Abstract
Purpose. One of the leading causes of irreversible blindness worldwide, age-related macular degeneration (AMD) is a progressive disorder leading to retinal degeneration. While several treatment options exist for the exudative form of AMD, there are currently no FDA-approved treatments for the more common nonexudative (atrophic) form. Mounting evidence suggests that mitochondrial damage and retinal pigment epithelium (RPE) cell death are linked to the pathogenesis of AMD. Human retinal progenitor cells (hRPCs) have been studied as a potential restorative therapy for degenerative conditions of the retina; however, the effects of hRPC treatment on retinal cell survival in AMD have not been elucidated. Methods. In this study, we used a cell coculture system consisting of hRPCs and AMD or age-matched normal cybrid cells to characterize the effects of hRPCs in protecting AMD cybrids from cellular and mitochondrial damage and death. Results. AMD cybrids cocultured with hRPCs showed (1) increased cell viability; (2) decreased gene expression related to apoptosis, autophagy, endoplasmic reticulum (ER) stress, and antioxidant pathways; and (3) downregulation of mitochondrial replication genes compared to AMD cybrids without hRPC treatment. Furthermore, hRPCs cocultured with AMD cybrids showed upregulation of (1) neuronal and glial markers, as well as (2) putative neuroprotective factors, responses not found when hRPCs were cocultured with age-matched normal cybrids. Conclusion. The current study provides the first evidence that therapeutic benefits may be obtainable using a progenitor cell-based approach for atrophic AMD. Our results suggest that bidirectional interactions exist between hRPCs and AMD cybrids such that hRPCs release trophic factors that protect the cybrids against the cellular and mitochondrial changes involved in AMD pathogenesis while, conversely, AMD cybrids upregulate the release of these neuroprotective factors by hRPCs while promoting hRPC differentiation. These in vitro data provide evidence that hRPCs may have therapeutic potential in atrophic AMD.
1. Introduction
Age-related macular degeneration (AMD), a progressive retinal condition, ranks as one of the principle causes of irreversible blindness across the world [1, 2]. Epidemiologic studies estimate that 10 million Americans suffer from AMD, comparable to the 12 million with cancer, and surpassing the 5 million with Alzheimer’s disease [3–6]. The pathogenesis involves two classifications of AMD, the atrophic (“dry”) form and the exudative (“wet”) form. Dry AMD is a chronic, progressive condition that begins asymptomatically with the extracellular deposition of insoluble drusen aggregates between Bruch’s membrane and the retinal pigment epithelium (RPE) [1, 3, 7]. In its advanced stage, this condition then evolves to geographic atrophy, which manifests with degeneration of the RPE and loss of photoreceptors that can cause severe blindness [1, 8]. The less common wet form of AMD emerges and often progresses rapidly in severity. This form is classified by choroidal neovascularization whereby immature blood vessels lead to bleeding and fluid leakage under the retina, causing a sudden loss of central vision [3]. Over 80% of AMD patients are classified as having the dry form, yet these patients may progress to wet AMD, causing more severe loss of vision [9]. Understanding the pathogenesis of AMD is complicated as it involves not only genetic predispositions but also at least four contributing processes, including lipofuscinogenesis, drusogenesis, localized inflammation, and choroidal neovascularization [9].
Therapy for wet AMD includes AREDS formulations and several effective anti-VEGF treatments that are FDA-approved [1, 10, 11]. On the other hand, while eating leafy green vegetables rich in antioxidants is widely recommended for dry AMD, there are no FDA-approved treatments for this condition [1, 12].
The retina is one of the highest oxygen-demanding tissues in the body and relies heavily on the mitochondrial production of ATP via oxidative metabolism [1, 13]. According to the endosymbiotic theory, the mitochondrion is an organelle that evolved from a bacterial ancestor and contains its own genome which is only transferred via the female germline [14, 15]. Mitochondrial DNA (mtDNA) is circular and double-stranded, coding for a variety of key proteins in oxidative phosphorylation [1, 15]. Due to its poor capacity for DNA repair, mtDNA is highly vulnerable to oxidative damage, leading to disruptions in energy metabolism. The result of this is oxidative stress, reduction in antioxidants, and ultimately RPE cell death. Aberrant mitochondrial function and consequent RPE cell death have been linked to a variety of ocular conditions, including AMD, diabetic retinopathy, and glaucoma [16–19].
