Early Hematopoietic Stem Cells Research Articles

Outcomes of Hematopoietic Stem Cell Transplantation in Patients With Thalassemia Major: How Do Anti-HLA Antibodies Impact?: The Impact of Anti-HLA Antibodies on Transplantation Outcomes in Thalassemia Major.

To investigate the effects of anti-human Leucocyte Antigen (HLA) antibody positivity on early hematopoietic stem cell transplantation (HSCT) results in patients with thalassemia major (TM). One hundred and twenty-four HLA-matched HSCTs were performed in patients with TM between 2015 and 2022. Ninety-one patients were screened for anti-HLA antibodies by testing panel reactive antigens (PRA). Demographic and transplantation characteristics of patients were recorded. The presence of PRA was tested with the Antibody Testing Assay (Luminex LIFECODES HLA Antibody Identification System). The number of PRA-positive patients was 54. There was no relationship between acute graft versus host disease (GVHD), chronic GVHD, grade of GVHD, and viral reactivation of the patients. However, platelet engraftment took around 3 days longer in the PRA-positive group (p = 0.05). The median number of erythrocyte transfusions was significantly higher in PRA-positive patients in the post-transplant period (p = 0.003), as was the median number of platelet transfusions (p = 0.003). Treosulfan conditioning increased the stable mixed chimerism (MC) rate by 3.8-fold (p = 0.011). In contrast, reduced rates of MC were found in patients who received matched unrelated donor cells or peripherally derived stem cells (p = 0.011 and p = 0.039, respectively) in the posttransplantation period in TM patients. PRA-positivity did not affect MC (p = 0.478). However, 80% of patients who had primary graft failure (n = 5; p = 0.59) and 75% of patients who died (n = 4) were PRA positive (p = 0.64), but these results were statistically insignificant due to the low number of patients. Anti-HLA antibodies primarily delayed platelet engraftment in TM patients and increased the erythrocyte and thrombocyte transfusion requirements. Although PRA positivity was more common in patients with primary graft failure or who died, there was no statistically significant impact of PRA positivity on chimerism, acute or chronic GVHD, viral activation, or mortality rates.

The E3 Ubiquitin Ligase Trip12 attenuates Wnt9a/Fzd9b signaling during hematopoietic stem cell development.

Wnt signaling is essential for both the development and homeostasis of diverse cellular lineages, including hematopoietic stem cells. Organism-wide, Wnt signals are tightly regulated, as overactivation of the pathway can lead to tumorigenesis. Although numerous Wnt ligands and Frizzled (Fzd) receptors exist, how particular Wnt/Fzd pairings are established and how their signals are regulated is poorly understood. We have previously identified the requirements of the cognate pairing of Wnt9a and Fzd9b for early hematopoietic stem cell proliferation. However, the specific signals governing activation, but equally important, the molecular mechanisms required to turn the signal 'off,' are unknown. Here, we show that the E3 ubiquitin ligase Trip12 (thyroid hormone receptor interactor 12) is specifically required to ubiquitinate the third intracellular loop of Fzd9b at K437, targeting it for lysosomal degradation. In contrast to other ubiquitin ligases described to regulate the cell surface availability of multiple Fzds broadly, our data indicate that Trip12 is selective for Fzd9b. We further demonstrate that this occurs through ubiquitination at K437 of Fzd9b in the third intracellular loop, ultimately leading to a decrease in Fzd9b receptor availability and in Wnt9a/Fzd9b signaling that impacts hematopoietic stem cell proliferation in zebrafish. Our results point to specific mechanisms driving the availability of different Fzd receptors. Determining how particular Fzd abundance is regulated at the membrane will be critical to developing specific therapies for human intervention.

A Multifunctional Nanostructured Hydrogel as a Platform for Deciphering Niche Interactions of Hematopoietic Stem and Progenitor Cells.

For over half a century, hematopoietic stem cells (HSCs) have been used for transplantation therapy to treat severe hematologic diseases. Successful outcomes depend on collecting sufficient donor HSCs as well as ensuring efficient engraftment. These processes are influenced by dynamic interactions of HSCs with the bone marrow niche, which can be revealed by artificial niche models. Here, a multifunctional nanostructured hydrogel is presented as a 2D platform to investigate how the interdependencies of cytokine binding and nanopatterned adhesive ligands influence the behavior of human hematopoietic stem and progenitor cells (HSPCs). The results indicate that the degree of HSPC polarization and motility, observed when cultured on gels presenting the chemokine SDF-1α and a nanoscale-defined density of a cellular (IDSP) or extracellular matrix (LDV) α4β1 integrin binding motif, are differentlyinfluenced on hydrogels functionalized with the different ligand types. Further, SDF-1α promotes cell polarization but not motility. Strikingly, the degree of differentiation correlates negatively with the nanoparticle spacing, which determines ligand density, but only for the cellular-derived IDSP motif. This mechanism potentially offers a means of predictably regulating early HSC fate decisions. Consequently, the innovative multifunctional hydrogel holds promise for deciphering dynamic HSPC-niche interactions and refining transplantation therapy protocols.

