Computational molecular phenotyping of retinal sheet transplants to rats with retinal degeneration.

Retinal progenitor sheet transplants have been shown to extend neuronal processes into a degenerating host retina and to restore visual responses in the brain. The aim of this study was to identify cells involved in transplant signals to retinal degenerate hosts using computational molecular phenotyping (CMP). S334ter line 3 rats received fetal retinal sheet transplants at the age of 24-40 days. Donor tissues were incubated with slow-releasing microspheres containing brain-derived neurotrophic factor or glial cell-derived neurotrophic factor. Up to 265 days after surgery, eyes of selected rats were vibratome-sectioned through the transplant area (some slices stained for donor marker human placental alkaline phosphatase), dehydrated and embedded in Eponate, sectioned into serial ultrathin datasets and probed for rhodopsin, cone opsin, CRALBP (cellular retinaldehyde binding protein), l-glutamate, l-glutamine, glutathione, glycine, taurine, γ-aminobutyric acid (GABA) and DAPI (4',6-diamidino-2-phenylindole). In large transplant areas, photoreceptor outer segments in contact with host retinal pigment epithelium revealed rod and cone opsin immunoreactivity whereas no such staining was found in the degenerate host retina. Transplant photoreceptor layers contained high taurine levels. Glutamate levels in the transplants were higher than in the host retina whereas GABA levels were similar. The transplant inner nuclear layer showed some loss of neurons, but amacrine cells and horizontal cells were not reduced. In many areas, glial hypertrophy between the host and transplant was absent and host and transplant neuropil appeared to intermingle. CMP data indicate that horizontal cells and both glycinergic and GABAergic amacrine cells are involved in a novel circuit between transplant and host, generating alternative signal pathways between transplant and degenerating host retina.

Retinal degeneration in two lines of transgenic S334ter rats.

Aim of this study was to examine synaptic connectivity changes in the retina and the location and rate of apoptosis in transgenic S334ter line-3 and line-5 rats with photoreceptor degeneration. Heterozygous S334ter-line-3 and line-5 at P11-13, P30, P60, P90 and several control non-dystrophic rats (Long Evans and Sprague-Dawley) at P60, were studied anatomically by immunohistochemistry for various cell and synaptic markers, and by PNA and TUNEL label.- S334ter line-3 exhibited the fastest rate of degeneration with an early loss of photoreceptors, with 1-2 layers remaining at P30, and only cones left at P60. Line-5 had 4-5 layers left at P30, and very few rods left at P60-90. In both lines, horizontal cell processes (including dendrites and axon) were diminished at P11-13, showing gaps in the outer plexiform layer (OPL) at P60, and at P90, almost no terminal tips could be seen. Bipolar cells showed a retraction of their dendrites forming clusters along the OPL. Synaptic terminals of A-II amacrine cells in the IPL lost most of their parvalbumin-immunoreactivity. The apoptosis rate was different in both lines. Line-3 rats showed many photoreceptors affected at P11, occupying the innermost part of the outer nuclear layer. Line-5 showed a lower number of apoptotic cells within the same location at P13. In summary, the S334ter line-3 rat has a faster progression of degeneration than line-5. The horizontal and bipolar terminals are already affected at P11-P13 in both models. Apoptosis is related to the mutated rhodopsin transgene; the first photoreceptor cells affected are those close to the OPL.

Visual restoration and transplant connectivity in degenerate rats implanted with retinal progenitor sheets.

