Send Orders for Reprints to
[email protected] Current Molecular Medicine 2013, 13, 1419-1431
1419
Subretinal Transplantation of Rat MSCs and Erythropoietin Gene Modified Rat MSCs for Protecting and Rescuing Degenerative Retina in Rats Y. Guan1,§, L. Cui2,§, Z. Qu2, L. Lu1, F. Wang1, Y. Wu2, J. Zhang1, F. Gao1, H. Tian1, L. Xu1, G. Xu3, W. Li*,1,4,¶, Y. Jin*,2,¶ and G.-T. Xu*,1,2,5,¶ 1
Department of Ophthalmology of Shanghai Tenth People’s Hospital, and Tongji Eye Institute, and Tongji Stem Cell Research Center, Tongji University School of Medicine, Shanghai, China 2
Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine, and Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China 3
Department of Ophthalmology, Second Affiliated Hospital of Suzhou University, Suzhou, China
4
Department of Ophthalmology, Drexel University College of Medicine, Philadelphia, PA, USA
5
Institute for Nutritional Sciences, Tongji University School of Medicine, Shanghai, China Abstract: For degenerative retinal diseases, like the acquired form exemplified by age-related macular degeneration (AMD), there is currently no cure. This study was to explore a stem cell therapy and a stem cellbased gene therapy for sodium iodate (SI)-induced retinal degeneration in rats. Three cell types, i.e., rat mesenchymal stem cells (rMSCs) alone, erythropoietin (EPO) gene modified rMSCs (EPO-rMSCs) or doxycycline (DOX) inducible EPO expression rMSCs (Tet-on EPO-rMSCs), were transplanted into the subretinal spaces of SI-treated rats. The rMSCs were prepared for transplantation after 3 to 5 passages or modified with EPO gene. During the 8 weeks after the transplantation, the rats treated with rMSCs alone or with two types of EPO-rMSCs were all monitored with fundus examination, fundus fluorescein angiography (FFA) and electroretinogram. The transplantation efficiency of donor cells was examined for their survival, integration and differentiation. Following the transplantation, labeled donor cells were observed in subretinal space and adopted RPE morphology. EPO concentration in vitreous and retina of SI-treated rats which were transplanted with EPO-rMSCs or Tet-on EPO-rMSCs was markedly increased, in parallel with the improvement of retinal morphology and function. These findings suggest that rMSCs transplantation could be a new therapy for degenerative retinal diseases since it can protect and rescue RPE and retinal neurons, while EPO gene modification to rMSCs could be an even better option.
Keywords: Erythropoietin, gene therapy, rMSCs, retinal degeneration, RPE, Tet-on.
INTRODUCTION Malfunction of retinal pigment epithelium (RPE) is essential for both acquired and inherited degenerative retinal diseases [1, 2]. Since there is no effective treatment, new approaches like gene therapy and cell or stem cell-based therapy have attracted considerable Address correspondence to these authors at the (W. Li) Department of Ophthalmology, Drexel University College of Medicine, 219 North Broad Street 3FL, Philadelphia, PA 19107, USA; Tel: 215-762-3937; Fax: 215-762-5600; E-mail:
[email protected]; (Y. Jin) Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine 225 South Chongqing Rd. #1 Bldg. Rm. 607, Shanghai, 200025, China; Tel: 86-21-6385-2591; Fax: 86-21-63852591; E-mail:
[email protected]; (G.-T. Xu) Department of Ophthalmology of Shanghai Tenth People’s Hospital, and Tongji Eye Institute, Tongji University School of Medicine, 1239 Siping Road, Medical School Building, Room 521, Shanghai, 200092, China; Tel: 86-21-6598-6358; Fax: 86-21-6598-6358; E-mail:
[email protected] §
Y. Guan and L. Cui are co-first authors. G.-T. Xu, Y. Jin and W. Li as corresponding authors who contributed equally to this study.
attention. Limited reports showed that subretinal transplantation of normal RPE patches derived either from allogeneic or xenogeneic sources could slow down retinal degeneration [3, 4]. The key factors hindering such cell therapies are the limited source of donor cells and low RPE harvesting efficiency in primary culture, in addition to other factors such as immunological rejection which may also attribute to the failure of such therapies [5, 6]. An alternative way to obtain RPE as donor cells is to differentiate stem cells into RPE. It could be done with either pluripotent stem cells like embryonic stem cells or adult stem cells such as mesenchymal stem cells (MSCs). The MSCs have been successfully induced to differentiate into matured, nonhematopoietic cells such as epithelial cells [7], as well as cells with neuronal characteristics [8, 9], including photoreceptors in vivo and in vitro [10]. In addition to their great potential, MSCs also have the advantages of fewer ethical concerns and less immune rejection.
