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Subretinal microglia support donor photoreceptor survival in rd1 mice

Stem Cell Research & Therapy volume 15, Article number: 436 (2024) Cite this article

Abstract

Purpose

To investigate the potential relationship between subretinal microglia and transplanted donor photoreceptors.

Methods

Photoreceptor precursors were transplanted into wild-type mice and rd1 mice by trans-scleral injection. Immunohistochemistry was employed to detect microglia and macrophages. PlX5622 feed was used to achieve microglia depletion and microglia repopulation. RNA-seq and qPCR were utilized to evaluate gene expression. Confocal microscopy was used to observe the interaction between microglia and donor photoreceptors.

Results

Donor photoreceptors survived in rd1 mice but not in wild-type mice after trans-scleral injection. The microglial cells closely interacted with donor cells. While donor cells failed to survive in rd1 mice after microglia depletion, they could survive following microglia repopulation. The RNA-seq analysis showed a pro-neurodevelopmental effect of sub-retinal microglia/RPE tissue in rd1 mice.

Conclusions

Subretinal microglia supported donor photoreceptor survival in rd1 mice.

Introduction

Inherited retinal diseases that lead to irreversible loss of photoreceptor cells afflict millions of people worldwide [1]. Currently, no effective treatments exist to prevent or slow down the progression of photoreceptor loss. One potential therapeutic approach is photoreceptor cell transplantation, which may re-establish the retinal neural circuits [2].

Over the last few decades, researchers have tried photoreceptor transplantation in different retinal degeneration models [3,4,5]. Studies in end-stage inherited retinal degeneration models, which excluded cytoplasmic exchange [6,7,8], provided evidence that visual function could be restored by generating new synaptic connections between donor and host cells [9, 10]. Compared to the early stage of retinal degeneration, which could be fixed by gene therapy, stem cell substitute therapy was more suitable for the end stage of retinal degeneration. However, the donor cell survival rates are poor [11, 12], and the number of surviving cells declines over time [13]. Studying graft protective strategies may be a critical step for this therapeutic method moving forward to clinical application.

The eye as a whole represents an immune-privileged environment, but immunosuppressants were still administered in most retinal pigment epithelium cell (RPE) transplantation trials. Without pharmaceutical inhibition of the immune reaction, acute rejection happened in both age-related retinal degeneration patients [14] and nonhuman primates [15, 16]. In photoreceptor transplantation, improved donor cell survival rates were achieved in some situations, such as using immune-defective host mice [17], immune modulation medicine [18], and a special stage of progenitor cells with immune modulation effects [19]. The possible reason could be that the subretinal space is only a “partially immune-tolerant” environment [20]. Immune modulation seems indispensable for stem cell transplantation to the subretinal space. However, a recent clinical trial showed that donor cells did not raise acute immune reactions in retinitis pigmentosa patients without immune inhibitor administration [21].

In retinal degeneration diseases, the retina becomes thinner, which increases the difficulty of subretinal injection by direct retina puncture. Instead of using trans-vitreous injection, trans-scleral injection showed many advantages in this situation, and by using this method, donor photoreceptor cells indeed survived in rd1 mice (a fast photoreceptor degenerating model with a PED6b mutation) without immune rejection [10]. Additionally, xenotransplantation of hiPSC-derived cone photoreceptors survived in Nrl−/− and Aipl1−/− retinal degeneration models without immunosuppressants [22]. Of note, trans-scleral injection pierces through Bruch’s membrane and retinal pigment epithelium, which breaks the physical immune-tolerance barrier. However, acute donor photoreceptor rejection was only reported in wild-type mice following trans-vitreous injection [23]. These results imply that the immune environment of the subretinal space may have greatly changed during retinal degeneration.

