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TISSUE-SPECIFIC STEM CELLS

Cells Isolated from Umbilical Cord Tissue Rescue Photoreceptors and Visual Functions in a Rodent Model of Retinal Disease

^aMoran Eye Center, University of Utah Health Science Center, Salt Lake City, Utah, USA;
^bCasey Eye Institute, Oregon Health & Sciences University, Portland, Oregon, USA
^cOphthalmology and Physiology, University of Alberta, Edmonton, Alberta, Canada;
^dCentocor, Johnson & Johnson Internal Ventures, Radnor, Pennsylvania, USA;
^eEthicon Inc., Center for Biomaterials and Advanced Technologies, Somerville, New Jersey, USA

Key Words. Retinal disease • Cell-based therapy • Royal College of Surgeons rat • Umbilical cord • Visual function • Photoreceptor

Correspondence: Sanjay Mistry, Ph.D., Centocor, Stem Cell Internal Venture, Radnor, Pennsylvania 19087, USA. Telephone: 610-651-6146; Fax: 610-651-6440; e-mail: smistry5{at}CNTUS.JNJ.com

Received on May 23, 2006; accepted for publication on October 13, 2006.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures Acknowledgments References

 
Progressive photoreceptor degeneration resulting from genetic and other factors is a leading and largely untreatable cause of blindness worldwide. The object of this study was to find a cell type that is effective in slowing the progress of such degeneration in an animal model of human retinal disease, is safe, and could be generated in sufficient numbers for clinical application. We have compared efficacy of four human-derived cell types in preserving photoreceptor integrity and visual functions after injection into the subretinal space of the Royal College of Surgeons rat early in the progress of degeneration. Umbilical tissue-derived cells, placenta-derived cells, and mesenchymal stem cells were studied; dermal fibroblasts served as cell controls. At various ages up to 100 days, electroretinogram responses, spatial acuity, and luminance threshold were measured. Both umbilical-derived and mesenchymal cells significantly reduced the degree of functional deterioration in each test. The effect of placental cells was not much better than controls. Umbilical tissue-derived cells gave large areas of photoreceptor rescue; mesenchymal stem cells gave only localized rescue. Fibroblasts gave sham levels of rescue. Donor cells were confined to the subretinal space. There was no evidence of cell differentiation into neurons, of tumor formation or other untoward pathology. Since the umbilical tissue-derived cells demonstrated the best photoreceptor rescue and, unlike mesenchymal stem cells, were capable of sustained population doublings without karyotypic changes, it is proposed that they may provide utility as a cell source for the treatment of retinal degenerative diseases such as retinitis pigmentosa.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures Acknowledgments References

 
Photoreceptor degeneration due either to an intrinsic genetic defect involving the photoreceptors themselves or to problems affecting the cells with which the photoreceptors interact presents as a major group of neurodegenerative diseases and is a leading cause of blindness in the developed world. It comprises age-related macular degeneration and retinitis pigmentosa and a number of associated diseases, which collectively account for visual impairment in more than nine million people in the U.S. alone [1]. There are few treatment options for these conditions. However, studies on animal models with similar patterns of photoreceptor loss have identified the therapeutic potential of several novel approaches directed at limiting the course of photoreceptor loss including growth factor injections [2], gene therapy [3], and cell-based therapies [4]. For cell-based therapies to be viable in translation to the clinic, there are several critical requirements to consider. They must be effective in preserving visual function; the cells must be easily attainable from an ethical cell source, be available in significant numbers, and ideally be in a form that requires little preparation or multiple surgeries. Finally, such cells should be well characterized, free of identifiable pathogens, demonstrate a normal karyotype through many passages, and ideally be nonimmunogenic, so avoiding potential safety concerns. Much of the previous work has used the Royal College of Surgeons (RCS) rat, an animal in which most photoreceptors degenerate between 21 and 100 days of age, due to a defect in the adjacent retinal pigment epithelium [5, 6]. Photoreceptors can be rescued by injection of freshly harvested retinal pigment epithelial (RPE) and iris pigment epithelial cells [7–9]. We have previously shown that RPE cell lines and Schwann cells can also rescue photoreceptors from degeneration and sustain a range of visual functions [10–15]. For translation to clinic, the ideal would be to provide an allogeneic cell source that could fulfill all of the aforementioned criteria. Here we have explored the potential of an ethical cell source derived from human umbilical cord tissue (hUTC). Recent work has reported on the molecular phenotype of cells derived from umbilical tissue and the ability of these cells to transform into neurons [16]. This study demonstrated that cells could reverse the effects of Parkinson-like lesions when injected into the striatum of rats. We have determined that hUTC can be readily expanded to yield at least 1 x 10¹⁷ cells from a single donor without karyotypic or phenotypic changes. The important issue was to see whether hUTC could be effective in containing or reversing the neurodegenerative events associated with photoreceptor loss. The efficacy of hUTC was tested after injection to the subretinal space of RCS rats, using functional measures and morphological criteria. The efficacy of hUTC was compared with two other expandable tissue-derived cell types, taken from placenta and bone marrow mesenchyme: human placental-derived cells (hPTC) were chosen because they represent a comparable cell source, whereas human bone marrow mesenchymal stem cells (hMSC) were used because they have been shown to be biologically active in the retina [17]. Injection of human adult dermal fibroblasts (hADF) and medium alone provided control material.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures Acknowledgments References

