Hematopoiesis of Human Induced Pluripotent Stem Cells Derived From Patients with Down Syndrome

Abstract 881

Trisomy 21, genetic hallmark of Down syndrome, is the most frequent human chromosomal abnormality. Infants and children with Down Syndrome (DS) are known to have some hematological disorders with an increased risk of developing leukemia. Ten to 20% of newborn with DS are diagnosed as neonatal preleukemic status, Transient Myeloproliferative Disorder (TMD), and approximately 30% of TMD patients are predisposed to acute megakaryoblastic leukemia (AMKL). Recently, acquired mutations in the N-terminal activation domain of the GATA1 gene, leading to expression of a shorter GATA1 isoform (GATA1s), have been reported in AMKL and TMD (Wechsler et al., 2002; Mundschau et al., 2003), but neither patients nor mice with germline mutations leading to expression of GATA1s developed AMKL and TMD in the absent of trisomy 21. These findings suggested that trisomy 21 itself directly contributes to the development of AMKL and TMD. However, the role of trisomy 21 in hematopoiesis, particularly in the human fetus remains poorly understood. To better understand the effects of trisomy 21 on hematopoiesis in embryonic stage and leukemogenesis, we employed human induced pluripotent stem cells (hiPSCs) derived from patients with DS (DS-hiPSCs). Six DS-hiPS and 5 hiPS cell lines (control) from healthy donors, which we used here, were all created from skin fibroblasts and reprogrammed by the defined 3 or 4 reprogramming factors (OCT3/4, KLF4, and SOX2, or c-MYC in addition to the 3 factors, respectively). We generated blood cells from DS-hiPSCs and controls with coculture system using murine aorta-gonad-mesonephros (AGM)-derived stromal cell line (Ma et al., 2009). The cells from hiPSCs were harvested at D11 or D12 of coculture and analyzed the presence of hematopoietic markers and the potentials of hematopoietic colony formation. In the experiments using hiPSCs reprogrammed by 3 factors, human CD34 expression in harvested cells from DS-hiPSCs or controls were detected 10.06 ± 4.35% and 3.04%, respectively. CD45 expression of CD34⁺ cells was small proportion in both DS-hiPSCs and controls. We next examined the hematopoietic colony formation. Both myeloid and erythroid colonies were detected. Number of colonies formed from DS-hiPSCs was 43.7±11.1 to 74.3±11.2 per an iPSC colony. It's approximately 2 to 3.5 folds numbers of control (p-value<0.05). Similar results were obtained in the experiments using hiPSCs reprogrammed by 4 factors. These results indicated that hiPSCs derived from patients with Down syndrome could differentiate into multiple hematopoietic cell lineages and the differentiation into hematopoietic lineage was promoted in DS patients. Further researches are under investigation to identify the responsible genes in trisomy 21 for acceleration of hematopoiesis with microarray analysis. Our study may contribute to understanding of the effects of trisomy 21 on hematopoiesis and effective use of patients derived hiPSCs in research and clinical application.