The aim of this study was to determine whether retinal progenitor layer transplants form synaptic connections with the host and restore vision. Donor retinal sheets, isolated from embryonic day 19 rat fetuses expressing human placental alkaline phosphatase (hPAP), were transplanted to the subretinal space of 18 S334ter-3 rats with fast retinal degeneration at the age of 0.8-1.3 months. Recipients were killed at the age of 1.6-11.8 months. Frozen sections were analysed by confocal immunohistochemistry for the donor cell label hPAP and synaptic markers. Vibratome slices were stained for hPAP, and processed for electron microscopy. Visual responses were recorded by electrophysiology from the superior colliculus (SC) in 12 rats at the age of 5.3-11.8 months. All recorded transplanted rats had restored or preserved visual responses in the SC corresponding to the transplant location in the retina, with thresholds between -2.8 and -3.4 log cd/m(2). No such responses were found in age-matched S334ter-3 rats without transplants, or in those with sham surgery. Donor cells and processes were identified in the host by light and electron microscopy. Transplant processes penetrated the inner host retina in spite of occasional glial barriers between transplant and host. Labeled neuronal processes were found in the host inner plexiform layer, and formed apparent synapses with unlabeled cells, presumably of host origin. In conclusion, synaptic connections between graft and host cells, together with visual responses from corresponding locations in the brain, support the hypothesis that functional connections develop following transplantation of retinal layers into rodent models of retinal degeneration.

Restoration of visual responses following transplantation of intact retinal sheets in rd mice.

PURPOSE: To correlate the functional outcomes with histologic findings following transplantation of fetal retinal sheets in rd mice, and to investigate the mechanisms of visual function restoration. METHODS: Twenty-one postnatal day 31-38 rd/rd (C3H/HeJ) mice were transplanted in one eye with retinal sheets (1.0 x 0.4 mm) obtained from embryonic day (E) 17 enhanced-green-fluorescent protein (eGFP) mice. Five mice underwent sham surgery without insertion of tissue. Four to five weeks after transplantation, visual responses to a light flash were recorded across the superior colliculus (SC) in seven eyes of seven transplanted mice that had clear corneas and lenses, and in all five sham surgery mice. Following the SC recording, the eyes were enucleated and processed for immunohistochemistry and examined using confocal microscopy. RESULTS: In three out of the seven eyes (43%), positive responses were recorded in the SC in an area topographically corresponding to the placement of the transplant in the host retina. No responses were recorded in the untreated eyes of 5-week-old and 9-week-old rd/rd mice, and in the 9-week-old sham surgery mice. In contrast, visual responses were recorded over the entire SC in normal eyes. The response onset latencies of the 3 transplanted mice with responses were similar to those of normal control mice. The organization of the graft did not appear to correlate as expected with the electrophysiology results, as eyes with well-organized, laminated grafts showed no response whereas the three light-responsive eyes had rosetted or disorganized grafts. All three light-responsive eyes demonstrated much higher levels of recoverin immunoreactivity in the host retina overlying the graft compared with untreated age-matched rd/rd mice. CONCLUSION: Restoration of the SC visual response does not appear to depend on a well-organized transplant in the rd mouse. Increased recoverin-staining in the host retina in light-responsive animals suggested that host cone rescue was the likely mechanism of vision restoration in this transplant model.

Alternative culture conditions for isolation and expansion of retinal progenitor cells.

PURPOSE: To investigate different in vitro model systems for retinal progenitor cell (RPC) isolation and expansion. METHODS: RPCs were isolated from embryonic day (E) 17 Long Evans rat retinas. Three different culture media: (1) modified serum free defined media (2) serum-containing media and (3) embryonic stem cell (ES)-conditioned media were used for RPC isolation and long term expansion. Expression of various cellular markers and cell morphologies were compared among the three culture systems at different passages by immunostaining and confocal microscopy. RESULTS: All three culture systems could maintain RPCs as nestin-positive cells (78-87%) after long-term in vitro expansion. However, RPCs appeared to proliferate faster in the serum-free culture system. The ES-conditioned media provided the best RPC survival. Cells appeared smaller at early passages compared with later passages. This morphology change occurred at P9-P10 in the serum-free medium, and at P5-P6 in the other two culture systems. CONCLUSIONS: The serum-free medium may be superior for preventing RPC differentiation during expansion.

Retinal transplants restore visually evoked responses in rats with photoreceptor degeneration.