A variety of cell- and gene-based therapies have emerged as possible restorative treatments for degenerative conditions of the retina that involve the loss of photoreceptors [20]. One reason for the interest in cell-based approaches relates to the poor innate regenerative capacity of the mammalian central nervous system, one consequence of which is that photoreceptor loss is irreversible [21]. Stem cell transplantation has recognized potential not only as a method of retinal cell replacement but also as a means of providing trophic support for host neurons, including photoreceptors [22]. For example, human embryonic stem cell-derived RPE cells have undergone clinical testing in dry AMD by Schwartz et al. [23, 24], while a different cell type, namely, hRPCs, has shown potential in the setting of photoreceptor neuroprotection and associated preservation of visual function in preclinical models of retinal degeneration [25]. In the latter example, the visual benefit is associated with the release of trophic factors from the transplanted hRPCs. Alternatively, RPCs may provide benefits through photoreceptor replacement. Those alternate strategies have led to early stage clinical trials of hRPCs in retinitis pigmentosa (RP) by jCyte (phase 1/2a NCT02320812, phase 2b NCT03073733) and ReNeuron (phase 1/2a NCT02464436).
To our knowledge, there have been no previous studies investigating the role of hRPCs in protecting AMD transmitochondrial ARPE-19 cybrid cells or AMD mitochondria. To better understand the mechanisms by which hRPCs interact with the RPE, we created our transmitochondrial cybrids via fusion of mitochondria-free ARPE-19 Rho0 cells with platelets, which contain an abundance of mitochondria, isolated from either AMD or age-matched normal patients. Previously, our group has shown that expression levels of RNA and proteins are significantly different in AMD cybrids compared to normal cybrids, despite having identical nuclei in all cell lines [26]. These expression changes are due to differences in mtDNA. Furthermore, our group has shown that AMD cybrids show increased mtDNA fragmentation, impaired levels of expression of mt transcription/replication genes, upregulated proapoptotic genes and proteins, increased mtROS levels, and decreased cellular viability in comparison to normal cybrids [1]. Our most recent studies revealed that the mitochondrial-derived peptide Humanin G and, separately, the antioxidant compound resveratrol protect AMD ARPE-19 cybrids from death [1, 10].
The current study uses a cell coculture system consisting of hRPCs and AMD cybrid cells to test the hypothesis that hRPCs would suppress the expression of harmful genes associated with AMD pathogenesis. We found that the coculture of hRPCs with AMD cybrids resulted in increased cellular viability and decreased expression levels of RNA of the apoptosis, autophagy, endoplasmic reticulum (ER) stress, and antioxidant pathways. Furthermore, coculture resulted in decreased expression of mitochondrial replication and biogenesis genes. Importantly, in examining the effects of AMD cybrids on hRPCs, we found that hRPCs responded to disease AMD cybrids with increased expression of neuroprotective factors and upregulation of glial and neuronal markers. This response was not found when cocultured with age-matched normal cybrids. Our results suggest that a bidirectional interaction occurs between hRPCs and AMD cybrids such that hRPCs release trophic factors that protect the RPE cells against the cellular changes involved in AMD pathogenesis, while AMD cybrids (with their damaged AMD mitochondria) promote the expression of neuroprotective factors by hRPCs as well as differentiation of the multipotent progenitor cells. Together, these in vitro data contribute to the mounting evidence that hRPC grafts carry potential as a candidate therapy for atrophic AMD.
2. Materials and Methods
2.1. Ethics Statement
Research involving human subjects was conducted according to the principles expressed in the Declaration of Helsinki. Informed written consent was obtained, and all research was approved by the Institutional Review Board of the University of California, Irvine (UCI IRB #2003-3131) and UC Irvine Human Stem Cell Research Oversight Committee (UCI hSCRO #2007-5935).
