Engraftment Of Hematopoietic Stem Cells Research Articles

Busulfan for Allogeneic Hematopoietic Stem Cell Transplantation in Children with Severe Aplastic Anemia: A Retrospective Study

Introduction: This retrospective study aimed to compare a range of conditioning regimens in children with severe aplastic anemia (SAA) undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) at the Seventh Medical Center of PLA General Hospital between January 2008 and June 2017. Methods: Patients were categorized into the Bu (Bu + Flu + Cy + ATG-F regimen) and control (Flu + Cy + ATG-F) groups, with a median follow-up time after HSCT of 3.5 (range, 3.1–6.2) and 3.7 (3.2–5.9) years in the Bu and control groups, respectively. Results: No differences were observed between the two groups regarding the median time of peripheral blood neutrophil and platelet engraftment (p = 0.538 and p = 0.491); the 28-day engraftment rates of neutrophils were similar (p = 0.199), although higher for platelets with Bu (p = 0.044). Additionally, graft failure was 0% and 20.0% in the Bu and control groups, respectively (p = 0.004). In both groups, the incidence of grades III–IV (or grades II–IV) acute graft-versus-host disease (GVHD) and chronic GVHD was not significantly different (p > 0.05). Moreover, the 3-year overall survival and failure-free survival did not show significant differences (p = 0.670 and p = 0.908). Discussion: In children with SAA undergoing allo-HSCT, conditioning regimen with Bu + Flu + Cy + ATG-F is capable of enhancing the myeloablation effect, promoting donor hematopoietic stem cell engraftment, and reducing the graft failure rate. Furthermore, it does not increase the incidence of complications, including GVHD.

Efficient manufacturing and engraftment of CCR5 gene-edited HSPCs following busulfan conditioning in nonhuman primates

Hematopoietic stem cell gene therapy has been successfully used for a number of genetic diseases and is also being explored for HIV. However, toxicity of the conditioning regimens has been a major concern. Here we compared current conditioning approaches in a clinically relevant nonhuman primate model. We first customized various aspects of the therapeutic approach, including mobilization and cell collection protocols, conditioning regimens that support engraftment with minimal collateral damage, and cell manufacturing and infusing schema that reflect and build on current clinical approaches. Through a series of iterative invivo experiments in two macaque species, we show that busulfan conditioning significantly spares lymphocytes and maintains a superior immune response to mucosal challenge with simian/human immunodeficiency virus, compared to total body irradiation and melphalan regimens. Comparative mobilization experiments demonstrate higher cell yield relative to our historical standard, primed bone marrow and engraftment of CRISPR-edited hematopoietic stem and progenitor cells (HSPCs) after busulfan conditioning. Our findings establish a detailed workflow for preclinical HSPC gene therapy studies in the nonhuman primate model, which in turn will support testing of novel conditioning regimens and more advanced HSPC gene editing techniques tailored to any disease of interest.

Reduced proliferation of bone marrow MSC after allogeneic stem cell transplantation is associated with clinical outcome.

Engraftment and differentiation of donor hematopoietic stem cells is decisive for the clinical success of allogeneic stem cell transplantation (alloSCT) and depends on the recipient's bone marrow (BM) niche. A damaged niche contributes to poor graft function after alloSCT; however, the underlying mechanisms and the role of BM multipotent mesenchymal stromal cells (MSC) are ill-defined. Upon multivariate analysis in 732 individuals, we observed a reduced presence of proliferation-capable MSC in BM aspirates from patients (N= 196) who had undergone alloSCT. This was confirmed by paired analysis in 30 patients showing a higher frequency of samples with a lack of MSC presence post-alloSCT compared with pre-alloSCT. This reduced MSC presence was associated with reduced survival of patients after alloSCT and specifically with impaired graft function. Post-alloSCT MSC showed diminished invitro proliferation along with a transcriptional antiproliferative signature, upregulation of epithelial-mesenchymal transition and extracellular matrix pathways, and altered impact on cytokine release upon contact with hematopoietic cells. To avoid invitro culture bias, we isolated the CD146+/CD45-/HLA-DR- BM cell fraction, which comprised the entire MSC population. The post-alloSCT isolated native CD146+MSC showed a similar reduction in proliferation capacity and shared the same antiproliferative transcriptomic signature as for post-alloSCT colony-forming unit fibroblast-derived MSC. Taken together, our data show that alloSCT confers damage to the proliferative capacity of native MSC, which is associated with reduced patient survival after alloSCT and impaired engraftment of allogeneic hematopoiesis. These data represent the basis to elucidate mechanisms of BM niche reconstitution after alloSCT and its therapeutic manipulation.

