Abstract Hematopoietic stem cells (HSC) with multilineage potential are critical for T cell reconstitution after allogeneic hematopoietic cell transplantation (allo-HCT). The Kit^lo HSC subset is enriched for multipotential precursors, but their T cell potential remains poorly characterized. Using a preclinical allo-HCT mouse model, we demonstrate that Kit^lo HSCs provide superior thymic recovery and T cell reconstitution, resulting in improved immune responses to post-transplant infection. Kit^lo HSCs with augmented bone marrow (BM) lymphopoiesis mitigate age-associated thymic alterations and enhance T cell recovery in middle-aged mice. Mechanistically, chromatin profiling reveals Kit^lo HSCs exhibiting higher activity of lymphoid-specifying transcription factors, such as, ZBTB1. Zbtb1 deletion diminishes HSC engraftment and T cell potential; by contrast, reinstating Zbtb1 in megakaryocytic-biased Kit^hi HSCs rescues hematopoietic engraftment and T cell potential in vitro and in vivo. Furthermore, age-associated decline in Kit^lo HSCs is associated with diminished T lymphopoietic potential in aged BM precursors; meanwhile, Kit^lo HSCs in aged mice maintain enhanced lymphoid potential, but their per-cell capacity is diminished. Lastly, we observe an analogous human BM KIT^lo HSC subset with enhanced lymphoid potential. Our results thus uncover an age-related epigenetic regulation of lymphoid-competent Kit^lo HSCs for T cell reconstitution. Subject terms: Lymphopoiesis, Gene regulation in immune cells, Autoimmunity, Ageing __________________________________________________________________ Hematopoietic stem cells (HSCs) differentiate into multiple lineages, with such capacity impacted by aging. Here the authors identify Kit^lo HSCs as a functionally distinct population that exhibits distinct lymphoid-primed chromatin landscapes, which drive enhanced lymphoid reconstitution capacity, and is altered in aged hosts. Introduction T lymphocytes play a crucial role in the adaptive immune response. A highly diverse T cell pool is required to recognize and eliminate foreign pathogens while also maintaining self-tolerance. The thymus continuously produces T cells, but with advancing age, its regenerative capacity declines^[70]1, leading to decreased thymic output and compromised T cell diversity^[71]2–[72]7. Furthermore, the thymus is also vulnerable to acute damage from infections, cancer therapies, and conditioning regimens for allogeneic hematopoietic cell transplantation (allo-HCT), which results in prolonged T cell lymphopenia, thereby increasing the risk of infections and cancer relapse, contributing to transplant-related complications and mortality^[73]8. For these reasons, early T cell reconstitution is a positive prognostic indicator of allo-HCT outcomes^[74]9,[75]10. Post-transplant T cell reconstitution requires a steady supply of hematopoietic stem cells (HSCs)-derived lymphoid progenitors^[76]11 as well as thymic recovery. Prior research has demonstrated that thymic integrity is dependent on thymocyte-stromal crosstalk, particularly in the thymic epithelial cell (TEC) compartment^[77]12–[78]14. Age-associated reduced lymphoid potential originating at the level of HSCs^[79]15, coupled with thymic decline^[80]1, contributes to delayed immune recovery. Therefore, identifying strategies to augment T lymphopoiesis and promote thymic regeneration is an unmet clinical need. Single-cell transplantation with tracking and sequencing of progeny^[81]16–[82]21 has uncovered heterogeneity in self-renewal capacity as well as lineage output among reconstituting HSCs- highlighting HSCs with multilineage versus lineage-biased potential. These studies showed that, while only a fraction of HSCs generate “balanced” multilineage output, the majority exhibit a diverse range of lineage potential following transplantation- varying in their contributions and reconstitution kinetics for each lineage, thereby highlighting their inherent biases. These lineage biases are intrinsically stable and maintained following transplantation^[83]22–[84]24. At the molecular level, these cell-autonomous features are mediated by epigenetic configuration and are divergent from their transcriptional state^[85]19,[86]25. Although this suggests the existence of a highly organized and predictable framework for lineage-restricted fates of long-term self-renewing HSCs, the molecular identities, gene regulatory networks, and functional implications of these differences governing lymphoid fate decisions remain unexplored. Kit^lo HSCs have previously been described to exhibit better self-renewal and multipotency, including improved T cell reconstitution in syngeneic transplant models^[87]26,[88]27. However, their thymic reconstituting ability, age-related changes, as well as the molecular basis for their enhanced T cell lymphoid potential are unknown. Here, we delineate the molecular mechanism underlying enhanced lymphoid potential in multilineage Kit^lo HSCs, identifying ZBTB1 as a critical transcription factor that governs their superior T cell reconstitution capacity. We demonstrate that the frequency of the Kit^lo subset declines with age and establish the pivotal role of these HSCs in orchestrating thymic recovery and immune reconstitution in both young and aged hosts. Through functional studies, we establish that ZBTB1 expression directly correlates with improved hematopoietic engraftment and thymic recovery following transplantation and is sufficient to rescue the lymphoid defect in aged HSCs. We further identify a corresponding human HSC subset with analogous enhanced lymphoid characteristics, demonstrating conservation of this marker to identify lymphoid-competent HSCs across species. This work builds on prior studies on lineage specification programs in HSCs and advances our fundamental understanding of potential molecular underpinnings governing lymphoid-primed HSCs. The identification of enhanced lymphoid-potential HSC subsets in human bone marrow, combined with emerging ex vivo expansion techniques, offers promising translational opportunities for improving immune regeneration after bone marrow transplantation while counteracting treatment-related immunosuppression and age-associated thymic decline. Results Reduced lymphoid output in aged HSCs impairs thymic recovery Previous studies have shown impaired lymphoid reconstitution with aged HSCs in a syngeneic transplant model, yet this has not been explored using an allogeneic setting. We used a preclinical allo-HCT model to analyze immune reconstitution of young and old donors. To evaluate if HSCs with differences in lymphoid progenitor production could impact thymic recovery, we transplanted LT HSCs (Long-Term Hematopoietic Stem Cells, Lineage^−CD34^−CD48^−CD150^+Sca-1^+cKit^+) cells from young adult (2-mo) or old (24-mo) C57BL/6 mice with rescue BM cells (CD45.1) into lethally irradiated young BALB/cJ recipients (Supplementary Fig. [89]1A). Because HSC-derived thymic progeny emerges within 5–6 weeks^[90]28, we established an 8-week harvest timepoint. This interval allowed sufficient time for HSC-derived progenitors to repopulate the thymus, thereby providing a better understanding of thymic regeneration dynamics and the efficacy of HSC transplantation in promoting thymic reconstitution. Consistent with prior reports, eight weeks post-HCT, we found that young HSCs supported balanced lineage reconstitution, while old HSCs exhibited significantly impaired T cell reconstitution in the peripheral blood (PB), despite comparable reconstitution patterns at four weeks (Supplementary Fig. [91]1B–E). Young HSCs demonstrated superior multilineage potential, particularly lymphoid reconstitution (LMPP/MPP4: lymphoid-primed multipotent progenitors and CLP: common lymphoid progenitors, Supplementary Fig. [92]1F, G), compared to old