A Non-Invasive Stem Cell Therapy Boosts Lymphopoiesis and Averts Age-Related Blood Diseases in Mice

A Non-Invasive Stem Cell Therapy Boosts Lymphopoiesis and Averts Age-Related Blood Diseases in Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A Non-Invasive Stem Cell Therapy Boosts Lymphopoiesis and Averts Age-Related Blood Diseases in Mice David Bryder, Anna Konturek-Ciesla, Qinyu Zhang, Shabnam Kharazi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4528815/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jun, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Hematopoietic stem cell (HSC) transplantation offers a cure for a variety of blood disorders, predominantly affecting the elderly; however, its application, especially in this demographic, is limited by treatment toxicity. In response, we developed a murine transplantation model based on low-intensity conditioning protocols using antibody-mediated HSC depletion. Initially, we identified significant age-related impediments to effective HSC engraftment. By optimizing HSC doses and non-toxic targeting methods, we could significantly enhance the long-term multilineage activity of the transplanted cells. We demonstrate that young HSCs, once transplanted, not only survive but thrive in aged hosts, dramatically improving hematopoietic output and ameliorating age-compromised lymphopoiesis. This culminated in a strategy that robustly mitigated disease progression in a genetic model of myelodysplastic syndrome. These results suggest that non-invasive HSC transplantation could fundamentally change the clinical management of age-associated hematological disorders, offering a novel, prophylactic tool to delay or even prevent their onset in elderly patients. Biological sciences/Stem cells/Ageing Biological sciences/Stem cells/Haematopoietic stem cells Biological sciences/Immunology/Bone marrow transplantation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Bone marrow transplantation (BMT) is a curative treatment for numerous blood and immune diseases 1 . BMT works by introducing healthy donor hematopoietic stem cells (HSCs) to individuals with defective or damaged hematopoiesis. These HSCs, in turn, regenerate the entire hematopoietic system and assure life-long hematopoiesis via their dual capacity for multilineage differentiation and self-renewal 2 . Despite the broad therapeutic potential of BMT, its adoption is constrained by health complications from current transplantation procedures. Successful BMT requires conditioning, which typically involves administering cytotoxic chemotherapy and/or total body irradiation (TBI). Conditioning eliminates host cells and provides space for newly transplanted HSCs, but comes at the price of deleterious side effects 3 . These include changes to the bone marrow (BM) architecture that may lead to long-term residual hematopoietic injury 4 and influence the fate of the transplanted HSCs 5 . To address the shortcomings of traditional conditioning regimens, alternative strategies have been developed to selectively eliminate hematopoietic stem and progenitor cells (HSPCs) from the BM while preserving non-hematopoietic cells. These strategies include the use of monoclonal antibodies to block essential survival signals for HSPCs 6 or to deliver lethal payloads specifically to these cells 7 . Additional non-invasive approaches, such as mobilization-based regimens, use agents like granulocyte colony-stimulating factor (G-CSF) and AMD3100 to disrupt HSPC-niche interactions, thereby vacating niches for transplanted cells 8 . Another method involves overcoming transplantation barriers by using higher doses of HSPCs 9 – 12 . Aging has profound effects on hematopoiesis that lead to an increased predisposition to a range of hematological shortcomings, including myelodysplasia and anemia 13 . Aging is associated with a decreased proportion of naive B and T cells and a corresponding increase in the frequency of memory-type B and T cells, which likely contribute to diminished immune responses to new antigens 14 . The decline in de novo production of new lymphocytes with age can, at least partially, be attributed to intrinsic changes accompanying the aging of HSCs 15 , 16 . Strategies aimed at re-instating more youthful hematopoiesis in aged individuals have, therefore, aimed to alter the function of HSCs in the aged setting 17 – 19 . However, such approaches often lead to only partial rejuvenation of aged HSCs. On the other hand, transplantation of young HSCs into aged hosts offers an opportunity to re-establish the entire hematopoietic system with young-like features. Practical limitations for this include an inability of aged individuals to cope with the devastating side effects from cytotoxic conditioning and the unresolved impact of host age/environment on the graft fate. Previous studies have, for instance, proposed that recipient age decreases the efficiency of homing and long-term engraftment of transplanted HSCs 20 , 21 , perhaps because of a more hostile pro-inflammatory BM microenvironment with age 22 . Here, we employed non-invasive transplantation methods to assess how recipients’ age affects transplantation success. While several key challenges needed to be overcome, we demonstrate the successful reinstatement of multilineage hematopoiesis from young HSCs in aged recipients. Finally, we present the potential of non-invasive conditioning to prevent the emergence of hematological malignancy in a model for myelodysplastic syndrome. RESULTS The aged bone marrow environment restrains HSC engraftment Our initial investigation aimed to evaluate the efficacy of CD45-SAP conditioning 7 in the context of aged hosts. Following administration of CD45-SAP (3 mg/kg) to young (2 months) and aged (16 months) C57BL/6-CD45.2 mice and analysis 8 days later (Fig. 1 a), we observed only marginal changes in overall peripheral blood (PB) white blood cell (WBC) counts in both groups (Fig. 1 b). More detailed assessments revealed transient reductions in platelets and hemoglobin of CD45-SAP-treated mice (Extended Data Fig. 1 a) and some noticeable changes in WBC distribution, with reduced lymphocyte and elevated myeloid cell counts (Extended Data Fig. 1 b). While splenic cellularity remained relatively constant following CD45-SAP-treatment, more evident reductions were observed in thymic cellularity (Fig. 1 c). The overall BM cellularity was also relatively unchanged (Fig. 1 d, left ), while in agreement with other studies 7 , 23 , we observed a pronounced decrease in the numbers of HSCs (Fig. 1 d, right ). In young mice, the effects on other multipotent and more lineage-restricted BM progenitors varied in a cell-type-specific manner, but many of these changes were less pronounced in aged mice (Extended Data Fig. 1 c). We next transplanted CD45-SAP-treated young and aged mice with HSPCs derived from young mice (Fig. 1 e). Donor-derived reconstitution was monitored in PB and by analyzing HSC chimerism in the BM at the experimental endpoint. While young recipients were effectively reconstituted, aged mice presented with only marginal donor-derived reconstitution in the PB (Fig. 1 f). This was further reflected in the levels of donor-derived HSCs (Fig. 1 g). To enhance reconstitution levels in aged hosts, we explored additional conditions in which CD45-SAP was co-injected with other selective immunotoxins and antibodies. These included treating aged mice with CD45-SAP in combination with CD8-SAP, CD4-SAP, or a B cell-specific antibody cocktail (Extended Data Fig. 1 d). However, neither of these treatments led to any evident enhancement in donor cell engraftment (Extended Data Fig. 1 e). We also expanded our investigation to include a combinatorial treatment with CD45-SAP and low-dose (200 cGy) TBI in young and aged mice (Extended Data Fig. 1 f). This aimed to understand whether this synergistic approach could enhance HSC engraftment while minimizing the toxic effects of higher-dose TBI. While this effectively enhanced the reconstitution levels in young recipients to achieve near-complete donor-derived chimerism, this approach was much less effective for aged mice (Extended Data Fig. 1 g). In summary, these results demonstrate that advanced age significantly impairs effective HSC engraftment and transplantation success in C57BL/6 mice. Ex vivo expanded HSCs effectively reconstitute multilineage hematopoiesis in young CD45-SAP-conditioned recipients We and others have previously established the efficacy of a polyvinyl alcohol (PVA)-based culture system in promoting murine HSC expansion. Notably, expanded HSCs enable a degree of HSC-derived reconstitution even in completely unconditioned hosts 11 , 12 . Given this, we explored the impact of larger quantities of HSCs on in vivo reconstitution outcomes in both non-conditioned and alternatively conditioned hosts. We expanded HSCs ex vivo for 21 days and transplanted equivalent fractions (EE) derived from either 100 or 500 HSCs into unconditioned young hosts (Fig. 2 a). Existing literature suggests that the BM niches available for engraftment in unconditioned hosts are limited, yet they are continuously made accessible through a process of niche recycling 9 . With this concept in mind, we examined the reconstitution outcome when the EE500 was subdivided into five separate fractions. Each of these fractions was then transplanted at weekly intervals to evaluate the possible advantage of spreading the transplantation over time (Fig. 2 a). In agreement with previous work 11 , we observed that all non-conditioned recipients of ex vivo expanded HSCs demonstrated durable long-term multilineage engraftment, although the lymphoid chimerism, and in particular for the B cell lineage, was not on par with the myeloid reconstitution (Fig. 2 b). EE500 resulted in higher engraftment compared to EE100, demonstrating a linear increase in myeloid lineage chimerism (19.0 ± 2.1 vs. 4.3 ± 2.0) (Fig. 2 b). However, dividing the EE500 graft into five weekly doses did not yield better results than a single bolus injection (Fig. 2 b). Subsequently, we evaluated the performance of ex vivo expanded HSCs in young hosts conditioned with CD45-SAP, comparing their behavior with that of hosts subjected to lethal (950 cGy) TBI (Fig. 2 c). As anticipated, lethal TBI led to near-complete multilineage donor-reconstitution (Fig. 2 d). CD45-SAP conditioning also resulted in prominent multilineage reconstitution, albeit with a lesser contribution to lymphoid lineages (Fig. 2 d). Crucially, an examination of HSC chimerism at the end of the experiment demonstrated reconstitution levels equivalent to those observed for myeloid lineages (Fig. 2 e), reinforcing that myeloid reconstitution serves as a dependable measure of ongoing HSC activity 24 . HSC transplantation into TBI-conditioned hosts detrimentally impacts their capacity to reconstitute secondary hosts 25 . To ascertain whether this also holds true for CD45-SAP-conditioned hosts, we conducted secondary transplantations of BM cells from the primary transplanted CD45-SAP-conditioned hosts. Two scenarios were considered: a) a non-competitive context where the transplanted cells in secondary hosts competed with the endogenous HSCs from the primary hosts, and b) a situation where the transplanted cells were mixed with an equal number of BM cells from young, untreated mice. BM cells harvested from primary TBI-treated recipients were included for comparison (Fig. 2 f). This disclosed that high reconstitution levels observed in primary CD45-SAP-treated hosts were sustained in the non-competitive setting (Fig. 2 g). Conversely, the reconstitution levels were markedly decreased upon competitive transplantation, mirroring the reduction in HSCs derived from primary TBI-conditioned hosts (Fig. 2 h). In summary, these results affirm prior studies, underscoring that while unconditioned wild-type (WT) hosts can attain long-term HSC-derived multilineage reconstitution, this necessitates significant quantities of HSCs 10 . Notably, pairing higher doses of HSCs with CD45-SAP conditioning dramatically enhanced the reconstitution outcomes. However, an intriguing parallel was noted with HSCs transplanted into TBI-conditioned hosts, where the process of transplantation itself appears to impair their potential for serial transplantation. Engraftment efficiency and functionality of transplanted young HSCs are maintained in aged hosts The interplay between HSCs and their environment encompasses intricate physiological interactions 26 . This complexity might be amplified considering the dynamics between transplanted young HSCs and aged host cells. Given our observed barrier in engrafting young HSCs in aged mice (Fig. 1 ), we entertained either reduced homing of young HSCs in aged recipients and/or an adverse environment in aged recipients for transplanted young HSCs. To approach these questions experimentally, we harvested HSCs from young mice, expanded them ex vivo , and labeled the expanded grafts with Cell Trace Violet (CTV) dye. The CTV-labeled cells were then transplanted into both unconditioned and CD45-SAP-conditioned young and aged hosts. This allowed for the assessment of engraftment and proliferation of CTV-labeled young HSCs 2–4 weeks post-transplantation (Fig. 3 a). Analyses of unconditioned hosts revealed that young-derived HSCs could be recovered from both young and aged recipients, but with a tendency for lower efficiency in aged hosts (1.5-fold, Fig. 3 b). More striking, but in agreement with the well-established expansion of HSCs associated with murine aging 2 , aged recipients exhibited a significantly increased number of host HSCs in the unconditioned setting (Fig. 3 b). When analyzing CD45-SAP-conditioned young and aged hosts, we observed similar amounts of recoverable young donor HSCs (Fig. 3 b). This demonstrated that compromised homing/engraftment in aged mice was unlikely to explain the inefficient reconstitution from young HSCs (Fig. 1 ). While CD45-SAP conditioning reduced the numbers of host HSCs in both young and aged mice (5.6- and 8.6-fold, respectively), aged recipients still harbored a notably higher number of host HSCs than young recipients (Fig. 3 b). CTV dye dilution revealed that transplanted HSCs exhibited similar proliferation kinetics in both young and aged unconditioned recipients (Fig. 3 c and 3 d). Even after CD45-SAP treatment, which accelerated HSC proliferation, this