Figure 1.
IL27Ra serves as a marker for function-compromised HSCs with myeloid bias. (A) The expression of indicated cytokine receptor between young (3 months) and aged (24-25 months) hematopoietic stem cells. (B) Representative plots from flow cytometry show the changes of indicated cytokine receptor on HSCs (Lineage− Sca1+ c-Kit+ CD150+ CD34−) from young (2 months) and aged (24-28 months) mice. (C) The left histogram depicts the number of HSCs and the right histogram depicts the number of IL27Ra+ HSCs and IL27Ra− HSCs between young (2 months) and aged mice (24-28 months). N = 5 mice per group, data are shown as mean ± SEM. (D) This scatterplot displays the percentage of IL27Ra+ cells in HSCs, MPPs, CMPs, common lymphoid progenitors (CLPs), megakaryocytic/erythroid progenitors (MEPs), and GMPs between young (2 months) and aged mice (24-28 months). N = 3-5 mice per group; data are shown as mean ± SEM. (E) The line plots depict the changes in peripheral blood chimerism in primary recipients transplanted with 10 IL27Ra− HSCs (defined as Y−, Young IL27Ra− HSC) or 10 IL27Ra+ HSCs (defined as Y+, Young IL27Ra+ HSC) freshly isolated from young (2 months) mice together with 2 × 105 competitor cells. Engraftment was examined at the indicated time points after transplantation. N = 7-8 mice per group; data are shown as mean ± SEM. (F) This histogram depicts the lineage distribution of myeloid cell, B cell, and T cell among donor-derived cells in peripheral blood 4 months after 10 young IL27Ra− HSCs (Y−) or 10 young IL27Ra+ HSCs (Y+) transplantation. N = 7-8 mice per group; data are shown as mean ± SEM. (G) The line plots depict the changes in peripheral blood chimera in primary recipients transplanted with 100 IL27Ra− HSCs (defined as O−, Old IL27Ra− HSC) or 100 IL27Ra+ HSCs (defined as O+, Old IL27Ra+ HSC) freshly isolated from 28-month-old mice together with 2 × 105 competitor cells. Engraftment was examined at the indicated time points after transplantation. N = 4-5 mice per group; data are shown as mean ± SEM. (H) This histogram depicts the lineage distribution of myeloid cell, B cell, and T cell among donor-derived cells in peripheral blood 4 months after 100 old IL27Ra− HSCs (O−) or 100 old IL27Ra+ HSCs (O+) transplantation. N = 4-5 mice per group; data are shown as mean ± SEM. (I) Experimental design for RNA-sequencing assay (see Methods). (J-L) These figures show the GSEA of cell aging-related genes, HSC fingerprints or neutrophil activation-related genes in Y− (Young IL27Ra− HSC) vs Y+ (Young IL27Ra+ HSC), or O− (Old IL27Ra− HSC) vs O+ (Old IL27Ra+ HSC). NES, normalized enrichment score; FDR-adjusted q values are provided. |NES| > 0.3 and q < 0.05 represent significant difference. (M) Experimental design of colony-forming assay for human HSCs (Lin− CD34+ CD38−). Two hundred freshly isolated IL27Ra− HSCs or IL27Ra+ HSCs from either young healthy people (ages 20, 22, and 29) or aged healthy people (ages 59, 61, and 63) were seeded in methylcellulose medium, and the colonies were counted 14 days later (see details in Methods). (N) This histogram shows the relative colony-forming capacity of human IL27Ra− and IL27Ra+ HSCs. GEMM, granulocyte, erythroid, macrophage, and megakaryocyte; GM, Colony-forming unit-granulocyte and macrophage; E, Burst-forming unit-erythroid and colony-forming unit erythroid. N = 3 individuals per group, clone numbers are normalized to IL27Ra− total numbers in each experiment, data are pooled from 3 independent experiments and shown as mean ± SEM; original data and calculation process are shown in supplemental Table 4. (O) This table depicts the percentage of successful long-term reconstitution in single HSC transplantation assay. Single IL27Ra− or IL27Ra+ HSC (KSL CD150+CD34−) from either young (2 months) or aged (24-28 months) mice was transplanted into lethally irradiated recipients (the number is indicated in the table) together with 2 × 105 competitor cells. Peripheral blood of recipient mice was analyzed 4 to 5 months after transplantation. If the chimera was 0.05% or more after transplantation, regardless of which lineage was reconstituted, mice were considered to be reconstituted with test donor cells.

