Fig 2: Rad21 deficiency increases self-renewal and leads to impaired and myeloid-skewed differentiation of HSPCs. (A–D) Freshly isolated LSK cells were infected with an shRNA against Rad21 shRad21 or a control virus (shLuci) in culture and transplanted into lethally irradiated mice together with noninfected LSK cells from the same culture. (A and B) The number of HSCs (GFP+CD150+CD34-LSK cells) derived from infected LSK cells in the primary recipients (A) and the secondary recipients (B) that were transplanted with total BM cells of the primary recipients at 4 mo after the first transplantation. The dots represent individual recipient mice (n = 4–5 mice per group per experiment, n = 3 independent experiments, one of the three repeat experiments is shown, the other experiments showed similar results). Statistical values were calculated by Welch’s t test after log transformation. (C) The chimerism of GFP+ cells from the primary recipients was analyzed by FACS at 4 mo after transplantation in the indicated populations. HSC: CD150+CD34-LSK; MPP: CD34+LSK; CMP: common myeloid progenitor (Lin-c-Kit+Sca-1-CD34+Fc?R-); GMP: granulocyte macrophage progenitor (Lin-c-Kit+Sca-1-CD34+Fc?R+); MEP: megakaryocyte erythrocyte progenitor (Lin-c-Kit+Sca-1-CD34-Fc?R-), CLP: common lymphoid progenitor (Lin-c-KitlowSca-1lowIL-7Ra+Flt3+), Lin-: lineage- cells; BM: total BM cells. n = 4–5 mice per group per experiment, three independent experiments, one of the three experiments is shown, the other experiments showed similar results. (D) PB cells from the primary recipients were analyzed by FACS. The histogram shows the percentage of myeloid and lymphoid cells in GFP+CD45+ cells in the PB at the indicated time points after transplantation (n = 4–5 mice per group for each experiment, n = 3 independent experiments, one of the three experiments is shown; the other experiments showed similar results). Statistical significance for myeloid cells was assessed by Welch’s t test after logit transformation of the data. (E and F) LSK cells from young (2 mo old) wild-type mice were transduced with shLuci or shRad21 virus followed by transplantation into 2–3-mo-old recipients. 8 wk after transplantation, recipient mice were sacrificed and total BM was transplanted into secondary recipients. 4 wk after secondary transplantation, BM cells were analyzed to investigate the chimerism of shLuci- or shRad21-LSK cells in BM. Chimerism of shLuci- or shRad21-infected LSK cells in PB was monitored every 4 wk after each round of transplantation. (E) The data show the relative change of GFP+ cells in PB from the recipients at indicated time points after transplantation. The initial transduction efficiency was 71% for shLuci-LSK and 54% for shRad21-LSK (n = 5 recipient mice per group, per experiment in the primary transplantation, n = 10 recipient mice per group, per experiment in the secondary transplantation, n = 2 independent experiments, one of the two experiments is shown; the other experiment showed a similar result). (F) The histograms depict the chimerism of GFP+ cells in the indicated populations of the secondary recipients at 4 wk after transplantation. The dots represent individual mice (n = 4 mice per group for each experiment, n = 2 independent experiments, one of the two experiments is shown, the other experiment showed a similar result). Data in all panels of this figure depict mean ± SD; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

Fig 3: Immuno-phenotypes of iHPCs after 10-day maturation (at day 21). Lin was defined as CD2-CD3-CD4-CD8-CD11b-Gr1-Ter119-CD19-NK1.1-TCR?d-. iHPCs were defined as Lin-c-kit+CD127+/CD135+/Sca1+.

Fig 4: M-ATOs Recapitulate T Cell Differentiation from Different Hematopoietic Subsets(A–C) Different subsets in the hematopoietic hierarchy from HSCs to T cell progenitors were isolated from the bone marrow and the thymus of C57BL/6 WT mice and seeded into M-ATOs. From the bone marrow: HSC (hematopoietic stem cell) (Lin-Sca1+cKit+CD48 CD150+IL7R-); MPP (multi-potent progenitor) (Lin-Sca1+c-Kit+CD48-CD150-IL7R-); LMPP (lymphoid-primed multi-potent progenitor) (Lin-Sca1+c-Kit+IL7R+Flk2+); and CLP (common lymphoid progenitor) (Lin-Sca1Loc-KitLo IL7R+Flk2+). From the thymus: ETP (Lin-CD4-CD8-c-Kithi CD44hiCD25-); DN2 (Lin-CD4-CD8-c-Kithi CD44hi CD25+); and DN3 (Lin-CD4-CD8-CD44-CD25+). Representative phenotypes of M-ATO-derived cells are shown at weeks 1 (A), 2 (B), and 6 (C). Data are representative of three biological replicates.(D) Frequencies of T cell populations shown as percentage of total CD45+ TCR?d- Lin- cells initiated from the different hematopoietic subsets in week 1, week 2, and week 6 M-ATOs. Error bars denote ± SD (n = 3 independent experiments).

Fig 5: Hematopoietic stem cells (HSCs) and other hematopoietic progenitors have altered cycling, global translation levels, and apoptosis in Rps12KO/+ bone marrow.(A) Representative flow cytometry gating of HSCs (Flk2-CD48-LSK) cell cycle stages (G0, G1, S/G2/M) distribution determined by DNA (Hoechst) and Ki67 levels. (B) Cell cycle stages distribution in HSCs and in indicated progenitor populations. Asterisks correspond to p values assessing significant differences in each cell cycle stage between Rps12KO/+ and Rps12+/+ mice (6- to 8-week-old littermates, +/+ n=4, and KO/+ n=3). (C) Representative flow cytometry histogram showing OPP intensity in RpS12KO/+ (green) and Rps12+/+ (gray) HSCs. (D, E) Median o-propargyl puromycin (OPP) intensity of the indicated bone marrow populations (6- to 8-week-old littermates, +/+ n=4, and KO/+ n=3). This analysis was repeated in 6- to 7-month-old mice with similar results. (F) Representative images of bone marrow cytospins showing the high number of apoptotic cells (arrows) in Rps12KO/+ samples. (G) Representative flow cytometry gating of LIN- population showing apoptotic populations as determined by AnexinV and PI staining. (H, I) Percentage of apoptotic (AnnexinV+) cells in LSK (Lin-cKit+Sca1+) and Myeloid progenitor (MPROG; Lin-cKit+Sca1-) populations (6- to 8-weeks-old littermates, +/+ n=4, and KO/+ n=3). Statistical analysis: quantifications represent mean +/- SEM, shown as the error bars, two-way ANOVA (B–F), and unpaired t-tests were performed to establish significance among populations between genotypes *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 6—source data 1.Hematopoietic population cycling, apoptosis, and translation.