Survival and growth of transplanted RGCs in the host retina

We previously studied the short-term transplantation of primary RGCs after intravitreal injection8, but longer-term assessment of axon and dendrite growth and electrophysiological integration past 7 days post-transplantation had not been attempted. To address this, 99.5% pure green fluorescent protein positive (GFP+) RGCs9,10 were transplanted bilaterally or unilaterally from early postnatal mice into the vitreous space of adult Sprague–Dawley rats, ages 1–3 months. In some transplantations (n=15 of total 152), transplanted (donor) RGCs migrated through the nerve fibre layer into the host ganglion cell layer (Fig. 1a). There was variability in the number of GFP+ RGC donor cells visible in the host retinas, ranging from 50 to more than 2,000 GFP+ RGCs per explanted host retina. Host eyes were fixed and treated with fluorescently labelled anti-GFP antibodies and, when biocytin was injected intracellularly, with fluorescent streptavidin to see more clearly the morphology of the transplanted cells. Axon-like projections extended from the individual GFP+ RGC cell bodies into the nerve fibre layer and towards the host optic nerve head (ONH) among unlabelled, host retinal axons. Many GFP+ RGCs had elaborate dendritic trees (Fig. 1b and Supplementary Fig. 1). Most (70%) transplanted RGCs were found to be positive for Brn3a, similar to the known distribution of Brn3a in rodent RGCs (Supplementary Fig. 2). In retinal cross-sections, dendrite-like neurites extended into the host inner plexiform layer (IPL; Fig. 1c). Thus, the transplanted RGCs had acquired three-dimensional morphologies similar to host RGCs and, in laminating within the IPL, appeared to be capable of integrating morphologically with the host retina. At a shorter, 1 week time point, growth cone-like structures directed towards the ONH were observed at the ends of transplanted RGCs’ GFP-labelled axons (Fig. 1d and Supplementary Fig. 3), indicating new, directed growth from transplanted cells.

Figure 1: Morphology of transplanted GFP+ RGCs in the host retina. (a) After 3 weeks, transplanted GFP+ RGCs were observed in host retinal explants with putative axons (arrowheads) extending towards the optic nerve head, ONH. (b) Elaborate dendrite architecture of donor RGCs with morphologies often seen in known subtypes of endogenous RGCs. (c) In retinal sections from host eyes, GFP+ RGCs were seen within the host ganglion cell layer, GCL, extending neurites (arrowheads) into the inner plexiform layer, IPL. (d) One week after transplantation, some GFP+ axons in the host retinal explants were observed with growth cone-like terminal endings (arrowheads) in the nerve fibre layer. Scale bars, 25 μm. Full size image

In 70 of 152 experiments, parallel cultures of donor cells were plated concurrently with transplantations on poly-D-lysine- and laminin-coated tissue culture plates in serum-free RGC growth medium9. Of these 70 experiments, successful transplantation in vivo occurred in 6 of the 14 eyes for which parallel cultures of donor cells survived in vitro, whereas transplantation failed in all 56 eyes for which parallel cultures of donor cells did poorly (P<0.0001 by Fisher’s Exact test, two-tailed; Table 1). The overall success rate with parallel cultures, 6 of 70, was comparable to the 9 of 82 with which parallel cultures were not made. Thus, although there may be several variables contributing to successful transplants, it is important to use donor cells that survive well.

