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The human bone marrow (BM) microenvironment involves hematopoietic and non-hematopoietic cell subsets organized in a complex architecture. Tremendous efforts have been made to model it in order to analyze normal or pathological hematopoiesis and its stromal counterpart. Herein, we report an original, fully-human in vitro 3D model of the BM microenvironment dedicated to study interactions taking place between mesenchymal stromal cells (MSC) and hematopoietic stem and progenitor cells (HSPC) during the hematopoietic differentiation. This fully-human Artificial Marrow Organoid (AMO) model is highly efficient to recapitulate MSC support to myeloid differentiation and NK cell development from the immature CD34?+?HSPCs to the most terminally differentiated CD15?+?polymorphonuclear neutrophils, CD64?+?monocytes or NKG2A-KIR2D?+?CD57?+?NK subset. Lastly, our model is suitable for evaluating anti-leukemic NK cell function in presence of therapeutic agents. Overall, the AMO is a versatile, low cost and simple model able to recapitulate normal hematopoiesis and allowing more physiological drug testing by taking into account both immune and non-immune BM microenvironment interactions.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-07717-9.

DEAD-box helicase 41 (DDX41) is implicated in germline (GL)-predisposed myeloid neoplasms, where pathogenic GL variants often lead to disease following the acquisition of a somatic variant in trans, most commonly p.R525H. However, the precise molecular mechanisms by which DDX41 variants contribute to the pathogenesis of myeloid neoplasms remain poorly understood, partly due to challenges in establishing cellular and animal models that faithfully recapitulate the human disease phenotype. This limitation highlights the necessity of directly analyzing primary human disease cells. In this case report, conducted to pursue this objective, we implemented single-cell RNA sequencing integrated with genotyping at the p.R525 locus in a myelodysplastic neoplasm (MDS) harboring both germline and somatic DDX41 variants, leveraging highly efficient Terminator-Assisted Solid-phase cDNA amplification and sequencing. We found that acquiring p.R525H induced G2/M cell cycle arrest selectively in colony-forming unit-erythroid cells, accompanied by R-loop accumulation, which impaired erythropoiesis through DNA damage. In hematopoietic stem and myeloid progenitor populations, gene expression profiles were largely similar between p.R525H-positive and -negative cells. However, ligand-receptor interaction and transcriptional regulation analyses suggested a non-cell-autonomous influence from p.R525H-expressing cells on GL variant-only cells. This interaction drove convergence toward a shared expression profile, highlighting an intricate interplay shaping the patient¡¯s MDS phenotype.

BACKGROUNDSepsis is a severe illness characterized by systemic and multiorgan reactive responses and damage. However, the impact of sepsis on the bone marrow, particularly on bone marrow mesenchymal stem cells (BMSCs), is less reported. BMSCs are critical stromal cells in the bone marrow microenvironment that maintain bone stability and hematopoietic homeostasis; however, the impairment caused by sepsis remains unknown.AIMTo investigate the effects of sepsis on BMSCs and the underlying mechanisms.METHODSBMSCs were obtained from healthy donors and patients with sepsis. We compared the self-renewal capacity, differentiation potential, and hematopoietic supportive ability in vitro. Senescence of septic BMSCs was assessed using ¦Â-galactosidase staining, senescence-associated secretory phenotype, intracellular reactive oxygen species levels, and the expression of P16 and P21. Finally, the changes in septic BMSCs after nicotinamide adenine dinucleotide (NAD) treatment were evaluated.RESULTSSeptic BMSCs showed decreased proliferation and self-renewal, bias towards adipogenic differentiation, and weakened osteogenic differentiation. Additionally, hematopoietic supportive capacity declines in sepsis. The levels of aging markers were significantly higher in the septic BMSCs. After NAD treatment, the proliferation capacity of septic BMSCs showed a recovery trend, with increased osteogenic and hematopoietic supportive capacities. Sepsis resulted in decreased expression of sirtuin 3 (SIRT3) in BMSCs, whereas NAD treatment restored SIRT3 expression, enhanced superoxide dismutase enzyme activity, reduced intracellular reactive oxygen species levels, maintained mitochondrial stability and function, and ultimately rejuvenated septic BMSCs.CONCLUSIONSepsis accelerates the aging of BMSCs, as evidenced by a decline in self-renewal and osteogenic capabilities, as well as weakened hematopoietic support functions. These deficiencies can be effectively reversed via the NAD/SIRT3/superoxide dismutase pathway.