HSCs. Furthermore, recipients of young HSCs demonstrated significantly greater thymic cellularity and enhanced reconstitution of precursor and mature thymocytes compared to old HSC recipients (Supplementary Fig. [93]1H, I). In conclusion, old HSCs have significantly less multilineage potential, but especially T lymphoid potential, than young HSCs in young allo-HCT recipients. Age-related decline in multilineage HSCs marked by low Kit expression Recent reports have shown remarkable functional heterogeneity of HSC subsets^[94]29–[95]34, highlighting discordance between current HSC phenotypic definitions to their molecular identities^[96]17–[97]19. To identify HSCs with an enriched multilineage program and gain insights into their transcriptional and epigenetic framework, we performed multiome single-cell RNA and ATAC sequencing on hematopoietic stem cells LT HSCs from 2-mo (young) or 24-mo (old) female mice. To define HSC subsets, we performed unsupervised Leiden cluster analysis on the multiome scRNA-seq data from our young cohort (Supplementary Fig. [98]2A, B). Using nomenclature and signatures derived from previous studies^[99]16,[100]17,[101]19,[102]35–[103]38, we mapped all unsupervised clusters into five main subsets (Supplementary Fig. [104]2C): Quiescent HSC (q-HSC) exhibited a low-output signature (Cdkn1c, Mpl, and Socs2) and shared transcriptional features with megakaryocytic-biased HSCs (Mgk-HSC), marked by elevated expression of stemness-associated genes including Mllt3, Hlf, Mecom, Txnip, Ifitm1, Msi2, Procr, Hoxa9, Ogt, Pim1, and Satb1. Multilineage HSCs (MLin-HSCs) were characterized by higher expression of multipotency genes (Zeb2, Pbx1, Runx3, and Tgfb1) and shared molecular signatures with proliferative HSCs (p-HSCs), including cell cycle regulators CDK6, Pola1, Hells, and Itga2b. Notably, we identified a novel Intermediate HSC population (Int-HSC) that displayed overlapping molecular features with Mgk-HSC, p-HSCs, and MLin-HSCs, distinguished by elevated expression of Stat5b, Jak2, Cdc42, and Ywhaz (related to LNK-JAK2 interaction). (Supplementary Data [105]1 and Supplementary Fig. [106]2D). To identify HSCs with multilineage potential, we compared multilineage enriched MLin-HSC versus low-output q-HSC subsets in young HSCs and queried for previously described bona fide HSC markers (Hlf, Kit, Neo1, Procr, CD244a, Pdzk1ip1, Ly9, Hoxb5). Hlf and Kit had significantly higher expression in q-HSCs compared to MLin-HSCs (Fig. [107]1A). Hlf has been previously implicated in HSC quiescence^[108]39–[109]41, whereas Kit enriches for platelet-biased HSCs^[110]27. To further analyze the composition of HSCs with varying Kit expressions, we defined Kit high and low HSC populations (Kit^hi and Kit^lo HSCs) based on the top and bottom 20% of Kit gene expression levels. We observed lower Kit expression in MLin-HSCs (Fig. [111]1B) and increased representation of q-HSC and MLin-HSCs within the Kit^lo HSCs (Fig. [112]1C and Supplementary Fig. [113]2E), consistent with prior reports of increased self-renewal capacity and multipotency in Kit^lo HSCs^[114]26,[115]27. Fig. 1. Age-related decline in multilineage HSCs marked by low Kit expression. [116]Fig. 1 [117]Open in a new tab A–H Multiome single-cell RNA and ATAC sequencing was performed on HSC (Lineage-Sca-1+cKit+ CD34-CD48-Flt3-CD150+) cells isolated from 2-mo (young) or 24-mo (old) female C57BL/6 mice. nTOTAL = 12,350 cells. A Volcano plot of DGE analysis between MLin-HSC vs. q-HSC subsets in young HSCs, highlighting bona fide HSC markers. B Violin plot for imputed Kit gene expression by annotated HSC subsets (as described in Fig. [118]S2C). C Frequency of computationally defined HSC subtypes in Kit^hi and Kit^lo subsets in young HSCs. D, E Combined Uniform manifold approximation and projection (UMAP) of young and old HSCs after Harmony batch correction, annotated by age (D) and annotated HSC subsets (q-HSC: Quiescent HSCs; Mgk-HSCs: Platelet-biased HSCs; Mlin-HSC: Multilineage HSCs; p-HSC: Proliferative HSCs; Int-HSC: Intermediate HSCs) (E). F Frequency of HSC subtypes in young and old HSCs. G Heatmap showing MAGIC-imputed gene expression values of bona fide HSC markers ordered by increasing Kit expression. H Violin plot for imputed Kit gene expression by age. Statistical analysis was performed using the Wilcoxon test. I Scaled change in frequency for each Kit HSC subset with age. J FACS analysis showing the frequency of Kit^hi and Kit^lo HSCs by age. All data are from n = 6 mice/group (young = 6; old = 6). Error bars represent mean ± SEM. **P < 0.01, ***P < 0.001. P-values calculated by two-way ANOVA. Source data are provided as a Source Data file, Source Data Fig. 1. To validate these RNA-based findings at the protein level and confirm the relationship between Kit abundance and HSC subset distribution, we analyzed a CITE-seq dataset generated on young LSKs ([119]GSE243197)^[120]42. We defined Kit high and low HSC populations (Kit^hi and Kit^lo HSCs) based on the top and bottom 30% of CD117 antibody-derived tag (ADT) reads, mirroring our gating thresholds to fractionate Kit HSC subsets. Consistent with our transcriptional analysis, we observed an increased representation of q-HSC and MLin-HSCs within the Kit^lo HSCs, while Kit^hi HSCs were enriched for Mgk-HSC, p-HSC, and Int-HSCs (Supplementary Fig. [121]2F and [122]G). We then sought to understand how Kit corresponds to previously defined HSC-lineage-biased markers (CD229, CD61, CD150, and Neogenin), which are known indicators of lineage bias. While CD150^lo and CD229+ mark lymphoid-biased HSCs^[123]23,[124]43, Neogenin+ identifies myeloid bias^[125]44, and CD61+ expression denotes quiescent HSCs^[126]45. We observed that Kit^lo HSCs express lymphoid-biased markers CD150^lo (Supplementary Fig. [127]3A–C) and CD229 (Supplementary Fig. [128]3G, H), though these markers significantly underrepresent Kit^lo HSCs (CD229+: ~10%; CD150lo: ~40%). In contrast, Kit^lo HSCs showed low expression of the myeloid-biased marker Neo1 (Neo1+: ~25%, Supplementary Fig. [129]3D–[130]F). Additionally, CD61-expressing HSCs are unable to distinguish Kit HSC subsets. (Supplementary Fig. [131]3G, [132]H). To assess for age-associated changes, we first compared the transcriptional profile of young and old HSCs. In our differential gene analysis, we found that HSCs from young mice had higher expression of genes related to cell cycle (Ccnd3, Cdc25a, Cdk1), and cell polarization (Arhgap15); conversely, old HSCs were enriched for expression of genes related to quiescence (Egr1, Junb, Jun, Jund, Fos), platelet differentiation (vWf, Clu, Itgb3, Fhl1 and Tgm2), and myeloid differentiation (Selp, Neo1) (Supplementary Data [133]2). To investigate age-related changes in HSC populations, we used our young HSC data as a reference to classify the HSC subtypes in old HSCs (Supplementary Fig. [134]2H). Subsequently, we generated a UMAP of our young and old datasets (Supplementary Fig. [135]2I and Fig. [136]1D) to visualize the post-ingestion results (Fig. [137]1E). While all HSC subsets were present in young and old cohorts (Fig. [138]1F), we notably observed an increased representation of MLin-HSCs, p-HSCs, and Int-HSCs in young HSCs. Conversely, q-HSCs and Mgk-HSCs were predominantly in old HSCs (Supplementary Fig. [139]S2J). Consistent with previous studies^[140]19,[141]30,[142]46, these findings suggest shifts in the composition of HSC subsets with aging, indicating a reduction in multilineage potential accompanied by an increase in platelet bias. Given the increased frequency of Mgk-HSCs in old HSCs and prior work^[143]27 demonstrating that Kit^hi HSCs are platelet-biased HSCs (Supplementary Fig. [144]2E, [145]J, and L), we hypothesized this could be attributed to an expansion in Kit^hi HSCs. We observed significantly higher Kit expression (Fig. [146]1G, H and