Potential Of Hematopoietic Stem Cells Research Articles

Lipoprotein metabolism mediates hematopoietic stem cell responses under acute anemic conditions

Hematopoietic stem cells (HSCs) react to various stress conditions. However, it is unclear whether and how HSCs respond to severe anemia. Here, we demonstrate that upon induction of acute anemia, HSCs rapidly proliferate and enhance their erythroid differentiation potential. In severe anemia, lipoprotein profiles largely change and the concentration of ApoE increases. In HSCs, transcription levels of lipid metabolism-related genes, such as very low-density lipoprotein receptor (Vldlr), are upregulated. Stimulation of HSCs with ApoE enhances their erythroid potential, whereas HSCs in Apoe knockout mice do not respond to anemia induction. VldlrhighHSCs show higher erythroid potential, which is enhanced after acute anemia induction. VldlrhighHSCs are epigenetically distinct because of their low chromatin accessibility, and more chromatin regions are closed upon acute anemia induction. Chromatin regions closed upon acute anemia induction are mainly binding sites of Erg. Inhibition of Erg enhanced the erythroid differentiation potential of HSCs. Our findings indicate that lipoprotein metabolism plays an important role in HSC regulation under severe anemic conditions.

Reprogramming of RNA m6A Modification Is Required for Acute Myeloid Leukemia Development.

Hematopoietic homeostasis is maintained by hematopoietic stem cells (HSCs), and it is tightly controlled at multiple levels to sustain the self-renewal capacity and differentiation potential of HSCs. Dysregulation of self-renewal and differentiation of HSCs leads to the development of hematologic diseases, including acute myeloid leukemia (AML). Thus, understanding the underlying mechanisms of HSC maintenance and the development of hematologic malignancies is one of the fundamental scientific endeavors in stem cell biology. N 6-methyladenosine (m6A) is a common modification in mammalian messenger RNAs (mRNAs) and plays important roles in various biological processes. In this study, we performed a comparative analysis of the dynamics of the RNA m6A methylome of hematopoietic stem and progenitor cells (HSPCs) and leukemia-initiating cells (LICs) in AML. We found that RNA m6A modification regulates the transformation of long-term HSCs into short-term HSCs and determines the lineage commitment of HSCs. Interestingly, m6A modification leads to reprogramming that promotes cellular transformation during AML development, and LIC-specific m6A targets are recognized by different m6A readers. Moreover, the very long chain fatty acid transporter ATP-binding cassette subfamily D member 2 (ABCD2) is a key factor that promotes AML development, and deletion of ABCD2 damages clonogenic ability, inhibits proliferation, and promotes apoptosis of human leukemia cells. This study provides a comprehensive understanding of the role of m6A in regulating cell state transition in normal hematopoiesis and leukemogenesis, and identifies ABCD2 as a key factor in AML development.

An epigenetically distinct HSC subset supports thymic reconstitution.

Hematopoietic stem cells (HSCs) with multilineage potential are critical for effective T cell reconstitution and restoration of the adaptive immune system after allogeneic Hematopoietic Cell Transplantation (allo-HCT). The Kit lo subset of HSCs is enriched for multipotential precursors, 1, 2 but their T-cell lineage potential has not been well-characterized. We therefore studied the thymic reconstituting and T-cell potential of Kit lo HSCs. Using a preclinical allo-HCT model, we demonstrate that Kit lo HSCs support better thymic recovery, and T-cell reconstitution resulting in improved T cell responses to infection post-HCT. Furthermore, Kit lo HSCs with augmented BM lymphopoiesis mitigate age-associated thymic alterations, thus enhancing T-cell recovery in middle-aged hosts. We find the frequency of the Kit lo subset declines with age, providing one explanation for the reduced frequency of T-competent HSCs and reduced T-lymphopoietic potential in BM precursors of aged mice. 3, 4, 5 Chromatin profiling revealed that Kit lo HSCs exhibit higher activity of lymphoid-specifying transcription factors (TFs), including Zbtb1 . Deletion of Zbtb1 in Kit lo HSCs diminished their T-cell potential, while reinstating Zbtb1 in megakaryocytic-biased Kit hi HSCs rescued T-cell potential, in vitro and in vivo . Finally, we discover an analogous Kit lo HSC subset with enhanced lymphoid potential in human bone marrow. Our results demonstrate that Kit lo HSCs with enhanced lymphoid potential have a distinct underlying epigenetic program.