2.2. Creation of AMD Transmitochondrial Cybrids
Transmitochondrial cybrids were created as previously described [19]. Polyethylene glycol fusion of mitochondria-free ARPE-19 (Rho0) cells and mitochondria-rich platelets isolated from AMD patients or age-matched normal (AMD, , years; normal, , years; ) was performed to create AMD and control cybrids (Figure 1). Epidemiology information for these patients is shown in Table 1. Successful fusion was confirmed with verification of the mtDNA haplogroup profile that compared the original blood sample to the newly created cybrid cell line. Passage 5 transmitochondrial cybrids were used for all experiments.
2.3. Isolation of Human Retinal Progenitor Cells
Isolation and culture of hRPCs were performed as previously described [27–29]. Human fetal eyes (17-20 weeks gestational age) were obtained from therapeutic termination of pregnancy. Neuroretina tissues were dissected out and mechanically and enzymatically dissociated by TrypLE (Life Technologies, Grand Island, NY, USA). These cells and cell clusters were then washed, plated, and expanded in vitro. At each passage and at isolation, cell number and viability were measured using Trypan blue.
2.4. Coculture of Transmitochondrial Cybrids with hRPCs
In each experiment, the AMD cybrids cells were cultured with or without hRPCs. 0.3 million of hRPCs were plated in fibronectin-coated Transwell cell culture inserts with 1 μm pore size (Fisher Scientific, Hampton, NH, USA) and grown in Advanced DMEM/F12 medium supplemented with N2, Glutamax, EGF (20 ng/ml), and bFGF (20 ng/ml) (Life Technologies, Grand Island, NY, USA) at 37°C. Concurrently, 0.5 million of AMD cybrids were plated in six-well plates and grown in DMEM/F12 cell culture media containing 10% FBS at 37°C for 24 hr recovery. After separated incubation for 24 hr, media for both hRPCs and cybrids were replaced with fresh hRPC media. Cell culture inserts containing hRPCs were transferred to the cybrid-containing six-well plates to begin coculture that was incubated at 37°C. After 48 hr coculture, the Transwell inserts containing hRPCs were removed, and hRPCs and cybrids were trypsinized for further experimentation. To study hRPC responses, hRPCs were plated alone or cocultured with cybrids derived from healthy individuals or patients with AMD.
2.5. Cell Count and Viability Assay
Following 48 hr culture, samples of trypsinized cocultured and control cybrids were exposed to Trypan blue dye and transferred to slides for cell-counting with the Countess Automated Cell Counter (Invitrogen, Carlsbad, CA, USA). The average numbers of live cells harvested from the treatment and control groups were compared to each other using an unpaired parametric -test.
2.6. RNA Extraction and cDNA Synthesis
RNA was extracted from coculture and control cybrids using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) following the manufacturer’s protocol. Following RNA quantification using NanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA), cDNA libraries were created by reverse transcription using a Superscript VILO Master Mix (Invitrogen, Carlsbad, CA, USA) or Omniscript RT Kit (QIAGEN Inc., Valencia, CA). cDNA was diluted and stored at -20°C.
2.7. Quantitative Reverse Transcription PCR (RT-qPCR)
RT-qPCR was performed using a StepOnePlus Real-Time PCR system and QuantStudio 6 Flex system (Applied Biosystems, Carlsbad, CA, USA). QuantiTect Primer Assays (QIAGEN) and Power SYBR Green PCR Master Mix (Life Technologies, Grand Island, NY, USA) were used. Table 2 lists the primer information in detail for genes associated with apoptosis (BAX, CASP3, CASP7, and CASP9), autophagy (ATG5, ATG12, LAMP2, LC3B, and PARK2), endoplasmic reticulum stress (DDIT3 and XBP1), antioxidant (GPX3, SOD2, and NQO1), and mitochondrial replication (POLG, POLRMT, and TFAM). A TaqMan assay system was used for hRPC markers: glial lineage (GFAP), neuronal lineage (MAP2), and neuroprotection (MDK, PTN, and FGF2). Samples were run in triplicate with HMBS and GAPDH, which were used as a housekeeping gene. The ΔΔCt method was used to calculate expression fold change between the treatment group and the control group for each cybrid line.