IL27Ra serves as a marker for function-compromised HSCs with myeloid bias. (A) The expression of indicated cytokine receptor between young (3 months) and aged (24-25 months) hematopoietic stem cells. (B) Representative plots from flow cytometry show the changes of indicated cytokine receptor on HSCs (Lineage− Sca1+ c-Kit+ CD150+ CD34−) from young (2 months) and aged (24-28 months) mice. (C) The left histogram depicts the number of HSCs and the right histogram depicts the number of IL27Ra+ HSCs and IL27Ra− HSCs between young (2 months) and aged mice (24-28 months). N = 5 mice per group, data are shown as mean ± SEM. (D) This scatterplot displays the percentage of IL27Ra+ cells in HSCs, MPPs, CMPs, common lymphoid progenitors (CLPs), megakaryocytic/erythroid progenitors (MEPs), and GMPs between young (2 months) and aged mice (24-28 months). N = 3-5 mice per group; data are shown as mean ± SEM. (E) The line plots depict the changes in peripheral blood chimerism in primary recipients transplanted with 10 IL27Ra− HSCs (defined as Y−, Young IL27Ra− HSC) or 10 IL27Ra+ HSCs (defined as Y+, Young IL27Ra+ HSC) freshly isolated from young (2 months) mice together with 2 × 105 competitor cells. Engraftment was examined at the indicated time points after transplantation. N = 7-8 mice per group; data are shown as mean ± SEM. (F) This histogram depicts the lineage distribution of myeloid cell, B cell, and T cell among donor-derived cells in peripheral blood 4 months after 10 young IL27Ra− HSCs (Y−) or 10 young IL27Ra+ HSCs (Y+) transplantation. N = 7-8 mice per group; data are shown as mean ± SEM. (G) The line plots depict the changes in peripheral blood chimera in primary recipients transplanted with 100 IL27Ra− HSCs (defined as O−, Old IL27Ra− HSC) or 100 IL27Ra+ HSCs (defined as O+, Old IL27Ra+ HSC) freshly isolated from 28-month-old mice together with 2 × 105 competitor cells. Engraftment was examined at the indicated time points after transplantation. N = 4-5 mice per group; data are shown as mean ± SEM. (H) This histogram depicts the lineage distribution of myeloid cell, B cell, and T cell among donor-derived cells in peripheral blood 4 months after 100 old IL27Ra− HSCs (O−) or 100 old IL27Ra+ HSCs (O+) transplantation. N = 4-5 mice per group; data are shown as mean ± SEM. (I) Experimental design for RNA-sequencing assay (see Methods). (J-L) These figures show the GSEA of cell aging-related genes, HSC fingerprints or neutrophil activation-related genes in Y− (Young IL27Ra− HSC) vs Y+ (Young IL27Ra+ HSC), or O− (Old IL27Ra− HSC) vs O+ (Old IL27Ra+ HSC). NES, normalized enrichment score; FDR-adjusted q values are provided. |NES| > 0.3 and q < 0.05 represent significant difference. (M) Experimental design of colony-forming assay for human HSCs (Lin− CD34+ CD38−). Two hundred freshly isolated IL27Ra− HSCs or IL27Ra+ HSCs from either young healthy people (ages 20, 22, and 29) or aged healthy people (ages 59, 61, and 63) were seeded in methylcellulose medium, and the colonies were counted 14 days later (see details in Methods). (N) This histogram shows the relative colony-forming capacity of human IL27Ra− and IL27Ra+ HSCs. GEMM, granulocyte, erythroid, macrophage, and megakaryocyte; GM, Colony-forming unit-granulocyte and macrophage; E, Burst-forming unit-erythroid and colony-forming unit erythroid. N = 3 individuals per group, clone numbers are normalized to IL27Ra− total numbers in each experiment, data are pooled from 3 independent experiments and shown as mean ± SEM; original data and calculation process are shown in supplemental Table 4. (O) This table depicts the percentage of successful long-term reconstitution in single HSC transplantation assay. Single IL27Ra− or IL27Ra+ HSC (KSL CD150+CD34−) from either young (2 months) or aged (24-28 months) mice was transplanted into lethally irradiated recipients (the number is indicated in the table) together with 2 × 105 competitor cells. Peripheral blood of recipient mice was analyzed 4 to 5 months after transplantation. If the chimera was 0.05% or more after transplantation, regardless of which lineage was reconstituted, mice were considered to be reconstituted with test donor cells.

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