An NAD+-dependent metabolic checkpoint regulates hematopoietic stem cell activation and aging

Abstract

How hematopoietic stem cells (HSCs) maintain metabolic homeostasis to support tissue repair and regeneration throughout the lifespan is elusive. Here, we show that CD38, an NAD⁺-dependent metabolic enzyme, promotes HSC proliferation by inducing mitochondrial Ca²⁺ influx and mitochondrial metabolism in young mice. Conversely, aberrant CD38 upregulation during aging is a driver of HSC deterioration in aged mice due to dysregulated NAD⁺ metabolism and compromised mitochondrial stress management. The mitochondrial calcium uniporter, a mediator of mitochondrial Ca²⁺ influx, also supports HSC proliferation in young mice yet drives HSC decline in aged mice. Pharmacological inactivation of CD38 reverses HSC aging and the pathophysiological changes of the aging hematopoietic system in aged mice. Together, our study highlights an NAD⁺ metabolic checkpoint that balances mitochondrial activation to support HSC proliferation and mitochondrial stress management to enhance HSC self-renewal throughout the lifespan, and links aberrant Ca²⁺ signaling to HSC aging.

Extended Data Fig. 1 |. CD38 is not required for HSC maintenance under homeostatic condition at young age.

a-i. Comparison of 3-month-old WT and CD38 KO mice. A. Gating strategy for HSCs. Enriched HSCs: Lin⁻c-Kit⁺Sca1⁺. Highly enriched HSCs: Lin⁻c-Kit⁺Sca1⁺CD150⁺CD48⁻. B, C. The number of enriched (p = 0.4210) (B) and highly enriched (p = 0.4955) (C) HSCs in the bone marrow were analyzed via flow cytometry. n = 6. D, E. Ki-67 (p = 0.2928) (D) and 7-AAD (p = 0.2349) (E) staining of HSCs were analyzed via flow cytometry. n = 6. F. Lineage differentiation in the peripheral blood was analyzed via flow cytometry. MNCs, mononuclear cells (p = 0.4013). n = 5 and 6. G, H. Complete blood count analyses (G, p = 0.4174; H, p = 0.1768). n = 6. I. Bone marrow cellularity normalized by body weight (p = 0.0718). n = 6 and 5. Data are presented as mean values +/− SE. ns: p > 0.05. One-sided student’s t test.

Extended Data Fig. 2 |. CD38 regulates Ca²⁺ signaling and mitochondrial activity upon HSC proliferation.

a. HSCs isolated from 3-month-old WT and CD38 KO mice were cultured in the presence of PVA for 7 days. Total cell number was counted by hemocytometer (p = 0.0026). n = 9. b. Flow cytometry analyses of Ki-67 staining of HSCs isolated from 3-month-old WT and CD38 KO mice 16 hours post pIpC treatment (p = 0.0229). n = 6. c. HSCs isolated from 3-month-old CD38 KO mice were transduced with lentivirus overexpressing CD38 or control lentivirus and were used as donors in competitive transplantation. The percentage of total donor-derived cells in the peripheral blood of the recipients was analyzed by flow cytometry (4 weeks p = 0.0652; 8 weeks p = 0.0059; 12 weeks p = 0.0085; 16 weeks p = 0.0260). n = 12. d-n. Noncompetitive transplantation was performed using bone marrow cells from 3-month-old WT and CD38 KO mice as donors. Donor-derived enriched HSCs from the recipient mice were compared. d, e. Relative abundances of metabolites were measured by LC-MS (D, p = 0.0273; E, p = 0.0105). n = 6 and 7(D). n = 10 (E). f. ATP levels (p = 0.0007). n = 4. g-n. Representative histograms and quantification for Fluo-4 (p = 0.0357) (G, H), Rhod-2 (p = 0.0291) (I, J), TMRM (p = 0.0389) (K, L), and ROS (p = 0.0367) (M, N) staining analyzed via flow cytometry. n = 6 and 5 (G, H). n = 5 and 6 (I, J), n = 6 (K, L), n = 5 (M, N). Data are presented as mean values +/− SE. *: p < 0.05. **: p < 0.01. ***: p < 0.001. One-sided student’s t test.

Extended Data Fig. 3 |. CD38 regulates Ca²⁺ signaling and mitochondrial activity upon HSC proliferation.

a–c. Flow cytometry analyses of Rhod-2 (p = 0.0049) (A), TMRM (p = 0.0400) (B), ROS (p = 0.0497) (C) staining of HSCs isolated from 3-month-old WT and CD38 KO mice 16 hours post pIpC treatment. n = 5 and 6 (A, C). n = 5 (B). d. Gating strategy. e, f. Flow cytometry analyses of TMRM (p = 0.0265) (E) and ROS (p = 0.0137) (F) staining of CD38^high and CD38^low HSCs isolated from 3-month-old WT mice cultured for 18 hours in the presence of cytokines. n = 6 and 5 (E). n = 6 (F). Data are presented as mean values +/− SE. *: p < 0.05. **: p < 0.01. One-sided student’s t test.

