|
Down syndrome (DS) presents a striking, clinically important pattern of erythroid abnormalities—neonatal polycythemia and persistent macrocytosis—that current models cannot mechanistically resolve. We propose a first-of-its-kind, human, non-animal platform that reconstructs the fetal liver–hematopoietic niche to uncover how trisomy 21 perturbs erythropoiesis. Using three in-house isogenic pairs of hiPSCs derived from individuals with mosaic DS, we will generate matched trisomic/disomic endothelial cells, fetal liver organoids, and CD34? HSPCs. This enables rigorous, genetically controlled dissection of cell-intrinsic versus niche-driven effects. Our approach integrates two synergistic aims. Aim 1 establishes a benchmarked in-vitro DS hematopoiesis model by differentiating isogenic HSPCs and quantifying lineage commitment, mitochondrial function, erythroid yield, and morphology. We will map in-vitro outputs (erythrocyte yield per input HSPCs, cell size) to clinical readouts (hematocrit, mean corpuscular volume), providing direct translational anchors. Aim 2 engineers a perfused, vascularized fetal liver organoid in an OrganoPlate® Graft and performs autologous co-culture with isogenic HSPCs to recreate the fetal niche. Niche-swap experiments (trisomic HSPCs in disomic liver organoids and vice versa) coupled to on-chip single-cell RNA-seq/ATAC-seq will causally separate hematopoietic-intrinsic from microenvironmental drivers. Functional assays (e.g., CFU output, permeability and tracer perfusion for vascular QC) complete a closed loop from multi-omics to phenotype. This project is deliberately high-risk/high-reward. It targets an underexplored developmental context where rodent models incompletely capture human DS hematology and fetal liver biology. Success would deliver a validated, human-specific, context-of-use platform for DS and other niche-modulated blood disorders (e.g., stress erythropoiesis under fetal-like hypoxia), advancing non-animal methods and enabling precision interrogation of pathways such as EPO signaling, heme metabolism, mitochondrial resilience, and cell-cycle regulation. Pre-specified success criteria, blinding, mixed-effects statistics, and stringent vascular QC (continuous, leak-free perfusion; macromolecular permeability window) ensure rigor and reproducibility. By engineering a vascularized fetal liver–hematopoietic niche with isogenic controls and causal niche-swap perturbations, this work can redefine how developmental erythropoiesis—and DS-associated dysregulation—are studied, positioning the field for targeted therapeutic discovery while replacing scarce fetal tissues and bypassing species-mismatch limitations.
|