Immunophenotypic features of early haematopoietic and leukaemia stem cells.

Many tumours are organised in a hierarchical structure with at its apex a cell that can maintain, establish, and repopulate the tumour-the cancer stem cell. The haematopoietic stem cell (HSC) is the founder cell for all functional blood cells. Like HSCs, the leukaemia stem cells (LSC) are hypothesised to be the leukaemia-initiating cells, which have features of stemness such as self-renewal, quiescence, and resistance to cytotoxic drugs. Immunophenotypically, CD34+CD38- defines HSCs by adding lineage negativity and CD90+CD45RA-. At which stage of maturation the further differentiation is blocked, determines the type of leukaemia, and determines the immunophenotype of the LSC specific to the leukaemia type. No apparent LSC phenotype has been described in lymphoid leukaemia, and it is debated if a specific acute lymphocytic leukaemia-initiating cell is present, as all cells are capable of engraftment in a secondary mouse model. In chronic lymphocytic leukaemia, a B-cell clone is responsible for uncontrolled proliferation, not a specific LSC. In chronic and acute myeloid leukaemia, LSC is described as CD34+CD38- with the expression of a marker that is aberrantly expressed (LSC marker), such as CD45RA, CD123 or in the case of chronic myeloid leukaemia CD26. In acute myeloid leukaemia, the LSC load had prognostic relevance and might be a biomarker that can be used for monitoring and as an addition to measurable residual disease. However, challenges such as the CD34-negative immunophenotype need to be explored.

Autophagy prevents graft failure during murine graft-versus-host disease

AbstractAutophagy is an intracellular survival process that has established roles in the long-term survival and function of hematopoietic stem cells (HSC). We investigated the contribution of autophagy to HSC fitness during allogeneic transplantation and graft-versus-host disease (GVHD). We demonstrate in vitro that both tumor necrosis factor and IL-1β, major components of GVHD cytokine storm, synergistically promote autophagy in both HSC and their more mature hematopoietic progenitor cells (HPC). In vivo we demonstrate that autophagy is increased in donor HSC and HPC during GVHD. Competitive transplant experiments demonstrated that autophagy-deficient cells display reduced capacity to reconstitute the hematopoietic system compared to wild-type counterparts. In a major histocompatibility complex-mismatched model of GVHD and associated cytokine dysregulation, we demonstrate that autophagy-deficient HSC and progenitors fail to establish durable hematopoiesis, leading to primary graft failure and universal transplant related mortality. Using several different models, we confirm that autophagy activity is increased in early progenitor and HSC populations in the presence of T-cell–derived inflammatory cytokines and that these HSC populations require autophagy to survive. Thus, autophagy serves as a key survival mechanism in HSC and progenitor populations after allogeneic stem cell transplant and may represent a therapeutic target to prevent graft failure during GVHD.

Age-Specific Induction of Mutant p53 Drives Clonal Hematopoiesis in Adult Mice Leading to Acute Myeloid Leukemia

