August 2016
Volume 57, Issue 10
Open Access
Retinal Cell Biology  |   August 2016
Secretion Profile of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium During Wound Healing
Author Affiliations & Notes
  • Whitney A. Greene
    Ocular Trauma Task Area US Army Institute of Surgical Research, Joint Base San Antonio-Fort Sam Houston, Texas, United States
  • Teresa A. Burke
    Ocular Trauma Task Area US Army Institute of Surgical Research, Joint Base San Antonio-Fort Sam Houston, Texas, United States
  • Elaine D. Por
    Ocular Trauma Task Area US Army Institute of Surgical Research, Joint Base San Antonio-Fort Sam Houston, Texas, United States
  • Ramesh R. Kaini
    Ocular Trauma Task Area US Army Institute of Surgical Research, Joint Base San Antonio-Fort Sam Houston, Texas, United States
  • Heuy-Ching Wang
    Ocular Trauma Task Area US Army Institute of Surgical Research, Joint Base San Antonio-Fort Sam Houston, Texas, United States
  • Correspondence: Whitney A. Greene, Ocular Trauma, US Army Institute of Surgical Research, 3698 Chambers Pass, Building 3611, JBSA Fort Sam Houston, TX 78234-7767, USA; whitney.a.greene2.vol@mail.mil
  • Heuy-Ching Wang, Ocular Trauma, US Army Institute of Surgical Research, 3698 Chambers Pass, Building 3611, JBSA Fort Sam Houston, TX 78234-7767, USA; heuy-ching.h.wang.civ@mail.mil
Investigative Ophthalmology & Visual Science August 2016, Vol.57, 4428-4441. doi:10.1167/iovs.16-19192
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      Whitney A. Greene, Teresa A. Burke, Elaine D. Por, Ramesh R. Kaini, Heuy-Ching Wang; Secretion Profile of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium During Wound Healing. Invest. Ophthalmol. Vis. Sci. 2016;57(10):4428-4441. doi: 10.1167/iovs.16-19192.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The purpose of this study was to characterize the secretion profile of induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. iPS-RPE was used to develop an in vitro wound healing model. We hypothesized that iPS-RPE secretes cytokines and growth factors which act in an autocrine manner to promote migration and proliferation of cells during wound healing.

Methods: iPS-RPE was grown in transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Levels of Ki-67, β-catenin, e-cadherin, n-cadherin, and S100A4 expression were analyzed by immunofluorescent labeling. Cell culture medium samples were collected from both the apical and basolateral sides of the transwells every 72 hours for 21 days. The medium samples were analyzed using multiplex ELISA to detect secreted growth factors and cytokines. The effects of conditioned medium on collagen gel contraction, cell proliferation, and migration were measured.

Results: iPS-RPE underwent epithelial-mesenchymal transition (EMT) during wound healing as indicated by the translocation of β-catenin to the nucleus, cadherin switch, and expression of S100A4. GRO, GM-CSF, MCP-1, IL-6, and IL-8 were secreted by both the control and the wounded cell cultures. VEGF, FGF-2, and TGFβ expression were detected at higher levels after wounding than those in control. The proteins were found to be secreted in a polarized manner. The conditioned medium from wounded monolayers promoted collagen gel contraction, as well as proliferation and migration of ARPE 19 cells.

Conclusions: These results indicate that after the monolayer is wounded, iPS-RPE secretes proteins into the culture medium that promote increased proliferation, contraction, and migration.

Immediately after injury, a cascade of events, including inflammation, new tissue formation, and tissue remodeling, is initiated to facilitate reconstruction of the wound. Release of cytokines and growth factors stimulates proliferation of fibroblasts, which migrate into the wound area and deposit extracellular matrix (ECM) components. Many growth factors have been shown to have beneficial effects during wound healing, including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and transforming growth factor-β (TGFβ).1,2 
Retinal pigment epithelium (RPE) is composed of pigmented cells that are nonproliferative under normal conditions3; however following injury to RPE, the cells undergo epithelial-mesenchymal transition (EMT) and convert to nonpigmented fibroblasts.4 The fibroblasts migrate into the vitreous space where they deposit ECM components to form epiretinal membranes.5 Contraction of epiretinal membranes causes secondary retinal detachment in a condition known as proliferative vitreoretinopathy (PVR).6,7 Currently, surgical removal of membranes is the only treatment option for PVR patients. Results may be anatomically successful, but visual outcomes are often poor with high recurrence rates.810 
RPE derived from induced pluripotent stem cells (iPS-RPE) displays the phenotype and functions of in vivo RPE and provides a physiologically relevant cell type with which to study wound healing.11,12 iPS-RPE was used to conduct in vitro wound healing assays. Expression and localization of β-catenin, e-cadherin, n-cadherin, and S100A4 were examined to confirm onset of EMT. Conditioned medium samples from control and wounded iPS-RPE monolayers were analyzed to detect cytokines and growth factors. The ability of the conditioned medium to stimulate collagen gel contraction, cell proliferation, and migration was measured. Results of these studies will provide insight into cellular events that lead to development of PVR following retinal injury. 
Methods
Cell Culture and Differentiation of iPS Cells
ARPE19 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco modified Eagle medium-F12 medium (DMEM-F12; Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS). iPS-RPE was derived as previously described.11,12 Briefly, human iPS cells (IMR-90-1;WiCell Research Institute, Madison, WI, USA) were cultured on Matrigel-coated (BD Biosciences, San Jose, CA, USA) in six-well plates and maintained in mTeSR1 medium (Stem Cell Technologies, Vancouver, BC, Canada). To initiate differentiation, The mTeSR1 medium was replaced with differentiation medium consisting of 10% knockout serum replacement (Life Technologies), 0.1 mM β-mercaptoethanol, 0.1 mM nonessential amino acids, 2 mM glutamine, and 10 μg/mL gentamicin in DMEM-F12 medium. Pigmented colonies were collected and expanded. Enriched iPS-RPE was cultured in fetal RPE (fRPE) medium composed of minimum essential medium, N1 supplement, glutamine, nonessential amino acids, 0.25 mg/mL taurine, 10 ng/mL hydrocortisone, 13 ng/mL triiodothyronine, and 15% FBS. iPS-RPE at passages 4 and 5 were used for all experiments. 
Wound Healing Assay
The experimental design for the wound healing assays and medium collection is shown in Figure 1. Briefly, iPS-RPE was seeded onto Matrigel-coated 30-mm2 transwell inserts with 0.45-μm pores (Corning Co., Corning, NY, USA). Cells were cultured in fRPE medium until fully confluent and pigmented, approximately 60 days. Fresh fRPE medium was added to transwells at 72 hours before initiation of the assay. On assay Day 0, medium was collected from apical and basolateral chambers and frozen at −20°C. Fresh medium was added to the transwells, and wounds were created using a sterile 200-μL pipet tip to scratch the monolayer. Every 72 hours until Day 21, conditioned apical and basolateral medium samples were collected and frozen at −20°C. Medium was replaced with the same volume of fresh medium at each time point. 
Figure 1
 