Supplementary Fig. [147]2M) in old HSCs, with an age-dependent reduction in the proportion of Kit^lo HSCs, accompanied by an increase in the frequency of Kit^hi HSCs (Fig. [148]1I). Flow cytometry analysis of young and old bone marrow confirmed a significant expansion of Kit^hi HSCs, accompanied by a concomitant decrease in Kit^lo HSCs in old HSCs (Supplementary Fig. [149]2N and Fig. [150]1J), consistent with our prior report^[151]47. Together, these findings indicate that the frequency of multilineage Kit^lo HSCs declines with age. Young Kit^lo HSCs exhibit enhanced T cell potential Next, we sought to determine the lymphoid potential of the HSC Kit subsets. To this end, we used the S17 lymphoid progenitor assay^[152]48 to assess for lymphoid progenitor output of purified Kit^hi and Kit^lo HSCs from young mice (Fig. [153]2A). Prior studies have elucidated the existence of functional heterogeneity among phenotypic Common Lymphoid Progenitors (CLPs) characterized by Ly6D expression^[154]49, resulting in the identification of two distinct subsets: All Lymphocyte Progenitors (ALPs), also referred to as functional CLPs, and B cell-biased Lymphocyte Progenitors (BLPs). Following a 12-day co-culture, we observed that young Kit^lo HSCs generated more ALPs and BLPs than Kit^hi HSCs (Fig. [155]2B). To further examine their T cell differentiation potential, we used murine artificial thymic organoids (M-ATO)^[156]50–[157]52 (Fig. [158]2A). Following an 8-week culture, we noted a substantial increase in overall ATO output from young Kit^l^o HSCs in comparison to Kit^hi HSCs (Fig. [159]2C). Fig. 2. Young Kit^lo HSCs exhibit enhanced T cell potential. [160]Fig. 2 [161]Open in a new tab A Experimental schema to evaluate in vitro lymphoid progenitor and T cell differentiation potential of Kit^hi (red) and Kit^lo HSCs (blue) from 2-mo (young) C57BL/6 mice using the S17 lymphoid assay and murine artificial thymic organoid (M-ATO), respectively. Enumeration of absolute number of lymphoid progenitor cells following 14 days in S17 lymphoid assay (B) and absolute number of T cell subsets following 8 weeks of culture in M-ATOs (C). Refer to Supplementary Fig. [162]13A, B for gating strategies to define the above populations. Aggregated data across 3 independent experiments, each performed in triplicate (Kit^lo = 3; Kit^hi = 3). D Experimental schema for competitive allogeneic HCT (allo-HCT) using Kit^hi (red) and Kit^lo HSCs (blue) from 2-mo (young) C57BL/6 mice with competitor bone marrow (BM) cells from B6.SJL-PtprcaPepcb/BoyJ mice were transplanted into lethally irradiated 7-week-old (young) BALB/cJ recipients. E–J Eight weeks after competitive HCT, (E) frequency of donor-derived chimerism (CD45.2/ H-2Kb) of mature lineages in the peripheral blood (PB, CD45+, Myeloid cell: Gr-1+CD11b+; B cell: B220+; T cell: CD3 + ), (F) Enumeration of absolute number of donor-derived cells in the BM for LSK cells (LT HSC: Lineage^−Sca-1^+cKit^+ CD34^−CD48^−Flt3^−CD150^+; ST HSC: Lineage^−Sca-1^+cKit^+ CD48^−Flt3^−CD150^−; MPP2: Lineage^−Sca-1^+cKit^+ CD48^+Flt3^−CD150^−; MPP3: Lineage^−Sca-1^+cKit^+ CD48^+Flt3^−CD150^+; MPP4/ LMPP: Lineage^−Sca-1^+cKit^+ Flt3^+CD150^−) and Common Lymphoid Progenitors cells (CLP: Lineage^−IL7Ra^+Flt3^+Sca^mid/lo Kit^lo). G–J Post-HCT thymi analysis for total thymic cellularity (G), H, I enumeration of absolute number of donor-derived cells for T cell precursors (ETP: Lineage^− CD4^− CD8^−CD44^+ CD25^−Kit^+; DN2: Lineage^− CD4^− CD8^−CD44^+ CD25^+; DN3: Lineage^− CD4^− CD8^−CD44^− CD25^+) (H), Mature T cells (DP: Lineage^− CD4^+ CD8^+; SP4: Lineage^− CD4^+ CD8^−; SP8: Lineage^− CD4^− CD8^+) (I), and analysis of thymic CD45- compartment for Thymic Epithelial Cells (TEC: CD45^− EpCAM^+), Cortical TEC (cTEC: CD45^− EpCAM^+UEA-1^lo 6C3^hi MHCII^hi/lo), Medullary TEC (mTEC: CD45^− EpCAM^+UEA-1^hi 6C3^lo MHCII^hi/lo^−), endothelial cells: CD45^− EpCAM^−Ter119 PDGFRα^− CD31^+ and fibroblasts: CD45^− EpCAM^−Ter119^−CD31^− PDGFRα^+) (J). Data for (E–I) are from n = 10–11 mice/group (Kit^hi = 10; Kit^lo = 11) and (J) from 9 mice/group (Kit^hi = 9; Kit^lo = 9), across two independent experiments. K Experimental schema to evaluate thymic function following competitive allo-HCT using young HSCs. L Frequency of donor-derived Recent Thymic Emigrants (RTE: CD3 + GFP + ) in the PB at 8 weeks post-HCT. Refer to Supplementary Fig. [163]13C for the gating strategy to define the above population. All data are from n = 5 mice/group (Kit^lo = 5; Kit^hi = 5). M Experimental schema to investigate the functional response of differential Kit-expressing HSC-derived T cells in secondary recipients to L. monocytogenes expressing chicken ovalbumin (LM-OVA) infection. N Spleen analysis of secondary recipients for frequency of OT1-derived chimerism of CD8+ T cells (OT1+CD8 + : CD45.1^+H2-K^bCD8^+) 7 days post-infection. Refer to Supplementary Fig. [164]13D for the gating strategy to define the above population. All data are from n = 10 mice/group, (Kit^lo = 10; Kit^hi = 10), across two independent experiments. O Experimental schema for competitive allogeneic HCT (allo-HCT) using Kit^hi (red) and Kit^lo HSCs (blue) from 2-mo (young) C57BL/6 mice with competitor bone marrow (BM) cells from B6.SJL-PtprcaPepcb/BoyJ mice transplanted into lethally irradiated 14-mo (Middle-aged) BALB/cJ recipients. P Frequency of donor-derived T cell chimerism at the indicated timepoints. Q, R Sixteen weeks after competitive HCT, enumeration of donor-derived CLPs (Q) and total thymic cellularity (R). Data for (P–R) are from n = 6–7 mice/group, (Kit^lo = 7; Kit^hi = 6), across two independent experiments. Refer to Supplementary Fig. [165]12 for gating strategies to define the above populations. Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P-values calculated by a nonparametric unpaired two-tailed Mann–Whitney U test. Panels A, D, K, M, and O were created in BioRender. Lab, K. (2025) [166]https://BioRender.com/fl4hwgnand [167]https://BioRender.com/oeh4i7x. Source data are provided as a Source Data file, Source Data Fig. 2. To further evaluate the reconstituting potential of Kit^lo HSCs, we competitively transplanted Kit^lo or Kit^hi HSCs from young C57BL/6 (CD45.2) mice with rescue BM cells (CD45.1) into lethally irradiated young BALB/cJ recipients (Fig. [168]2D). Eight weeks after allo-HCT, we found that Kit^lo HSCs generated significantly better B and T cell reconstitution, while myeloid reconstitution was comparable (Fig. [169]2E, Supplementary Fig. [170]4A, [171]B). Concordant with a prior report of better self-renewal ability^[172]27, we found that Kit^lo HSCs demonstrated increased LT HSC reconstitution (Fig. [173]2F). In support of multilineage potential, we observed that Kit^lo HSC recipients had higher lymphoid progenitor reconstitution (including LMPP and CLP, Fig. [174]2F, G), along with comparable myeloid precursor output (including MPP3: Multipotent Progenitor 3, CMP: Common Myeloid Progenitor, GMP: Granulocyte-Monocyte Progenitor, MEP: Megakaryocyte-Erythrocyte Progenitor, Fig. [175]2F and Supplementary Fig. [176]4C). To evaluate whether enhanced lymphoid precursor output could contribute to thymic reconstitution, we concurrently evaluated the recipient thymi. We found that Kit^lo recipients demonstrated enhanced thymic recovery, characterized by significantly higher thymic cellularity and reconstitution of precursor and mature thymocytes (Fig. [177]2H–I). Notably, in support of a critical role for thymocyte-stromal crosstalk in preserving thymic architecture^[178]12–[179]14, we also found higher thymic epithelial cells (TECs), specifically medullary TECs, and endothelial cell recovery in Kit^lo reconstituted thymi (Fig. [180]2J). We next examined donor-derived lymphoid progenitors for differential expression of molecules involved in homing to the thymus (CCR7, CCR9, and