Background: Acute Myeloid Leukemia (AML) is a devastating hematological malignancy commonly observed in the elderly population. Mutations in TP53 can give rise to clonal hematopoiesis (CH), a condition preceding leukemia, where mutated hematopoietic stem cells (HSCs) proliferate without developing overt malignancy. The mechanisms underlying progression of TP53 mutant CH to AML and lymphoma remain incompletely understood. Through the creation of a traceable conditional Trp53-mutant CH model and complementary genetic models, we demonstrate that the age of HSCs is critical in determining whether the transformation leads to myeloid or lymphoid malignancy. Methods: To induce p53 mutation in early hematopoietic stem cells, we generated two cohorts of mice, one with Vav-Cre-mediated recombination of a conditional Trp53 allele ( Vav-Cre; Trp53 fl/wmR172H) and another with recombination of homozygous conditional floxed alleles ( Vav-Cre; Trp53 fl/fl), allowing us to compare the effects of p53 mutation versus complete loss of p53 in hematopoietic stem cells. To investigate the impact of Trp53 mutation on adult HSCs and their clonal evolution into myeloid malignancies, we developed an inducible mouse model driven by Mx1-cre. The inducible conditional mouse model allowed us to induce a somatic Trp53 mutation in HSCs of mice at 4 months of age, which is equivalent to young adult humans, and track the recombined cells using the mTmG allele ( Fig. 1A). Results: Mice with Trp53 mutations and Trp53 deletion introduced early in life developed lymphoma with complete penetrance, indicating that early-age mutation or deletion of Trp53 is more likely to lead to lymphoma development. However, when a somatic Trp53 mutation was induced in adult HSCs ( Mx1-Cre;Trp53 wmR172H/fl;mTmG mice), it resulted in myeloid-biased hematopoiesis leading to AML. Fluorescence microscopy analysis of the bone marrow (BM) clearly showed the presence of clonal clusters of GFP+ cells ( Fig. 1B). These GFP+ cells formed distinct colonies ( Fig. 1C) and were detectable in the peripheral blood ( Fig. 1D). The population of Trp53 mutant (GFP+) cells increased over time ( Fig. 1E). Flow cytometry analysis of peripheral blood after three months revealed an increased myeloid population defined as CD11b+ ( Fig. 1F). While the p53 wild-type group showed relatively stable population levels over time, the p53 mutant population displayed a significant expansion with increasing duration following pIpC injection. CyTOF analysis using a panel of 31 antibodies showed a slight increase in the myeloid lineage in p53 mutant and null mice compared to p53 wild-type, but no significant expansion of a specific stem/progenitor cell population in p53 mutant GFP+ cells was observed. After a one-year follow-up period, the Mx1-Cre;Trp53 wmR172H/fl;mTmG mice developed various myeloid malignancies (11 out of 25 mice), including AML, acute erythroid leukemia (AEL), and myeloid sarcoma ( Fig 1G). Peripheral blood smears analysis revealed the presence of large blast cells expressing GFP ( Fig. 1H). Characterizing the AML revealed a significant expansion of myeloid cells not only in the BM but also in the liver and spleen, indicating widespread dissemination of the disease ( Fig. 1I). Immunofluorescence analysis of the BM confirmed the accumulation of the mutant p53 in GFP+ cells ( Fig. 1J). Among the mice, 8 exhibited AML, 2 had AEL, and 2 presented a mixed disease phenotype consisting of both AML and AEL in the Mx1-Cre;Trp53 wmR172H/fl;mTmG cohort. The AML cells were identified as CD11b+Ly6G+CD3- ( Fig. 1K), while the AEL cells were characterized as CD34+CD71+ ( Fig. 1L). Conclusion: This data demonstrates the importance of age in the development of p53 mutant myeloid malignancies, and that p53 mutations in adult HSCs lead to the development of myeloid, but not lymphoid malignancies, highlighting the different mechanisms underlying the development of these types of malignancies, and suggest potential preventative or therapeutic strategies for TP53-mutant AML.

Early Autologous Hematopoietic Stem Cells (AHSC) Boost (HCB) to Overcome Immune Effector Cell-Associated Hematotoxicity (ICAHT) after CAR T-Cell Therapy for Lymphoma: A Single Center Experience