Association of myeloid-derived suppressor cells with hematopoietic recovery after high-dose chemotherapy in multiple myeloma

Myeloid-derived suppressor cells (MDSCs) play an important role in the immune response regulation in many pathologies, primarily in malignant tumors, but their role in the hematopoietic stem cell engraftment and the hematopoietic recovery after high-dose chemotherapy and autologous stem cell transplantation remains practically unexplored. This study is aimed at studying the correlation between the number of MDSC subpopulations and blood parameters at the stage of hematopoietic recovery after high-dose chemotherapy and autologous hematopoietic stem cell transplantation in patients with multiple myeloma (MM). Circulating MDSCs were assessed at the stage of leukopenia recovery (absolute leukocyte count in peripheral blood (PB) > 1 x 109/L) by flow cytometry. The number of transplanted CD34+CD45+ hematopoietic stem cells was 4.38 x 106/kg (IQR (3.1—5.6) x 106/kg). The duration of recovery from leukopenia varied from 8 to 18 days (Me 12 days). The number of MDSCs at the engraftment was not associated with the number of CD34+ cells/kg in the graft. The relative number of monocytic MDSCs (M-MDSCs, CD14+HLA-DRlow/-) directly correlated with the number of monocytes at the stage of recovery from leukopenia (R = 0.417, p = 0.002). Granulocytic MDSCs (PMN-MDSCs, Lin-HLA-DR-CD33+CD66b+) were characterized by an inverse correlation with the number of monocytes (R = -0.493, p = 0.0003) while the association with the absolute number of neutrophils was weak (R = 0.273, p = 0.048). The number of lymphocytes at the stage of recovery from leukopenia had an inverse correlation with PMN-MDSCs (R = -0.347, p = 0.014) and did not correlate with M-MDSCs. When analyzing the duration of leukopenia, an inverse correlation with this indicator was revealed for the percentage and absolute number of M-MDSCs (R = -0.347, p = 0.018 and R = -0.469, p = 0.0008, respectively). Multiple regression analysis showed dependence of the lymphopenia duration on the proportion of circulating M-MDSCs (p = 0.014) and the number of transplanted CD34+ cells/kg (p = 0.032). According to the data of multivariate analysis of variance, the number of transplanted CD34+ cells/kg and the number of M-MDSCs were significant factors for the duration of leukopenia. At the same time, such clinical parameters as the depth of response and minimal residual disease status before high-dose chemotherapy and hematopoietic stem cell transplantation, as well as the MM stage, did not affect the duration of hematopoietic recovery. Thus, the obtained results indicate the association of a higher number of M-MDSCs with a shorter duration of leukopenia after high-dose chemotherapy with autologous stem cell transplantation and indicate a positive role of M-MDSCs in hematopoietic recovery in the early post-transplant period in patients with MM.

Mitochondrial Transfer to Host Cells from Ex Vivo Expanded Donor Hematopoietic Stem Cells.

