Retinal degenerative diseases are one of the main causes of complete blindness in aged population. In this study, we compared the therapeutic potential for retinal degeneration of human mesenchymal stem cells derived from abdominal subcutaneous fat (ABASCs) or from orbital fat (OASCs) due to their accessibility and mutual embryonic origin with retinal tissue, respectively. OASCs were found to protect RPE cells from cell death and were demonstrated to increase early RPE precursor markers, while ABASCs showed a raise in retinal precursor marker expression. Subretinal transplantation of OASCs in a mouse model of retinal degeneration led to restoration of the RPE layer while transplantation of ABASCs resulted in a significant restoration of the photoreceptor layer. Taken together, we demonstrated a lineage-specific therapeutic effect for either OASCs or ABASCs in retinal regeneration.
Blindness and vision impairment affect more than 2 billion people worldwide and will inevitably continue to increase due to population age. Oxidative stress, inflammation, and degeneration of retinal cells are notably implicated in the development of several eye pathologies, in particular, in age-related macular degeneration (AMD) [
The retinal pigment epithelium (RPE) is crucial for the retinal function, as it participates in nutrition, protection against photo/light oxidation, secretion of proteins required for retinal homeostasis [
Mesenchymal stem cells (MSCs) are especially attractive population for cell therapy purposes, besides the fact that they can serve as an autologous cell source or even allogenic cell source [
To this end, we studied the phenotypic characterization of OASCs, and we evaluated their protective effect on RPE cell survival when exposed to oxidative stress and their cytokine secretion profile when compared to ABASCs. Next, we studied the ability of OASCs to differentiate toward RPE compared to ABASCs. Finally, we investigated the long-term effect of subretinal transplantation of ABASCs and OASCs in a mouse model of retinal degeneration.
OASCs positively expressed classic MSC markers (CD29:
Characterization of OASCs by surface phenotype and differentiation potential. (a)–(c) OASCs were immunostained for CD29, CD31, CD34, CD45, CD73, and CD105 and analyzed by FACS. OASCs exhibited classic MSC phenotype when compared to ABASCs. (b, c) CD34 expression in OASCs was evaluated and compared to that observed in ABASCs. (d, e) Multipotency of OASCs shown by staining for Oil Red O and Alizarin Red and markers for adipocytes and osteocytes. Data are represented as
We assessed the protective role of OASC–conditioned medium (CM) on RPE cells previously exposed to H2O2 as described for ABASCs-CM [
Paracrine activity of OASCs and ABASCs. (a, b) OASCs rescue RPE cells under oxidative stress. RPE cells were incubated with CM or with non-CM for 48 hours, followed by exposure to H2O2 (1 mM, 7 h). Cells were harvested, and cell death rate was analyzed by PI staining followed by flow cytometer analysis. Data are represented as
Mesenchymal stem cells exert their effect on their microenvironment by paracrine release [
OASCs/ABASCs and pRPE were prelabeled with CFSE and CellTrace Violet, respectively (Figures
Differentiation of OASCs to RPE like cells. (a, b) Coculture of p-RPE cells and OASCs. (a) Prestaining of cells: OASCs were marked with CSFE (green color), and RPE mature cells were marked with Violet Cell Trace (blue color). (b) FACS sorter: three distinct populations are defined after coculture: RPE cells, OASCs and OASCs, and RPE cells which were fused together. Autofluorescent cells were excluded according to negative and positive controls of labeled cells. (c) Evidence of differentiation of OASCs to RPE like cells by RT-qPCR. Results are presented as
Differentiation potential of ABASCs to retinal precursors cells. (a, b) Coculture of p-RPE and ABASCs. (a) Prestaining of cells: ABASCs were marked with CSFE (green color), and RPE mature cells were marked with Violet Cell Trace (blue color). (b) FACS sorter: three distinct populations are defined after coculture: RPE, ABASCs, RPE and ABASCs which were fused together. (c) Fusion of cells as shown by one cell expressing both Violet Cell Trace (RPE tracer) and CFSE (OASC tracer). (d) Evidence of differentiation potential of ABASCs to retinal precursors cells by immunofluorescence. (A) Sorted ABASCs expressing cytoplasmic OTX2. (B) Sorted ABASCs in CFSE showing nuclear localized PAX6 expression. (C, D) Positive control of sorted RPE cells in Violet Cell Trace showing nuclear localized OTX2 expression and nuclear localized PAX6 expression (E, F) negative controls of undifferentiated ABSCs does not show any OTX2 expression and PAX6 expression. CT: cell trace.
