Single-Cell Transcriptomics Reveal Altered Hematopoietic Mechanisms Driven By T21 and GATA1s

Introduction Trisomy 21 (T21) is associated with baseline erythrocytosis and thrombocytopenia and risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia of Down syndrome (ML-DS). TAM and ML-DS are characterized by mutations in the transcription factor GATA1, resulting in the truncated isoform GATA1s (G1S). We previously found that T21 increases erythropoiesis, while G1S severely impairs erythroid development but enhances megakaryocyte (MK) proliferation (Byrska-Bishop et al, JCI 2015). To better understand how T21 and G1S each impact hematopoiesis, we performed single-cell RNA sequencing (scRNA-seq) of multipotent and late hematopoietic progenitor cells (HPCs) differing only by chromosome 21 and/or G1S status. Methods Four isogenic human induced pluripotent stem cell (iPSC) lines differing only by chromosome 21 (euploid vs. T21) and/or GATA1 (WT vs. G1S) status underwent hematopoietic differentiation by embryoid body formation. Cells were collected at 3 timepoints: on day 7 (D7), CD41 +235 + multipotent HPCs were purified by flow cytometry and on days 9 (D9) and 11 (D11), late HPCs biased to a single lineage were collected. Cells were sequenced by scRNA-seq and underwent quality control followed by clustering and analysis using Seurat. Clusters were manually annotated based on expression of 141 key hematopoietic genes, including aggregated expression of lineage markers. Early vs. committed lineage clusters were determined based on the relative expression of lineage-specific markers. Within each lineage, differentially expressed genes (DEGs) associated with a genetic background were assessed using the Wilcoxon rank-sum test. The χ² test was used to compare categorical data. A 2-tailed p or adjusted p