Exploring hematopoietic stem cell population in human milk and its benefits for infants: A scoping review

Objective: To comprehensively explore hematopoietic stem cells (HSCs) in human milk, understanding their molecular markers, isolation methods, benefits for infants, and potential medical applications. Methods: We conducted a scoping literature review following the PRISMA-ScR guidelines. This review included studies investigating HSCs in human milk, utilizing molecular markers such as CD34+, CD113+, and CD117+ for characterization. Both in vitro and in vivo studies exploring the morphology, function, and clinical implications of these cells were considered. The diverse range of papers reviewed were indexed in PubMed, Science Direct, Scopus, Sage Journals, and Google Scholar, published between 2010 and 2023. Results: This scoping review explored 577 articles and selected 13 studies based on our inclusion criteria, focusing on HSCs in human milk. Most studies dilute samples prior to HSC isolation, followed by detection using markers such as CD34+, CD113+, and CD117+, with flow cytometry serving as the primary analysis tool, focusing on their isolation and detection methods. While no definitive benefits have been conclusively established, there is a strong belief in the potential of HSCs to positively impact infant immunity, growth, and tissue repair. Conclusions: This review presents significant evidence supporting the presence of HSCs in human milk, identified by markers such as CD34+, CD113+, and CD117+. These cells show considerable potential in enhancing infant health, including immunity, tissue repair, cognitive development, and gastrointestinal health. Despite methodological variations in isolation and detection techniques, the collective findings underscore the potential clinical relevance of HSCs in human milk. Moreover, this review highlights the non-invasive accessibility of human milk as a source of HSCs and emphasizes the need for further research to unlock their therapeutic potential.

Fetal liver macrophages contribute to the hematopoietic stem cell niche by controlling granulopoiesis.

During embryogenesis, the fetal liver becomes the main hematopoietic organ, where stem and progenitor cells as well as immature and mature immune cells form an intricate cellular network. Hematopoietic stem cells (HSCs) reside in a specialized niche, which is essential for their proliferation and differentiation. However, the cellular and molecular determinants contributing to this fetal HSC niche remain largely unknown. Macrophages are the first differentiated hematopoietic cells found in the developing liver, where they are important for fetal erythropoiesis by promoting erythrocyte maturation and phagocytosing expelled nuclei. Yet, whether macrophages play a role in fetal hematopoiesis beyond serving as a niche for maturing erythroblasts remains elusive. Here, we investigate the heterogeneity of macrophage populations in the murine fetal liver to define their specific roles during hematopoiesis. Using a single-cell omics approach combined with spatial proteomics and genetic fate-mapping models, we found that fetal liver macrophages cluster into distinct yolk sac-derived subpopulations and that long-term HSCs are interacting preferentially with one of the macrophage subpopulations. Fetal livers lacking macrophages show a delay in erythropoiesis and have an increased number of granulocytes, which can be attributed to transcriptional reprogramming and altered differentiation potential of long-term HSCs. Together, our data provide a detailed map of fetal liver macrophage subpopulations and implicate macrophages as part of the fetal HSC niche.

Long-term follow-up of cancer and catastrophic diseases in hematopoietic stem cell donors: a comprehensive matched cohort study.