PURPOSE: To assess whether transplantation of intact sheets of fetal retina with retinal pigment epithelium (RPE) into a retina with photoreceptor degeneration restores visually evoked responses. METHODS: Sheets of fetal retina with RPE were transplanted into the subretinal space of Royal College of Surgeons (RCS) rats at 37 to 69 days of age. Sixty-three days to 10 months after transplantation, multiunit visual responses were recorded in the superior colliculus (SC) of transplanted rats, age-matched untransplanted rats, and rats with sham surgery. RESULTS: In 19 of 29 RCS rats with transplants, visually evoked responses were recorded from and restricted to a small area of the SC that corresponds topographically to the portion of the retina in which the transplant was placed. Outside of this area, no visual responses were evoked. Visually evoked responses were never recorded in age-matched, nontransplanted RCS rats. Visually evoked responses were recorded in 6 of 13 RCS rats with sham surgery, but these responses were significantly different from responses in rats with transplants. CONCLUSIONS: These results demonstrate that this transplantation technique restores visually evoked responses in the brain. Although the underlying mechanism is unknown, we propose that the central visual response results from increased synaptic efficacy within the host retina. If it can be established that functional connections between the transplant and the host retina produce the effect, then it would indicate that the technique could be explored as a therapeutic strategy in some diseases of retinal degeneration.

Selective photoreceptor damage in albino rats using continuous blue light. A protocol useful for retinal degeneration and transplantation research.

PURPOSE: To develop a retinal degeneration model with selective photoreceptor loss and RPE sparing, to be used as recipient for evaluating retinal transplants. METHODS: Albino rats were exposed to blue light, continuously, for 1-7 days (24-168 h) in a specially designed cage. Eyes were histologically analyzed at periods between 2 h and 8 months after the light exposure. Electroretinograms (ERGs) were recorded from some rats at 12-216 days after exposure. Using behavioral methods, visual thresholds of some rats were determined before exposure and re-measured between 18 and 52 days following exposure. RESULTS: Apoptotic nuclei appeared exclusively in the photoreceptor layer after 1-5 days exposure to blue light. Light microscopy revealed that 2-4 days of light exposure reduced the outer nuclear layer (normally eight to ten rows) to 1 row of cells in the central retina and to two to three rows in the periphery, both in the superior and the inferior retina. Average ERG a- and b-wave amplitudes of light-damaged rats were both reduced by about 98%. Visual performance in the behavioral test was substantially impaired. CONCLUSIONS: Continuous exposure of albino rats to moderate blue light for 2-5 days selectively eliminates most of the photoreceptors while leaving the RPE initially intact.

Preliminary report: indications of improved visual function after retinal sheet transplantation in retinitis pigmentosa patients.

PURPOSE: To report indications of new visual function after retinal transplantation in two blind patients with retinitis pigmentosa. METHODS: Intact sheets of fetal retina (15 and 17 weeks gestational age) were transplanted subretinally (between the neurosensory retina and the retinal pigment epithelium) near the fovea in the left eye of a 23-year-old white man (Patient A) and in the left eye of a 72-year-old white woman (Patient B), both with autosomal-recessive retinitis pigmentosa. RESULTS: Postoperatively, at 6 and 5 months, respectively, both patients reported new visual sensation in the visual field corresponding to the transplant. In both patients, the visual sensation continued to be present after transplantation, at 12 and 8 months, respectively. In Patient A, a transient multifocal electroretinography (mfERG) response was observed in the transplant area 4 months postoperatively but was not detectable in Patient A at 6.0 and 9.5 months post-retinal transplantation. In Patient B, no positive mfERG responses were seen up to 5 months postoperatively. No rejection (presenting as cystoid macular edema, macular pucker, and extensive intraretinal edema with disrupted retinal pigment epithelium) to the transplanted tissue was seen up to 13 months in Patient A and 9 months in Patient B by fluorescein angiography. CONCLUSION: Transplantation of intact sheets of fetal human retina in two patients with retinitis pigmentosa was not associated with evidence of transplant rejection. Subjective improvement and an indication of objective improvement 4 months postoperatively were seen in Patient A, and subjective improvement only was seen in Patient B.