Sustained cytopenias designed as Immune Effector Cell-Associated Hematotoxicity (ICAHT) represent one of the most frequent side effect of CAR-T-cell therapy (30 to 90 % patients in recent real life studies) resulting in significant morbidity, mortality and resources consumption. We report single center experience of early reinjection of previously cryopreserved AHSC (HCB) in that setting. From July 2021 until January 2023, 27 patients received CAR T-cell therapy (25 Axi-Cel, 2 Brexu-Cel) after Fludarabine and Cyclophosphamide lymphodepletion for relapsed/refractory large B-cell lymphomas (24), mantle lymphoma (2), or follicular lymphoma (1). Median age was 62 years (19-75). CAR-HEMATOTOX score was ≥ 2 for 46% of patients. All patients received Filgrastim from D0. Hematopoietic recovery was longitudinally evaluated until D90 or censoring for relapse and/or subsequent cytotoxic treatment: severe (CTAE grade ≥ 3) neutropenia, anemia or thrombopenia occurred respectively for 88, 44 and 76% of patients until D28, and 60, 24 and 60% until D90. Median duration of severe neutropenia was 7 days (0-39) and 28 days for severe thrombocytopenia (0-118). Prolonged neutropenia occurred in 18/25 evaluable patients, mostly with a biphasic course (intermittent recovery and aplastic phenotype for 14 and 4 patients respectively). CAR-HEMATOTOX score was significantly correlated with sustained thrombopenia (p = 0,005) but only marginally with ICAHT (p = 0,054) and not with severe neutropenia (p = 0,44) . We observed significant correlation of severe ICAHT with progressive disease at CAR T-cell infusion (p = 0,015), platelets nadir < 30 giga/l before D28 (p < 0,005) and CAR T-cell expansion (> 200/µl) at D7 (p = 0,023). 7 patients received HCB (no conditioning) at a median D24 (15-41) post CAR T-cell infusion: after D14 for patients with aplastic phenotype, or for cytopenic recurrence for patients with biphasic course. Filgrastim was pursued or reintroduced until neutrophil recovery (> 1 giga/l) which occurred after a median of 10 days (4-16); platelet recovery (> 20 giga/l and > 50 giga/l) occurred after a median of respectively 12 (8-14) and 17 (11-22) days. If we consider D0 as CAR T-cell infusion, neutrophil recovery occurred at median D37 (22-49) and platelet recovery (> 50 giga/l) at median D37 (26-63). Median complete hematopoietic reconstitution (defined as PMNC > 1 giga/l, and Hb > 100 g/l, and PLT > 100 giga/l) was significantly shorter (median 23 days, 14-30) for patients receiving HCB compared with patients with prolonged cytopenia (> 28 days) who do not receive HCB (60 days, p = 0,018). Transfusion and hematopoietic growth factors (HGF) consumption was significantly reduced after HCB but we observe no difference between 2 groups considering hospitalization, EFS or OS. 2 patients receiving HCB experienced late recurrence of cytopenia (after D90) requiring intermittent HGF support but no transfusion or hospitalization. Discussion - Perspectives Recent case series highlighted clinical feasibility and safety and of this approach across a broad population of pediatric and adult patients: we validate this strategy in a more homogeneous and systematic practice. As study population is small, we failed to demonstrate that earlier application of an HCB is associated with superior survival outcomes as shown by others. We agree with recent updated recommendation of experts panel considering application of HCB without prior conditioning chemotherapy for grade ≥ 3 ICAHT beyond D14 (so considered early) if AHSC graft is readily available. Prophylactic AHSC harvest in high-risk candidates has been proposed but collection process may add to already high logistic burden and costs of CAR T-cell therapy in the lymphoma setting. Conversely, cryopreserved ASHC is mainly available for patients with myeloma and this approach would be readily proposed and useful for around 20% patients as recently described. Gagelmann N et al. Hematopoietic stem cell boost for persistent neutropenia after CAR T-cell therapy: a GLA/DRST study. Blood Adv. 2023;7(4):555 Rejeski K et al, Immune Effector Cell-Associated Hematotoxicity (ICAHT): EHA/EBMT Consensus Grading and Best Practice Recommendations. Blood. 2023 doi: 10.1182/blood.2023020578 Davis JA et al. Efficacy and safety of CD34+ Stem Cell Boost for Delayed hematopoietic Recovery After BCMA Directed CAR T-cell Therapy. Transplant Cell Ther. 2023 doi: 10.1016/j.jtct.2023.05.012

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.

Early Molecular Steps in Human Bone Marrow Stem Cell Differentiation Trajectories

Understanding the molecular signatures of human hematopoietic stem/progenitor cells (HSCPs) and gene network changes leading to differentiation induction are of utmost importance to achieve therapeutic hematopoietic stem cell (HSC) expansion. The identification of surface markers has improved the prospective isolation of human bone marrow HSCs over the last decades, but the isolated population still remains very heterogeneous. Single cell sequencing approaches have the potential to unbiased assort stem cells along their differentiation stages and trajectories, and to decipher molecular markers in differentiation hierarchies. However, whole transcriptome single cell sequencing often suffers from inadequate detection of low-expressed genes with increased drop-outs. To address this limitation and to provide sensitive mRNA quantification in human HSPCs, we developed a selected targeted gene panel (600 genes) in combination with 50 protein markers to cover most relevant genes/proteins of early HSPC differentiation and cell cycle status. We employed single cell CITE-Seq (cellular indexing of transcriptomes and epitopes by sequencing) based multi-omic profiling to >60.000 high-quality FACS-enriched human bone marrow HSPCs (CD34 + and CD34 +CD38 - cells) from 15 healthy donors representing young, mid and old age groups. Here, we demonstrate the most comprehensive molecular map of early human HSC differentiation and provide evidence of early branching points into lineage trajectories across different age groups using Slingshot pseudotime analysis. Our data demonstrate that the HSC branched into two main lineages, with the first branching point giving rise to megakaryocytic and erythroid progenitors (MKP/ERP), followed by lymphoid and myeloid progenitors (LMP/MDP). As expected, the most immature HSCs were defined by high expression of CD34, CD90, CRHBP, HOPX, HLF, MLLT3. However, we observed a number of hitherto undescribed HSC markers. The MKP/ERP lineage was defined by GATA1, TFRC, CD36, MPL and VWF expression. Furthermore, we found CD274, the Programmed Cell Death receptor ligand 1, to be selectively expressed in the MKP cluster. Re-clustering and trajectory analysis of the most immature CD34 +CD38 -/low cells confirmed early lineage commitment, suggesting that an MKP/ERP development does not include myeloid or lymphoid progenitors. A similar pattern was observed for all age groups. Interestingly, we observed increased expression of novel marker genes ( HLA-E, DLK1, ADGRG6, ADGRG1, MMRN1) and proteins (CD137 and CD273) in the most immature HSCs. Besides a gradual upregulation of CD38 along differentiation, differentiated cells showed lower CD45 levels. Furthermore, MKP/ERP committed cells showed a downregulation of SPINK2. TradeSeq analysis defined the continuous single cell expression changes in genes and markers upon early differentiation and lineage commitment, allowing the detection of decision-making gene networks. Our data adds further evidence to the revised model of hematopoiesis that challenges the dogma of a common myeloid progenitor giving rise to erythroid, megakaryocytic and myeloid cells. Furthermore, we see a structured hierarchical organization with a sequential loss of lineage potential and not an early commitment of uni-lineage restricted precursors. We report on the upregulation of ADGRG6, ADGRG1, DLK1, HLA-E, MMRN1 CD137 and CD273 in the most immature HSCs. Molecular and functional characterization of CD273 hi and low HSCs are presented in an adjacent report by our group at this meeting (Schmachtel et al.). In conclusion, we provide a valuable resource for the continuous regulation of genes and surface proteins along early differentiation of very immature human HSCs.

