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. 2010 Apr;51(4):2208-18.
doi: 10.1167/iovs.09-4447. Epub 2009 Nov 5.

Embryonic retinal cells and support to mature retinal neurons

Jennifer J Stanke  1 , Andy J Fischer
Affiliations

Embryonic retinal cells and support to mature retinal neurons

Jennifer J Stanke et al. Invest Ophthalmol Vis Sci. 2010 Apr.

Abstract

Purpose. There is a paucity of neuron replacement studies for retinal ganglion cells. Given the complex phenotype of these neurons, replacement of ganglion cells may be impossible. However, transplanted embryonic cells could provide factors that promote the survival of these neurons. The authors sought to determine whether transplanted embryonic retinal cells from various stages of development influence the survival of mature ganglion cells Methods. Acutely dissociated retinal cells, obtained from chick embryos, were transplanted into the vitreous chamber of posthatch chicken eyes after the ganglion cells were selectively damaged. Eight days after transplantation, numbers of ganglion cells were determined Results. Embryonic retinal cells from embryonic day (E)7, E10, and E11 promoted the survival of ganglion cells, whereas cells from earlier or later stages of development or from other tissue sources did not. The environment provided by the posthatch eye did not support the proliferation of the embryo-derived cells, unlike the environment provided by culture conditions. Furthermore, cells that migrated into the retina failed to express neuronal or glial markers; those that remained in the vitreous formed aggregates of neuronal and glial cells Conclusions. The environment provided within the mature retina does not support the differentiation and proliferation of retinal progenitors. Furthermore, embryo-derived cells likely produce secreted factors that promote the survival of damaged ganglion cells. Therefore, embryonic retinal cells could be applied as a cell-based survival therapy to treat neurodegenerative diseases of the retina.

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Figures

Figure 1.
Figure 1.
Transplanted cells that migrate into NMDA-damaged retinas fail to express neuronal markers. (a–m) retinal progenitors (obtained from E5 chick embryos) infected with RCAS-GFP and transplanted into the postnatal chicken eye that has been damaged by NMDA. (a–d) Representative images of transplanted cells that migrated to the retina. (e–m) Embryo-derived cells that migrated into the retina and acquired neuronal morphology but did not express markers of mature neurons, such as HuC/D (e–g), neurofilament (h–j), and Pax6 (k–m). (n–t) Representative images of E5 retinal cells infected with adenovirus serotype 5 CMV-eGFP (Ad5-eGFP) that migrated into the postnatal retinas that were injured with NMDA. Arrows: GFP+, embryo-derived cells that migrated to the host retina. (n) Small VCM labeled with antibodies to HuC/D (red) and GFP (green). Arrows: transplanted cells within the VCM that express the neural marker HuC/D. (o–q) Representative micrographs of transplanted cells that migrated into the pecten. These cells were labeled with antibodies to HuC/D and GFP. (r–t) Transplanted cell that migrated into the retina that failed to express HuC/D. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 2.
Figure 2.
Colchicine-mediated death of ganglion cells. (a) Injection and harvesting paradigm for colchicine-induced retinal damage. (b–g) One to 6 days after colchicine-treatment at P2, transverse sections of central retina were labeled with antibodies to CC3 (red). (h–s) Flat-mount retinas labeled with antibodies to Brn3a (green) and CC3 (red). Representative micrographs were taken from dorsal (h–j), central (k–m), nasal (n–p), and temporal (q–s) regions of the retina from uninjured P2 retina (h, k, n, q), retina 4 days after P2 colchicine injection (i, l, o, r), or from retinas 10 days after colchicine injection (j, m, p, s). Arrows: CC3+ cells. IPL, inner plexiform layer; GCL, ganglion cell layer.
Figure 3.
Figure 3.
Cells derived from embryonic retina, OT, and WB remain as masses of cells within the vitreous. (a) Injection and harvesting paradigm for experiments involving colchicine-induced retinal damage and transplantation of cells from GFP-transgenic embryos. (b) Representative micrograph of transplanted cells (arrow) adherent to the pecten (arrowheads). (c) Representative micrograph of aggregates of transplanted cells in the vitreous. Numbers of proliferating retinal cells significantly decreased after transplantation into the vitreous. (d–i) Representative micrographs of proliferating cells exposed to BrdU for 4 hours immediately before fixation. Cells were labeled with DAPI (blue) and with antibodies to GFP (green) and BrdU (red). (d) Cultured E7 retinal cells. (e, f) Sections through an aggregation of transplanted retinal cells adherent to the pecten. Arrows: BrdU+ cells. Yellow lines: border between the transplanted cells and the pecten. (g) Section through a VCM from transplanted E7 retinal cells. (h) Section through a VCM of transplanted E5 optic tectal cells. (i) Section through a VCM of transplanted E5 WB cells. (j) Histogram of the percentages of cells that incorporate BrdU in the vitreous after transplantation (black), in vitro (gray), and in ovo (blue). The percentage of proliferating cells is represented on the y-axis, and the developmental stage of the cells is represented on the x-axis. (j, insets) Percentage of proliferating cells from WB and OT. (k) Histogram of the percentages of neuronal markers in culture compared with transplanted cells within the VCM. Scale bars, 50 μm. **P ≤ 0.01; ***P ≤ 0.001.
Figure 4.
Figure 4.
Transplanted embryonic cells form a reaggregated, semilaminated masses of cells within the vitreous. (a–c) VCM resulting from E8 retinal transplanted cells. Transverse sections through VCM were labeled with antibodies to HuC/D (red), BrdU (turquoise), and GFP (green). (d) Micrograph of colchicine-injured retina from an eye that received a transplant, illustrating the distribution of HuC/D. (e–i) VCM resulting from an E8 retinal transplant labeled with antibodies to AP2-α (red), calretinin (turquoise), and GFP (green). (h, i) Micrographs of colchicine-injured retina illustrating the distribution of AP2-α (h) and calretinin (i). (j–l) VCM resulting from an E10 retinal transplant labeled with antibodies to visinin (red), BrdU (turquoise), and GFP (green). (m) Micrograph of colchicine-injured retina illustrating the distribution of visinin. (n, o) Micrographs of an E10 retinal VCM labeled with antibodies to 2M6 (red) and DAPI (blue). (p) Micrograph of colchicine-injured retina illustrating the normal distribution of 2M6. DNA strand breaks were detected using the TUNEL (red) reaction on VCM sections from E10 retinal transplants (q). (c, l, arrows) Neuronal marker-positive cells. Arrowheads: BrdU+, neuronal marker-negative nuclei. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars: (d; also applies to h, i, m, p) 50 μm; (q; also applies to a, e, j, n) 100 μm.
Figure 5.
Figure 5.
Transplanted embryonic retinal cells promote the survival of ganglion cells in colchicine-treated eyes. (a, b) Representative confocal micrographs of Brn3a+ ganglion cells in central regions of colchicine-treated retinas from eyes that received vehicle (a) or transplants of E10 retinal cells (b). (c) Histogram illustrating the difference (mean ± SD for treated − control) in the numbers of ganglion cells per 0.45 mm2 of retinas from eyes that received control injections and those that received transplants. The source of the transplanted cells included the embryonic retina (gray bars), OT (black bars), and WB (white bars). The developmental stage at which the donor cells were harvested and the region of the retina in which cells were counted are indicated along the x-axis. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
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