Mitochondrial dysfunction is observed in various conditions, from metabolic syndromes to mitochondrial diseases. Moreover, mitochondrial DNA (mtDNA) transfer is an emerging mechanism that enables the restoration of mitochondrial function in damaged cells. Hence, developing a technology that facilitates the transfer of mtDNA can be a promising strategy for the treatment of these conditions. Here, we utilized an ex vivo culture of mouse hematopoietic stem cells (HSCs) and succeeded in expanding the HSCs efficiently. Upon transplantation, sufficient donor HSC engraftment was attained in-host. To assess the mitochondrial transfer via donor HSCs, we used mitochondrial-nuclear exchange (MNX) mice with nuclei from C57BL/6J and mitochondria from the C3H/HeN strain. Cells from MNX mice have C57BL/6J immunophenotype and C3H/HeN mtDNA, which is known to confer a higher stress resistance to mitochondria. Ex vivo expanded MNX HSCs were transplanted into irradiated C57BL/6J mice and the analyses were performed at six weeks post transplantation. We observed high engraftment of the donor cells in the bone marrow. We also found that HSCs from the MNX mice could transfer mtDNA to the host cells. This work highlights the utility of ex vivo expanded HSC to achieve the mitochondrial transfer from donor to host in the transplant setting.

Hematopoietic Jagged1 is a fetal liver niche factor required for functional maturation and engraftment of fetal hematopoietic stem cells

Notch signaling is essential for the emergence of definitive hematopoietic stem cells (HSCs) in the embryo and their development in the fetal liver niche. However, how Notch signaling is activated and which fetal liver cell type provides the ligand for receptor activation in HSCs is unknown. Here we provide evidence that endothelial Jagged1 (Jag1) has a critical early role in fetal liver vascular development but is not required for hematopoietic function during fetal HSC expansion. We demonstrate that Jag1 is expressed in many hematopoietic cells in the fetal liver, including HSCs, and that its expression is lost in adult bone marrow HSCs. Deletion of hematopoietic Jag1 does not affect fetal liver development; however, Jag1-deficient fetal liver HSCs exhibit a significant transplantation defect. Bulk and single-cell transcriptomic analysis of HSCs during peak expansion in the fetal liver indicates that loss of hematopoietic Jag1 leads to the downregulation of critical hematopoietic factors such as GATA2, Mllt3, and HoxA7, but does not perturb Notch receptor expression. Exvivo activation of Notch signaling in Jag1-deficient fetal HSCs partially rescues the functional defect in a transplant setting. These findings indicate a new fetal-specific niche that is based on juxtracrine hematopoietic Notch signaling and reveal Jag1 as a fetal-specific niche factor essential for HSC function.

Humanized mouse models with endogenously developed human natural killer cells for in vivo immunogenicity testing of HLA class I-edited iPSC-derived cells

Human induced pluripotent stem cells (hiPSCs) genetically depleted of human leucocyte antigen (HLA) class I expression can bypass T cell alloimmunity and thus serve as a one-for-all source for cell therapies. However, these same therapies may elicit rejection by natural killer (NK) cells, since HLA class I molecules serve as inhibitory ligands of NK cells. Here, we focused on testing the capacity of endogenously developed human NK cells in humanized mice (hu-mice) using MTSRG and NSG-SGM3 strains to assay the tolerance of HLA-edited iPSC-derived cells. High NK cell reconstitution was achieved with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs) followed by the administration of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15Rα). Such “hu-NK mice” rejected HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes and T cells, but not HLA-A/B-knockout, HLA-C expressing HPCs. To our knowledge, this study is the first to recapitulate the potent endogenous NK cell response to non-tumor HLA class I-downregulated cells in vivo. Our hu-NK mouse models are suitable for the non-clinical evaluation of HLA-edited cells and will contribute to the development of universal off-the-shelf regenerative medicine.