Hematopoietic stem cell (HSC) transplantation, using either bone marrow (BM) or peripheral blood stem cells (PBSC), is a well-established therapy for various hematologic and non-hematologic diseases. However, the long-term health outcomes after HSC donation remain a major concern for several potential donors. Thus, we aimed to conduct a matched cohort study of 5003 unrelated donors (1099 BM and 3904 PBSC) and randomly selected 50,030 matched controls based on age, sex, and resident area from the donor registry between 1998 and 2018. The medical insurance claims of all the participants were retrieved from the Taiwan National Health and Welfare Data Science Center after de-identification. Our findings revealed no differences in the incidence of cancer, death, and catastrophic diseases between HSC donors and matched healthy participants during long-term follow-up. Kaplan-Meier curves depicting the cumulative incidence of cancer and overall mortality throughout the follow-up period also demonstrated similar outcomes between donors and non-donors. In conclusion, our results indicate that HSC donation, whether through BM or PBSC, is safe and not associated with an increased risk of cancer, death, or catastrophic diseases. These findings provide valuable information for counseling potential HSC donors and for long-term management of HSC donor health.

Recruiting refugees and migrants as potential hematopoietic stem cell donors to serve patients of comparable ethnicities with rare human leucocyte antigen patterns - The BluStar.NRW project in North Western Germany

Currently, approximately 19 million people with a migration background live in Germany. The majority of those descend from regions where the population has a genetically different distribution of HLA antigens when compared to the HLA frequencies usually found in North Western Europe. In case of severe haematological disorders of these individuals, allogeneic stem cell transplantation may be the treatment of choice. However, finding appropriate histocompatible hematopoietic stem cell donors continues to be a major challenge. If no matching sibling donors are available, there are only few suitable donors with a similar genetic background available in international blood stem cell donor registries.The “BluStar.NRW” project aimed to recruit new blood and hematopoietic stem cell donors with a migration background and to noticeably increase the number of suitable donors for patients within this group. Since December 2017, a total number of 9100 blood and stem cell donors with a migration background were recruited and typed for this project. HLA typing for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 was performed by Next Generation Sequencing. We assessed the proportion of rare alleles according to HLA frequency tables, as defined by a frequency of <1:1000. The rare HLA allele frequencies according to HLA frequency tables of the BluStar.NRW cohort were compared with a matched control donor cohort: Rare HLA-A, -B, -C, -DRB1 and -DQB1 alleles occurred three times more frequent than in the control group, but rare HLA-DPB1 alleles occurred more frequently in the control cohort. This difference was highly significant for all HLA alleles (p < 0.0001 for HLA-A, -B, -C, -DRB1, -DPB1; p = 0.0002 for HLA-DQB1). In addition, the distribution of rare alleles differed between the two groups. To date, 29 work-ups were initiated, 12 PBSC, one BM and three DLI were collected so far out of the BluStar.NRW cohort. The apheresis probability is twofold higher (0.18% vs. 0.07%) compared to the control group which clearly shows a serious medical need. However, 13 work-ups were cancelled in the BluStar.NRW donor cohort which represents an almost twice as higher cancellation rate (45% vs. 25%).This single registry analysis with a large sample cohort clearly indicates that hematopoietic stem cell donors with a migration background represent an adequate donor pool to serve patients of comparable ethnicity.

The novel HLA-A*74:46 allele detected in a potential hematopoietic stem cell donor.

One nucleotide substitution in codon 67 of HLA-A*74:41 results in the novel allele, HLA-A*74:46.

Decline in Cardiolipin in Hematopoietic Stem Cell during Aging Alters Their Regenerative Potential

