Differential gene expression analysis of trisomy 21 and euploid hematopoietic progenitor cells derived from human pluripotent stem cells.

Children with trisomy 21 (T21) are highly predisposed to acute myeloid leukemia, which is preceded by a pre-leukemic transient abnormal myelopoiesis driven by truncating mutations in GATA1 (GATA1s). We investigated gene expression profiles in T21 and isogenic euploid hematopoietic progenitor cells (HPCs) derived from human induced pluripotent stem cells (iPSCs), in combination GATA1s and STAG2 mutations associated with T21 myeloid leukemia. We identified upregulation of interferon alpha/gamma responses and ribosome subunit pathways in gene set enrichment analysis in T21 versus euploid CD45+ hematopoietic cells, whereas pathways regulating genome integrity including mitotic spindle and G2/M checkpoint were downregulated. GATA1s in T21 cells led to downregulation of heme metabolism and apoptosis pathways as well as a further significant increase in the average expression of genes on chromosome 21. These results suggest that T21 and GATA1s mutations cooperate to disrupt normal HPC functions and cause myeloid lineage skewing. We introduced mutations in exon 2 of GATA1 by CRISPR/Cas9 to generate GATA1s-mutant single clones in T21 and isogenic euploid human iPSCs (PMID: 23045682). A STAG2 mutant clones was generated in the T21-GATA1s line by disrupting exon 5 using CRISPR/Cas9. Hematopoietic differentiation was performed using STEMdiff Hematopoietic Kit (STEMCELL #05310) following manufacturer’s protocol to derive HPCs. RNA was purified using RNeasy Micro Kit (Qiagen) and submitted for bulk RNA-seq on Illumina NextSeq platform using single-end 75bp setting. Reads were mapped to hg19 genome using Rsubread (2.12.0) in R (4.2.2). Differential analyses were performed using edgeR (3.40.2). Submitter states that missing raw data will be deposited in dbGaP