2.8. Mitochondrial DNA Copy Number
AMD cybrids () were plated in six-well plates, and the total DNA was isolated after 48 h coculture using a DNA extraction kit (PUREGENE, QIAGEN, Valencia, CA). In order to determine mtDNA copy numbers with or without hRPC treatment, qPCR was performed using the TaqMan gene expression assays (Cat. # 4369016) with 18S gene to represent nuclear DNA and mt-ND2 gene to represent mtDNA (Cat. # 4331182, Thermo Fisher Scientific). Relative mtDNA copy numbers were determined using the ΔΔCt method.
2.9. Statistical Analysis
Results between treatment and control groups were analyzed for differences by performing one-sample -tests on the expression fold change values from the five cybrid lines for each gene, comparing the values with a hypothetical value of 1 (representing a hypothesis of no difference between the coculture and control groups). Expression fold changes were calculated using . Accordingly, fold values above 1 indicate upregulation of the gene compared to control, while fold values below 1 indicate downregulation of the gene compared to control. Statistical significance was determined at value < 0.05. The fold changes and values for comparison of differential gene expression, mitochondrial copy number, and cellular viability are shown in Table 3.
For assessing the hRPC response to diseased AMD cybrids vs. healthy age-matched normal cybrids, the relative quantification () was utilized. Tukey -test and Student -test (unpaired) were used for values between two groups, with significance determined at . All statistical analyses were performed using Prism, version 7.0 (GraphPad Software Inc.) (Figures 2–4) or JMP (Figure 5).
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3. Results
3.1. Comparison of AMD Cybrids Cocultured with hRPCs (Treatment) versus AMD Cybrids without hRPCs (Control)
3.1.1. AMD Cybrids Cocultured with hRPCs Exhibit Increased Cellular Viability
At 48 h, AMD cybrids cocultured with hRPCs demonstrated a significant increase in viability compared to the control AMD cybrids (Figure 2). The mean number of live cybrid cells harvested per well in the hRPC coculture group was , while the control AMD cybrids had ().
3.1.2. Coculture of AMD Cybrids with hRPCs Decreases Gene Expression of Apoptosis, Autophagy, ER Stress, and Antioxidant Genes in AMD Cybrids
The qRT-PCR was performed to determine the effect of hRPC coculture on the expression of genes involved in cellular damage and death pathways in AMD cybrids. Apoptosis, autophagy, ER stress, and antioxidant genes were downregulated in AMD cybrids cocultured with hRPCs compared to control AMD cybrids grown without hRPC coculture. Two of four apoptosis genes measured were significantly decreased in the treatment group compared to untreated control AMD cybrids (assigned the value of 1 and represented as the dotted line in the figures: BAX (, ), CASP3 (, ), CASP7 (, ), and CASP9 (, )) (Figure 3(a)). Four of five autophagy genes measured showed significantly lower expression levels in the treatment group compared to control: ATG5 (, ), ATG12 (, ), LAMP2 (, ), LC3B (, ), and PARK2 (, ) (Figure 3(b)). Two of two ER stress genes measured were significantly lower in the treatment group compared to the control: DDIT3 (, ) and XBP1 (, ) (Figure 3(c)). Two of three antioxidant genes measured were expressed significantly less in the treatment group compared to the control: GPX (, ), SOD2 (, ), and NQO1 (, ) (Figure 3(d)).
3.1.3. Effect of hRPCs on AMD Cybrid Mitochondria
Two of three mitochondrial replication genes had significantly lower expression levels in AMD cybrids cocultured with the hRPC cells compared to the control and untreated AMD cybrids (Figure 4(a)): POLG (, ), POLRMT (, ), and TFAM (, ). Mitochondrial DNA copy number (copy number relative to , , Figure 4(b)) was not significantly different between the hRPC treatment and control groups (assigned value of 1, dotted line in graph).