 
Animals
Experiments were performed on 80 pigmented dystrophic RCS rats (rdy–/p+), which were housed with a 12-hour light/dark cycle. Nondystrophic congenic rats (rdy+/p+) were available for comparison purposes. All procedures were approved and monitored by the University of Utah Institutional Animal Care and Use Committee and have been conducted in accordance with the Policies on the Use of Animals and Humans in Neuroscience Research, revised and approved by the Society for Neuroscience in January 1995.

Donor Cell Preparation
Four human-derived donor cell types have been evaluated—hUTC, hPTC, hADF, and hMSC. Both the hUTC and hPTC were sourced in-house, and hADF and hMSC were obtained from Cambrex (Walkersville, MD, www.cambrex.com).

Isolation and Culture of Human Umbilical- and Placenta-Derived Cells.   Human umbilical cords and placentas were obtained with donor consent following live births from the National Disease Research Interchange (Philadelphia, PA). Tissues were minced and enzymatically digested. After almost complete digestion with a Dulbecco's modified Eagle's medium (DMEM)-low glucose (Lg) (Invitrogen, Carlsbad, CA, www.invitrogen.com) medium containing a mixture of 50 U/ml collagenase (Sigma, St. Louis, www.sigmaaldrich.com), the cell suspension was filtered through a 70-µm filter, and the supernatant was centrifuged at 350g. Isolated cells were washed in DMEM-Lg several times and plated at a density of 5,000 cells per cm² in T75 flasks (Corning, Corning, NY, www.corning.com) in DMEM-high glucose (Hg), 15% (vol/vol) defined fetal bovine serum (FBS; HyClone, Logan, UT, www.hyclone.com), 0.1% (vol/vol) nonessential amino acids, β-mercaptoethanol (Sigma) + 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). After cryopreservation, cells were thawed and seeded on gelatin (Sigma)-coated flasks in growth medium (consisting of DMEM-Lg containing 15% (vol/vol) FBS, 0.001% (vol/vol) β-mercaptoethanol, and 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were cultured under standard conditions in atmospheric oxygen with 5% carbon dioxide at 37°C. When cells reached approximately 70% confluence after initial seeding they were passaged using trypsin/EDTA (Gibco, Grand Island, NY, www.invitrogen.com) every 3–4 days and reseeded at a density of 5,000 cells per cm² onto gelatin-coated flasks. The process of gelatin coating entailed adding a solution of 2% (wt/vol) gelatin (porcine Type B: 225 Bloom; Sigma) into tissue culture flasks for a minimum of 20 minutes at room temperature. Gelatin was then aspirated, and the flasks were washed at least two times with phosphate-buffered saline (PBS) prior to cell culture use. Cells were harvested after 10 passages (approximately 20 population doublings) and cryopreserved in supplemented growth medium containing 10% (vol/vol) DMSO (Sigma) in a programmable rate freezer (Thermo Forma, Marietta, OH, www.thermoforma.com).

Culture of Mesenchymal Stem Cell and Adult Dermal Fibroblasts.   hMSC were cultured per the manufacturer's recommendations and cryopreserved as above after five passages of growth. hADF were cultured in DMEM-high glucose with 10% FBS (vol/vol) and were cryopreserved as above after 10 passages.

Karyology, Viral Pathogens, and Mycoplasma Testing.   Cytogenetic analysis of metaphase cells with G banding performed by the Center for Human & Molecular Genetics at the New Jersey Medical School, Newark, NJ. Viral pathogen testing for hUTC, hPTC, hADF, and hMSC was performed by reverse transcription-polymerase chain reaction for HIV 1/2, HTLV I/II, HBV, HCV, EBV, and CMV by AppTec (St. Paul, MN). Mycoplasma testing was performed on actively expanding cell cultures by Bionique (Saranac Lake, NY, www.bionique.com).

Flow Cytometry Analysis
Briefly, adherent hUTC cells in flasks were washed in phosphate buffered saline (PBS, Gibco) and detached with Trypsin/EDTA. Cells were harvested, centrifuged, and resuspended in 3% (vol/vol) FBS in PBS at a cell concentration of 1 x 10⁷ per milliliter (all antibodies in Fig. 2 purchased from BD Pharmingen, San Diego, www.bdbiosciences.com/index_us.shtml except for IgG controls, which were obtained from Sigma). Five µl of antibody was added to 100 µl of cell suspension per manufacturer's specifications (1:20 final concentration), and the cells were incubated in the dark for 30 minutes at 4°C. After incubation, cells were washed with PBS and centrifuged to remove unbound antibody. Cells were resuspended in 500 µl of PBS and analyzed by flow cytometry using FACSCalibur (Becton, Dickinson and Company, Franklin Lakes, NJ, www.bd.com).