similarity persisted across different age environments. To further evaluate the functionality of young HSCs exposed to an aged environment, we isolated HSCs from young mice, expanded them ex vivo , and labeled them with CTV dye. We then transplanted these cells into unconditioned young and aged mice. Six weeks later, CTV-positive HSCs were extracted from the primary hosts and competitively transplanted into TBI-conditioned recipients (Fig. 3 e). These experiments demonstrated efficient long-term multilineage reconstitution from isolated HSCs, irrespective of whether the cells were obtained from young or aged primary hosts (Fig. 3 f). Taken together, these results establish that young HSCs can successfully engraft in an aged environment, which does not inherently harm HSCs or hinder their ability for proliferation or long-term multilineage reconstitution. Young HSCs support youthful hematopoietic characteristics upon transplantation into aged recipients Although young HSCs successfully reconstituted aged recipients, the reconstitution was limited (Fig. 3 ). We hypothesized that residual endogenous HSCs might restrict this process (Fig. 3 b). Therefore, more thorough elimination of the host’s aged HSCs could potentially enhance hematopoiesis from transplanted young HSCs. Recent studies suggest that mobilizing endogenous HSCs could be a viable strategy to coax these cells out of their supportive niches within the BM, thereby creating vacant niches for transplanted HSCs 8 , 27 . Therefore, our subsequent experiments evaluated the reconstitution levels of EE100 young HSCs after CD45-SAP conditioning, either alone or in combination with a G-CSF/AMD3100-based mobilization protocol (Fig. 4 a). Examination of PB 18 weeks post-transplantation showed that aged mice undergoing the combined CD45-SAP/mobilization-based conditioning had over a two-fold increase in donor-derived multilineage reconstitution compared to those conditioned with CD45-SAP alone (Fig. 4 b). BM HSC analysis revealed nearly four times more recoverable donor HSCs in mice receiving the combined treatment than in those with CD45-SAP conditioning only (Fig. 4 c). Consistent with earlier data (Fig. 3 b), CD45-SAP significantly reduced host HSC levels, with further reductions observed in mobilized recipients (Fig. 4 d). We next examined the characteristics of young HSC-derived hematopoiesis and its interplay with the host's aged-derived hematopoiesis. We employed multi-parameter flow cytometry to stage hematopoiesis in the BM and, given the notable impact of aging on lymphopoiesis 14 , conducted detailed examinations of B and T cell compartments in the spleen and thymus. Our analysis also included a small cohort of young recipients undergoing the same transplant procedure. To explore the relationship between transplanted and host HSCs and their differentiated progeny, we compared the chimerism levels of HSC progeny to those of BM HSCs. This consistently demonstrated that chimerism in progeny from young HSCs was significantly higher than in host-derived cells in aged recipients, but less pronounced in young hosts (Fig. 4 e). For the lymphoid lineages, the contribution to the early stages of differentiation (MPP Ly and CLPs) in aged recipients was almost five times higher than that of the HSCs themselves (Fig. 4 e). Further examination of the B cell lineage revealed a slightly lower chimerism at the later B cell progenitor stages, although differentiation into these stages was still considerable higher than that observed for the age-derived cells. By contrast, early B cell progenitors in young recipients were predominantly host-derived (Fig. 4 e). Examination of the thymus revealed higher chimerism across all evaluated T cell subsets compared to cells derived from the host. Importantly, this difference was significantly more pronounced in aged recipients relative to their younger counterparts (Fig. 4 e). Additional assessment of splenic B and T cells showed that while the overall chimerism from the young donor was greater than that for BM HSCs, these levels were generally not as high as those observed in the primary hematopoietic organs associated with these lineages (Fig. 4 e). Aging has been reported to correlate with the accumulation of a specific B cell subset known as age-associated B cells (ABCs) 28 , 29 . Therefore, we examined the presence and origins of ABCs, in conjunction with traditional follicular and marginal zone (MZ) B cells analysis (Fig. 4 f). Although a small proportion of ABCs originated from the young donor, the overwhelming majority of these cells were host-derived (Fig. 4 f and 4 g). Conversely, donor cells effectively generated follicular B cells, while their contribution to the MZ B cell compartment was similar to the host-derived cells (Fig. 4 f and 4 g). Next, we examined the distribution of more mature T cell subsets within the young to aged chimeras. Aging associates with an increased frequency of both central and effector memory cells, alongside a corresponding decrease in naive T cells 14 . Our analysis revealed that naive CD4 and CD8 T cells from host/aged-derived cells accounted for only about 5% and 15%, respectively, in stark contrast to the roughly 40% chimerism in both subsets derived from young HSCs (Fig. 4 h and 4 i). This underscores the potential to significantly boost the production of naive CD4 and CD8 T cells in aged mice. A recent study suggested that selectively depleting aged HSCs through antibody-mediated targeting could mitigate age-related lymphoid deficiencies and potentially enhance immune function in the elderly 30 . Using CD45-SAP for specific HSC depletion (Fig. 1 ), we compared mature B and T cell subsets in aged mice with those in unmanipulated aged controls (Extended Data Fig. 2 a). We observed significant reductions in host memory CD4 and CD8 T cells, follicular B cells, and a decrease in ABCs post-treatment. Although there was no increase in naive T cell production, this treatment showed promise in boosting immature B cell generation from aged host cells (Extended Data Fig. 2 b-d). Overall, these findings indicate that depleting host HSCs and transplanting young donor cells not only endows their progeny with youthful hematopoietic traits in aged recipients but also that the CD45-SAP treatment contributes to reverting the composition of the aged host's adaptive immune cells to a more youthful-like state. Molecular stability in early young donor-derived lymphoid progenitors exposed to aging bone marrow Age-related decline in lymphopoiesis can be linked to reduced production of early lymphoid progenitors, including MPP Ly 15 , 16 . Because this subset was effectively regenerated from young donor cells in aged recipients (Fig. 4 e), our subsequent analysis examined the molecular features of these cells. For this, we performed RNA-sequencing of donor- and host MPP Ly cells from young and aged recipients (Fig. 4 e). Somewhat unexpectedly, principal component analysis failed to separate between donor and host MPP Ly across age groups (Extended Data Fig. 3 a) and differential gene expression analysis revealed only 17 upregulated genes in donor MPP Ly from aged mice (Extended Data Fig. 3 b). Similarly, analysis of host cells identified merely 16 upregulated genes upon aging (Extended Data Fig. 3 c), without association to any MSigDB pathway (Extended Data Fig. 3 d). This sharply contrasted with aged HSCs from an independent study 31 , which, using a similar analytical approach, displayed pronounced differences from their young counterparts, characterized by a distinct aging signature (Extended Data Fig. 3 e-f) 32 . Together, these results revealed no significant differences in the transcriptomic signatures of donor MPP Ly cells, even when exposed to an aging environment. This corroborates our functional data, which demonstrates that transplanting young HSCs effectively regenerates hematopoiesis with youthful characteristics in aged hosts (Fig. 4 ). Non-invasive BM conditioning followed by transplantation mitigates disease progression in a mouse model of myelodysplastic syndrome In our final experiments, we examined how CD45-SAP conditioning and HSC transplantation affect the development of age-associated hematological malignancies in the NUP98-HOXD13 (NHD13 tg ) transgenic mouse model, which predisposes to myelodysplastic syndrome (MDS) and acute leukemia 33 . Our experimental layout entailed monitoring the disease evolution in NHD13 tg mice for their entire lifespan (up to 24 months, n = 20). This group was juxtaposed against a cohort of NHD13 tg mice that underwent CD45-SAP conditioning and transplantation with 10 7 WT BM cells when they were 2 months old (n = 9). We also incorporated a small group (n = 5) of WT littermate mice as a control (Fig. 5 a). Mice subjected to CD45-SAP conditioning and transplantation exhibited high-level donor multilineage reconstitution four months post-transplantation (Fig. 5 b). None of the aged WT mice developed hematological malignancies during the 2-year observation period. In contrast, many untreated NHD13 tg mice began showing signs of diverse hematological diseases, including both myelo- and lymphoproliferative disorders and acute myeloid and T-cell leukemia, after six months of age. Although not every case could be conclusively diagnosed, many of the unclassified conditions were associated with pronounced thymic hyperplasia. Overall, the incidence of disease in transplanted mice was significantly lower compared to their non-transplanted counterparts. Among the NHD13 tg mice, 75% (15 out of 20) developed hematological malignancies, compared to only 33% (3 out of 9) of those receiving WT cell transplants (Fig. 5 c). Furthermore, while 25% (5 out of 20) of NHD13 tg mice developed acute leukemia, none of the transplanted mice did (Fig. 5 d). DISCUSSION In this work, we explored non-invasive BM conditioning as a regimen for providing aged recipients with HSCs from younger donors. Traditional conditioning commonly employs varied levels of TBI. However, TBI associates with systemic side effects that are poorly tolerated by aged recipients 34 , emphasizing the need for alternative conditioning approaches. While BMT is a well-established clinical procedure, many aspects concerning successful HSC reconstitution remain elusive. The recognition that HSCs inhabit niches crucial for regulating their function has led to a central hypothesis suggesting the necessity for these niches to be available for the seeding of transplanted HSCs 35 . Moreover, it has been proposed that a scarcity of such niches could limit HSC engraftment. Consistent with this notion, a temporary mobilization combined with transplantation at the mobilization peak has been recently proposed to enhance reconstitution in otherwise unconditioned hosts 8 . Interestingly, additional suggestions propose that repeated mobilization and transplantation may not only result in significant hematopoiesis derived from younger sources but also extend lifespan 27 . In line with the effectiveness of this conditioning method, we confirmed that combining it with CD45-SAP significantly enhances reconstitution in aged hosts. Still, the concept of niche recycling could be more complex, given recent findings that suggest it is possible to efficiently reconstitute unconditioned hosts with massive numbers of infused HSCs 10 . However, the requirement to achieve high-level engraftment - necessitating HSCs from nearly 300 mice - poses severe experimental limitations 10 . Instead, we capitalized on recent breakthroughs in HSC ex vivo expansion 11 . This approach facilitated donor chimerism in young unconditioned hosts, which could be significantly boosted in combination with CD45-SAP-mediated conditioning. As part of our work, we tried to reassess the concept of niche recycling by comparing the contribution from one dose of HSCs to the same dose infused over five separate occasions. This resulted in high overall reconstitution levels that, however, were not further elevated in the setting of repetitive transplantation. This contrasts with previous studies suggesting increased graft contribution with constantly vacated niches 9 . We attribute this deviation to the need for extremely high quantities of HSCs, not previously attainable, to saturate the available niches of an unconditioned recipient 10 . One of our key initial findings was that aged mice presented significant barriers to effective engraftment. However, by addressing the limitations associated with donor HSCs number and by combining two different non-invasive conditioning techniques, we achieved substantial hematopoiesis from transplanted young HSCs in aged recipients. It has been hypothesized that aging associates with changes in BM niches, potentially impacting HSC function 36 . Additionally, aging may contribute to other systemic changes that could compromise HSC functionality 37 , 38 . If such age-related alterations are significant, introducing young HSCs into aged recipients might not effectively restore youthful hematopoietic function. Nonetheless, aged individuals still possess HSC clones capable of contributing to multilineage hematopoiesis when transplanted into younger hosts 15 . This suggests that aging may limit the contribution from these clones, as studies have shown that eliminating senescent cells 17 or specifically targeting functionally aged HSCs 30 can enhance host HSC function. Our findings further support this notion in two significant ways. Firstly, we demonstrate that young HSCs transplanted into aged hosts exhibit behavior akin to their performance in a normal environment. Secondly, our research reveals that the CD45-SAP treatment utilized in our studies mitigates some of the age-related decline in host HSC function, particularly concerning adaptive immune components. Aging is widely recognized to result in dampened adaptive immunity and, in particular, neo-responses 39 , making the restoration of robust lymphopoiesis in aged hosts a primary objective. We illustrate that young HSCs can facilitate highly efficient BM B lymphopoiesis in aged recipients, challenging the idea that the aged environment inherently exerts negative influences on this differentiation pathway 40 . Similarly, we observed a notable increase in thymic seeding and/or expansion, which correlated with dramatically elevated numbers of naive peripheral T cells. These findings