Fig. 4: PEMP generate HLA-DR^neg HBC-like cells in vitro.

A Schematic depicting experimental design for PEMP single-cell culture assay. B Boxplot depicting total colony-forming efficiency of adult peripheral blood GMP (Granulo-myeloid progenitors) and PEMP. GMP n = 3 donors, PEMP n = 6 donors. P-value calculated by two-tailed Mann–Whitney test. Exact p value = 0.0238. C Boxplot depicting mean number of CD45⁺ cells per colony per donor. GMP n = 3 donors, PEMP n = 6 donors. P value calculated by two-tailed Mann–Whitney test. Exact p value = 0.0238. D Representative flow cytometry analysis of a single-cell PEMP-derived colony which yielded CD14⁺ cells. E Representative flow cytometry analysis of a single-cell PEMP-derived colony which yielded megakaryocytes (Mega), erythroid cells (Ery) and granulocytes (Gran). F Donut plot showing lineage potential of PEMP defined by single-cell culture and flow cytometry analysis. ‘Immature’ denotes CD45⁺ colonies which did not contain any other defined immune cell lineages. (n = 6 donors, k = 42 colonies generated). G Stacked bar charts showing lineage potential of PEMP for each donor profiled. PCW age of the donors are added as labels above the bar chart. H Analysis of CD163 and FOLR2 expression in CD14⁺ colony derived from single PEMP and GMPs via flow cytometry, and I boxplot quantification of co-expression for all CD14⁺ colonies profiled (GMP n = 3 donors, k = 61 colonies) (PEMP n = 6 donors, k = 25 colonies). P value calculated by two-tailed Mann–Whitney test. P value <0.0001. J Representative Giemsa-stained cytospins of cells from CD14⁺ colonies derived from a single PEMP. Scale bars, 50 μm. Representative images from n = 4 donors. K Analysis of HLA-DR expression in CD14⁺ colony derived from single PEMPs and GMPs via flow cytometry. Each histogram represents a colony derived from an independent donor. L Boxplot quantification of HLA-DR expression in CD14⁺ colony derived from single PEMPs and GMPs via flow cytometry for all colonies profiled (GMP n = 3 donors, k = 61 colonies) (PEMP n = 6 donors, k = 25 colonies). P value calculated by two-tailed Mann–Whitney test. Exact p value = <0.0001. M Boxplot depicting median HLA-DR expression of all CD14⁺ colonies per donor. (GMP n = 3 donors) (PEMP n = 6 donors). All boxplot centre lines represent the median, with box limits showing the upper and lower quartiles, and whiskers denoting minimum and maximum values. Exact p value = 0.0238. *P ≤ 0.05, **P ≤ 0.01; ***P ≤ 0.001, ****P ≤ 0.0001.