Experimental design to analyze secretion profile of iPS-RPE during wound healing. Culture medium was collected from both the apical and basolateral chambers of the transwells every 72 hours from Day 0 until Day 21. Culture medium samples were then subjected to conventional and multiplex ELISA to compare the secretion profiles of the control unscratched cells compared to the scratched cells.
Figure 1
 
Experimental design to analyze secretion profile of iPS-RPE during wound healing. Culture medium was collected from both the apical and basolateral chambers of the transwells every 72 hours from Day 0 until Day 21. Culture medium samples were then subjected to conventional and multiplex ELISA to compare the secretion profiles of the control unscratched cells compared to the scratched cells.
Immunofluorescence and Microscopy
iPS-RPE wound healing assay was performed as described above. Images were acquired every 72 hours from Day 0 until Day 21, using light microscopy (model CK2; Olympus, Center Valley, PA, USA) at 40× magnification. To measure surface area of transwells occupied by iPS-RPE during wound healing, images were examined using ImageJ software (http://imagej.nih.gov/ij; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). Images were converted to 8-bit gray scale, and thresholds were established to identify objects for analysis. Percentage of total surface area of transwells occupied by cells was converted square millimeters based on known surface area of transwells. Total surface area calculations were used to determine final concentrations of secreted factors for each time point. In separate experiments, expression levels of Ki-67, β-catenin, e-cadherin, n-cadherin, and S100A4 were visualized by immunofluorescent labeling. iPS-RPE cells were fixed in 4% paraformaldehyde on Days 0 and 21 and processed for immunofluorescence following standard protocol. Ki-67, β-catenin, e-cadherin, n-cadherin, and S100A4 primary antibodies were purchased from Abcam (Cambridge, MA, USA). Secondary antibodies were purchased from Molecular Probes (Invitrogen, Grand Island, NY, USA). Images were acquired using fluorescence microscopy (model BX53; Olympus). 
ELISA to Detect Secreted Proteins
Cell culture medium samples collected from wound healing assays were analyzed by ELISA and multiplex ELISA to detect secreted proteins. HCYTOMAG-60K-14 human cytokine/chemokine 14-plex panel, TGFBMAG-64K-03 3-plex panel, HAGP1MAG-12K-02 angiogenesis/GF panel, HCYTOMAG-60K-07 human cytokine panel, HNDG2MAG-36K human degenerative disease panel 2 (EMD Millipore, Billerica, MA, USA), and human PDGF-CC Quantikine ELISA kits (R&D Systems, Minneapolis, MN, USA) were used according to the manufacturers' directions. Minimum detectable concentrations (MDC) for each analyte are shown in Table 1. Data was analyzed with Bio-Plex data Pro software (Bio-Rad, Hercules, CA, USA). Heat maps were generated to show changes in protein secretion over time. The amount of analyte detected at each time point was used to calculate the mean for that analyte. Time points with higher amounts of analyte relative to the mean were assigned the color red. Time points with lower amounts of analyte relative to the mean were assigned the color green. Time points unchanged from the mean were assigned the color black. 
Table 1
 
Minimum Detectable Concentrations of Growth Factors and Cytokines Analyzed in This Study
Table 1
 