Tissue-resident microglia act as sentinels in the central nervous system and play an important role in the innate immune response [24]. While microglia are normally distributed in the ganglion cell layer, inner plexiform layer, and outer plexiform layer of the retina, they move into the subretinal space during retinal degeneration [25]. These microglia may direct the initial response to transplanted grafts [26]. Evidence of microglial protection of photoreceptor cells has been reported in many disease conditions, such as retinal detachment [27], light-induced retinal damage, and P23H−/− retinal degeneration mouse models [28]. Whether microglia in the subretinal space can protect donor photoreceptors is unknown. In this study, we explored the possible relationship between microglia and donor photoreceptors.

Methods

Animals

All animal studies were approved by the Animal Ethics Committee at the West China Hospital and were performed with adherence to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The work has been reported in line with the ARRIVE guidelines 2.0. C57BL/6J mice were purchased from the Experimental Animal Center of Kunming Medical University. C3HPde6b mice, also referred to as rd1 mice, were obtained from Vitalstar Biotechnology Co.,Ltd. (Beijing, China). Rho-icre mice (Cyagen, China) were crossed with tdTomatofloxed mice (Cyagen, China) to generate rho-tdTomato mice. Rho gene is robustly expressed by rod photoreceptors and codes for visual pigment rhodopsin. The animals were raised at room temperature with a 12-hour light/12-hour dark cycle and standard mouse food and drinking water.

Three-month-old wild-type (n = 9) and rd1 (n = 9) mice received photoreceptor precursor transplantation in one eye and a sham injection in another eye. The transplantation experiments were performed three separate times, with three mice each time and their sex randomly assigned to each time. One wild-type mice was died during the study. Necropsy didn’t determine the cause of death. For depletion of microglia, wild-type (n = 3)and rd1 mice (n = 3) were administered dietary PLX5622 (an inhibitor of colony-stimulating factor-1 receptor, 1200 mg/kg feed, M18090601, Moldiets, Biopike, Inc., China), starting from one week before the transplantation to the end of the experiment. For microglia repopulation, wild-type (n = 3) and rd1 mice (n = 3) mice were given PLX5622 feed for one week, which was then replaced by AIN-76 A control diet for another three weeks before transplantation [29].

Donor cell preparation and fluorescence-activated cell sorting (FACS)

The neonatal mice(P4-P7) were anesthetized by chilling on ice, followed immediately by decerebration and harvest of retinas. Retina digestion was performed using the Papain Dissociation System (Worthington Biochemical Corp., Lakewood, NJ). In brief, the retinas were placed in a papain solution for 15 min at 37 °C. After this incubation, the retinas were triturated to dissociate them into single cells. The papain reaction was stopped with the addition of ovomucoid albumin inhibitor, and cells were collected by centrifugation.

Cell sorting was performed with a FACSAria SORP (Becton, Dickinson and Company) fitted with a 561 nm yellow-green laser to excite tdTomato-positive cells, which were collected.

Transplantation

The recipient mice (three months old)were anesthetized with a mixture of ketamine and xylazine. Trans-scleral transplantation was performed by making a tunnel in the sclera with a 31-gauge needle, and the donor cell suspension was injected through this tunnel using a blunt 30-gauge needle. One microliter of cell suspension (1 × 105 cells/µl) was slowly injected into the subretinal space. Balance salt solution was injected to the fellow eye as sham injection. Erythromycin ointment was applied to the eye after surgery. Steroids or immunosuppressants were not used in this study. Two weeks after transplantation, the mice were euthanized to collect ocular tissues.

Immunofluorescence

We performed immunostaining as previously reported [30]. In brief, eyeballs were fixed in 4% paraformaldehyde at 4 °C for 1 h, then dehydrated in 30% sucrose overnight, and embedded in optimal cutting temperature compound(OCT). Cryostat sections were cut at 10 μm and collected on glass slides, then stored at -20 °C. The researchers were blind to.