PSGL1)^[181]53. We observed significantly higher expression of CCR7 on Kit^lo-derived lymphoid progenitors (CLP) (Supplementary Fig. [182]4D). In addition, we analyzed thymic supernatants and found no differences in levels of thymopoietic ligands, IL22^[183]54 and RANKL^[184]55 (Supplementary Fig. [185]4E). Finally, we found significantly higher secondary lymphoid organ (spleen) reconstitution in Kit^lo recipients (Supplementary Fig. [186]4F-[187]G). To evaluate long-term reconstitution, we performed additional harvests at 20 weeks following HCT. We observed similar trends in higher BM lymphopoiesis, thymic reconstitution, and thymic stromal recovery in Kit^lo recipients (Supplementary Fig. [188]5A–[189]H). Having established Kit^lo HSC’s superior thymic reconstitution capacity, we next investigated how this HSC subset with enhanced lymphoid potential compares to previously defined lymphoid-biased HSC populations. Our immunophenotypic characterization studies of Kit^lo HSCs (Supplementary Fig. [190]3) revealed modest enrichment of lymphoid-biased CD229+ HSCs (~10%). In comparison, we observed a 40% enrichment for a previously described lymphoid-biased, CD150^lo HSCs^[191]23, but their relative contribution to thymic reconstitution compared to Kit^lo HSCs has not been directly assessed. Thus, we generated mix chimeras of Kit^lo HSCs and CD150^lo HSCs in an equal competition transplant (Supplementary Fig. [192]6A, [193]B). We observed significantly better T cell and thymic reconstitution with Kit^lo HSCs compared to CD150^lo HSCs in both PB and thymus (Supplementary Fig. [194]6C, [195]G, [196]H). To determine whether these markers identify overlapping or distinct HSC populations with potential synergistic effects on lymphoid potential, we performed competitive transplants of Kit^loCD150^lo HSCs versus CD150^lo HSCs chimeras. These studies revealed better T cell and thymic repopulating potential of Kit^loCD150^lo HSCs (Supplementary Fig. [197]6C, [198]G, [199]H), suggesting Kit expression provides additional lymphoid potential beyond that conferred by low CD150 expression alone. Finally, to investigate whether Kit^lo better identifies multipotent HSCs within myeloid-biased CD150^hi HSCs, we examined our CD150^hi-CD150^hiKit^lo chimeras (Supplementary Fig. [200]6C, [201]G, [202]H). In line with prior observations, we observed that, while CD150^hi HSCs exhibit decreased T cell output compared with CD150^lo HSCs, CD150^hiKit^lo HSCs possess superior thymic repopulating potential compared to CD150^hi HSCs (Supplementary Fig. [203]6C), revealing a previously unrecognized functional heterogeneity within traditionally myeloid-biased HSC populations. We observed similar trends in B cell reconstitution across Kit^lo and CD150^lo HSCs (Supplementary Fig. [204]6D), though not reaching statistical significance. In contrast, CD150^hi HSC subsets exhibited robust myeloid reconstitution compared to Kit^lo and CD150^lo HSC subsets, consistent with their myeloid-biased potential (Supplementary Fig. [205]6E). Notably, the improved thymocyte repopulation with CD150^loKit^lo and Kit^lo HSCs was reflected in superior bone marrow lymphopoiesis with increased LMPP and CLP compared to CD150^lo HSCs (Supplementary Fig. [206]6F), while the enhanced thymocyte repopulating potential of CD150^hiKit^lo HSCs compared to CD150^hi HSC subsets, despite comparable CLP reconstitution (Supplementary Fig. [207]6F), is consistent with previously described CLP-independent pathways of T cell differentiation^[208]56,[209]57. To assess thymic output, we used the RAG2-GFP model^[210]58, which allows the analysis of recent thymic emigrants (RTEs) (Fig. [211]2K). We observed higher numbers of PB GFP + T cells in Kit^lo vs Kit^hi recipients (Fig. [212]2L). Additionally, to assess whether this increased number of PB T cells in Kit^lo recipients results in increased functionality (Fig. [213]2M), we analyzed response to ovalbumin engineered Listeria Monocytogenes (LM-OVA) and observed a significant increase in OT1+CD8+ T cells (Fig. [214]2N). A key element in the post-HCT recovery of thymic function is the influx of bone marrow-derived lymphoid progenitors, also known as thymic seeding progenitors (TSPs)^[215]11. Furthermore, the significance of restoring the bone marrow-thymus axis in mitigating thymic involution has been underscored through various strategies aimed at augmenting BM lymphopoiesis, including sex-steroid ablation^[216]59,[217]60 and administration of Ghrelin^[218]61, among other approaches^[219]62. We therefore hypothesized that robust BM lymphopoiesis driven by Kit^lo HSCs could potentially counteract age-related alterations within the thymic microenvironment, consequently ameliorating the decline in T cell output. To this end, we competitively transplanted Kit^lo or Kit^hi HSCs from young C57BL/6 mice (CD45.2) with rescue BM cells (CD45.1) into lethally irradiated 14-month-old (Middle-aged) BALB/cJ recipients (Fig. [220]2O). Sixteen weeks post-transplantation, we observed significantly higher numbers of peripheral T and B cells in Kit^lo recipients (Fig. [221]2P and Supplementary Fig. 4[222]I, [223]J). This was attributed to increased numbers of Kit^lo HSC-derived lymphoid progenitors (including LMPP/MPP4 and CLPs, Supplementary Fig. [224]4J and Fig. [225]2Q), while numbers of myeloid precursors were comparable (MPP3, CMP, GMP, and MEP, Supplementary Fig. [226]4J, [227]K). In our parallel analysis of the thymi of Kit^lo vs Kit^hi recipients, we observed increased numbers of thymocytes and TECs (Fig. [228]2R and Supplementary Fig. [229]4L–[230]N). Collectively, these findings establish the potential of Kit^lo HSCs for lymphopoiesis and to improve thymic recovery and peripheral T cell reconstitution in recipients of various ages. Kit^lo HSCs exhibit distinct epigenetic features that facilitate lymphoid potential Prior work has shown that immunophenotypically defined HSC clones with differential lymphoid output have distinct epigenetic signatures, with no discernible patterns in terms of gene expression^[231]19,[232]25,[233]63. To identify the gene regulatory network orchestrating the lymphoid potential differences in HSCs, we analyzed our single-cell ATAC-seq (scATAC-seq) component from our multiome dataset (Supplementary Fig. [234]7A–[235]D). Through unsupervised clustering, we identified 6 clusters, including young and old HSCs (Fig. [236]3A, B). Distinct HSC subsets identified via scRNA-seq analyses (Fig. [237]1B) are situated in varied regions on the scATAC-seq UMAP (Fig. [238]3C, D). Notably, MLin-HSC and Kit^lo HSC intersect with Cluster 3 from the scATAC-seq clusters (Fig. [239]3E). Fig. 3. Kit^lo HSCs exhibit distinct epigenetic features that facilitate lymphoid potential. [240]Fig. 3 [241]Open in a new tab A UMAP of 6 unsupervised HSC clusters identified by tile-based scATAC-seq analysis after Harmony batch correction. B–D Annotated by age (B), HSC subsets (C), and Kit subsets (D) on the scATAC-seq UMAP. E scATAC-seq cluster composition by HSC subsets (left) and Kit subsets (right). Color scale denotes correlation values between the ATAC clusters and cell annotations, generated using ArchR. F, G Matrix plots showing motif enrichment identified by ChromVAR (F) and gene accessibility (G) for lymphoid-specifying transcription factors for Kit subsets in young and old HSCs. Z-scores denote chromVAR motif enrichment in (F) and gene accessibility scores (peak matrix values) that have been row-scaled in (G), generated using ArchR. We characterized the differentially accessible regions (DARs) of chromatin that defined the HSC subsets and found 2816 DARs in cluster C3 (MLin-HSC and Kit^lo enriched) compared to cluster C6 (q-HSC and Kit^hi enriched) (Supplementary Fig. [242]7E). Next, to determine the differential activity and accessibility of lymphoid-specifying