Minimum Detectable Concentrations of Growth Factors and Cytokines Analyzed in This Study
Cell Proliferation Assay
ARPE19 cells were seeded at 2 × 103 cells per well in 96-well plates in DMEM plus 10% FBS. After 16 hours, culture medium was replaced with 100 μL per well of conditioned medium collected from apical chambers of the wound healing assay. Fresh fRPE medium was included as a negative control. After 48 hours, cell proliferation was determined by measuring DNA content by using CyQuant cell proliferation kit (Life Technologies). Results were read using a multiwell plate reader (Synergy MX model; BioTek, Winooski, VT, USA) set at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. 
Collagen Gel Contraction Assay
Ability of conditioned medium from the apical chambers of the wound healing assay to induce contraction was measured using commercially available three-dimensional (3D) collagen gel contraction assays (Cell BioLabs, San Diego, CA, USA). Aliquots of 2 × 105 ARPE19 cells/mL were suspended in 2 mg/mL soluble collagen. One milliliter per well of collagen cell solution was placed in a 24-well plate and incubated for 1 hour at 37°C. Gels were released from sides of the well using 200-μL pipet tips. One milliliter of conditioned medium collected from the wound healing assay was added to each well. Fresh fRPE medium was included as a negative control. Collagen gels were observed using a fluorescent lightbox equipped with a 60-W bulb. The gels were measured in millimeters immediately after release (Time 0) and again at 48 hours after release. Contraction was determined by subtracting the 48-hour measurement from the Time-0 measurement of each gel. Results are percentage of contraction at 48 hours post release. 
Migration and Invasion Assay
Cell migration was measured using a transwell-based cell migration assay. Aliquots of 3 × 104 ARPE19 cells were seeded onto Biocoat invasion chambers (Corning) with 8.0-μm pores in 24-well plates. One milliliter of conditioned medium collected during the wound healing assay was placed in the basolateral chamber, and 500 μL of DMEM-F12 medium without FBS was placed in the upper chamber. Cells were incubated at 37°C. At 24 hours, both the upper and the lower chambers were fixed with 4% paraformaldehyde. Cells were labeled with 4′,6-diamidino-2-phenylindole to enable visualization of cell nuclei. Chambers were imaged using fluorescence microscopy at 200× magnification. Total number of cells that had migrated from upper to lower chambers was quantified. 
Statistical Analysis
Each experiment was performed twice. A minimum of two replicates was analyzed for each experiment. Data are mean ± SEM. To analyze changes in secretion over time, all post wound measurements were compared to baseline Day-0 measurements by repeated measures 2-way ANOVA using Prism software (GraphPad, La Jolla, CA, USA). All other statistical parameters were evaluated with paired Student t-tests for comparison between two groups. Statistical significance was set at a P value of <0.05. 
Results
iPS-RPE Wound Healing Assay
iPS-RPE was grown on transwells until pigmented and fully confluent. Cell monolayers were scratched to induce a wound. The response of the iPS-RPE to wounding was monitored by light microscopy for 21 days. As seen in Figure 2, within 3 days of injury, cells began to migrate into the wound area. Within 21 days of wounding, large numbers of iPS-RPE had transdifferentiated, obtained fibroblastic phenotype, and migrated into the wound area. Some areas of the wound were filled completely with fibroblasts. Brightfield images were further analyzed to measure total surface area occupied by iPS-RPE before wounding and during the wound healing process. As seen in Figure 2B, 100% of the transwell surface area was occupied by iPS-RPE at the time of wounding. Induction of the wound reduced the surface area of the monolayers from an average of 29.98 mm2 before scratch to 20.31 mm2 after scratch. Over the course of the wound healing assay, the surface area of the monolayers gradually increased to an average 25.59 mm2 by Day 21. 
Figure 2
 
Wound healing assay with iPS-RPE. iPS-RPE grown to confluency on transwells were scratched to create a wound. Wound healing was monitored for 21 days. Images were acquired at an original magnification of 40× (A). Images were analyzed using ImageJ software to determine total surface area occupied by iPS-RPE before and during the wound healing assay. (B) Surface area of the monolayers is shown in square millimeters.
Figure 2
 
Wound healing assay with iPS-RPE. iPS-RPE grown to confluency on transwells were scratched to create a wound. Wound healing was monitored for 21 days. Images were acquired at an original magnification of 40× (A). Images were analyzed using ImageJ software to determine total surface area occupied by iPS-RPE before and during the wound healing assay. (B) Surface area of the monolayers is shown in square millimeters.
Results shown in Figure 2 indicate that the cells repopulated the wound region, which eventually led to wound closure. To understand the mechanism of repopulation, we examined cell proliferation by immunofluorescent labeling using the cell proliferation marker Ki-67.13 As seen in Figure 3A, before wounding, Ki-67 was not detectable. However, after wounding, cells at the wound edge were positive for Ki-67, indicating that iPS-RPE re-enters the cell cycle and proliferates. These results suggest that iPS-RPE is undergoing EMT, the process in which quiescent nonproliferating epithelial cells convert to motile, proliferative mesenchymal phenotype. EMT can be triggered by various stimuli such as growth factor exposure or loss of contact inhibition, such as after an injury. Either growth factor exposure or loss of contact inhibition can trigger EMT by inducing translocation of β-catenin from the plasma membrane to the nucleus, where it activates transcription of pro-EMT genes.1417 To confirm EMT was initiated after wounding, we performed immunofluorescence analysis to visualize localization of β-catenin. As seen in Figure 4, on Day 0 pre-wounding, β-catenin was localized to cell membranes and cell–cell junctions. However within 14 days after wounding, β-catenin translocated from the plasma membrane to nuclei in cells at the wound edge (Figs. 4B, 4C). In addition to β-catenin translocation, other alterations in protein expression occurred during EMT, including loss of e-cadherin, increased n-cadherin (cadherin switch),18 and increased S100A4 expression.19,20 To further confirm that EMT is involved in wound healing response of iPS-RPE, we analyzed cells by using immunofluorescence to detect e-cadherin, n-cadherin, and S100A4. As seen in Figure 5A, e-cadherin was localized to plasma membranes in control cells, whereas scratched cells exhibited lower levels of and less organized e-cadherin. Although e-cadherin expression was reduced, n-cadherin expression increased after wounding (Fig. 5B). In addition, as seen in Figure 5C, wounding also induced an increase in S100A4 expression. Furthermore, morphology of the cells at the wound edge and in the wound region was noticeably different from not only unscratched control cells but also from cells at the center of the monolayer, away from the wound. Cells at the wound edge had converted from hexagonal, pigmented phenotype to the unpigmented fibroblast phenotype (Figs. 4B, 5). In total, these results suggest that iPS-RPE initiated EMT to facilitate migration and proliferation of cells after wounding. 
Figure 3
 
