Fig 2: In vivo lineage potential of human foetal HHyPs. a Experimental strategy for isolation and in vivo characterisation of foetal hepatobiliary hybrid progenitors (HHyPs) by transplantation beneath the renal capsules of immunodeficient NOD scid gamma (NSG) mice. b 2D t-SNE visualisation of single cells isolated from foetal human liver coloured by FACS gating population (left panel) and transcript expression of ALB and MCAM overlaid on the 2D t-SNE space of human foetal liver scRNA-seq analysis (right panel). Expression is Log10(TPM). c Gating scheme for the isolation of distinct foetal human liver populations based on expression of CD235a, CD45, EpCAM, NCAM and MCAM. d Hematoxylin and eosin (H&E) staining in tissue cross-sections of implant regions 4 weeks post renal capsule transplantation of human foetal liver FACS populations. Scale bars represent 100 µm. e Immunofluorescence (IF) co-staining of CK19 (green) and ALB (red) in implant regions of HHyPs post 4 weeks transplantation and matched control mouse adult liver. Slides counterstained in DAPI (cyan). Scale bar represents 25 µm. f IF staining of FAH (red) and HNF4A (green) in implant region of HHyPs 4 weeks post transplantation. Slides counterstained in DAPI (cyan). Scale bars represent 25 µm. g IF co-staining of TROP-2 (white) and ALB (red) in implant region of HHyPs 4 weeks post transplantation. Slides counterstained in DAPI (cyan). Scale bars represent 25 µm. t-SNE t-distributed stochastic neighbour embedding, FACS fluorescence-activated cell sorting, TPM transcripts per million

Fig 3: Successive emergence of primitive and definitive hematopoietic populations during differentiation of human iPSCs. (A) Representative flow cytometry plots for CD34, CD45 and CD235a expression in gated CD43+CD45+/- hematopoietic cells at various days of differentiation. Gates shown include: CD34-CD45-CD235a+ primitive erythroid progenitors (red), CD34hiCD45lo multilineage HSPCs (black), and CD34lo/-CD45+ hematopoietic progenitors (gray). The complete gating strategy is shown in Fig. S1B. (B) Percentage of hematopoietic populations with immunophenotypes classified above within the CD43+CD45+/- hematopoietic fraction at various days of differentiation (n = 3). (C) CFU counts per 1000 sorted hematopoietic cells at various days of differentiation (n = 6). Insets at the bottom are representative CFU plates demonstrating multilineage HSPCs at day 10 and almost exclusive production of erythroid progenitors at day 21 of culture. (D) Percentage of ß-globin series expression in erythroid cells differentiated from day 7 and 17 supernatant cells (n = 3), measured by RP-HPLC. (E) Absolute numbers of CD43+CD45+ hematopoietic cells with a CD34+CD38-CD45RA-CD90+CD49f+ immunophenotype arising from 20 to 35 iPSC clusters (one well of a 12-well plate) at various days of differentiation (n = 3). In panels (B) to (E), results are displayed as mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 4: Expansion of EpCAM+/NCAM+/TROP2- human foetal liver HHyPs in the renal capsule. a 2D t-SNE visualisation of single cells isolated from foetal and adult human liver coloured by TROP-2 gene expression. Expression is Log10(TPM). b Schematic of hepatic and BEC lineage restriction of HHyPs based on NCAM/TROP-2 expression. c Gating scheme for the isolation of distinct foetal human liver populations based on expression of CD235a, CD45, EpCAM, NCAM and TROP2. d Hematoxylin and eosin (H&E) staining in tissue cross-sections of explant region for 4-week post renal capsule transplantation. Scale bars represent 250 µm. e Immunofluorescence (IF) staining of FAH (red) in explant region of CD235a-/CD45-/EpCAM+/NCAM+/TROP2- HHyPs 4-week post transplantation. Slides counterstained in DAPI (cyan). Scale bars represent 75 µm. t-SNE t-distributed stochastic neighbour embedding, TPM transcripts per million