Immunohistochemistry
Human UTC and hPTC were harvested and plated onto gelatin-precoated tissue culture plastic. Upon reaching 70%–90% confluence, adherent cultures of hUTC and hPTC were fixed in cold 4% paraformaldehyde for 15 minutes at room temperature. Briefly, cultures were washed with PBS and exposed to a protein-blocking solution containing PBS, 4% (vol/vol) goat serum (Invitrogen), and 0.3% (vol/vol) Triton X-100 (Sigma) for 30 minutes to access intracellular antigens. Primary antibodies, diluted in blocking solution, were then applied to the cultures for a period of 1 hour at room temperature (anti-vimentin, 1:200, and anti- smooth muscle actin, 1:400, Sigma). Next, primary antibody solutions were removed, and cultures were washed with PBS prior to application of secondary antibody solutions (1 hour at room temperature) containing block along with the appropriate isotype specific antibody; in these cases, goat anti-mouse IgG1-Alexa 488 (1:250 Molecular Probes, Eugene, OR, http://probes.invitrogen.com) for vimentin or goat anti-mouse IgG-Texas Red (1:250, Molecular Probes) for smooth muscle actin. Cultures were then washed with PBS and 10 µM 4',6-diamidino-2-phenylindole (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using the appropriate fluorescence filter on a Nikon inverted epifluorescent microscope (Nikon, Lake Placid, NY, www.nikonusa.com). In all cases, positive staining represented fluorescence signal above control staining where the entire procedure outlined above was followed except the application of a primary antibody solution (no 1° control).

Assessment of Trophic Factor Secretion Using Enzyme-Linked Immunosorbent Assay
Cryopreserved hUTC, hPTC, and hADF at passage 10 were thawed, washed, counted, and seeded at 375,000 viable cells per 75-cm² flask containing 15 ml of growth medium and then were cultured for 24 hours. The medium was changed to a serum-free DMEM-Lg containing 0.1% (wt/vol) bovine serum albumin (Sigma), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen) for 8 hours. Conditioned serum-free medium was collected at the end of incubation and stored at –20°C after centrifugation at 14,000g for 5 minutes. To estimate the number of cells in each flask, cells were detached using trypsin/EDTA (Gibco) and counted using a hemocytometer. Brain-derived neurotrophic factor (BDNF) and fibroblast growth factor (FGF)2 releases were measured using Search Light Proteome Arrays (Pierce Biotechnology Inc, Rockford, IL, www.piercenet.com). IL-6 was measured using an enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN). Vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF1), and transforming growth factor (TGF)2 were also measured. All assays followed the instructions provided by the manufacturer.

Cell Injections
All cells except hMSC were expanded in culture for 10 passages prior to freezing. Cells were thawed and expanded for one further passage prior to injection. The MSC were cultured for five passages and then frozen. They were also expanded for one further passage prior to cell injection. At age P21–P23, dystrophic RCS rats under xylazine-ketamine anesthesia received subretinal injections of a suspension of cells (2 x 10⁴ cells per eye) via a trans-scleral injection into the upper temporal retina area of one eye, as previously described [10]. Twenty-three rats received hUTC grafts, 15 received hMSC, 8 received hPTC, and 10 received hADF. A separate group of animals (n = 10) received hUTC injections using cells that had been prelabeled with bromodeoxyuridine (BrdU) specifically for identifying donor cells. Finally an additional group of rats received injections of hUTC (n = 8) or medium alone (n = 6) into the vitreous cavity to examine whether cell location in the eye was crucial to their efficacy. All animals received daily dexamethasone injections (1.6 mg/kg, intraperitoneal, starting on the day of surgery) for 2 weeks and were maintained on cyclosporine-A (Bedford Labs, Bedford, MA, www.bedfordlabs.com) administered in the drinking water (210 mg/l; resulting blood concentration: 250–300 µg/l [11]) from 1 to 2 days prior to cell injection until animals were euthanized. Within each group of animals, 3–4 eyes received injections of medium alone. The unoperated eye provided further baseline data. Although the majority of rats survived until at least 100 days of age (P100), the BrdU prelabeled group was taken for histology from P30 onwards.

Electroretinogram Responses
The dark-adapted electroretinogram (ERG) response was recorded as previously described [18]. A double flash protocol was used to isolate cone responses. A conditioning flash was followed 1 second later by a probe flash. The role of the conditioning flash in this paradigm is to saturate rods transiently so that they are rendered unresponsive to the probe flash [19–21]. The intensity of the conditioning flash for complete rod bleaching was set to 1.4 log candela seconds (cds)/m² for all tests. A mixed b-wave was obtained by presenting the probe flash alone, that is, without being preceded by a conditioning flash. The response to the probe flash (1.4 log cds/m²), preceded by the conditioning flash, was taken as reflecting cone-driven activity, and allowed derivation of the rod contribution. Averages of 3–5 traces (set at 2 minutes apart to assure recovery of rod responsiveness) were sufficient to obtain clear responses. Special care was taken to maintain the electrode placement in a consistent position in all animals.