transcription factors (TFs) in the identified Kit^hi and Kit^lo HSC subsets, we analyzed chromatin accessibility variation within TF motifs using chromVAR and gene accessibility. Compared to Kit^hi HSCs, we found increased gene accessibility and enrichment for Zinc finger motifs, specifically Zbtb1 and Zbtb7a, in Kit^lo DARs, which are implicated in the lymphoid differentiation program^[243]19,[244]64–[245]66 (Fig. [246]3F, G). In summary, we found that Kit^lo HSCs exhibit an epigenetic program, emphasizing an enrichment of lymphoid-specifying transcription factors, in support of their heightened lymphoid potential. ZBTB1 regulates HSC multipotency and drives thymic reconstitution potential Among members of the ZBTB gene family, Zbtb1 plays a pivotal role in T cell development^[247]66–[248]68 and lymphoid reconstitution^[249]66. However, its role in regulating lymphoid potential in HSCs remains unknown. We hypothesized that Zbtb1 loss impairs the lymphoid potential of Kit^lo HSCs. To this end, we used the CRISPR-Cas9 system to generate Zbtb1-deficient Kit^lo HSCs (Fig. [250]4A) and confirmed a decrease in ZBTB1 by flow cytometry and Western blot analyses (Supplementary Fig. [251]8A). Zbtb1-deficient Kit^lo HSCs showed significantly reduced in vitro T cell potential (Fig. [252]4B). To evaluate the in vivo reconstituting potential, we competitively transplanted young recipients with control or Zbtb1 knockout (KO) Kit^lo HSCs. Sixteen weeks after allo-HCT, we found that Zbtb1-KO recipients exhibited decreased reconstitution across all lineages in the PB and impaired regeneration of the HSPC compartment, including LT HSCs (Fig. [253]4C, D and Supplementary Fig. [254]8B). Furthermore, consistent with reduced BM lymphopoiesis in Zbtb1-KO HSC recipients, we observed decreased thymic reconstitution (Fig. [255]4E and Supplementary Fig. [256]8C). Fig. 4. ZBTB1 regulates HSC multipotency and drives thymic reconstitution potential. [257]Fig. 4 [258]Open in a new tab A Experimental schema for T cell differentiation assay and competitive allogeneic HCT with Zbtb1-deficient Kit^lo HSCs generated with 2-mo young Rosa26Cas9-eGFP KI mice. B Frequency of T cell subsets following 6 weeks of culture in M-ATOs. Refer to Supplementary Fig. [259]13B for gating strategies to define the above populations. Aggregated data across three independent experiments, each performed in duplicates (Control = 3; Zbtb1-KO = 3). C–E Competitive allogeneic HCT with Zbtb1-deficient Kit^lo HSCs generated with 2-mo young Rosa26Cas9-eGFP KI mice. Sixteen weeks after competitive HCT, frequency of donor-derived chimerism of mature lineages in the peripheral blood as described in Fig. [260]2E (C), donor-derived chimerism of LSK and MP cell subsets in the BM (D), and donor-derived chimerism of T cell precursor thymocyte and mature T cell subsets in the thymus (E). All data are from n = 6 mice/group, (Control = 6; Zbtb1-KO = 6), across two independent experiments. F Experimental schema for T cell differentiation assay and competitive allogeneic HCT with Zbtb1-OE Kit^hi HSCs from young mice. G Frequency of T cell subsets following 6 weeks of culture in M-ATO of young Kit^hi HSCs. Refer to Supplementary Fig. [261]13B for gating strategies to define the above populations. Aggregated data across three independent experiments, each performed in duplicates (Control = 3; Zbtb1-OE = 3). H–J Competitive allogeneic HCT with Zbtb1-OE Kit^hi HSCs generated with young mice. Sixteen weeks after competitive HCT, frequency of donor-derived chimerism of mature lineages in the peripheral blood as described in Fig. [262]2E (H), donor-derived chimerism of LSK and MP cell subsets in the BM (I), and donor-derived chimerism of T cell precursor thymocyte and mature T cell subsets in the thymus (J). All data are from n = 6 mice/group, (Control = 6; Zbtb1-OE = 6), across two independent experiments. Refer to Supplementary Fig. [263]12 for gating strategies to define the above populations. K Previously identified Zbtb1 targets^[264]69 and Notch1-interacting proteins^[265]71 enriched in Kit^lo HSCs. L Pathway enrichment analysis for non-Notch1 Zbtb1 targets enriched in Kit^lo HSCs by gseapy()^[266]104 using GO_Biological_Process_2023 libraries. Bubble plot showing representative pathways. Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P-values calculated by nonparametric unpaired two-tailed Mann–Whitney U test. Panels (A) and (F) were created in BioRender. Lab, K. (2025) [267]https://BioRender.com/zy2p156. Source data are provided as a Source Data file, Source Data Fig. 4. Next, we questioned whether reinstating Zbtb1 expression in megakaryocytic-biased, Kit^hi HSCs is sufficient to rescue their lymphoid defect. To test our hypothesis, we overexpressed Zbtb1 cDNA in Kit^hi HSCs (Fig. [268]4F and Supplementary Fig. [269]8D) and we observed a rescue of their T cell potential when compared to Kit^hi controls, thus partially phenocopying Kit^lo HSCs (Fig. [270]4G) as well as improved lymphoid reconstitution in vivo (Fig. [271]4H). Comparing competitively transplanted control or Zbtb1-overexpressed (OE) Kit^hi HSCs, we observed increased trilineage reconstitution, increased chimerism at the level of the Multipotent Progenitor 2 (MPP2), MPP4, and CLPs, thymic reconstitution, and better thymic stromal recovery, in Zbtb1-OE recipients (Fig. [272]4H–J; Supplementary Fig. [273]8E–G). Taken together, our data demonstrates that ZBTB1 is required for normal HSPC function, multilineage engraftment, and lineage bias. Previous studies have identified ZBTB1 targets that facilitate Notch-mediated T cell differentiation in lymphoid progenitors^[274]69 and support chromatin remodeling following DNA damage^[275]70. Given Zbtb1 motif enrichment and enhanced T cell differentiation in Kit^lo HSCs, we hypothesized that ZBTB1 targets mediating lymphoid differentiation gene programs are also enriched in Kit^lo HSCs. To this end, we queried Kit^lo enriched genes for Notch1-interacting proteins^[276]71. We observed ~50% overlap of Notch1-partners enriched in Kit^lo HSCs, of which 60% were ZBTB1 targets identified in a previous CHIP-seq study^[277]69 (Fig. [278]4K). Furthermore, we identified an additional subset of ZBTB1 targets (33%) that were not associated with the Notch1 interactome (Supplementary Data [279]3). To further query for non-Notch-related biological pathways, we performed pathway analysis and identified a strong enrichment for gene sets related to chromatin remodeling and RNA stability (Fig. [280]4L, Supplementary Data [281]4). This data demonstrates that ZBTB1 contributes to an epigenetic regulatory program that facilitates T cell differentiation in primitive Kit^lo HSCs through instructive and permissive mechanisms. Old Kit^lo HSCs exhibit enhanced T cell potential To determine whether Kit^lo HSCs retain their lymphoid potential with age, we evaluated the lymphoid potential of old Kit subsets. Remarkably, even in the case of old mice, we noted a substantial increase in lymphoid progenitor and ATO output from old Kit^lo HSCs compared to Kit^hi HSCs (Fig. [282]5A, B), although modest when compared to the young HSCs. This finding is consistent with prior reports of proliferative defects in aged T cell precursors contributing to decreased thymopoiesis^[283]72–[284]74. Fig. 5. Old Kit^lo HSCs exhibit enhanced T cell potential. [285]Fig. 5 [286]Open in a new tab A, B Evaluation of in vitro lymphoid progenitor and T cell differentiation potential of Kit^hi (red) and Kit^lo HSCs (blue) from or 24-mo (old) C57BL/6 mice using the S17 lymphoid assay and murine artificial thymic organoid (M-ATO), respectively, as shown in Fig. [287]2A. Enumeration of absolute number of lymphoid progenitor cells following 14 days in S17 lymphoid assay (A) and absolute number of T cell subsets following 8 weeks of culture in M-ATOs (B). Refer to