IL-6–GP130 signaling protects human hepatocytes against lipid droplet accumulation in humanized liver models

Liver steatosis is an increasing health issue with few therapeutic options, partly because of a paucity of experimental models. In humanized liver rodent models, abnormal lipid accumulation in transplanted human hepatocytes occurs spontaneously. Here, we demonstrate that this abnormality is associated with compromised interleukin-6 (IL-6)-glycoprotein 130 (GP130) signaling in human hepatocytes because of incompatibility between host rodent IL-6 and human IL-6 receptor (IL-6R) on donor hepatocytes. Restoration of hepatic IL-6-GP130 signaling, through ectopic expression of rodent IL-6R, constitutive activation of GP130 in human hepatocytes, or humanization of an Il6 allele in recipient mice, substantially reduced hepatosteatosis. Notably, providing human Kupffer cells via hematopoietic stem cell engraftment in humanized liver mice also corrected the abnormality. Our observations suggest an important role of IL-6-GP130 pathway in regulating lipid accumulation in hepatocytes and not only provide a method to improve humanized liver models but also suggest therapeutic potential for manipulating GP130 signaling in human liver steatosis.

Selective Bcl-2 inhibition promotes hematopoietic chimerism and allograft tolerance without myelosuppression in nonhuman primates.

Hematopoietic stem cell transplantation (HSCT) has many potential applications beyond current standard indications, including treatment of autoimmune disease, gene therapy, and transplant tolerance induction. However, severe myelosuppression and other toxicities after myeloablative conditioning regimens have hampered wider clinical use. To achieve donor hematopoietic stem cell (HSC) engraftment, it appears essential to establish niches for the donor HSCs by depleting the host HSCs. To date, this has been achievable only by nonselective treatments such as irradiation or chemotherapeutic drugs. An approach that is capable of more selectively depleting host HSCs is needed to widen the clinical application of HSCT. Here, we show in a clinically relevant nonhuman primate model that selective inhibition of B cell lymphoma 2 (Bcl-2) promoted hematopoietic chimerism and renal allograft tolerance after partial deletion of HSCs and effective peripheral lymphocyte deletion while preserving myeloid cells and regulatory T cells. Although Bcl-2 inhibition alone was insufficient to induce hematopoietic chimerism, the addition of a Bcl-2 inhibitor resulted in promotion of hematopoietic chimerism and renal allograft tolerance despite using only half of the dose of total body irradiation previously required. Selective inhibition of Bcl-2 is therefore a promising approach to induce hematopoietic chimerism without myelosuppression and has the potential to render HSCT more feasible for a variety of clinical indications.

Abstract 6354: A new tumor-bearing humanized mouse model to evaluate the efficacy of bispecific T cell engager and monoclonal checkpoint antibodies