Relative mRNA expression of cocultured OASCs compared to non cocultured OASCs control is presented in Figure
To better understand whether or not OASCs have an advantage in differentiation to RPE, we compared the differentiation capacity of ABASCs to that of OASCs under the same differentiation protocol. To this end, ABASCs were cocultured with p-RPE cells as previously described in methods. After their coculture with p-RPE cells, ABASCs were able to express the eye-field and neural markers PAX6 and OTX2 exhibiting a potential of early differentiation to retinal precursors. It was observed that PAX6 was expressed in sorted ABASCs in the nuclear region (Figure
To determine the potential of ABASCs and of OASCs in mending retinal injury caused by oxidative stress, we injected those cells to the subretinal space in a sodium iodate mice model, known to describe a retinal degeneration [
Transplanted ASCs were detected in the subretinal space at day 0 by hematoxylin and eosin staining (H&E), thus confirming accuracy of transplantation to the desired location (supplementary Figure
ONL thickness and rhodopsin intensity were previously shown to be reduced after a week of 50 mg/kg sodium iodate [
ONL thickness as well as rhodopsin intensity was significantly higher in ABASC-treated mice, when compared to the PBS-treated group. Also, ONL thickness was higher in ABASC-treated mice, when compared to the OASC-treated group. No significant difference was found with ONL thickness or rhodopsin intensity in OASC-treated mice (Figures
Retinal layers 21 days post transplantation of ABASCs and OASCs in the NaIO3 mice model. (a) Immunohistochemistry pictures of mice retinas for ONL quantification and rhodopsin intensity by DAPI and rhodopsin staining in ABASCs and OASCs transplanted mice versus controls. Yellow arrows indicate the PR layer. (b) Immunohistochemistry pictures of the mice RPE layer after RPE65 and DAPI staining in ABASC and OASC transplanted mice versus controls. Yellow arrows indicate the RPE layer. (c) Graphic summary of RPE65 intensity evident by RPE65. (d) Graphic summary of rhodopsin intensity evident by rhodopsin staining. (e) Graphic summary of ONL thickness evident by DAPI staining. (f) Graphic summary of INL thickness evident by DAPI staining. ONL: outer nuclear layer; INL: inner nuclear layer; RGC: retinal ganglion layer; PR: photoreceptor; RPE: retinal pigment epithelium.
RPE65 is often used as a marker of RPE functionality [
We demonstrated basal CD45+ cell infiltration in NaIO3 mice prior to transplantation of OASCs and ABASCs (Figure
Immune response 21 days post transplantation of ABASCs and OASCs in the NaIO3 mice model. (a) Immunohistochemistry pictures of CD45+ staining in NaIO3-treated mice compared to nontreated mice revealed the infiltration of CD45+ cells in the retinas of NaIO3-treated mice compared to controls. (b) Immunohistochemistry pictures of CD45-Iba1 costaining. (b, c) Quantification of the number of CD45+ cells revealed a higher infiltration of leucocytes in ABASC-treated mice compared to PBS treated mice (
In this study, we characterized the phenotype of OASCs, we demonstrated the protective effect of OASCs on RPE cell death in the setting of oxidative stress, and defined their cytokine secretion profile compared to that of ABASCs. Next, we showed that OASCs were able to express early RPE markers, while ABASCs expressed less specific retinal markers, after coculture with primary RPE cells. Finally, we demonstrated a lineage specific therapeutic effect of OASC and ABASC transplantation in a mouse model of retinal degeneration.
