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发表于 2009-3-5 10:47 |只看该作者 |倒序浏览 |打印

a The Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA;
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b Yale University, Department of Bioenginering, New Haven, Connecticut, USA;
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c Children’s Hospital of Orange County and University of California Irvine, Orange, California, USA;

d Massachusetts Institute of Technology, Department of Chemical Engineering Cambridge, Massachusetts, USA4 P" A0 Z5 V/ s; y  Q: t9 Z% K
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Key Words. Retinal transplantation ? Retinal progenitor cells ? Cell survival ? Biodegradable polymer ? Retinal regeneration
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Correspondence: Michael J. Young, Ph.D., The Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, Massachusetts 02111, USA. Telephone: 617-912-7419, Fax: 617-912-0101; e-mail: mikey@vision.eri.harvard.edu
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ABSTRACT

The two major clinical subtypes of retinal degeneration (RD) are retinitis pigmentosa and age-related macular degeneration (ARMD). A hallmark of these diseases is photoreceptor cell degeneration resulting in visual loss. No effective restorative treatment exists for either subtype. Recently, the transplantation of stem and progenitor cells has shown promise as a strategy for photoreceptor replacement . Many mammalian tissues, including the retina, contain stem or progenitor cells that can be isolated, propagated, and grafted to animal models of retinal degeneration . The goal of these studies is to either replace or preserve the function of photoreceptors in the affected eye.

Previously, it has been reported that brain-derived progenitor cells can migrate and differentiate into cells expressing markers of mature neurons and glia when grafted to the retina of mice and rats with RD . Despite incorporation into the host retina and morphological similarities to various retinal cell types, in each of these studies the transplanted cells failed to express retina-specific markers. In an attempt to overcome this hurdle, retinal progenitor cells (RPCs) have been isolated from two different derivatives of the embryonic eye cup: the ciliary epithelium and the neuroretina. These RPCs have the capacity to differentiate into photoreceptors and other cells of retinal lineage. However, poorsurvival of grafted cells remains a significant barrier to functional cell replacement. Similar problems have been seen in other cell-based therapies, such as intracerebral grafts in animal models  and patients with Parkinson’s disease . Moreover, bolus injection of cells into the subretinal space does not result in a well-organized photo-receptor layer of the type needed for high visual acuity./ F; b9 ^" u9 @, d

We have applied tissue engineering techniques to this problem by using biodegradable polymers as a substrate for RPC grafts. Tissue engineering has arisen, in part, to address the shortage of tissues and organs available for transplantation . It has shown promise in the repair of bladder , cartilage , and skin  defects and is being evaluated for a range of additional clinical applications. Moreover, poly (lactic-co-glycolic acid) (PLGA)/poly (L-lactic acid) (PLLA) polymers exhibit a high degree of biocompatibility in the brain  and spinal cord . Tissue engineering does not yet allow the construction of a functional retina de novo, but polymers have the potential to address several critical, unsolved problems in retinal transplantation. We hypothesize that an organized graft, containing aligned and polarized cells, is more readily achieved by seeding the cells onto a PLLA/PLGA polymer scaffold before transplantation. The polymer construct could also increase control over graft size and placement. Importantly, donor cell survival may be improved because cell death, leakage, and migration from the injection site occur when RPCs are delivered as a single-cell suspension .
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Biodegradable polymers are attractive in tissue engineering applications for several reasons, including availability and ease of manufacture. The polymers can be easily processed into a variety of structures, and degradation can be readily controlled. These constructs have FDA approval for use in several applications . Here we develop a biodegradable polymer/retinal progenitor composite graft that provides an effective method for transplantation of progenitor cells to the subretinal space of RD mice by significantly improving survival, control of delivery, and cellular differentiation compared with injection of dissociated cells.

