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Additional file 2: Containing supplementary data about CK3 and CK12 expression in post-thaw p1 hPSC-LSCs (Supplementary Fig. S1), ���Np63�� expression in hPSC-LSCs derived from the ABCG2+ cell population (Supplementary Fig. S2) and the Supplementary Movie S3 showing the wound closure of p1 hPSC-derived LSCs versus the tissue-derived porcine LSCs

Figure S1. Description of three distinct stem cell population phenotypes emerging during the hPSC-LSC differentiation, separated by their expression patterns for ABCG2 and ∆Np63α. Representative cells from different populations are marked with symbols to the IF images of d10 and d24 cytospin samples. Scale bars, 100 μm. Cell nuclei counterstained with DAPI (blue). Data are presented with the representative hESC line Regea08/017. (DOCX 742 kb)

Figure S2. (A) Representative flow cytometry graphs of the negative controls, isotype controls, and ABCG2-stained hPSC-LSC samples in different time points. (B) Morphology and ABCG2/p63α expression of day 11 sorted ABCG2-positive hPSC-LSCs after continued culture (17 days) in CnT-30 medium and on LN-521/Col IV (B). Scale bar, 100 μm. Cell nuclei counterstained with DAPI (blue). BF: brightfield, FACS: fluorescence-activated cell sorting. Data are presented with the representative hESC line Regea08/017. (DOCX 1668 kb)

Figure S4. (A) Additional characterization of OCT3/4, PAX6, CK14, and CK15 expression at d24 in the novel CnT-07+ENRC maintenance condition. (B) Characterization of Ki67/ABCG2 protein expression at d11, as well as after continued culture in CnT-07+ENRC or CnT-30 at d21. In both panels, cell nuclei counterstained with DAPI (blue) and scale bars, 100â Îźm. Data are presented for the hESC lines Regea11/013 (A) and Regea08/017 (B). (DOCX 5938 kb)

Supplementary materials and methods. (DOCX 65 kb)

(DOCX 19 kb)

Supplementary dataset 1. Optimization of hPSC-RPE differentiation. (DOCX 36 kb)

Supplementary dataset 2. Optimization of hPSC-LESC differentiation. (DOCX 42 kb)

Figure S3. Characterization of putative LSC marker expression during hPSC-LSC differentiation for hiPSC line UTA.04607.WT. (A) Representative morphology and protein expression of the cultures at selected time points. Scale bars, 100 μm for all images in the same column. Cell nuclei counterstained with DAPI (blue). (B) Marker expression differences in the d10 and d24 populations. Five images per sample and a minimum of 600 cells per time point were analyzed for each marker from cytospin samples. (C) p63α and ABCG2 expression in d10 and d24 hPSC-LSCs. Five images per sample and a minimum of 3 000 cells per time point were analyzed from cytospin samples. (D) The level of ABCG2 protein expression in UD-hPSCs and in d10 and d24–26 hPSC-LSCs, analyzed with flow cytometry. (G) The ABCG2 mRNA expression levels in UD-hPSCs and in d10 and d24 hPSC-LSCs analyzed with qRT-PCR. All quantitative data are presented as the mean + SD and n marks the individual cell differentiation batches serving as biological replicates. Statistical analysis in (D) was carried out using the Mann-Whitney U test. *P ≤ 0.05. (DOCX 4413 kb)

Table S2. Distribution of ABCG2-positive and ABCG2-negative cells in Regea08/017 d10 hPSC-LSC subpopulations marked by selected cell surface markers. (DOCX 19 kb)

Figure S3. Characterization of putative LSC marker expression during hPSC-LSC differentiation for hiPSC line UTA.04607.WT. (A) Representative morphology and protein expression of the cultures at selected time points. Scale bars, 100 μm for all images in the same column. Cell nuclei counterstained with DAPI (blue). (B) Marker expression differences in the d10 and d24 populations. Five images per sample and a minimum of 600 cells per time point were analyzed for each marker from cytospin samples. (C) p63α and ABCG2 expression in d10 and d24 hPSC-LSCs. Five images per sample and a minimum of 3 000 cells per time point were analyzed from cytospin samples. (D) The level of ABCG2 protein expression in UD-hPSCs and in d10 and d24–26 hPSC-LSCs, analyzed with flow cytometry. (G) The ABCG2 mRNA expression levels in UD-hPSCs and in d10 and d24 hPSC-LSCs analyzed with qRT-PCR. All quantitative data are presented as the mean + SD and n marks the individual cell differentiation batches serving as biological replicates. Statistical analysis in (D) was carried out using the Mann-Whitney U test. *P ≤ 0.05. (DOCX 4413 kb)

Full detailed descriptions for the hPSC differentiation towards LSCs and standard hPSC-LSC culture, establishment and maintenance of the ABCG2-positive hPSC-LSC culture, immunofluorescence characterization protocol, flow cytometry analysis and fluorescence-activated cell sorting protocol, quantitative RT-PCR protocol, and cell surface antigen screening with the LEGENDScreenâ ˘ Kit. (DOCX 29 kb)

Table S1. Primary and secondary antibodies used in this study. (DOCX 22 kb)

Table S2. Distribution of ABCG2-positive and ABCG2-negative cells in Regea08/017 d10 hPSC-LSC subpopulations marked by selected cell surface markers. (DOCX 19 kb)

Full detailed descriptions for the hPSC differentiation towards LSCs and standard hPSC-LSC culture, establishment and maintenance of the ABCG2-positive hPSC-LSC culture, immunofluorescence characterization protocol, flow cytometry analysis and fluorescence-activated cell sorting protocol, quantitative RT-PCR protocol, and cell surface antigen screening with the LEGENDScreenâ ˘ Kit. (DOCX 29 kb)

Table S1. Primary and secondary antibodies used in this study. (DOCX 22 kb)

Additional file 1: Containing detailed descriptions of the fluorescence activated cell sorting of ABCG2+ hPSC-LSCs and further differentiation of p1 hPSC-LSCs

Additional file 1: Containing detailed descriptions of the fluorescence activated cell sorting of ABCG2+ hPSC-LSCs and further differentiation of p1 hPSC-LSCs