A Comparison of Myelopoiesis from Induced Pluripotent Stem Cells with a Mutation in ELANE between Cyclic Neutropenia and Severe Congenital Neutropenia

The ELANE is known as the responsible gene for both cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). However, relations between mutations in the ELANE gene and abnormal myelopoiesis in the different phenotype of these diseases still remain unclear. It has been reported that induced pluripotent stem cell (iPSC) from an individual patient with SCN (SCN-iPSC) demonstrated maturation arrest of myeloid progenitor cells and poor response to granulocyte-colony stimulating factor as similarly observed in patient's bone marrow cells. Thus, the study on myelopoiesis using disease specific iPSC seems to provide disease pathogenesis as a novel in vitro experimental model.

In this study, we established iPSC line from an individual patient with CyN (CyN-iPSC) with heterozygous mutation in ELANE gene (Exon5, R191Q point mutation). Then we compared myelopoiesis among healthy Control-iPS (253G1), SCN-iPS (Exon5, C194X point mutation) , and CyN-iPSC. Undifferentiated colonies derived from CyN-iPSC were staind with pluripotency markers (OCT3/4 and NANOG). CyN-iPSC retained a normal karyotype and ELANE locus mutation of the original samples.

In vitro myelopoiesis was examined by using a serum- and feeder-free monolayer hematopoietic culture system. iPSC colonies were cultured on growth factor-reduced Matrigel-coated cell culture dishes in modified Tenneille Serum Replacer 1 (mTeSR™1) medium (StemCell Technologies, Inc.), containing BSA, rh bFGF, rh TGFβ, Lithium Chloride, Pipecolic acid, GABA. Medium was replaced every four days. Then medium was changed to StemPro®-34 SMF Complete Medium plus nutrient supplement (Life technologies Corp.). The iPSC were cultured with BMP4 (80 ng/mL) for four days, and then replaced with VEGF165 (80 ng/mL), bFGF (25.7 ng/mL), and SCF (100 ng/mL) on Day 4. On Day 6, cytokines were replaced with a combination of SCF (50 ng/mL), IL-3 (50 ng/mL), and G-CSF (50 ng/mL). Medium was replaced every 3 - 4 days.

No significant difference in the ratio of proliferating CD33+ cells were noted between CyN-iPSCs and Control-iPSCs. CyN-iPSCs showed less capability in the proliferation and maturation for CD15+ cells on days 20 to 40 than Control-iPSCs. The decreased number of CD15+ cells derived from CyN-iPSc implies the defect in mature neutrophil survival. In contrast, CD15+ / CD33+ cells derived from SCN-iPSCs were hardly observed in this culture condition, suggesting the defects of proliferation and maturation in SCN-iPSCs.

We next examined the colony formation of CD34+ cells derived from CyN-iPSCs, Control-iPSCs, and SCN-iPSCs. CD34+ cells were obtainded at the day 12 of primary culture of iPSCs and purified by cell sorting using FACS-Aria®. No significant differences in the number of G-colony and GM-colony between CD34+ cells from CyN-iPSCs and Control-iPSCs. In contrast, CD34+ cells from SCN-iPSCs gave rise to the significantly decreased number of G-colony and GM-colony.

The observations of myeloid proliferation/maturation and colony formation of CD34+ cells were almost compatible with those obtained from bone marrow cells in patients with SCN and CyN. Furthermore, neutrophils differentiated from CyN-iPSCs showed the excessive cell death, whereas SCN-iPSCs presented the defective myelopoiesis. These results suggest that the analyses using CyN-iPSCs and SCN-iPSCs may be useful tool for investigating the relation of gene mutation and pathophysiology in both diseases.