Hematopoietic Stem Cells (HSCs) are known for their regenerative potential. This property allowed the use of HSC in bone marrow transplantation to treat hematological disorders such as leukemia. However, aging influences these characteristics of HSCs. The major hallmarks of aging are limited self-renewal and reconstitution capacity resulting in HSC exhaustion in aged individuals. Mitochondrial metabolism and activity are active drivers of HSC fate decisions and data from our lab shows that mitochondria in aged HSCs have increased sphericity with a polarized mitochondrial network and a lower mitochondrial membrane potential. An important aspect of mitochondrial functions is the lipid composition of their membranes. A lipid trafficking assay showed an atypical pattern of lipid incorporation by mitochondria in aged HSCs suggesting that mitochondrial lipids become abnormal upon aging. The overall content of Cardiolipin (CL) which is a signature mitochondrial lipid, found exclusively in the inner mitochondrial membrane was also reduced during aging. This was done with flow cytometry using a CL marker NAO (Non-Acridine Orange). Tafazzin encoded by the gene TAZ is crucial for remodeling CL into its mature form which is required for maintaining mitochondrial functions. Mutations in the TAZ gene have been known to cause a reduction in the synthesis of mature CL which is often associated with HSCs functional defect. Western Blot analysis showed a considerable decrease in Taz protein expression in aged stem and progenitor cells (HSPC) compared to young HSPC. To understand the effect of reduced Taz expression on HSC functions, we used a doxycycline inducible, sh-RNA mediated TAZ knock-down (KD) mouse model. Bone marrow analysis indicates that the frequency of HSC and multipotent progenitors decreases significantly in 6-month-old TAZ KD mice compared to wild type (WT) mice. Our data shows that following 5FU induction TAZ KD mice have a significant reduction in the multipotent progenitor and HSC population on day 10 and day 21 post 5FU, indicating TAZ KD decrease the regenerative potential of HSCs under stress conditions. Further pointing towards the significance of cardiolipin in HSC regeneration. TAZ KD mice also have abnormal peripheral blood recovery post 5FU mainly affecting the platelets. We next examined the repopulation potential of HSCs isolated from TAZ KD mice using serial competitive transplantation assays. While primary transplanted mice of TAZ KD HSCs exhibited similar donor-cell chimerism compared to mice transplanted with WT HSCs, about 50-60%, secondary recipient of TAZ KD cells had significantly lower donor-cell chimerism than secondary recipients of WT cells. Further, donor-derived HSC frequency in the bone marrow of recipients of TAZ KD HSCs 24 weeks following engraftment was also significantly lower than recipients of WT HSCs. Hence, Taz-deficiency causes a defect in HSC self-renewal under regenerative conditions. We next evaluated whether restoring cardiolipin levels in mid-aged HSCs could rescue their functions. For this, mid-aged HSC were incubated for 3 days under conditions that maintain HSC functions in vitro with Alcar (Acetyl-L-Carnitine), which promotes CL production. In lipid incorporation assay, Alcar supplementation restored mitochondrial membrane potential, mitochondrial organization and lipid incorporation of mid-aged HSCs. In competitive transplant assay, while mid-aged WT HSC treated with vehicle gave rise to a myeloid-bias graft, Alcar supplemented mid-aged WT HSC gave rise to a more balanced graft with increased ratio lymphoid to myeloid cells in the peripheral blood 16 weeks following transplantation, albeit the rescue was partial. This study indicates that HSC mitochondrial membrane lipids become abnormal with aging which causes a decline in HSC function and highlights the critical role of mitochondrial cardiolipin in HSC functions. It implies that metabolite supplementation is a viable option for restoring age-related HSC functional defects.

Mitochondria-Mediated (MAVS) Innate Immune Signaling Drives Inflammation-Induced Bone Marrow Failure

