Stem Cell Reports Repor t Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model Alvaro Plaza Reyes,1,5 Sandra Petrus-Reurer,1,2,5 Liselotte Antonsson,1 Sonya Stenfelt,1 Hammurabi Bartuma,2 Sarita Panula,1 Theresa Mader,1 Iyadh Douagi,3 Helder André,2 Outi Hovatta,1,4,6 Fredrik Lanner,1,6,* and Anders Kvanta2,6 1Department of Clinical Sciences, Intervention and Technology, Karolinska Institutet, 14186 Stockholm, Sweden of Clinical Neuroscience, Section for Ophthalmology and Vision, St. Erik Eye Hospital, Karolinska Institutet, 11282 Stockholm, Sweden 3Department of Medicine, Center for Hematology and Regenerative Medicine, Karolinska Institutet, 14157 Stockholm, Sweden 4Cell Therapy Department, Nova Southeastern University, Fort Lauderdale, FL 33314, USA 5Co-first author 6Co-senior author *Correspondence: fredrik.lanner@ki.se http://dx.doi.org/10.1016/j.stemcr.2015.11.008 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2Department SUMMARY Human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model. INTRODUCTION Age-related macular degeneration (AMD), the most common cause of severe vision loss in the Western world, occurs in wet (neovascular) and dry (degenerative) forms. In early dry AMD, the retinal pigment epithelium (RPE) becomes dysfunctional, whereas end-stage disease, geographic atrophy (GA), is characterized by degeneration of RPE and photoreceptors (Bhutto and Lutty, 2012). The RPE is a monolayer of polarized cells that constitutes the outer blood-retina barrier and performs central tasks in the eye, e.g., light adsorption, secretion of growth factors, and phagocytosis of photoreceptor outer segments (POS) (Sparrow et al., 2010). The apical surface harbors microvilli that interact with the light-sensitive POS, whereas the basolateral surface adheres to Bruch’s membrane (BM), which in turn separates the RPE from the underlying choroid. Subretinal transplantation of RPE cells derived from human embryonic stem cells (hESC) could potentially be used as replacement therapy in GA (Schwartz et al., 2012, 2015). However, a critical question is whether donor cells integrate into the host RPE and support the overlying photoreceptors. Experimental transplantations of hESC-RPE have only been conducted in small-eyed rodent models (Carido et al., 2014; Idelson et al., 2009; Lund et al., 2006; Vugler et al., 2008). Functional effects in these models are however non-specific and surgical techniques, instrumentation, and imaging methods differ from those applied in humans, limiting their use as preclinical models (e.g., Pinilla et al., 2009). We have recently described a damage model in the large-eyed rabbit that exhibits typical GA changes including photoreceptor loss and RPE alterations (Bartuma et al., 2015). Several hESC-RPE derivation protocols have been described with the common limitation of relying on culture steps that involve xeno- or human-feeder cells or use medium components that are either undefined or not xeno-free (Klimanskaya et al., 2004; Lane et al., 2014; Osakada et al., 2009; Pennington et al., 2015; Vaajasaari et al., 2011). Recently, we described a defined and xeno-free clonal culture of hESC using recombinant human laminin (rhLN) and E-cadherin (Rodin et al., 2014a, 2014b). Encouraged by this work, we set out to evaluate whether rhLN-matrix could support efficient hESC-RPE differentiation. BM, the RPE basement membrane, contains four LNs, LN-111, LN-332, LN-511, and LN-521, that adhere to the RPE via specific integrins (Aisenbrey et al., 2006). In the present study, we show that rhLNs are more effective in supporting xeno-free and defined differentiation compared with exogenous matrix such as gelatin, used in Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors 9 (legend on next page) 10 Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors ongoing clinical studies (Schwartz et al., 2012). Moreover, we demonstrate that suspension transplantations of rhLN-521-hESC-RPE integrate as polarized subretinal monolayers that rescue overlying photoreceptors from induced damage. We conclude that rhLN-521 effectively supports differentiation of clinically compliant hESC-RPE, and presents evidence of efficient long-term functional integration of hESC-RPE in a large-eyed disease model. RESULTS Robust Induction of Primary Pigmented Cells Using Suspension Differentiation in Defined Medium We have recently developed a xeno-free hESC derivation and culture methodology, using rhLN-521-based matrix and a xeno-free and defined NutriStem hESC XF medium containing basic fibroblast growth factor (bFGF) (Rodin et al., 2014a, 2014b). As FGF removal is critical for RPE differentiation (Pittack et al., 1997), we decided to evaluate whether our hESC culture methodology could efficiently support hESC-RPE differentiation following bFGF removal. hESCs were cultured on rhLN-521 and manually scraped to produce a suspension culture of embryoid bodies (EBs) in NutriStem hESC XF without bFGF (Figures 1A and 1B). This culture robustly supported formation of pigmented structures resembling optical vesicles (OVs) at as early as 3 weeks of differentiation (Figures 1A and 1B). At this stage, we observed an average of 0.3 OVs per EB, which in the subsequent weeks increased to 0.8 per EB (Figure 1C). This efficiency of OV induction is well in line with previous reports (Idelson et al., 2009), confirming that our xeno-free culture medium efficiently supports the first stage of hESCRPE differentiation. rhLN-521 Efficiently Supports Homogeneous Expansion of Pigmented and Functional hESC-RPE Endogenous BM contains four LNs: LN-111, LN-332, LN511, and LN-521. Consequently, we decided to compare subsequent expansion and maturation of primary pigmented cells on gelatin or rhLNs found in the endogenous BM. The pigmented OVs were mechanically cut out using a scalpel and dissociated into single cells. Cells were seeded through a cell strainer onto gelatin or LN-coated dishes. Three days following plating, it was clearly observable that LN-521 had the best performance, with 69% plating efficiency compared with 8% in gelatin-coated cultures (Table S1). Pigmentation was initially lost in all cultures, but was progressively reestablished from day 21 (Figure 1D), as previously described. Interestingly, time-lapse microscopy showed that cells on rhLN-511 and rhLN-521 were highly migratory forming uniform monolayers throughout the wells (Figures 1D–1F and Movie S1), while progressively maturing into pigmented hexagonal cells. This correlates well with a previous study showing that the same subtype of integrin receptors recognizes LN-511 and LN-521 (Aisenbrey et al., 2006). Cells on gelatin were migratory, but tended to stay in tight colonies and failed to fully cover the plate even after 77 days (Figures 1D–1F and S1A). Transcriptional analysis showed similar profiles in hESCRPE differentiated on each of the five substrates with Figure 1. Xeno-Free and Defined RPE Differentiation of hESC to hESC-RPE (A) Differentiation protocol scheme. Confluent hESC cultures were scraped and cultured in low-attachment plates with