Human bone scaffolds support clonal persistence and functional retention of MDS-derived CD34+ cells in a 3D in vitro bone marrow model Free

Introduction:

Myelodysplastic neoplasms (MDS) are clonal hematopoietic disorders with ineffective hematopoiesis and risk of progression to acute myeloid leukemia, predominantly affecting elderly patients. Modeling disease-specific interactions for understanding pathogenesis and therapeutic vulnerabilities in MDS remains challenging. The bone marrow (BM) niche is essential for the maintenance and regulation of hematopoietic stem and progenitor cells (HSPCs). In MDS, alterations in this microenvironment drive ineffective hematopoiesis and clonal evolution. Murine xenografts lack human microenvironmental features and raise ethical concerns, while 2D or static co-cultures fail to mimic BM architecture. To address this, we used a perfusion bioreactor and compared synthetic hydroxyapatite scaffolds with decellularized human trabecular bone to establish a humanized 3D BM niche for functional MDS studies. We hypothesized that bone scaffolds better preserve perivascular stromal niches, particularly CD146⁺/Nestin⁺/NG2⁺ pericytes known to regulate HSC quiescence, explaining superior hematopoietic support compared to hydroxyapatite.

Methods:

Decellularized trabecular scaffolds from femoral heads were compared to commercial hydroxyapatite scaffolds. Scaffolds were pre-seeded with mesenchymal stromal cells (MSCs) and stromal vascular fraction for four weeks, then seeded with CD34⁺ BM cells from four MDS patients (MDS-EB2, MDS-EB-1, MDS-MLD, and MDS-EB-2). Samples were obtained with informed consent and ethics approval. Cultures were maintained in a “U-CUP” bioreactor under perfusion for 21 days. Analyses included flow cytometry, histology, colony-forming unit (CFU) assays, targeted sequencing, and single-cell RNA sequencing (scRNA-seq) to assess hematopoietic retention, functional output, clonal stability, and stromal composition. scRNA-seq was performed on bone scaffolds using 10x Genomics Chromium and analyzed with Seurat/Azimuth for reference annotation.

Results:

Human bone scaffolds demonstrated superior retention of viable CD34⁺ cells, including primitive CD34⁺CD38⁻ and CD34⁺CD38⁻CD45RA⁻CD90⁺ subsets as compared to hydroxyapatite (19 % vs. 2 %, p < 0.0001). CFU assays confirmed 6-fold higher total colony counts in bone scaffolds versus hydroxyapatite, and targeted sequencing after 21 days revealed stable variant allele frequencies of patient-specific, disease-defining mutations (ASXL1, TET2, TP53, EZH2 and RUNX1). Niche cells such as MSCs and pericytes remained detectable in all scaffold types. Hematoxylin and eosin staining revealed a 1.6-fold greater density of hematopoietic clusters per mm² in bone scaffolds as compared to hydroxyapatite scaffolds after 21 days of culture. scRNA-seq yielded 41,000 high-quality cells across five U-CUP cultures after quality control (87 % of raw cells retained), enabling robust characterization of stromal and hematopoietic compartments. scRNA-seq confirmed transcriptional homology between hematopoietic and stromal cell populations from the bone scaffold and native human BM. Further profiling identified four distinct stromal subtypes, including Nestin⁺NG2⁺ perivascular cells and quiescent MSC-like fibroblasts, closely resembling those found in native human BM and known to regulate long-term HSC maintenance. In line with this, flow cytometry analysis demonstrated a significant enrichment of CD146⁺ pericyte-like stromal cells in bone scaffolds compared to hydroxyapatite, supporting the hypothesis that preservation of perivascular niches contributes to the enhanced hematopoietic support observed in native bone scaffolds.

Conclusion:

Decellularized human bone scaffolds provide a robust and physiologically improved platform for ex vivo modeling of patient-specific MDS hematopoiesis. By preserving clonal architecture and recapitulating key stromal subtypes, this humanized 3D BM niche offers a powerful tool for dissecting disease-specific cell–niche interactions and enables translational applications in personalized medicine and preclinical drug testing for hematological malignancies. The enrichment of perivascular stromal cells further highlights a potential mechanistic link between niche composition and hematopoietic support, opening avenues for targeted niche engineering in future studies. This platform may be adapted to study other hematological malignancies or normal hematopoiesis, offering a human-specific alternative to current xenograft approaches.