The retinal sections were incubated in blocking solution(5% donkey serum) for 1 h and then incubated with primary antibodies at 4 °C overnight. The following primary antibodies were used in the study: CD68 (1:400,#97778, Cell Signaling Technology), Iba1 (1:200, #019-19741, Wako), Galectin-3 (1:200, #sc-53127, Santa Cruz), F4/80(1:400, #71299, Cell Signaling Technology), CD11b (1:200, 101,209, Biolegend). Subsequently, the slides were incubated with a secondary antibody for 1 h at room temperature in the dark. Finally, the slides were incubated with 4’6-diamidino-2-phenylindole (Sigma Aldrich Corp.) and mounted with Mowiol mounting medium. For the retinal mounts, the tissue was rocked at 4 °C overnight for primary antibody staining and incubated for three hours for secondary antibody staining. Stained retinas were analyzed using a Nikon A1RMP confocal microscope. Figures show projection images of 10 μm stacks or single confocal sections. The tdTomato-labeled donor cell was captured with its spontaneous signal.

The number of surviving donor cells per section was determined by counting both tdTomato-positive and rhodopsin-positive cells in every 1 in 4 serial sections. This was multiplied by the total number of sections that encompassed the injection site to give an estimate of the mean number of surviving cells per eye [31].

RPE cell and microglia isolation and RNA extraction

We modified the simultaneous RPE isolation and RNA extraction protocol described by Cynrhia Xin Zhao et al. [32]. In brief, the posterior eye cup was transferred into 200 µl of RNAprotect Cell Reagent (Qiagen cat.76526) and incubated for 10 min at room temperature. The tube was briefly agitated to ensure that most of the RPE cells were released, and then the eye cup was removed. If rd1 mice were used, subretinal microglial cells were released at the same time. Centrifugation was performed for 5 min at 600 g to pellet the cells, which were then subjected to total RNA extraction using the RNeasy Micro Kit (Qiagen cat.74004) per the manufacturer’s instructions. Lysis buffer was added, and the sample was homogenized. The sample was then centrifuged for 2 min. The supernatant and 70% ethanol(1:1volume) were mixed and then transferred to an RNeasy MinElute spin column. In the final step, DNase I was used to digest DNA, and the RNA was eluted with RNase-free water.

The cDNA libraries were prepared using the Illumina TruSeq RNA sample preparation kit, and the qualities were assessed using an Agilent 2100 Bioanalyzer. For sequencing, the cDNA libraries were loaded onto an Illumina Hiseq 2500 at Novogene (Beijing, China). The following differential expression gene (DEG) analysis and expression enrichment analysis were completed via the Novomagic cloud platform.

For real-time PCR, cDNA was synthesized from 1 µg of total RNA using a PrimeScript RT reagent kit (Takara Biotechnology, Dalian, China). To quantify the cDNA, real-time PCR was performed on a qTOWER 2.2 system (Analytik Jena, Germany). The PCR amplification was conducted in a volume of 20 µl using EvaGreen Supermix (Bio-Rad Laboratories Inc., Hercules, CA, USA). To determine mRNA expression, all samples were tested in duplicate, and the average Ct values were used for quantification. The mRNA expression was normalized to the endogenous reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers used in this study were provided in supplementary Table 1.

Cell culture

According to the approved euthanasia procedure, adult mice were deeply anesthetized with the mixture of ketamine and xylazine, followed immediately by decerebration. Eyes of three-month-old rd1 and wild-type mice were enucleated and kept in ice-cold HBSS medium before dissection. The cornea and the lens were removed, then the RPE-choroid-scleral complex (RCSC) was separated from the retina and radially cut into four pieces. The flattened pieces were embedded in growth factor-reduced Matrigel in the 24-well plates on ice and incubated at 37 °C for 30 min. After polymerizing, DMEM: F12 medium was added into the wells and collected after 24 h as conditional medium. Photoreceptor precursors were suspended in wild-type or rd1 conditional medium, added with N2 supplement, and 1% FBS. Cells were plated at a density of 1 × 105 cells/cm2 on growth factor-reduced Matrigel in the 24-well plates. After 24 h of culture, the surviving photoreceptor precursor cells were imaged by fluorescence microscope. The number of tdTomato-positive cells was counted using ImageJ software.

Statistics

Statistical analysis was performed by using the GraphPad Prism software (Version 9.5.0, GraphPad Prism Software, Inc., San Diego, CA, USA). Unpaired Student’s t test was used for comparisons between two groups. The threshold for significance was set at p < 0.05.