Abstract T cell-based immunotherapies such as bispecific T- cell engagers (BiTE) and checkpoint inhibitors (CIs) have been developed rapidly for cancer treatment, but each has their own limitations in the clinic. Combination therapies are an emerging approach in ongoing clinical trials to enhance the drug efficacy of monotherapy. Here we described a new preclinical animal model using immunodeficient NSG™-SGM3-IL15 mice engrafted with human CD34+ hematopoietic stem cells (HSC) to evaluate the combination treatment of EGFRXCD3 BiTE and two CIs (anti-PD-1 pembrolizumab and anti-PD-L1 avelumab). NSG™-SGM3-IL15 mice, which were created by crossing NSG™-SGM3 with NSG™-IL15, develop a more complete humane immune system after HSC engraftment. Humanized NSG™-IL15 mice have a higher peripheral NK cell frequency, and NK cells are functional with ADCC activity when compared to NSG™ (Aryee et al., 2022 FASEB). Here we show that humanized NSG™-SGM3-IL15 mice show improved human CD33+ myeloid progenitor cells and near physiological levels of NK cells and Tregs in peripheral blood when compared to humanized NSG™-SGM3. Triple negative breast cancer MDA-MB-453 cell line is PD-L1 positive, expresses low levels of EGFR and has a low baseline tumor immune cell infiltration in xenograft using humanized NSG™ mice. Previously we have shown BiTE efficacy on this tumor model. Here tumor-bearing humanized NSG™-SGM3-IL15 mice from two donors were used to evaluate the efficacy and immune responses from the BiTE treatment in combination with CIs. For donor 0348, monotherapy BiTE and avelumab didn’t result in significant tumor growth reduction when compared to PBS control whereas the combination treatment significantly reduced tumor growth. BiTE treatment alone resulted in a significant increase in CD3+ T cell infiltration into tumor and higher frequency of CD69+ CD4 T and CD8 T cells. PD-1 expression on the tumor infiltrating lymphocytes and PD-L1 expression on tumor cells increased significantly. BiTE and avelumab combination treatment further increased human CD45+ and CD3+ T cell infiltrations with activated T cell phenotypes in tumor. For the second donor 0313, BiTE alone or in combination with the CIs didn’t show anti-tumor growth activity. Flow cytometry analysis showed that the immune response to BiTE treatment was less pronounced in this donor, especially there were little change in lymphocyte infiltration and CD4+ T cell population in tumor. In this study, we demonstrated donor-dependent response to combination treatment of BiTE and avelumab and found that the responding donor showed an increase in T cell infiltration and activation in tumor after the treatment. The data suggested beneficial effects from inhibition of PD-1/PD-L1 axis when used in combination with an immunotherapy targeting tumor antigen. The potential involvement of NK cells in this combination therapy remained to be examined. Citation Format: Li-Chin Yao, Danying Cai, Destanie Rose, Guoxiang Yang, Mingshan Cheng, Michael A. Brehm, Dale L. Greiner, Leonard D. Shultz, James G. Keck. A new tumor-bearing humanized mouse model to evaluate the efficacy of bispecific T cell engager and monoclonal checkpoint antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6354.

Abstract 5204: Preclinical models for translational immuno-oncology research: Rare patient-derived xenografts on humanized mice

Abstract Background The preclinical evaluation of novel immune checkpoint modulators is dependent on models with functional human immune cells. In previous experiments, we have demonstrated, that we can use either peripheral blood mononuclear cells (PBMC), subset of immune cells like NK cells or hematopoietic stem cells (HSC) to establish a humanized immune system on highly immunodeficient 1st and 2nd generation NOG mice with functional T-, B-, and NK cells, as well as monocytes and dendritic cells. Furthermore, we determined PD-L1 expression as a predictive marker and target for immunotherapy on different patient-derived xenografts (PDX). By co-transplantation of rare PDX, like leukemia or Nuclear-protein-in-testis (NUT) carcinoma, we successfully generated a fully human tumor-immune-cell model in mice. Finally, we evaluated the functionality of the model by the treatment with checkpoint inhibitors like Ipilimumab (Ipi), Nivolumab (Nivo) or Pembrolizumab (Pembro). In parallel, we investigated the functionality of the human immune cells and evaluated concepts for combination therapies i.e. with chemotherapy or radiation. Methods HSC-humanized mice were generated by i.v. HSC transplantation and engraftment of immune cells was monitored by FACS analysis. For PBMC-humanized mice, immune cells were implanted i.v.. PDX models from different entities (leukemia or NUT carcinoma) were transplanted on HSC-humanized (HIS) mice and treated with Ipi, Nivo and Pembro alone or in combination with radiation. Blood and tumor samples were analysed by FACS for immune cell infiltration and activation. Results The transplanted HSC showed engraftment in mice with proliferation and differentiation. 14 weeks after HSC inoculation up to 20% of the human immune cells in the blood were T-cells, characterized by a high PD-1 expression. We have transplanted PDX from different tumor entities on HSC-humanized mice. Most of the investigated PDX (>70%) successfully engrafted on humanized mice and showed no significant difference in tumor growth compared to growth on non-humanized mice. However, for some PDX we observed a delayed tumor growth or a complete rejection. Engraftment delay seems to correlate with the PD-L1 expression of PDX (the higher PD-L1, then the higher growth delay). Furthermore, our results demonstrate the functionality of the engrafted human immune cells against some PDX. Treatment with Ipi, Nivo or Pembro led to a minor tumor growth delay. Response to checkpoint inhibitors showed a correlation to innate immune response and PD-L1 expression of PDX and could further be increased by combination with radiotherapy. Conclusions Our humanized immune-PDX models enable appropriate preclinical translational research on tumor immune biology and the evaluation of new therapies and combinations, as well as the identification and validation of biomarkers for immune therapy, especially in rare PDX models. Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Wolfgang Walther, Jens Hoffmann. Preclinical models for translational immuno-oncology research: Rare patient-derived xenografts on humanized mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5204.

PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime

PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime

The fatty acid elongase Elovl6 is crucial for hematopoietic stem cell engraftment and leukemia propagation

The fatty acid elongase Elovl6 is crucial for hematopoietic stem cell engraftment and leukemia propagation

Bioluminescence Imaging and ICP-MS Associated with SPION as a Tool for Hematopoietic Stem and Progenitor Cells Homing and Engraftment Evaluation.

Bone marrow transplantation is a treatment for a variety of hematological and non-hematological diseases. For the transplant success, it is mandatory to have a thriving engraftment of transplanted cells, which directly depends on their homing. The present study proposes an alternative method to evaluate the homing and engraftment of hematopoietic stem cells using bioluminescence imaging and inductively coupled plasma mass spectrometry (ICP-MS) associated with superparamagnetic iron oxide nanoparticles. We have identified an enriched population of hematopoietic stem cells in the bone marrow following the administration of Fluorouracil (5-FU). Lately, the cell labeling with nanoparticles displayed the greatest internalization status when treated with 30 µg Fe/mL. The quantification by ICP-MS evaluate the stem cells homing by identifying 3.95 ± 0.37 µg Fe/mL in the control and 6.61 ± 0.84 µg Fe/mL in the bone marrow of transplanted animals. In addition, 2.14 ± 0.66 mg Fe/g in the spleen of the control group and 2.17 ± 0.59 mg Fe/g in the spleen of the experimental group was also measured. Moreover, the bioluminescence imaging provided the follow up on the hematopoietic stem cells behavior by monitoring their distribution by the bioluminescence signal. Lastly, the blood count enabled the monitoring of animal hematopoietic reconstitution and ensured the transplantation effectiveness.

An improved medium formulation for efficient ex vivo gene editing, expansion and engraftment of hematopoietic stem and progenitor cells

Gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR-Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat hematological disorders, and clinical trials using these tools are now under way. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and their invitro expansion for clinical application. In this study, we assessed the effect of different culture medium compositions on the ability of HSPCs to proliferate and undergo homology-directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during exvivo culture. We demonstrated that by supplementing the culture medium with stem cell agonists and by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both invitro and invivo, with a beneficial balance between preservation of stemness and cell expansion. Overall, the implementation of this optimized exvivo HSPC culture protocol can improve the efficacy, feasibility, and applicability of gene editing as a key step to unlocking the full therapeutic potential of this powerful technology.

Severe hematopoietic stem cell inflammation compromises chronic granulomatous disease gene therapy

X-linked chronic granulomatous disease (CGD) is associated with defective phagocytosis, life-threatening infections, and inflammatory complications. We performed a clinical trial of lentivirus-based gene therapy in four patients (NCT02757911). Two patients show stable engraftment and clinical benefits, whereas the other two have progressively lost gene-corrected cells. Single-cell transcriptomic analysis reveals a significantly lower frequency of hematopoietic stem cells (HSCs) in CGD patients, especially in the two patients with defective engraftment. These two present a profound change in HSC status, a high interferon score, and elevated myeloid progenitor frequency. We use elastic-net logistic regression to identify a set of 51 interferon genes and transcription factors that predict the failure of HSC engraftment. In one patient, an aberrant HSC state with elevated CEBPβ expression drives HSC exhaustion, as demonstrated by low repopulation in a xenotransplantation model. Targeted treatments to protect HSCs, coupled to targeted gene expression screening, might improve clinical outcomes in CGD.

