The outer nuclear layer (ONL), normally consisting of five to six layers, was reduced to a single layer in PBS-injected eyes (Determine?4A), whereas eyes transplanted with rhLN-521-hESC-RPE had preserved ONL and POS (Physique?4B). 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 Rabbit Polyclonal to US28 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. and and displayed Monepantel as relative to undifferentiated hESCs. Bars represent Monepantel means SEM from three impartial experiments. (H) Flow cytometry analysis of MITF expression on hESC-RPE cells produced 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 impartial 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 4C were used as unfavorable controls. Bars represent Monepantel means SD from three impartial experiments. (L) TER measurements of hESC-RPE cells produced 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 impartial experiments. Scale bars: B, D, E, 500?m. See also Figure?S1. rhLN-521 Efficiently Supports Homogeneous Growth of Pigmented and Functional hESC-RPE Endogenous BM contains four LNs: LN-111, LN-332, LN-511, and LN-521. Consequently, we decided to compare subsequent growth 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 (Physique?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 1DC1F and Movie S1), while progressively maturing into pigmented hexagonal cells. This correlates well with Monepantel a previous study showing that this 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 1DC1F and S1A). Transcriptional analysis showed comparable profiles in hESC-RPE differentiated on each of the five substrates with reduction of pluripotency-associated transcripts 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 (Physique?S1B). RPE differentiation was evident with expression of bestrophin 1 (BEST1), RPE-specific protein 65?kDa (RPE65), and premelanosome protein (PMEL) (Physique?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 1ICK and S1DCS1G). 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 junction integrity of our hESC-RPE monolayer on LN-111, LN-511, and LN-521 Monepantel in a time-dependent manner, but not.