suggest that introducing young HSCs can significantly enhance thymic function in older recipients despite the pronounced thymic involution with age. Lastly, we aimed to evaluate whether the CD45-SAP-mediated conditioning could impact hematological disease progression. By leveraging the NHD13 tg mouse model, we discovered that the introduction of young HSCs into asymptomatic NHD13 tg mice led to a noteworthy decrease in hematological disease occurrence and, strikingly, to a complete inhibition of acute leukemia development. As at least some host hematopoiesis remains after CD45-SAP conditioning and transplantation, we interpret these results to illustrate that young and genetically intact HSCs can function as a tumor suppressor, perhaps through a cell competition mechanism reminiscent of the Scribble mutation in Drosophila 41 . In conclusion, we here tried to shed light on the potential of non-invasive BM conditioning as an effective strategy to introduce young HSCs into aged hosts. Crucially, we have demonstrated that young HSCs can enhance adaptive immune cell generation, even within an aged milieu, and we highlight the potential of combined non-invasive conditioning with HSC transplantation to hinder the progression of age-related hematological disorders. MATERIALS AND METHODS Mice All experiments involved young (2–4 months) and aged (16–20 months) C57BL/6-CD45.2, C57BL/6-CD45.1 or F1 C57BL/6-CD45.1/CD45.2 mice obtained from Jackson Laboratory, Janvier Labs, Taconic Bioscience or generated in house. NHD13 tg mice 33 (RRID: IMSR_JAX:010505) were obtained from Jackson Laboratory. All analyses were performed on female mice, except in Fig. 2 f and Fig. 5 , which involved both males and females. For transplantation of tdTomato + HSCs (Fig. 1 and Extended Data Fig. 1 ), cells were isolated from Fgd5 CreERT2/+ ; Rosa26 LSL − tdTomato/+ mice, generated by crossing Fgd5-ZsGreen-2A-CreERT2 mice 42 (RRID: IMSR_JAX:027789) to Rosa26-LoxP-STOP-LoxP-tdTomato (RRID: IMSR_JAX:007905) mice. tdTomato-labeling in Fgd5 -expressing HSCs was induced with tamoxifen (Sigma Aldrich, 10 mg/ml, resuspended in peanut oil) by intraperitoneal injections at 50 mg/kg 15 . Mice were housed in the Animal Facility at the Biomedical Center of Lund University in environment-enriched conditions with 12-hour light-dark cycles and water and food provided ad libitum . All experimental procedures were approved by a local ethical committee (permits M186-15 and 16468/2020). Animal procedures Bleeding and isolation of bone marrow, thymus, and spleen cells Peripheral blood was collected from the tail vein into EDTA-coated tubes (Sarstedt) or 2% (v/v) FBS/PBS with heparin (Leo Pharma, 5000 IE/ml diluted 1:500). Complete blood count was determined using Sysmex KX-21N and XQ-320 analyzers. For BM cell isolation, mice were euthanized by cervical dislocation, and femurs, tibias and hip bones were collected from both hind legs. Bones were crushed in ice-cold 2% (v/v) FBS/PBS. For isolation of thymus and spleen cells, organs were dissociated using a plunger and 70 µm strainer in ice-cold 2% (v/v) FBS/PBS. Single-cell suspensions were centrifuged at 400g for 10 min and filtered through 70 µm cell strainers prior to sample processing. Immunotoxin preparation and conditioning The CD45-SAP immunotoxin was prepared as previously described 7 . Biotinylated anti-CD45.2 antibodies (clone 104, Sony Biotechnology) were mixed with streptavidin-saporin conjugate (Advanced Targeting Systems, Lot #132–178 and #201 − 151) at a 1:1 molar ratio. In all experiments, CD45-SAP was administered at 3 mg/kg 8 days before analysis or transplantation, except in cohorts of young and aged animals in Fig. 4 e and Extended Data Fig. 3 , which received CD45-SAP at 60 µg/mouse 20 weeks after transplantation. For CD4-SAP and CD8-SAP treatment, biotinylated anti-CD4 (clone GK1.5, Sony Biotechnology) or anti-CD8a (clone 53 − 6.7, Sony Biotechnology) were combined with streptavidin-saporin conjugate and injected at 0.5 mg/kg 2–3 days before transplantation. All immunotoxins were diluted in PBS and administered intravenously. B cell depletion was performed as previously reported 43 by intraperitoneal injection of rat anti-mouse CD19 and CD22 (clones 1D3 and CY34.1 respectively; BioXCell) and rat anti-mouse B220 (clone RA3-6B2; eBioscience) antibodies at 150 µg/mouse. After 48 hours, mice received intraperitoneal injections of anti-rat kappa light chain (clone MAR18.5; BioXCell) at 150 µg/mouse. For TBI, mice were sublethally (200 cGy) or lethally (950 cGy) irradiated one day or four hours before transplantation, respectively. All mice received antibiotic prophylaxis (Ciprofloxacin, HEXAL, 125 mg/l in drinking water) for two weeks following conditioning. G-CSF/AMD3100 mobilization Mice received subcutaneous injections of recombinant human G-CSF (Zarzio) at 125 µg/kg every 12 hours for two days. Eighteen hours after the last G-CSF injection, mice received AMD3100 at 5 mg/kg in PBS (Sigma). One hour following the AMD3100 injection, mice were transplanted with ex vivo expanded HSCs. Transplantation All transplantations were performed through tail vein injection. See Supplementary Table 1 and corresponding figure legends for detailed experimental descriptions. For HSPC transplantation in Fig. 1 and Extended Data Fig. 1 , tdTomato + HSCs were isolated by FACS from tamoxifen-induced Fgd5 CreERT2/+ ; Rosa26 LSL − Tomato/+ mice and 500 cells were injected alongside 500,000 BM cells from C57BL/6-CD45.1 mice into each young and aged recipient. For transplantation of ex vivo expanded HSCs, cultured cells were collected, washed, and filtered/FACS-purified prior to transplantation. In all experiments, cells from cultures were pooled, and each recipient received an equal portion of the same input HSCs. For secondary transplantation (Fig. 2 f), 3x10 6 BM cells from primary recipients were either non-competitively transplanted or mixed with whole BM-derived from C57BL/6-CD45.1/CD45.2 mice at a 1:1 ratio (3x10 6 cells of respective fraction per mouse) and transplanted into TBI-treated C57BL/6-CD45.1/CD45.2 secondary hosts. Cells from primary recipients were pooled before transplantation. For in vivo proliferation tracking, cultured cells and CD4-enriched splenocytes isolated from C57BL/6-CD45.1 or C57BL/6-CD45.2 mice were labeled with CTV dye as previously described 12 . EE100 HSCs were co-transplanted with 2x10 6 splenocytes into each C57BL/6-CD45.2 or C57BL/6-CD45.1/CD45.2 unconditioned or CD45-SAP treated recipient. Disease classification in NHD13 tg mice Classification of hematopoietic diseases was based on the following criteria: Myelodysplastic syndrome was characterized by WBC WBC > 20; acute myeloid and lymphoid leukemia were defined by a WBC > 50 and their respective myeloid- or lymphoid-lineage assignments as determined by flow cytometry. Ex vivo HSC expansion Ex vivo HSC expansion was performed as described before 11 . Briefly, 96-well flat-bottom plates were coated with 100 ng/ml fibronectin (Sigma) for at least 1 hour. HSCs (Lineage-cKIT + SCA-1 + CD48-CD150 + CD201 high ) were isolated from young mice and cultured for 21 days in F12 medium (Gibco) supplemented with 1% insulin–transferrin–selenium–ethanolamine (Gibco), 10 mM HEPES (Gibco), 1% penicillin/streptomycin/glutamine (Gibco), 0.1% PVA (87–90%-hydrolyzed, Sigma), 10 ng/ml mouse SCF (Peprotech) and 100 ng/ml mouse TPO (Peprotech) at 37°C with 5% CO 2 . Media changes were performed every 2 days starting from day 5 after sorting. Cells were split when reaching 80–90% confluency. Flow cytometric analysis and FACS sorting Fluorescently labeled and biotinylated antibodies used in this study are listed in Supplementary Table 2. Stainings that included Brilliant Violet-conjugated reagents were supplemented with 10% Brilliant Stain Plus Buffer (BD). The cells were stained in 2% (v/v) FBS/PBS for 30 min at 4ºC in the dark, unless otherwise stated. For PB analysis, erythrocytes were sedimented with 1% Dextran T500 (Sigma-Aldrich) at 37°C for 30 min. The remaining erythrocytes were then lysed with ammonium chloride solution (STEMCELL Technologies) for 3 min at room temperature. Cells were stained in 2% (v/v) FBS/PBS with 2 mM EDTA (Vwr) and antibodies against TER119, CD19, CD11b, Gr1, NK1.1 and CD3. HSPC analysis was performed on whole BM or cKIT-enriched cells. For cKIT enrichment, BM cells were stained with anti-cKIT-APC or anti-cKIT-APCeFluor780 antibody, followed by incubation with anti-APC MicroBeads (1:20, Miltenyi Biotec) for 30 min. Magnetic separation was performed using LS or MS columns and a manual separator, according to manufacturer’s instructions (Miltenyi Biotec). In all stainings, except for myelo-erythroid cell subsets, cells were pre-incubated with Fc-block (1:50, BioXCell) for 15 min prior to antibody staining. For HSPC analysis, cells were stained with antibodies against lineage markers (B220, Gr1, TER119, NK1.1, CD3), SCA-1, cKIT, CD150, CD48, CD201 and in some experiments also against CD135 and CD127. Myelo-erythroid progenitors were identified using lineage markers (B220, Gr1, TER119, NK1.1, CD3), SCA-1, cKIT, CD150, CD105, CD16/32 and CD41. B cell progenitors were identified using lineage markers (Gr1, TER119, NK1.1, CD3), CD19, B220, IgM, CD93, CD43 and cKIT. For thymocyte analysis, cells were stained with antibodies against CD19, B220, CD3, CD4 and CD8. For mature B and T cells, splenocytes were stained with antibodies against CD19, CD93, CD23, CD21/35, CD43, CD11b and CD11c for B cell lineage or Gr1, CD4, CD8, CD3, CD44 and CD62L for T cell lineage. In transplantation experiments, all staining panels included antibodies against CD45.1 and CD45.2 to monitor chimerism levels. HSC isolation by FACS was performed on cKIT-enriched BM cells stained with the HSPC antibody cocktail described above. For isolation of cultured HSCs, cells were stained against lineage markers (Fcer1a, B220, Gr1, TER119, NK1.1, CD3) and CD201. Prior to analysis or cell sorting, cells were filtered and incubated with propidium iodide (1:1000, Invitrogen) to exclude dead cells. All flow cytometry and FACS experiments were performed at Lund Stem Cell Center FACS Core Facility (Lund University). Flow cytometric analyses were conducted on LSRFortessa or Fortessa-X20, and cell sorting was performed on FACSAria III or FACSSymphony S6 instruments (Becton Dickinson). Data was analyzed using FlowJo v.10.8.1 (Treestar). RNA-sequencing and bioinformatic analysis Young (3 months) and aged (16 months) G-CSF/AMD3100-treated mice were transplanted with ex vivo expanded HSCs. After 20 weeks, mice were treated with CD45-SAP at 20 µg/mouse to deplete host BM cells and hematopoietic reconstitution was monitored for additional 16 weeks. At the endpoint, MPP Ly cells (Lineage-cKIT + SCA-1 + IL7Ra-CD135 + CD150-) were isolated from the donor (young) and host (young or aged) BM fractions for bulk RNA-sequencing analysis. Cell isolation was performed in two batches, with each batch consisting of donor and host cells from both young and aged recipients. cDNA library preparation and sequencing were performed at Single Cell Discoveries (Utrecht, the Netherlands) using a modified CEL-Seq2 protocol. Briefly, 200 MPP Ly cells were isolated by FACS, and total RNA was extracted using TRIzol reagent (Invitrogen). mRNA was reverse transcribed, barcoded, pooled, and amplified with an in vitro transcription. The resulting RNA was fragmented and used to generate cDNA sequencing libraries with Truseq Small RNA adapters (Illumina). Libraries were paired-end sequenced on a NovaSeq X Plus instrument using a 10B Reagent kit (100 cycles) and the following read configuration: R1 = 26 cycles, i7 = 6 cycles, R2 = 60 cycles (Illumina). After sequencing, data were demultiplexed and mapped to the mouse GRCm38 reference genome using STARsolo v.2.7.10b software. Reads mapping to multiple locations were discarded. Subsequent analyses were performed in R v.4.2.1, including normalization and differential gene expression using DESeq2 v.1.36.0 package 44 . PCA plot was generated using the vst() function of DESeq2. Differentially expressed genes (DEGs) were identified based on an adjusted p-value < 0.1. Venn diagrams were generated using Venny v.2.0.2 ( https://bioinfogp.cnb.csic.es/tools/venny/index2.0.2.html ), and pathway analysis was conducted using Enrichr ( https://maayanlab.cloud/Enrichr/ ). For analysis of bulk RNA-seq data of young and aged HSCs 31 , the count table was obtained from GEO and processed using the DESeq2 pipeline. DEGs with an adjusted p-value < 0.1 were compared with an HSC aging signature derived from Flohr Svendsen et al. 32 . The odds ratio was computed using a hypergeometric distribution for a total of 21,955 genes, and the p-value was calculated using Fisher’s exact test. Statistical analysis Data were analyzed and visualized using Microsoft Excel v.16.66.1 (Microsoft), Prism 9 v.9.3.1 (GraphPad) and R v.4.2.1 software. Results are presented as mean ± SEM, unless otherwise stated. Experiments were repeated as indicated in figure legends, with n denoting the number of independent biological repeats. Two-group comparison with normally distributed data employed a two-tailed t -test with Welch correction, while not normally distributed data were analyzed using the Mann-Whitney U test. Statistical analyses were unpaired, except in Fig. 4 g and 4 i, which involved paired comparisons. Multiple group comparisons were assessed by one-way ANOVA with post hoc Tukey correction. Specific tests used are indicated in the corresponding figure legends. Declarations Acknowledgments: We thank Single Cell Discoveries for their assistance with bulk RNA-sequencing and S. Soneji for suggestions on bioinformatic analysis. We acknowledge the expertise and assistance of the staff at the Lund University Animal Facility and Lund Stem Cell Center FACS Facility. Funding: The study was generously supported by the Tobias Foundation (Tobias Prize to D.B.), the Swedish Research Council grant 2022-00932 (D.B.), the Swedish Cancer Society 211470Pj (D.B.), the Swedish Pediatric Leukemia Foundation PR2022-0091 (D.B.) and by the Royal Physiographic Society of Lund foundation 42335 and 43250 (A.K-C.) and 42331 and 43043 (Q. Z.). Author contributions: Conceptualization: A.K-C., D.B., Methodology: A.K.-C., Q.Z., Investigation: A.K.-C., Q.Z., S.K., Visualization: A.K.-C., D.B., Funding acquisition: A.K.