A large single-center cohort of bare lymphocyte syndrome: Immunological and genetic features in Turkey.

Major histocompatibility complex class II (MHC-II) deficiency or bare lymphocyte syndrome (BLS) is a rare, early-onset, autosomal recessive, and life-threatening inborn error of immunity. We aimed to assess the demographic, clinical, laboratory, follow-up, and treatment characteristics of patients with MHC-II deficiency, together with their survival. We retrospectively investigated 21 patients with MHC-II deficiency. Female/male ratio was 1.63. The median age at diagnosis was 16.3 months (5 months-9.7 years). Nineteen patients (90.5%) had parental consanguinity. Pulmonary diseases (pneumonia, chronic lung disease) (81%), diarrhoea (47.6%), and candidiasis (28.6%) were common. Four (19%) had autoimmunity, two developed septic arthritis, and three (14%) developed bronchiectasis in the follow-up. Three patients (14%) had CMV viraemia, one with bilateral CMV retinitis. Eight (38.1%) had lymphocytopenia, and four (19%) had neutropenia. Serum IgM, IgA, and IgG levels were low in 18 (85.7%), 15 (71.4%), and 11 (52.4%) patients, respectively. CD4+ lymphocytopenia, a reversed CD4+/CD8+ ratio, and absent/low HLA-DR expressions were detected in 93.3%, 86.7%, and 100% of the patients, respectively. Haematopoietic stem cell transplantation (HSCT) was performed on nine patients, and four died of septicaemia and ARDS after HSCT. The present median age of patients survived is 14 years (1-31 years). Genetic analysis was performed in 10 patients. RFX5 homozygous gene defect was found in three patients (P1, P4 and P8), and RFXANK (P2 and P14) and RFXAP (P18 and P19) heterozygous gene defects were found in each two patients, respectively. This large cohort showed that BLS patients have severe combined immunodeficiency (SCID)-like clinical findings. Flow cytometric MHC-II expression study is crucial for the diagnosis, differential diagnosis with SCID, early haematopoietic stem cell transplantation (HSCT), and post-HSCT follow-up. Genetic studies are required first for matched family donor evaluation before HSCT and then for genetic counselling.

Spatial and temporal expression pattern of somatostatin receptor 2 in mouse

Somatostatin (SST) is an inhibitory polypeptide hormone that plays an important role in a variety of biological processes. Somatostatin receptor 2 (SSTR2) is the most widely expressed somatostatin receptor. However, the specific cell types expressing Sstr2 in the tissues have not been investigated. In this study, we detected the expression pattern of SSTR2 protein in mouse at different development stages, including the embryonic 15.5 days and the postnatal 1, 7, 15 days as well as 3 and 6 months, by multicolour immunofluorescence analyses. We found that Sstr2 was expressed in some specific cells types of several tissues, including the neuronal cells and astrocytes in the brain, the mesenchymal cells, the hematopoietic cells, the early hematopoietic stem cells, and the B cells in the bone marrow, the macrophages, the type Ⅱ alveolar epithelial cells, and the airway ciliated cells in the lung, the epithelial cells and the neuronal cells in the intestine, the hair follicle cells, the gastric epithelial cells, the hematopoietic stem cells and the nerve fibre in the spleen, and the tubular epithelial cells in the kidney. This study identified the specific cell types expressing Sstr2 in mouse at different developmental stages, providing new insights into the physiological function of SST and SSTR2 in several cell types.