Can subretinal microphotodiodes successfully replace degenerated photoreceptors?

The idea of implanting microphotodiode arrays as visual prostheses has aroused controversy on its feasibility from the moment it appeared in print. We now present results which basically support the concept of replacing damaged photoreceptors with subretinally implanted stimulation devices. Network activity in degenerated rat retinae could be modulated through local electrical stimulation in vitro. We also investigated the long term stability and biocompatibility of the subretinal implants and their impact on retinal physiology in rats. Ganzfeld electroretinograms and histology showed no significant side effect of subretinal implants on retinal function or the architecture of the inner retina.

Photoreceptor function of retinal transplants implicated by light-dark shift of S-antigen and rod transducin.

The aim was to demonstrate functional properties of transplanted histologically normal photoreceptors. Subretinal intact-sheet transplants of fetal E17-E20 rat retinas to light-damaged albino rat eyes were fixed in light or dark, 2 to 42 weeks after transplantation, and stained immunohistochemically for certain phototransduction proteins. In light adapted transplants, transducin was predominantly found in inner segments of parallel-organized photoreceptors. Transducin shifted to the outer segments with dark-adaptation. S-antigen distribution was opposite to transducin. Rhodopsin distribution did not change. The shift of signal transduction proteins correlated to the light conditions indicates that normal phototransduction processes were established in photoreceptors of transplanted retinal sheets.

Successful cotransplantation of intact sheets of fetal retina with retinal pigment epithelium.

PURPOSE: Many retinal diseases, such as macular degeneration, affect both retinal pigment epithelium (RPE) and photoreceptors. Therefore, retinal repair may require transplantation of both tissues together as a cograft. METHODS: As recipients of retina-RPE cografts, 7- to 10-week-old albino Royal College of Surgeons rats that lose their photoreceptors because of a pigment epithelium defect were used. Freshly harvested intact sheets of RPE with neural retina from pigmented normal rat fetuses were gel embedded for protection and transplanted into the subretinal space. RESULTS: After 6 to 7 weeks, with the support of the cografted RPE sheet, transplanted photoreceptors developed fully in organized parallel layers in the subretinal space. Immunohistochemistry for rhodopsin, rod alpha-transducin, and S-antigen and peanut agglutinin labeling for cone interphotoreceptor matrix domains suggested that the photoreceptors in the graft were capable of normal function. CONCLUSIONS: Freshly harvested intact sheets of fetal RPE and retina, transplanted together into the subretinal space, can develop a normal morphology. Such transplants have the potential to benefit retinal diseases with dysfunctional RPE and photoreceptors.

Intact sheets of fetal retina transplanted to restore damaged rat retinas.

PURPOSE: The aim of this study was to establish a model for morphologic retinal reconstruction after destruction of photoreceptors. METHODS: Rat embryos were prelabeled by injection of bromodeoxyuridine (BrdU) into timed pregnant rats on 2 to 6 consecutive days. Pieces of fetal retinas (embryonic day [E] 17 to E22) were embedded in growth factor-reduced matrigel for protection and stored in medium on ice. With the use of a custom-mnade implantation tool, trimmed embedded pieces were placed into the subretinal space of albino rats whose photoreceptors had been damaged by continuous exposure to blue light for 3 to 4 days. RESULTS: Donor cells were unequivocally identified by the BrdU label. Approximately 25% of transplants in the subretinal space developed parallel layers, with photoreceptor outer segments facing the host pigment epithelium. Transplants developed rosettes if host pigment epithelium had been damaged, if trauma to the donor tissue occurred during preparation or transplantation, and if the donor tissue was misplaced into the choroid or into the epiretinal space on top of the host retina. If the surgery was performed more than 4 weeks after the light damage, continued degeneration of the host retina caused secondary pigment epithelium damage, and transplants did not develop parallel layers of photoreceptor outer segments. CONCLUSIONS: After transplantation to the subretinal space of a degenerated retina, gel-protected fetal retina can develop parallel layers and photoreceptor outer segments in contact with host pigment epithelium. Transplants can develop good fusion with the inner retina of a photoreceptor-deficient recipient.

