Anders Bergström

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.

Neuronal markers in rat retinal grafts

Rat E15 retina was grafted to the retina of adult rat hosts. After varying survival times (1 week-6 months), grafts were stained by immunohistochemistry for neurofilament 160 kDa (NF), HPC-1 (an amacrine cell marker), choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD) and somatostatin-28 (SS-28). The first differentiating graft amacrine cells (cholinergic and dopaminergic) could be seen 1 week after transplantation (corresponding to postnatal day 1 = P1). The inner plexiform layer of the graft started to differentiate at 2 weeks (corresponding to P8) seen by HPC-1 and GAD staining. ChAT, TH and SS-28 immunostaining revealed an abnormal lamination pattern in the graft inner plexiform layer. Also by 2 weeks, the outer plexiform layers of the graft contained NF-immunoreactive horizontal cells. No NF-stained retinal ganglion cells could be observed in the graft. Five and 7 weeks after grafting, the transplants had obtained the same staining intensity with different markers as the host retina.

Transplantation of embryonic retina to the subretinal space in rabbits

Embryonic rabbit retina can be transplanted to the subretinal space of adult rabbit with a new method, which gives a high rate of successful short-term transplants. Embryonic (stage E 15) neural retina cells were injected through an incision just behind the sclerocorneal border with a thin (inner diameter 0.15-0.4 mm, outer diameter 0.3-0.5 mm) plastic tube attached to a specially designed instrument, by which the length of the protruding plastic tip could be controlled. The retina was penetrated from the vitreous side and the donor tissue was injected into the subretinal space. The cells survived in the host for at least 5 months, although the long-term survival rate tended to decrease. The transplanted cells matured and differentiated, forming an approximation of the layered, retinal structure with some anomalies (e.g. rosettes). The subretinal location offers an interesting and convenient way of studying the development of retinal cell transplants in rabbits. Large transplants can be produced, and the risk for failures due to erroneous vitreous placement is small.

Function and Structure in Retinal Transplants

Embryonic mammalian donor retina transplanted into the subretinal space of a mature host develops into a graft with wellorganized, but atypical retinal structure. We tested the effect of this organization on rabbitto-rabbit graft functional properties, isolating the graft to avoid contamination of graft responses by host retinal activity. Transient ON or ON-OFF spike-like responses and local electroretinograms (L-ERGs) were recorded simultaneously via a single electrode on the graft surface. These response components depended on stimulus diameter, sometimes in a way indicating antagonistic center-surround receptive field organization and spatial tuning (43%). Other times, the responses were an increasing function of stimulus diameter which saturated for large spots (57%). Response amplitudes were transplantation surgery is to be done with therapeutic aims.

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 Bruchs 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)

REVERSED RATIO OF COLOR-SPECIFIC CONES IN RABBIT RETINAL CELL TRANSPLANTS

Recently, we have reported on the emergence of various retinal cell types in embryonic rabbit retina transplanted to adult rabbits. When comparing the relative numbers of the spectrally different cone types in the transplants to those in the host or age-matched control retinas, a surprising shift was observed. While in the normal rabbit retina the middle-wavelength-sensitive (M) cones are considerably more abundant than the short-wave-sensitive (S) cones, the S/M cone ratio was found to be the opposite in the graft. The number of rosettes containing only S-cones in high density was found to be considerably higher than that of M-cone rich rosettes. The number of S-cones also exceeded that of the M-cones in each rosette that contained both cell types. Our results were obtained from the systematic immunocytochemical analysis of 15 different transplants derived from transplantations of embryonic rabbit retinas into adult hosts of the same species. The emergence and proportion of the two cone types were followed between 14 and 63 days after transplantation (between 29 and 78 postconceptional days of the donor tissue). Sections from various parts of the transplants were reacted with the monoclonal antibodies COS-1 and OS-2, specific for the middle- and short-wavelength-sensitive cones, respectively. The explanation for the reverse cone ratio in these transplants is not known yet, however, the observed phenomenon may indicate differences between the specification of the two basic cone types.

