Down syndrome-related transient abnormal myelopoiesis is attributed to a specific erythro-megakaryocytic subpopulation with GATA1 mutation

概要

Down syndrome-related transient abnormal myelopoiesis (TAM) is a temporal preleukemic state presenting the marked elevation of blast cells at birth.1 Although somatic GATA1 mutations are known to be a cause of TAM, it is still unclear in which immature hematopoietic progenitor cell (HPC) subpopulation they most strongly evoke abnormal proliferation.2 Considering the different properties of hematopoietic cells from different species, it is necessary to establish an appropriate human model applicable for studying TAM and its embryonic definitive hematopoietic origin. Recent pluripotent stem cell (PSC)-based models have reported the recapitulation of TAM phenotypes and suggested the implication of a specific gene locus on chromosome 21 in GATA1 mutation-driven perturbated hematopoiesis.3-5 However, little is known about which progenitor cells the subpopulation with abnormal myelopoiesis are primarily associated with the abnormal myelopoiesis due to the lack of chronological analysis following hematopoietic differentiation. In order to overcome this point, we previously used our step-wise hematopoietic differentiation system to trace the progenitors from mesoderm to lineage-committed HPC via very immature multipotent progenitors.6,7

In this study, we first examined the GATA1-dependent hematopoietic property using two independent isogenic trisomy 21 (Ts21) PSC pairs (Figure 1A): one is a TAM patient-blast-derived induced PSC (iPSC) pair (Online Supplementary Figure S1) 8 and the other is a human embryonic stem cells (ESC) pair with or without GATA1 mutation.9 We confirmed that the obtained genome-edited clones have no additional karyotype abnormality other than Ts21. No off-target mutations in the entire exonic sequence of GATA1 gene were observed by Sanger sequencing. GATA1-wild-type clones (WT-clones) expressed both full-length and short GATA1, while GATA1-mutated clones (G1s-clones) expressed only short GATA1. These G1s-clones showed abnormal hematopoiesis such as a decrease in CD71bright+CD42bCD235a+ erythroid and CD41+ CD42b+ CD235amegakaryocytic cells in step-wise hematopoietic differentiation (Online Supplementary Figure S2). On the other hand, CD34- CD235aCD41- CD43+ CD45+ myeloid-lineage cells from G1s-clones showed an increase not only in multi-lineage culture (Online Supplementary Figure 2AB) but also in myeloid-specific culture (data not shown).

In order to identify the relevant HPC subpopulation inducing these differences, we traced the differentiation efficacy and expression profiles using an hematopoiesisfocused PCR array during step-wise hematopoiesis (Figure 1B; Online Supplementary Figure 3; Online Supplementary Table S1).10 In mesodermal and early hematopoietic differentiation, WT- and G1s-clones showed similar efficiencies in the patterns of phenotype transition. KDR+ CD34+ hemoangiogenic progenitors were similarly observed in both clones on day 4, followed by CD34+ CD43+ HPC on day 6 (Online Supplementary Figure S4). On the other hand, later phase progenitors on day 9 were significantly different between WT- and G1s-clones. The frequency of CD34+ CD43+ CD235a- HPC was significantly higher in G1s-clones (Figure 1C). The day 9 HPC were further categorized into three subpopulations based on the expressions of CD11b (a representative marker of myeloid cells), CD71 (erythroid and megakaryocytic progenitors), and CD41 (megakaryocytic progenitors) as CD34+ CD43+ CD235a- CD11b+ CD41- (designated here as P-mye) (Figure 1D), CD34+ CD43+ CD235a-CD11bCD71+ CD41- (P-erymk41(-)) (Figure 1E), and C D 3 4 + C D 4 3 + C D 2 3 5 a - C D 1 1 b - C D 7 1 + C D 4 1 + (P-erymk41(+ )) (Figure 1F), respectively. Regarding the P-mye frequency, no significant difference was observed between WT- and G1s-clones at this stage. On the other hand, the P-erymk41(-) and P-erymk41(+) frequencies showed a significant difference between WT- and G1sclones, but in opposite directions: P-erymk41(-) was increased in G1s-clones and P-erymk41(+) in WT-clones. These data suggested the presence of some pivotal pathways in these two immature subpopulations that caused significant differences in later development, that is, increased myeloid lineages and impaired megakaryocytic maturation in G1s-clones on day 16. Thus, our observations indicated that GATA1 mutation distinctly affected the properties of late-stage CD34+ P-erymk progenitors rather than earlier-stage CD34+ progenitors.

In order to narrow down the responsible factors and their target subpopulations in day 9 CD34+ CD43+ CD235a- HPC, we performed correlation analysis using the first approximation to compare each subpopulation frequency on day 9 and the resultant lineage-committed cell number on day 16. As a result, only the P-erymk41(+) subpopulation on day 9 showed a significantly positive correlation with the number of both mature megakaryocytic cells on day 16 in WT-clones and immature megakaryoblastic cells in G1s-clones (Figure 1G, second and third panels from the left), and also showed a positive correlation with erythroid cells, which were observed only in WT-clones (Figure 1G, leftmost panel). These results suggested that P-erymk41(+) is most closely related to the normal erythroid and megakaryocytic differentiation of WT-clones and the impaired maturation of megakaryocytic lineage in G1s-clones. On the other hand, regarding myeloid lineages, no subpopulations showed a significant correlation in either WT- or G1s-clones (Figure 1G, fourth and fifth panels from the left), indicating that myeloid lineage cells on day 16 could not be attributed to a single HPC subpopulation on day 9 under erythroid-megakaryocytic differentiation condition.

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