Fiber and synaptic connections between embryonic retinal transplants and host retina.

The aim of this study was to investigate (a) whether embryonic retinal transplants can sprout fibers into a lesioned adult host retina and (b) if these fibers established synaptic connections with the host. Embryonic rat (E16-22) or human (9-13 weeks) retinal cells were transplanted to adult rats. Normal Long-Evans rats received rat transplants. The hosts for human transplants were athymic nude rats. After varying survival times (3 to 11 months), animals were perfused with 4% paraformaldehyde (sometimes with added 0.1% glutaraldehyde). Glass microneedles, coated with DiI (a carbocyanine dye) were placed into the transplants which were then stored at room temperature in 2% paraformaldehyde for 3-15 months. This filled the cells that had processes in the area where the needle had been placed. Gelatin-embedded eyecups were cut on a vibratome. DiI-labeled transplant cells exhibited fiber outgrowth into the host retina. After photoconversion of the dye to an electron-dense precipitate, these neuronal processes could be followed with better resolution than with fluorescence. Occasionally, host cells could also be labeled by DiI placed into the graft, indicating fiber ingrowth of host fibers into the transplants. Selected photoconverted sections were embedded for electron microscopy. Synapses could be found along transplant processes that had grown into the host inner plexiform layer. These results indicate that neuronal fibers originating from embryonic retinal transplants form synapses in the host retina.

Co-transplantation of embryonic retina and retinal pigment epithelial cells to rabbit retina.

The retinal pigment epithelium (RPE) is important for normal development of the neural retina. We sought to investigate whether cografting RPE cells affected the differentiation and survival of retinal grafts. Pigmented embryonic day 16 (E16) rabbit retina was dissected either with or without attached RPE and injected into a lesion site in retinas of young adult rabbit hosts. Each host obtained a pure retina graft in one eye and a retina/RPE cograft in the other. Animals were sacrificed after 4, 8 and 12 weeks. After 4 weeks, grafts (1-2 mm in diameter) were seen in both experimental groups at the lesion site or in the subretinal space. However, 8 and 12 weeks after transplantation, the graft survival rate decreased. The grafts developed cell layers in folded sheets and many rosettes (a rosette consists of photoreceptors and cells of other retinal layers around a central lumen defined by an outer limiting membrane). Cografts of retina with RPE had areas of more distinct cell lamination than transplants of pure retina. Grafted RPE cells were organized in clusters of cells surrounded by extracellular matrix and often associated with blood vessels. If the extracellular matrix of RPE cell clusters was outside the rosettes close to inner retinal layers in the graft, transplant Müller cell endfeet developed an inner limiting membrane. Müller cell endfeet could also be observed in subretinal transplants attached to the denuded Bruch's membrane of the host. In 12-week grafts, when RPE cell clusters were inside rosettes, the surrounded photoreceptors survived better. No RPE effect could be seen if single RPE cells were dispersed among retinal donor cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplantation of embryonic retinal donor cells labelled with BrdU or carrying a genetic marker to adult retina.

After transplantation of embryonic retinal cells to injured adult retina, it is often difficult to distinguish donor from host cells. To overcome this problem, two methods were applied: labelling donor cells with the nuclear marker bromodeoxyuridine (BrdU) and use of transgenic donor tissue. BrdU was injected into timed-pregnant rats on 2 or 3 consecutive days. The donor embryos were taken 1-4 days later for transplantation. The BrdU-labelled donor tissue was examined in transplants sampled up to 1 year after grafting. Labelled donor cells were specifically identified in the transplants and in the interface with the adjacent host retina. The varying intensities of cell labelling indicated differences in the initial uptake of BrdU in the S-phase, or the dilution of the label by cell divisions after BrdU injection. The best labelled cells were presumably the ones that stopped dividing shortly after injection of BrdU. As controls, the normal development of BrdU-labelled retinas from the offspring of females that had been BrdU-injected at E16 and E17 and not used for transplantation was studied. Near the time of birth, clones of labelled cells were radially distributed. In the mature retina, labelled cells were seen in all retinal layers. Embryonic retina derived from transgenic (NSE-lacZ) mice was transplanted to 'nude', immunodeficient rats (xenografts). These transgenic mice contain the Escherichia coli beta-galactosidase gene, coupled to the promoter for neuron-specific enolase (NSE). Thus, all retinal donor cells that contain NSE could be identified by histochemistry or immunohistochemistry. The donor cells expressing the transgene could be detected several months after transplantation.