3.2. Comparison of hRPCs Cocultured with AMD Cybrids (Treatment) versus hRPCs Cocultured with Normal Cybrids (Control)
3.2.1. hRPC Response to Diseased AMD Cybrids versus Healthy Normal Cybrids
The qRT-PCR was performed to determine the differential gene expression of hRPCs when cocultured with AMD cybrids compared to coculture with age-matched normal cybrids. hRPCs responded to diseased AMD cybrids through upregulation of markers of neuronal (MAP2, , , Figure 5(a)) and glial lineage (GFAP, , , Figure 5(b)). Furthermore, hRPCs responded to the diseased AMD cybrids with elevated expression levels of putative neuroprotective factors: MDK (, , Figure 5(c)), PTN (, , Figure 5(d)), and FGF2 (, , Figure 5(e)). On the other hand, hRPCs responded to healthy cybrids with minimal changes that were not statistically significant.
4. Discussion
Cell therapy is an emerging therapeutic strategy for various forms of retinal degeneration, most of which are currently untreatable, and in recent years, a number of early stage clinical trials have been initiated [30, 31]. Compared to more conventional approaches, one of the challenges facing cell therapy is to delineate the mechanism of action, which can be complex and difficult to assess using established techniques. This is particularly true in the setting of cytoprotection mediated by innate paracrine effects, such as retinal neuroprotection induced by RPCs. In this study, we use a novel in vitro coculture system of hRPCs combined with a transmitochondrial ARPE-19 cybrid model of AMD to investigate the effects of human retinal progenitor cells on gene expression changes and cellular damage seen in AMD. Through cell-based assays and molecular biology techniques, we found that hRPCs suppressed gene expression changes seen in AMD pathogenesis and protected AMD cybrids from cellular damage and death. Additionally, our data showed that hRPCs respond to AMD cybrids through cellular differentiation and increased expression of putative neuroprotective factors. These findings reveal the existence of two-way signaling between hRPCs and AMD cybrids that has potential therapeutic significance, particularly for the use of hRPCs in dry AMD.
Previous studies using mtDNA-deficient Rho0 ARPE-19 cells have shown that mitochondrial dysfunction plays a role in altering nuclear gene expression related to drusen deposition, inflammation, lipid receptors, and extracellular matrix proteins [1, 32]. This suggests that the oxidative damage to mtDNA in AMD is implicated in disease pathogenesis. The current study utilized the same host cell line of mitochondria-free Rho0 ARPE-19 cells to create transmitochondrial cybrids containing mitochondria isolated from either AMD patients or age-matched normal patients. Our group previously found that AMD transmitochondrial cybrids showed decreased cellular viability, reduced mtDNA copy numbers, decreased expression of mitochondrial transcription/replication genes, and upregulated gene and protein expression of autophagy, apoptosis, and ER stress compared to cybrids possessing age-matched normal mitochondria [1]. Consequently, these gene expression changes related to RPE cell damage and death in AMD cybrids are attributed to the diseased AMD mitochondria since the nuclei are identical in all of the cybrid cell lines. We previously showed these cybrids to be reliable, personalized models for each patient that are useful for screening mitochondria-targeting drugs [1]. A variety of studies have examined the roles of various treatments as therapeutic targets for AMD, including antithyroid drugs, autophagy regulation, pigment epithelium-derived factor (PEDF), and antioxidant compounds such as esculetin [33–36]. Our most recent studies revealed that the mitochondrial-derived peptide Humanin G and, separately, the antioxidant compound resveratrol protect AMD ARPE-19 cybrids from death [1, 10].