Supplementary Fig. [288]13A, B for gating strategies to define the above populations. Aggregated data across 3 independent experiments, each performed in triplicate (Kit^lo = 3; Kit^hi = 3). C Experimental schema for competitive allogeneic HCT (allo-HCT) using Kit^hi (red) and Kit^lo (blue) HSCs from 22–24-mo (old) C57BL/6 mice with competitor bone marrow (BM) cells from B6.SJL-PtprcaPepcb/BoyJ mice transplanted into lethally irradiated young BALB/cJ recipients. D–H Eight weeks after competitive HCT, enumeration of absolute number of donor-derived T cells in the PB (Kit^hi = 9; Kit^lo = 9) (D), LSK cell subsets and CLP cells (Kit^hi = 10; Kit^lo = 10) (E), total thymic cellularity (Kit^hi = 9; Kit^lo = 9). F Donor-derived T cell precursor thymocyte subsets (Kit^hi = 9; Kit^lo = 9) (G), stromal subsets within thymic CD45-compartment (Kit^hi = 9; Kit^lo = 9) (H). Aggregated data across two independent experiments. I Experimental schema to evaluate thymic function following competitive allo-HCT using old HSCs. J Frequency of donor-derived Recent Thymic Emigrants (RTE: CD3^+GFP^+) in the PB at 8 weeks post-HCT. Refer to Supplementary Fig. [289]13C for the gating strategy to define the above population. All data are from n = 5 mice/group (Kit^lo = 5; Kit^hi = 5). K Experimental schema for competitive allogeneic HCT (allo-HCT) using Kit^hi (red) and Kit^lo HSCs (blue) from 22–24-mo (old) C57BL/6 mice with competitor bone marrow (BM) cells from B6.SJL-PtprcaPepcb/BoyJ mice transplanted into lethally irradiated middle-aged BALB/cJ recipients. L–N Eight weeks after competitive HCT, enumeration of the absolute number of donor-derived T cells in PB (L), donor-derived CLPs in the BM as defined in Fig. [290]3D (M), and thymic cellularity (N). All data are from n = 5 mice/group (Kit^lo = 5; Kit^hi = 5). Refer to Supplementary Fig. [291]12 for gating strategies to define the above populations. O Frequency of T cell subsets following 6 weeks of culture in M-ATO of old Kit^hi HSCs following Zbtb1 OE. Refer to Supplementary Fig. [292]13B for gating strategies to define the above populations. Aggregated data across three independent experiments, each performed in duplicates (Control = 3; Zbtb1-OE = 3). Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P-values calculated by a nonparametric unpaired two-tailed Mann–Whitney U test. Panels (C), (I), and (K) were created in BioRender. Lab, K. (2025) [293]https://BioRender.com/oeh4i7x. Source data are provided as a Source Data file, Source Data Fig. 5. Next, to test their in vivo potential, we competitively transplanted either Kit^lo or Kit^hi HSCs from old C57BL/6 (CD45.2) mice with rescue BM (CD45.1) cells into lethally irradiated young BALB/cJ recipients (Fig. [294]5C). At eight weeks post-transplantation, we noted significantly higher numbers of T cells in the peripheral blood of Kit^lo vs Kit^hi recipients. (Fig. [295]5D and Supplementary Fig. [296]9A). Consistent with our experiments using HSCs from young donors, we observed in the BM of Kit^lo vs Kit^hi recipients significantly higher numbers of LT HSCs, LMPPs and CLPs (Fig. [297]5E) with comparable myeloid potential (Fig. [298]5E and Supplementary Fig. [299]9B). Furthermore, mirroring our findings in HSCs from young mice, we observed in recipients of old Kit^lo HSCs enhanced overall thymic cellularity, thymocyte reconstitution, and recovery of TECs and endothelial cells (Fig. [300]5F–H and Supplementary Fig. [301]9C), higher RTE output (Fig. [302]5I, J), and increased CD4+ and CD8+ T cells in the spleen (Supplementary Fig. [303]9C). At 20-weeks post-HCT, we observed similar trends of enhanced reconstitution in old Kit^lo HSC recipients, confirming their long-term reconstituting potential (Supplementary Fig. [304]9F-M). Next, to examine whether old HSC subsets with preserved lymphoid could augment aged thymic recovery, we competitively transplanted middle-aged recipients with old Kit^lo or Kit^hi donors (Fig. [305]5K). We observed increased peripheral T cells (Fig. [306]5L and Supplementary Fig. [307]9E) and CLPs in the BM (Fig. [308]5M), but a modest, non-significant increase in thymic cellularity (Fig. [309]5N) in Kit^lo recipients. Like our Zbtb1 functional studies with young Kit^hi HSCs, we observed a rescue in T cell potential in Zbtb1-overexpressed old Kit^hi HSCs (Fig. [310]5O). Finally, to directly compare the per cell functionality of Kit^lo HSCs in the context of aging, we generated mixed chimeras combining Kit^lo HSC cells from both young and old mice in an equal competition transplant (Supplementary Fig. [311]10A). Consistent with our in vitro findings (Fig. [312]5A, B), we observed an overall decrease in lymphoid reconstitution within the PB, BM, and thymus with old Kit^lo HSCs compared to their young counterparts (Supplementary Fig. [313]10B–E). Pathway analysis revealed decreased enrichment of chromatin remodeling-related gene sets (Supplementary Fig. [314]S10F, Supplementary Data [315]2 and [316]5). Additionally, we noted decreased accessibility and expression of Zbtb1 (Supplementary Fig. [317]10G–I) in Old Kit^lo HSCs, indicating an impaired epigenetic program for lymphoid differentiation. Remarkably, ectopic Zbtb1 expression in old Kit^lo HSCs significantly increased HSC and lymphoid-biased HSPCs, including CLPs, and enhanced peripheral T cell reconstitution (Supplementary Fig. [318]10J–L). These findings demonstrate that old Kit^lo HSCs exhibit preserved lymphoid potential and contribute to thymic recovery, albeit with reduced output. Additionally, forced ZBTB1 expression in old HSCs can improve HSC and lymphoid-biased engraftment potential. KIT^lo human BM HSCs exhibit enhanced lymphoid potential Informed by a recent study that mapped the molecular profiles of human thymic seeding progenitors (TSPs) to their transcriptional counterparts within the HSC compartment^[319]75, we next sought to identify a comparable human HSC subset with lymphoid potential. Interrogating a CITE-Seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) dataset^[320]76 generated from young and old human bone marrow (BM) samples (Fig. [321]6A–C and Supplementary Fig. [322]11A), we mapped our mouse Kit^lo gene signature onto human HSCs and noted a significant enrichment in young BM (Fig. [323]6D, E). We observed lower protein expression of KIT (CD117) on young HSCs by using antibody-derived tag (ADT) reads (Supplementary Fig. [324]11B). To validate our in-silico findings, we next assessed KIT expression in human BM samples (young: 20–40 years; middle-age/ old: >40 years). In our FACS analysis, we found that phenotypic HSCs (p-HSCs: CD34+CD38-CD10-CD45RA-CD90+) showed the lowest KIT expression (Supplementary Fig. [325]11C), with higher KIT levels on old p-HSCs (Fig. [326]6F). Moreover, we noted a statistically significant association between age and the composition of KIT subsets in p-HSCs, indicating an increase in KIT^hi and a decrease in KIT^lo HSCs, aligning with our observations in mice. (Fig. [327]6G). Fig. 6. KIT^lo human BM HSCs exhibit enhanced lymphoid potential. [328]Fig. 6 [329]Open in a new tab A–E Young and old human BM CITE-seq dataset generated and published by Sommarin et al.