Results

Donor photoreceptors can survive in rd1 mice following trans-scleral injection

Using the trans-scleral injection method, we transplanted photoreceptor precursors into the subretinal space of both wild-type and rd1 mice, and examined them two weeks later. In rd1 mice, both surviving and dead cells could be found, and the number of surviving donor cells was 3419±2359 (n = 9) (Fig. 1A and B). However, no surviving cells were found in wild-type mice (n = 8), which was different from the transplantation result by trans-vitreous injection as we previously reported [30]. When comparing the failure cases in wild-type and rd1 mice following trans-scleral transplantation, the dead cells showed differences in morphology (Fig. 1A). While the dead cells seemed to be engulfed by macrophages in wild-type mice, only cell debris with lower fluorescence intensity was found in rd1 mice (Fig. 1C).

Fig. 1
figure 1

Photoreceptor survival and death following transplantation. A. Donor cells survived (left), died (middle) in end-stage rd1 mice, and died(right) in wild-type mice. B. The number of surviving donor cells in rd1 mice and wild-type mice. C. In rd1 mice, the fluorescence intensity of surviving donor cells was significantly higher than that of dead cell debris. * p < 0.05, ** p < 0.01

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Microglia but not macrophages surround donor cells in rd1 mice

To determine the underlying mechanism, we performed immunostaining detection. In wild-type mice, as expected, a large number of CD68 + macrophages were observed engulfing the graft in the subretinal space. In rd1 mice, macrophages were detected only in the choroid rather than the subretinal space with either surviving or dead donor cells (Fig. 2A).

Fig. 2
figure 2

Macrophage and microglia detection after transplantation. A. In rd1 mice, macrophages (CD68+) were observed in the choroid when donor cells either survived (left) or died (middle). In wild-type mice, dead donor cells were engulfed by macrophages (right). Scale bar = 10 μm. B. In trans-scleral injection group, while microglial cells surrounded surviving donor cells in rd1 mice (i), they could hardly be detected around dead cell debris (ii). In wild-type mice, microglia/macrophages engulfed dead donor cells (iii). Following trans-vitreous injection, both dead (arrows) and surviving donor cells (arrowheads) were found in the subretinal space. While macrophages/microglia engulfed the dead cells (arrows), microglia were not close to surviving cells (iv). C. A Comparison of chemokines expression between wild-type and rd1 mice showed a significant increase in the mRNA levels of ccl2, ccl3 and cxcl2 in rd1 mice. D. The comparison of cytokine levels between wild-type and rd1 mice showed no significant difference. E. No significant differences were found in oxidative stress factors between wild-type and rd1 mice. * p < 0.05, ** p < 0.01, *** p < 0.001

Full size image

As microglia migrate to the subretinal space during retinal degeneration, we investigated their distribution after photoreceptor precursors were transplanted to the same space. In rd1 mice, microglial cells were found close to the surviving donor cells, but they could hardly be identified around dead cell debris (Fig. 2Bi and Bii). Of note, although a great number of CD68 + macrophages/microglia were found in RPE mounts of rd1 mice (Supplementary Fig. 1), the microglia close to the graft were CD68 negative. In wild-type mice, microglia/macrophages could be found engulfing dead donor cells after either trans-scleral or trans-vitreous transplantation (Fig. 2Biii and Biv). In rd1 mice, the macrophages in the choroid may reflect the systemic immune reaction to foreign cells after trans-scleral injection, but the bone marrow-derived macrophages did not infiltrate the subretinal space.

Next, we extracted mRNA from RPE and attached subretinal cells after transplantation. Cytokines (TNFα, IL1β, IFNγ), chemokines (ccl2,ccl3,ccl4,cxcl2,cx3cl1) and oxidative stress factors (Prdx1, Sod1, Gpx4, mt-Atp6, Gstp1) were compared between rd1 and wild-type mice. To our surprise, the expression of inflammatory cytokines and oxidative stress factors was not elevated in rd1 mice. Only some chemokines (ccl2,ccl3,cxcl2) were increased in rd1 mice (Fig. 2C, C and E). Taken together, these results indicated that microglia closely interacted with the surviving donor cells in rd1 mice, and they didn’t exhibit phagocytosis or an inflammatory response to the graft.