¶
1-5/13 $58.00+.00
© 2013 Bentham Science Publishers
1420
Current Molecular Medicine, 2013, Vol. 13, No. 9
Guan et al.
In animal models, therapeutic genes expressed in lentiviral or adenoviral vector have been proven to be effective and long-term interventions for neurological disorders [11] and inherited retinal degenerations [12]. Therefore, a gene therapy employing MSCs, a cellbased gene therapy, ought to be a more effective approach.
1083 (Sigma, USA) and suspended at a concentration 7 of 110 /mL. The cells were then incubated for 1 day, and the non-adherent cells were removed by replacing the medium. The rMSCs were used in experiments after three to five passages.
Erythropoietin (EPO) has been realized as an endogenous retinal survival factor [13], in addition to its erythropoietic function. Recently, it has been reported that many pathologic conditions could be improved by EPO therapy in animal models [14-16]. Our previous work showed that intravitreal injection of EPO protected retinal cells in diabetic rats, and helped in maintaining the functions of retina and blood-retinal barrier (BRB) [17]. Clinically, the application of such procedure improved the vision and reduced retinal thickness in 5 patients with persistent diabetic macular edema [18]. However, the therapeutic effect of a single intravitreal injection of EPO lasted only 4 to 6 weeks [17], and the patients have to be repeatedly injected to maintain the efficacy [18]. We proposed that, in addition to potential therapeutic effects of stem cells like MSCs themselves, EPO produced by transplanted EPO gene modified MSCs as donor cells could also serve as a long term, consistent or regulatory supply of EPO in the eyes, to eliminate multiple injections. Meanwhile, a conditional EPO gene expression, like drug-inducible gene expression system, should also be studied [19].
To produce a uniform cell population, primary cultured rMSCs were passaged 3~5 times before analysis by fluorescent flow cytometry. The cells were dissociated with 1mM EDTA, washed in 4 ml of phosphate buffered saline (PBS) containing 0.1% sodium azide and 1% BSA, and incubated with antibodies against CD29, CD11b, CD45, CD90 (Alexa ® Fluor 488, Biolegend, USA), Flk-1 and CD31 (Santa Cruz, USA) on ice for 30 minutes. The cells were analyzed using a flow cytometer (FCM, FACS Calibur and CellQuest Pro Software, Becton-Dickinson, USA).
In the present study, we employed a novel idea to explore the efficacy of subretinal transplantation of EPO gene modified rat MSCs (rMSCs) in a rat model of retinal degeneration, by determining the ability of rMSCs to differentiate into RPE cells and their situation in the niche of the host subretinal space, and by demonstrating the better therapeutic effect of EPO expressing rMSCs on retinal morphology and visual functions than rMSCs alone.
MATERIALS AND METHODS Animal Models The animals were maintained under Shanghai Institutes for Biological Sciences (SIBS) approved animals care conditions and treated according to the regulations in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. SpragueDawley (SD) and Brown Norway (BN) rats, 6–8 weeks old and weighing between 180 and 200 g, were purchased from Shanghai Laboratory Animal Co. Ltd (SLAC). Retinal degeneration was induced by a single intravenous injection of 50 mg/kg of Sodium Iodate (SI) (Sigma, USA) [20, 21]. The animal eyes were monitored with fundus examination, fluorescein angiography [22] and electroretinogram (ERG). Isolation and Cultivation of rMSCs The rMSCs were isolated from bone marrow aspirates of male adult SD rats using a method described previously [23, 24], with minor modification that the nucleated cells were isolated by HISTOPAQUE
Characterization of rMSCs
In Vitro Differentiation of rMSCs into RPE For induction of neural sphere formation, rMSCs were cultivated in DMEM supplemented with 10% FBS, and transferred to Neurobasal medium supplemented with B27 (2%), N2 (1%), bFGF (20 ng/ml), EGF (20 ng/ml) and 1% FBS (Invitrogen GIBCO, USA and Peprotech, USA) for 7 days. The cells were then dissociated, plated on Collagen IV (Sigma, USA) coated dishes, and co-cultured with the RPE determination medium (Supplementary Material) for another 7 days. The rMSCs were characterized by RTPCR and immunocytochemistry using RPE specific markers such as RPE65 and Cralbp (Abcam, USA), following the manufacturer’s instructions. Phagocytosis experiment was described previously [25] with some modifications (Supplementary Material). Modification of EPO Expressing rMSCs EPO gene modified rMSCs (EPO-rMSCs) and Teton EPO-rMSCs were prepared by infecting the rMSCs with either pRRL-hPGK-rmEPO or pLVCT-rmEPOtTRKRAB lentiviral-pseudotyped viral particles (Supplementary Material). To monitor the EPO expressed in the in vivo and in vitro systems, the amounts of EPO in cell culture medium and tissue samples were quantified by RT-PCR and ELISA, according to the manufacturer’s instructions (Quantikine, R&D, USA). Co-Culture of Retinal Cell Lines or Photoreceptors with EPO-rMSCs Conditional Medium Several cell lines were used in this study. R28 and RGC5 represent rat mixed retinal neural cells (R28) and retinal ganglion cells (RGC5) respectively. ARPE19 is a human RPE cell line. ECV represents endothelial cells of vessel. Primary mouse photoreceptors were also used in cell culture to study the neurotrophic effects of conditional medium (CM) from EPO-rMSCs (Supplementary Material). Glutamic acid (5 mM) was used as stress, and added into the medium for 2 hours before the medium was replaced by the CM. After another 24 hours culture, different
EPO Modified rMSCs Therapy for Retina Degeneration
analyses like the MTT cell proliferation assay were carried out. Transplantation of rMSCs or EPO Gene-Modified rMSCs Donor cells were transplanted into subretinal space as previously described [26]. The procedure for labeling the cell with CM-DiI (Invitrogen, Molecular Probes, USA) was performed according to the manufacture’s recommendation. In Tet-on EPO-rMSCs rat experiments, 0.2 g/l Doxycycline (Sigma, USA) and 5% sucrose were added into drinking water. In the 6 present study, 10 cells/eye were used for subretinal transplantation. ERG Examination ERG recordings, with a Bio-2000 system (BEIAO, China), were performed following the procedures described by Sauve Y, etc. [27]. After SI injection and rMSC transplantation, corneal ERG recordings were obtained weekly from both eyes of the rats until the rats were killed. The parameters of ERG consisted of average amplitude of a-wave and b-wave (ANa, APb) and oscillatory potentials (OPs). Since OPs have been proven as sensitive indicators of abnormalities in retinal circulation [28], OPs tests were applied to monitor the changes in retinal circulation, while a- and b-waves were examined to reflect the disorders in retinal neurons. The ERGs of the 6 rats (12 eyes) from the same group at each examination time point were superimposed. Immunostaining and Histology For immunochemistry analysis, retina samples were examined with the following antibodies: Keratin, ZO-1 (Invitrogen, USA, GFAP (Abcam, USA), MAP2 (Chemicon, USA), Opsin (Sigma, USA), CD11b, CD29, CD45, CD90, RPE65 and Cralbp. To prepare retinal flatmount, animals were anesthetized and perfused with 50 mg/ml FITC-dextran (Sigma, USA) first, and then the retinas were collected at various time points and prepared for whole mounting. Retinas were fixed with 4% PFA and blocked in DAKO for preservation at 4 degree. Retinal thickness was measured by method previously described [17]. In Situ Detection of Cell Death in Retina by TUNEL Assay The TUNEL assay was performed using In Situ Cell Death Detection Kit (Roche, USA), following the manufacture’s instructions. Examination of BRB Permeability BRB permeability was evaluated according to the method previously described [17]. Statistical Analysis We used SPSS 11 (SPSS, Chicago, IL) for Windows to conduct statistical analyses. All results are
Current Molecular Medicine, 2013, Vol. 13, No. 9
1421
presented as mean ± SD. Comparisons between groups were analyzed by t test (2-sided) or One-way ANOVA followed by the post hoc Dunnett's test for experiments with more than 2 groups (Levene's tests for equal variance). Dunnett's T3 test was used as post hoc test comparison for the analysis of unequal variances (Welch's and Brown–Forsythe's test). Differences were considered statistically significant at P < 0.05.
RESULTS Establishment of SI-Induced Retinal Degeneration Model in Rats SI-induced retinal degeneration model was established in both pigmented BN rats and albino SD rats, and verified by functional and morphological examinations focusing on RPE death and subsequent retinal degeneration. In comparison with the evenly distributed pigmentation in the eye fundus of normal control rats, the fundus photos detected severe retinal hyperpigmentations and hypopigmentations (typical mosaic-like fundus) in SI-induced BN rats 2 months after SI treatment (Fig. 1a, top). Simultaneously, FFA examination revealed stronger transmitted fluorescence from choroidal capillaries in SI-treated rats (Fig. 1a, bottom), indicating the RPE damage. The functional examination of the rat retina with ERG recording showed quick reduction in the amplitude of waves in SI-treated rat. On day 1 post SI injection, the amplitudes of ERG were already dropped down. On day 14, only very weak ERG waves could be recorded (Fig. 1b, *P