Optomotor Recordings for Acuity Thresholds
In the optomotry apparatus used in this study, an image of a rotating cylinder covered with a vertical sine wave grating was presented in virtual three-dimensional (3D) space on four computer monitors arranged in a square [22, 23]. Rats standing unrestrained on a platform in the center of the square tracked the grating with reflexive head movements [24]. The spatial frequency of the grating was clamped at the viewing position by repeatedly recentering the "cylinder" on the head of the test subject. Acuity was quantified by increasing the spatial frequency of the grating until an optomotor response could no longer be elicited.

Luminance Threshold Recording
To assess visual sensitivity, we recorded single and multiunit activity in the superficial layers of the superior colliculus (SC) using a modification of a procedure we developed in previous work [25]. For each of the 15–20 positions recorded over the surface of the SC, a discrete receptive field for that position was localized, and the brightness of a flashing spot, 3°-diameter projected on a hemisphere, was varied with neutral density filters until a response was obtained that was double the background activity. This was defined as the luminance threshold level.

Histology
Rats were given a lethal dose of sodium pentobarbital (Sigma) and perfused with PBS. The eyes were removed and immersed in 2% (wt/vol) paraformaldehyde for 1 hour, infiltrated with sucrose, and embedded in OCT (Optimal Cutting Temperature). Coronal sections (10 µm) were cut on a cryostat. Five series were collected. One series was stained with cresyl violet (Sigma) for assessing injection site and retinal lamination and for recognizing cellular patterns that might suggest abnormalities, such as second order changes or tumors. The rest were stained with antibodies: human-specific nuclear marker—MAB1281 (1:300, Chemicon)—for donor cells; anti-bromodeoxyuridine (1:1,000, Sigma) for BrdU-prelabeled donor cells; recoverin (1:3,000, Dr. McGinnis, University of Oklahoma) for photoreceptors and on-cone bipolar cells; cone arrestin (1:500, Drs. Zhu and Craft, University of California) for cone photoreceptors; PKC (1:1,000, Sigma) for rod bipolar cells; mGluR6 (1:2000, Neuromics) for photoreceptor postsynaptic sites associated with on-bipolar cell dendrites. The protocols for processing human-specific nuclear marker and BrdU (Sigma) were conducted according to the manufacturers' data sheets: other antibodies were processed as previously described [15]. The cells were visualized by using Vector Nova RED (Vector Labs, Burlingame, CA, www.vectorlabs.com). All sections were lightly counterstained with cresyl violet before being mounted with Vector mount (Vector). Photographs were taken using the Image-pro-Plus program; montage pictures were achieved using Photoshop. For confocal images, the pinholes were 75 µm, and the width of optical sections was 1 µm. Final images were obtained from the projections of 6–8 single frames. The TIFF images were produced in Adobe Photoshop.

Statistical Analysis
Average data are presented as mean ± SE or mean ± SD. We used t-test or Mann-Whitney for comparisons unless otherwise indicated (Statview). Significance is designated as p < .05.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures Acknowledgments References

 
In Vitro Characterization of Umbilical-, Placental-, and Skin-Derived Fibroblast Cells
All cells used were tested prior to utilization and were shown to have normal karyotypes and no detectable mycoplasma or known viral pathogens. Human umbilical tissue-derived cells (hUTC) and placental tissue-derived cells (hPTC) were isolated, examined by phase contrast imaging, and serially passaged to examine their expansion potential. Like hPTC, isolated hUTC grew as adherent cells with a fibroblastic morphology that was evident as early as passage one and maintained throughout culture until senescence (Fig. 1A, 1B). Both hUTC and hPTC were determined to expand readily for up to 40 passages. By contrast, hMSC senesced after 8 passages, and hADF senesced after 15 passages (Fig. 1C). The hMSC results were very similar to those reported in a previous study [26]. Immunohistochemical staining results indicated that both hUTC and hPTC populations were positive for CD90 (a confirmation of flow results), vimentin, and smooth muscle actin (data not shown).

View larger version (63K): [in this window] [in a new window]   Figure 1. Morphology and long-term growth profile of hUTC, hPTC, hADF, and hMSC. (A, B): Phase contrast images (A, x40; B, x200) of hUTC at 20 population doublings (PD) just prior to transplantation. As a population, hUTC cell morphology appeared to be homogeneous at all passages. Likewise, hPTC shared a similar fibroblastic morphology (data not shown). (C): Long-term growth profile of hUTC, hPTC, hADF, and hMSC. Although hMSC undergo senescence at early passage 8 PD (total cells generated = 5 x 107 cells), hADF continue to grow until 26 PD (total cells generated 3 x 1013 cells), exhibiting the classic property of Hayflick's limit. hUTC and hPTC, however, continue to expand beyond 40 PD, reaching in excess of 1 x 1017 cells. Abbreviations: hADF, human adult dermal fibroblasts; hMSC, human mesenchymal stem cells; hPTC, human placental-derived cells; hUTC, human umbilical tissue cells.

