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. 2007 Jun 29:13:1045-57.

Isolation of retinal progenitor and stem cells from the porcine eye

Ping Gu  1 , Laura J Harwood, Xiaohong Zhang, Mildred Wylie, W James Curry, Tiziana Cogliati
Affiliations

Isolation of retinal progenitor and stem cells from the porcine eye

Ping Gu et al. Mol Vis. .

Abstract

Purpose: Retinal progenitor cells (RPCs) and retinal stem cells (RSCs) from rodents and humans have been isolated and characterized in vitro. Transplantation experiments have confirmed their potential as tools for cell replacement in retinal degenerative diseases. The pig represents an ideal pre-clinical animal model to study the impact of transplantation because of the similarity of its eye to the human eye. However, little is known about porcine RPCs and RSCs. We aimed to identify and characterize in vitro RPCs and RSCs from porcine ocular tissues.

Methods: Cells from different subregions of embryonic, postnatal and adult porcine eyes were grown in suspension sphere culture in serum-free medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Growth curves and BrdU incorporation assays were performed to establish the proliferative capacity of isolated porcine retina-derived RPCs and ciliary epithelium (CE)-derived RSCs. Self-renewal potential was investigated by subsphere formation assays. Changes in gene expression were assayed by reverse transcription polymerase chain reaction (RT-PCR) at different passages in culture. Finally, differentiation was induced by addition of serum to the cultures and expression of markers for retinal cell types was detected by immunohistochemical staining with specific antibodies.

Results: Dissociated cells from embryonic retina and CE at different postnatal ages generated primary nestin- and Pax6-immunoreactive neurosphere colonies in vitro in numbers that decreased with age. Embryonic and postnatal retina-derived RPCs and young CE-derived RSCs displayed self-renewal capacity, generating secondary neurosphere colonies. However, their self-renewal and proliferation capacity gradually decreased and they became more committed to differentiated states with subsequent passages. The expansion capacity of RPCs and RSCs was higher when they were maintained in monolayer culture. Porcine RPCs and RSCs could be induced to differentiate in vitro to express markers of retinal neurons and glia.

Conclusions: Porcine retina and CE contain RPCs and RSCs which are undifferentiated, self-renewing and multipotent and which show characteristics similar to their human counterparts. Therefore, the pig could be a useful source of cells to further investigate the cell biology of RPCs and RSCs and it could be used as a non-primate large animal model for pre-clinical studies on stem cell-based approaches to regenerative medicine in the retina.

