Multipotent repopulating HSCs/MPPs and quiescent CD71⁺ HSC-like cells with restricted erythroid/megakaryocyte differentiation potential coexist in steady-state PB. (A) Frequency of repopulating cells in PB HSCs/MPPs calculated by using Extreme Limiting Dilution Analysis⁷³ statistics at 8 weeks (2 experiments, n = 17) and 16 weeks (2 experiments, n = 10) posttransplantation. The table indicates doses of cells injected and number of NSG mice with human cell engraftment in their BM (see Methods). (B) Ratio of early megakaryocyte/erythroid/mast cell/basophil (MEMB, sum cluster 2, 12) to early myeloid (My,sum cluster 6, 9) progenitors in all tissues. (C-D) Colonies derived from single phenotypic HSCs/MPPs from BM (n = 913 single cells from 7 samples), spleen (SPL; n = 234 single cells from 3 samples), and PB (n = 3 034 single cells; 27 independent PBs over 16 experiments) seeded into medium supporting Ery, Meg, lymphoid, and My differentiation (see Methods). (C) Frequency of colonies containing Ery and/or Meg cells for each tissue as assessed by flow cytometry. (D) Relationship between the percentage of all Ery-, Meg-, and My-containing colonies and the proportion of CD71⁺CD34^lo cells within the phenotypic PB HSC/MPP pool. Linear regression and 95% confidence interval are indicated by solid line and shaded area, respectively. n = 17 PBs. (E) Representative pseudocolor plot for flow cytometry isolation of CD71^– and CD71⁺ HSCs/MPPs in PB gated on phenotypic HSCs/MPPs (CD19^–CD34⁺CD38^–CD45RA^– cells as defined in supplemental Figure 1D). (F) Percentage of colonies generated by CD71^– (n = 872 single cells; 15 independent PBs) and CD71⁺ (n = 1 109 single cells; 18 independent PBs) HSCs/MPPs. P values comparing CD71^– and CD71⁺ HSC/MPP colony output are shown. Two-tailed Mann-Whitney test. (G) Serial replating of PB CD71^– or CD71⁺ HSCs/MPPs and CD71⁺ MEPs (E-MEPs) in methylcellulose medium. Colony numbers per indicated number of seeded cells after first (left) and secondary (right) plating are shown. n = 4 PBs over 3 experiments. Paired, 2-tailed t test. *P < .05, **P < .01. (H) Ratio of NSG mice engrafted to total mice tested at the indicated time points after transplantation of CD71^– and CD71⁺ phenotypic PB HSCs/MPPs. P values comparing engraftment of CD71^– and CD71⁺ HSCs/MPPs were determined by 2-tailed Fisher’s exact test and are shown below each time point. (I) Frequency of repopulating cells within all phenotypic PB HSCs/MPPs (same as Figure 6A) and CD71^– HSCs/MPPs at 8 weeks after transplantation using Extreme Limiting Dilution Analysis statistics. (J) Percentage of CD71⁺ cells within the phenotypic HSC/MPP pool of BM (n = 8), SPL (n = 4), and PB (n = 65). One-way analysis of variance; Tukey’s multiple comparison. Data in panels B and C are given as median ± 95% confidence interval; Kruskal-Wallis and Dunn’s multiple comparison tests. Data in panels F, G, and J are given as mean ± standard deviation. BFU-E, burst forming unit erythroid; CFU-E, colony forming unit erythroid; GEMM, granulocyte, erythrocyte, monocyte, megakaryocyte; GM/M granulocyte monocyte/monocyte; LD; living donor; n.d., not determined; NK, natural killer; OD, organ donor; ns, not significant (P > .05); undiff, undifferentiated.