-C., Q.Z., D.B., Project administration: A.K.-C., D.B., Supervision: D.B., Writing – original draft: A.K.-C., D.B., Writing – review & editing: A.K.-C., D.B. Competing interests: Authors declare that they have no competing interests. Data and materials availability: Bulk RNA-sequencing data has been deposited in the GEO database under the accession number GSE267079 and will be available as of the publication date. No original code was developed in this study. All data are available in the main text or the supplementary materials. References Niederwieser, D. , et al. One and a half million hematopoietic stem cell transplants: continuous and differential improvement in worldwide access with the use of non-identical family donors. Haematologica 107 , 1045-1053 (2022). Bryder, D., Rossi, D.J. & Weissman, I.L. Hematopoietic stem cells: the paradigmatic tissue-specific stem cell. Am J Pathol 169 , 338-346 (2006). Gyurkocza, B. & Sandmaier, B.M. Conditioning regimens for hematopoietic cell transplantation: one size does not fit all. Blood 124 , 344-353 (2014). Wang, Y., Schulte, B.A., LaRue, A.C., Ogawa, M. & Zhou, D. Total body irradiation selectively induces murine hematopoietic stem cell senescence. 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Supplementary Files KonturekCiesla.et.alSupplementNat.Med240601akc.docx Cite Share Download PDF Status: Published Journal Publication published 02 Jun, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4528815","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":317636941,"identity":"4d89071b-75ed-4d54-b953-e5fb3d2bfacc","order_by":0,"name":"David Bryder","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIie3PPWsCMRjA8Sc8oEta5w7SfoQcNwlav4oScDqxo0OHA8GtdBX0Q7RL5kggtwRcXUW4yUFwUTja5sINLhc7Fpo/5GXID54AhEJ/sL5dkkJhjwHAEYCkzRskSh3RjpBFSfAGYdJuFJQjSH9FMhPJA+gJy7g+9ZJuO0XKyHnqISZh6xUUnQ+T8+VYjOKSIDW+wUYDZf/C2DaJcSzU0BEy95BNXhJlyeSEHfHtCLl8eciWy4okiERIR+AurSfRYi/XK6bZg8lj8iZ4PMfGi6K6nvRbw9nxMC3Yfcb3cBHP7ffm7HN3fq0n1XgAT7K6N+yS9U+vevRMHwqFQv+8HxguV8xSFeweAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-8761-4237","institution":"Lund University, Medical Faculty, Department of Laboratory Medicine","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"","lastName":"Bryder","suffix":""},{"id":317636942,"identity":"af7f6790-fc62-42a2-9880-0a314c7809ad","order_by":1,"name":"Anna Konturek-Ciesla","email":"","orcid":"https://orcid.org/0000-0003-1746-930X","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Konturek-Ciesla","suffix":""},{"id":317636943,"identity":"fcdeca2c-1ca3-4c43-9ab5-6b14636374de","order_by":2,"name":"Qinyu Zhang","email":"","orcid":"https://orcid.org/0000-0001-8292-938X","institution":"Lund University","correspondingAuthor":false,"prefix":"","firstName":"Qinyu","middleName":"","lastName":"Zhang","suffix":""},{"id":317636944,"identity":"f6376dfc-1dcf-4922-9ca9-69e2482b8ed2","order_by":3,"name":"Shabnam Kharazi","email":"","orcid":"","institution":"Lund University","correspondingAuthor":false,"prefix":"","firstName":"Shabnam","middleName":"","lastName":"Kharazi","suffix":""}],"badges":[],"createdAt":"2024-06-04 14:35:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4528815/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4528815/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-60464-3","type":"published","date":"2025-06-02T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60173244,"identity":"04f893fa-ca6e-4e7d-8207-99decf8e28e7","added_by":"auto","created_at":"2024-07-12 15:20:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":588524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe aged bone marrow environment restrains HSC engraftment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Experimental design for Fig. 1b-d. Young (2 months) and aged (16 months) C57BL/6-CD45.2 mice received an intravenous injection of CD45-SAP (3 mg/kg), and hematopoietic cell subsets were analyzed after 8 days. Untreated mice served as controls. n = 3-4 mice/group. (\u003cstrong\u003eb\u003c/strong\u003e) Peripheral blood (PB) white blood cell (WBC) counts in untreated and CD45-SAP-treated mice. (\u003cstrong\u003ec\u003c/strong\u003e) Spleen and thymic cellularity in untreated and CD45-SAP-treated mice. (\u003cstrong\u003ed\u003c/strong\u003e) Bone marrow cellularity (\u003cem\u003eleft\u003c/em\u003e) and absolute HSCs numbers (\u003cem\u003eright\u003c/em\u003e) in young and aged untreated and CD45-SAP-treated mice. (\u003cstrong\u003ee\u003c/strong\u003e) Experimental design for Fig. 1f-g. Young (2 months) and aged (16 months) C57BL/6-CD45.2 mice received CD45-SAP (3 mg/kg). Eight days after treatment, the mice were transplanted with HSPCs derived from young (2-4 months) mice. n = 5-10 mice/group. (\u003cstrong\u003ef\u003c/strong\u003e) Donor-derived reconstitution in PB of young and aged recipients. (\u003cstrong\u003eg\u003c/strong\u003e) Absolute numbers of donor- (\u003cem\u003eleft\u003c/em\u003e) and host-derived (\u003cem\u003eright\u003c/em\u003e) HSCs in young and aged mice 17 weeks after transplantation. In (b-d) and (g), each point indicates values for individual mice. Shown is mean ± SEM. Statistical significance was determined using unpaired two-sided \u003cem\u003et\u003c/em\u003e-test with Welch correction. See also Supplementary Table 1.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4528815/v1/9686e2fab5bb177ca7ae4748.png"},{"id":60174227,"identity":"454a2943-dffb-4563-beef-cf5b04e767c9","added_by":"auto","created_at":"2024-07-12 15:28:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":868411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEx vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expanded HSCs effectively reconstitute multilineage hematopoiesis in young CD45-SAP-conditioned recipients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Experimental design for Fig. 2b. HSCs were isolated from young (2-3 months) C57BL/6-CD45.2 and C57BL/6-CD45.1 mice, \u003cem\u003eex vivo\u003c/em\u003e expanded for 21 days and transplanted into young (3-5 months) unconditioned C57BL/6-CD45.1/CD45.2 recipients. Total cultures with equivalent expansions from 100 input HSCs were transplanted via a single injection (1 x EE100). For 5 x EE100 and 1 x EE500, fractions of Lineage-EPCR+ cells from cultures initiated with 100 or 500 input HSCs were transplanted over five weekly injections or via a single injection, respectively. n = 4-5 mice/group. (\u003cstrong\u003eb\u003c/strong\u003e) Donor-derived reconstitution from EE100 and EE500 cultured HSCs in indicated PB lineages 16 weeks after transplantation. n = 5-10 mice/group. Statistical significance was determined by one-way ANOVA with Tukey post-hoc test. (\u003cstrong\u003ec\u003c/strong\u003e) Experimental design for Fig. 2d-e. 100 HSCs were isolated from C57BL/6-CD45.1 mice, expanded \u003cem\u003eex vivo\u003c/em\u003e for 21 days, and transplanted into young (2-3 months) C57BL/6-CD45.2 recipients treated with CD45-SAP (3 mg/kg) or total body irradiation (TBI, 950 cGy). For TBI-treated mice, cultured cells were transplanted together with 500,000 BM cells from C57BL/6-CD45.2 mice. n = 5-8 mice/group. (\u003cstrong\u003ed\u003c/strong\u003e) Reconstitution kinetics for indicated PB lineages in CD45-SAP and TBI-treated mice. (\u003cstrong\u003ee\u003c/strong\u003e) Frequency (\u003cem\u003eleft\u003c/em\u003e) and absolute numbers (\u003cem\u003eright\u003c/em\u003e) of donor-derived HSCs in CD45-SAP and TBI-treated mice 20 weeks after transplantation. n = 5-8 mice/group. (\u003cstrong\u003ef\u003c/strong\u003e) Experimental design for Fig. 2g-h. BM cells from primary recipients (Fig. 2e) were serially transplanted into young (2-3 months) TBI-treated C57BL/6-CD45.1/CD45.2 secondary hosts. For non-competitive transplantation, 3x10\u003csup\u003e6\u003c/sup\u003e BM cells from CD45-SAP-treated primary recipients were transplanted. In a competitive setting, donor cells were mixed with competitor BM-derived from C57BL/6-CD45.1/CD45.2 mice at a 1:1 ratio prior to transplantation. (\u003cstrong\u003eg\u003c/strong\u003e) Donor-derived PB reconstitution 16 weeks after non-competitive secondary transplantation of BM cells from primary CD45-SAP-treated hosts. n = 5. (\u003cstrong\u003eh\u003c/strong\u003e) Donor-derived PB reconstitution 16 weeks after competitive secondary transplantation of BM cells from CD45-SAP or TBI-treated primary hosts. n = 5/group. In (b), (e) and (h), data points indicate values for individual mice. For (d-e) and (h), statistical significance was determined using unpaired two-sided \u003cem\u003et\u003c/em\u003e-test with Welch correction. Shown is mean ± SEM. See also Supplementary Table\u0026nbsp;1.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4528815/v1/b1f391ed824070649dc94e59.png"},{"id":60173243,"identity":"9be5aefd-9fcd-4931-9b89-94276593df7e","added_by":"auto","created_at":"2024-07-12 15:20:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":630483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe engraftment efficiency and functionality of transplanted young HSCs are maintained in aged hosts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Experimental design for Fig. 3b-d. HSCs were isolated from young (2-3 months) mice and \u003cem\u003eex vivo\u003c/em\u003eexpanded for 21 days. Cultured cells were next labeled with Cell Trace Violet (CTV) dye and transplanted into unconditioned or CD45-SAP-treated young (2-3 months) and aged (16-17 months) recipients at EE100 cells/mouse. HSC engraftment and CTV labeling were analyzed 2-4 weeks after transplantation. n = 4-5 mice/group.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Absolute numbers of donor and host HSCs in unconditioned (\u003cem\u003eleft\u003c/em\u003e) and CD45-SAP-treated (right) young and aged recipients. Statistical significance was determined using Mann-Whitney U test.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ec\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eRepresentative histograms depicting CTV labeling in donor-derived HSCs in unconditioned (\u003cem\u003eleft\u003c/em\u003e) and CD45-SAP-treated (\u003cem\u003eright\u003c/em\u003e) young and aged recipients 2 weeks after transplantation.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ed\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eFrequency of undivided and divided donor HSCs in unconditioned (\u003cem\u003eleft\u003c/em\u003e) and CD45-SAP-treated \u003cem\u003e(right\u003c/em\u003e) young and aged recipients. Statistical significance was determined using unpaired two-sided \u003cem\u003et\u003c/em\u003e-test with Welch correction.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ee\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eExperimental design for Fig. 3f. HSCs were isolated from young (2-3 months) mice and \u003cem\u003eex vivo\u003c/em\u003e expanded as in (a). Cultured cells were next labeled with CTV dye and transplanted into unconditioned young (2-3 months) and aged (16-17 months) recipients at EE100 cells/mouse. Undivided HSCs were extracted from primary hosts 6 weeks after transplantation and competitively transplanted into young (2 months) TBI-treated secondary recipients. (\u003cstrong\u003ef\u003c/strong\u003e) Hematopoietic reconstitution from HSCs isolated from young and aged primary recipients. Values in parenthesis indicate the frequency of mice with multilineage (M+B+T) and lymphoid (B+T) reconstitution 16 weeks after transplantation. In (b) and (d), points indicate values for individual mice. See also Supplementary Table 1.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4528815/v1/98bb4ebb43eae9228127d866.png"},{"id":60173246,"identity":"4c3c91cb-b7e8-4c83-a8c0-6992c48d7fea","added_by":"auto","created_at":"2024-07-12 15:20:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1419627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYoung HSCs support youthful hematopoietic characteristics upon transplantation into aged recipients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Experimental design. Aged (16 months) mice were treated with CD45-SAP with or without G-CSF/AMD3100 and transplanted with \u003cem\u003eex vivo\u003c/em\u003eexpanded young HSCs (EE100 cells/mouse). n = total 6-8 mice/group. (\u003cstrong\u003eb\u003c/strong\u003e) Young HSC-derived PB reconstitution in aged hosts 18 weeks after transplantation. (\u003cstrong\u003ec-d\u003c/strong\u003e) Absolute numbers of donor (c) and host (d) HSCs in aged recipients treated with CD45-SAP with or without G-CSF/AMD3100. In (d), HSC numbers in age-matched untreated controls were assessed for comparison (n = 5). (\u003cstrong\u003ee\u003c/strong\u003e) Chimerism levels in indicated hematopoietic cell subsets relative to donor (young)- and host (aged)-derived HSCs in aged recipients (\u003cem\u003eleft\u003c/em\u003e). Young recipients were used for comparison (\u003cem\u003eright\u003c/em\u003e). p-value for each comparison is presented. n = total 4-10 mice/group. (\u003cstrong\u003ef\u003c/strong\u003e) Representative flow cytometric profiles of splenic B cell subsets within donor (young) and host (aged) cell fractions. ABC – age-associated B cells; MZ – marginal zone B cells; FO – follicular B cells. (\u003cstrong\u003eg\u003c/strong\u003e) Frequency of splenic ABC, MZ and FO cells within donor (young)- and host (aged) CD19+ cell fractions. (\u003cstrong\u003eh\u003c/strong\u003e) Representative flow cytometric profiles of splenic CD4+ T cell subsets within donor (young) and host (aged) cell fractions. CM – central memory; EM – effector memory. (\u003cstrong\u003ei\u003c/strong\u003e) Frequency of splenic naive, CM and EM T cells within donor (young)- and host (aged) CD4+ (\u003cem\u003eleft\u003c/em\u003e) and CD8+ (\u003cem\u003eright\u003c/em\u003e) cell fractions. In (b-e), (g) and (i), points indicate values for individual mice. Statistical significance was determined by unpaired two-sided \u003cem\u003et\u003c/em\u003e-test with Welch correction in (b-c) and (e), one-way ANOVA with Tukey test in (d), Wilcoxon matched-pairs signed rank test in (g) and paired two-sided \u003cem\u003et\u003c/em\u003e-test in (i). Shown is mean ± SEM.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4528815/v1/48a1e1afea750089b95ba507.png"},{"id":60173247,"identity":"4db2b88a-4981-48fb-9dba-0494339f2912","added_by":"auto","created_at":"2024-07-12 15:20:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":296047,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNon-invasive BM conditioning followed by transplantation mitigates disease progression in a mouse model of myelodysplastic syndrome.