Proliferation of iPS-RPE during wound healing. iPS-RPE before (upper panel) and 21 days after (lower panel) wounding were analyzed for expression of the cell proliferation marker Ki-67 (A). Original magnification 100×. The average number of Ki-67-positive cells per field of view is indicated in the bar graph (B).
Figure 3
 
Proliferation of iPS-RPE during wound healing. iPS-RPE before (upper panel) and 21 days after (lower panel) wounding were analyzed for expression of the cell proliferation marker Ki-67 (A). Original magnification 100×. The average number of Ki-67-positive cells per field of view is indicated in the bar graph (B).
Figure 4
 
iPS-RPE undergoes EMT during wound healing. The cells at the wound edge were labeled to visualize β-catenin (red) (A). The translocation of β-catenin from the plasma membrane to the cytoplasm and nucleus at Day 14 indicates the process of EMT at the wound edge (B). Original magnification 200×. To enable visualization of nuclear beta-catenin, cells from the wound edge (B) are shown in the insets (C).
Figure 4
 
iPS-RPE undergoes EMT during wound healing. The cells at the wound edge were labeled to visualize β-catenin (red) (A). The translocation of β-catenin from the plasma membrane to the cytoplasm and nucleus at Day 14 indicates the process of EMT at the wound edge (B). Original magnification 200×. To enable visualization of nuclear beta-catenin, cells from the wound edge (B) are shown in the insets (C).
Figure 5
 
Expression of EMT markers by iPS-RPE during wound healing. Expression levels of e-cadherin (A), n-cadherin (B), and S100A4 (C) were examined by immunofluorescence analysis. Control cells before wounding are shown in the upper panels of each figure. Cells in the wound region at 21 days post scratch are shown in lower panels. Original magnification (A) 400×; (B) 200×; (C) 100×.
Figure 5
 
Expression of EMT markers by iPS-RPE during wound healing. Expression levels of e-cadherin (A), n-cadherin (B), and S100A4 (C) were examined by immunofluorescence analysis. Control cells before wounding are shown in the upper panels of each figure. Cells in the wound region at 21 days post scratch are shown in lower panels. Original magnification (A) 400×; (B) 200×; (C) 100×.
Secretion of Cytokines and Growth Factors During Wound Healing
The experiments described in Figures 2 through 5 were conducted without addition of additional growth factors to cell culture medium, suggesting that wounding induces secretion of cytokines and growth factors. Secreted factors may act in an autocrine fashion to stimulate EMT, leading to proliferation and migration of cells into the wound area. To test this hypothesis, iPS-RPE was grown on transwells until pigmented and fully confluent. Cell monolayers were scratched to induce a wound. Culture medium samples were collected every 72 hours from both the apical and basolateral chambers of transwells. Conditioned medium was analyzed using conventional ELISA and multiplex ELISA to detect secreted growth factors and cytokines. As shown in Figure 6, VEGF, MCP-1, IL-8, and IL-6 were detected in conditioned medium from iPS-RPE. Secretion of these factors occurred in a polarized fashion, as VEGF, MCP-1, and IL-6 were found predominately in apical chambers of both the control and wounded cells, whereas IL-8 was detected at higher levels in basolateral chambers. VEGF secretion peaked at 3 days after wounding, as 247.16 ± 56.78 pg/mL/mm2 was detected in medium collected from apical chambers compared to 73.61 ± 43.22 pg/mL/mm2 from same-day matched controls (Fig. 6B). Apical secretion of MCP-1 also increased after wounding compared to that in control cells, with 21.36 ± 1.29 pg/mL/mm2 versus 15.46 ± 2.75 pg/mL/mm2 detected on Day 3. Basolateral secretion of MCP-1 increased after wounding, with 12.94 ± 2.17 pg/mL/mm2 detected in medium from scratched cells compared to same-day matched controls (7.42 ± 0.09 pg/mL/mm2) on Day 3. In addition, apical secretion of IL-8 decreased after wounding (141.97 ± 4.38 fg/mL/mm2 vs. 252.88 ± 7.38 fg/mL/mm2, respectively; Day 12), whereas basolateral secretion increased on Day 3 and then returned to baseline. Overall secretion of IL-6 was increased after wounding, with highest levels detected on Days 6 and 9 in apical and basolateral chambers, respectively (Figs. 6B, 6C). Secretion patterns of other growth factors and chemokines, including EGF, FGF-2, GM-CSF, GRO, PDGF-A/B, BMP-9, HGF, MIP-4, MIP-1α, MDC, IL-17α, IL-3, IFNγ, eotaxin, and PEDF were also analyzed. As seen in Figure 7, GRO, GM-CSF, and FGF-2 were detected predominantly in the apical chamber. Generally, secretion of GRO, GM-CSF, and FGF-2 increased after wounding, with higher levels detected in apical and basolateral chambers of wounded cells (Figs. 7B, 7C). PEDF was secreted by both the control and the wounded cells, without any significant differences in secretion after wounding (data not shown), whereas EGF, PDGF-A/B, BMP-9, HGF, MIP-4, MIP-1α, MDC, IL-17α, IL-3, IFNγ, and eotaxin were not detectable in medium from either control or wounded cells (data not shown). 
Figure 6
 