NutriStem / (bFGF removed) to form EBs with Rock inhibitor during the first 24 hr. First pigmentation (OV-like structures) appears following week 3. At week 5, pigmented structures are cut out with a scalpel and dissociated into single cells. Cells are plated on different substrates and cultured until homogeneous pigmentation is reached (week 9). RPE cells are dissociated to a single-cell suspension for subretinal injection in the rabbit eye. (B) hESC-EB with pigmented OV-like structures. (C) Quantification of pigmented OV formation per starting EB during differentiation. Bars represent means ± SD from three independent experiments. (D) Differentiation on gelatin and LN-111, LN-332, LN-511, and LN-521 following 21 and 32 days. See also Movie S1. (E) Images depicting the migratory tracks of ten random cells during the first 7 days of culture on the different conditions. (F) Bar graph showing the average length migrated by cells growing on the different substrates. Three individual wells with ten cells analyzed in each were quantified over a period of 7 days using time-lapse imaging. Bars represent means ± SD. (G) Transcriptional analysis of hESC-RPE differentiated on the different substrates. Values are normalized to RPLPO and GAPDH and displayed as relative to undifferentiated hESCs. Bars represent means ± SEM from three independent experiments. (H) Flow cytometry analysis of MITF expression on hESC-RPE cells grown on the different substrates for 29 days. (I and J) Polarized secretion of VEGF and PEDF in hESC-RPE. Bars represent means ± SEM from three independent experiments. (K) Phagocytosis of fluorescein isothiocyanates (FITC)-labeled POS by hESC-RPE on the different substrates. hESC-RPE cells incubated with FITC-labeled POS at 4 C were used as negative controls. Bars represent means ± SD from three independent experiments. (L) TER measurements of hESC-RPE cells grown on the different substrates. The TER value for undifferentiated hESCs (fully confluent plate) is shown for comparison (dashed line). Bars represent means ± SEM from three independent experiments. Scale bars: B, D, E, 500 mm. See also Figure S1. Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors 11 Figure 2. Morphology and Marker Expression of rhLN-521-hESC-RPE (A) Bright-field image of hESC-RPE cells grown on rhLN-521 acquiring hexagonal shape before pigmentation. (B) Mature and highly pigmented hESC-RPE cells displaying hexagonal morphology. (C–F) Expression of key RPE markers: cellular retinaldehyde-binding protein (CRALBP), zonula occludens protein-1 (ZO-1), Na/ K-ATPase, and Bestrophin1 (Best1) by immunostaining. (G and H) Z-stack confocal projections showing polarized expression of ZO-1 and Na/K-ATPase markers on the apical side of the hESC-RPE. Scale bars: A, B, 100 mm; C–H, 20 mm. reduction of pluripotency-associated transcripts OCT3/4 and NANOG, together with robust expression of neuroectoderm transcripts sex-determining region Y-box 9 protein (SOX9) and paired box 6 (PAX6). Low expression levels of paired box 3 (PAX3) and endothelin receptor B (EDNRB) transcripts eliminated the possibility of contaminating melanocytes in any of the substrates (Figure S1B). RPE differentiation was evident with expression of bestrophin 1 (BEST1), RPE-specific protein 65 kDa (RPE65), and premelanosome protein (PMEL) (Figure 1G). However, more sensitive single-cell analysis of mature RPE purity through flow cytometry for microphthalmia-associated transcription factor (MITF) and BEST1 showed more homogeneous expression on all LNs compared with gelatin (Figures 1H and S1C). Functionally, all cultures showed polarized secretion of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), as well as active phagocytosis of POS (Figures 1I–K and S1D–S1G). hESC-RPE only secreted PEDF from week 5 and not earlier (data not shown). We found that hESC-RPE growing on LN-332 and gelatin displayed lower levels of PEDF secretion compared with those growing in all the other tested conditions. Also, interestingly, transepithelial electrical resistance (TER) measurements proved the functional tight 12 Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors junction integrity of our hESC-RPE monolayer on LN111, LN-511, and LN-521 in a time-dependent manner, but not on LN-332 and gelatin (Figure 1L). This observation is in line with the fact that RPE cells did not manage to form a continuous monolayer when growing on these two substrates (Figures 1D and S1A). hESC-RPE seeded on LN-521 reached values of 180 U cm2 after 31 days, indicative of a functionally mature monolayer. Extended analysis confirmed that rhLN-521-hESC-RPE cultures acquired a pigmented and hexagonal morphology (Figures 2A and 2B), and they were also shown to be uniformly positive for cellular retinaldehyde-binding protein (CRALBP) and BEST1 with clear apical polarization of zonula occludens protein 1 (ZO-1) and Na/K-ATPase (Figures 2C–2H). hESC-RPE Transplantation into Albino Rabbits For transplantation, we chose the albino rabbit with an eye size approximately 70% of that the human eye. All major retinal and subretinal layers were clearly detectable using cross-sectional spectral domain optical coherence tomography (SD-OCT) (Figures S2A and S2B). We next transplanted suspensions of rhLN-521-hESCRPE into the subretinal space. Pigmentation was not evident by ophthalmoscopy but a thickened and irregular RPE/BM layer was detected by SD-OCT 1 week after Figure 3. In Vivo Integration of rhLN521-hESC-RPE (A) H&E-stained section 1 week after transplantation demonstrates the presence of lightly pigmented cells as a continuous monolayer (arrows) between the POS and the underlying choriocapillaris. (B) Subretinal human (NuMA-positive) cells detected in the injected region. Panels show NuMA (B1), DAPI (B2) and NuMA with DAPI (B3). (C) A heavily pigmented subretinal monolayer of transplanted cells is detected in the injected region 8 weeks after transplantation. (D and E) 8 weeks after transplantation, the integrated NuMA-positive rhLN-521-hESCRPE cells are polarized with basolateral BEST1 expression. BF, bright field. Panels show BEST1 with BF (D1) and BEST1 only (D2). (F–H) 34 weeks after transplantation, a heavily pigmented monolayer is present in the injected region displaying RPE65-positive cells and engulfment of rhodopsinpositive particles. Dashed lines represent individual nuclei. (I) The non-pigmented native RPE layer (arrows) of an untreated area is shown for comparison. Scale bars: A–C, 10 mm; D, 100 mm; E–I, 10 mm. transplantation (Figure S2C). Histologic analysis demonstrated a monolayer of lightly pigmented cells that integrated into the host RPE overlaid by well-preserved photoreceptors (Figure 3A). Positive immunostaining for human nuclear mitotic apparatus protein (NuMA) confirmed the human origin of the cells (Figure 3B). Eight weeks after transplantation, monolayers of cells had become heavily pigmented and acquired a polarized phenotype as demonstrated by basolateral expression of BEST1 in the injected area (Figures 3C–3E and S2E). Importantly, all NuMA-positive cells were also pigmented