Bone marrow failure syndromes (BMFS) are life threatening diseases, characterized by impaired fitness of hematopoietic stem cells (HSC) and ineffective hematopoiesis causing the absence of one or more hematopoietic lineages in the peripheral blood. Substantial clinical data indicate that hyperactivity of inflammatory cytokines, including TNFα, IL-6, and transforming growth factor-β (TGFβ), directly contribute to BMF. Inflammatory challenges result in irreversible HSC functional decline, including decreased repopulation potential and myeloid-bias differentiation. We recently showed that the combination of inflammatory challenges, including polyinosinc:polycytidilic acid (pIC - a double stranded RNA [dsRNA]) plus high TGFβ causes chronic pancytopenia, anemia, expanded hematopoietic stem and progenitor cell pools, and enhanced bone marrow dysplasia, mimicking a myelodysplastic syndrome (MDS) that developed 3-4 months after pIC challenge (Javier et.al. Haematologica 2022). However, the mechanisms behind this association and/or causality are still ill-defined. Here, we show that mitochondrial antiviral signaling (MAVS) protein can mediate the negative effect of pIC and TGFβ on HSC functions and BMF/ MDS development. Differential gene expression analyses in HSCs suggested that pIC plus TGFβ altered expression of nuclear-encoded mitochondrial genes. Mitochondria serve as platform for the activation of MAVS and downstream innate immune signaling in response to dsRNA. We thus examined the role of mitochondria and MAVS in pIC plus TGFβ-induced BMF/MDS. We show that overexpression TGFβ and pIC together cause alterations in mitochondrial proteins and membrane potential as well as enhanced caspase1 activity, but not the necroptotic signals p-RIP1 and p-RIP3, in HSCs. pIC exposure caused an increase in MAVS expression, and a redistribution into large aggregates that localize onto mitochondria in HSCs, indicative of MAVS activation (two days after pIC exposure in vivo). The MAVS aggregates resolved into smaller and dispersed clusters by 3 months after pIC exposure in control mice. In contrast, in HSCs from TGFβ overexpressing mice, MAVS aggregates persisted 3 months after pIC exposure. To determine the impact of augmented MAVS in dsRNA+TGFβ-driven BMF, we used MAVS deficient mice crossed with TGFβ overexpressing mice. We found that absence of MAVS protein restored normal white blood cell count, including neutrophils and lymphocytes, restored normal red blood cell but not platelet counts in TGFβ overexpressing mice after pIC challenge, compared to controls. Remarkably, MAVS deficiency partially rescued the dysplastic features of bone marrow myeloid cells in TGFβ overexpressing mice after pIC challenge. MAVS-deficiency also reduced the increased caspase1 activity and lowers mitochondria membrane potential in HSCs of these mice. In serial competitive transplant studies, MAVS-deficiency conferred HSC repopulation advantage. These findings suggest that pIC+TGFβ overexpression mediated BMF/MDS phenotype depends on chronic activation of MAVS. Because MAVS aggregation depends on mitochondrial fusion, we used genetic loss of the mitochondrial fission regulator Drp1. Drp1-deficiency caused severe mitochondrial aggregation in HSCs; this was associated with sustained MAVS aggregation. In response to pIC, Drp1-deficient mice exhibited increased caspase-1 activity, increased mitochondrial membrane potential and increase in HSC numbers 6 months after pIC challenges. Drp1 loss resulted in anemia and mild pancytopenia. Interestingly, Drp1-deficient bone marrow myeloid progenitors exhibited severe dysplastic features after pIC challenged. These findings suggest that chronic mitochondrial and MAVS aggregation are sufficient to drive BMF/MDS phenotype. Finally, we investigated the levels of epigenetic markers (acetyl H3K27 and methyl H3 Lys4) in HSCs after pIC stress. H4K4me3 levels were decreased in HSCs long-term after pIC challenges [6 months], this was further decreased in Drp1-deficient HSCs. In conclusion, these findings indicate that non-genetic factors contribute to the onset of BMF/MDS. Mitochondria and MAVS protein together mediate an innate immune mechanism that drives BMF/MDS. This study opens the possibility to target mitochondria-related proteins as a therapeutic strategy to restore ineffective hematopoiesis due to chronic inflammation.

