Reduced Production of Mature Neutrophils From Induced Pluripotent Stem Cells Derived From a Severe Congenital Neutropenia Patient with HAX1 Gene Deficiency

Abstract 2402

Induced pluripotent stem (iPS) cells are reprogrammed somatic cells with embryonic stem (ES) cell-like characteristics. As iPS cells can be generated from somatic cells of patients with a certain disease, they are expected to be a novel model to study pathogenesis of various diseases. Recently, we established a neutrophil differentiation system from human iPS cells (Morishima T, et al. J Cell Physiol. 2011). In an attempt to apply the system to investigate pathophysiology of neutrophil-affected disorders, we generated iPS cells from a severe congenital neutropenia (SCN) patient with HAX1 gene deficiency.

The patient was an 11-year-old boy with severe congenital neutropenia as well as developmental delay and epilepsy. DNA sequence analysis revealed HAX1 gene mutation in exon 2 (Matsubara K, et al. Haematologica. 2007). Four iPS cell lines were generated from skin fibroblasts of the patient by retroviral overexpression of the three or four transcription factors Oct3/4, Sox2, and Klf4, with or without c-Myc. These patient-derived iPS cell lines showed human ES cell like morphology and could be maintained under human ES cell culture condition. They also expressed typical human ES cell markers and were capable of differentiating into the cell lineages and tissues representing three germ layers by teratoma formation in vivo. These cells had normal karyotype and short tandem repeat analysis indicated that they were derived from parental skin fibroblast. DNA sequencing analysis of the iPS cell lines identified the same mutation carried in the parental skin fibroblasts, thus confirmed that we had established the HAX1 deficiency patient-specific iPS cells (HAX1-iPSCs).

Next these HAX1-iPSCs and the healthy-person derived iPS cells were differentiated into neutrophils in vitro using feeder-free culture protocols established in our laboratory. In this culture system, small human iPS cell clumps were cultured on the matrigel-coated dish with recombinant cytokines and without any feeder cells and fetal calf serum. Around day 25 of culture, mature neutrophils were obtained as floating cells. Morphologically, the majority of HAX1-iPSCs-derived cells were classified into myeloblast or promyelocyte stage and there were only a few mature neutrophils. The proportion of mature neutrophils was only less than 10% in HAX1-iPSCs-derived cells whereas more than 40% in normal control. Flow cytometric analysis revealed that the percentage of immature CD34 positive cells was significantly higher and that of myeloid-committed CD11b positive cells was lower in the HAX1-iPSCs-derived cells than normal control. Immunocytochemical analysis for neutrophil specific granules showed that lactoferrin- and gelatinase-positive cells decreased in the HAX1-iPSCs-derived cells compared with normal control, confirming that HAX1-iPSCs-derived cells contained less mature neutrophils than normal control. Apoptosis assay by Annexin V staining revealed that HAX1-iPSCs-derived cells showed higher percentage of Annexin V-positive cells compared with normal control.

Overall, these HAX1 deficiency patient-specific iPS cell lines recapitulate the hematological phenotype in the patient. These results indicate that patient-derived iPS cells provide us a novel disease model and make a contribution to the understanding of the pathophysiology of the diseases that affect neutrophils.