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Experimental design for Fig. 5b-d. Young (2 months) NHD13 transgenic (tg) mice were treated with CD45-SAP and transplanted with 10x10\u003csup\u003e6\u003c/sup\u003e BM cells derived from C57BL/6-CD45.1 mice. Untreated NHD13\u003csup\u003etg\u003c/sup\u003e and wild-type (WT) littermates served as controls. Mice were monitored for disease development and donor-derived chimerism for up to 24 months of age. n = 5-20 mice/group. (\u003cstrong\u003eb\u003c/strong\u003e) Donor-derived reconstitution in indicated PB lineages 16 weeks after transplantation. Points indicate values for individual mice. Shown is mean ± SEM. (\u003cstrong\u003ec\u003c/strong\u003e) Frequency of mice that developed hematological disease. Statistical significance was determined using Fisher’s exact test. (\u003cstrong\u003ed\u003c/strong\u003e) Disease type of individual mice. Values in parentheses indicate frequencies.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4528815/v1/88831c32e7838307541de8f3.png"},{"id":83811474,"identity":"5adeea1f-0f1d-4384-8716-12011a051890","added_by":"auto","created_at":"2025-06-03 07:05:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4933427,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4528815/v1/ff22613f-b7e4-41d5-83e6-476f62f5fc4b.pdf"},{"id":60173248,"identity":"cfdac06e-bbef-43d6-a648-d40016231625","added_by":"auto","created_at":"2024-07-12 15:20:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1310547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"KonturekCiesla.et.alSupplementNat.Med240601akc.docx","url":"https://assets-eu.researchsquare.com/files/rs-4528815/v1/233ed4e5d3e85c08419cc949.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A Non-Invasive Stem Cell Therapy Boosts Lymphopoiesis and Averts Age-Related Blood Diseases in Mice","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eBone marrow transplantation (BMT) is a curative treatment for numerous blood and immune diseases\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. BMT works by introducing healthy donor hematopoietic stem cells (HSCs) to individuals with defective or damaged hematopoiesis. These HSCs, in turn, regenerate the entire hematopoietic system and assure life-long hematopoiesis via their dual capacity for multilineage differentiation and self-renewal\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite the broad therapeutic potential of BMT, its adoption is constrained by health complications from current transplantation procedures. Successful BMT requires conditioning, which typically involves administering cytotoxic chemotherapy and/or total body irradiation (TBI). Conditioning eliminates host cells and provides space for newly transplanted HSCs, but comes at the price of deleterious side effects\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. These include changes to the bone marrow (BM) architecture that may lead to long-term residual hematopoietic injury\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and influence the fate of the transplanted HSCs\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo address the shortcomings of traditional conditioning regimens, alternative strategies have been developed to selectively eliminate hematopoietic stem and progenitor cells (HSPCs) from the BM while preserving non-hematopoietic cells. These strategies include the use of monoclonal antibodies to block essential survival signals for HSPCs\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e or to deliver lethal payloads specifically to these cells\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Additional non-invasive approaches, such as mobilization-based regimens, use agents like granulocyte colony-stimulating factor (G-CSF) and AMD3100 to disrupt HSPC-niche interactions, thereby vacating niches for transplanted cells\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Another method involves overcoming transplantation barriers by using higher doses of HSPCs\u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAging has profound effects on hematopoiesis that lead to an increased predisposition to a range of hematological shortcomings, including myelodysplasia and anemia\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Aging is associated with a decreased proportion of naive B and T cells and a corresponding increase in the frequency of memory-type B and T cells, which likely contribute to diminished immune responses to new antigens\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The decline in \u003cem\u003ede novo\u003c/em\u003e production of new lymphocytes with age can, at least partially, be attributed to intrinsic changes accompanying the aging of HSCs\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Strategies aimed at re-instating more youthful hematopoiesis in aged individuals have, therefore, aimed to alter the function of HSCs in the aged setting\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, such approaches often lead to only partial rejuvenation of aged HSCs. On the other hand, transplantation of young HSCs into aged hosts offers an opportunity to re-establish the entire hematopoietic system with young-like features. Practical limitations for this include an inability of aged individuals to cope with the devastating side effects from cytotoxic conditioning and the unresolved impact of host age/environment on the graft fate. Previous studies have, for instance, proposed that recipient age decreases the efficiency of homing and long-term engraftment of transplanted HSCs\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, perhaps because of a more hostile pro-inflammatory BM microenvironment with age\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we employed non-invasive transplantation methods to assess how recipients\u0026rsquo; age affects transplantation success. While several key challenges needed to be overcome, we demonstrate the successful reinstatement of multilineage hematopoiesis from young HSCs in aged recipients. Finally, we present the potential of non-invasive conditioning to prevent the emergence of hematological malignancy in a model for myelodysplastic syndrome.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eThe aged bone marrow environment restrains HSC engraftment\u003c/h2\u003e \u003cp\u003eOur initial investigation aimed to evaluate the efficacy of CD45-SAP conditioning\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e in the context of aged hosts. Following administration of CD45-SAP (3 mg/kg) to young (2 months) and aged (16 months) C57BL/6-CD45.2 mice and analysis 8 days later (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), we observed only marginal changes in overall peripheral blood (PB) white blood cell (WBC) counts in both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). More detailed assessments revealed transient reductions in platelets and hemoglobin of CD45-SAP-treated mice (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and some noticeable changes in WBC distribution, with reduced lymphocyte and elevated myeloid cell counts (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile splenic cellularity remained relatively constant following CD45-SAP-treatment, more evident reductions were observed in thymic cellularity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The overall BM cellularity was also relatively unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, \u003cem\u003eleft\u003c/em\u003e), while in agreement with other studies\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, we observed a pronounced decrease in the numbers of HSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, \u003cem\u003eright\u003c/em\u003e). In young mice, the effects on other multipotent and more lineage-restricted BM progenitors varied in a cell-type-specific manner, but many of these changes were less pronounced in aged mice (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eWe next transplanted CD45-SAP-treated young and aged mice with HSPCs derived from young mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Donor-derived reconstitution was monitored in PB and by analyzing HSC chimerism in the BM at the experimental endpoint. While young recipients were effectively reconstituted, aged mice presented with only marginal donor-derived reconstitution in the PB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). This was further reflected in the levels of donor-derived HSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eTo enhance reconstitution levels in aged hosts, we explored additional conditions in which CD45-SAP was co-injected with other selective immunotoxins and antibodies. These included treating aged mice with CD45-SAP in combination with CD8-SAP, CD4-SAP, or a B cell-specific antibody cocktail (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). However, neither of these treatments led to any evident enhancement in donor cell engraftment (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eWe also expanded our investigation to include a combinatorial treatment with CD45-SAP and low-dose (200 cGy) TBI in young and aged mice (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). This aimed to understand whether this synergistic approach could enhance HSC engraftment while minimizing the toxic effects of higher-dose TBI. While this effectively enhanced the reconstitution levels in young recipients to achieve near-complete donor-derived chimerism, this approach was much less effective for aged mice (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eIn summary, these results demonstrate that advanced age significantly impairs effective HSC engraftment and transplantation success in C57BL/6 mice.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEx vivo\u003c/b\u003e \u003cb\u003eexpanded HSCs effectively reconstitute multilineage hematopoiesis in young CD45-SAP-conditioned recipients\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe and others have previously established the efficacy of a polyvinyl alcohol (PVA)-based culture system in promoting murine HSC expansion. Notably, expanded HSCs enable a degree of HSC-derived reconstitution even in completely unconditioned hosts\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Given this, we explored the impact of larger quantities of HSCs on \u003cem\u003ein vivo\u003c/em\u003e reconstitution outcomes in both non-conditioned and alternatively conditioned hosts.\u003c/p\u003e \u003cp\u003eWe expanded HSCs \u003cem\u003eex vivo\u003c/em\u003e for 21 days and transplanted equivalent fractions (EE) derived from either 100 or 500 HSCs into unconditioned young hosts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Existing literature suggests that the BM niches available for engraftment in unconditioned hosts are limited, yet they are continuously made accessible through a process of niche recycling\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. With this concept in mind, we examined the reconstitution outcome when the EE500 was subdivided into five separate fractions. Each of these fractions was then transplanted at weekly intervals to evaluate the possible advantage of spreading the transplantation over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn agreement with previous work\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, we observed that all non-conditioned recipients of \u003cem\u003eex vivo\u003c/em\u003e expanded HSCs demonstrated durable long-term multilineage engraftment, although the lymphoid chimerism, and in particular for the B cell lineage, was not on par with the myeloid reconstitution (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). EE500 resulted in higher engraftment compared to EE100, demonstrating a linear increase in myeloid lineage chimerism (19.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 vs. 4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). However, dividing the EE500 graft into five weekly doses did not yield better results than a single bolus injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eSubsequently, we evaluated the performance of \u003cem\u003eex vivo\u003c/em\u003e expanded HSCs in young hosts conditioned with CD45-SAP, comparing their behavior with that of hosts subjected to lethal (950 cGy) TBI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). As anticipated, lethal TBI led to near-complete multilineage donor-reconstitution (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). CD45-SAP conditioning also resulted in prominent multilineage reconstitution, albeit with a lesser contribution to lymphoid lineages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Crucially, an examination of HSC chimerism at the end of the experiment demonstrated reconstitution levels equivalent to those observed for myeloid lineages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), reinforcing that myeloid reconstitution serves as a dependable measure of ongoing HSC activity\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHSC transplantation into TBI-conditioned hosts detrimentally impacts their capacity to reconstitute secondary hosts\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. To ascertain whether this also holds true for CD45-SAP-conditioned hosts, we conducted secondary transplantations of BM cells from the primary transplanted CD45-SAP-conditioned hosts. Two scenarios were considered: a) a non-competitive context where the transplanted cells in secondary hosts competed with the endogenous HSCs from the primary hosts, and b) a situation where the transplanted cells were mixed with an equal number of BM cells from young, untreated mice. BM cells harvested from primary TBI-treated recipients were included for comparison (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). This disclosed that high reconstitution levels observed in primary CD45-SAP-treated hosts were sustained in the non-competitive setting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Conversely, the reconstitution levels were markedly decreased upon competitive transplantation, mirroring the reduction in HSCs derived from primary TBI-conditioned hosts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eIn summary, these results affirm prior studies, underscoring that while unconditioned wild-type (WT) hosts can attain long-term HSC-derived multilineage reconstitution, this necessitates significant quantities of HSCs\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Notably, pairing higher doses of HSCs with CD45-SAP conditioning dramatically enhanced the reconstitution outcomes. However, an intriguing parallel was noted with HSCs transplanted into TBI-conditioned hosts, where the process of transplantation itself appears to impair their potential for serial transplantation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEngraftment efficiency and functionality of transplanted young HSCs are maintained in aged hosts\u003c/h2\u003e \u003cp\u003eThe interplay between HSCs and their environment encompasses intricate physiological interactions\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. This complexity might be amplified considering the dynamics between transplanted young HSCs and aged host cells. Given our observed barrier in engrafting young HSCs in aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), we entertained either reduced homing of young HSCs in aged recipients and/or an adverse environment in aged recipients for transplanted young HSCs.