Donor photoreceptor cells did not survive after microglia depletion

To eliminate microglia/macrophages, mice were fed with PLX5622, a CSF1R inhibitor, one week before transplantation. After microglia depletion, we transplanted photoreceptor precursors, and only cell debris was found in rd1 mice(n = 6) (Fig. 3A). No microglia and/or macrophages were detected around dead cell debris. To exclude the direct toxicity of PLX5622 to photoreceptor cells in vivo, we compared the thickness of the outer nuclear layer of wild-type mice with or without PLX5622 treatment, and no statistically significant difference was detected (Supplementary Fig. 2). Next, we transplanted donor cells to rd1 mice after microglia repopulation. Survived donor cells were detected, and microglial cells were close to them (Fig. 3B). These results indicated that subretinal microglial cells played an important role in graft survival in rd1 mice.

Fig. 3
figure 3

Microglia depletion and repopulation. A. In rd1 mice with microglia depletion, only dead cell debris was found in the subretinal space. B. After microglia repopulation, surviving graft could be found, and microglial cells were close to donor cells (Separate channels were inserted). C. In wild-type mice, macrophages could still be detected following PLX5622 treatment. Some donor cells survived, while others were engulfed by macrophages. D. After microglia/macrophage repopulation, all donor cells were engulfed

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In microglia-depleted wild-type mice, macrophages/microglia could still be detected after transplantation. In this situation, part of the donor cells were survived and some of them were engulfed by macrophages (Fig. 3C). After microglia/macrophage repopulation, all the donor cells were dead (Fig. 3D). These results indicated that immune modulation is critical for graft survival in wild-type mice.

Pro-neurodevelopmental effect of sub-retinal microglia

During retinal degeneration in rd1 mice, microglia migrate to the subretinal space and can be detected in RPE mounts. Microglial cells were supposed to be collected during RPE cell isolation. Thus, we extracted mRNA from the RPE of rd1 mice fed with or without PLX5622, and RNA-seq analysis was performed (log2fold change > 2, padj < 0.05). In the comparison between rd1 mice with or without microglia depletion, the expression levels of microglial markers (siglech, cx3cr1, p2ry12, and fcrl3) were higher in the latter group (Fig. 4A). In addition, markers associated with both M1 microglia (CD74 and havcr2) and M2 microglia (Mrc1 and CD163) were elevated. In the differentially expressed gene (DEGs) analysis, positive regulation of immune response (GO:0050778)(18/117 genes), regulation of defense response (GO:0031347) (18/117 genes), adaptive immune response (GO:0002250) (16/117 genes), and other immune reaction-related pathways were enriched in the rd1 mice without PLX5622 treatment (Fig. 4B). These results support the feasibility of this method to analyze subretinal microglia and indicate that the net effect of subretinal microglia was proinflammatory.

Fig. 4
figure 4

RNA-seq analysis of RPE and subretinal cells. A. Comparison between rd1 mice fed with and without PLX5622. The latter showed elevated microglia-related marker genes; B. Cluster analysis of upregulated genes between rd1 mice with and without PLX5622 treatment; C. Cluster analysis of upregulated genes between rd1 and age-matched wild-type mice; D. Comparison of genes in the axonogenesis cluster (GO:0007409) between rd1 and wild-type mice; E. Cluster analysis of upregulated genes between PLX5622-treated rd1 and wild-type mice; F. Comparison of the survival rate of photoreceptor cells in wild-type conditional medium and rd1 conditional medium