Extended Data Fig. 4 |. Effect of CD38 on Ca²⁺ signaling and mitochondrial activity under homeostatic condition.

a-h. Comparison of enriched HSCs derived from 3-month-old WT and CD38 KO mice. A, B. Representative histograms (A) and quantification (p = 0.3041) (B) for Fluo-4 staining analyzed via flow cytometry. MFI, mean fluorescence intensity. n = 6. c, d. Representative histograms (C) and quantification (D) for Rhod-2 staining analyzed via flow cytometry (p = 0.2955). n = 6. e, f. Representative histograms (E) and quantification (F) for ROS staining analyzed via flow cytometry (p = 0.3350). n = 6. g, h. Representative histograms (G) and quantification (H) for TMRM staining analyzed via flow cytometry (p = 0.1819). n = 6. Data are presented as mean values +/− SE. ns: p > 0.05. One-sided student’s t test.

Extended Data Fig. 5 |. MCU is not required for HSC maintenance under homeostatic condition at young age.

a-d. Comparison of 7-month-old WT and MCU KO mice. A. HSC number in the bone marrow analyzed via flow cytometry (p = 0.2488). n = 6. B. 7-AAD staining of HSCs analyzed via flow cytometry (p = 0.2157). n = 6. C. Lineage differentiation in the peripheral blood analyzed via flow cytometry. MNCs, mononuclear cells (p = 0.2453). n = 10 and 11. D. Bone marrow cellularity normalized by body weight (p = 0.1160). n = 6. Data are presented as mean values +/− SE. ns: p > 0.05. One-sided student’s t test.

Extended Data Fig. 6 |. MCU is required to support HSC proliferation.

a, b. Competitive transplantation using HSCs isolated from 7-month-old WT and MCU KO mice as donors. The percentage of donor-derived HSCs in the bone marrow of the recipients (p = 0.0267) (A) and the percentage of donor-derived cells in the peripheral blood of the recipients (4 weeks p = 0.0425; 8 weeks p = 0.0101; 12 weeks p = 0.0142; 16 weeks p = 0.0090) (B) were quantified by flow cytometry. n = 6 (A). n = 14 (B). c-g. Enriched HSCs isolated from 3-month-old WT and MCU KO mice were cultured in the presence of cytokines. BrdU incorporation (p = 0.0477) (C), 7-AAD (p = 0.0984) (D), Rhod-2 (p = 0.0031) (E), TMRM (p = 0.0445) (F), and ROS (p = 0.0278) (G) staining of HSCs were analyzed by flow cytometry. n = 6 (C, D). n = 5 (E-G). Data are presented as mean values +/− SE. *: p < 0.05. **: p < 0.01. ns: p > 0.05. One-sided student’s t test.

Extended Data Fig. 7 |. CD38 regulates HSC function via Ca²⁺ signaling and mitochondrial activity.

a-d. HSCs from 3-month-old WT and CD38 KO mice were treated with or without MCU shRNA for 48 hours in culture in the presence of cytokines before flow cytometry analyses of Rhod2 staining (Ctrl, p = 0.0239; MCU shRNA, p = 0.4907) (A), TMRM staining (Ctrl, p = 0.0334; MCU shRNA, p = 0.0720) (B), HSC number (Ctrl, p = 0.0002; MCU shRNA, p = 0.2252) (C), and colony forming assay (Ctrl, p = 0.0250; MCU shRNA, p = 0.2180) (D). n = 3. Data are presented as mean values +/− SE. *: p < 0.05. ***: p < 0.001. ns: p > 0.05. One-sided student’s t test.

Extended Data Fig. 8 |. CD38 is upregulated in HSCs during aging.

a. Schematic illustration of the NAD⁺ metabolic pathways. b-g. Comparison of HSCs isolated from young (3 months old) and old (24 months old) WT mice. B. Quantitative real-time PCR analyses of NAD⁺ metabolic enzymes (PARP1, p = 0.4431; NAMPT, p = 0.1517; NAPRT, p = 0.1546; NMNAT3, p = 0.3542; QPRT, p = 0.0419; CD38, p = 0.0010). n = 6 and 7 (CD38). n = 3 and 4 (other enzymes). C, D. Representative histograms (C) and quantification (D) for CD38 staining analyzed via flow cytometry (p = 0.0472). n = 4. E-G. Flow cytometry analyses of Fluo-4 (p = 0.0269) (E), TMRM (p = 0.0005) (F), and ROS (p = 0.0001) (G) staining. n = 5. Data are presented as mean values +/− SE. *: p < 0.05. **: p < 0.01. ***: p < 0.001. ns: p > 0.05. One-sided student’s t test.