4.1. Role of hRPCs in Protecting AMD Cybrids from Cellular Damage and Death
In order to prevent cellular and mitochondrial damage seen in AMD, we hypothesized that coculturing transmitochondrial AMD cybrids with hRPCs would protect the cybrids from cellular damage and death. In that regard, AMD and normal cybrids were cocultured with hRPCs, and the cellular viability was measured. As hypothesized, coculture of AMD cybrids with hRPCs led to a significant increase in numbers of viable cells (), confirming that hRPCs protected AMD cybrids from mitochondria-driven RPE cell death. These results are consistent with previous findings that media from hRPCs inhibited RPE cell death in vitro, suggesting that hRPCs secrete antiapoptotic molecules that rescue RPE cells from oxidative damage [37]. Furthermore, Luo et al. found that hRPCs transplanted into the eyes of RCS rats had improved visual acuity and higher cell counts in the outer nuclear layer compared to vehicle-treated control eyes [28].
After confirming the cytoprotective effects of hRPCs on AMD cybrids, we then used qRT-PCR to investigate the role of hRPCs on gene expression changes related to RPE cell death in these cybrids. Gene expression levels of apoptosis, autophagy, ER stress, and antioxidant genes were significantly downregulated in AMD cybrids cocultured with hRPCs compared to control AMD cybrids without hRPC coculture. These findings suggest that coculture with hRPCs prevented the upregulation of genes involved in cellular damage and death pathways in AMD cybrids. Our findings are in agreement with the protective effects of other stem cells on the degenerating retina, such as induced pluripotent stem cell-derived RPE cells [38].
We next examined the effects of hRPC coculture on AMD cybrid mitochondria. AMD cybrids cocultured with hRPCs showed significant downregulation of POLG and TFAM genes, which are involved in mtDNA replication. Moreover, the mtDNA copy numbers were similar in the hRPC-treated AMD cybrid and untreated AMD cybrids. These findings suggest that the beneficial effects that hRPCs had on the AMD cybrids did not involve increased mtDNA replication and/or mitochondrial biogenesis. It is likely that the hRPCs may modulate the AMD mitochondrion via its other known functions, including changes in oxidative phosphorylation and bioenergetics, or the retrograde signaling (mitochondria to nucleus) that regulates apoptosis along with inflammation pathways and calcium homeostasis. Further studies will be needed to determine the underlying mechanism(s) by which hRPCs rescue the cybrids possessing damaged AMD mitochondria. Other studies have reported reversal of mitochondrial dysfunction in retinal ischemia rats and RPE cells via intravenous mesenchymal stem cells (MSCs) and coculture with MSCs, respectively [39]. Mansergh et al. used retinal progenitor cells as cell therapy to successfully preserve retinal function in Leber’s hereditary optic neuropathy, the most prevalent primary mitochondrial disorder [40]. Our findings show that hRPCs are capable of protecting the cybrid cell lines that contain dysfunctional AMD mitochondria, but mechanisms of action are unclear at this time.
4.2. Role of AMD Cybrids in hRPC Neuronal/Glial Differentiation and Neuroprotection
Having confirmed the restorative effects of hRPCs on AMD cybrids, we then found that hRPCs responded to AMD cybrids with increased expression of putative neuroprotective factors and upregulation of glial and neuronal markers. Importantly, age-matched normal cybrids were not capable of stimulating retinal progenitor cells in a similar way. Therefore, it appears that the presence of mitochondria from AMD was adequate to recruit hRPCs for protection. Our data suggest that at least some stem-like cells are capable of rescuing RPE cells and that this effect can be induced or amplified by signals from the target cell. In addition to neuroprotection, RPCs might be useful for cell replacement. One application of this approach is suggested by Bartsch et al. from their findings that subretinal transplantation of premature retinal cells not only integrated into the outer nuclear layer but also differentiated into mature photoreceptors [41]. Our results showing hRPC upregulation of glial and neuronal markers indicate that these cells begin to lose multipotency when cocultured with AMD cybrids. While this data does not address possible differentiation into photoreceptors, the induction of differentiation seen could provide benefits in terms of therapeutic safety for a strictly neuroprotective approach by limiting the proliferation of transplanted hRPCs.