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Next, we compared rd1 mice with age-matched wild-type mice. The top differences under GO enrichment included passive transmembrane transporter activity (GO:0022803), synaptic membrane (GO:0097060), channel activity (GO:0015267), and regulation of trans-synaptic signaling (GO:0099177) (Fig. 4C). Biological processes such as axon development (GO:0061564) and axonogenesis (GO:0007409) were also upregulated in rd1 mice (Fig. 4D and Supplementary Fig. 3). These results may explain why neurite-like structures of donor photoreceptors could be found in rd1 but not in wild-type mice after transplantation (Supplementary Fig. 4). Next, we compared PLX5622-treated rd1 with wild-type mice. DEGs showed that sensory perception, eye development, and visual perception were the top different biological processes under GO enrichment instead of axonogenesis-related biological processes, confirming that these processes are microglia-related (Fig. 4E). Considering that the net effect of microglia was proinflammatory, the pro-neurodevelopmental effect was probably an indirect effect of microglia.

In a cell culture study, we collected conditional medium from RPE-choroid-sclera tissue of wild-type and rd1 mice, respectively. The survival rate of photoreceptor precursor cells cultured in different conditional media was compared. The latter showed a significantly higher survival rate than the former(Fig. 4F).This result supported the RNA-seq analysis.

Discussion

Transplantation of RPE cells has been reported in several clinical trials [33,34,35]. Immunosuppressants were commonly administered in these studies, although RPE itself can modulate the immune reaction to some extent. In photoreceptor transplantation, immune inhibition was also performed in most experiments. However, these studies still face problems such as a low survival rate and chronic immune rejection [36]. This is one of the most critical problems impeding the progression of photoreceptor replacement therapy. Researchers generated NOG-rd1/NOG-rd10 mice and IL2r-gamma-/- mice, which reduced the numbers of B and T lymphocytes and lacked natural killer cells, promoting the survival rate of donor cells [17, 37]. These studies indicated the importance of immune modulation in photoreceptor transplantation. In recent studies, however, donor RPE was reported to survive without using an immunosuppressant when transplanted to retinitis pigmentosa patients [21]. Xenotransplantation of human stem cell-generated cone photoreceptors was also reported to survive in Nrl-/- and Aipl1-/- mice without immune inhibition [22]. The degenerated mice seem to favor the survival of grafts, which sheds light on a more delicate immune modulation method.

To explore the underlying mechanism, we studied the method of trans-scleral injection as first described by Mandeep Singh et al. [10]. As the intraocular environment exhibits immune tolerance, avoiding the destruction of the blood-eye barrier was key to successful transplantation in trans-vitreous injection in wild-type mice. Trans-scleral injection, which penetrates the RPE-Bruch membrane complex, definitely breaks the outer blood-ocular barrier. However, using this method, the donor cells survived in rd1 mice [10].

The RPE serves not only as a physical barrier but also as a source of immune-suppressive molecules, contributing to the immune-privileged status of the eye [38]. Penetrating the RPE/Bruch’s membrane resulted in donor cell immune-rejection in wild-type mice, emphasizing the importance of RPE integrity in maintaining immune privilege. Compared to wild-type mice, one critical difference of retinal degenerated mice is that microglia migrate to the subretinal space [39]. In our study, we found that microglial cells protected the donor cells from immune attack even after the break of the physical barrier in rd1 mice. While microglial cells were found close to the graft, macrophages were restricted to the choroid. This is consistent with previous observations in inherited and light-induced retina degeneration models, in which tissue-resident microglia dominate the subretinal space and limit bone marrow derived macrophage infiltration [39]. This explains why trans-scleral injection didn’t result in acute immune rejection in rd1 mice following photoreceptor transplantation.

Early studies have reported that microglia are related to rosette formation in neonatal neuronal retina sheet transplantation [40]. We investigated whether microglia also play a role in the survival of isolated photoreceptor donor cells. In our study, microglia closely interacted with surviving donor cells but were not detected in dead grafts in rd1 mice. Importantly, these microglial cells were negative for CD68 and galectin-3 (Supplementary Fig. 5), suggesting a non-phagocytic and non-inflammatory feature. Microglia have been reported to monitor and protect neuronal function by generating specialized connections [41]. We observed that many processes of microglia reached out to interact with donor photoreceptor precursors (Supplementary Fig. 6). The transplantation result in rd1 mice with microglia depletion and repopulation further confirmed the critical role of subretinal microglial cells in graft survival. Taken together, these findings indicate that subretinal microglia interact with photoreceptor grafts and correlate with a better survival rate.

