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

2011; Elsevier BV; Volume: 118; Issue: 21 Linguagem: Inglês

10.1182/blood.v118.21.911.911

1528-0020

Stella T. Chou, Daniel VanDorn, Yu Yao, Deborah L. French, Mitchell J. Weiss,

Immunodeficiency and Autoimmune Disorders

Abstract 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.Table.Average number of colonies per 5000 CD41+/235+/43+ progenitors plated in methylcellulose assays for BFU-E and CFU-GM, and Megacult assay for CFU-MK. Standard deviation in parenthesesGenotype#patients#replicatesBFU-ECFU-GMCFU-MkEuploid31031 (+26)138 (+70)468 (+135)T2138417* (+201)52* (+32)574 (+211)T21+GATA1s5140** (+0)394** (+245)754 (+657)*p<0.05, euploid vs T21,**p<0.05 T21 vs T21+GATA1s. Disclosures: No relevant conflicts of interest to declare.