^[330]76 A–C UMAP of CD34+ young and old human BM annotated with HSC subsets (A), and age (B, C) HSC cluster composition by age. D Kit^lo gene signature (top 50 marker genes from 239 mouse genes identified with human orthologues) was overlaid on human HSC UMAP. E Violin plot for Kit^lo gene score by age. Statistical analysis was performed using the Wilcoxon test. F, G FACS analysis of human BM samples. F Violin plot of KIT protein expression on phenotypic HSCs (p-HSC: CD34 + CD38-CD10-CD45RA-CD90 + ) by age (young: 20–40 yrs; MA/old: >40 yrs). G Scatterplot showing distribution of KIT^hi and KIT^lo HSC subsets within p-HSCs in human BM samples with varying age. The two-tailed P-values generated for (G) were based on a single multivariable generalized estimating equation (GEE) logistic model. The model was adjusted for age, KIT^lo and KIT^hi groups, and their interaction, with an exchangeable working correlation structure to account for the matched data structure. H Experimental schema to evaluate in vitro T cell and lympho-myeloid potential of human BM HSC subsets based on differential KIT expression using human artificial thymic organoids (H-ATO) and MS5 assay, respectively. I Following 8–10-weeks of culture, enumeration of CD19+ B cells and T cell subsets within CD34+ T cell precursors (Early Thymic Progenitors, ETP: CD34^+CD1a^−CD7^−; CD1a^neg- ProT: CD34^+CD1a^−CD7^+; CD1a^pos-ProT: CD34^+CD1a^+CD7^+), and mature T cells (DP: CD34^−CD5^+CD7^+CD4^+CD8^+; SP4: CD34^−CD5^+CD7^+CD4^+CD8^−; SP8: CD34^−CD5^+CD7^+CD4^−CD8^+). Refer to Supplementary Fig. [331]10E for gating strategies to define the above populations. Aggregated data from 8 independent BM donors, each performed in duplicates, across two independent experiments (KIT^lo = 8; KIT^hi = 8). J Following 4 weeks of culture, enumeration of Lineage output (Neutrophil-Granulocytes: hCD45+CD15+; monocyte-macrophages: hCD45+CD14+; B cells: hCD45+CD19+; NK cells: hCD45+CD56+) after culturing 200 KIT^lo and KIT^hi HSCs.^79Aggregated data from 5 independent BM donors, each performed in triplicate or triplicates, across two independent experiments (KIT^lo = 5; KIT^hi = 5). Refer to Supplementary Fig. [332]10F for gating strategies to define the above populations. K Our model illustrates that hematopoietic stem cells (HSCs) with distinct lymphoid-primed chromatin states and differential ZBTB1 activity, which determines their lymphoid potential, can be distinguished by their CD117/KIT expression. In young bone marrow, Kit^lo HSCs (blue) with high ZBTB1 activity exhibit enhanced lymphoid potential, thymic recovery, and T-cell reconstitution, in contrast to Kit^hi HSCs (red) with reduced lymphoid potential. The age-related decline in lymphoid potential results from both a proportional shift toward Kit^hi HSCs and a global decrease in ZBTB1 activity across all HSC populations. Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P-values calculated either by nonparametric paired two-tailed Mann–Whitney U test (I and J), unpaired Mann–Whitney U test (F). Panels (H) and (K) were created in BioRender. Lab, K. (2025) [333]https://BioRender.com/fl4hwgn. Source data are provided as a Source Data file, Source Data Fig. 6. Cord blood (CB) and BM have previously been fractionated based on KIT levels^[334]77,[335]78. To evaluate the T cell potential of these human BM HSC subsets, we generated artificial human thymic organoids (H-ATO) using purified KIT^hi or KIT^lo HSCs from human BM samples (Fig. [336]6H and Supplementary Fig. [337]11D; Supplementary Data [338]6). In concordance with our mouse in vitro studies, following 8–10 weeks of culture, we observed significantly higher output of precursor and mature T cells from KIT^lo HSCs-derived ATOs compared to KIT^hi HSCs (Fig. [339]6I). To further evaluate the multilineage potential of KIT HSC subsets, we performed a lympho-myeloid differentiation assay, as previously described^[340]79. While we observed comparable myeloid differentiation potential, Kit^lo HSCs exhibited significantly more B cells and a non-significant increase in NK cell differentiation potential (Fig. [341]6J and Supplementary Fig. [342]11E). Collectively, these results identify a previously uncharacterized subset of human BM HSCs, KIT^lo HSCs – which exhibit enhanced lymphoid potential. Discussion Recent studies have uncovered significant heterogeneity within the HSC compartment and their role in hematopoiesis. This heterogeneity has implications for disease development and treatment. Of note, certain aspects of HSC heterogeneity challenge the established hematopoietic models, with lineage biases and lineage-restricted cells impacting self-renewal dynamics^[343]80,[344]81. Decisions regarding lympho-myeloid fate are established at multiple levels of the hematopoietic hierarchy: HSCs^[345]22,[346]23,[347]82, MPP, LMPP, and more mature GMPs and CLPs. At the level of HSCs, the process of epigenetic imprinting confers a distinct advantage for lymphoid priming within multilineage HSCs, as well as maintaining these cell-autonomous features imposed at steady state, even under conditions of stress^[348]19,[349]25. Here, we report on the enhanced T cell potential of Kit^lo HSCs, observed in both mice and humans. Through combining multiomic single-cell sequencing and functional analyses utilizing a preclinical allo-HCT model, we demonstrate the augmented potential of Kit^lo HSCs for T cell lymphopoiesis and to support thymic recovery, independent of age. During the early post-transplant period, we noted a relative increase in DN3 thymocytes relative to other thymocyte precursor subsets. This observation is consistent with previous studies reporting that DN3 thymocytes undergo compensatory expansion to support early post-transplant thymopoiesis, with minimal contribution from T cell precursor thymocytes (ETP and DN2)^[350]11,[351]83,[352]84. Further investigation into the mechanisms driving these compensatory processes and their implications for immune function is critical to our understanding of post-transplant immune reconstitution. Previous studies have identified several markers to identify lineage-biased HSCs (lymphoid-biased: CD150^lo, CD229+, vWF-; myeloid-biased: Neogenin+; megakaryocytic-biased: vWF+). However, these previous studies did not assess the utility of these markers in fractionating aged HSCs or characterizing human HSCs. Our comparative analysis demonstrates that Kit expression provides crucial complementary information to CD150 for identifying functionally distinct HSC populations. While both Kit^lo and CD150^lo HSCs support lymphoid reconstitution, Kit^lo HSCs exhibit superior thymic reconstitution capacity. Notably, CD150^loKit^lo HSCs showed enhanced T cell and thymic repopulating potential compared to CD150^lo HSCs alone, indicating that Kit expression provides additional resolution for identifying HSCs with robust T cell potential. Further, CD150^hiKit^lo HSCs exhibited enhanced thymic reconstitution compared to CD150^hi HSCs, confirming that Kit expression identifies functionally distinct HSCs even within conventionally defined subsets. Although CD150^hi HSCs repopulate thymocyte precursors, they exhibit a T cell differentiation block beyond the DN2 stage, suggesting their myeloid-biased state may impact terminal thymocyte differentiation. The functional differences stem from distinct developmental trajectories: Kit^lo and CD150^lo HSCs follow classical CLP-dependent differentiation pathways, while CD150^hiKit^lo HSCs achieve superior thymic reconstitution via a CLP-independent T cell differentiation route. This developmental heterogeneity reinforces the value of KIT as a marker for identifying HSC subsets with diverse yet efficient T cell reconstitution strategies—findings with potential clinical relevance. We further show that Kit is a reliable marker to prospectively identify multilineage or lymphoid-primed Kit^lo HSCs in aged BM. Our findings extend to human biology and identify an analogous KIT^lo HSC subset with enhanced lymphoid potential in adult BM, suggesting translational relevance in human hematopoiesis, although further functional characterization is warranted to definitively establish their clinical significance. Cumulatively, our studies show that low-expressing Kit HSCs more robustly identify lymphoid-primed HSCs compared to other immunophenotypic-based definitions. This marker system is conserved across species, thus providing a simple fractionating strategy to isolate lineage-biased HSC populations. Mechanistically, Kit^lo