Extended Data Fig. 9 |. CD38 inactivation prevents HSC aging.

a-h. Comparison of HSCs isolated from 24-month-old WT and CD38 KO mice. A, B. RNA-sequencing analyses of enriched HSCs. Volcano plot summarizing the differentially expressed genes (Red: upregulated in CD38 KO. Blue: downregulated in CD38 KO. P adj<0.1. Benjamini-Hochberg procedure). Genes related to ribosomal proteins are labeled (A). Gene ontology analysis for the biological functions of differentially expressed genes (B). n = 2. C, D. Flow cytometry analyses of TMRM (p = 0.0076) (C) and ROS (p = 0.0058) (D) staining. n = 10 (C). n = 10 and 12 (D). E. Quantitative real-time PCR analysis of HSP60 (p = 0.0022). n = 3. F-H. Staining for 7-AAD (p = 0.0029) (F), activated caspase 1 (p = 0.0254) (G), and Ki-67 (p = 0.1974) (H) were analyzed by flow cytometry. n = 6 (F). n = 6 and 5 (G). n = 5 and 6 (H). Data are presented as mean values +/− SE. *: p < 0.05. **: p < 0.01. ns: p > 0.05. One-sided student’s t test.

Extended Data Fig. 10 |. MCU inactivation prevents HSC aging.

a-e. Comparison of 24-month-old WT and MCU KO mice. A, B. Complete blood count analyses (A, p = 0.0190; B, p = 0.0122). n = 8. C. Bone marrow cellularity normalized by body weight (p = 0.0181). n = 7 and 8. D, E. Competitive transplantation using HSCs isolated from 24-month-old WT and MCU KO mice as donors. The percentage of donor-derived cells in the peripheral blood of the recipients (4 weeks p = 0.0419; 8 weeks p = 0.0001; 12 weeks p = 0.0023; 16 weeks p = 0.0010) (D) and donor-derived lineage differentiation in the peripheral blood of the recipients (p = 0.0002) (E) were determined by flow cytometry. n = 12 and 9. f. A working model. CD38 catalyzes synthesis of Ca²⁺ second messengers from NAD⁺. At young age, CD38 is required for HSC proliferation through activating Ca²⁺ signaling and mitochondrial metabolism. NAD⁺ metabolism is required for activating sirtuins and suppressing mitochondrial stress. The balanced NAD⁺ metabolism and Ca²⁺ signaling ensure sufficient mitochondrial activation and adequate mitochondrial stress resistance. At old age, CD38 is upregulated, tipping the balance toward increased Ca²⁺ signaling and decreased NAD⁺ metabolism, and leading to uncontrolled mitochondrial activation and stress. Data are presented as mean values +/− SE. *: p < 0.05. **: p < 0.01. ***: p < 0.001. One-sided student’s t test.

Fig. 1 |. CD38 is required to support HSC proliferation at young age.

a–c, Enriched HSCs isolated from 3-month-old WT and CD38 KO mice were cultured in the presence of cytokines. The number of HSCs (P = 0.0113) (a), BrdU incorporation (P = 0.0215) (b) and 7-AAD staining (P = 0.3356) (c) of HSCs were analyzed using flow cytometry. a,b, n = 6. c, n = 6 and 5. d–f, Competitive transplantation using HSCs isolated from 3-month-old WT and CD38 KO mice as donors. The percentage of donor-derived HSCs in the BM of the recipients (P = 0.0144) (d), donor-derived lineage differentiation in the peripheral blood (P = 0.2442) (e) and the percentage of donor-derived cells in the peripheral blood of the recipients (4 weeks, P < 0.0001; 8 weeks, P = 0.0017; 12 weeks, P = 0.0018; 16 weeks, P = 0.0039) (f) were analyzed using flow cytometry. d, n = 5 and 6. e,f, n = 11 and 12. g,h, Noncompetitive transplantation using whole-BM cells isolated from 3-month-old WT and CD38 KO mice as donors. Ki-67 staining (P = 0.0442) (g) and 7-AAD staining (P = 0.1802) (h) of donor-derived HSCs were analyzed using flow cytometry 1 month after transplantation. g, n = 5. h, n = 6. i, The homing ability of BM cells from 3-month-old WT and CD38 KO mice 16 h after transplantation was analyzed using flow cytometry (P = 0.2659). n = 6. Data are presented as the mean ± s.e. *P < 0.05, **P < 0.01, ***P < 0.001, NS P > 0.05. A one-sided Student’s t-test was used. MNC, mononuclear cell.