and BEST1-positive. Pigmented rhLN-521-hESC-RPE monolayers with preservation of the neurosensory retina were further observed for up to 34 weeks (Figures 3F, S2C, and S2D). Donor cells were positive for the specific RPE marker RPE65 and cyto- plasmic rhodopsin suggestive of maintained phagocytic activity (Figures 3G and 3H). In Vivo Photoreceptor Rescue by hESC-RPE In the albino rabbit, subretinal injection of PBS alone creates a GA-like phenotype including photoreceptor loss (Bartuma et al., 2015). We therefore tested whether transplanted rhLN-521-hESC-RPE possessed photoreceptor rescue capacity. The outer nuclear layer (ONL), normally consisting of five to six layers, was reduced to a single layer in PBS-injected eyes (Figure 4A), whereas eyes transplanted with rhLN-521-hESC-RPE had preserved ONL and POS (Figure 4B). Integration of transplanted cells was further necessary for ONL rescue as outer retinal thickness (ORT) measurements in eyes with minimal or no integration were Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors 13 Figure 4. Photoreceptor Rescue by Transplanted rhLN-521-hESC-RPE (A) PBS-induced photoreceptor degeneration is shown as a hyperreflective circle and outer retinal thinning by infrared-confocal scanning laser ophthalmoscopy (A1) and SD-OCT (A2) respectively. Green arrow, SDOCT scan plane. A H&E stained section of the bleb transition zone (arrowhead, A3) is shown with the normal (A4) and degenerated (arrowheads, A5) photoreceptor layer magnified. ORT is indicated (arrow). (B) Two months after transplantation, large pigmented areas are shown (B1) with integrated rhLN-521-hESC-RPE shown as a hyperreflective band (between arrowheads, B2). The overlying neurosensory retina is well preserved as demonstrated by SD-OCT (B2) and histology (B3, B4). ORT is indicated (arrow). (C–E) ORT is significantly greater in eyes with integrated (defined as a pigmented area >2.5 mm2) than with non-integrated (defined as a pigmented area <2.5 mm2) rhLN-521-hESC-RPE. The ORT for PBS injection alone is adapted from Bartuma et al. (2015) and shown for comparison (dashed line, E). Bars represent means ± SD from 14 injected eyes. See also Supplemental Experimental Procedures. Scale bars: A1, A2, B1, B2, 200 mm; A3, A4, A5, B3, B4, 50 mm. **p < 0.01 (2-sided Student’s t test). See also Figures S2 and S3. similar to those with PBS treatment alone (Figures 4C–4E). Importantly, undifferentiated hESCs and fibroblasts that formed transient cell aggregates were not protective of photoreceptor loss (Figures S3A and S3B). Thus, the photoreceptor rescue model showed evidence of both specificity (i.e., non-RPE cells were ineffective) and sensitivity (i.e., only integrated RPE cells were effective) for functional RPE. DISCUSSION Our differentiation method based on rhLN-521 completely eliminates undefined and animal-based products throughout the process from derivation of hESCs to differentiated functional hESC-RPE. Using our approach, we can avoid microbial contamination, including new agents that have not yet been identified. In addition, we can prevent the identified risk of rejection potentially brought by 14 Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors non-human proteins into the cells during culture and/or differentiation (Aisenbrey et al., 2006). Key to our methodology is the combined use of xeno-free and defined culture medium with a relevant extracellular matrix. BM supporting the RPE contains collagen types I, II, and IV, fibronectin, heparin sulfate proteoglycans, and LNs: LN-111, LN-332, LN-511, and LN-521 (Campochiaro et al., 1986; Martin et al., 2005). As recombinant mouse LN-111 has been shown to support RPE differentiation (Rowland et al., 2013) and we have shown that LN-521 supports derivation and culture of hESCs (Rodin et al., 2014b), we set out to evaluate LNs in a xeno-free and defined differentiation to functional RPE. In accordance with previous observations, we found that rhLN-511 and rhLN-521 supported the highest degree of migration (Aisenbrey et al., 2006), which allowed the cells to spread out evenly in the culture plates. Interestingly, TER assessment from human adult retinal explants and immortalized RPE cell lines has been reported to reach 148 and 100 U cm2, respectively (Hornof et al., 2005). In our culture, cells seeded on LN111, LN511, and especially on LN521 appear to be as good as human eye explants and superior to immortalized RPE cell lines in creating highly polarized monolayers. Gelatin-coated cultures instead generated tight and irregularly shaped colonies, which failed to fully cover the plate and displayed a poor monolayer functional integrity. All substrates generated cells with similar transcriptional in vitro profiles, but single-cell analysis of MITF and BEST1 revealed significant heterogeneity in gelatin cultures compared with all LN cultures. Expanding cells in suboptimal culture conditions have the inherent risk of selecting for growth-promoting genetic abnormalities. It was therefore encouraging that seeding efficiency on rhLN-521 was 69%, compared with 8% on gelatin. From these in vitro observations, we conclude that rhLN-521 not only eliminates the need for undefined or xeno-derived matrix components but is in fact a more supportive and suitable culture matrix for hESC-RPE. Upon transplantation, integrated cells initially displayed reduced pigmentation, which was progressively reestablished, similar to what was observed during in vitro hESC-RPE derivation. Concomitantly, integration of donor cells varied both between animals and between eyes of the same animal. A putative explanation is xeno-graft rejection, as failure of integration correlated with signs of immunoreaction including subretinal cell infiltration, retinal atrophy, and donor cell loss. Optimization of our immunosuppressive protocol may thus overcome this variation. In addition, integration may depend on the state of the native RPE as transplanted cells could adhere better to a denuded BM as shown in mice with sodium iodate-induced RPE atrophy (Carido et al., 2014). Accordingly, we have shown that subretinal injection alone causes disturbed RPE morphology and partial RPE loss (Bartuma et al., 2015). In this study, we transplant hESC-RPE to a large-eyed animal. Human and rabbit eyes are of similar size, the main advantage compared with previously studied mouse and rat models (Carido et al., 2014; Lund et al., 2006). Transplantation in rodents requires high cell concentrations (i.e., typically 50,000 cells/ml or more) and a transscleral approach through the choroid (Vugler et al., 2008). The outer blood-retinal barrier is thus compromised, potentially triggering an inflammatory response. Moreover, high cell concentrations and limited surgical control may cause misdirection, multilayering, and clumping of transplanted cells. Indeed, several publications have shown that photoreceptor rescue is neither RPE specific nor correlated with an intact donor cell layer (eg. Pinilla et al., 2009). The large-eyed rabbit allowed us to perform a surgical technique with instrumentation identical to a clinical setting (el Dirini et al., 1992). The model also permitted high-reso- lution in vivo tracking of transplanted cells and monitoring of the overlying neurosensory retina through time. Using this methodology, we demonstrated subretinal monolayers of rhLN-521-hESC-RPE that remained for up to 8 months. Furthermore, integrated cells possessed in vivo functionality including phagocytic activity and rescue of photoreceptors from induced degeneration. Importantly, this effect showed both specificity and sensitivity as non-RPE and non-integrated RPE cells were ineffective. Suspension transplants have been frequently used in rodent models (Carido et al., 2014; Idelson et al., 2009; Lund et al., 2006; Vugler et al., 2008) and ongoing clinical studies (Schwartz et al., 2012, 2015). Concerns about this delivery method were raised with the main criticism being possible multilayering of donor cells leading to inefficient integration (Schwartz et al., 2012). As an alternative, transplantation of cells as prepolarized sheets with or without a supporting biomatrix has been suggested (Diniz et al., 2013; Stanzel et al., 2014). However, these large transplants are surgically demanding and may lead to retinal scarring and outer retinal degeneration (Kamao et al., 2014; Stanzel et al., 2014). In the present study, we noted minimal retinal scarring and a well-preserved neurosensory retina overlying the transplanted monolayer. Moreover, by using suspension transplants, we obtained monolayers up to ten times the size of RPE sheets with a typical size of 2–2.5 mm2 (Kamao et al., 2014; Stanzel et al., 2014). We demonstrate that a minimally invasive surgical procedure in a large-eyed disease model can achieve high-yield functional long-term hESC-RPE integration with photoreceptor preservation. These findings have important implications for ongoing and future clinical studies for the development of a safe and efficient cell replacement therapy for GA. EXPERIMENTAL PROCEDURES Cell Culture and Differentiation hES line HS980 was established and cultured under xeno-free and defined conditions on rhLN-521, and passaged as previously described (Diniz et al., 2013; Rodin et al., 2014b). For differentiation, cells were scraped and cultured in low-attachment plates, 5–7 3 104 cells/cm2 in custom-made NutriStem hESC XF medium without bFGF and transforming growth factor b. Rho-kinase inhibitor was included during the first 24 hr. Following differentiation, pigmented areas were cut out using a scalpel, dissociated, passed through a 20G needle, and plated at a density of 0.6–1.2 3 104 cells/cm2. Subretinal Transplantation and In Vivo Imaging Dissociated hESC-RPE cells were injected (50 ml, 50,000 cells) subretinally using a transvitreal pars plana technique. SD-OCT and confocal scanning laser ophthalmoscopy was performed to obtain Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors 15 horizontal cross-sectional b scans and en face fundus in vivo images, respectively. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, three figures, one table, and one movie and can be found with this article online at http://dx.doi.org/10.1016/j. stemcr.2015.11.008. AUTHOR CONTRIBUTIONS A.P.R., S.P.R., H.B. I.D., and A.K. performed the experiments. L.A., S.S., S.P., and T.M. contributed to the in vitro differentiation methodology. H.B. and H.A. contributed to the animal work. A.P.R., S.P.R., O.H., F.L., and A.K. planned the experiments, analyzed the data, and wrote the manuscript. ACKNOWLEDGMENTS This work was supported by grants from the Swedish Research Council (VR), Ragnar Söderberg Foundation, Swedish Foundation for Strategic Research, Stockholm County Council (ALF project), Ögonfonden and Cronqvist Foundation. This study was performed at the WIRM flow cytometry facility, supported by Knut och Alice Wallenbergs Stiftelse (KAW) and the Live Cell Imaging Unit/Nikon Center of Excellence, supported by KAW, VR, Centre for Innovative Medicine, and the Jonasson donation. Diniz, B., Thomas, P., Thomas, B., Ribeiro, R., Hu, Y., Brant, R., Ahuja, A., Zhu, D., Liu, L., Koss, M., et al. (2013). Subretinal implantation of retinal pigment epithelial cells derived from human embryonic stem cells: improved survival when implanted as a monolayer. Invest. Ophthalmol. Vis. Sci. 54, 5087–5096. el Dirini, A.A., Wang, H.M., Ogden, T.E., and Ryan, S.J. (1992). Retinal pigment epithelium implantation in the rabbit: technique and morphology. Graefes Arch. Clin. Exp. Ophthalmol. 230, 292–300. Hornof, M., Toropainen, E., and Urtti, A. (2005). Cell culture models of the ocular barriers. Eur. J. Pharm. Biopharm. 60, 207–225. Idelson, M., Alper, R., Obolensky, A., Ben-Shushan, E., Hemo, I., Yachimovich-Cohen, N., Khaner, H., Smith, Y., Wiser, O., Gropp, M., et al. (2009). Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. Cell Stem Cell 5, 396–408. Kamao, H., Mandai, M., Okamoto, S., Sakai, N., Suga, A., Sugita, S., Kiryu, J., and Takahashi, M. (2014). Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application. Stem Cell Rep. 2, 205–218. Klimanskaya, I., Hipp, J., Rezai, K.A., West, M., Atala, A., and Lanza, R. (2004). Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. Cloning Stem Cells 6, 217–245. Received: February 21, 2015 Revised: November 16, 2015 Accepted: November 18, 2015 Published: December 24, 2015 Lane, A., Philip, L.R., Ruban, L., Fynes, K., Smart, M., Carr, A., Mason, C., and Coffey, P. (2014). Engineering efficient retinal pigment epithelium differentiation from human pluripotent stem cells. Stem Cells Transl. Med. 3, 1295–1304. REFERENCES Lund, R.D., Wang, S., Klimanskaya, I., Holmes, T., Ramos-Kelsey, R., Lu, B., Girman, S., Bischoff, N., Sauve, Y., and Lanza, R. (2006). Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats. Cloning Stem Cells 8, 189–199. Aisenbrey, S., Zhang, M., Bacher, D., Yee, J., Brunken, W.J., and Hunter, D.D. (2006). Retinal pigment epithelial cells synthesize laminins, including laminin 5, and adhere to them through alpha3- and alpha6-containing integrins. Invest. Ophthalmol. Vis. Sci. 47, 5537–5544. Bartuma, H., Petrus-Reurer, S., Aronsson, M., Westman, S., André, H., and Kvanta, A. (2015). In vivo imaging of subretinal blebinduced outer retinal degeneration. Invest. Ophthalmol. Vis. Sci. 56, 2423–2430. Bhutto, I., and Lutty, G. (2012). Understanding age-related macular degeneration (AMD): relationships between the photoreceptor/retinal pigment epithelium/Bruch’s membrane/choriocapillaris complex. Mol. Aspects Med. 33, 295–317. Campochiaro, P.A., Jerdon, J.A., and Glaser, B.M. (1986). The extracellular matrix of human retinal pigment epithelial cells in vivo and its synthesis in vitro. Invest. Ophthalmol. Vis. Sci. 27, 1615– 1621. Carido, M., Zhu, Y., Postel, K., Benkner, B., Cimalla, P., Karl, M.O., Kurth, T., Paquet-Durand, F., Koch, E., Munch, T.A., et al. (2014). Characterization of a mouse model with complete RPE loss and its use for RPE cell transplantation. Invest. Ophthalmol. Vis. Sci. 55, 5431–5444. 16 Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors Martin, M.J., Muotri, A., Gage, F., and Varki, A. (2005). Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat. Med. 11, 228–232. Osakada, F., Jin, Z.B., Hirami, Y., Ikeda, H., Danjyo, T., Watanabe, K., Sasai, Y., and Takahashi, M. (2009). In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction. J. Cell Sci. 122, 3169–3179. Pennington, B.O., Clegg, D.O., Melkoumian, Z.K., and Hikita, S.T. (2015). Defined culture of human embryonic stem cells and xenofree derivation of retinal pigmented epithelial cells on a novel, synthetic substrate. Stem Cells Transl. Med. 4, 165–177. Pinilla, I., Cuenca, N., Martinez-Navarrete, G., Lund, R., and Sauvé, Y. (2009). Intraretinal processing following photoreceptor rescue by non-retinal cells. Vision Res. 49, 2067–2077. Pittack, C., Grunwald, G.B., and Reh, T.A. (1997). Fibroblast growth factors are necessary for neural retina but not pigmented epithelium differentiation in chick embryos. Development 124, 805–816. Rodin, S., Antonsson, L., Hovatta, O., and Tryggvason, K. (2014a). Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions. Nat. Protoc. 9, 2354–2368. ation and Stargardt’s macular dystrophy: follow-up of two openlabel phase 1/2 studies. Lancet 385, 509–516. Rodin, S., Antonsson, L., Niaudet, C., Simonson, O.E., Salmela, E., Hansson, E.M., Domogatskaya, A., Xiao, Z., Damdimopoulou, P., Sheikhi, M., et al. (2014b). Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xenofree environment. Nat. Commun. 5, 3195. Sparrow, J.R., Hicks, D., and Hamel, C.P. (2010). The retinal pigment epithelium in health and disease. Curr. Mol. Med. 10, 802–823. Rowland, T.J., Blaschke, A.J., Buchholz, D.E., Hikita, S.T., Johnson, L.V., and Clegg, D.O. (2013). Differentiation of human pluripotent stem cells to retinal pigmented epithelium in defined conditions using purified extracellular matrix proteins. J. Tissue Eng. Regen. Med. 7, 642–653. Schwartz, S.D., Hubschman, J.P., Heilwell, G., Franco-Cardenas, V., Pan, C.K., Ostrick, R.M., Mickunas, E., Gay, R., Klimanskaya, I., and Lanza, R. (2012). Embryonic stem cell trials for macular degeneration: a preliminary report. Lancet 379, 713–720. Schwartz, S.D., Regillo, C.D., Lam, B.L., Eliott, D., Rosenfeld, P.J., Gregori, N.Z., Hubschman, J.P., Davis, J.L., Heilwell, G., Spirn, M., et al. (2015). Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degener- Stanzel, B.V., Liu, Z., Somboonthanakij, S., Wongsawad, W., Brinken, R., Eter, N., Corneo, B., Holz, F.G., Temple, S., Stern, J.H., et al. (2014). Human RPE stem cells grown into polarized RPE monolayers on a polyester matrix are maintained after grafting into rabbit subretinal space. Stem Cell Rep. 2, 64–77. Vaajasaari, H., Ilmarinen, T., Juuti-Uusitalo, K., Rajala, K., Onnela, N., Narkilahti, S., Suuronen, R., Hyttinen, J., Uusitalo, H., and Skottman, H. (2011). Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells. Mol. Vis. 17, 558–575. Vugler, A., Carr, A.J., Lawrence, J., Chen, L.L., Burrell, K., Wright, A., Lundh, P., Semo, M., Ahmado, A., Gias, C., et al. (2008). Elucidating the phenomenon of HESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation. Exp. Neurol. 214, 347–361. Stem Cell Reports j Vol. 6 j 9–17 j January 12, 2016 j ª2016 The Authors 17 Stem Cell Reports, Volume 6 Supplemental Information Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model Alvaro Plaza Reyes, Sandra Petrus-Reurer, Liselotte Antonsson, Sonya Stenfelt, Hammurabi Bartuma, Sarita Panula, Theresa Mader, Iyadh Douagi, Helder André, Outi Hovatta, Fredrik Lanner, and Anders Kvanta SUPPLEMENTAL FIGURES AND LEGENDS Figure S1. Morphology of hESC-RPE on gelatin and quantification of phagocytic activity, related to Figure 1. (A) hESC-RPE growing on gelatin fail to cover the whole surface even after long-term cultures. hESC-RPE on gelatin after 7 days (A1), 35 days (A2) and 77 days (A3) in culture. (B) Transcriptional analysis for melanocytic markers PAX3 (C1) and EDNRB (C2) of hESCRPE differentiated on the different substrates. Values are normalized to RPLPO and GAPDH and displayed as relative to undifferentiated hESC. Normal human epidermal melanocytes (NHEM) were used as a positive control. Bars represent mean±SEM, from three independent experiments. (C) Flow cytometry analysis of BEST1 expression on hESC-RPE cells grown on the different substrates for 47 days. (D-E) Phagocytosis of FITC-labeled POS by hESC-RPE growing on rhLN-521, after overnight incubation at 4°C (D) and 37°C (E). The former used as negative control, since active-phagocytosis is temperature-dependent. (D1 and E1) Maximum intensity projection image depicting internalized FITC-labeled POS in green and phalloidin staining of hESCRPE in red. (D2 and E2) Output image of phagocyted FITC-labeled POS generated by the CellProfiler software. (D3 and E3) Z-stack confocal projections of the area delimited by arrows in D1 and E1. (F-G) Phagocytosis of FITC-labeled POS by hESC growing on rhLN-521, after overnight incubation at 4°C and 37°C. The former used as negative control, since active-phagocytosis is temperature-dependent. The images correspond to maximum intensity projections depicting internalized FITC-labeled POS in green (not present) and hESC bounderies shown in red by wheat germ agglutinin staining. Scale bars: (A1) = 200μm (A2, A3) = 500μm; (D1, D2, E1, E2, F, G) = 100μm; (D3, E3) = 20μm. Figure S2. In vivo imaging of the normal albino rabbit posterior segment and longterm integration of hESC-RPE, related to Figure 3. (A) En-face IR-cSLO reflectance shows the choroidal vasculature (A1). Green arrow = SDOCT scan plane. The corresponding cross-sectional SD-OCT b-scan of the neurosensory retina and choroid is shown (A2). (B) On higher magnification the individual layers of the neurosensory retina, RPE/BM, and underlying choroid are distinguished (B1). The corresponding histologic structures are shown (HE staining) (B2). GCL (ganglion cell layer), IPL (inner plexiform layer), INL (inner nuclear layer), OPL (outer plexiform layer), ONL (outer nuclear layer), OLM (outer limiting membrane), EZ (ellipsoid zone), OS (outer segments), RPE (retinal pigment epithelium), BM (Bruch’s membrane), CC (choriocapillaris). (C) Pigmentation is absent in the subretinal bleb area (dashed circle) one week after transplantation (C1). Green arrow = SD-OCT scan plane. Simultaneous SD-OCT scan shows a thickened hyperreflective RPE/BM (between closed arrowheads). Magnification of the boxed area shows an irregular layer representing putative rhLN-521-hESC-RPE (open arrows) beneath the hyporeflective outer segments. Twelve weeks (C2), and 34 weeks (C3) after transplantation of rhLN-521-hESC-RPE multicolor-cSLO and SD-OCT scans demonstrate stable pigmented donor cell integration (between arrowheads) and a wellpreserved overlying photoreceptor layer. Green arrow = SD-OCT scan plane. *Indicates the corresponding region of the multicolor-cSLO images in C2 and C3. (D) HE staining of the corresponding histologic section confirms presence of a preserved neurosensory retina (D1) and pigmented hESC-RPE monolayer (D2). (E) 8 weeks after transplantation, uninjected areas do not present pigmented cells (E1) and show negative staining for both NuMA and BEST1 (E2). (BF = bright field). Scale bars: Scale bars: (A1, A2, C) = 200µm; (B1) = 100µm; (B2, D) = 50µm; (E1, E2) = 10µm. Figure S3. Undifferentiated hESC or fibroblasts do not rescue photoreceptors, related to Figure 4. (A-B) Transplanted undifferentiated hESC (A) or human fibroblasts (B) form transient hyperreflective subretinal aggregates as demonstrated by SD-OCT. ORT (arrows) overlying the transplanted bleb area is reduced as depicted by comparing the respective magnified (boxed) areas 1 week and 4 weeks after transplantation. Note that cell aggregates have disappeared after 4 weeks. The bleb margin is indicated (arrowheads). Scale bars = 200µm. SUPPLEMENTAL MOVIES Movie S1, related to Figure 1. Time-lapse phase contrast imaging of the hESC-RPE growing on gelatin, rhLN-111, -332, 511 or -521. Videos are the result of time-lapse stack images taken every hour during the first 21 days after cell seeding. SUPPLEMENTAL TABLES Supplemental Table S1, related to Figure 1. SUPPLEMENTAL EXPERIMENTAL PROCEDURES Cell Culture Human embryonic stem cell lines HS980, HS975 and HS983a were derived and cultured under xeno-free and defined conditions according to the previously described method. [Rodin 2014b]. The cells were maintained by clonal propagation on LN-521 (Biolamina) in NutriStem hESC XF medium (Biological Industries), in a 5% CO2/5% O2 incubator and passaged enzymatically at 1:10 ratio every 5-6 days. For passaging, confluent cultures were washed twice with PBS without Ca2+ and Mg2+ and incubated for 5 min at 37°C, 5% CO2/5% O2 with TrypLE Select (GIBCO, Invitrogen). The enzyme was then carefully removed and the cells were collected in fresh pre-warmed NutriStem hESC XF medium by gentle pipetting to obtain a single cell suspension. The cells were centrifuged at 1100 rpm for 4 min, the pellet resuspended in fresh prewarmed NutriStem hESC XF medium and cells plated on a freshly rhLN-521 coated dish. Two days after passage the medium was replaced with fresh prewarmed NutriStem hESC XF medium and changed daily subsequently. GFP-labeled H9 human embryonic stem cell (hESC) line cells were maintained in mTESR media (StemCell Technologies) on Matrigel (BD Biosciences) coated plates (1:60) until confluence. Normal human dermal fibroblasts (NHDF) from adult origin (Lonza) were maintained in DMEM media (LifeTechnologies) with 10% FBS (LifeTechnologies) until confluence. In vitro differentiation Pluripotent stem cells were cultured to confluence on rhLN-521 and manually scraped to produce embryoid bodies (EBs) using a 1000 µl pipette tip. The EBs were then cultured in suspension in low attachment plates (Corning) at a density of 5-7x104 cells/cm2. Differentiation was performed in custom-made NutriStem hESC XF medium in which bFGF and TGFβ have been eliminated with media change twice a week. 10 μM Rho-kinase inhibitor (Y-27632, Millipore) was added to the suspension cultures only during the first 24h. Following five weeks differentiation, pigmented areas were mechanically cut out of the EBs using a scalpel. Cells were then dissociated using TrypLE Select, followed by flushing through a 20G needle and syringe. Cells were seeded through a cell strainer (ø 40 μm, BD Bioscience) on LN-coated dishes at a cell density of 0.6-1.2x104 cells/cm2 and fed twice a week with the same differentiation medium referred above. Time-lapse microscopy The behavior of hESC-RPE on the different substrates was monitored using the Cell-IQ live imaging system (Chip-Man Technologies Ltd.) equipped with a 10x phase contrast objective, an automated stage and an integrated incubator (37°C, 5% CO2). After OVs dissociation, hESC-RPE were seeded in triplicates on the different substrates: Gelatin (0.1%, Sigma), rhLN-111, -332, -511, and -521 (all 20μg/mL, BioLamina). On day 1 after seeding, the plates were transferred to the live cell imaging equipment. Images were acquired for both the center (2x2 image grids) and the periphery (single images) of every well. Every region of interest was monitored every hour for 21 days. Cell migration was assessed for every time-lapse image stacks using NIS-Elements v.4.0 (Nikon). For each stack 10 cells were randomly chosen and manually tracked during the first 7 days of imaging. The length and trajectory of the tracks followed by the different cells was used to compare the migration capabilities of hESC-RPE among the different substrates. Results are presented as mean ±SD (standard deviation). Quantitative real-time PCR Total RNA was isolated using the RNeasy Plus Mini Kit and treated with RNase-free DNase (both from Qiagen). cDNA was synthesized using 1 µg of total RNA in 20 µl reaction mixture, containing random hexamers and Superscript III reverse transcriptase (GIBCO Invitrogen), according to the manufacturer’s instructions. Taq-polymerase together with Taqman probes (Life Technologies) for RPLPO (cat. no. 4333761F), GAPDH (cat. no. 4333764F), NANOG (cat. no. Hs02387400_g1), POU5F1/OCT4 (cat. no. Hs03005111_g1), SOX9 (cat. no. Hs01001343_g1), PAX6 (cat. no. Hs01088112_m1), BEST1 (cat. no. Hs00188249_m1), RPE65 (cat. no. Hs01071462_m1) PMEL (cat. no. Hs00173854_m1), PAX3 (cat no. Hs00240950_m1) and EDNRB (cat no. Hs00240747_m1) were used. Samples were subjected to real-time PCR amplification protocol on StepOne™ real-time PCR System (Applied Biosystems). Biological triplicates were performed for every condition and technical duplicates were carried for each reaction. Results are presented as mean ±SEM (standard error of the mean). Flow Cytometry hESC-RPE growing on the tested substrates were dissociated into single cells using TrypLE Select after 1 month in culture. Cells were stained with violet LIVE/DEAD fixable stain kit (Invitrogen) following the manufacturer instructions. Samples were then fixed for 15 min in 4% methanol free formaldehyde (Polysciences) and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. Mouse anti-MITF (Abcam ab3201, clone [D5]) and mouse anti-BEST1 (Millipore, MAB5466) primary antibodies were used at a concentration of 10 μg/mL, diluted in 2% FBS, 0.1% Tween-20 (Sigma). Cell were incubated with the primary antibodies on ice for 30 min. Indirect immunostaining was completed using Alexa Fluor 488 Donkey anti-mouse IgG secondary antibody at a concentration of 2 μg/mL (Life Technologies A21202) on ice for another 30 min. Fluorescence minus one (FMO) controls were included for each condition to identify and gate negative and positive cells. Stained cells were analyzed using a BD LSRFortessa flow cytometer equipped with 488 nm, 561 nm, 405 nm and 640 nm lasers (BD Biosciences). Analysis of the data was carried out using FlowJo v.10 software (Tree Star). Enzyme-Linked Immunosorbent Assay (ELISA) hESC-RPE were cultured on Transwell membranes (0.33 cm2, Millipore) coated with different substrates. Supernatants from both the hESC-RPE apical and basal sides (meaning upper and lower compartments of the Transwell, respectively) were collected 60 hours after the medium was changed. VEGF and PEDF secretion levels were measured in triplicates for each condition with commercially available human VEGF and PEDF ELISA Kits (VEGF: Cat#DVE00, R&D Systems; PEDF: Cat#RD191114200R, BioVendor), in accordance with the instructions of the manufacturers, after 4.5 and 7 weeks of culture, respectively. The optical density readings were measured using SpectraMax 250 Microplate Reader (MolecularDevices). Results are presented as mean ±SEM (standard error of the mean). Phagocytosis assay In order to assess the phagocytic activity of our hESC-RPE, an in vitro assay using FITClabelled photoreceptor outer segments (POS) was performed. FITC-labelled bovine POS were isolated and kindly given by Dr. E.F. Nandrot from Institut de la Vision, Paris (Parinot et al., 2014). For that purpose, hESC-RPE were cultured on Transwell membrane (0.33 cm2, Corning) coated with different substrates for one month after seeding. Cells were incubated at 37°C or 4°C for 16 hours with 2.42x106 thawed POS/Transwell diluted in DMEM or CO2 independent media (both from LifeTechnologies), respectively. After incubation, cells were quenched with Trypan Blue Solution 0.2% (GIBCO, Invitrogen) for 10 minutes at room temperature, fixed with 4% methanol free formaldehyde (Polysciences) at room temperature for 10 min and permeabilized with 0.3% Triton X-100 in DPBS for 15 min. Rhodamine phalloidin staining (1:1000, 20 min at room temperature, Life Technologies) was used to visualize the cell boundaries. Nuclei were stained with Hoechst 33342 (1:1000, 20 min at room temperature, Invitrogen). Undifferentiated hESC were used as a negative control. hESC were cultured under the same conditions and incubated also at 37°C and 4°C with 2.42x106 thawed POS/Transwell diluted in DMEM or CO2 independent media. Wheat Germ Agglutinin-Alexa Fluor® 594 Conjugate (1:200, 20 min at room temperature, Invitrogen) was used in to visualize the cell boundaries of hESC. Images were acquired with Zeiss LSM710-NLO point scanning confocal microscope. Postacquisition analysis of the pictures was performed using IMARIS (Bitplane). Total number of engulfed FITC-POS per condition was quantified with the custom-made pipeline developed within cell image analysis software CellProfiler (Broad Institute). Results are presented as mean ±SD (standard deviation). Transepithelial resistance measurements Transepithelial electrical resistance (TER) RPE cells plated on Transwells (0.33 cm2, Millipore) was measured using the Millicell Electrical Resistance System volt-ohm meter (Millicell ERS-2, Millipore), according to the manufacturer’s instructions. Cultures were equilibrated outside the incubator at room temperature for 15-20 min before the experiment. Measurements were performed in unchanged culture media in triplicates for each condition, at three different positions of each well. Averages were used for further analysis. The background resistance was determined from a blank culture insert in the same media coated with the corresponding substrate but without cells, and subtracted from the respective experiment condition. Measurements are reported as resistance in ohms times the area in square centimeter (Ω*cm2). Results are presented as mean ±SEM (standard error of the mean). Immunofluorescence Protein expression of mature hESC-RPE monolayers was assessed with immunofluorescence. Cells were fixed with 4% methanol free formaldehyde at room temperature for 20 min, followed by permeabilization with 0.3% Triton X-100 in Dulbecco’s phosphate-buffered saline (DPBS) for 10 min and blocking with 4% fetal bovine serum (FBS) and 0.1% Tween-20 in DPBS for 1 hour. Primary antibodies were diluted to the specified concentrations in 4% FBS, 0.1% Tween-20, DPBS solution: Bestrophin 1 (BEST1) (1:100, Millipore MAB5466), Zonula occludens-1 (ZO-1) (1:100, Invitrogen 40-2200), cellular retinaldehyde-binding protein (CRALBP) (1:250, Abcam ab15051, clone [B2]) and alpha 1 sodium/potassium ATPase (Na/K-ATPase) (1:200, Abcam ab7671, clone [464.6[). The primary antibodies were incubated overnight at 4°C followed by 2 hours incubation at room temperature with secondary antibodies: Alexa Fluor 647 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-mouse IgG (both from Life Technologies, A31573 and A21202, respectively) diluted 1:1000 in 4% FBS, 0.1% Tween-20, DPBS solution. Nuclei were stained with Hoechst 33342 (1:1000, Invitrogen). Images were acquired with Zeiss LSM710-NLO point scanning confocal microscope. Post-acquisition analysis of the pictures was performed using IMARIS (Bitplane). Animals After approval by the Northern Stockholm Animal Experimental Ethics Committee 17 New Zealand white albino rabbits (provided by Lidköpings rabbit farm, Lidköping, Sweden) aged 5 months, weighing 3.5 to 4.0 kg, where used in this study. All experiments were conducted in accordance with the Statement for the Use of Animals in Ophthalmic and Vision Research. Subretinal transplantation hESC-RPE monolayers were washed with PBS, incubated with TrypLE, and dissociated to single cell suspension in a similar manner as here described for hESC, using a 5% CO2 incubator. Cells were then counted in a Neubauer hemocytometer chamber using 0.4% trypan blue (GIBCO, Invitrogen). For subretinal injection, a hESC-RPE single cell suspension was centrifuged at 2400 rpm for 4 min, the medium was carefully removed, and the cell pellet was resuspended in freshly filter-sterilized PBS to a final concentration of 1000 cells/μL. The cell suspension was then aseptically aliquoted into 100 μL units and kept on ice until surgery. hESC were washed with PBS, incubated with Accutase (GIBCO, Invitrogen) for 2 min at 37°C, centrifuged at 1000 rpm for 5 min, the medium was carefully removed, and the pellet was resuspended in freshly filter-sterilized PBS to a final concentration of 1000 cells/μL. The cell suspension was then aseptically aliquoted into 400 μL units and kept on ice until surgery. NHDF cells were washed with PBS, incubated with Trypsin 1x (LifeTechnologies) for 5 min at 37°C, centrifuged at 1000 rpm for 5 min, the medium was carefully removed, and the pellet resuspended in freshly filter-sterilized PBS to a final concentration of 1000 cells/μL. The cell suspension was then aseptically aliquoted into 400 μL units and kept on ice until surgery. Animals were put under general anesthesia by intramuscular administration of 35 mg/kg ketamine (Ketaminol, 100 mg/ml, Intervet) and 5 mg/kg xylazine (Rompun vet. 20 mg/ml, Bayer Animal Health), and the pupils were dilated with a mix of 0.75% cyclopentolate / 2.5% phenylephrine (APL). Microsurgeries were performed on both eyes using a 3-port 25G transvitreal pars plana technique (Alcon Accurus, Alcon Nordic). 