Notch Signaling Is Required for Extramedullary Hematopoiesis in the Adult Liver

During embryonic development, hematopoietic stem cells (HSC) in the fetal liver undergo expansion and self-renewal. Fetal HSCs migrate to the bone marrow (BM) niche towards birth and remain there during adulthood. Extramedullary hematopoiesis (EMH), or hematopoiesis outside of the BM, predominantly occurs in the fetal liver or when the adult BM niche ceases to be a site for functional hematopoiesis. The mechanism by which the adult liver becomes reactivated as a site of EMH for itinerant HSCs is unknown. Our project is investigating EMH in the adult liver by inducing acute hemolytic anemia in adult mice that induces egress and homing of hematopoietic progenitors to the liver and spleen. Our work demonstrates that long-term HSCs isolated from the portal vein in the adult liver can serially reconstitute lethally irradiated mice. Thus, the adult liver serves as a potential reservoir for long-term, functional HSCs in the presence of a damaged BM niche. In pursuit of a mechanism, we focused on Notch-signaling which is an evolutionarily conserved pathway that has been shown to play a critical role in HSC emergence and fetal liver development. When comparing WT liver donors to Notch-deficient (Notch1 +/ΔTAD) or Notch knockout (Notch1 ffVavCre +) liver donors, hematopoietic transplants reveal that Notch mutant progenitors fail to efficiently reconstitute irradiated recipients. Using an HSC-specific a-catulin-GFPmouse model, we are able to track the expression of GFP + liver-originating HSCs after hematopoietic reconstitution. We show that mice reconstituted by EMH-activated a-catulin-GFP liver HSCs are able to provide long-term reconstitution in lethally irradiated recipients and resume residence in the bone marrow niche of recipients. Additionally, we see distinct localization of GFP + HSCs localized near sinusoids throughout the EMH liver niche using whole-mount imaging. We have validated receptor-ligand binding interactions of the Notch1 receptor with ligand-expressing hematopoietic cells in the adult liver. Using intracellular flow cytometry after EMH in the adult liver, we see a 3-fold increase in mean fluorescent intensity of GATA2 in HSPCs (hematopoietic stem cell progenitors) when compared to our bone marrow and isotype control mice. We demonstrate that Notch-driven GATA2 expression is a crucial regulator of self-renewal and functional maintenance of stemness during EMH of HSCs in the adult liver. Our findings reveal a developmentally conserved Notch signaling mechanism is reactivated in the adult liver to retain the functional potential and maintenance of HSCs as they adapt to inhospitable conditions in the bone marrow niche.

Myelodysplastic Syndrome Hematopoietic Stem Cells Can Undergo Differentiation Trajectory Rewiring to Escape Venetoclax Vulnerability