Treatment of RPE with conditioned medium of OASCs resulted in salvage of RPE from cell death induced by oxidative stress. These data are in line with a previous study conducted by our group that demonstrated a protective effect of ABASCs on RPE cell death [
We demonstrate an increase of the early RPE marker expression in OASCs and a raise in less specific retinal lineage marker expression in ABASCs. Immunocytochemistry studies revealed specific nuclear staining to PAX6 and OTX2 in OASCs, implying to obvious lineage direction of OASCs towards RPE [
Moreover, it was previously shown that stem cells could memorize epigenetic marks from their origin [
To note, we found a population of cell fusion of RPE with ABASCs and with OASCs. Although cell fusion was a rare phenomenon of less than 0.7% of cases, it may have some implications. First, this may explain why we found increase in RPE65 transcript in the qRT-PCR study only a week after the differentiation study, since RPE65 is known as a mature RPE markers and usually upregulated later during the course of differentiation [
As opposed to a recent report by Kuriyan and colleagues, in which transplantation of nonselected mixture of whole adipose tissue cells was injected to the vitreous of AMD patients and caused devastating results, the population of stem cells transplanted in this study was carefully segregated from the whole adipose tissue by a two-step procedure. First, mesenchymal stem cells were isolated from the adipose tissue by collagenase digestion. Second, cells were seeded on tissue culture plate and further purified with specific medium which allowed the selection of purified MSCs. MSC phenotype was then verified by FACS analysis, as mentioned in methods. Moreover, purified MSCs were injected to the subretinal space rather than to the vitreous cavity to reduce the risk of retinal detachment as observed by Kuriyan and colleagues [
It is known that NaIO3 injection causes degeneration of ONL and INL [
We observed higher CD45+/Iba1+ cell infiltration in retinal layers after ABASC treatment compared to the PBS group and to OASC group. The implication of microglial cells is correlated with neuroinflammation which triggers the increase of the CD45 expression [
We demonstrated preservation of the RPE layer in mice treated with OASCs compared to mice treated with ABASCs or controls of PBS. RPE is one of the contributors to the integrity of the outer blood retinal barrier (oBRB). RPE also exhibits immunosuppressive properties by paracrinely preventing the traffic of cells and proteins from the blood to the retinal region [
The purpose of the study and the procedures used were presented to all of the subjects, and a signed informed consent was obtained from each. This study was approved by the ethics committee for clinical trials of Tel Aviv Sourasky Medical Center and was conducting according to the Declaration of Helsinki.
Subcutaneous abdominal human adipose tissue was harvested from 4 healthy patients with a mean age of
Characterization of cultured OASCs was performed at passages 3-4 as follows: after reaching 100% confluence, cells were trypsinized and collected in FACS tubes in aliquots (
OASCs at passage 3 were studied to determine their ability to differentiate into osteocytes and adipocytes.
Following rescue studies as described above, RPE cells at passage three were harvested with 0.25% trypsin/0.05% EDTA (Biological Industries). Cells (
Cytokine secretion of OASCs was studied by cytokine antibody array on collected culture medium and compared to that of ABASCs. Conditioned medium was prepared as described above; briefly, ASCs’ medium was changed to serum-free medium, following 48 hours of incubation, and the medium was collected and centrifuged at 4500 rpm for 5 min and supernatants stored at −80°C until they were assayed. Levels of medium released cytokines and growth factors were measured by RayBio® Human Cytokine Antibody Array G10 (Thermo-Fisher) according to manufacturer’s protocol.
We evaluated the differentiation potential of OASCs to RPE using a coculture system [
After cell sorting of the cells, we identified OASC-derived RPE cells, and we characterized the level of differentiation by qRT-PCR using primers (Table
The gene expression profile of OTX2, PAX6, SIX3, RPE65, and KLF4 in OASCs after costaining with primary human RPE was quantified using qRT-PCR in order to detect differentiation markers. Results were compared to a negative control-OASCs which were non c-cultured with RPE. We also quantified the expression of Ck-
For detection of retinal precursors markers at the protein level, sorted cells and controls were plated on 13 mm coverslips in a 24 well-plate (Falcon) overnight, respectively, in a mixture medium (50% of DMEM 10% FBS/50% of free-FBS RtEGM) or in DMEM containing 10% of FBS and incubated at 37°C in a humidified atmosphere (respectively, 5 and 8% of CO2). The cells were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton™ X-100 for 5 minutes, blocked overnight with PBS diluted 2% BSA, and labeled with rabbit primary antibody anti-OTX1/2 (OTX1 is not expressed in RPE and photoreceptors [
Wild-type C57BL mice received intraperitoneal (IP) injection of 50 mg/kg of sodium iodate (NaIO3) (Sigma-Aldrich) (
Mice received cyclosporine in drinking water for a week after the transplantation, at a concentration of 210 mg/l. Animal handling and experiments were performed following institutional care guidelines with the approval of the Tel Aviv Sourasky Medical Center Animal Ethics Committee.