MATERIALS AND METHODS
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Formulation of Polymer Scaffolds
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Scaffolds were fabricated using the solid-liquid-phase separation technique leading to pores oriented normal to the plane of the scaffold as desired for mimicking the polarized cytoarchitecture of the retina. The pore structure was largely uniform, with diameters of approximately 35–50 μm. The polymer solution (PLGA in dioxane) was cast onto slides and frozen on ice at 0～C, leading to the precipitation of the polymer solute from the solid dioxane phase. The dioxane was then sublimated, leaving the PLGA with a unique pore architecture as a direct artifact of the dioxane crystallization. A wide range of pore architectures can be obtained by controlling the concentration of dioxane, the amount of undercooling, and the thermal gradient. Our goal was to seed the scaffolds with RPCs; thus, relatively large, oriented pores were desired. We used very little undercooling (11～C), coupled with the promotion of nucleation at the glass surface via contact with a copper coil at –80～C. The solid phase was observed to grow along the glass slide and then normal to the glass slide. After sublimation of the dioxane, we produced polymers with a large, oriented, and reproducible pore structure (Figs. 1A–1C).0 S) I! g9 U* s. `8 i7 _3 ~
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Figure 1. Polymer structure, differentiation, and characterization of donor cell line on polymer composite graft. Scanning electron microscope of the polymer substrate (A, B, C) before and (D, E) after seeding with RPCs. The polymer/RPC composite grafts were cut into rectangular pieces of 0.5 x 0.3 mm and viewed under fluorescein isothiocyanate illumination under (F) low and (G) high magnification. The polymer composite grafts were cut into smaller pieces for transplantation, and some of these pieces were then analyzed immunocytochemically using epifluorecent microscopy. Constitutive GFP expression (I, L, O, R, U), antibody/Cy3 immunoreactivity, Ki67 (J), nestin (M), GFAP (P), Map2 (S), and PKC- (V), and merged images (K, N, Q, T, W). Arrows indicate cells coexpressing these labels. (I–K): GFP  RPCs coex-pressed Ki67 on the composite graft. (L–N): GFP  RPCs coexpressed nestin on the polymer composite graft. Some cells coexpressed (O–Q) GFAP, (R–T) Map2, or (U–W) PKC- on the composite graft. Most of the RPCs on the composite grafts continued to express Ki67 and nestin, although some cells now expressed neuronal and astrocytic markers before transplantation. (H): Polymer composite grafts were EGF  at 1 week after transplantation to subretinal space in vivo. Polymer incubated with media containing EGF were EGF  (X-I-X-III), whereas polymers pretreated with 5% poly vinyl alcohol before EGF incubation were EGF– (Y-I-Y-III) after 1 week in vitro. Abbreviations: EGF, epidermal growth factor; GFAP, glial fibrillary acidic protein; PKC, protein kinase C; RPC, retinal progenitor cell.1 B" z+ x: s8 O. [

Characterization of Donor Cells In Vitro
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When grown on conventional substrates in media supplemented with EGF, GFP-transgenic RPCs exhibited high levels of endogenous green fluorescence (Fig. 2A) and maintained an undifferentiated state characterized by ubiquitous Ki67 and nestin immunoreactivity (Figs. 2B, 2C). Cells could be maintained in this state for up to 1 year. To examine differentiation in vitro, media without EGF was supplemented with 10% FBS. After 2 weeks of culture under differentiation conditions, the cells were analyzed immunocytochemically. The number of Ki67  cells markedly decreased (data not shown), and subpopulations expressed GFAP, Map2, PKC-, recoverin, or rhodopsin. These markers are consistent with differentiation into rod photoreceptors, bipolar cells, and Muller glia, all of which are known to be born late in retinogenesis. Interestingly, these immunopositive cells also showed morphological evidence of differentiation into rod photoreceptor and bipolar cell types (Fig. 2). No immunocytochemical or morphological evidence of early-born cell types (e.g., cones or retinal ganglion cells) was observed. These data indicate that RPCs derived from neonatal mice have the intrinsic potential to differentiate into late-born retinal cell types.