Secretion of VEGF, MCP-1, IL-8, and IL-6 by iPS-RPE during wound healing. Heat map depiction of multiplex ELISA results (A). Bar graphs represent the levels of VEGF, MCP-1, IL-8, and Il-6 detected in medium from the apical chamber (B) and basolateral chamber (C). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Figure 6
 
Secretion of VEGF, MCP-1, IL-8, and IL-6 by iPS-RPE during wound healing. Heat map depiction of multiplex ELISA results (A). Bar graphs represent the levels of VEGF, MCP-1, IL-8, and Il-6 detected in medium from the apical chamber (B) and basolateral chamber (C). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Figure 7
 
Secretion of GRO, GM-CSF, and FGF-2 by iPS-RPE during wound healing. Heat map depiction of multiplex ELISA results (A). Bar graphs represent the levels of GRO, GM-CSF, and FGF-2 detected in medium from the apical chamber (B) and basolateral chamber (C). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Figure 7
 
Secretion of GRO, GM-CSF, and FGF-2 by iPS-RPE during wound healing. Heat map depiction of multiplex ELISA results (A). Bar graphs represent the levels of GRO, GM-CSF, and FGF-2 detected in medium from the apical chamber (B) and basolateral chamber (C). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Secretion of PDGF-C During Wound Healing
Although PDGF-A/B was not detected in either control or wounded cell medium, PDGF-C was detected in medium from both the control and wounded cells (Fig. 8A). Notably, although apical secretion of PDGF-C was increased after wounding on Day 9, wounded cells secreted significantly less PDGF-C to the basolateral chamber than same-day matched controls from Days 3 to 21. Specifically, only 129.42 ± 39.0 fg/mL/mm2 was detected in basolateral medium from scratched cells compared to 279.65 ± 40.3 fg/mL/mm2 detected in medium from same-day matched controls at Day 15 (Fig. 8B). 
Figure 8
 
Secretion of PDGF-C by iPS-RPE during wound healing. Bar graphs depict the results of ELISA to detect PDGF-C in the apical chamber (A) and basolateral chamber (B). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Figure 8
 
Secretion of PDGF-C by iPS-RPE during wound healing. Bar graphs depict the results of ELISA to detect PDGF-C in the apical chamber (A) and basolateral chamber (B). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Secretion of TGFβ During Wound Healing
Secretion of TGFβ by iPS-RPE was also analyzed (Fig. 9). All three isoforms of TGFβ were detected in medium from control and wounded cells. However, apical secretion of TGFβ-1 and TGFβ-2 increased in response to wounding. Apical secretion of TGFβ-1 began to increase within three days after wounding, with 17.2 ± 0.15 pg/mL/mm2 detected compared to 10.5 ± 0.35 pg/mL/mm2 from same-day matched controls, and remained elevated until the final time point at Day 21. Secretion of TGFβ-2 was highest at three days after scratch, with 21.65 ± 0.85 pg/mL/mm2 detected in apical chambers, and remained elevated until Day 21. As seen in Figure 9C, basolateral secretion levels of TGFβ-1 (8.31 ± 0.03 pg/mL/mm2 vs. 5.7 ± 0.14 pg/mL/mm2, respectively) and TGFβ-2 (22.44 ± 0.69 pg/mL/mm2 vs. 16.10 ± 0.07 pg/mL/mm2) were also significantly altered by wounding. Finally, apical secretion of TGFβ-3 showed an increase at Day 3 after wounding compared to same-day matched controls (90.13±5.19 pg/mL/mm2 vs. 46.38 ± 0.85 pg/mL/mm2, respectively), whereas basolateral secretion was not affected by wounding. 
Figure 9
 
Secretion of TGFβ isoforms by iPS-RPE during wound healing. Heat map depiction of multiplex ELISA results (A). Bar graphs depicting TGFβ-1, -2, and -3 isoforms detected in culture medium from the apical chamber (B) and basolateral chamber (C). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Figure 9
 
Secretion of TGFβ isoforms by iPS-RPE during wound healing. Heat map depiction of multiplex ELISA results (A). Bar graphs depicting TGFβ-1, -2, and -3 isoforms detected in culture medium from the apical chamber (B) and basolateral chamber (C). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Effects of Conditioned Medium From Wound Healing Assay on Cell Proliferation, Invasion/Migration, and Contraction
Results described thus far indicate that iPS-RPE secretes increased amounts of cytokines and growth factors after wounding. Apical secretion of several growth factors and cytokines peaked on Day 3 after wounding; therefore apical medium (AM) from Day 3 (D3AM) from both control (CA) and scratched (SA) cells was used to examine the effects on cell proliferation, cellular contractility, and migration. To analyze effects on cell proliferation, ARPE19 cells were seeded onto 96-well plates and exposed to D3AM or fresh medium. After 48 hours, cells were analyzed for total DNA content. As seen in Figure 10A, D3AM from wounded iPS-RPE increased proliferation of ARPE19 cells as indicated by increased DNA content. D3AM was further analyzed for effects on cellular contractility in collagen gel contraction assays. ARPE19 cells embedded in collagen gels were exposed to D3AM. Gels were measured on Day 0 and again at 48 hours. Percentage of gel contraction was determined by comparing 48-hour measurements to baseline measurements. As seen in Figure 10B, D3AM from wounded iPS-RPE increased the ability of ARPE19 cells to induce contraction of collagen gels. Finally, to examine effects of D3AM on cell migration, ARPE19 cells were seeded onto invasion/migration assay transwells. After 24 hours exposure to D3AM, the number of cells that had migrated through the membrane to the basolateral chamber was quantified. Results shown in Figure 10C indicate that exposure to D3AM from wounded iPS-RPE increased the ability of ARPE19 cells to degrade ECM and migrate through porous membranes. 
Figure 10
 
