Abstract 730

Severe congenital neutropenia (SCN) is a rare disorder characterized by severe neutropenia present at birth, an arrest of neutrophilic differentiation at the promyelocyte or myelocyte stage, and a propensity to develop acute myeloid leukemia and myelodysplasia. Mutations of the ELA2 gene encoding neutrophil elastase have been identified in majority cases of SCN. To date, more than 50 distinct mutations of the ELA2 gene have been reported in patients with SCN. However, the mechanisms by which these mutations disrupt neutrophil development are unclear.

To understand the roles of ELA2 mutation in SCN, we established three human induced pluripotent stem (iPS) cell clones (SPN0101, SPN0102 and SPN0103) from bone marrow stromal cells of a patient having heterozygous mutation in ELA2 gene by transfection with retrovirus vector whichexpressed human OCT3/4, SOX2, KLF4, and c-MYC (SCN-iPS cells). The silencing of exogenous genes and the capability to differentiate into three germ lines by teratoma formation were confirmed in the three SCN-iPS cell clones.

We first examined the hematopoietic differentiation of SCN-iPS and control iPS cells which were generated from healthy donors by the same method to SCN-iPS cells, using coculture system with murine embryonic aorta-gonad-mesonephros region-drived stromal cell line (AGM-3 cells) (Ma F. et al., PNAS, 2009). The cocultured cells were harvested at day 12 and analyzed for the hematopoietic colony formation by 10,000 CD34+ cells which were separated using magnetic beads system. There was no difference between SCN-iPS and control iPS cells in the number and size of erythroid and mixed-lineage colonies (erythroid colonies; 11.5±3.7 in SCN-iPS cells and 10.3±4.5 in control and mixed-lineage colonies; 25.5±8.0 in SCN-iPS cells and 18.7±4.2 in control). However, the number of myeloid colonies derived from SCN-iPS cells was significantly smaller than that in control (48.0±21.3 in SCN-iPS cells and 206.7±37.6 in control, p<0.01), and their size was tiny as compared to that in control. The myeloid colonies derived from SCN-iPS cells consisted of monocytic cells and a few immature myeloid cells whereas those from control iPS cells contained a substantial number of mature neutrophils. Furthermore, since SCN patients need higher dose of granulocyte colony-stimulating factor (G-CSF) treatment to increase neutrophil numbers and avoid recurrent life-threatening bacterial or fungal infections, we compared the sensitivity of myeloid colony formation to G-CSF between SCN-iPS and control iPS cells. The number and size of myeloid colonies derived from control iPS cells reached a plateau at a concentration of 1 ng/mL G-CSF, while those from SCN-iPS cells were enhanced in association with the escalated concentrations of G-CSF up to 1,000 ng/mL although the number of the colonies was fewer, and their size was smaller than control even at 1,000 ng/mL of G-CSF. The large colonies containing more than 100 cells were formed from control iPS cells at 1 ng/mL of G-CSF, but SCN-iPS cells at higher doses of 100 ng/mL.

We next examined the neutrophil development in more detail in suspension culture. 10,000 CD34+ cells derived from SCN-iPS and control iPS cells were cocultured with AGM-3 stromal cells in the presence of various cytokines including 10 ng/mL of G-CSF, but not erythropoietin to exclude the growth of erythroid cells. The cells from control iPS cell increased 23.3-fold for 2 weeks, and most of the cultured cells were neutrophils. By contrast, the cells from SCN-iPS cells gradually decreased, and contained few neutrophils, but mainly monocytic cells.

These results indicated thatthe development of neutrophils was selectively impaired in hematopoiesis derived from SCN-iPS cells, although other lineages of cells, such as erythroid and monocytic cells, were not affected, and that the stimulation of high concentration of G-CSF compensated the impaired neutrophil development to some extent in SCN-iPS cells. The present study demonstrated that hematopoiesis derived from SCN-iPS cells reflected the pathophysiological status of SCN patients including neutrocytopenia and its improvement by G-CSF treatment, and established SCN-iPS cells could contribute to understand the pathophysiology and leukemogenesis in SCN. Further researches are under investigation to clarify the relationship between ELA2 mutation and clinical status in SCN including leukemogenesis.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.