Figure 2. Differentiation and characterization of donor cells in vitro. RPCs formed GFP  neurospheres when grown in serum-free media supplemented with EGF. (A): Viewed under fluorescein isothiocyanate illumination. RPCs were plated on eight-well slides coated with laminin and were cultured in the absence of EGF and the presence of 10% fetal bovine serum for (B, C) 1 day or (D–H) 14 days. The cells were stained for (B) Ki67, (C) nestin, (D) GFAP, (E) Map2, (F) PKC-, (G) recoverin, and (H) rhodopsin. At day 1, cells expressed (B) Ki67 and (C) nestin. At day 14, some cells differentiated morphologically into specific cell types expressing (D) GFAP, (E) Map2, (F) PKC-, (G) recoverin, and (H) rhodopsin. Abbreviations: EGF, epidermal growth factor; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; KC, protein kinase C; RPC, retinal progenitor cell." p0 Y# |0 y! P

Attachment and Incorporation of RPCs onto the Polymer Substrate, H3 o- `" B( j, Y8 `
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The solid-liquid-phase separation technique using dioxane was able to generate polymers with the appropriate pore structure for the seeding of RPCs. The pore architecture can be seen in SEM images (Figs. 1A–1C) before the addition of RPCs. The scaffolds are approximately 95% porous, and the pore size is estimated to be 35–50 μm in diameter. The parameters of the seeding procedure described in this work were developed to ensure the maximum seeding density without induction of cell death. We used a two-phase procedure in which one side of the polymer was seeded first and the other 3 days later, followed by an additional 4 days in culture. We determined that this method leads to a composite graft in which RPCs were fully incorporated yet not overcrowded such that significant cell death could occur. The seeded polymers are shown in SEM images (Figs. 1D, 1E) and under GFP illumination (Figs. 1F, 1G).+ N! p$ e! Z( O! K& ^$ [; n. a

Characterization of Donor Cells on Polymer Scaffolds Before Grafting

Polymer/RPC composites were analyzed immunocytochemically to determine whether culturing RPCs on a PLGA substrate induces changes in gene expression indicative of differentiation. Polymer/RPC composites were cultured for 7 days and then cut into 0.5 x 0.3-mm fragments, as used for transplantation, and examined for the cellular markers listed above. Most RPCs expressed Ki67 and nestin (Figs. 1I–1N), although at a lower level than that seen when the same cells were grown under identical conditions but without the PLGA substrate. A subpopulation of cells expressed the more mature markers GFAP, Map2, and PKC- (Figs. 1O–1W); however, there was no evidence for expression of the retina-specific markers recoverin or rhodopsin. These data indicate that most of the RPCs cultured on a PLGA scaffold remain relatively undifferentiated before grafting, whereas a subset expresses early markers of more mature neurons or glia.+ w1 @+ T7 s' Z7 L5 M! G

Biodegradable Polymers Stained for EGF In Vivo and In Vitro8 L" @1 ]* W! `* ?
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Sections were stained with EGF antibody at 1, 2, and 4 weeks after transplantation to subretinal space of B6 mice. We found EGF  staining throughout the polymer at all time points (Fig. 1H). We also found EGF  staining of the polymer after 1 week in culture with EGF (Figs. 1X-I–X-III). No EGF  staining was detected when the polymer was treated with 5% PVA (Figs. 1Y-I–Y-III). These results suggest that the biodegradable polymer used here can adsorb the key cell survival factor EGF in both an in vitro model and in an in vivo transplantation model. Furthermore, this EGF adsorption is dependent on the hydrophobic properties of the polymer, as treatment with PVA eliminates this effect. The presence of the EGF, and perhaps other factors, on the biodegradable polymer may in part underlie the increased cell survival seen in these experiments.) z, ?2 Z8 j% U
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The Fate of RPCs Transplanted as a Polymer Composite Graft to the Subretinal Space
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Survival and integration (defined as morphological incorporation) of grafted RPCs was evaluated by fundus examination as well as immunohistochemical analysis of tissue sections (Figs. 3A–3H). Intraocular GFP  profiles were identified at 1, 2, and 4 weeks after transplantation using in vivo fluorescence microscopy (Figs. 3E–3H). Survival and integration of grafted RPCs in the retina of nonimmunosuppressed host animals were confirmed by histological analysis. In syngeneic C57/Bl6 mice and rho–/– mice, surviving RPCs were found in all recipient eyes at all three points (total, 76/76; 100%). GFP-expressing RPCs frequently migrated into the host retina, where they showed signs of morphological integration. At 1, 2, and 4 weeks after transplantation, integrated RPCs were found in 83%, 100%, and 90% of adult rho–/– mice and 85%, 87%, 87% of adult C57/Bl6 mice, respectively (Table 1).- B6 L  k2 S& l0 f7 V9 j0 g' e# G