Microglia are commonly thought to be related to inflammation and harmful to donor cell survival. Most of these experiments were carried out in wild-type mice, where microglia in their static form would perform surveillance and scavenging roles. However, in the degenerated retina, subretinal microglia influenced by the RPE may suppress their immune activity and display an anti-inflammatory phenotype [42]. E.G.O’Koren et al. reported the presence of protective subretinal microglia by single-cell sequencing in light-induced and genetically defective retinal degeneration models [25]. However, unlike the high levels of Gal3, Cd68 and Lpl found in the previous study [28], the Gal3-negative microglia in our study seemed to play a major role(Supplementary Fig. 5). When facing transplanted donor photoreceptor precursors, the protective microglia may originate from another subgroup or the same subgroup exhibiting different features in different situations. Additionally, it is important to note that the single-cell data used in the above study were acquired by dissociating the whole retina rather than isolating cells from the subretinal space, which could potentially lead to a different conclusion.

Extracted mRNA from RPE and subretinal cells was used to perform RNA-seq analysis. In the comparison between rd1 and age-matched wild-type mice, elevated genes were enriched in biological pathways such as axonogenesis. This is probably the reason why neurite-like structures of donor cells could be commonly found in rd1 mice but not in wild-type mice. By using PLX5622 to eliminate microglia, the pro-neurodevelopment environment was diminished, confirming its association with microglia. Given that the net effect of microglia was proinflammatory, the pro-neurodevelopment effect was probably indirectly generated by microglia, which may be another reason why donor photoreceptor can survive in the subretinal space of rd1 mice. Isolated photoreceptor cells had a better survived rate in the conditional medium derived from rd1 than from wild-type mice, which also supports the RNA-seq results.

Despite obtaining better results in rd1 mice, it is important to illustrate that the total number of surviving donor cells was low, and the standard deviation was large (3419±2359). Regarding to isolation, sorting and transplantation, all the procedures were performed under equal conditions. Transplanted organs are exposed to various types of cellular stresses that compromise tissue viability [43]. When comparing organs or tissues, isolated cells may have an even lower resistance to various stresses. Furthermore, to avoid confounders, glucocorticoids and immunosuppressants were not administered, which may amplify the influence of surgical trauma and local inflammation [44].

In conclusion, subretinal microglia were supportive of the survival of donor photoreceptor in rd1 mice.

Data availability

The RNA-seq data generated by this study have been deposited in Sequence Read Archive (SRA) with the BioProject number PRJNA1135536. Other data generated or analyzed in this study are included in this published article.

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Acknowledgements

We declare that we have not used Artificial Intelligence in this study.

Funding

This study was supported by the National Natural Science Foundation of China (Grant no.82171070) (Author Chen Liang).

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  1. Qinjia Ren and Fang Lu contributed equally to this work.

Authors and Affiliations

  1. Department of Ophthalmology, West China Hospital, Sichuan University, Cheng Du, Sichuan, China

    Qinjia Ren, Fang Lu, Ruwa Hao, Yingying Chen & Chen Liang

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  1. Qinjia Ren
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Contributions

RQJ, HRW and CYY performed cell transplantation, immunofluorescence, qRT-PCR and other animal studies. LF supervised the work and reviewed the manuscript. LC directed the project, was involved in all aspects of the project, and wrote the manuscript.

Corresponding author

Correspondence to Chen Liang.

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Ethical approval

The project “The mechanism and function study of subretinal microglia/macrophage in photoreceptor precursor transplantation” were approved by the Animal Ethics Committee at the West China Hospital. Approval number: 2021961 A. Approval date:2021.3.4.

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Ren, Q., Lu, F., Hao, R. et al. Subretinal microglia support donor photoreceptor survival in rd1 mice. Stem Cell Res Ther 15, 436 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-04052-0

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Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512