Mice were euthanized on day zero and three weeks following subretinal transplantation; the eyes were enucleated and fixed in 4% formaldehyde (Merck, Darmstadt, Germany) overnight. The eyes were washed in PBS and then incubated for cryoprotection with 30% sucrose in PBS overnight at 4°C. Fixed tissue was embedded in OCT Tissue Freezing Medium (Scigen Scientific, Gardena, CA, USA) and frozen on dry ice. Cross-sections (10
Frozen slides were stained with hematoxylin and eosin (H&E). For immunofluorescence staining, sections were washed in PBS for 20 minutes and blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich), 1% normal goat serum (NGS) (Invitrogen, no. 31872), and 0.5% Triton X-100 (Sigma-Aldrich) in PBS for 1 hour at room temperature. The sections were incubated overnight with anti-RPE65 rabbit monoclonal primary antibody (ab231782, 1 : 200), anti-CD45 clone IBL-5/25 Rat monoclonal antibody (EMD Millipore, Merck, Darmstadt, Germany, 1 : 50), Mouse anti-rhodopsin monoclonal antibody (EMD Millipore, Merck, Darmstadt, Germany, 1 : 200), Iba1, and rabbit antibody (Wako, Osaka, Japan, 1 : 1000), in blocking solution at 4°C. The slides were washed three times with PBS-0.5% Triton X-100 (PBST), incubated with Alexa Fluor 488 Goat anti-rabbit, Alexa Fluor 546 Goat-anti-rat, and Alexa Fluor 546 Goat anti-mouse (Invitrogen, 1/200) in PBST for 1 h at room temperature in blocking solution, and washed again three times with PBST. The sections were then incubated with the nuclear dye DAPI (Molecular Probes, Thermo Fisher Scientific) for 10 minutes, washed twice in PBS, and mounted in ImmunoMount (Thermo Scientific, 9990412).
Pictures were carried out with a Zeiss LSM 700 confocal microscope in a blinded manner. Analysis was performed by ImageJ software. RPE65 and rhodopsin intensity and quantification of positive CD45 and Iba1 cells were performed by measuring the fluorescence intensity compared to the image’s background. Also, the thickness of ONL and INL was evaluated by measuring length of ONL and INL layers (stained with DAPI) in at least 6 points of the layer. The sections shown were selected at the same distance to the optic disc and in the same retinal quadrant.
Statistical analyses were performed using GraphPad Prism software (version 9.0, GraphPad Software, San Diego, California). The Mann–Whitney test was used to analyze nonparametric data. For the mice results, a Kruskal-Wallis nonparametric test was used to identify differences between the three groups. Statistical significance was accepted for
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This study was approved by the Tel Aviv Sourasky Medical Center Institutional Review Board (Helsinki Committee, approval number 920120440). All the experimental procedures involving patients in this study were approved by the Tel Aviv Sourasky Medical Center Institutional Review Board (Helsinki Committee, approval number 920120440).
Written or verbal informed consent was obtained from all patients; the study conforms to the principles outlined in the Declaration of Helsinki.
This study was presented in the Association for Research in Vision and Ophthalmology (ARVO) Annual Meeting Abstract in June 2021.
The authors declare no competing interests regarding the publication of this paper.
B.K. wrote the paper; B.K., S.W. and I.N. conducted the experiments; A.B. and B.K designed the experiments. Bryan Krief and Shira Weisthal Algor contributed equally to this work.
This study was supported by grants from the Moxie Foundation (260375) and from Orion grant (213310).
Table S1: primers used in quantitative RT-PCR experiments. Supplementary Figure 1: detection of cells in the subretinal space on the day of ASC transplantation in the NaIO3 mice model.