Beyond hemostasis: Factor VIII deficiency impacts  hematopoietic stem cells development in zebrafish

Abstract Introduction Hemophilia A (HA) is a monogenic bleeding disorder caused by coagulation Factor (F) VIII deficiency. Recent studies suggest that FVIII function may extend beyond hemostasis. In a previous study, we utilized morpholino antisense oligonucleotides to create a hemophilic zebrafish model; we proved that genetically corrected hematopoietic stem cells rescued excessive HA bleeding. Surprisingly, we discovered that f8 deficiency impacted the hematopoietic niche vasculature and reduced blood cells in zebrafish. Therefore, we decided to explore in depth the role of FVIII in hematopoiesis. Methods We knocked down f8 through morpholino injections (f8-KD) in zebrafish wild-type and transgenic zygotes, representing myeloid Tg(mpx:EGFP), early and mature red erythroid Tg(gata1:DsRed) lineages, and vasculature Tg(fli1:EGFP). We followed their maturation until 7 days-larval stage in single and crossed Tg lines. Structural changes were measured with fluorescent microscope images and live videos (ZEISS Celldiscoverer 7 microscope) from 20 to 77 hours post-fertilization. RNA-seq analyses and Flow cytometry sorting of fluorescent cells were performed at different maturation stages (17 hours pf until 7 days pf) in f8 knockdown and controls whole larvae and sorted myeloid, erythroid and vascular cells (6-25 larvae/condition in multiple experiments). To analyze transcriptomics, we used danRerLib, a newly developed Python package that combines ortholog of human annotations to enable functional enrichment analyses for KEGG and GO pathways (Schwartz et al., 2024). Finally, we performed a 14-days colony-forming units (CFU) assays with CD34+ hematopoietic cells (n=3 healthy donors) exposed to increasing concentrations of rFVIII (0, 0.62, 10, and 20 IU/mL) for 6 hours, before the assay. Results Differential gene expression analyses revealed substantial transcriptomic changes across erythroid, myeloid and vascular lineages at multiple timepoints in f8-knockdown (KD) samples compared to controls. Pathway enrichment analyses of mpx+ f8-KD cells showed that f8 deficiency was associated with extracellular matrix (ECM) reorganization, enrichment in tight junction/adherence pathways. Gata1+ f8-KD cells were marked by apoptosis and compensatory increased hemoglobin and red blood cell metabolism. Fli1+ f8-KD cells exhibited apoptotic and stress signals. Time-lapse videos of Tg crossed gata1-fli1 larvae recorded from day 1 to 4 post fertilization (pf) proved a significant retainment of gata1+ cells in the caudal hematopoietic niche, in the absence of f8 and at the same time a vascular delayed and disrupted maturation. Flow cytometry analyses of dissociated larvae sorted for gata1+, mpx+ and fli1+ cells at 5 and 7 days confirmed an imbalance in myeloid-erythroid stem cell maturation and an increased myeloid output at the expenses of the erythroid lineage. To understand if FVIII directly impacts hematopoiesis, we exposed in-vitro healthy CD34+ stem cells to increasing levels of FVIII for 6 hours. A subsequent 14 days CFU-assay showed a proportional increase in BFU-E (Burst-Forming Unit-Erythroid) and CFU-GM (Colony-Forming Unit-Granulocyte, Macrophage) p=0.006 and 0.0278, respectively in linear regression analyses. Conclusions Our findings demonstrate a clear influence of FVIII on hematopoiesis. Its deficiency leads to significant retention of stem cells within the zebrafish hematopoietic niche, suggesting an impaired mobilization. This is accompanied by an impaired myelo-erythropoiesis skewed towards myelopoiesis, observed macroscopically and supported by transcriptomic data. Furthermore, CD34+ human cells exposed to increasing doses of FVIII generated a linear increase in BFU-E erythroid and CFU-GM myeloid colonies, suggesting that the role of FVIII on hematopoietic stem cell maturation is -at least partially- direct. This skewed lineage development may contribute to explain the anemia observed in HA patients.