Disease-Specific Induced Pluripotent Stem Cells Recapitulate The Pathophysiology Of Familial Platelet Disorder With Predisposition To Acute Myelogenous Leukemia
Authors: Arai Shunya , Iiduka Hiromitsu , Takayama Naoya , Masashi Miyauchi , Taoka Kazuki , Kataoka Keisuke , Hosoi Masataka , Kumano Keiki , Eto Koji , Kurokawa Mineo 
Published: October 2019

Abstract
Familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) is an autosomal dominant disorder and is characterized by inherited thrombocytopenia and a lifelong risk of development of hematological malignancies. About 30 pedigrees of FPD/AML have been reported in the literature, when the first FPD/AML pedigree was reported. Heterozygous inherited RUNX1 mutations as the cause of this disorder was confirmed. Mutations of RUNX1 observed in FPD/AML are heterogeneous and tend to be specific to individual families. An individual mutation is thought to cause a functional loss of the RUNX1 protein with an individual severity, accounting for the variable phenotypes of FPD/AML among pedigrees. Mutations that cause haploinsufficiency are most frequent, though some mutations are predicted to result in dominant-negative effects. Indeed, the ability to gain more insight into the pathogenesis of FPD itself or leukemia progression of FPD/AML has been hampered by small patient numbers and the heterogeneity of the disease. Human induced pluripotent stem cells (iPSCs) derived from somatic cells hold promise to develop research models, especially for rare inherited and acquired diseases. Therefore, we obtained samples from a 45-year old female FPD/AML patient, who had inherited RUNX1 R174X mutation. She was diagnosed as acute myeloid leukemia at the age of 41 and received matched unrelated bone marrow transplantation, and was in remission for three years. Using primary dermal fibroblasts, we established induced pluripotent stem cells with heterozygous RUNX1 R174X mutation (FPD-iPSCs), by two-rounds of infection with pMX retrovirus encoding Oct4, Sox2, Klf4 and c-Myc. The morphology of FPD-iPSCs looked like embryonic stem cells. The endogenous stem cell genes were expressed. And the RUNX1 mutation derived from the primary patient cells was confirmed in the FPD-iPSCs. If iPSCs which harbor genomic abnormalities specific for the diseases are established, we can differentiate them and obtain the genetically abnormal disease cells continuously. To investigate the differentiation capacity of FPD-iPSCs for hematopoietic lineages, we first differentiated FPD-iPSCs into hematopoietic progenitor cells (HPCs) within the ‘unique sac-like structures’, which were called iPS-sacs, by co-culture in the presence of VEGF with C3H10T1/2 cells, a murine stromal cell line. After 15 days of culture, FPD-iPSCs produced the almost normal number of hematopoietic progenitors. It is an interesting observation because primitive hematopoiesis is impaired in murine RUNX1 deficient models, while adult hematopoietic stem cells/progenitors rather increase in number. CD34+/CD43+ hematopoietic progenitor cells (FPD-HPCs) propagated in the iPS-sacs were collected and analyzed about hematopoietic lineage differentiation capacity. First, colony forming capacity of FPD-HPCs was measured using CD34+/CD43+ cells by semisolid culture supplemented with SCF, GM-CSF, IL-3, G-CSF, and EPO. FPD-HPCs showed normal erythroid and myeloid colony forming capacities. On the other hand, megakaryocytic colony forming assay revealed that FPD-HPCs formed a significantly lower number of CD41+ CFU-Mk colonies than HPCs from normal iPSCs in culture supplemented with IL-3, IL-6 and TPO. Also, we analyzed the in vitro megakaryocyte differentiation of the FPD-HPCs on C3H10T1/2 cells with SCF, MGDF (megakaryocyte growth and differentiation factor) and heparin. Cytospin showed CD41a+/CD42b+ mature megakaryocytes from FPD-HPCs were morphologically normal. However, the ratio of CD41a+/CD42b+ megakarocytes to CD41a+/CD42b- immature megakaryocytes were reduced, showing that RUNX1 R174X blocks megakaryocytic maturation. We conclude that FPD-HPCs derived from FPD-iPSCs recapitulate the pathophysiology of FPD/AML on a cellular level. FPD-iPSCs would be an attractive tool to elucidate the pathogenesis of this disease, including leukemic transformation caused by additional somatic mutations.

Disclosures:
Kurokawa: Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.