Culture medium from the iPS-RPE wound healing assay increases cell proliferation, collagen gel contraction, and migration. ARPE19 cells were treated with apical medium from the scratched (SA D3) and control (CA D3) cells collected on Day 3 after wounding. Cells grown in fresh fRPE medium were included as controls. Cell proliferation was analyzed by CyQuant to measure DNA content (A). Collagen gels were seeded with 2.5 × 105 ARPE19 cells. Gels were incubated with SA D3 and CA D3. Cells grown in fresh fRPE medium were included as controls. The area of the gels was measured after 48 hours to determine the amount of contraction caused by exposure to culture medium (B). ARPE19 cells (3 × 104 cells/well) were seeded onto invasion/migration assay transwells, with a pore size of 0.8 μm. SA D3 and CA D3 were added to the apical chamber. Cells treated with DMEM F12 medium without FBS were included as controls. After 24 hours' incubation, cells were labeled with DAPI to visualize nuclei. Cells that had migrated to the basolateral side were visualized by fluorescence microscopy. Five random fields of view were counted for each transwell (C). *Statistically significant results, set at P < 0.05.
Figure 10
 
Culture medium from the iPS-RPE wound healing assay increases cell proliferation, collagen gel contraction, and migration. ARPE19 cells were treated with apical medium from the scratched (SA D3) and control (CA D3) cells collected on Day 3 after wounding. Cells grown in fresh fRPE medium were included as controls. Cell proliferation was analyzed by CyQuant to measure DNA content (A). Collagen gels were seeded with 2.5 × 105 ARPE19 cells. Gels were incubated with SA D3 and CA D3. Cells grown in fresh fRPE medium were included as controls. The area of the gels was measured after 48 hours to determine the amount of contraction caused by exposure to culture medium (B). ARPE19 cells (3 × 104 cells/well) were seeded onto invasion/migration assay transwells, with a pore size of 0.8 μm. SA D3 and CA D3 were added to the apical chamber. Cells treated with DMEM F12 medium without FBS were included as controls. After 24 hours' incubation, cells were labeled with DAPI to visualize nuclei. Cells that had migrated to the basolateral side were visualized by fluorescence microscopy. Five random fields of view were counted for each transwell (C). *Statistically significant results, set at P < 0.05.
Discussion
Injury to the epithelial cell monolayer disrupts cell–cell contact, and the subsequent loss of contact inhibition results in a cascade of cellular events leading to initiation of EMT.2124 Because RPE cells are quiescent when confluent, their ability to respond to wounding suggests that they produce factors that stimulate EMT. To understand these autocrine effects, we have characterized the secretion profile of iPS-RPE during wound healing. Results of this analysis, summarized in Table 2, provide important insights into the source for cytokines and growth factors during wound healing. Factors identified in this study function as chemoattractants (IL-6, IL-8, GRO, GM-CSF, and MCP-1) and mitogens (VEGF, PDGF-C, FGF-2, and TGFβ). These results are in agreement with those of published reports of protein secretion by RPE as well as in other wound healing models. For example, IL-6 is a proinflammatory cytokine that has been previously shown to be constitutively expressed and released by RPE cells.25 VEGF has also been shown to be constitutively secreted by RPE; however, after wounding, VEGF was detected at higher levels in the apical chambers, with the highest level detected at 3 days after wounding. Furthermore, our analysis revealed constitutive secretion of PDGF-C, although neither PDGF-A nor -B was detectable in control or scratched cell medium from either apical or basolateral chambers. This finding aligns with that of previous studies that reported increased PDGF-C but not PDGF-A/B in vitreous fluid in animal models of PVR and in human PVR patients.26 
Table 2
 
Summary of Results
Table 2
 
Summary of Results
Published studies of PVR and RPE responses to wound healing have implicated TGFβ as a profound inducer of EMT and subsequent migration and proliferation that contribute to the pathology of PVR.2730 However, most in vitro studies used exogenously applied TGFβ to activate cellular pathways leading to EMT.24,3133 Assays conducted in the current study were performed without addition of exogenous TGFβ, yet the cells were able to respond to wounding by activating EMT. The three TGFβ isoforms encoded by the human genome34 have distinct and overlapping functions during wound healing. They have been shown to be mitogenic for fibroblasts but inhibit proliferation of most other cell types.2 TGFβ-1 and -2 have been detected in vitreous fluid,28,35 with higher amounts detected in patients with ocular fibroproliferative disease such as PVR and proliferative diabetic retinopathy. TGFβ-1 and -2 are pro-fibrotic,27,36 whereas TGFβ-3 reduces expression of fibrotic proteins and promotes deposition of ECM.37,38 All three isoforms of TGFβ were found in conditioned medium from iPS-RPE. These results are complementary to those revealed in a recent report of secretion of TGFβ-2 by primary RPE and RPE derived from human embryonic stem cells (hESC-RPE).39 In that study, secretion of TGFβ-2 decreased as the cells grew to confluence, suggesting that contact inhibition and the integrity of cell–cell junctions regulate secretion of TGFβ-2 by RPE. 
Although this study was conducted in vitro, using stem cell-derived RPE, knowledge gained from these experiments can be used not only to identify factors that drive wound healing but also to understand timing of protein secretion during wound healing. To our knowledge, this study is the first to reveal the dynamic changes in protein secretion after injury to RPE in vitro. As detailed in Table 3, highest levels of VEGF, GRO, FGF-2, and TGFβ-1 and -2 were detected at 3 days post wound, whereas the highest levels of GM-CSF were detected from Days 6 to 12, and the highest levels of IL-6 were detected on Day 9. In addition, injury to the monolayer induced significant reductions in secretion of IL-8 and PDGF-C, with lowest levels detected on Day 15. Although minor fluctuations in secretion were observed in unscratched control cells, they were not as great as the changes induced by wounding of the monolayers. 
Table 3
 
