Single-cell transcriptomics reveal synergistic and antagonistic effects of GATA1s and T21 on hematopoiesis

Trisomy 21 (T21), or Down syndrome (DS), is associated with baseline macrocytic erythrocytosis, thrombocytopenia, and neutrophilia, as well as transient abnormal myelopoiesis (TAM) and myeloid leukemia of DS (ML-DS). TAM and ML-DS blasts both arise from an aberrant megakaryocyte-erythroid progenitor and exclusively express GATA1s, the truncated isoform ofGATA1, while germlineGATA1smutations in a non-T21 contextleadto congenital cytopenia(s) without a leukemic predisposition. This suggests that T21 and GATA1s perturb hematopoiesis independently and synergistically in hematopoietic progenitors (HPCs), but this is challenging to study due to limited access to relevant human progenitorpopulations. To dissect individual developmental impacts, we used single-cell RNA-sequencing to interrogate HPCs from isogenic human induced pluripotent stem cells differing only by chromosome 21 and/orGATA1status. These HPCs were surprisingly heterogeneous and displayed lineage skew dictated by T21 and/or GATA1s. T21 and GATA1s each disrupted temporal regulation of lineage-specific transcriptional programs and specifically perturbed cell cycle genes. Trajectory inference revealed that GATA1s nearly eliminated erythropoiesis, slowed MK maturation, and promoted myelopoiesis in the euploid context, while in T21 cells, GATA1s competed with the enhanced erythropoiesis and impaired megakaryopoiesis driven by T21 and give rise to immature erythrocytes, MKs, and myeloid cells. The use of isogenic cells revealed distinct transcriptional programs that can be attributed specifically to T21 and GATA1s, and how together they result in HPC proliferation at the expense of maturation, consistent with a pro-leukemic phenotype.