Table 1 Healthy donor cell population may contribute to successful transplantation. Full size table

Variable numbers of GFP+ RGCs were visible in the recipient retinas removed from the host eyes; for the two quantities of cells injected, there were paradoxically more cells on average when fewer RGCs were injected into the eye (Fig. 2a). The highest retention of donor cells was ∼7%, but values closer to 1% were more typical (Fig. 2a). Owing to the low number of successful transplants at each time point, it was not possible to determine whether the number of retained donor cells declined with time after transplantation, but of the cells retained after 1–4 weeks, more than 70% of GFP+ RGCs in the retina were healthy, as judged by 4,6-diamidino-2-phenylindole (DAPI)-stained nucleus morphology (Fig. 2b–e). More than 60% of GFP+ RGCs observed in the host retinas extended axon-like neurites after 1–4 weeks (Fig. 2f). Donor RGCs also extended dendrite-like processes and the number of transplanted cells with visible dendrites was significantly higher than those without visible processes; almost 80% of the retained GFP+ RGCs had dendrite-like processes, some elaborate. Although there is little precedent for it, we addressed whether fusion of transplanted cells with host cells could explain the RGC-like morphologies and functional properties acquired by GFP+ transplanted RGCs. In whole-mounted retinas that allowed clear observation of individual nuclei with DAPI staining (Fig. 2b–d; n=7 retinas), we counted nuclei associated with individual GFP+ cells to evaluate the presence of fused cells. Similar images were obtained from sectioned retinas (Fig. 1c). Using confocal microscopy of whole mounts, we found only single nuclei and no double nuclei (Fig. 2g), indicating that none of the labelled cells resulted from cell fusion.

Figure 2: Transplanted GFP+ RGCs survive and extend neurites in the host retina. (a) Retention of donor GFP+ RGCs 3–4 weeks after successful intravitreal transplantations, with significantly different mean values shown as red lines (*P<0.05 by t-test, two-sided). (b–d) Representative images from recipient, DAPI-stained retinal whole mounts showing nucleus morphologies (blue) and the corresponding green GFP-merged images of donor RGCs; examples of punctate, unhealthy nuclei (arrowheads) and large, circular, healthy nuclei (arrow) are marked. (e) Based on DAPI morphology, 70% of the retained GFP+ RGCs were judged to be healthy, and (f) over 60% extended axon-like neurites in the host retina. (g) Of nearly 500 GFP+ RGCs counted, all had single nuclei and none with double or fused nuclei were observed in the host retinas (n=7). (h,i) For GFP+ cells, the fraction of cells with neurites was constant (r=0.99), independent of the number of cells transplanted; the number of neurites reaching the optic nerve head (ONH) within the same quadrant increased linearly with cell number (correlation coefficient r=0.95), indicating that neurite growth too was not influenced by transplanted cell density. Scale bars, 20 μm. Full size image

The number of GFP+ cells with visible neurites was linearly proportional to the density of GFP+ cells in a given quadrant (Fig. 2h), as was the number of GFP+ neurites that reached the ONH (Fig. 2i), suggesting that donor cells grew neurites and axon-like projections independently of donor cell density. The regression line correlating density of retained donor cells and extension of neurites up to the ONH (Fig. 2i) could be affected by bundling of axons at higher densities, lowering the numbers of neurites counted; indeed, the correlation coefficient was greater (r=0.96) when the highest density was omitted.

Morphology and dendrite stratification of transplanted RGCs

Transplant recipient retinas were stained with anti-GFP antibodies to show donor RGCs including the architecture and stratification of their dendrites. RGCs with brightly labelled dendrites and distinct arbours were imaged using confocal microscopy, traced using Adobe Photoshop and their morphologies analysed. Similar to previous studies of labelled RGCs and RGC subtypes11,12, dendrite arbours had varied shapes and extent (Fig. 3a). Similar numbers of transplanted donor RGCs stratified in the inner or outer laminae of the IPL (over 40% each), and fewer (almost 15%) were bistratified (Fig. 3b). Monostratifying-inner, monostratifying-outer and bistratifying RGCs were separately analysed, measuring their dendritic field diameter (Fig. 3c), soma diameter (Fig. 3d), distance of peak intercepts by Sholl analysis from the cell body (Fig. 3e,f). We found that RGCs stratifying in the inner lamina tended to have large dendritic fields (>400 μm) compared with RGCs stratifying in the outer lamina, where most dendritic field diameters ranged within 200–300 μm. In contrast, the majority of RGCs stratifying in the inner lamina had smaller cell bodies (15–20 μm) compared with most of the RGCs stratifying in the outer lamina (20–25 μm); bistratified RGCs showed a broader distribution across these sizes (Fig. 3d). As one measure of complexity, we counted the number of intercepts made by dendrite branches at different distances from the cell body using Sholl analysis. Most of the cells had peak intercepts between 100 and 150 μm from the cell body in both inner and outer lamina-stratifying RGCs. These data highlight the large variability in dendrite architecture of transplanted RGCs, suggesting that either various morphological subtypes survive the transplantation process, or that transplanted cells take on a variety of subtype morphologies when integrating into the local host environment.