Fig. 2 |. CD38 regulates Ca²⁺ signaling and mitochondrial activity on HSC proliferation.

a–l, Enriched HSCs isolated from 3-month-old WT and CD38 KO mice were cultured in the presence of cytokines. a–c, RNA-seq analyses of enriched HSCs. a, Volcano plot summarizing differentially expressed genes (red, upregulated in CD38 KO; blue, downregulated in CD38 KO. P_adj < 0.1. Benjamini–Hochberg-corrected). Genes related to HSC proliferation are labeled. b,c, Gene set enrichment analysis (GSEA) for mitochondria (b) and cell cycle (c). n = 2. d–g, [U-¹³C]glucose was administered to the HSC culture. Relative abundances of total metabolites (succinate, P = 0.0399 (d); fumarate, P = 0.0273 (e)) and ¹³C-labeled metabolites (malate, P = 0.0362 (f); fumarate, P = 0.0076 (g)) in enriched HSCs were measured using liquid chromatography–mass spectrometry (LC–MS). n = 4. h, ATP levels in enriched HSCs (P = 0.0193). n = 3. i–l, The staining for Fluo-4 (P = 0.0071) (i), Rhod-2 (P = 0.0245) (j), TMRM (P = 0.0027) (k) and ROS (P = 0.0272) (l) of HSCs was analyzed using flow cytometry. i,k,l, n = 5 and 6. j, n = 6. Data are presented as the mean ± s.e. *P < 0.05. **P < 0.01. A one-sided Student’s t-test was used. MFI, mean fluorescence intensity; NES, normalized enrichment score.

Fig. 3 |. CD38 inactivation prevents HSC aging.

a–h, Comparison of 24-month-old WT and CD38 KO mice. a, NAD⁺ levels in enriched HSCs (P = 0.0454). n = 5 and 6. b,c, White blood cell (WBC) (b) and lymphocyte (c) analyses (P = 0.0182 (b); P = 0.0193 (c)). n = 8 and 7. d, Lineage differentiation in peripheral blood was analyzed using flow cytometry (P = 0.0392). n = 6 and 7. e, BM cellularity normalized according to body weight (P = 0.0472). n = 6 and 8. f–h, Competitive transplantation using HSCs isolated from 24-month-old WT and CD38 KO mice as donors. Donor-derived HSC engraftment in the BM (P = 0.0311) (f), the percentage of total donor-derived cells in peripheral blood (4 weeks, P = 0.0647; 8 weeks, P = 0.0313; 12 weeks, P = 0.0090; 16 weeks, P = 0.0104) (g) and donor-derived lineage differentiation in the peripheral blood (P = 0.0107) (h) of the recipients were analyzed using flow cytometry. f, n = 6. g,h, n = 12 and 14. Data are presented as the mean ± s.e. *P < 0.05. **P < 0.01. A one-sided Student’s t-test was used.

Fig. 4 |. 78c treatment reverses HSC aging.

a–o, Twenty-four-month-old WT mice were treated with or without 78c for 8 weeks. a, Schematic illustration of the experimental setup and timeline. b, NAD⁺ levels in enriched HSCs (P = 0.0002). n = 6. c–h, Lymphocyte and WBC analyses of young and aged mice before treatment (c,f) (P = 0.0009 (c); P = 0.0561 (f)) and aged mice after treatment for 5 weeks (d,g) (P = 0.0387 (d); P = 0.0888 (g)) or 8 weeks (e,h) (P = 0.0142 (e); P = 0.0459 (h)). c,f, n = 10 and 25. d,g, n = 9. e,h, n = 10 and 9. i, BM cellularity normalized according to body weight (P = 0.0485). n = 6. j, Lineage differentiation in peripheral blood was analyzed using flow cytometry (P = 0.0474). n = 9. k, Lineage-biased HSCs in the BM were analyzed using flow cytometry. Myeloid-biased HSCs: Lin⁻c-Kit⁺Sca1⁺CD135⁻CD34⁻CD150^hi. Lineage-balanced HSCs: Lin⁻c-Kit⁺Sca1⁺CD135⁻CD34⁻CD150^lo (P = 0.0029). n = 6. l,m, Colonies formed in a colony-forming assay using BM cells (l) and quantification (P = 0.0038) (m). n = 6 and 5. n,o, Staining for TMRM (P = 0.0490) (n) and ROS (P = 0.0362) (o) of HSCs was analyzed using flow cytometry. n, n = 5 and 6. o, n = 5. Data are presented as the mean ± s.e. *P < 0.05, **P < 0.01, ***P < 0.001. A one-sided Student’s t-test was used. Ctrl, control.