Dynamic Changes in Protein Secretion During Wound Healing
Table 3
 
Dynamic Changes in Protein Secretion During Wound Healing
Finally, this study demonstrates that secreted growth factors and cytokines can promote proliferation, contractility, and migration of target cells. Several factors identified in this study, particularly TGFβ, PDGF-C, and VEGF, have been implicated in promotion of cell proliferation, contractility, and migration. This study examined secretion of over 20 different cytokines, chemokines, and growth factors during wound healing, but the potential contribution of unidentified factors to cellular activation after injury must also be considered. The effects on cell activation are not likely due to the effects of a single factor but to synergistic effects of multiple cytokines and growth factors working in concert to activate RPE cells, initiate EMT, and promote wound healing. In terms of treating pathological healing conditions such as PVR, these results suggest that targeting a single factor may not be effective. A multifactorial approach targeting critical regulators of cellular responses to injury will increase chances of therapeutic success. 
Acknowledgments
The authors thank Dallas Golden for technical assistance with stem cell culture. 
This work was presented, in part, at the Association for Research in Vision and Ophthalmology (ARVO) Annual Meeting, Denver, CO, May 2015. 
Supported by US Army Clinical Rehabilitative Medicine Research Program, Military Operational Medicine Research Program, National Research Council (WAG, RRK). 
The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. 
Disclosure: W.A. Greene, None; T.A. Burke, None; E.D. Por, None; R.R. Kaini, None; H.-C. Wang, None 
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Figure 1
 
Experimental design to analyze secretion profile of iPS-RPE during wound healing. Culture medium was collected from both the apical and basolateral chambers of the transwells every 72 hours from Day 0 until Day 21. Culture medium samples were then subjected to conventional and multiplex ELISA to compare the secretion profiles of the control unscratched cells compared to the scratched cells.
Figure 1
 
Experimental design to analyze secretion profile of iPS-RPE during wound healing. Culture medium was collected from both the apical and basolateral chambers of the transwells every 72 hours from Day 0 until Day 21. Culture medium samples were then subjected to conventional and multiplex ELISA to compare the secretion profiles of the control unscratched cells compared to the scratched cells.
Figure 2
 
Wound healing assay with iPS-RPE. iPS-RPE grown to confluency on transwells were scratched to create a wound. Wound healing was monitored for 21 days. Images were acquired at an original magnification of 40× (A). Images were analyzed using ImageJ software to determine total surface area occupied by iPS-RPE before and during the wound healing assay. (B) Surface area of the monolayers is shown in square millimeters.
Figure 2
 
Wound healing assay with iPS-RPE. iPS-RPE grown to confluency on transwells were scratched to create a wound. Wound healing was monitored for 21 days. Images were acquired at an original magnification of 40× (A). Images were analyzed using ImageJ software to determine total surface area occupied by iPS-RPE before and during the wound healing assay. (B) Surface area of the monolayers is shown in square millimeters.
Figure 3
 
Proliferation of iPS-RPE during wound healing. iPS-RPE before (upper panel) and 21 days after (lower panel) wounding were analyzed for expression of the cell proliferation marker Ki-67 (A). Original magnification 100×. The average number of Ki-67-positive cells per field of view is indicated in the bar graph (B).
Figure 3
 
Proliferation of iPS-RPE during wound healing. iPS-RPE before (upper panel) and 21 days after (lower panel) wounding were analyzed for expression of the cell proliferation marker Ki-67 (A). Original magnification 100×. The average number of Ki-67-positive cells per field of view is indicated in the bar graph (B).
Figure 4
 
iPS-RPE undergoes EMT during wound healing. The cells at the wound edge were labeled to visualize β-catenin (red) (A). The translocation of β-catenin from the plasma membrane to the cytoplasm and nucleus at Day 14 indicates the process of EMT at the wound edge (B). Original magnification 200×. To enable visualization of nuclear beta-catenin, cells from the wound edge (B) are shown in the insets (C).
Figure 4
 
iPS-RPE undergoes EMT during wound healing. The cells at the wound edge were labeled to visualize β-catenin (red) (A). The translocation of β-catenin from the plasma membrane to the cytoplasm and nucleus at Day 14 indicates the process of EMT at the wound edge (B). Original magnification 200×. To enable visualization of nuclear beta-catenin, cells from the wound edge (B) are shown in the insets (C).
Figure 5
 
Expression of EMT markers by iPS-RPE during wound healing. Expression levels of e-cadherin (A), n-cadherin (B), and S100A4 (C) were examined by immunofluorescence analysis. Control cells before wounding are shown in the upper panels of each figure. Cells in the wound region at 21 days post scratch are shown in lower panels. Original magnification (A) 400×; (B) 200×; (C) 100×.
Figure 5
 
Expression of EMT markers by iPS-RPE during wound healing. Expression levels of e-cadherin (A), n-cadherin (B), and S100A4 (C) were examined by immunofluorescence analysis. Control cells before wounding are shown in the upper panels of each figure. Cells in the wound region at 21 days post scratch are shown in lower panels. Original magnification (A) 400×; (B) 200×; (C) 100×.
Figure 6
 
Secretion of VEGF, MCP-1, IL-8, and IL-6 by iPS-RPE during wound healing. Heat map depiction of multiplex ELISA results (A). Bar graphs represent the levels of VEGF, MCP-1, IL-8, and Il-6 detected in medium from the apical chamber (B) and basolateral chamber (C). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Figure 6
 
Secretion of VEGF, MCP-1, IL-8, and IL-6 by iPS-RPE during wound healing. Heat map depiction of multiplex ELISA results (A). Bar graphs represent the levels of VEGF, MCP-1, IL-8, and Il-6 detected in medium from the apical chamber (B) and basolateral chamber (C). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Figure 7
 
Secretion of GRO, GM-CSF, and FGF-2 by iPS-RPE during wound healing. Heat map depiction of multiplex ELISA results (A). Bar graphs represent the levels of GRO, GM-CSF, and FGF-2 detected in medium from the apical chamber (B) and basolateral chamber (C). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Figure 7
 
Secretion of GRO, GM-CSF, and FGF-2 by iPS-RPE during wound healing. Heat map depiction of multiplex ELISA results (A). Bar graphs represent the levels of GRO, GM-CSF, and FGF-2 detected in medium from the apical chamber (B) and basolateral chamber (C). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Figure 8
 
Secretion of PDGF-C by iPS-RPE during wound healing. Bar graphs depict the results of ELISA to detect PDGF-C in the apical chamber (A) and basolateral chamber (B). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Figure 8
 
Secretion of PDGF-C by iPS-RPE during wound healing. Bar graphs depict the results of ELISA to detect PDGF-C in the apical chamber (A) and basolateral chamber (B). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Figure 9
 
Secretion of TGFβ isoforms by iPS-RPE during wound healing. Heat map depiction of multiplex ELISA results (A). Bar graphs depicting TGFβ-1, -2, and -3 isoforms detected in culture medium from the apical chamber (B) and basolateral chamber (C). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Figure 9
 
Secretion of TGFβ isoforms by iPS-RPE during wound healing. Heat map depiction of multiplex ELISA results (A). Bar graphs depicting TGFβ-1, -2, and -3 isoforms detected in culture medium from the apical chamber (B) and basolateral chamber (C). All post wound measurements were compared to baseline Day 0 measurements by repeated measures 2-way ANOVA. *Statistically significant results, set at P < 0.05.
Figure 10
 
Culture medium from the iPS-RPE wound healing assay increases cell proliferation, collagen gel contraction, and migration. ARPE19 cells were treated with apical medium from the scratched (SA D3) and control (CA D3) cells collected on Day 3 after wounding. Cells grown in fresh fRPE medium were included as controls. Cell proliferation was analyzed by CyQuant to measure DNA content (A). Collagen gels were seeded with 2.5 × 105 ARPE19 cells. Gels were incubated with SA D3 and CA D3. Cells grown in fresh fRPE medium were included as controls. The area of the gels was measured after 48 hours to determine the amount of contraction caused by exposure to culture medium (B). ARPE19 cells (3 × 104 cells/well) were seeded onto invasion/migration assay transwells, with a pore size of 0.8 μm. SA D3 and CA D3 were added to the apical chamber. Cells treated with DMEM F12 medium without FBS were included as controls. After 24 hours' incubation, cells were labeled with DAPI to visualize nuclei. Cells that had migrated to the basolateral side were visualized by fluorescence microscopy. Five random fields of view were counted for each transwell (C). *Statistically significant results, set at P < 0.05.
Figure 10
 
Culture medium from the iPS-RPE wound healing assay increases cell proliferation, collagen gel contraction, and migration. ARPE19 cells were treated with apical medium from the scratched (SA D3) and control (CA D3) cells collected on Day 3 after wounding. Cells grown in fresh fRPE medium were included as controls. Cell proliferation was analyzed by CyQuant to measure DNA content (A). Collagen gels were seeded with 2.5 × 105 ARPE19 cells. Gels were incubated with SA D3 and CA D3. Cells grown in fresh fRPE medium were included as controls. The area of the gels was measured after 48 hours to determine the amount of contraction caused by exposure to culture medium (B). ARPE19 cells (3 × 104 cells/well) were seeded onto invasion/migration assay transwells, with a pore size of 0.8 μm. SA D3 and CA D3 were added to the apical chamber. Cells treated with DMEM F12 medium without FBS were included as controls. After 24 hours' incubation, cells were labeled with DAPI to visualize nuclei. Cells that had migrated to the basolateral side were visualized by fluorescence microscopy. Five random fields of view were counted for each transwell (C). *Statistically significant results, set at P < 0.05.
Table 1
 
Minimum Detectable Concentrations of Growth Factors and Cytokines Analyzed in This Study
Table 1
 
Minimum Detectable Concentrations of Growth Factors and Cytokines Analyzed in This Study
Table 2
 
Summary of Results
Table 2
 
Summary of Results
Table 3
 
Dynamic Changes in Protein Secretion During Wound Healing
Table 3
 
Dynamic Changes in Protein Secretion During Wound Healing
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