Stemness‐related genes revealed by single‐cell profiling of naïve and stimulated human CD34+ cells from CB and mPB

Hematopoietic stem cells (HSCs) from different sources show varied repopulating capacity, and HSCs lose their stemness after long-time ex vivo culture. A deep understanding of these phenomena may provide helpful insights for HSCs. Here, we applied single-cell RNA-seq (scRNA-seq) to analyse the naïve and stimulated human CD34+ cells from cord blood (CB) and mobilised peripheral blood (mPB). We collected over 16 000 high-quality single-cell data to construct a comprehensive inference map and characterised the HSCs under a quiescent state on the hierarchy top. Then, we compared HSCs in CB with those in mPB and HSCs of naïve samples to those of cultured samples, and identified stemness-related genes (SRGs) associated with cell source (CS-SRGs) and culture time (CT-SRGs), respectively. Interestingly, CS-SRGs and CT-SRGs share genes enriched in the signalling pathways such as mRNA catabolic process, translational initiation, ribonucleoprotein complex biogenesis and cotranslational protein targeting to membrane, suggesting dynamic protein translation and processing may be a common requirement for stemness maintenance. Meanwhile, CT-SRGs are enriched in pathways involved in glucocorticoid and corticosteroid response that affect HSCs homing and engraftment. In contrast, CS-SRGs specifically contain genes related to purine and ATP metabolic process, which is crucial for HSC homeostasis in the stress settings. Particularly, when CT-SRGs are used as reference genes for the construction of the development trajectory of CD34+ cells, lymphoid and myeloid lineages are clearly separated after HSCs/MPPs. Finally, we presented an application through a small-scale drug screening using Connectivity Map (CMap) against CT-SRGs. A small molecule, cucurbitacin I, was found to efficiently expand HSCs ex vivo while maintaining its stemness. Our findings provide new perspectives for understanding HSCs, and the strategy to identify candidate molecules through SRGs may be applicable to study other stem cells.

Transient regulation of RNA methylation in human hematopoietic stem cells promotes their homing and engraftment

Enhancing the efficiency of hematopoietic stem cell (HSC) homing and engraftment is critical for cord blood (CB) hematopoietic cell transplantation (HCT). Recent studies indicate that N6-methyladenosine (m6A) modulates the expression of mRNAs that are critical for stem cell function by influencing their stability. Here, we demonstrate that inhibition of RNA decay by regulation of RNA methylation, enhances the expression of the homing receptor chemokine C-X-C receptor-4 (CXCR4) in HSCs. We show that YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), a m6A reader and FTO α-ketoglutarate dependent dioxygenase (FTO), a m6A eraser play an opposite role in this process. Through screening, we identified several FDA-approved compounds that regulate the expression of YTHDF2 and FTO in CB CD34+ cells. We show that transient downregulation of YTHDF2 or activation of FTO by using these compounds inhibits CXCR4 decay in CB HSCs and promotes their homing and engraftment. Our results demonstrate a novel regulation strategy to enhance the function of CB HSCs and provide a translational approach to enhance the clinical efficacy of HCT.

Human growth hormone supplement promotes human lymphohematopoietic cell reconstitution in immunodeficient mice.

Aim: To investigate the potential of human growth hormone (hGH) to improve human hematopoietic reconstitution in humanized mice. Materials & methods: Immunodeficient mice were conditioned by total body irradiation and transplanted with human CD34+ fetal liver cells. Peripheral blood, spleen and bone marrow were harvested, and levels of human lymphohematopoietic cells were determined by flow cytometry. Results: Supplementation with hGH elevated human lymphohematopoietic chimerism by more than twofold. Treatment with hGH resulted in significantly increased reconstitution of human B cells and myeloid cells in lymphoid organs, enhanced human erythropoiesis in the bone morrow, and improved engraftment of human hematopoietic stem cells. Conclusion: hGH supplementation promotes human lymphohematopoietic reconstitution in humanized mice.