Figure 3: Transplanted RGCs exhibit varied morphologies and dendrite architecture. (a) Representative tracings of GFP+ transplanted RGCs demonstrating the types of morphologies seen 3–4 weeks post transplantation. (b) Schematic representation of the distribution of mono-stratifying and bi-stratifying transplanted RGCs observed in the host retinas. (c–f) Comparison of distribution of different morphological properties in transplanted RGCs: dendritic field diameter (c), soma diameter (d), peak intercepts in each cell against distance from cell bodies by Sholl analysis (e,f). Full size image

After transplantation, donor RGCs extended individual axons radially along the nerve fibre layer towards the ONH (Fig. 1a). To test whether these axons traversed the optic nerve and extended towards the various brain targets of RGCs, we sectioned and stained the optic nerves (n=3) and brains (n=1) from a small subset of transplanted animals. Preliminary results show that at 4 weeks after unilateral transplantation, numerous GFP+ axons were visible in the optic nerve sections, some extending into the optic chiasm (Supplementary Fig. 4a). We did not observe any GFP+ axons in optic nerves from uninjected eyes. Using volumetric analysis (n=6), we estimated that on average, ∼140 axons grew into the host optic nerves at 1 month post transplantation. In all the sections examined (n=6, 2 sections for each of the 3 optic nerves), we did not notice obvious branching or growth cones indicating termination within the optic nerve. Axons were also seen crossing to the contralateral optic tract at the optic chiasm, growing up the optic tract and terminating prominently in the dorsal and ventral lateral geniculate nucleus and the superior colliculus (SC; Supplementary Fig. 4b,d). Comparison with AAV-GFP-labelled RGC axon tracts and terminals in the brain (Supplementary Fig. 4c,e) showed that axon terminals from transplanted donor cells exhibit exuberant growth and extend into deeper layers of the SC, behaviour that is not characteristic of the mature visual system. Donor RGC axon terminals were observed in the anterior portion of the host SC closer to the midline, the expected area of projection based on the ventral temporal localization of the transplanted RGCs in the corresponding host retina13. In all the brain sections examined, no GFP+ axons were visible in brain regions outside the visual pathways. These preliminary data indicate that some of the donor RGCs that survive and extend axons are capable of reaching usual RGC targets in the brain, but may overshoot their local addresses, at least at 4 weeks.

Synapse formation and light responses in transplanted RGCs

Sections of recipient retinas with transplanted cells were stained for GFP and the synaptic markers synaptophysin and PSD95. Synaptic puncta within the sections were identified as regions of co-localization between these pre- and post-synaptic markers using Volocity software (Perkin-Elmer). Synaptic puncta were identified associated with GFP+ dendrites from transplanted RGCs visible within the 30-μm sections that were analysed (Fig. 4a,b and Table 2), suggesting that the transplanted RGCs made morphological synapses with the host retina following intravitreal injection. To test for functional synapses upon the transplanted cells, electrical recordings were made from them at various times after transplantation. For recording, host retinas were acutely explanted and biocytin-filled patch pipettes applied to donor cells, identifiable by their GFP fluorescence (Fig. 5a,b). The recorded patched cells, filled with biocytin, were fixed and stained using labelled avidin to show their extent and labelled anti-GFP+ antibody to confirm their identity as donor RGCs (Fig. 5c). Observed at 1 week, donor cells were electrically excitable, firing action potentials in response to depolarizing current stimulation (Fig. 5d). However, we found that the minimum current needed to elicit action potentials was ∼80 pA for transplanted RGCs compared with ∼20 pA for host RGCs (n=5 each; P<0.05 by Student’s t-test, two-sided). Spontaneous action potential firing and synaptic events were observed in these cells, indicating communication with other cells after transplantation.