Nutritional Assessment in Early Allogenic Hematopoietic Stem Cell Transplant Patients, a Cross-Sectional Study

Background Our study aims to comprehensively assess nutrition status and malnutritional prevalence in early allogenic hematopoietic stem cell transplant (allo-HSCT) patients. Methods This single-center, cross-sectional study included 171 patients within the 90 days post-transplantation (from September 2019 to April 2020). Data collected included demographic, 3 day 24-h diet record, a Patient-Generated Subjective Global Assessment (PG-SGA) tool, laboratory tests, anthropometric indices, and body composition. Results One hundred and seventy-one patients with a mean age of 37.8 ± 11.3 and a male to female ratio of 102 to 69 were included. According to PG-SGA, 115 (67.3%) indicated the critical need for nutritional intervention and symptom management (PG-SGA score > 9). Forty-three (43.3%) of patients had experienced insufficient intakes of energy according to a 24-h diet record. Our study found that 120 (70.2%) patients had a body fat percentage and high triacylglycerol (64.9%). Reduced free fat mass index and low hand-grip strength were found in 133 (77.78%) and 104 (60.81%), respectively. The prevalence of malnutrition was 24.6% and the prevalence of sarcopenia was 13.5%. Conclusion Although the prevalence was not high, this research has demonstrated a high risk of malnutrition and a lower muscle mass in early allo-HSCT. Furthermore, our study confirmed body composition assessment would be an excellent way to identify malnutrition precisely.

Acute kidney injury and chronic kidney disease in umbilical cord blood transplant recipients.

Acute kidney injury (AKI) is a frequent early complication post hematopoietic stem cell transplant (HSCT), associated with high morbidity and mortality. Cord blood transplant (CBT) recipients are potentially exposed to more nephrotoxic insults, compared to patients undergoing HSCT from other donor sources. We aimed to identify risk factors for AKI in patients undergoing CBT. We also aimed to identify the impact of AKI on chronic kidney disease (CKD) and survival outcomes by one-year post-CBT. Adults and children who underwent a first CBT at our Institution were retrospectively evaluated. AKI was staged according to Kidney Disease Improving Global Outcomes (KDIGO) definitions. Cox regression models were used to estimate the association of demographic factors and post-CBT parameters with the cause-specific hazard of AKI. We identified 276 patients. Median age was 32 years, 28% (77/276) were children (<18 years) and 129 (47%) were white. A myeloablative conditioning regimen was administered to 243 patients (88%) and 248 (90%) received cyclosporine for GVHD prophylaxis. One-hundred and eighty-six patients (67%) developed AKI by day 60 post-transplant, with 72 (26%) developing severe AKI (stage 2 and 3). In a multivariable analysis, each increase in bilirubin level of 1 mg/dL was associated with a 23% increase in the risk of severe AKI (adjusted HR 1.23, 95% CI 1.13 - 1.34, p<.0001). Conversely, systemic steroid administration appeared to be protective of severe AKI (unadjusted HR 0.36, 95% CI 0.18 - 0.72, p=.004) in a univariate model . Two-hundred-forty-seven patients were evaluable at the one-year time point. Among those, 100 patients (40%) developed CKD one-year post-CBT. Severe AKI was associated with a higher hazard of non-relapse mortality (adjusted HR=3.26, 95% CI 1.65-6.45, p=.001) and overall mortality (adjusted HR=2.28, 95% CI 1.22-4.27, p=.01). AKI is a frequent complication after CBT and is associated with worse outcomes. Questions remain as to the mechanism of the protective role of steroids on kidney function in the setting of CBT.

The Zinc Transporter SLC39A10 Plays an Essential Role in Embryonic Hematopoiesis.

The role of zinc in hematopoiesis is currently unclear. Here, SLC39A10 (ZIP10) is identified as a key zinc transporter in hematopoiesis. The results show that in zebrafish, Slc39a10 is a key regulator of the response to zinc deficiency. Surprisingly, both slc39a10 mutant zebrafish and hematopoietic Slc39a10-deficient mice develop a more severe form of impaired hematopoiesis than animals lacking transferrin receptor 1, a well-characterized iron gatekeeper, indicating that zinc plays a larger role than iron in hematopoiesis, at least in early hematopoietic stem cells (HSCs). Furthermore, it is shown that loss of Slc39a10 causes zinc deficiency in fetal HSCs, which in turn leads to DNA damage, apoptosis, and G1 cell cycle arrest. Notably, zinc supplementation largely restores colony formation in HSCs derived from hematopoietic Slc39a10-deficient mice. In addition, inhibiting necroptosis partially restores hematopoiesis in mouse HSCs, providing mechanistic insights into the requirement for zinc in mediating hematopoiesis. Together, these findings indicate that SLC39A10 safeguards hematopoiesis by protecting against zinc deficiency-induced necroptosis, thus providing compelling evidence that SLC39A10 and zinc homeostasis promote the development of fetal HSCs. Moreover, these results suggest that SLC39A10 may serve as a novel therapeutic target for treating anemia and zinc deficiency-related disorders.