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Figures

Figure 1
Figure 1
Identification of retinal progenitor cells and retinal stem cells in the developing and mature retina and ciliary epithelium. A-C: light microphotographs of H&E stained 15 μm retina cryosections. At PN14 (B) retinal histology was characteristic of the mature (PN150) retina (C) with each of the nuclear and plexiform layers readily identifiable. Mature outer segments of the photoreceptors (brackets) were already evident in the PN14 retina. The apparent detachment of the retina in C is a histological artifact. D-F: confocal fluorescent microphotographs of retina and G-I: of ciliary body (CB) cryosections (15 μm) immunostained with anti-nestin antibody (green). At E60 nestin immunoreactivity was observed in the ganglion cell layer (GCL; arrowheads), (developing) inner nuclear layer (dINL; thick arrows), neuroblast layer (NBL; thin arrows), and in the inner plexiform layer (IPL). At PN14 (E) nestin immunostaining was observed in the GCL (arrowheads) and IPL (asterisk). By PN150 (F) nestin immunoreactivity was observed in fibers in the GCL and IPL (asterisks), and in sparse cells in the GCL (arrowhead). At both E60 (G) and PN14 (H) nestin immunoreactivity was observed in cells distributed within the CB epithelium (thin arrows in insets at bottom). A higher percentage of cells with intense nestin immunostaining were observed in the E60 CB (G). Nestin immunoreactivity was not detected in the PN150 CB (I). Insets in G-I represent higher magnification images of the marked areas. Labeled stromal cells in G most likely represent migrating precursors of neural crest origin. The red line in I corresponds to propidium iodide (PI) staining in the adjacent lens. Nuclei were counterstained with PI (red). Scale bars: A, G-I, 50 μm; D-F, 100, B-C, 200 μm. ONL represents outer nuclear layer, OPL represents outer plexiform layer.
Figure 2
Figure 2
Primary sphere formation in serum-free medium in vitro. A: single cells from primary cultures of dissociated E60 retina-derived cells and B: of 3 week old ciliary epithelium (CE)-derived cells at day 0. Dissociated CE cultures comprise pigmented and non-pigmented cells (B). C, D: primary sphere colonies at day 7 after plating, showing pigmented spheres in CE-derived RSC cultures (D). Scale bars represent 200 μm. E, F: number of primary spheres formed from retina (E) and CE-derived (F) primary cultures at different ages. Three week old CE cultures generated more primary spheres than those from 15 and 45 week old pigs (F). Data are expressed as mean±SD from three independent experiments.
Figure 3
Figure 3
Subsphere formation and gene expression changes with passage. A, B: subcultured retinal stem cell (RSC) spheres express the undifferentiated retinal cell markers nestin (A) and Pax6 (B) by immunohistochemistry (IHC). Scale bars represents 100 μm. Nuclei were stained with propidium iodide. C, D: dissociated retinal progenitor cell (RPC) and RSC spheres generated secondary spheres when grown in suspension. Data are expressed as mean±SD from three independent experiments. E, F: RT-PCR analysis of RNA from RPC (E) and RSC (F) spheres at different passages. M indicates molecular weight marker lane. (-) indicates PCR amplification using cDNA synthesis reactions without reverse transcriptase. β-actin was used as internal control.
Figure 4
Figure 4
BrdU incorporation of retinal progenitor cell, retinal stem cell, and neural stem cell spheres at different passages. A: 84% and 92% of cells within retinal progenitor cell (RPC) and retinal stem cell (RSC) spheres, respectively were positive for BrdU at P1. The proportion of BrdU-positive cells decreased with increasing passages to less than 50% at P3 for RPCs and P9 for RSCs. The proportion of BrdU-positive cells within brain NSC-spheres remained constant at around 96% from P1 to P9. For each plot the shaded profile shows counts of cells after BrdU labeling detected by FACS, the white profile represents counts of control cells reacted with secondary antibody only. Individual values at each passage are plotted in B.
Figure 5
Figure 5
Multipotentiality of retinal stem cells and retinal progenitor cells. Retinal progenitor cells (RPCs) and retinal stem cells (RSCs) were plated on coverslips coated with poly-D-lysine and incubated in differentiation medium for 2 weeks. A: phase contrast microphotograph of serum-treated RPCs showing cells with small bodies and elongated dendritic processes (thin arrows), some apparently connected (thick arrows) as well as cells with large, polygonal shapes (arrowhead) to indicate morphological changes associated with neuronal and glial differentiation. B-I, L: RSCs maintained in differentiation medium for 2 weeks were fixed and immunostained with antibodies to: nestin (B); Pax6 (retinal progenitors, amacrine cells; C); GFAP (glial cells; D); neurofilament-M (RGCs, interneurons; E); HuC/HuD (horizontal, amacrine cells; F); recoverin (cone and rod photoreceptors; G); rhodopsin (rod photoreceptors; H); Islet-1 (bipolar, amacrine cells I); and calbindin (horizontal, amacrine, RGCs; L). J-K: RPCs maintained in differentiated medium for two weeks were fixed and immunostained with antibodies to: recoverin (J), and rhodopsin (K). Cells expressing the same marker differentiated in clusters. Thus, microphotographs of recoverin and rhodopsin immunostaining in RPCs and RSCs are not for quantitative comparison and are not representative of the counts reported in Figure 6. Nuclei were stained with propidium iodide. Scale bars: A, C, and F represents 100 μm; B, D-E, and G-K represents 50 μm.
Figure 6
Figure 6
Quantification of E60 retina-derived retinal progenitor cells and 3 week old ciliary epithelium-derived retinal stem cells displaying distinct immunoreactivity after serum-induced differentiation in vitro. Dissociated retinal progenitor cell (RPC) and retinal stem cell (RSC) spheres were incubated in differentiation medium for two weeks, fixed, and immunostained with the indicated antibodies. Quantification was performed by recording the number of immunopositive cells over the number of nuclei counterstained with PI in random fields. Two hundred-1,000 cells for each immunostaining reaction for each culture were counted. Differentiated cells manifested retinal neural phenotypes in different proportions as indicated. A relatively large percentage of cells in both cultures displayed GFAP immunoreactivity. Data represent the mean±SD of three independent experiments.
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