\u003c/p\u003e \u003cp\u003eTo approach these questions experimentally, we harvested HSCs from young mice, expanded them \u003cem\u003eex vivo\u003c/em\u003e, and labeled the expanded grafts with Cell Trace Violet (CTV) dye. The CTV-labeled cells were then transplanted into both unconditioned and CD45-SAP-conditioned young and aged hosts. This allowed for the assessment of engraftment and proliferation of CTV-labeled young HSCs 2\u0026ndash;4 weeks post-transplantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalyses of unconditioned hosts revealed that young-derived HSCs could be recovered from both young and aged recipients, but with a tendency for lower efficiency in aged hosts (1.5-fold, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). More striking, but in agreement with the well-established expansion of HSCs associated with murine aging\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, aged recipients exhibited a significantly increased number of host HSCs in the unconditioned setting (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eWhen analyzing CD45-SAP-conditioned young and aged hosts, we observed similar amounts of recoverable young donor HSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). This demonstrated that compromised homing/engraftment in aged mice was unlikely to explain the inefficient reconstitution from young HSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). While CD45-SAP conditioning reduced the numbers of host HSCs in both young and aged mice (5.6- and 8.6-fold, respectively), aged recipients still harbored a notably higher number of host HSCs than young recipients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eCTV dye dilution revealed that transplanted HSCs exhibited similar proliferation kinetics in both young and aged unconditioned recipients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Even after CD45-SAP treatment, which accelerated HSC proliferation, this similarity persisted across different age environments.\u003c/p\u003e \u003cp\u003eTo further evaluate the functionality of young HSCs exposed to an aged environment, we isolated HSCs from young mice, expanded them \u003cem\u003eex vivo\u003c/em\u003e, and labeled them with CTV dye. We then transplanted these cells into unconditioned young and aged mice. Six weeks later, CTV-positive HSCs were extracted from the primary hosts and competitively transplanted into TBI-conditioned recipients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). These experiments demonstrated efficient long-term multilineage reconstitution from isolated HSCs, irrespective of whether the cells were obtained from young or aged primary hosts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eTaken together, these results establish that young HSCs can successfully engraft in an aged environment, which does not inherently harm HSCs or hinder their ability for proliferation or long-term multilineage reconstitution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eYoung HSCs support youthful hematopoietic characteristics upon transplantation into aged recipients\u003c/h2\u003e \u003cp\u003eAlthough young HSCs successfully reconstituted aged recipients, the reconstitution was limited (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We hypothesized that residual endogenous HSCs might restrict this process (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Therefore, more thorough elimination of the host\u0026rsquo;s aged HSCs could potentially enhance hematopoiesis from transplanted young HSCs.\u003c/p\u003e \u003cp\u003eRecent studies suggest that mobilizing endogenous HSCs could be a viable strategy to coax these cells out of their supportive niches within the BM, thereby creating vacant niches for transplanted HSCs\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Therefore, our subsequent experiments evaluated the reconstitution levels of EE100 young HSCs after CD45-SAP conditioning, either alone or in combination with a G-CSF/AMD3100-based mobilization protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExamination of PB 18 weeks post-transplantation showed that aged mice undergoing the combined CD45-SAP/mobilization-based conditioning had over a two-fold increase in donor-derived multilineage reconstitution compared to those conditioned with CD45-SAP alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). BM HSC analysis revealed nearly four times more recoverable donor HSCs in mice receiving the combined treatment than in those with CD45-SAP conditioning only (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Consistent with earlier data (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), CD45-SAP significantly reduced host HSC levels, with further reductions observed in mobilized recipients (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eWe next examined the characteristics of young HSC-derived hematopoiesis and its interplay with the host's aged-derived hematopoiesis. We employed multi-parameter flow cytometry to stage hematopoiesis in the BM and, given the notable impact of aging on lymphopoiesis\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, conducted detailed examinations of B and T cell compartments in the spleen and thymus. Our analysis also included a small cohort of young recipients undergoing the same transplant procedure.\u003c/p\u003e \u003cp\u003eTo explore the relationship between transplanted and host HSCs and their differentiated progeny, we compared the chimerism levels of HSC progeny to those of BM HSCs. This consistently demonstrated that chimerism in progeny from young HSCs was significantly higher than in host-derived cells in aged recipients, but less pronounced in young hosts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eFor the lymphoid lineages, the contribution to the early stages of differentiation (MPP Ly and CLPs) in aged recipients was almost five times higher than that of the HSCs themselves (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Further examination of the B cell lineage revealed a slightly lower chimerism at the later B cell progenitor stages, although differentiation into these stages was still considerable higher than that observed for the age-derived cells. By contrast, early B cell progenitors in young recipients were predominantly host-derived (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eExamination of the thymus revealed higher chimerism across all evaluated T cell subsets compared to cells derived from the host. Importantly, this difference was significantly more pronounced in aged recipients relative to their younger counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Additional assessment of splenic B and T cells showed that while the overall chimerism from the young donor was greater than that for BM HSCs, these levels were generally not as high as those observed in the primary hematopoietic organs associated with these lineages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eAging has been reported to correlate with the accumulation of a specific B cell subset known as age-associated B cells (ABCs)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Therefore, we examined the presence and origins of ABCs, in conjunction with traditional follicular and marginal zone (MZ) B cells analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Although a small proportion of ABCs originated from the young donor, the overwhelming majority of these cells were host-derived (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). Conversely, donor cells effectively generated follicular B cells, while their contribution to the MZ B cell compartment was similar to the host-derived cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eNext, we examined the distribution of more mature T cell subsets within the young to aged chimeras. Aging associates with an increased frequency of both central and effector memory cells, alongside a corresponding decrease in naive T cells\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Our analysis revealed that naive CD4 and CD8 T cells from host/aged-derived cells accounted for only about 5% and 15%, respectively, in stark contrast to the roughly 40% chimerism in both subsets derived from young HSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). This underscores the potential to significantly boost the production of naive CD4 and CD8 T cells in aged mice.\u003c/p\u003e \u003cp\u003eA recent study suggested that selectively depleting aged HSCs through antibody-mediated targeting could mitigate age-related lymphoid deficiencies and potentially enhance immune function in the elderly\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Using CD45-SAP for specific HSC depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), we compared mature B and T cell subsets in aged mice with those in unmanipulated aged controls (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). We observed significant reductions in host memory CD4 and CD8 T cells, follicular B cells, and a decrease in ABCs post-treatment. Although there was no increase in naive T cell production, this treatment showed promise in boosting immature B cell generation from aged host cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d).\u003c/p\u003e \u003cp\u003eOverall, these findings indicate that depleting host HSCs and transplanting young donor cells not only endows their progeny with youthful hematopoietic traits in aged recipients but also that the CD45-SAP treatment contributes to reverting the composition of the aged host's adaptive immune cells to a more youthful-like state.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMolecular stability in early young donor-derived lymphoid progenitors exposed to aging bone marrow\u003c/h2\u003e \u003cp\u003eAge-related decline in lymphopoiesis can be linked to reduced production of early lymphoid progenitors, including MPP Ly\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Because this subset was effectively regenerated from young donor cells in aged recipients (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), our subsequent analysis examined the molecular features of these cells. For this, we performed RNA-sequencing of donor- and host MPP Ly cells from young and aged recipients (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Somewhat unexpectedly, principal component analysis failed to separate between donor and host MPP Ly across age groups (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) and differential gene expression analysis revealed only 17 upregulated genes in donor MPP Ly from aged mice (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Similarly, analysis of host cells identified merely 16 upregulated genes upon aging (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), without association to any MSigDB pathway (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). This sharply contrasted with aged HSCs from an independent study\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, which, using a similar analytical approach, displayed pronounced differences from their young counterparts, characterized by a distinct aging signature (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-f)\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTogether, these results revealed no significant differences in the transcriptomic signatures of donor MPP Ly cells, even when exposed to an aging environment. This corroborates our functional data, which demonstrates that transplanting young HSCs effectively regenerates hematopoiesis with youthful characteristics in aged hosts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eNon-invasive BM conditioning followed by transplantation mitigates disease progression in a mouse model of myelodysplastic syndrome\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn our final experiments, we examined how CD45-SAP conditioning and HSC transplantation affect the development of age-associated hematological malignancies in the NUP98-HOXD13 (NHD13\u003csup\u003etg\u003c/sup\u003e) transgenic mouse model, which predisposes to myelodysplastic syndrome (MDS) and acute leukemia\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur experimental layout entailed monitoring the disease evolution in NHD13\u003csup\u003etg\u003c/sup\u003e mice for their entire lifespan (up to 24 months, n\u0026thinsp;=\u0026thinsp;20). This group was juxtaposed against a cohort of NHD13\u003csup\u003etg\u003c/sup\u003e mice that underwent CD45-SAP conditioning and transplantation with 10\u003csup\u003e7\u003c/sup\u003e WT BM cells when they were 2 months old (n\u0026thinsp;=\u0026thinsp;9). We also incorporated a small group (n\u0026thinsp;=\u0026thinsp;5) of WT littermate mice as a control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMice subjected to CD45-SAP conditioning and transplantation exhibited high-level donor multilineage reconstitution four months post-transplantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). None of the aged WT mice developed hematological malignancies during the 2-year observation period. In contrast, many untreated NHD13\u003csup\u003etg\u003c/sup\u003e mice began showing signs of diverse hematological diseases, including both myelo- and lymphoproliferative disorders and acute myeloid and T-cell leukemia, after six months of age. Although not every case could be conclusively diagnosed, many of the unclassified conditions were associated with pronounced thymic hyperplasia.