Fig 2: ER stress induction and cytokine signals affect CD244 expression(A) Expression patterns of CD244 and CD48 on KSL cells after in vitro culture in Stemspan SFEM medium supplemented with 100 ng/mL mSCF and 100 ng/mL hTPO for 7 days, and with or without 0.5 μg/mL Tunicamycin or 100 μM TUDCA. Representative FACS plots are shown.(B) Summary of the flow cytometry analyses. Frequencies of KSL cells, CD48- fraction in KSL population, and CD244−CD48- fraction in KSL population are shown. Mean ± SD from two independent experiments (n = 5–11) are displayed. Significance was calculated using one-way ANOVA within each population. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.(C) A heatmap showing frequencies of CD244−CD48−KSL cells after 7 days’ culture with various SCF/TPO concentrations. Mean values of Z score (n = 3) in each fraction are displayed.

Fig 3: CD244 expression divides CD48−KSL cells into functionally distinct subpopulations after in vitro culture(A) Experimental design of the in vitro culture experiment. One hundred CD48−KSL cells were sorted from BM of young mice and cultured in Stemspan SFEM medium supplemented with 100 ng/mL mSCF and 100 ng/mL hTPO for 7 days.(B) Expression patterns of CD244 and CD48 on the cell surface of fresh and 14 days cultured KSL cells. Representative FACS plots on KSL population are shown.(C and D) qRT-PCR analysis for HSC-related genes and mast cell-related genes in CD244+CD48−KSL cells compared with the CD244+CD48−KSL counterpart. Representative FACS plot and gating of CD244+CD48−KSL cells (green) and CD244−CD48−KSL cells (red) on 7 days cultured KSL cells are shown in (C). Relative expression levels of genes in CD244+CD48−KSL cells to CD244−CD48−KSL are shown in (D).(E) Competitive reconstitution assay. After 7 days’ culture, two subpopulations were sorted and 1,000 of CD244- or 1,500 of CD244+CD48−KSL cells were separately transplanted into lethally irradiated recipient mice (seven mice) with 2 × 105 total BM cells (competitor). Chimerism was monitored by analyzing PB every month. Significance was calculated using Student's t test at each time point. Mean ± SD from two independent experiments (n = 7) are displayed. ∗∗∗∗p < 0.0001.(F) Lineage balance of donor-derived cells in the PB of recipient mice. Mean ± SD from two independent experiments (n = 6) are displayed. ∗∗p < 0.01, ∗∗∗∗p < 0.0001. Each color represents different lineages.(G) Analysis of BM from engrafted mice after 16 weeks. Chimerism in each cell fraction is shown. Significance was calculated using Student's t test. Mean ± SD from two independent experiments (n = 6) are displayed. ∗∗p < 0.01.(H and I) Limiting dilution assay for CD244−CD48−KSL cells. CD244−CD48−KSL cells were re-sorted after 7 days’ in vitro culture and 200, 40, or 10 cells were transplanted to recipient mice in a competitive manner. Chimerism above 1% was judged as successful engraftment. The frequency of functional HSC was calculated using ELDA (http://bioinf.wehi.edu.au/software/elda/). Chimerism of individual mice is shown in (I).

Fig 4: Gene expression changes in CD48−KSL cells upon in vitro culture and aging(A) Experimental design of the gene expression analysis. Five hundred CD48−KSL cells were sorted from BM of young (10 weeks old, YF) mice. Cells were also cultured in Stemspan SFEM medium supplemented with 100 ng/mL mSCF and 100 ng/mL hTPO for 14 days, and CD48−KSL cells were re-sorted.(B) Volcano plot showing differentially expressed genes between YF and YC. Significant difference was defined as p < 0.001 and log2 fold change < −2 (blue) or >2 (red).(C) Heatmaps showing a list of differentially expressed genes. Significant difference was defined as p < 0.0001 and log2 fold change < −3 or >3. Blue arrowheads indicate CD markers.(D) Gene set enrichment analysis (GSEA) of the microarray data comparing YF and YC. NES, normalized enrichment score; NOM p-val, nominal p value; FDR q-val, false discovery rate q-value. See also Table S1.

Fig 5: Phc2 deficiency leads to immune cell deficiency without intrinsic defects in HSPCs. a Representative images of spleens and thymi from WT and KO mice. n = 6. b Normal architecture of the thymus and spleen of the KO mouse. Representative hematoxylin and eosin staining images of spleens and thymi from WT and KO mice. n = 6. Scale bars: 200 µm. c Absolute number of immune cells in the BM, thymus (Thy), spleen (SP), and liver of WT or KO mice. n = 6. d Relative ratio of BM-resident hematopoietic lineage cells between WT and KO mice. n = 5. LSK, Lin-Sca-1+c-kit+ cells; LT-HSC, long-term HSC; SLAM-HSC, Lin-CD41-CD48-CD150+ cells; ST-HSC, short-term HSC; MPP, multipotent progenitors; CMP, common myeloid progenitors; GMP granulocyte/monocyte progenitors; MEP, megakaryocyte/erythrocyte progenitors; CLP, common lymphoid progenitors; Myeloid, myeloid cells. e Cell cycle status of BM-resident LSK cells from WT and KO BM. n = 4. f Percentage of apoptosis for BM-resident LSK cells from WT and KO mice. n = 3. g BM-resident clonogenic progenitors from WT and KO mice were assessed by CFU assays. n = 5. CFU-GM, CFU-granulocyte and/or macrophage; BFU-E, erythroid burst-forming units, CFU-GEMM, CFU-granulocyte/erythrocyte/monocyte/megakaryocyte. h Absolute number of BM-resident B cell subpopulations from WT and KO mice (n = 6). Pro/pre, pro-B cells/pre-B cells; Immature, immature B cells. Statistical significance was assessed by two-tailed Student’s t-test. **P < 0.01. All data are presented as the mean ± SEM. Source data are provided as a Source Data File