Primary Myelofibrosis with Extramedullary Hematopoiesis – A Case Report with a Review of Literature

Primary myelofibrosis (PMF) is the least common of all myeloproliferative neoplasms (MPNs), characterized by a neoplastic transformation of early hematopoietic stem cells, predominantly megakaryocytes and granulocytes. The disease shows gradual evolution from an initial prefibrotic stage to an overt fibrotic stage. Janus kinase (JAK) 2, CALR, and MPL mutations are most common in nonchronic myeloid leukemia MPNs but are not always present. Ineffective marrow hematopoiesis leads to extramedullary hematopoiesis and associated symptoms such as splenomegaly, hepatomegaly, anemia, and pro-inflammatory cytokines-induced constitutional symptoms. The WHO criteria, 2016, consider the combination of clinical, morphological, and molecular genetics features for the diagnosis of the condition. Currently, Dynamic International Prognostic Scoring System is most widely used to predict the prognosis. Here, we report the case of a 63-year-old male diagnosed with the rare disease PMF with extramedullary hematopoiesis and bleeding gastric varices. PMF was diagnosed by bone marrow biopsy showing diffuse fibrosis, positive JAK2-V617F mutation in genetic analysis, negative Philadelphia chromosome, pancytopenia, splenomegaly, and raised leukocyte dehydrogenase.

Association between human leukocyte antigen and immunosuppressive treatment outcomes in Chinese patients with aplastic anemia.

Activated cytotoxic T cells (CTLs) recognize the auto-antigens presented on hematopoietic stem/progenitor cells (HSPCs) through class I human leukocyte antigen (HLA) molecules and play an important role in the immune pathogenesis of aplastic anemia (AA). Previous reports demonstrated that HLA was related to the disease susceptibility and response to immunosuppressive therapy (IST) in AA patients. Recent studies have indicated that specific HLA allele deletions, which helped AA patients to evade CTL-driven autoimmune responses and escape from immune surveillance, may lead to high-risk clonal evolution. Therefore, HLA genotyping has a particular predictive value for the response to IST and the risk of clonal evolution. However, there are limited studies on this topic in the Chinese population. To explore the value of HLA genotyping in Chinese patients with AA, 95 AA patients treated with IST were retrospectively investigated. The alleles HLA-B*15:18 and HLA-C*04:01 were associated with a superior long-term response to IST (P = 0.025; P = 0.027, respectively), while the allele HLA-B*40:01 indicated an inferior result (P = 0.02). The allele HLA-A*01:01 and HLA-B*54:01 were associated with high-risk clonal evolution (P = 0.032; P = 0.01, respectively), and the former had a higher frequency in very severe AA (VSAA) patients than that in severe AA (SAA) patients (12.7% vs 0%, P = 0.02). The HLA-DQ*03:03 and HLA-DR*09:01 alleles were associated with high-risk clonal evolution and poor long-term survival in patients aged ≥40 years. Such patients may be recommended for early allogeneic hematopoietic stem cell transplantation rather than the routine IST treatment. HLA genotype has crucial value in predicting the outcome of IST and long-term survival in AA patients, and thus may assist an individualized treatment strategy.

Study of Hematopoietic Stem Cells in Patients with Extensive Burns

Introduction. Assessment of tissue regeneration, including that at the cellular and subcellular levels, appears to be one of the important trends in the complex treatment of patients with extensive burns. It is known that hematopoietic stem cells (HSCs) contribute to tissue restoration and regeneration through paracrine effects or direct cell differentiation, being a central component of post-burn anemia recovery and capable of forming not only blood cells, but also other types of cells. Notably, the role of these cells in burn injury has not been studied yet.The aim of the study was to investigate in dynamics the content of hematopoietic stem cells of different phenotypes in patients with extensive burns in the process of complex treatment. Methods. Hematopoietic stem cells and their subpopulations in peripheral blood samples were analyzed on a FACSCalibur flow cytometer (Becton Dickinson, USA) using the CellQuest program and CD45/CD34/CD38 and CD45/CD34/CD133 monoclonal antibody panels (BD, USA). The results obtained were statistically processed using the GraphPad Prism 7.0 program (USA), results were considered statistically significant at p0.05.Results. Hematopoietic stem cells and their subpopulations were studied at different stages of the complex treatment in 25 patients with a large burn area, ​​more than 30% of the body surface. The comparison group consisted of 15 healthy volunteers. Upon admission to the Burn Center, a group of severely burned patients revealed a significant deep deficiency of both total HSCs CD45+34+ (p=0.0002) and their subpopulations CD45dim34+38+ (p=0.019), with predominantly early precursors of hematopoiesis CD45dim34+38- (p=0.0001) and CD45dim34+133+ (p=0.0002). In the course of the complex treatment, including surgical necrectomy and autodermoplasty of burn wounds, there was observed normalization of total HSCs CD45+34+ (0.050.012%, p=0.031) and a subpopulation of early HSCs CD45dim34+38- (0.0390.009%, p=0.016) in 20 days of treatment in the group of burn patients. There was a significant increase of mature CD45dim34+133- HSCs (p=0.0380) as a result of treatment, while the deficit of the more differentiated population of CD45dim34+38+ HSCs did not fully recover (p=0.272).Conclusion. The firstly detected modulations in the content of hematopoietic stem cells of different phenotypes in patients with extensive burns may reflect the state of compensatory-adaptive reactions of hematopoiesis in the course of the complex treatment. The data obtained may support the predictive use of HSC subcellular markers to assess the regenerative potential in burn wounds healing, including that during staged surgical treatment and autodermoplasty, and to predict the development of local and general complications of burn disease.