Figure 4: Transplanted RGCs form multiple synapses within the host retina. (a) Section from recipient host retina 4 weeks post transplantation showing GFP+ transplanted RGCs in blue and synaptic puncta in white. (b) Magnified view of a portion of a GFP+ dendrite with visible synaptic puncta (white, arrows). Scale bar, 25 μm. Full size image

Table 2 The total number of synaptic puncta associated with GFP+ RGCs in three separate sections. Full size table

Figure 5: Transplanted GFP+ RGCs respond to light stimulation of the host retina. (a–c) Retina during whole-cell patch clamping of a transplanted cell within the host retina, viewed under infrared illumination with differential interference contrast optics (IR-DIC) (a), and after fixation with the recorded cell labelled with GFP in green (b) and with biocytin in red (c) Scale bar, 25 μm. (d) Transplanted GFP+ RGCs exhibit electrical excitability when stimulated with injected current (60 pA for 200 ms). (e,g,i) Light-evoked responses recorded from different GFP+ RGCs in host retinas after 2–4 weeks include an ON response (e), an OFF response (g), and an ON–OFF response (i). (f,h) Example traces of light-evoked responses from endogenous, host ON RGC (f) and OFF RGC (h) exhibiting robust firing with similar stimulation. Light flashes are 1 s; horizontal marks before the traces positioned at −50 and −60 mV. In all traces, black arrows indicate depolarization/action potential firing and white arrows indicate hyperpolarization, black bars under each electrical trace correspond to duration of light stimulation. Full size image

Brief flashes of light were used to test whether the transplanted RGCs responded to stimulation of the host retinal explant and had become functionally connect to it. Acutely explanted host retinas were dark-adapted and viewed under infrared illumination during whole-cell patch clamping of GFP+ donor RGCs. ON-, OFF- and ON–OFF-type responses were observed in different donor RGCs (Fig. 5e–g, respectively; n=5 GFP+ RGCs, from three separate host retinas), similar to the known diversity of physiological responses for RGCs14,15,16. ON-like responses were recorded from two donor cells in separate retinas at 1 and 2 weeks post transplantation, and in each case the response adapted to the first stimulus, demonstrating high susceptibility to adaptation of the ON response (Fig. 5e). This was in sharp contrast to robust light responses observed in host RGCs under similar stimulation conditions, with little adaptation between stimuli (Fig. 5e). Another of the recorded GFP+ RGCs exhibited OFF-type response, hyperpolarizing at light onset and firing multiple action potentials at light offset, with some progressive weakening with repetitive stimulation (Fig. 5f). This was different from the action potentials in host OFF RGCs at light offset, which were maintained, without appreciable adaptation to repeated stimuli (Fig. 5f). ON–OFF-type light responses were elicited from two other GFP+ RGCs 2 weeks after transplantation, again showing adaptation for the ON response, yet responding to each light offset (Fig. 5g). Neither the short response latencies nor the ON or OFF responses matched expected properties for intrinsically photosensitive RGCs17,18,19,20. In addition, OFF-type responses elicited from some transplanted cells make it less likely that the recorded responses came from melanopsin-containing RGCs. These results indicate that transplanted primary RGCs retain electrical excitability, make synapses and connect functionally with the host retina. However, these synapses may be weaker than the established synapses of host RGCs, as indicated by their adaptation and less robust firing.