In terms of neuroprotection, the basic fibroblast growth factor (bFGF) is a cytokine with known trophic effects in the retina, including rescue of photoreceptors in the RCS rat model [42]. Midkine (MDK) is a cytokine known to play an important role in retinal development [43]. MDK has also been reported to rescue photoreceptors [44] and appears to play a role in modulating the local tissue response to retinal injury [45]. Pleiotrophin (PTN) is a related cytokine that is highly expressed by human neural progenitor cells, including hRPCs [46].
Taken together, these data suggest that a bidirectional interaction exists between hRPCs and AMD cybrids such that hRPCs release trophic factors that are protective against the cellular changes involved in AMD pathogenesis, while AMD cybrids provide signals that result in hRPC differentiation and elevated expression of trophic factors.
4.3. New Paradigm for Using a Stem Cell-Based Approach for Atrophic AMD
The mechanism of action by which hRPC coculture exerts a cytoprotective effect on AMD cybrids remains to be elucidated. One possible mechanism may be through protection of elevated numbers of mitochondria. Previous studies demonstrated that AMD mitochondria and primary RPE mitochondria can be rescued by pretreatment with the mitochondrial-derived peptide Humanin [1, 47]. However, our study showed that hRPC coculture did not upregulate mtDNA replication genes or increase mtDNA copy numbers. Another possible mechanism aligns with the early stem cell theory based on stem cell differentiation in the transplantation site, leading to replacement of damaged tissue [40]. Enthusiasm for pursuing this mode of action has tended to wane in recent years in favor of an alternative mechanism involving a paracrine mode of action [48–50]. The premise of the paracrine theory is that stem cell-secreted therapeutic trophic factors provide benefit to injured host tissue via enhancement of natural repair processes, structural repair through physical contact, exertion of a cytoprotective effect (as suggested by decreased gene expression of RPE cell death), and secretion of cytokines, other extracellular proteins, or exosomes. Of note, findings from the current study provide the first evidence that therapeutic-like benefits may be obtained using a stem-cell based approach in atrophic AMD. A combination of hRPC-secreted trophic factors may be aiding recovery from AMD mitochondria-induced RPE damage and therefore may provide a candidate therapy for atrophic AMD. Identification of such paracrine factors would permit testing for therapeutic benefits independent from cell transplantation, which could bypass cell sourcing and a variety of other issues pertaining to cell transplantation.
In conclusion, this study demonstrates the protective role of hRPCs against cell death in AMD transmitochondrial cybrids. Simultaneously, AMD cybrids promote the differentiation of hRPCs and upregulate their expression of putative neuroprotective factors. Our findings support the hypothesis that hRPCs provide a significant cell survival effect with high potential as a candidate therapy for the treatment of atrophic AMD. These results also highlight the bidirectional interaction between hRPCs and AMD cybrids via secretion of specific trophic factors, whose potential beneficial properties should be investigated in future studies. Furthermore, this type of coculture method might have broader use in the setting of assay development, not only for characterization of paracrine interactions and cell therapy product testing, but also for personalized medicine, e.g., to predict individual responses to a particular cell-based drug product. In addition to hRPCs, the method could potentially be adapted to the testing of a range of drug/cell products as well as cellular models of disease indications, not limited to retinal or eye diseases.
Data Availability
The data used to support the findings of this study are available from the corresponding author upon reasonable request.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Authors’ Contributions
Jeffrey J. Yu and Daniel B. Azzam contributed equally to this work.
Acknowledgments
This work was supported by the Discovery Eye Foundation; Polly and Michael Smith; Edith and Roy Carver; Iris and B. Gerald Cantor Foundation; Max Factor Family Foundation; and NEI R01 EY0127363 (MCK). This work was supported in part by an Unrestricted Departmental Grant from Research to Prevent Blindness. We acknowledge the support of the Institute for Clinical and Translational Science (ICTS) at the University of California, Irvine. J. J. Yu is an Arnold and Mabei Beckman Fellow in Retinal Degeneration.