View larger version (41K): [in this window] [in a new window]   Figure 2. Human umbilical-derived cells (hUTC) express CD10 (A), CD13 (B), CD44 (E), CD73 (G), CD90 (H), and HLA-A, -B, and -C (J), but lack expression of CD31 (C), CD34 (D), CD45RA (F), CD117 (I), and HLA-DR, -DP, -DQ (K) by flow cytometry. Isotype-matched IgG controls are shown with non-shaded curves, and hUTC cell curves are shown shaded. A minimum of 10,000 events was recorded in all cases. Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin.

hUTC.   In nondystrophic rats, recorded under similar conditions, an a-wave of around 628 µV was achieved and a b-wave of 1,371 µV [14]. Previous work has documented the deterioration of ERG responsiveness in the pigmented RCS line used in this study. The time points for ERG recordings in the present study were chosen because at P60, the a-wave ERG response is normally lost in RCS rats, whereas by P100, the b-wave response has been lost [27, 28], allowing graft-related effects to be recognized over background performance. By P60, hUTC achieved a low-level rescue of ERG a-wave responses, but this failed to reach significance over sham (medium only) levels. However, mixed b-wave responsiveness was significantly better than in sham-injected and untreated rats (123 ± 46 µV for hUTC vs. 25 ± 20 µV for sham, p < .01). hUTC also showed significant rescue of the rod and cone b-waves over shams. The b-wave amplitudes dropped between P60 and P90 from 9% to 6% of nondystrophic values. Composite data for P90 are shown in Figure 3A, whereas an individual record is shown in Figure 4A. An a-wave could still be elicited, whereas the b-wave amplitude had reduced to 79 ± 42 µV, but still significantly higher than either sham or untreated eyes (p < .01). As evident in Figure 3A, both rod and cone b-wave amplitudes were each significantly better than in sham-injected eyes.

View larger version (29K): [in this window] [in a new window]   Figure 3. Functional assessments, comparing hUTC and hMSC with sham-injected rats. (A): ERG recordings obtained under scotopic conditions at P90, showing amplitudes of a-wave, mixed, and rod-and-cone-attributable b-waves: maximal amplitudes. Significance of cell injected against shams is indicated by double asterisk, p < .01, or single asterisk, p < .05. There was no difference between hUTC and hMSC (p > .05); Mann-Whitney nonparametric test. Values presented as mean + SEM. (B): Optomotor acuity threshold levels show that hUTC and hMSC are significantly better than sham-injected rats (p < .01), but hUTC and hMSC were not significantly different from one another, nor were sham and untreated. Mann-Whitney non-parametric test, values presented as mean + SEM. (C, D): Luminance thresholds recorded from the superior colliculus at approximately P100. The data presented here showed percentage of area that has been rescued at different threshold levels. Each curve (mean ± SD) shows the percent of retinal area (y-axis) where the visual threshold is less than corresponding value on x-axis (log units). Asterisks indicate the points at which the curves for grafted and sham-operated eyes are statistically different (t-test, p < .05). Abbreviations: ERG, electroretinogram; hMSC, human mesenchymal stem cells; hUTC, human umbilical tissue cells.

View larger version (22K): [in this window] [in a new window]   Figure 4. Individual records from a single animal that had received hUTC cell injection at P21. (A): Individual electroretinogram traces for full response (left), and after double flash, cone response (middle record), and rod-derived response (left) recorded at P60 and P90. (B): Visual threshold measurements were made with microelectrode penetrations in multiple sites over superior colliculus (SC) in grafted rat at P100. Thresholds recorded at each site in SC (black circles) contralateral (animal's left SC) and ipsilateral (right SC) to the grafted eye are shown. It shows that visual thresholds recorded with stimulation of the grafted eye were significantly lower than those with untreated eye, where visual responses in many points could not be elicited at all. (C): A horizontal section from the dorsal retina of the animal whose performance is detailed in (A) and (B). A substantial length of photoreceptor rescue (small dark cells seen in the outer nuclear layer) can be seen. Scale bar = 200 µm.

Luminance threshold responses were recorded at various points across the SC in a subset of the rats tested as above. This measure provided indication of relative sensitivity of the retina across the visual field and therefore efficacy of the cell treatment in local rescue of visual function. In foundation studies [25], threshold levels were less than 0.6 log units over a background of 0.02 cd/m² in normal rats and around 3.0 log units or higher in dystrophics at P100 (Fig. 4B, non-grafted side). In cell-injected RCS rats, although the normal retinal topographic order was retained, response sensitivity was not uniform across the visual map, being best in the retinal region that received the cell injection (Fig. 4B, grafted side of input from the eye giving the ERG response in Fig. 4A). Although such recordings showed response sensitivity at individual points across the visual field, data could be reduced for comparison purposes between groups to show the area of the SC giving threshold responses at various luminance levels. Such reduced data are shown in Figure 3C. They show that 18% of the area of the SC gave thresholds <1.2 log units, and 45% gave <1.7 log units against shams in which 0% gave <1.2, and 3% gave <1.7 log units. Differences between cell-injected and shams were significant at many points, indicated by asterisks, in Figure 3C (p < .05, t-test).