Human embryonic retinal cell transplants in athymic immunodeficient rat hosts.

This study investigates the possibility to use the athymic "nude" rat as a host for the transplantation of human embryonic retinal cells without immunosuppression. The long-term development of such transplants is compared with results from our earlier study that used immunosuppressed rats, and showed transplant immunoreactivity for S-antigen. Several additional cell markers have been included: rhodopsin, rod (alpha-transducin, neuron-specific enolase (NSE), synaptophysin (SYN), cone-specific opsins, vimentin, cellular retinaldehyde binding protein (CRALBP), glial fibrillary acidic protein (GFAP), rat major histocompatibility antigen class II (MHC-II) and a rat macrophage marker (Ox-42). Human retinal cells (9-13 wk postconception) were transplanted to the eyes of 28 athymic rats. Host rats were kept in microisolator cages for up to 48 wk after surgery. Host immune response and the development of the transplants were studied using histology, immunohistochemistry and electron microscopy. When using retinas of donors 9-11 wk postconception, transplants grew to 2-3 mm in diameter with many rosettes, in 31 of 35 eyes. Transplants derived from donors 12-13 wk postconception did not survive as well (8 out of 11 eyes), were smaller and less organized. All transplants fused well with the host retina, better than corresponding transplants to immunosuppressed rat hosts. Most transplants appeared to be healthy, even after long survival times, and only occasionally were MHC-II positive macrophages observed in transplants or host retinas. All retinal layers were observed, except for an inner limiting membrane on the vitreous surface. The oldest transplants (34-57 wk total age = donor age + time after surgery) exhibited well developed photoreceptors, rods and cones, with inner and outer segments. SYN-staining showed the development of inner and outer plexiform layers. Although many cones stained for SYN and NSE, few were immunoreactive for red-green or blue opsin. Most rods became immunoreactive for S-antigen and rhodopsin. Transplant Müller cells stained for vimentin and CRALBP. Immunoreactivity for GFAP developed slowly and was not completely expressed in all transplant Müller cells until 44 wk total age. Nude rats offer an excellent model for the study of human retinal xenografts without the negative effects of immunosuppression. Compared to immunosuppressed rats, transplantation to nude rats gives consistent results and superior long-term survival of hosts and transplants.

Photoreceptor and glial markers in human embryonic retina and in human embryonic retinal transplants to rat retina.