Electron microscopy of human first trimester and rat mid-term retinal cell transplants with long development time

The ultrastructure of rat to rat and human to rat long-term transplants of retinal cell transplants has been investigated. The human transplants were examined at 30 to 41 weeks of total age after conception. Rat homotransplants were nine to ten weeks of age after conception in four cases and 20 weeks in one case. Xenotransplanted rats were immunosuppressed with Cyclosporin A.Both xeno-and homotransplants can develop in the epiretinal or the subretinal space. The development is often heterogeneous. Photoreceptor cells can form both inner and outer segments as well as synaptic terminals. In regions corresponding to the inner plexiform layer, the adult complement of synapses has been seen, including advanced features like serial synapses as well as reciprocal synapses at bipolar cell dyads. Incompletely differentiated synapses of both amacrine and bipolar cell types have been observed, especially in rat epiretinal transplants. Ganglion cell processes have not been identified with certainty. Transplants from ...

Ultrastructure of Retinal Cells Transplanted to the Rabbit Choroid

Purpose: Allogenic rabbit-to-rabbit retinal cell transplants survive in the choroid, which is not as expected because it has not been shown that this is an immune-privileged site. We have therefore examined the ultrastructure of such transplants, looking for features that might explain the phenomenon. Methods: Rabbit retinal tissue fragment transplants were produced with previously described methods. The donor age was 15 days and the transplants were examined by standard electron microscopy when the transplants were 1–2 months (3 transplants) or 3–4 months old, of postconception age (3 transplants). Results: The transplants survived and developed as expected from previous observations. Rosettes were seen, but they were not as common as in transplants produced with the same technique in the subretinal space of rabbits. Photoreceptor outer segments were not seen in the transplants. At 1 month, there was an incomplete sheath of Müller cells around the transplants, and a complete one at 3–4 months. There was also a well-developed basement membrane around the transplant at 3–4 months, but less so at 1 month. Blood vessels did not enter the transplant. The fenestrations in the choriocapillaris were not affected as long as the pigment epithelium was normal. Conclusions: The enclosure of the transplants by Müller cells might help to insulate them from the immune system of the host, but it is a late phenomenon and it is not likely to have much effect for the first few weeks after the transplantation. We suspect that either the rabbit choroid is an immune-privileged site, even though there is no previous direct evidence for this, or that the retinal tissue itself is responsible for the prolonged survival at this site.

Functional Properties of Subretinal Transplants in Rabbit

There has been a rapid growth in research focused on retinal transplantation as a strategy for restoration of or rescue of function in retinas rendered afunctional by disease or trauma. This strategy has been partially stimulated by recent transplantation efforts in brain related to neurodegenerative diseases such as Parkinson’s, Huntington’s, and Alzheimer’s. These latter efforts have been hindered by a dearth of information regarding the specific properties of the inputs, intrinsic processing and outputs within the brain underlying these diseases. This is especially significant when attempting to study correlated functional and structural properties of the implanted tissue. In contrast to such studies, this research utilizes the retina, of which there is greater knowledge about the inputs (complex light patterns), intra-retinal processing and anatomical micro-circuitry, and the output (patterns of ganglion cell activity). Our research demonstrates, for the first time, organized, complex physiological function and its underlying neuronal microcircuitry in mammalian subretinal transplants.

Characteristics of embryonic retina transplanted to rat and rabbit retina

When aggregates of immature retina are transplanted to lesioned adult retina, the donor cells reorganize themselves into folded sheets and rosettes. With the exception of retinal ganglion cells and an inner limiting membrane, most cell types and all layers develop, corresponding to the normal retina. The transplants can integrate with the adult host retina without the presence of glial barriers. Mouse and human donor retinas have been transplanted to immunosuppressed rat hosts with long-term survival. Transplants of embryonic retina cografted with RPE can contain an apparent ganglion cell layer and an inner limiting membrane, which are not observed in transplants of retina alone. Embryonic rat donor retinas can be transplanted successfully after long-term storage in liquid nitrogen.