Patient-Derived Induced Pluripotent Stem Cells Reveal Distinct Hematopoietic Defects Conferred by Trisomy 21 and Truncated GATA-1

Abstract 911

Infants with Down syndrome (DS, trisomy 21, T21) frequently exhibit hematological abnormalities including polycythemia and/or thrombocytopenia. About 10% of DS infants develop transient myeloproliferative disease (TMD), which usually self-resolves. However, approximately 30% of affected patients develop acute megakaryoblastic leukemia (AMKL) by age 4 years. Both TMD and AMKL are accompanied by somatic GATA1 gene mutations that give rise to GATA-1s (for “short”), an amino-truncated protein lacking amino acids 1–81. Thus, DS-associated AMKL requires at least three sequentially occurring genetic abnormalities in hematopoietic cells: germline T21, somatic GATA1 mutations in fetal progenitors, and postnatal mutations in additional, currently unidentified genes. To analyze this malignant progression step by step and better understand T21-associated hematopoietic abnormalities, we created induced pluripotent stem cells (iPSCs) from DS subjects (n=3), TMD blasts (n=5) and controls (n=3). All iPSC lines exhibited signature features of pluripotency and retained their relevant genotypes: T21, T21+GATA1s and normal euploid. We compared the blood-forming capacities of iPSC lines by generating embryoid bodies in defined medium containing hematopoietic cytokines. Stage-matched embryoid bodies of each genotype produced similar numbers CD41+/235+/43+ hematopoietic progenitors capable of erythroid, myeloid and megakaryocytic differentiation. However, in methylcellulose colony assays, progenitors from DS iPSCs contained 13.5-fold increased numbers of burst forming unit erythroid (BFU-E) progenitors compared to control iPSCs (p=.009) (Table). While the absolute numbers of colony forming unit-megakaryocytes (CFU-MK) were similar between DS and wild type iPSC-derived progenitors (p=0.21), the CFU-MK:CFU-myeloid ratio was increased in progenitors from DS iPSCs (p=0.014). Thus, DS iPSC-derived hematopoietic progenitors exhibit increased propensity for erythro-megakaryocytic differentiation, similar to what occurs in DS fetal liver (Chou et al; Tunstall-Pedoe et al, Blood v112, 2008). In contrast, CD41+/235+/43+ progenitors from all 5 DS TMD iPSC lines studied (T21/GATA1s) exhibited complete absence of erythroid developmental potential in liquid culture and methylcellulose colony assays (p<.001), despite robust production of myeloid and megakaryocytic cells. We confirmed this observation by comparing the hematopoietic potential of iPSCs generated from TMD blasts (T21/GATA1s) and normal blood cells (T21/GATA1wt) of the same DS patient (n = 2 different individuals). In each case, acquisition of the GATA1s mutation selectively blocked erythropoiesis and tended to increase megakaryopoiesis. Thus, the amino terminus of GATA-1, absent in GATA-1s, is required for primitive (yolk-sac type) erythropoiesis, the developmental stage that is recapitulated in our iPSC differentiation protocols. In agreement, loss of the GATA-1 amino terminus inhibits primitive erythropoiesis in mice (Li et al, Nature Genetics v37, 2005). Our findings illustrate distinct hematopoietic defects conferred by T21 and GATA-1s, and suggest how these might synergize in TMD and AMKL. More generally, our studies illustrate how analysis of patient-derived iPSCs can be used to analyze genetic blood disorders, particularly those that arise during fetal development.