At the end of functional testing, retinas were prepared for morphological examination. (Fig. 5G–5J). These retinas were supplemented by additional ones from animals in which the cells were labeled with bromodeoxyuridine prior to transplantation and harvested at shorter survival times.

View larger version (71K): [in this window] [in a new window]   Figure 5. Anatomical examination of retinal sections taken from dystrophic Royal College of Surgeons rats under a range of conditions. Sections in A–F show the relative rescue of the photoreceptors in the outer nuclear layer at P100; sections; G–J show donor cell labeling at P35. All cell injections were at P21. (A): Section of retina, rescued by a subretinal injection of hUTC. (B): Section of retina after injection of hPTC. (C): Section of retina after hADF injection, showing minimal rescue around injection site (arrow) (D): Untreated retina for comparison. (E): Section of retina after hMSC injection showing small area of rescue (indicated by arrows). (F): Section showing only local photoreceptor rescue following vehicle injection (indicated by arrows). (G): hUTC immunostaining by human nuclear marker. (H): hUTC prelabeled with bromodeoxyuridine. (I): High power view of cells labeled in (G). (J): High power view of cells labeled in (H). (K): Retinal section with intravitreal injection at P100 showing rescued photoreceptors. Scale bars equal 50 µm for (A–H) and (K) and 20 µm for (I) and (J).

In unoperated dystrophic eyes, the photoreceptor layer became reduced from a layer, 10 cells deep at P21 to a single discontinuous layer at P100+ (Fig. 5D). Sham-injected retinas showed local rescue around the injection site covering at the most 3%–5% of the retinal length in a single section (Fig. 5F). The cell-injected retinas showed extensive photoreceptor rescue involving approximately 30% of the retinal length through the dorsal half of the retina: the outer nuclear layer (ONL) was up to 5–6 cells deep (Figs. 5A and 6. Antibodies against cone arrestin (Fig. 6A, 6B), recoverin (Fig. 6C, 6D), and mGluR6 (Fig. 6E, 6F) showed that both rods and cones were rescued and that markers of synaptic connectivity were preserved in the outer plexiform layer, suggesting that connections between the photoreceptors and the cells of the inner retina were sustained.

View larger version (86K): [in this window] [in a new window]   Figure 6. Confocal images showing specific antibody staining in P100 retinas after hUTC injections at P21. Images are shown for the area of best rescue and in the same retina, distant from the area of graft rescue effect. (A, B): Cone arrestin staining in the rescued area (A) and distant from the graft (B); cone photoreceptors were preserved with inner and outer segments in (A), whereas in (B) the cone morphology is no longer evident. (C, D): Recoverin staining in the rescued area (C), showing preserved ONL with inner segments, with only a discontinuous layer of photoreceptors in the nonrescued area (D). (E, F): Double stained with protein kinase C (green) and mGluR6 (red) in rescued area (E) and distant from graft (F). Scale bar equals 50 µm. Abbreviations: DZ, debris layer; IPL, inner plexform layer; INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OS, outer segment.

hMSC.   hMSC showed a quite similar level of functional rescue to hUTC, but in most cases morphological rescue was localized to a more limited area of retina.

By P60, hMSC achieved a low level of rescue of ERG a-wave responses, but as with hUTC this failed to reach significance over sham levels (Fig. 3A). However, mixed b-wave responsiveness was significantly better than in sham-injected and untreated rats (81 ± 21 µV vs. 25 ± 20µV, p < .01). Although like hUTC there was significant rescue of the rod b-wave over shams, the cone b-wave differed in not being significantly higher than in shams. As with hUTC, the b-wave dropped with time to 64 ± 31 µV at P90, still significantly higher than either sham or untreated eyes (p < .01). Although this figure was lower than that recorded for hUTC, the difference was not significant.

A mean acuity threshold of 0.46 c/d was recorded at P100 (Fig. 3B). Although this mean was higher than that achieved with hUTC, the difference was not significant. The best animal had a threshold response of 0.58 c/d, close to that seen in normal nondystrophic rats (0.62 c/d).

Luminance thresholds recorded in these animals showed a smaller area of visual representation with lower thresholds than in hUTC-treated RCS rats (3% of the SC input at <1.2 log units over background and 30% at <1.7 log units), but again the difference did not reach significance. The results are illustrated in Figure 3D.