Myelodysplastic syndromes (MDS) are incurable neoplasms that arise from early hematopoietic stem cells (HSCs). The standard of care for MDS is hypomethylating agent (HMA) therapy. However, the disease invariably fails these agents, resulting in fatal cytopenias or progression to acute myeloid leukemia (AML) with a dismal prognosis. We previously showed that the cellular hierarchy of MDS HSCs predicts the mechanisms of progression after HMA failure and can guide the design or choice of specific therapeutic approaches targeting these cells (Ganan-Gomez et al., Nature Medicine 2022). MDS HSCs in one of two differentiation states, long-term HSCs or lymphoid-primed multipotent progenitors, have a “common myeloid progenitor (CMP) pattern” or “granulocytic-monocytic progenitor (GMP) pattern” of differentiation. HSCs in these 2 differentiation states persist throughout the disease course and, driven by the recurrent activation of BCL2- or NF-κB-mediated survival pathways, respectively, expand at disease progression. In our preclinical studies, pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden, which suggests that only patients with “CMP pattern” MDS may benefit from venetoclax-based therapies. Accordingly, patients with “CMP pattern” MDS, whose disease progressed after frontline HMA failure, enrolled in clinical trials of venetoclax-based therapy (NCT04160052, NCT04550442, or NCT04655755; n=28: 12 “CMP pattern” MDS and 16 “GMP pattern” MDS) had a shorter cumulative time to complete remission (P=0.012) and a longer relapse-free survival duration (14.0 months vs 5.2 months) than those with “GMP pattern” MDS did. Consistent with the hypothesis that venetoclax-based therapy selectively targets different HSC populations after HMA therapy failure, bone marrow (BM) HSC frequencies were significantly decreased at the time of remission after venetoclax-based therapy in “CMP pattern” MDS patients, but not in those with “GMP pattern” MDS. These results explain why “GMP pattern” MDS are resistant to venetoclax therapy. However, 8 of the 12 patients with “CMP pattern” MDS relapsed during venetoclax treatment and/or progressed to AML after an initial remission. In 2 patients with “CMP pattern” MDS, sequential flow cytometry and next generation sequencing analyses showed that the HSC hierarchy switched to “GMP pattern” MDS before venetoclax failure. This immunophenotypic hierarchical change was associated with the detection or expansion of STAG2- or RUNX1-mutant clones with isolated trisomy 8. To evaluate whether the immunophenotypic switch resulted from the distinct differentiation potential of early HSCs or from the aberrant expression of cell surface markers, we performed sequential single-cell RNA sequencing analyses of BM mononuclear cells isolated from the 2 “CMP pattern” MDS patients whose disease changed hierarchy before venetoclax failure. Our analyses revealed that after undergoing significant depletion during disease remission, HSCs expanded at therapy failure. Differential expression analysis showed that the expansion of STAG2- or RUNX1-mutant clones in the HSC compartment not only rewired MDS HSCs' differentiation state towards a granulo-monocytic-biased trajectory but also changed HSCs' survival dependency from BCL2-mediated anti-apoptotic pathways to TNFa-induced pro-survival NF-κB signaling, thus enabling HSCs to evade the cytotoxic effects of venetoclax (Fig. 1). The anti-apoptotic gene MCL1, a direct transcriptional target of NF-κB, was also significantly upregulated in HSCs at the time of progression, which suggests that the combination of venetoclax with an MCL1 inhibitor can overcome secondary failure to venetoclax. Together, these results reveal a novel mechanism of venetoclax resistance. HSCs under venetoclax therapy undergo survival pressure, which results in the expansion or acquisition of clones carrying specific mutations that change HSCs' survival dependencies (Fig. 2). Our study suggests that MDS patients receiving venetoclax-based therapy should be monitored at the molecular level and enrolled in clinical trials of other targeted agents before their disease undergoes clonal evolution and HSC hierarchical rewiring.

Characterization of the novel HLA-C*07:402:02 allele detected in a potential hematopoietic stem cell donor.

The novel HLA-C*07:402:02 allele was characterized using next-generation sequencing technology.

Recognition of the novel HLA-A*24:602 allele detected in a potential hematopoietic stem cell donor.

The novel HLA-A*24:602 allele differs by one nucleotide change from HLA-A*24:02:01:01.

Immunogenetic characteristics of hematopoietic stem cell donors representing the most numerous ethnic groups in Russia