\u003c/p\u003e \u003cp\u003eOverall, the incidence of disease in transplanted mice was significantly lower compared to their non-transplanted counterparts. Among the NHD13\u003csup\u003etg\u003c/sup\u003e mice, 75% (15 out of 20) developed hematological malignancies, compared to only 33% (3 out of 9) of those receiving WT cell transplants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Furthermore, while 25% (5 out of 20) of NHD13\u003csup\u003etg\u003c/sup\u003e mice developed acute leukemia, none of the transplanted mice did (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this work, we explored non-invasive BM conditioning as a regimen for providing aged recipients with HSCs from younger donors. Traditional conditioning commonly employs varied levels of TBI. However, TBI associates with systemic side effects that are poorly tolerated by aged recipients\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, emphasizing the need for alternative conditioning approaches.\u003c/p\u003e \u003cp\u003eWhile BMT is a well-established clinical procedure, many aspects concerning successful HSC reconstitution remain elusive. The recognition that HSCs inhabit niches crucial for regulating their function has led to a central hypothesis suggesting the necessity for these niches to be available for the seeding of transplanted HSCs\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Moreover, it has been proposed that a scarcity of such niches could limit HSC engraftment. Consistent with this notion, a temporary mobilization combined with transplantation at the mobilization peak has been recently proposed to enhance reconstitution in otherwise unconditioned hosts\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Interestingly, additional suggestions propose that repeated mobilization and transplantation may not only result in significant hematopoiesis derived from younger sources but also extend lifespan\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In line with the effectiveness of this conditioning method, we confirmed that combining it with CD45-SAP significantly enhances reconstitution in aged hosts. Still, the concept of niche recycling could be more complex, given recent findings that suggest it is possible to efficiently reconstitute unconditioned hosts with massive numbers of infused HSCs\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, the requirement to achieve high-level engraftment - necessitating HSCs from nearly 300 mice - poses severe experimental limitations\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Instead, we capitalized on recent breakthroughs in HSC \u003cem\u003eex vivo\u003c/em\u003e expansion\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This approach facilitated donor chimerism in young unconditioned hosts, which could be significantly boosted in combination with CD45-SAP-mediated conditioning.\u003c/p\u003e \u003cp\u003eAs part of our work, we tried to reassess the concept of niche recycling by comparing the contribution from one dose of HSCs to the same dose infused over five separate occasions. This resulted in high overall reconstitution levels that, however, were not further elevated in the setting of repetitive transplantation. This contrasts with previous studies suggesting increased graft contribution with constantly vacated niches\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. We attribute this deviation to the need for extremely high quantities of HSCs, not previously attainable, to saturate the available niches of an unconditioned recipient\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOne of our key initial findings was that aged mice presented significant barriers to effective engraftment. However, by addressing the limitations associated with donor HSCs number and by combining two different non-invasive conditioning techniques, we achieved substantial hematopoiesis from transplanted young HSCs in aged recipients.\u003c/p\u003e \u003cp\u003eIt has been hypothesized that aging associates with changes in BM niches, potentially impacting HSC function\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Additionally, aging may contribute to other systemic changes that could compromise HSC functionality\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. If such age-related alterations are significant, introducing young HSCs into aged recipients might not effectively restore youthful hematopoietic function. Nonetheless, aged individuals still possess HSC clones capable of contributing to multilineage hematopoiesis when transplanted into younger hosts\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This suggests that aging may limit the contribution from these clones, as studies have shown that eliminating senescent cells\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e or specifically targeting functionally aged HSCs\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e can enhance host HSC function. Our findings further support this notion in two significant ways. Firstly, we demonstrate that young HSCs transplanted into aged hosts exhibit behavior akin to their performance in a normal environment. Secondly, our research reveals that the CD45-SAP treatment utilized in our studies mitigates some of the age-related decline in host HSC function, particularly concerning adaptive immune components.\u003c/p\u003e \u003cp\u003eAging is widely recognized to result in dampened adaptive immunity and, in particular, neo-responses\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, making the restoration of robust lymphopoiesis in aged hosts a primary objective. We illustrate that young HSCs can facilitate highly efficient BM B lymphopoiesis in aged recipients, challenging the idea that the aged environment inherently exerts negative influences on this differentiation pathway\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Similarly, we observed a notable increase in thymic seeding and/or expansion, which correlated with dramatically elevated numbers of naive peripheral T cells. These findings suggest that introducing young HSCs can significantly enhance thymic function in older recipients despite the pronounced thymic involution with age.\u003c/p\u003e \u003cp\u003eLastly, we aimed to evaluate whether the CD45-SAP-mediated conditioning could impact hematological disease progression. By leveraging the NHD13\u003csup\u003etg\u003c/sup\u003e mouse model, we discovered that the introduction of young HSCs into asymptomatic NHD13\u003csup\u003etg\u003c/sup\u003e mice led to a noteworthy decrease in hematological disease occurrence and, strikingly, to a complete inhibition of acute leukemia development. As at least some host hematopoiesis remains after CD45-SAP conditioning and transplantation, we interpret these results to illustrate that young and genetically intact HSCs can function as a tumor suppressor, perhaps through a cell competition mechanism reminiscent of the Scribble mutation in \u003cem\u003eDrosophila\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, we here tried to shed light on the potential of non-invasive BM conditioning as an effective strategy to introduce young HSCs into aged hosts. Crucially, we have demonstrated that young HSCs can enhance adaptive immune cell generation, even within an aged milieu, and we highlight the potential of combined non-invasive conditioning with HSC transplantation to hinder the progression of age-related hematological disorders.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eAll experiments involved young (2\u0026ndash;4 months) and aged (16\u0026ndash;20 months) C57BL/6-CD45.2, C57BL/6-CD45.1 or F1 C57BL/6-CD45.1/CD45.2 mice obtained from Jackson Laboratory, Janvier Labs, Taconic Bioscience or generated in house. NHD13\u003csup\u003etg\u003c/sup\u003e mice\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e (RRID: IMSR_JAX:010505) were obtained from Jackson Laboratory. All analyses were performed on female mice, except in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, which involved both males and females. For transplantation of tdTomato\u0026thinsp;+\u0026thinsp;HSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), cells were isolated from \u003cem\u003eFgd5\u003c/em\u003e\u003csup\u003eCreERT2/+\u003c/sup\u003e; \u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003eLSL\u0026thinsp;\u0026minus;\u0026thinsp;tdTomato/+\u003c/sup\u003e mice, generated by crossing Fgd5-ZsGreen-2A-CreERT2 mice \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e (RRID: IMSR_JAX:027789) to Rosa26-LoxP-STOP-LoxP-tdTomato (RRID: IMSR_JAX:007905) mice. tdTomato-labeling in \u003cem\u003eFgd5\u003c/em\u003e-expressing HSCs was induced with tamoxifen (Sigma Aldrich, 10 mg/ml, resuspended in peanut oil) by intraperitoneal injections at 50 mg/kg\u003csup\u003e15\u003c/sup\u003e. Mice were housed in the Animal Facility at the Biomedical Center of Lund University in environment-enriched conditions with 12-hour light-dark cycles and water and food provided \u003cem\u003ead libitum\u003c/em\u003e. All experimental procedures were approved by a local ethical committee (permits M186-15 and 16468/2020).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAnimal procedures\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003eBleeding and isolation of bone marrow, thymus, and spleen cells\u003c/h2\u003e \u003cp\u003ePeripheral blood was collected from the tail vein into EDTA-coated tubes (Sarstedt) or 2% (v/v) FBS/PBS with heparin (Leo Pharma, 5000 IE/ml diluted 1:500). Complete blood count was determined using Sysmex KX-21N and XQ-320 analyzers. For BM cell isolation, mice were euthanized by cervical dislocation, and femurs, tibias and hip bones were collected from both hind legs. Bones were crushed in ice-cold 2% (v/v) FBS/PBS. For isolation of thymus and spleen cells, organs were dissociated using a plunger and 70 \u0026micro;m strainer in ice-cold 2% (v/v) FBS/PBS. Single-cell suspensions were centrifuged at 400g for 10 min and filtered through 70 \u0026micro;m cell strainers prior to sample processing.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunotoxin preparation and conditioning\u003c/h2\u003e \u003cp\u003eThe CD45-SAP immunotoxin was prepared as previously described\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Biotinylated anti-CD45.2 antibodies (clone 104, Sony Biotechnology) were mixed with streptavidin-saporin conjugate (Advanced Targeting Systems, Lot #132\u0026ndash;178 and #201\u0026thinsp;\u0026minus;\u0026thinsp;151) at a 1:1 molar ratio. In all experiments, CD45-SAP was administered at 3 mg/kg 8 days before analysis or transplantation, except in cohorts of young and aged animals in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, which received CD45-SAP at 60 \u0026micro;g/mouse 20 weeks after transplantation. For CD4-SAP and CD8-SAP treatment, biotinylated anti-CD4 (clone GK1.5, Sony Biotechnology) or anti-CD8a (clone 53\u0026thinsp;\u0026minus;\u0026thinsp;6.7, Sony Biotechnology) were combined with streptavidin-saporin conjugate and injected at 0.5 mg/kg 2\u0026ndash;3 days before transplantation. All immunotoxins were diluted in PBS and administered intravenously. B cell depletion was performed as previously reported\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e by intraperitoneal injection of rat anti-mouse CD19 and CD22 (clones 1D3 and CY34.1 respectively; BioXCell) and rat anti-mouse B220 (clone RA3-6B2; eBioscience) antibodies at 150 \u0026micro;g/mouse. After 48 hours, mice received intraperitoneal injections of anti-rat kappa light chain (clone MAR18.5; BioXCell) at 150 \u0026micro;g/mouse.\u003c/p\u003e \u003cp\u003eFor TBI, mice were sublethally (200 cGy) or lethally (950 cGy) irradiated one day or four hours before transplantation, respectively. All mice received antibiotic prophylaxis (Ciprofloxacin, HEXAL, 125 mg/l in drinking water) for two weeks following conditioning.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eG-CSF/AMD3100 mobilization\u003c/h2\u003e \u003cp\u003eMice received subcutaneous injections of recombinant human G-CSF (Zarzio) at 125 \u0026micro;g/kg every 12 hours for two days. Eighteen hours after the last G-CSF injection, mice received AMD3100 at 5 mg/kg in PBS (Sigma). One hour following the AMD3100 injection, mice were transplanted with \u003cem\u003eex vivo\u003c/em\u003e expanded HSCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTransplantation\u003c/h2\u003e \u003cp\u003eAll transplantations were performed through tail vein injection. See Supplementary Table\u0026nbsp;1 and corresponding figure legends for detailed experimental descriptions. For HSPC transplantation in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, tdTomato\u0026thinsp;+\u0026thinsp;HSCs were isolated by FACS from tamoxifen-induced \u003cem\u003eFgd5\u003c/em\u003e\u003csup\u003eCreERT2/+\u003c/sup\u003e; \u003cem\u003eRosa26\u003c/em\u003e\u003csup\u003eLSL\u0026thinsp;\u0026minus;\u0026thinsp;Tomato/+\u003c/sup\u003e mice and 500 cells were injected alongside 500,000 BM cells from C57BL/6-CD45.1 mice into each young and aged recipient. For transplantation of \u003cem\u003eex vivo\u003c/em\u003e expanded HSCs, cultured cells were collected, washed, and filtered/FACS-purified prior to transplantation. In all experiments, cells from cultures were pooled, and each recipient received an equal portion of the same input HSCs. For secondary transplantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), 3x10\u003csup\u003e6\u003c/sup\u003e BM cells from primary recipients were either non-competitively transplanted or mixed with whole BM-derived from C57BL/6-CD45.1/CD45.2 mice at a 1:1 ratio (3x10\u003csup\u003e6\u003c/sup\u003e cells of respective fraction per mouse) and transplanted into TBI-treated C57BL/6-CD45.1/CD45.2 secondary hosts. Cells from primary recipients were pooled before transplantation. For \u003cem\u003ein vivo\u003c/em\u003e proliferation tracking, cultured cells and CD4-enriched splenocytes isolated from C57BL/6-CD45.1 or C57BL/6-CD45.2 mice were labeled with CTV dye as previously described\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. EE100 HSCs were co-transplanted with 2x10\u003csup\u003e6\u003c/sup\u003e splenocytes into each C57BL/6-CD45.2 or C57BL/6-CD45.1/CD45.2 unconditioned or CD45-SAP treated recipient.