Morphologically, the hMSC group showed much poorer rescue, covering an area only slightly larger than after sham injection (Fig. 5E). This occupied a maximum of 8% of the retinal length in a single section, in all but one retina, extending 2–3 cells deep over less than 20% of the dorsal retina. In the one exception there was somewhat more extensive rescue of approximately 3–4 cells deep over 20% of the retinal length. No evidence of untoward pathology or tumor formation was seen.

hPTC.   Eyes with hPTC injections responded poorly and had limited morphological rescue. ERG responses recorded at P60, gave an a-wave amplitude level of 20 ± 20 µV, and a mixed b-wave of 81 ± 72 µV; by P90 an ERG response could no longer be recorded. Luminance threshold responses were also recorded from the SC. In accord with the ERG, luminance threshold recording from the SC revealed that hPTC grafts provided significantly higher minimal threshold values over smaller SC areas, in contrast to hUTC and hMSC grafts and records were similar to those recorded in sham-injected rats.

Anatomical examination at P100 showed local photoreceptor rescue around the injection site, but this was not nearly as substantial as with hUTC grafts. There was no untoward pathology.

hADF.   ERG responses were poor in this group; the a-wave amplitude was 27 ± 18 µV, and the mixed b-wave was 92 ± 31 µV at P60; neither was recordable by P90.

Histological examination revealed no photoreceptor rescue at P100 other than immediately around the injection site. The appearance was much the same as in a medium injection control (Fig. 5C).

Mechanism of Action
The precedent observations show that hUTC grafts are more effective at sustaining the full range of indices recorded in this study. How they may work is clearly a point of interest. Here we have examined whether they might be effective in delivering growth factors and whether they might function at a distance rather than necessarily through cell contact-mediated events.

ELISA experiments examined the secretion of several neurotrophic factors and found that hUTC secreted significantly higher levels of IL-6 (2,522 ± 1,515 pg per hour per million cells), FGF2 (92 ± 59 pg per day per million cells), and BDNF (238 ± 131 pg per day per million cells) compared with cultures of hPTC, which secreted IL-6 (115 ± 152 pg per hour per million cells) and small amounts of BDNF (17 ± 24 pg per hour per million cells) and hADF, which were only demonstrated to secrete IL-6 (61 pg per hour per 10⁶ cells). Further characterization of the hUTC populations utilized in this study demonstrated that these cells did not secrete factors such as VEGF, SDF1, or TGFβ2. As controls in this assay hADF were tested and were demonstrated to secrete low levels of VEGF (29 pg/hour ± 2) and SDF1 (19 pg/hour ± 1). hADF did not secrete detectable levels of TGFβ2. Given their superior performance over the other two cell types, the higher levels of expression of two known neurotrophic factors by hUTC is consistent with the idea that the cells might function by delivery of neurotrophic agents. Such findings have also been identified in recent work characterizing the molecular profile of other cells derived from umbilical cord on a larger range of factors utilized in a Parkinson study [16]. Previous studies have determined that injection of IL-6, FGF2, or [29, 30] transplantation of genetically engineered cells that secrete BDNF [31] can rescue photoreceptor function. These data suggest that neurotrophic factors can impact photoreceptor survival. In the current study we were unable to observe any direct changes to the vasculature or differentiation of the injected cell populations into retinal cells. The possibility that they might function by action at a distance was tested in vivo by injecting cells into the vitreous cavity. Such cells were distributed within the vitreous but did not appear to invade the retina. Significant photoreceptor survival was achieved (Fig. 5K). In cases where the cell density was not too high in the vitreous, improved optomotor performance over controls was found. Taken together these observations suggest that diffusible factors are an important route whereby hUTC can sustain photoreceptors.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures Acknowledgments References

 
The results show that both hUTC and hMSC were effective in sustaining visual function for several months after injection into the subretinal space of RCS rats but show that hPTC and hADF were little better than medium alone. The anatomical studies showed that a larger area of rescue was achieved in the hUTC group than with most of the animals in the hMSC group. The other cell types gave only slightly greater rescue around the injection site than was found in medium alone sham-operated rats.

The three functional tests used here assayed different aspects of vision. The corneal full field ERG effectively averages the response across the whole retina and computes the average degree and area of rescue. This means that if a small area of retina is rescued, the ERG will give a low amplitude response, although vision may be effective in that focal area. In addition, ERG responses as recorded here do allow the dissection of rod and cone responses. Here both hUTC and hMSC grafts rescued rod-driven functions, but the hMSC failed to show significant rescue of the cone b-wave over sham performance. The luminance threshold studies monitor response sensitivity across the retinal area, providing a more specific indication of the area of rescued responsiveness and the amount of rescue at individual points across the visual field, giving a result similar to that achieved clinically using a Humphrey perimeter test. Under the conditions used here, recordings were made within the luminance range in which cone activity is likely to predominate. The optomotor response gives spatial acuity, using a subcortically driven visual function, which appears to be dependent on rod-driven input [32]. It should be noted that the optimal acuity is somewhat lower than that recorded using other tests such as the visual water test [13]. However because it is a sensitive and reliable measure for showing differences between treated and untreated animals, it is useful for indicating relative change between different treatments, although it does not record absolute acuity.