Figure 3. Fundus examination after composite graft transplantation. Fundus pictures of the same C57/Bl6 mouse at (A) 2 weeks and (B) 4 weeks. Fundus pictures of the same rho–/– mouse at (C) 2 weeks and (D) 4 weeks. The arrowheads show the optic nerve head, and arrows show the same retinal vessel. (E–H): In each eye, surviving RPCs were confirmed to be GFP  by fundus examination via fluorescent microscopy. (I): A large number of RPCs migrated into the host retina. Hematoxylin and eosin staining after operation at (J) 1, (K) 2, and (L) 4 weeks. (J–L): These polymers in the subretinal space were gradually degraded at 1, 2, and 4 weeks, with pores increasing in size over this period (arrows). Abbreviation: RPC, retinal progenitor cell.

Table 1. Survival and integration of grafted retinal progenitor cells in the eye of nonimmunosuppressed host animals at 1, 2, and 4 weeks after transplantation+ o- N6 N/ L+ `+ x
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Polymer Degradation

The breakdown of RPC-seeded PLGA scaffolds was examined in cryosections and compared at 1, 2, and 4 weeks after transplantation to the subretinal space. Gradual breakdown of the polymer component of the composite grafts was seen over the course of this period (Figs. 3J–3L). Pores progressively increased in size and merged, such that at 4 weeks, very little of the polymer scaffold remained. Although the average overall thickness did not change substantially, degradation was observed as an increase in the pore size, such that large channels were observed in the polymers at 2 and 4 weeks after implantation (Figs. 3K, 3L).
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Migration and Differentiation of Cells Delivered as Polymer/RPC Composite Grafts

At 1, 2, and 4 weeks after transplantation, RPCs migrated into retinal laminae adjacent to the graft and showed morphological evidence of neuronal differentiation (Figs. 3I, 4). GFP  donor cells coexpressed several markers indicative of phenotypic maturation, including NF200, GFAP, PKC-, recoverin, and rhodopsin (Figs. 4, 5). Expression of these markers increased after transplantation, with rhodopsin and recoverin becoming more intense and widespread by the 4-week time point. Interestingly, rhodopsin was expressed by RPCs grafted to rho–/– recipients, a finding not seen in previous work using single-cell suspension grafts in this model .
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Figure 4. Migration and differentiation of RPCs from polymer composite grafts into C57/Bl6 mice retina. Epifluorescent (A–F) and confocal (G–O) images of the expression of neural and photorecep-tor markers by RPCs after polymer composite grafting to the eye of normal adult C57/Bl6 mice, seen at 2 and 4 weeks after grafting; (A, D, G, J, M) constitutive GFP expression, (B) antibody/Cy3 immunoreactivity for NF200, (E) GFAP, (H) PKC-, (K, N) recoverin, and (C, F, I, L, O) merged images. (A–C): NF-200–coexpressing RPCs extended neural fibers. (D–F): GFAP-coexpressing RPCs migrated from the composite graft into the host retina, and some RPCs in the polymer also expressed GFAP. (G–I): PKC-–coexpressing RPCs were found in the host inner nuclear layer. Recoverin-coexpressing RPCs were found in the (J–L) composite graft and the (M–O) host retina of C57/Bl6 mice at 2 and 4 weeks after transplantation. Abbreviations: GFAP, glial fibrillary acidic protein; PKC, protein kinase C; RPC, retinal progenitor cell.
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Figure 5. Migration and differentiation of RPCs from polymer composite grafts into the rho–/– retina. Confocal (A–F, J–L) and epifluorescent (G–I) images of the expression of neural and photoreceptor markers by RPCs after polymer composite grafting to the eye of adult rho–/– mice at 2 and 4 weeks after grafting. Constitutive GFP expression (A, D, G, J), antibody/Cy3 immunoreactivity for recoverin (B, E), rhodopsin (H, K), and merged images (C, F, I, L). Recoverin-coexpressing RPCs were found in the retina of rho–/– mice at (A–C) 2 weeks and (D–F) 4 weeks after transplantation. Rhodopsin-coexpressing RPCs were found in rho–/– mice at (G–I) 2 weeks and (J–L) 4 weeks. Abbreviation: RPC, retinal progenitor cell.

