Recapitulation of Transient Abnormal Myelopoiesis Using Patient Derived iPSCs
Authors: Nishinaka-Arai Yoko , Niwa Akira , Osawa Mitsujiro , Nakahata Tatsutoshi , Saito Megumu K 
Published: October 2019

Abstract
Down syndrome (DS) is characterized by the trisomy of chromosome 21 and complicated with multi-organ dysfunctions including the hematopoietic system. Among them, myeloproliferative disorder is known as a particular feature of the abnormality in hematopoiesis. At birth, about 10% of DS newborns show an extreme increase in blast cell number of peripheral blood and bone marrow, which is called transient abnormal myelopoiesis (TAM), because the blasts spontaneously disappear within 3 months. Morphologically, blast cells in TAM are similar to those typically found in acute megakaryoblastic leukemia (AMKL). Genetic analysis of blasts in TAM usually shows mutation in GATA1 gene. After spontaneous remission of TAM, 20 to 30% of TAM patients develop AMKL within several years. This type of AMKL is especially called DS related-AMKL (DS-AMKL).
This leukemogenic transition from TAM to AMKL is considered to be a typical model of multi-step tumorigenesis. In this model, we deal with the initial part of TAM development in relationship with trisomy 21 and GATA1 mutation, especially focusing on how GATA1 mutation promotes TAM development and why hematopoietic progenitors with GATA1 mutation prevail during embryonic hematopoiesis only in the cells with trisomy 21. In order to address these unsolved issues, we have established a strictly controlled human induced pluripotent stem cell (iPSC) lines derived from DS patients with or without TAM.
In this study, to recapitulate the phenotype of TAM and to specify the differentiation stage affected in hematopoietic cells in TAM patients, we differentiated established isogenic iPSC lines into megakaryocytes and erythrocytes in a step-wise manner. For this purpose, we employed two-dimensional differentiation system and compared the frequency of hematopoietic progenitor cells at various stages. For megakaryocytic lineage, we traced their differentiation as follows; hematopoietic progenitor cells committed to megakaryocytic lineage (day 9, CD34+CD41a+CD42b-CD235a-), megakaryoblasts (day 16, CD34-CD41a+CD42b-CD235a-) and promegakaryocytes (day 16, CD34-CD41a+CD42b+CD235a-). For erythrocytic differentiation, CD71+CD235a+ cells were defined as erythroid-committed hematopoietic cells.
On nine days after the initiation of hematopoietic differentiation (day 9), the frequency of CD41a+CD235a- cells showed no significant differences irrespective of the status of chromosome 21 and GATA1 genotype. However, on the day 16, while the frequency of promegakaryocytes significantly decreased in GATA1-mutated iPSCs, megakaryoblasts, an earlier stage cells than promegakaryocytes, were increased in GATA1-mutated iPSCs. These data suggest that megakaryocytic maturation is arrested in GATA1-mutated iPSCs at the stage of megakaryoblasts. In GATA1-non-mutated clones, iPSCs with trisomy 21 yielded erythroid-committed CD71+CD235a+ cells more frequently than those with disomy 21. However, in GATA1-mutated clones, either trisomy 21 or disomy 21 iPSC clones never yielded the erythroid-committed cells.
Taken these results together, we suspected that these in vitro phenotypes observed in both erythroid and megakaryocytic lineages were caused by the impairment of fate decision in their progenitor cells.
In conclusion, we successfully recapitulated the phenotypes of TAM in vitro in regard to the abnormal differentiation into megakaryocytic and erythroid lineages. We noticed that the in vitro phenotype were associated with the GATA1 genotype and the ploidy of chromosome 21. Considering these results, analyses of the megakaryocytic and erythroid progenitor cells, such as CMP, MEP and Mk-p, are important to determine which stage of progenitors is responsible for the impairment of hematopoietic cell maturation and subsequent TAM development. Moreover, we believe that the recapitulated TAM model using iPSCs is helpful for the comprehensive understanding of pathogensis in TAM.

Disclosures
No relevant conflicts of interest to declare.