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. Author manuscript; available in PMC: 2022 Mar 1.
Published in final edited form as: Nature. 2021 Aug 11;597(7875):256–262. doi: 10.1038/s41586-021-03795-7

Extended Data Fig. 5 ∣. The bone marrow microenvironment influences HSC lineage output.

Extended Data Fig. 5 ∣

a, Schematic of experimental approach for transplanting freshly isolated HSCs from fetal liver or 24-month-old mice into either 2-month-old or 24-month-old lethally irradiated mice. b, BMD in 2-month-old and 24-month-old lethally irradiated mice transplanted with fetal liver (FL) HSCs or HSCs from 24-month-old mice 8 weeks after haematopoietic reconstitution (E15 FL into old mice, n = 6; n = 5, all other groups). BM, bone marrow. c, Callus index of recipient mice at day 14 after fracture induced at the 8-week time point after transplantation (E15 FL groups, n = 5; 24-mo BM groups, n = 4). d, Representative FACS-gating strategy for myeloid (GR1+) and lymphoid (B and T cells) cells in peripheral blood after haematopoietic reconstitution with GFP-donor HSCs (gated from TER119−, live cells). e, Representative bone marrow FACS-gating strategy of GFP+ donor-derived cells for haematopoietic lineage tree populations. f, Peripheral blood analysis for donor chimerism after haematopoietic reconstitution of 2-month-old and 24-month-old mice with young HSCs. g, BM analysis of donor-derived (GFP+) HSC lineage cell populations by flow cytometry. Two-way ANOVA with Bonferroni post-hoc test. h, Representative TRAP-staining and GFP-fluorescence images (same section) from day-10 fracture calluses of 2-month-old and 24-month-old mice reconstituted with GFP-labelled HSCs from 2-month-old mice. i, Quantification of the total area of TRAP+GFP+ regions in sections of fracture calluses of mice (n = 3 per age group).j, Flow cytometric analysis of lymphoid and myeloid cell types in 6-day co-cultures (no SSCs, n = 4; 2-mo, n = 5; 24-mo, n = 5). One-way ANOVA with Tukey’s posthoc test for comparison of more than two groups. k, Peripheral blood analysis for donor chimerism after haematopoietic reconstitution with co-cultured haematopoietic cells. Two-way ANOVA with Bonferroni post-hoc test. l, Bone marrow analysis of co-cultured donor-derived (GFP+) HSC lineage cell populations by flow cytometry (no SSCs, n = 3; 2-mo, n = 4; 24-mo, n = 3). One-way ANOVA with Tukey’s post-hoc test for comparison of more than two groups. Comparison of 2-month-old versus 24-month-old groups by two-sided Student’s t-test adjusted for non-normality (Mann–Whitney test) or unequal variances (Welch’s test) where appropriate. One-way ANOVA with Tukey’s post-hoc test. All data are mean + s.e.m. For exact P values, see Source Data. Scale bar, 100 μm.