There is presently only a limited amount of work exploring the use of renewable cell sources as possible treatments for photoreceptor degeneration. One study injected mouse ES-cell-derived neural precursor cells into the subretinal space of young rhodopsin knockout mice, using a similar approach to the present study. Although morphological rescue was achieved in some mice, teratomas developed in 50% of the eyes by 8 weeks after injection [33]. More recent work has shown, however, that teratomas are not an obligatory consequence of using ES-derived RPE cells [34]. Lineage negative hematopoietic stem cells have also been shown to rescue photoreceptors when injected intravitreally in rd1/rd1 (retinal degeneration) mice, perhaps by indirect effects via vascular reconstruction [35], but they did not achieve robust preservation of ERG responses, the only functional test explored. Here we found no evidence of untoward pathological manifestations associated with injection of any of the cell types used here, including the hUTC and hMSC. Donor cells showed no evidence of tumor-like transformations nor was the orderly array of the host retina adversely affected. Most important, both cell types demonstrated robust functional efficacy using several tests of visual function, although the hMSC showed less robust anatomical rescue. One very important factor when considering the criteria laid out at the beginning, with respect to applicability in the clinic, was whether large-scale cell expansion might be possible. In this study it was demonstrated that hUTC could replicate through many passages without karyotypic changes or senescence, whereas we and others [36] have found that unmodified hMSC senesce after several passages. For this reason as well as the better anatomical rescue observed and the better cone ERG rescue, hUTC may provide more utility as an allogeneic cell therapy approach to treat human retinal disease.

Recent work [16] utilizing other umbilical cells has demonstrated that cells from this tissue can take on a neuronal phenotype when injected into the brain. This was not found in the present study following injection in the eye. Furthermore, detection of human nuclear marker in the brain showed that the umbilical cells showed rapid diminution in numbers, even faster than that seen in the present study. Whether this was the result of immunological mismatch or to a biological factor associated with the cells was not clear. Deterioration in cell numbers was also seen after injection of the human-derived RPE cell line, ARPE-19, into the subretinal space of RCS rats.

Clearly, the question arises as to why a cell not normally present in the retina might be so effective. Although hUTC do not appear to transform into neurons, one likely explanation is that the cells serve as a source of neurotrophic factors. hUTC in culture demonstrated higher expression levels of neurotrophic factors, including IL-6 and BDNF, than hPTC and hADF. Although not explored here, other work has shown that hMSC can also secrete a number of cytokines including IL-6 [37]. These among other neurotrophic factors have been shown to be effective in rescuing photoreceptors in a number of different retinal degeneration models after direct injection into the vitreous or via viral vector delivery [38–43]. In addition, Schwann cells [12, 31, 44], either alone or transfected to make additional growth factors, appear to function as a local cell-based factor delivery system, rescuing photoreceptors after injection into the subretinal space. In the present study hUTC were effective not only after subretinal injection but also with intravitreal delivery, indicating that rescue is likely to be achieved through diffusible factors rather than contact-mediated activities. We have not examined the potential phagocytic activity of the donor cells here, but the intravitreal studies show that it is not necessary for the introduced cells to show such activity to be effective. However, whether they affect the phagocytic capability of the host RPE cells has not been investigated.

The target of the action is likely to be complex. Growth factor receptors have been found on a number of retinal cells. Although enigmatically in the rat, CNTF receptors are not located on photoreceptors [45], even though CNTF has been shown in this animal to be effective in preserving rods [46]. A second issue is that in studies on dark and light adaptation [47], it has been found with rescue achieved with other cell implants in RCS rats that although rods are preserved, they do not function at low luminance levels. However, their presence does appear to contain the further degeneration of cones. The functional tests used here depend heavily on continued cone rescue. Together, these studies argue for the possibility of an indirect effect on cone preservation, which for patients with outer retinal degenerative diseases is the most important first goal.

One important aspect of using hUTC is that the preservation of visual function occurred without needing to genetically alter the cells, including engineering them to over express growth factors. This, combined with the ability to generate large numbers of these cells at a central site, provides for a simpler cell therapy approach making hUTC a viable candidate for clinical use in treating retinal degeneration. In a clinical/commercial setting, a major requirement would be that hUTC should be able to survive as allogeneic grafts. Here we have examined efficacy as xenogeneic grafts with immune suppression but have not yet examined their ability to survive as allografts.

	CONCLUSION

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures Acknowledgments References

 
The demonstrated efficacy presented here in the RCS model, which has a human orthologue [48], suggests that hUTC may provide utility in treating patients with retinal degeneration, such as that observed in the retinitis pigmentosa patient population, but that this capability is achieved without the cells transforming into neurons.

	DISCLOSURES

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures Acknowledgments References

 
I.H., D.M., and S.M. own stock in and receive support from Centocor Research and Development Inc. F-Y.C., A.G., A.H., and T.K. own stock in and receive support from Ethicon Inc.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures Acknowledgments References

 
We acknowledge the technical support provided by Elena Budko, Nicholas Bischoff, and Jennifer Hunter; Agnes Seyda, Jeff K. Kennedy, and Stephanie Goldman. R.D.L. is recipient of a Research to Prevent Blindness Senior Scientific Investigator Award.

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Top Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosures Acknowledgments References

 

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