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDisease classification in NHD13\u003csup\u003etg\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eClassification of hematopoietic diseases was based on the following criteria: Myelodysplastic syndrome was characterized by WBC\u0026thinsp;\u0026lt;\u0026thinsp;5 and anemia and/or thrombocytopenia. Lympho- and myeloproliferation were identified by 50\u0026thinsp;\u0026gt;\u0026thinsp;WBC\u0026thinsp;\u0026gt;\u0026thinsp;20; acute myeloid and lymphoid leukemia were defined by a WBC\u0026thinsp;\u0026gt;\u0026thinsp;50 and their respective myeloid- or lymphoid-lineage assignments as determined by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEx vivo HSC expansion\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEx vivo\u003c/em\u003e HSC expansion was performed as described before\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Briefly, 96-well flat-bottom plates were coated with 100 ng/ml fibronectin (Sigma) for at least 1 hour. HSCs (Lineage-cKIT\u0026thinsp;+\u0026thinsp;SCA-1\u0026thinsp;+\u0026thinsp;CD48-CD150\u0026thinsp;+\u0026thinsp;CD201\u003csup\u003ehigh\u003c/sup\u003e) were isolated from young mice and cultured for 21 days in F12 medium (Gibco) supplemented with 1% insulin\u0026ndash;transferrin\u0026ndash;selenium\u0026ndash;ethanolamine (Gibco), 10 mM HEPES (Gibco), 1% penicillin/streptomycin/glutamine (Gibco), 0.1% PVA (87\u0026ndash;90%-hydrolyzed, Sigma), 10 ng/ml mouse SCF (Peprotech) and 100 ng/ml mouse TPO (Peprotech) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Media changes were performed every 2 days starting from day 5 after sorting. Cells were split when reaching 80\u0026ndash;90% confluency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometric analysis and FACS sorting\u003c/h2\u003e \u003cp\u003eFluorescently labeled and biotinylated antibodies used in this study are listed in Supplementary Table\u0026nbsp;2. Stainings that included Brilliant Violet-conjugated reagents were supplemented with 10% Brilliant Stain Plus Buffer (BD). The cells were stained in 2% (v/v) FBS/PBS for 30 min at 4\u0026ordm;C in the dark, unless otherwise stated. For PB analysis, erythrocytes were sedimented with 1% Dextran T500 (Sigma-Aldrich) at 37\u0026deg;C for 30 min. The remaining erythrocytes were then lysed with ammonium chloride solution (STEMCELL Technologies) for 3 min at room temperature. Cells were stained in 2% (v/v) FBS/PBS with 2 mM EDTA (Vwr) and antibodies against TER119, CD19, CD11b, Gr1, NK1.1 and CD3.\u003c/p\u003e \u003cp\u003eHSPC analysis was performed on whole BM or cKIT-enriched cells. For cKIT enrichment, BM cells were stained with anti-cKIT-APC or anti-cKIT-APCeFluor780 antibody, followed by incubation with anti-APC MicroBeads (1:20, Miltenyi Biotec) for 30 min. Magnetic separation was performed using LS or MS columns and a manual separator, according to manufacturer\u0026rsquo;s instructions (Miltenyi Biotec).\u003c/p\u003e \u003cp\u003eIn all stainings, except for myelo-erythroid cell subsets, cells were pre-incubated with Fc-block (1:50, BioXCell) for 15 min prior to antibody staining. For HSPC analysis, cells were stained with antibodies against lineage markers (B220, Gr1, TER119, NK1.1, CD3), SCA-1, cKIT, CD150, CD48, CD201 and in some experiments also against CD135 and CD127. Myelo-erythroid progenitors were identified using lineage markers (B220, Gr1, TER119, NK1.1, CD3), SCA-1, cKIT, CD150, CD105, CD16/32 and CD41. B cell progenitors were identified using lineage markers (Gr1, TER119, NK1.1, CD3), CD19, B220, IgM, CD93, CD43 and cKIT.\u003c/p\u003e \u003cp\u003eFor thymocyte analysis, cells were stained with antibodies against CD19, B220, CD3, CD4 and CD8. For mature B and T cells, splenocytes were stained with antibodies against CD19, CD93, CD23, CD21/35, CD43, CD11b and CD11c for B cell lineage or Gr1, CD4, CD8, CD3, CD44 and CD62L for T cell lineage. In transplantation experiments, all staining panels included antibodies against CD45.1 and CD45.2 to monitor chimerism levels.\u003c/p\u003e \u003cp\u003eHSC isolation by FACS was performed on cKIT-enriched BM cells stained with the HSPC antibody cocktail described above. For isolation of cultured HSCs, cells were stained against lineage markers (Fcer1a, B220, Gr1, TER119, NK1.1, CD3) and CD201. Prior to analysis or cell sorting, cells were filtered and incubated with propidium iodide (1:1000, Invitrogen) to exclude dead cells.\u003c/p\u003e \u003cp\u003eAll flow cytometry and FACS experiments were performed at Lund Stem Cell Center FACS Core Facility (Lund University). Flow cytometric analyses were conducted on LSRFortessa or Fortessa-X20, and cell sorting was performed on FACSAria III or FACSSymphony S6 instruments (Becton Dickinson). Data was analyzed using FlowJo v.10.8.1 (Treestar).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRNA-sequencing and bioinformatic analysis\u003c/h2\u003e \u003cp\u003eYoung (3 months) and aged (16 months) G-CSF/AMD3100-treated mice were transplanted with \u003cem\u003eex vivo\u003c/em\u003e expanded HSCs. After 20 weeks, mice were treated with CD45-SAP at 20 \u0026micro;g/mouse to deplete host BM cells and hematopoietic reconstitution was monitored for additional 16 weeks. At the endpoint, MPP Ly cells (Lineage-cKIT\u0026thinsp;+\u0026thinsp;SCA-1\u0026thinsp;+\u0026thinsp;IL7Ra-CD135\u0026thinsp;+\u0026thinsp;CD150-) were isolated from the donor (young) and host (young or aged) BM fractions for bulk RNA-sequencing analysis. Cell isolation was performed in two batches, with each batch consisting of donor and host cells from both young and aged recipients. cDNA library preparation and sequencing were performed at Single Cell Discoveries (Utrecht, the Netherlands) using a modified CEL-Seq2 protocol. Briefly, 200 MPP Ly cells were isolated by FACS, and total RNA was extracted using TRIzol reagent (Invitrogen). mRNA was reverse transcribed, barcoded, pooled, and amplified with an \u003cem\u003ein vitro\u003c/em\u003e transcription. The resulting RNA was fragmented and used to generate cDNA sequencing libraries with Truseq Small RNA adapters (Illumina). Libraries were paired-end sequenced on a NovaSeq X Plus instrument using a 10B Reagent kit (100 cycles) and the following read configuration: R1\u0026thinsp;=\u0026thinsp;26 cycles, i7\u0026thinsp;=\u0026thinsp;6 cycles, R2\u0026thinsp;=\u0026thinsp;60 cycles (Illumina).\u003c/p\u003e \u003cp\u003eAfter sequencing, data were demultiplexed and mapped to the mouse GRCm38 reference genome using STARsolo v.2.7.10b software. Reads mapping to multiple locations were discarded. Subsequent analyses were performed in R v.4.2.1, including normalization and differential gene expression using DESeq2 v.1.36.0 package\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. PCA plot was generated using the \u003cem\u003evst()\u003c/em\u003e function of DESeq2. Differentially expressed genes (DEGs) were identified based on an adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.1. Venn diagrams were generated using Venny v.2.0.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinfogp.cnb.csic.es/tools/venny/index2.0.2.html\u003c/span\u003e\u003cspan address=\"https://bioinfogp.cnb.csic.es/tools/venny/index2.0.2.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and pathway analysis was conducted using Enrichr (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://maayanlab.cloud/Enrichr/\u003c/span\u003e\u003cspan address=\"https://maayanlab.cloud/Enrichr/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e For analysis of bulk RNA-seq data of young and aged HSCs\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, the count table was obtained from GEO and processed using the DESeq2 pipeline. DEGs with an adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.1 were compared with an HSC aging signature derived from Flohr Svendsen et al.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The odds ratio was computed using a hypergeometric distribution for a total of 21,955 genes, and the p-value was calculated using Fisher\u0026rsquo;s exact test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed and visualized using Microsoft Excel v.16.66.1 (Microsoft), Prism 9 v.9.3.1 (GraphPad) and R v.4.2.1 software. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, unless otherwise stated. Experiments were repeated as indicated in figure legends, with n denoting the number of independent biological repeats. Two-group comparison with normally distributed data employed a two-tailed \u003cem\u003et\u003c/em\u003e-test with Welch correction, while not normally distributed data were analyzed using the Mann-Whitney U test. Statistical analyses were unpaired, except in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei, which involved paired comparisons. Multiple group comparisons were assessed by one-way ANOVA with post hoc Tukey correction. Specific tests used are indicated in the corresponding figure legends.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank Single Cell Discoveries for their assistance with bulk RNA-sequencing and S. Soneji for suggestions on bioinformatic analysis. We acknowledge the expertise and assistance of the staff at the Lund University Animal Facility and Lund Stem Cell Center FACS Facility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The study was generously supported by the Tobias Foundation (Tobias Prize to D.B.), the Swedish Research Council\u0026nbsp;grant 2022-00932 (D.B.), the Swedish Cancer Society 211470Pj (D.B.), the Swedish Pediatric Leukemia Foundation PR2022-0091 (D.B.)\u0026nbsp;and by the Royal Physiographic Society of Lund foundation\u0026nbsp;42335 and\u0026nbsp;43250 (A.K-C.) and\u0026nbsp;42331\u0026nbsp;and\u0026nbsp;43043\u0026nbsp;(Q. Z.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e Conceptualization: A.K-C., D.B., Methodology: A.K.-C., Q.Z., Investigation: A.K.-C., Q.Z., S.K., Visualization: A.K.-C., D.B., Funding acquisition: A.K.-C., Q.Z., D.B., Project administration: A.K.-C., D.B., Supervision: D.B., Writing – original draft: A.K.-C., D.B., Writing – review \u0026amp; editing: A.K.-C., D.B.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e Authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e Bulk RNA-sequencing data has been deposited in the GEO database under the accession number GSE267079 and will be available as of the publication date. No original code was developed in this study. All data are available in the main text or the supplementary materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eNiederwieser, D.\u003cem\u003e, et al.\u003c/em\u003e One and a half million hematopoietic stem cell transplants: continuous and differential improvement in worldwide access with the use of non-identical family donors. \u003cem\u003eHaematologica\u003c/em\u003e \u003cstrong\u003e107\u003c/strong\u003e, 1045-1053 (2022).\u003c/li\u003e\n \u003cli\u003eBryder, D., Rossi, D.J. \u0026amp; Weissman, I.L. Hematopoietic stem cells: the paradigmatic tissue-specific stem cell. \u003cem\u003eAm J Pathol\u003c/em\u003e \u003cstrong\u003e169\u003c/strong\u003e, 338-346 (2006).\u003c/li\u003e\n \u003cli\u003eGyurkocza, B. \u0026amp; Sandmaier, B.M. 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Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. \u003cem\u003eGenome Biol\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 550 (2014).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4528815/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4528815/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHematopoietic stem cell (HSC) transplantation offers a cure for a variety of blood disorders, predominantly affecting the elderly; however, its application, especially in this demographic, is limited by treatment toxicity. In response, we developed a murine transplantation model based on low-intensity conditioning protocols using antibody-mediated HSC depletion. Initially, we identified significant age-related impediments to effective HSC engraftment. By optimizing HSC doses and non-toxic targeting methods, we could significantly enhance the long-term multilineage activity of the transplanted cells. We demonstrate that young HSCs, once transplanted, not only survive but thrive in aged hosts, dramatically improving hematopoietic output and ameliorating age-compromised lymphopoiesis. This culminated in a strategy that robustly mitigated disease progression in a genetic model of myelodysplastic syndrome. These results suggest that non-invasive HSC transplantation could fundamentally change the clinical management of age-associated hematological disorders, offering a novel, prophylactic tool to delay or even prevent their onset in elderly patients.\u003c/p\u003e","manuscriptTitle":"A Non-Invasive Stem Cell Therapy Boosts Lymphopoiesis and Averts Age-Related Blood Diseases in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-12 15:20:44","doi":"10.21203/rs.3.rs-4528815/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"86c0544a-a215-4c51-977c-8fd28abee703","owner":[],"postedDate":"July 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":33590276,"name":"Biological sciences/Stem cells/Ageing"},{"id":33590277,"name":"Biological sciences/Stem cells/Haematopoietic stem cells"},{"id":33590278,"name":"Biological sciences/Immunology/Bone marrow transplantation"}],"tags":[],"updatedAt":"2025-06-03T07:05:22+00:00","versionOfRecord":{"articleIdentity":"rs-4528815","link":"https://doi.org/10.1038/s41467-025-60464-3","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-06-02 04:00:00","publishedOnDateReadable":"June 2nd, 2025"},"versionCreatedAt":"2024-07-12 15:20:44","video":"","vorDoi":"10.1038/s41467-025-60464-3","vorDoiUrl":"https://doi.org/10.1038/s41467-025-60464-3","workflowStages":[]},"version":"v1","identity":"rs-4528815","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4528815","identity":"rs-4528815","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}