Journal: bioRxiv

Article Title: Extensive monolayer formation depends on a subpopulation of transplanted human iPSC-derived RPE cells

doi: 10.1101/2024.10.15.618147

Figure Lengend Snippet: Identification and validation of cell surface sorting markers for iPSC-derived P1 RPE cells (A) P1 RPE cells were screened for expression of 371 antibodies directed against cell surface markers using flow cytometry. Hits were identified based on correlation to side scatter (SSC) intensity as proxy for pigmentation, here shown for CD54 (ICAM-1) (positive correlation) and PSA-NCAM (negative correlation). (B) Combinatorial staining for CD54 and PSA-NCAM reliably discriminates SSC-low non-target (unpigmented, backgated in blue) and SSC-high target cells (pigmented, backgated in orange). (C) Immunofluorescent staining of RPE cells in culture shows cells expressing PSA-NCAM but little CD54 mostly in regions lacking pigmentation and proper cobblestone morphology (areas highlighted by dashed lines). Co-expression of CD54 and PSA-NCAM appears to occur primarily in regions of intermediate pigmentation levels and mixed cobblestone morphology (area highlighted by solid line) (D) Flow scatter and corresponding transmitted light microscopy images for unsorted (UNS), CD54 + /PSA-NCAM - -enriched (ENR) and CD54 + /PSA-NCAM - -reduced (RED) cell fractions. Cells were sorted using the MACSQuant Tyto cell sorter to maximize target cell purity. Replated fractions reveal that pigmented cells can be efficiently enriched with the dual marker strategy. (E) CD54 + /PSA-NCAM - sorting of frozen and freshly harvested cells consistently yielded a target fraction of high purity. Circles show single re-analysis measurements, solid points their average. Scale bars: 100 µm (D), 50 µm (C).

Article Snippet: P1 RPE cells derived from CRTD1 iPSC were screened for expression of 371 antibodies directed against cell surface markers using the flow cytometry-based MACS® Marker Screen, anti-human, version 01 (Miltenyi Biotec, 130-110-055) according to the manufacturer’s instructions.

Techniques: Biomarker Discovery, Derivative Assay, Expressing, Flow Cytometry, Staining, Light Microscopy, Marker

Journal: bioRxiv

Article Title: Extensive monolayer formation depends on a subpopulation of transplanted human iPSC-derived RPE cells

doi: 10.1101/2024.10.15.618147

Figure Lengend Snippet: CD54 + /PSA-NCAM - RPE cells show increased monolayer formation after transplantation. (A – C) Flat-mounted eyecups that received P1 RPE single cell suspensions which were either unsorted (UNS; A), enriched (ENR, B), or reduced (RED, C) in CD54 + /PSA-NCAM - cells show large areas of donor cells identified by Human Nuclei staining (graft region outlined with dotted lines). While in UNS (A, A’) and RED (C) samples the proportion of area with cell clusters (visible through intense Human Nuclei staining) is high, in ENR transplanted retinas the proportion of monolayer with typical cobblestone morphology as seen by staining for F-actin is high (B, B’, B’’). Human nuclei visibility in properly structured monolayers is concealed due to high pigmentation of donor cells (B’’; for single z-planes see figures S4E and F), but better visible at monolayer edges (B’). Magnification of an RPE-depleted area without donor cells is shown in C’. (D) Outlines of monolayer cells from B’’ as detected with the REShAPE tool( Ortolan et al , 2022 ) show the possibility to analyse the grafts in flatmount view. (E-G) REShAPE analysis of the flatmounts A-C (corresponding REShAPE cell outline maps see figure S4G-I). (E) reveals a highly increased number of monolayer cells in the ENR graft in comparison to UNS or RED (UNS: 7453, ENR: 37607, RED: 6921). ENR monolayer cells on average appear larger than UNS or RED monolayer cells (F; mean areas: UNS 57 µm 2 , ENR 79 µm 2 , RED 55 µm 2 , violin plots of all cells detected with overlaid box-plots showing median and 25% quartiles, black lines showing the mean). In all three conditions human monolayer cells have mostly 5 or 6 neighboring cells, with the ENR graft having the highest ratio of cells with 5-7 neighbors (G; UNS: 67%, ENR: 82%, RED: 69%). (H) Monolayers formed in all conditions show a polarized morphology with basal collagen IV (COL4) and apical ATPase (ATP1A1) expression. Human nuclei are located basally, while pigmentation (dark in DIC images) appears mainly apically. Large, highly pigmented, round structures are frequently detected in clusters (see also A’) and sometimes in monolayer areas, likely representing pigment-filled macrophages (compare also figure S5A). (I, J) Quantification of monolayer proportion after transplantation of RPE cells from two separate differentiation rounds. While in both rounds the CD54 + /PSA-NCAM - ENR donor cell fractions robustly generated similarly high proportions of monolayer (ENR - I : 82.5% ± 3.5%; n=7; ENR – J: 69.5% ± 6.9%; n=4), the amount of monolayer generated by UNS (UNS - I: 67.3% ± 3.5%; n=5; UNS - J: 23.1% ± 3.3%, n=7) and RED cell fractions (RED - I: 55.2% ± 8.1%; n=6; RED - J: 6.9% ± 1.3%, n=9; Kruskal-Wallis with pairwise Wilcoxon test) strongly varied, correlating with different amounts of CD54 + /PSA-NCAM - cell purity within the different fractions (see also figure S4A for purity data related to and figure S4B for purity data related to ). (K) Quantification of monolayer proportion after transplantation of RPE cells from a third differentiation round, sorted with conventional flow cytometry (UNS: 17.9% ± 3.8%, n = 5; ENR: 55.3% ± 12.2%, n = 4; RED: 21.5% ± 7.2%, n = 4; Kruskal-Wallis with pairwise Wilcoxon test). Scale bars, 500 µm (A, B, C), 50 µm (A’, B’, B’’, C’, D) and 20 µm (H). Data are represented as mean ± SEM.

Article Snippet: P1 RPE cells derived from CRTD1 iPSC were screened for expression of 371 antibodies directed against cell surface markers using the flow cytometry-based MACS® Marker Screen, anti-human, version 01 (Miltenyi Biotec, 130-110-055) according to the manufacturer’s instructions.

Techniques: Transplantation Assay, Staining, Comparison, Expressing, Generated, Flow Cytometry