Cell Survival in Polymer/RPC Composite Grafts Versus Single-Cell Suspensions

RPCs were counted before grafting and at 1, 2, and 4 weeks after transplantation to the subretinal space as a composite graft or single-cell suspension. Before transplantation, each polymer graft contained approximately 6,000 cells (mean ± SD, 15,800 ± 672; n = 7), and this same number of cells was used for the single-cell suspension grafts. Because a significant drawback of single-cell suspensions is reflux of cells from the injection site, we also counted the number of cells present immediately after injection. An average of 9,232 cells (57.7% of those grafted) were still present in recipients analyzed at day 0 (n = 6), indicating that 43.3% of the grafted cells were either lost due to reflux or did not survive the transplantation procedure. We then assessed the number of surviving cells in the polymer composite grafts at 1, 2, and 4 weeks after transplantation and found 91% (n = 6), 96% (n = 6), and 78% (n = 6) survival at these time points, respectively (Fig. 6A). Results for single-cell suspension grafts, calculated relative to the day-0 result (9,232), showed survival of 12.7% (n = 6), 9.9% (n = 6), and 8.1% (n = 6) at 1, 2, and 4 weeks (Fig. 6A). These data show that the percent survival of RPCs delivered as a polymer composite graft is approximately 10-fold higher than cell-suspension grafts after 4 weeks (p
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Figure 6. Integration and cell survival of RPCs. Comparison of polymer composite grafts and single-cell suspensions. (A): Cell numbers were compared with composites before transplantation and in eyes after composite transplantation. At 1, 2, and 4 weeks after transplantation, 91% (n = 6), 96% (n = 6), and 78% (n = 6) of the cells survived in the eye. To examine the number of cells transplanted, cell numbers at 1, 2, and 4 weeks were compared with cell numbers at day 0. At 1, 2, and 4 weeks after single-cell suspension transplantation, 12.7% (n = 6), 9.9% (n = 6), and 8.1% (n = 6) survived in the retina. These data show that polymer composite grafts can deliver 10-fold more cells compared with single-cell suspension transplantation. *p

TUNEL  Cells Are Greatly Reduced with Polymeric Delivery Methods; D7 k( @  X8 O: Q1 e, k% z, `& t

We assessed the number of TUNEL  cells in cell culture preparations that were analogous to the transplantation methods used in this study. The preparation that used a conventional glass needle to seed (rather than inject) RPCs resulted in an extremely high rate of cell death (57.8% TUNEL ) at 3 days after seeding onto culture slides (Fig. 6B). In contrast, when we used the pipette method of seeding onto either culture slides, or polymer substrates, the rate of TUNEL  staining was decreased to 1.56% and 14.9%, respectively. These results indicate that traumatic cell death associated with cell injection is greatly reduced by using a large-bore pipette rather than a small-bore glass needle and that cell death is approximately 9.5% higher when seeding onto the biodegradable polymers compared with standard culture-treated slides.* G5 |7 u, y( w+ v
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DISCUSSION

This study was supported by grants from the Siegal Foundation (to M.J.Y., E.L., R.L.), the Minda de Gunzburg Center for Retinal Transplantation (to M.J.Y.), the NEI (09595, to M.J.Y.), the Hoag Foundation (to H.K.), and the Department of Defense (to M.J.Y.).' X+ t7 a) B- V! R: [+ b

DISCLOSURES% R/ a/ A1 A* f! k/ c$ J

The authors indicate no potential conflicts of interest.  s  s% N( u5 T/ K* t

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