Long term follow-up after haematopoietic stem cell transplantation for mucopolysaccharidosis type I-H: a retrospective study of 51 patients

Mucopolysaccharidosis type I-H (MPS I-H) is a rare lysosomal storage disorder caused by α-L-Iduronidase deficiency. Early haematopoietic stem cell transplantation (HSCT) is the sole available therapeutic option to preserve neurocognitive functions. We report long-term follow-up (median 9 years, interquartile range 8–16.5) for 51 MPS I-H patients who underwent HSCT between 1986 and 2018 in France. 4 patients died from complications of HSCT and one from disease progression. Complete chimerism and normal α-L-Iduronidase activity were obtained in 84% and 71% of patients respectively. No difference of outcomes was observed between bone marrow and cord blood stem cell sources. All patients acquired independent walking and 91% and 78% acquired intelligible language or reading and writing. Intelligence Quotient evaluation (n = 23) showed that 69% had IQ ≥ 70 at last follow-up. 58% of patients had normal or remedial schooling and 62% of the 13 adults had good socio-professional insertion. Skeletal dysplasia as well as vision and hearing impairments progressed despite HSCT, with significant disability. These results provide a long-term assessment of HSCT efficacy in MPS I-H and could be useful in the evaluation of novel promising treatments such as gene therapy.

CO134 Systematic Review of the Impact of Early Versus Late Diagnosis and/or Hematopoietic Stem Cell Transplantation (HSCT) for Severe Combined Immunodeficiency (SCID)

HSCT is considered the gold standard treatment for infants with SCID. This study aimed to determine whether or not age at diagnosis and or HSCT may impact clinical outcomes.

Regulation of myeloid and lymphoid cell development by O-glycans on Notch.

Notch signaling via NOTCH1 stimulated by Delta-like ligand 4 (DLL4) is required for the development of T cells in thymus, and NOTCH2 stimulated by Notch ligand DLL1 is required for the development of marginal zone (MZ) B cells in spleen. Notch signaling also regulates myeloid cell production in bone marrow and is an essential contributor to the generation of early hematopoietic stem cells (HSC). The differentiation program in each of these cellular contexts is optimized by the regulation of Notch signaling strength by O-glycans attached to epidermal growth factor-like (EGF) repeats in the extracellular domain of Notch receptors. There are three major types of O-glycan on NOTCH1 and NOTCH2 - O-fucose, O-glucose and O-GlcNAc. The initiating sugar of each O-glycan is added in the endoplasmic reticulum (ER) by glycosyltransferases POFUT1 (fucose), POGLUT1/2/3 (glucose) or EOGT (GlcNAc), respectively. Additional sugars are added in the Golgi compartment during passage through the secretory pathway to the plasma membrane. Of particular significance for Notch signaling is the addition of GlcNAc to O-fucose on an EGF repeat by the Fringe GlcNAc-transferases LFNG, MFNG or RFNG. Canonical Notch ligands (DLL1, DLL4, JAG1, JAG2) expressed in stromal cells bind to the extracellular domain of Notch receptors expressed in hematopoietic stem cells and myeloid and lymphoid progenitors to activate Notch signaling. Ligand-receptor binding is differentially regulated by the O-glycans on Notch. This review will summarize our understanding of the regulation of Notch signaling in myeloid and lymphoid cell development by specific O-glycans in mice with dysregulated expression of a particular glycosyltransferase and discuss how this may impact immune system development and malignancy in general, and in individuals with a congenital defect in the synthesis of the O-glycans attached to EGF repeats.