HSCs Engraft in a Stochastic Pattern and Form Clonal Pools after Transplantation

Hematopoietic stem cells (HSCs) are considered the only cell type capable of self-renewing and generating all blood lineages. HSCs are further assumed to be rare, barely proliferating, and not contributing to short-term hematopoietic recovery after transplantation. Although widely accepted, individual definitions have been challenged. Tracking of murine HSCs showed continuous contribution during steady-state hematopoiesis. Similarly, single HSCs can reconstitute the entire murine host illustrating extensive proliferative capabilities without immediate exhaustion while maintaining long-term persistence. Studying these findings in humans is challenging. Knowledge about the engraftment kinetics and contribution of human HSCs during recovery is limited to clinical trials of gene therapy patients enrolled in clonal tracking studies using retroviral integration site analysis (ISA). However, the tracking of human HSCs remains under-investigated due to the limited number of specimens possible to obtain, especially during the early phases of reconstitution where blood cell counts are low. In addition, clone tracking is impacted by technical limitations. While ISA is reliable to monitor the potential outgrowth of dominant clones, low sensitivity and high error rates require significant data exclusion and sophisticated statistical tests to ensure data reliability (Adair et al. 2020, Molecular Therapy MCD). Lack of sensitivity and loss of low abundance clones can be overcome by increasing the frequency of sampling (high density) as well as repeated sampling, aspects hard to achieve in patients. To investigate the clonal dynamics in the early phases of hematopoietic reconstitution and determine the onset of HSC contribution, we performed high-density sampling for ISA in nonhuman primates (NHPs). In the first month of hematopoietic recovery, weekly blood samples were taken to enhance data density. We applied a new clonal tracking approach to reliably detect the contribution of multipotent long-term persisting HSCs during neutrophil recovery even in samples with low cellularity during early recovery. Animals were followed for 4.5 years to collect HSC clone signatures, and multipotent stem cell clones were retrospectively identified in early samples. Finally, observed changes in clonal diversity in vivo were used to inform a simulation of hematopoietic reconstitution to determine the temporal involvement of HSCs. Contribution of multipotent long-term persisting HSCs clones was detected in the very first blood samples taken 2 to 3 weeks post-transplant during neutrophil recovery. HSCs became the dominant source for mature blood cells in the peripheral blood as early as 50 days post-transplant. Based on these finding, simulations predicted that hematopoietic recovery is primarily HSC driven, differentiating HSC clones are the main source of blood production during recovery, and that long-term persisting HSC clones expand forming clonal pools. The ability of HSCs to symmetrically expand was confirmed ex vivo in colony-forming cells assays as well as in vivo using ISA and tracking of expressed barcodes. As predicted by the simulation, we observed a rapid decline of clonal diversity and individual HSCs symmetrically expand forming clonal pools. Here, we show evidence that long-term persisting multipotent HSCs actively contribute during early hematopoietic reconstitution after transplantation. Enhanced sampling showed that multipotent HSCs produce neutrophils during recovery, are the main source of mature blood cells as early as 50 days post-transplant, and symmetrically divide to form clonal pools long-term. Most importantly, observed changes in the clonal diversity during early recovery suggest a stochastic HSC engraftment and differentiation pattern rather than a bi-phasic reconstitution initially driven by short-term progenitors. Early blood production is driven by these HSC clones that are not predetermined but stochastically contribute to a limited number of lineages before they disappear - a population of cells previously incorrectly identified as lineage-restricted short-term engrafting progenitors. Consequently, we here propose a revised model of hematopoietic reconstitution and provide novel insights into HSC biology with direct implications for the design of HSC transplantation as well as gene therapy studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal