Transplantation of young gut microbiota increased functional HSCs in mice. (A) Flowchart for limiting-dilution transplantation assay (LDA) to determine the frequency of functional HSCs in FMT-AA mice and FMT-YA mice. (B-C) Representative flow cytometry plots and line graph of donor-derived CD45.2⁺ reconstitution in CD45.1⁺ mice receiving the highest cell doses at 16 weeks after transplantation in primary LDA. (D) HSC frequency determined by primary LDA. (E) Frequency in TNCs and absolute cell number of donor-derived HSPCs in BM from recipient mice in primary LDA. (F-G) Representative flow cytometry plots and line graph of donor-derived CD45.2⁺ reconstitution in CD45.1⁺ mice receiving the highest cell doses at 16 weeks after transplantation in secondary LDA. (H) HSC frequency determined by secondary LDA. (I) Frequency in TNCs and absolute cell number of donor-derived HSPCs in BM from recipient mice in secondary LDA. (J) Flowchart for clonogenic assay and transplantation assay of purified LT-HSCs. (K) Number of total colonies measured for LT-HSCs in FMT-AA mice and FMT-YA mice in methylcellulose. (L) Line graph of donor-derived CD45.2⁺ reconstitution in CD45.1⁺ mice at 16 weeks after HSC transplantation. (M) Absolute cell numbers of donor-derived HSPCs in BM from recipient mice in HSC transplantation assay. Graphs show mean ± SEM, with statistical significance determined by Student t test (n = 6 per group). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. Med, medium; ST-HSC, short-term HSC.