The purpose of this study was to compare the development of photoreceptor and glial cells in human embryonic retinal transplants with the development of normal human embryonic retina (13-20 weeks postconception). Human embryonic retinal cells (donor age 6-11 weeks postconception) were transplanted to the retinas of adult immunosuppressed rat hosts. Host animals were killed when the transplants were of 13-37 weeks total age (donor age+survival time after surgery). Immunohistochemistry was performed with antibodies specific for neuron-specific enolase (NSE), synaptophysin (SYN), cone-specific opsins, rhodopsin, rod alpha-transducin, S-antigen, vimentin, cellular retinaldehyde-binding protein (CRALBP) and glial fibrillary acidic protein (GFAP). With regards to photoreceptors, NSE and SYN immunoreactive cones were seen in transplants from 14-16 weeks of age, but cone opsin immunoreactivity was not seen until 25 weeks. Developing graft rods became S-antigen immunoreactive at 17-18 weeks. At 20 weeks, inner segments and some cell somas of graft rods stained faintly for alpha-transducin and rhodopsin. At 31 and 37 weeks, inner and outer rod segments were intensely labelled for the rod-specific antigens. The grafts exhibited areas of varying maturation with different staining intensities. Concerning the glial cells, vimentin immunoreactivity was seen in the earliest transplants studied (total age 14-16 weeks), but only in stages older than 19 weeks was the immunoreactivity of graft Müller cells comparable in intensity to those of the host retina. Host Müller cells were immunoreactive for GFAP near the lesion site at all times. At 20 weeks, some GFAP immunoreactive processes were seen inside the graft, apparently coming from the host retina. At 25 weeks, faintly stained Müller cells intrinsic to the graft were observed, indicating a gliosis within the graft. Graft Müller cells were first seen to express CRALBP immunoreactivity at 19-20 weeks and, at 25 weeks, intense immunoreactivity was seen in the transplant, mostly in regions near the host. In the transplants only the Müller cells were stained, whereas both Müller and retinal pigment epithelium cells were CRALBP immunoreactive in the host retina. The development of human embryonic retinal transplants appears to parallel approximately normal in utero development. Transplant cones, rods and Müller cells all express their cell-specific proteins. The photoreceptors develop both inner and outer segments and contain several essential proteins for processing light. The transplants can reach a degree of maturity comparable to newborn retina.

Ultrastructural circuitry in retinal cell transplants to rat retina.

The development of five transplants of fetal retinal tissue to adult rat eyes was examined with the electron microscope. The transplants were of 9 to 10 weeks total age after conception in four cases and 20 weeks in one case. They were at stage E15 when transplanted. Transplants developed in both the epiretinal and subretinal spaces. The transplants were heterogeneously developed with some parts showing almost normal differentiation and others little. Subretinal transplants examined in this study were more developed than epiretinal grafts. Photoreceptor cells developed both inner and outer segments. Their synaptic terminals possessed output ribbon synapses with postsynaptic processes similar to those seen in normal retinas. In regions corresponding to the inner plexiform layer, the adult complement of synapses was seen, including advanced features such as serial synapses as well as reciprocal synapses at bipolar cell dyads. Incompletely differentiated synapses of both the amacrine and bipolar cell types were often observed, especially in the rat epiretinal transplants. Ganglion cell processes could not be identified with certainty. Although transplant cells were adjacent to host photoreceptor cells and pigment epithelium, obvious specializations or interactions were not observed. The experiments suggest that embryonic rat retinal cell transplants develop most or perhaps all of the structural components and neuronal circuitry necessary to transduce light and process some visual information.

Ultrastructure of human retinal cell transplants with long survival times in rats.

Human fetal retinas (6-12 weeks post-conception) were obtained from elective abortions, transplanted to rat retinas and examined by electron microscopy. The oldest transplants that form the basis of this report were obtained 40 and 41 total weeks post-conception. The host rats were immunosuppressed with cyclosporin A. The transplants developed according to their intrinsic, genetically determined timetable. The development was heterogeneous with some parts showing almost normal differentiation and others, little. Both rods and cones developed with inner and outer segments and synaptic terminals. In regions corresponding to the inner plexiform layer, bipolar cell processes were seen in the typical dyad arrangement. Likewise, amacrine cell processes formed typical conventional synapses. Serial synapses were seen, engaging amacrine cell synapses as well as a few reciprocal synapses at the bipolar cell dyads. Monad-type synaptic complexes, a sign of immaturity, were common in bipolar cell processes. Similarly, incompletely differentiated synapses of both the amacrine and bipolar cell types were often observed. Ganglion cell processes could not be identified with certainty. A structure with morphological characteristics similar to the inner limiting membrane was noted to form inside the transplant. Both epi-retinal and sub-retinal transplants were obtained. Transplant cells touched host photoreceptor cells or pigment epithelium without any obvious specializations. The host pigment epithelium microvilli were absent adjacent to the graft. However, graft cells did appear in the host retina, and nerve cell processes were observed to cross the membrane separating the transplant and host.