Development of criteria for the optimal donor selection based on the analysis of the allo-HSCT results, high-resolution HLA typing for the donor and recipient resulted in decreased incidence of immunological complications, primarily an acute «graft-versus-host reaction». However, due to the pronounced allelic polymorphism of the main histocompatibility complex (MHC) genes, the search for an optimal donor is ineffective in a number of patients. To increase the chances of selecting a donor for patients with rare HLA genotypes, the hematopoietic stem cell donor registries recruit the persons of various nationalities. An increased number of donors from different ethnic groups provide a broader immunogenetic diversity of the donor cohort. Currently, the registry of hematopoietic stem cell donors at the Russian Research Institute of Hematology and Transfusiology includes representatives of 49 nationalities, most of which, are considered themselves Russians. The third largest ethnic group in the registry comprises Tatars. The purpose of this study is a comparative analysis of immunogenetic characteristics of potential hematopoietic stem cells donors in the registry, who have self-identified as Russians and Tatars. As a result of the study, we have not found significant differences in frequencies of HLA allelic groups in the compared cohorts, a trend for higher frequency of the HLA-B*27 group was noted in Tatars. However, significant differences have been revealed for the distribution of HLA haplotypes in Russians and Tatars. The most common HLA haplotype among Tatars was A*02-B*44-DRB1*07, being much less common in Russians (4.61% vs 0.55%, p = 0.002). HLA haplotype A*03-B*13-DRB1*07, belonging to the ten most common among Tatars, was significantly less frequently detected in Russians (1.62% vs 0.08%, p = 0.026). HLA haplotype A*03-B*08-DRB1*03 was also significantly more common in Tatars compared to Russians (1.42% vs 0.06%, p = 0.026). HLA haplotypes A*02-B*18-DRB1*11, A*02-B*15-DRB1*04, A*02-B*15-DRB1*13, presented in Russians at a frequency of &gt; 1%, were not determined among the tested Tatars. HLA haplotypes A*31-B*58-DRB1*04, A*24-B*44-DRB1*01, presented in Tatars at a frequency of &gt; 1%, were not detectable in Russians. The results of our study indicate a need for recruiting more representatives of the Tatar ethnicity to the registry, thus increasing immunogenetic diversity of the donor pool and resulting into increased chances of compatible unrelated donor selection for the patients with HLA haplotypes, which are now underrepresented in our registry.

Nomenclature for factors of the HLA system, update January, February, and March 2023.

Nomenclature for factors of the HLA system, update January, February, and March 2023.

Characterization of the novel HLA-B*27:05:57 allele detected in a potential hematopoietic stem cell donor.

The novel HLA-B*27:05:57 allele differs by two nucleotide changes from HLA-B*27:05:02:01.

Characterization of the novel HLA-A*03:444 detected in a potential hematopoietic stem cell donor.

The novel HLA-A*03:444 allele was characterized using next generation sequencing technology.

Abstract 351: Developing A Human Bone Marrow Organoid Culture System To Study The Diabetic Immune System

Objectives: There is limited understanding on how diabetes negatively impacts wound healing after ischemic tissue damage. Murine models for limb ischemia and myocardial infarction have demonstrated that recruitment of bone marrow hematopoietic stem cell (HSC)-derived immune cells at all stages of ischemic tissue remodeling and healing. By leveraging our lab’s three-dimensional (3D) bone marrow organoid culture system used to study human HSC, we aim to study the effect of diabetes on immune cell development. Methods: Surgically discarded bone marrow tissue samples from patients undergoing lower extremity amputation for non-healing ulcer or tissue loss was used to develop our bone marrow 3D organoid culture system. Human HSCs are identified using fluorescence-tagged antibodies against cell surface markers. Hematopoietic potential of bone marrow-derived HSCs was tested using colony forming unit assay. In vitro immune cell differentiation into monocyte and macrophage was performed. Bulk RNA libraries were sequenced on the NextSeq 500 system (Illumina) and analysis done on DeBrowser. Results: Our novel human bone marrow co-culture system provides a method for bone marrow cell expansion from primary human bone marrow tissue explants. De novo sprouting cells can be observed growing from bone marrow tissue explants harvested from non-diabetic and diabetic donors. Using fluorescence-activated cell sorting analysis, we identified a trend towards reduced HSC numbers in diabetic when compared to non-diabetic donors. HSC derived from both diabetic and non-diabetic bone marrow tissue explants were able to undergo hematopoiesis and immune cell differentiation. Bulk RNA sequencing demonstrates distinct gene expression profiles of the cells expanded from non-diabetic and diabetic bone marrow cells. Conclusions: Our 3D bone marrow organoid culture system will allow for future comparative studies between the non-diabetic and diabetic immune system. The preliminary data suggest that there are gene expression profile differences between bone marrow progenitor cells from diabetic and non-diabetic donors. These gene expression differences may attribute to alterations in the HSC number and immune cell development observed in diabetes.

Identification of the novel HLA-C*12:349 allele in a potential hematopoietic stem cell donor.

The novel HLA-C*12:349 allele was characterized using next generation sequencing technology.