25G trocars were inserted 1 mm from the limbus and an infusion cannula was connected to the lower temporal trocar. The cell suspension was drawn into a 1 mL syringe connected to an extension tube and a 38G polytip cannula (MedOne Surgical Inc). Without infusion or prior vitrectomy the cannula was inserted through the upper temporal trocar. After proper tip positioning, ascertained by a focal whitening of the retina, 50 μL of cell suspension (equivalent to 50.000 cells) was injected slowly subretinally approximately 6 mm below the inferior margin of the optic nerve head, forming a uniform bleb that was clearly visible under the operating microscope. Care was taken to maintain the tip within the bleb during the injection to minimize reflux. After instrument removal light pressure was applied to the self-sealing suture-less sclerotomies. No post-surgical topical steroids or antibiotics were given. Animals were treated in three separate cohorts. In the first cohort (7 animals receiving 521derived hESC-RPE) 4 animals received 2 mg (100 μL) of intravitreal triamcinolone (Triescence, Alcon Nordic) immediately after surgery, one animal received systemic cyclosporine-A (a descending dose of 20-5 mg/kg/d sc) (Teva Sweden AB), one animal received both triamcinolone and cyclosporine-A, and one animal was maintained without immunosuppression. On post-operative examination, none of the eyes showed signs of extra- or intra-ocular infection or inflammation except for one animal (one eye) that had a uveitis one week after surgery and was sacrificed. A second animal was sacrificed 6 weeks post-transplantation after signs of cyclosporine-A toxicity (hematuria). Analysis of cohort one showed no beneficial effect of cyclosporine-A on donor cell integration (defined as appearance of pigmented areas within 4 weeks post-transplantation) whereas several animals treated with only triamcinolone had successful integration. The second cohort (6 animals receiving 521-derived hESC-RPE) was therefore treated with intravitreal triamcinolone alone without any post-operative complications. The third cohort received either undifferentiated hESC (2 animals) or fibroblasts (2 animals) with post-operative triamcinolone. In animals kept for long-term evaluation, intravitreal triamcinolone was readministered every 3 months. Spectral domain-optical coherence tomography (SD-OCT) Anesthetized rabbits were placed in an adjustable mount. A commercial Spectralis HRA + OCT device (Heidelberg Engineering) with the Heidelberg Eye Explorer Software (version 1.9.10.0) was used to obtain horizontal cross-sectional b-scans of hESC-RPE treated animals. The Spectralis has a real-time motion tracking system that minimizes eye motion artifacts. At least 3 cross-sectional OCT scans were obtained with simultaneous infraredconfocal scanning laser ophthalmoscope (IR-cSLO) reflectance reference images representing the upper, central and lower portion of the transplanted area. The best overall image quality was obtained when the OCT setting was on high-speed acquisition with at least 50 averaged automatic real-time images. En-face fundus images were obtained by IRor multicolor cSLO (a composite of three simultaneously acquired color cSLO images). These modalities have a higher contrast level compared to conventional fundus camera photos. The total area of the pigmented lesions was estimated manually on IR-cSLO images using the built-in measuring tool of the Spectralis software. Photoreceptor rescue measurements We previously defined ORT as the SD-OCT distance between the inner nuclear layer (INL) and RPE using the ImageJ software (http://imagej.nih.gov/ij/) (Bartuma et al., 2015). ORT of treated regions was obtained from a section overlying at least 500 μm of continuously integrated cells (mean of 10 random measurements). ORT of non-treated control regions was obtained from the same scan 500 μm outside the bleb (mean of 5 random measurements). Relative ORT was then calculated as the ratio between treated and nontreated retina. Distances were normalized to the 200 μm scale bar of the original image. All measurements were done one month post-transplantation, or at the nearest time-point after. A 2-sided Student’s T-test was performed comparing the relative ORT between eyes with integrated and with non-integrated rhLN-521-derived hESC-RPE (defined as a pigmented area >2.5 mm2). Results are presented as mean ±SD (standard deviation). Histology and tissue immunostaining Immediately after sacrifice by intravenous injection of 100 mg/kg pentobarbital (Allfatal vet. 100 mg/ml, Omnidea), the eyes were enucleated and the bleb injection area marked with green Tissue Marking Dye (TMD) (Histolab Products). An intravitreal injection of 100 μL fixing solution (FS) consisting of 4% buffered formaldehyde (Solvenco AB) was made before fixation in FS for 24-48 hours, and embedding in paraffin. 4 μm serial sections were made through the TMD-labeled area and every 4 sections were stained with hematoxylin-eosin (HE). Images were captured with an Iphone 4S mounted to a bright-field microscope (Zeiss Axioskop 40, CarlZeiss). For immunostaining, slides were deparaffinized in xylene, dehydrated in graded alcohols, and rinsed with dH2O and Tris Buffered Saline (TBS, pH 7.6). Antigen retrieval was done in 10 mM citrate buffer (trisodium citrate dihydrate, Sigma-Aldrich, pH 6.0) with 1:2000 Tween20 (Sigma-Aldrich) at 96°C for 30 min, followed by 30 min cooling at room temperature. Slides were washed with TBS and blocked for 30 min with 10% Normal Donkey Serum (Abcam) diluted in TBS containing 5% (w/v) IgG and protease free bovine serum albumin (Jackson Immunoresearch) in a humidified chamber. Primary antibodies diluted in blocking buffer, were incubated overnight at 4°C: human nuclear mitotic apparatus protein (NuMA) (1:200, Abcam ab84680), BEST1 (1:200, Millipore MAB5466), RPE65 (1:200, Abcam ab78036, clone [401.8B11.3D9]) and Rhodopsin (1:2000, Millipore MAB5356). Secondary antibodies (Alexa Fluor 555 donkey anti-rabbit IgG A31572 and Alexa Fluor 647 donkey antimouse IgG A31571, both from Life Technologies) diluted 1:200 in blocking buffer, were incubated 1 hour at room temperature. Sections were mounted with vector vectashield with DAPI mounting medium (Vector Laboratories) in a 24x50 mm coverslip. Images were taken with Zeiss LSM710-NLO point scanning confocal microscope. Post-acquisition analysis of the pictures was performed using ImageJ software. In order to enhance the signal of the NuMA, TSA-Plus-Cyanine3 System was used (Perkin Elmer Life Sciences). An extra blocking step of 30 min with 3% H2O2 in Methanol was added prior to serum blocking followed by incubation with NuMA. Secondary antibody chicken antirabbit HRP (1:200, Santa Cruz sc-2963) diluted in the serum blocking buffer was incubated for 30 min, followed by 8 min incubation with Tyramide-Cy3 according to manufacturer’s instructions. Images were taken with an Olympus IX81 fluorescence microscope and postacquisition analysis of the pictures was performed using ImageJ software. REFERENCES Parinot, C., Rieu, Q., Chatagnon, J., Finnemann, S.C., and Nandrot, E.F. (2014). Large-scale purification of porcine or bovine photoreceptor outer segments for phagocytosis assays on retinal pigment epithelial cells. J Vis Exp 94, e52100, doi:10.3791/52100. Kruse, P.F., and Patterson, M.K. (1973). Tissue culture: methods and applications; Section IV: Replicate Culture Methods; Chapter 3, Leonard Hayflick (Academic Press, New York).