In the present study we show
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 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.
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 non-human proteins into the phospholipase a2 inhibitor 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 Ω 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).