HSCs exhibit differential expression and activity of lymphoid-specifying transcription factors (TFs), including Zbtb1, indicating their epigenetic program towards a lymphoid fate. We speculate this relationship between Kit signaling and ZBTB1-mediated HSC function operates at multiple levels: transcriptionally, via direct regulation of the Zbtb1 promoter by KIT signaling effectors; post-transcriptionally, through RNA-binding proteins that stabilize Zbtb1 mRNA; and post-translationally, via E3 ligases that modulate protein stability. Studies in mouse models have shown that disruptions in Zbtb1 (Zbtb1-mutant^[353]66 and Zbtb1-KO^[354]67) result in T cell developmental defects and severe combined immunodeficiency phenotypes, with no differences within the stem and progenitor compartment, in native hematopoiesis^[355]66,[356]67. While competitive transplantation studies with Zbtb1-mutants demonstrated additional defects in B and NK cell reconstitution with loss-of-function Zbtb1-mutants^[357]66, similar transplantation studies were not performed with Zbtb1-KO mice^[358]67, which did not rule out the possibility of additional reconstitution defects in the complete absence of ZBTB1. Indeed, we identify ZBTB1 as an essential transcription factor governing hematopoietic stem and progenitor engraftment and lymphoid specification in phenotypic HSCs. Our in vivo experiments reveal that ZBTB1 manipulation produces lineage-specific outcomes. Progenitor analyses showed that ZBTB1 alteration disproportionately affects lymphoid (LMPP and CLP) versus myeloid lineages (CMP, GMP, and MEP) (8-fold versus 2.5-fold decrease with ZBTB1 knockout; 5-fold versus 2-fold increase with ZBTB1 overexpression). These effects were not proportional to the observed differences within the HSC compartment (~2.5–3-fold decrease in ZBTB1-KO; ~1.5-fold increase with ZBTB1 overexpression). This was further reflected in overall differences in cellularity, with Zbtb1-KO resulting in a 65% reduction in thymic cellularity compared to only a 10% decrease in bone marrow cellularity, while Zbtb1 overexpression increased thymic cellularity by 45% versus a 20% increase in bone marrow. We observed an age-related functional decline in Kit^lo HSCs during aging, characterized by competitive disadvantage in old Kit^lo HSCs and reduced Zbtb1 expression. This aging phenotype was further marked by increased expression of the multipotency repressor Ezh1^[359]85. as well as age-associated markers Selp^[360]29 and Neo1^[361]86 in old Kit^lo HSCs, which previous studies have linked to HSC functional impairment. Importantly, ZBTB1’s lymphoid-specifying role is further highlighted in our aging experiments, where Zbtb1 overexpression in aged Kit^lo HSCs enhanced lymphoid reconstitution 3.5-fold while myeloid chimerism remained statistically unchanged, demonstrating a clear lineage-specific effect. Supporting these in vivo findings, our artificial thymic organoid assays—a cell-autonomous readout of T cell differentiation potential—demonstrate that Zbtb1 overexpression specifically rescues the lymphoid defects in both young and aged megakaryocytic-biased Kit^hi HSCs, increasing T cell output by 5-fold. Consistent with prior research, our study suggests ZBTB1 modulates Notch-dependent pathways^[362]69 – critical for HSPC function and lymphoid fate decisions. Additionally, our in vivo experiment with aged HSCs uncovers a previously unrecognized role for ZBTB1 in influencing broader HSC repopulation capacity across multiple progenitor populations. This function likely regulates cellular survival programs^[363]32,[364]87, thereby promoting overall HSPC fitness. Collectively, our findings demonstrate a dual role of ZBTB1 in enhancing HSPC function and lymphoid specification in HSCs. Further mechanistic studies are needed to fully delineate which ZBTB1-specific dependencies mediate its HSC effects in the context of self-renewal versus lineage specification. Examining the direct role at the HSC stage, or if its expression could be indicative of epigenetic states that then facilitate ZBTB1 function at later developmental stages. Future work will also address whether ZBTB1 could be used to augment the generation of mature lymphoid lineages from human BM precursors, with potential therapeutic implications for addressing age-related immune decline. Long-term reconstituting CD34+KIT^lo adult human BM cells have been previously reported^[365]78. In contrast, fractionating phenotypic HSCs in Cord Blood (CB) based on KIT levels showed reduced reconstitution of KIT^int HSCs compared to KIT^hi HSCs^[366]77. These contrasting findings between adult bone marrow and cord blood suggest that the functional importance of KIT signaling varies depending on developmental stage. In our study, combining transcriptional, immunophenotypic, and functional analyses of human BM samples, we identified a KIT^lo subset within immunophenotypically defined HSCs with enhanced lymphoid potential, highlighting its translational relevance in human biology. Further functional characterization is warranted to definitively establish the clinical significance of these observations. This understanding is crucial as prospective HSC isolation with subsequent ex vivo expansion^[367]88,[368]89, holds promise for enhancing immune regeneration after bone marrow transplantation, effectively counteracting treatment-related immunosuppression, and age-associated thymic decline. Methods Animal use Young female mice (6–10 weeks of age): C57BL/6J (CD45.2/H-2Kb, JAX#000664), B6.SJL-PtprcaPepcb/BoyJ (CD45.1, JAX# 002014), and B6(C)-Gt (ROSA)26Sor^em1.1(CAG^−^cas9*^−^EGFP) Rsky/J (Rosa26^Cas9 KI, JAX#028555) mice were purchased from The Jackson Laboratory (JAX). CD45.1 (Stem)-CD45.2 chimeric female mice were obtained from Dr. Joseph Sun. Aged C57BL/6 (CD45.2/H-2Kb) mice (23–24 months of age) were obtained from the National Institute on Aging (Baltimore, MD). Aged female mice (ranging between 18 and 24 months of age) correlate with humans ranging between 56 and 69 years of age. For recipients, we either used young (6–10 weeks of age) BALB/cJ (H-2Kd, JAX#000651) or generated middle-aged BALB/cJ mice (ranging between 14 and 16 months of age) by initially purchasing young BALB/cJ (H-2Kd) mice from JAX and subsequently allowing them to age under controlled conditions in our facility. Middle-aged mice (ranging between 14 and 16 months of age) correlate with humans ranging between 40 and 60 years of age. For consistency across all our transplantation experiments, only female recipient mice were used. RAG2-EGFP-CD45.1 chimeric female mice were generated by crossing FVB-Tg (RAG2-EGFP)1Mnz/J (JAX# 005688) and B6.SJL-PtprcaPepcb/BoyJ (CD45.1, JAX# 002014). Young TCR-OT1 (C57BL/6-Tg (TcraTcrb)1100Mjb/J, JAX#003831) transgenic female mice were obtained from Dr. Andrea Schietinger. All mice were kept under barrier, specific pathogen-free facility (cohousing of 3–5 mice per cage, chow and water ad libitum, 12 h-light cycle [6:00 pm: off]) and were allowed to acclimatize in our vivarium for at least 10-14 days before experiments. All the animal experiments were approved, and mice were euthanized with CO2 gas inhalation under an MSKCC Institutional Animal Care and Use Committee-approved protocol (IACUC). Cell lines To provide a microenvironment that supports lymphoid progenitors in vitro, we cocultured purified HSCs from young and old B6 bone marrow with the S17 stromal cells^[369]48, which were originally obtained from K. Dorshkind (UCLA). To support in vitro T cell differentiation, we cocultured purified HSCs from young and old B6 bone marrow and human BM with MS5-mDLL4 or MS5-hDLL4 stromal cells^[370]50, which were obtained from Dr. Gay Crooks (UCLA). Subsequently, both cell lines were maintained in our laboratory as described in the original references.