft into established aggregate structures. The neurite outgrowth-promoting phenotype observed in Crx(-/-) recipients in vivo is recapitulated in donor-host aggregate cocultures, demonstrating the utility of this surrogate in vitro approach. The removal of Müller glia from host aggregates reduced donor cell neurite outgrowth, identifying a role for this cell type in donor-host signaling. Although disruption of chondroitin sulfate proteoglycans in aggregates had no effect on the neurite outgrowth of donor photoreceptors, disruption of Rho/ROCK signaling enhanced outgrowth. Collectively, these data show a novel role of Crx, Müller glia, and Rho/ROCK signaling in controlling neurite outgrowth and provide an accessible in vitro model that can be used to screen for factors that regulate donor-host connectivity. Stem Cells 2019;37:529-541.

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.

Establishment of a cone photoreceptor transplantation platform based on a novel cone-GFP reporter mouse line.

We report successful retinal cone enrichment and transplantation using a novel cone-GFP reporter mouse line. Using the putative cone photoreceptor-enriched transcript Coiled-Coil Domain Containing 136 (Ccdc136) GFP-trapped allele, we monitored developmental reporter expression, facilitated the enrichment of cones, and evaluated transplanted GFP-labeled cones in wildtype and retinal degeneration mutant retinas. GFP reporter and endogenous Ccdc136 transcripts exhibit overlapping temporal and spatial expression patterns, both initiated in cone precursors of the embryonic retina and persisting to the adult stage in S and S/M opsin(+) cones as well as rod bipolar cells. The trapped allele does not affect cone function or survival in the adult mutant retina. When comparing the integration of GFP(+) embryonic cones and postnatal Nrl(-/-) 'cods' into retinas of adult wildtype and blind mice, both cell types integrated and exhibited a degree of morphological maturation that was dependent on donor age. These results demonstrate the amenability of the adult retina to cone transplantation using a novel transgenic resource that can advance therapeutic cone transplantation in models of age-related macular degeneration.

Inclusion of the viral anti-apoptotic molecule M11L in DNA vaccine vectors enhances HIV Env-specific T cell-mediated immunity.

A current goal of vaccine development against human immunodeficiency virus (HIV) is to develop a strategy that stimulates long-lasting memory T-cell responses, and provides immediate cytotoxicity in response to viral challenge. We demonstrate that the viral antiapoptotic molecule M11L promotes cellular immune responses to the HIV envelope protein. Coexpression of M11L in vitro inhibits gp140-mediated apoptosis and increases gp140 expression levels. Mice primed with M11L-pHERO DNA, followed by vCP205 boosting, exhibit significantly greater HIV-specific T-cell responses. Moreover, M11L synergizes with CpG motifs to augment anti-HIV responses and stimulates robust expansion of central memory and effector memory CD8(+) T-cells. Inclusion of M11L in a DNA vector increases the magnitude of T-cell responses, and promotes the generation of memory T-cells that provide rapid-responding CTL responses. This vaccine strategy may facilitate the generation of an efficacious vaccine for HIV, and other chronic diseases that require enhanced cell-mediated immunity, including HCV and metastatic cancer.

Current Status for High Titre Poxvirus Stock Preparation in CEF Under Serum-Free Medium Conditions: Implication for Vaccine Development.

In light of the recent detection of BSE in North America and its endemic nature in other regions of the world, there is a real need to employ cell culture conditions that do not require any animal-derived material. Here we report the use of an ultra-low protein serum-free medium (VP-SFM, Invitrogen) for the amplification of poxviruses in primary chicken embryo fibroblasts (CEF). We compared the amplification of four different poxviruses (canarypox, modified Ankara Virus (MVA), vaccinia virus strain Copenhagen and myxoma strain Lausanne) in three different media: DMEM 10%, DMEM 2% and serum-free medium VP-SFM. VP-SFM is a serum-free, ultra-low protein medium containing no proteins or peptides of human or animal origin designed to support the replication of viruses and the production of recombinant proteins and monoclonal antibodies. Our results show that high titre poxvirus stocks can be prepared in VP-SFM equivalent to that prepared in serum containing medium.

CPG ODN allows lower dose of antigen against hepatitis B surface antigen in BALB/c mice.

We have evaluated alum, immunostimulatory cytosine guanine dinucleotide-containing oligodeoxy-nucleotides (CPG ODN), and an alum/CPG ODN combination as adjuvants with hepatitis B surface antigen, to compare their potential to allow lower doses of antigen to be used for induction of humoral responses. BALB/c mice were immunized by intramuscular injection with 0.01, 0.1 or 1.0 micro g recombinant hepatitis B surface antigen without adjuvant or with alum and/or CPG ODN added. When given without adjuvant or with alum, each 10-fold decrease in amount of antigen resulted in a similarly reduced titre of antibody against hepatitis B surface antigen. In contrast, CPG ODN, on its own or combined with alum, allowed high anti-hepatitis B surface antigen titres even with a 1000-fold reduction in amount of antigen. These findings may have important immunological and economical consequences for vaccine development.