Exploring the Pathogenesis of Down Syndrome-Related Myeloproliferative Disorders Using iPSCs

Down syndrome (DS) is a congenital syndrome due to the trisomy of chromosome 21. Transient myeloproliferative disorder (TMD) is its hematopoietic complication, affecting approximately 10% of DS-neonates. TMD is characterized by transient abnormal proliferation of blastic cells, and importantly, all TMD blasts bear mutations in GATA1 gene. Although TMD usually resolves spontaneously within 3 months after birth, twenty to thirty percent of TMD patients develop acute megakaryoblastic leukemia (AMKL) within several years afterward. This leukemogenic transition is considered as a good model for multi-step tumorigenesis. According to this putative multi-step model, the first hit should be additional chromosome 21, the second one is mutations on GATA1 gene which is requisite to the onset of TMD, and the “unknown” third hits are required for the progression into AMKL. However, it is still unclear that 1) how GATA1 mutation promotes TMD development, 2) what kinds of third hit are required for the onset of AMKL, and 3) why GATA1-mutated progenitors prevails during embryonic hematopoiesis only in trisomy 21 patients?

In order to address these issues, a strictly controlled isogenic cell panels that can reproduce human emboryonic hematopoietic development is needed. Human induced pluripotent stem cells (iPSCs) derived from DS patients are a promising platform for this, but so far there is no report regarding GATA1-mutated TMD-associated iPSCs. Therefore, we set out to establish an iPSC panel that covers each genomic status of chromosome 21 and GATA1 gene. For this, we established both GATA1 mutant and wildtype clones from both trisomy 21 and disomy 21 clones. And we also established TMD patient derived iPSCs.

First, we established isogenic iPSCs derived from EB virus immortalized B-lymphocytes of 2 mosaic DS patients. Frequency of trisomy 21 cells evaluated by FISH analysis and G-banding were 93% and 94% for each patient. We reprogrammed these cells by introducing 5 episomal vectors, pCE-hOCT3/4, pCE-hSK, pCE-hUL, pCE-mp53DD and pCXB-EBNA1, under feeder free condition. We genotyped each iPSC clones by digital-PCR analysis and found that the frequency of trisomy iPSC clones were comparable to that of trisomy cells in original EBV immortalized B-lymphocytes. There is no morphological difference between disomy and trisomy iPSC clones in both patients. We next introduced disease-associated GATA1 mutation into established isogenic trisomy and disomy iPS clones using transcription activator-like effector nuclease (TALEN) technology. We introduced a frameshift mutation in exon 2, which causes premature termination of the full-length transcript originated from 1^st ATG and exclusively produces the shorter isoform of GATA1 (GATA1s) transcribed from 2^nd ATG.

Next, we obtained peripheral blood mononuclear cells (PBMCs) from a TMD patient in order to establish TMD-blast-derived iPSCs. Eighty-nine percent of nuclear cells in the PBMC fraction was CD117+CD45+ blastic cells, whereas only 5.6% were non-blast cells including CD3 positive T-lymphocytes, CD11b positive myeloid lineage cells and CD19 positive B-lymphocytes. The CD117+ cells showed TMD/AMKL blast-like appearance such as coarse choromatin pattern with nucleolus and bleb-like structures. We sorted out CD45+CD117+ blastic cells and CD45+CD117- non-blastic cells and successfully established iPSC clones from both populations.

In conclusion, we successfully established a comprehensive panel of iPSC clones for evaluating the hematopoietic consequence associated with the GATA1 genotype and the ploidy of chromosome 21. We are currently evaluating hematopoietic differentiation potential of each clone and exploring the underlying pathophysiology of TMD/AMKL by using this platform. We believe that comprehensive understanding of TMD and AMKL pathogenesis provides a fruitful insight into our understanding of human leukemogenesis.