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Trisomy 21-Associated Defects in Human Primitive Hematopoiesis Revealed Through Induced Pluripotent Stem Cells

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Trisomy 21-Associated Defects in Human Primitive Hematopoiesis Revealed Through Induced Pluripotent Stem Cells Trisomy 21-associated defects in human primitive hematopoiesis revealed through induced pluripotent stem cells Stella T. Choua,1, Marta Byrska-Bishopb, Joanna M. Toberc, Yu Yaoa, Daniel VanDorna, Joanna B. Opalinskaa, Jason A. Millsd, John Kim Choie, Nancy A. Speckc, Paul Gadued,e, Ross C. Hardisonb, Richard L. Nemirofff, Deborah L. Frenchd,e, and Mitchell J. Weissa,1 aDivision of Hematology, eDepartment of Pathology and Laboratory Medicine, and dCenter for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; bDepartment of Biochemistry and Molecular Biology, Center for Comparative Genomics and Bioinformatics, Pennsylvania State University, University Park, PA 16802; and cAbramson Family Cancer Institute and Department of Cell and Developmental Biology, and fDepartment of Obstetrics and Gynecology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 Edited* by Stuart H. Orkin, Children’s Hospital and the Dana Farber Cancer Institute, Harvard Medical School and Howard Hughes Medical Institute, Boston, MA, and approved September 14, 2012 (received for review July 5, 2012) Patients with Down syndrome (trisomy 21, T21) have hematologic hematopoiesis is unknown and difficult to examine in human tissues abnormalities throughout life. Newborns frequently exhibit ab- at such early stages of embryogenesis. Moreover, murine models for normal blood counts and a clonal preleukemia. Human T21 fetal DS only partially recapitulate the hematopoietic abnormalities ob- livers contain expanded erythro-megakaryocytic precursors with served in humans (14–16). As an alternative approach, we examined enhanced proliferative capacity. The impact of T21 on the earliest the effects of T21 on embryonic hematopoiesis by studying human stages of embryonic hematopoiesis is unknown and nearly impos- induced pluripotent stem cells (iPSCs) with germ-line T21. sible to examine in human subjects. We modeled T21 yolk sac iPSCs generated by reprogramming somatic cells resemble ES hematopoiesis using human induced pluripotent stem cells (iPSCs). cells in their ability to self-renew in culture and generate nu- Blood progenitor populations generated from T21 iPSCs were merous mature cell types (17). In vitro differentiation of ES/iPSCs present at normal frequency and proliferated normally. However, recapitulates the ontogeny of hematopoiesis (18, 19). Human their developmental potential was altered with enhanced eryth- ES/iPSCs can be differentiated into erythroid cells expressing mainly ε-orγ/β-globins, likely reflecting primitive and definitive ropoiesis and reduced myelopoiesis, but normal megakaryocyte hematopoietic lineages, respectively (20–24). However, the cul- production. These abnormalities overlap with those of T21 fetal ture conditions and mechanisms that specify these distinct de- livers, but also reflect important differences. Our studies show that fi fi velopmental outcomes are not well de ned or understood. We T21 confers distinct developmental stage- and species-speci chema- analyzed iPSC lines from seven individuals (four T21, three eu- topoietic defects. More generally, we illustrate how iPSCs can pro- ploid) using an in vitro differentiation protocol optimized for vide insight into early stages of normal and pathological human primitive hematopoiesis (20). Our results further define the scope development. of T21 abnormalities during early human embryogenesis and illustrate that the associated hematopoietic defects are species- own syndrome (DS, trisomy 21, T21) affects many tissues, in- and developmental stage-specific. More generally, our findings Dcluding blood (1). Many DS neonates exhibit erythrocytosis, illustrate the power of iPSCs for studying the consequences of thrombocytopenia, and leukocytosis (2, 3). Approximately 10% genetic disorders on human ontogeny, particularly the earliest of DS newborns exhibit a clonal preleukemia, termed transient stages that are least accessible via primary tissue samples. myeloproliferative disease (TMD), which progresses to acute megakaryoblastic leukemia (AMKL) in ∼30% of cases. Both TMD Results and AMKL are accompanied by somatic mutations in the GATA1 Generation of iPSCs. iPSCs were derived from four T21 and three gene, causing production of an amino-truncated form of the tran- euploid control subjects (Table S1) by reprogramming somatic scription factor GATA-1 (reviewed in refs. 4 and 5). Importantly, tissues using four retroviruses, encoding OCT4, SOX2, KLF4,or somatic GATA1 mutations do not predispose to leukemia without MYC individually (17), or by a single polycistronic lentivirus en- T21 (6), and the most common DS blood abnormalities occur coding all four genes regulated by a doxycycline-inducible pro- without GATA1 mutations. Thus, T21 influences blood formation moter (25). Consistent experimental results were obtained using independently, particularly during embryogenesis (7, 8). These iPSC clones derived from different cell types and reprogramming derangements likely predispose to TMD/AMKL and could also vectors. All clones used in this study exhibited typical morphol- contribute to the early fetal demise that occurs throughout gesta- ogy, cell-surface expression of pluripotency markers (Tra1-60, tion in approximately one-third of T21 pregnancies (1). For these Tra1-81, SSEA3, SSEA4, KIT, KDR), expression of endogenous reasons, it is of biological and medical importance to fully define the pluripotency genes (ABCG2, DNMT3B, NANOG, OCT4, REX1), effects of T21 on blood formation at all stages of human ontogeny. During mammalian development, hematopoiesis occurs in multiple waves that differ with respect to timing, origin, and the Author contributions: S.T.C., J.M.T., P.G., D.L.F., and M.J.W. designed research; S.T.C., types of blood cells produced (reviewed in refs. 9 and 10). At about J.M.T., Y.Y., D.V., J.B.O., and J.A.M. performed research; S.T.C., M.B.-B., J.M.T., Y.Y., week 3 of human gestation, “primitive” blood cells produced by D.V., J.B.O., J.A.M., J.K.C., N.A.S., P.G., R.C.H., R.L.N., D.L.F., and M.J.W. analyzed data; the embryonic yolk sac are released into circulation. By weeks 4– and S.T.C., M.B.-B., J.M.T., N.A.S., R.C.H., R.L.N., D.L.F., and M.J.W. wrote the paper. MEDICAL SCIENCES 5, “definitive” progenitors emerge from the yolk sac and begin to The authors declare no conflict of interest. seed the fetal liver. After wk 5, hematopoietic stem cells emerge *This Direct Submission article had a prearranged editor. in the aorta-gonad-mesonephros (AGM) region and colonize the Data deposition: The microarray data reported in this paper have been deposited in the fetal liver, which becomes the major site of blood production Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. until birth, when hematopoiesis shifts to bone marrow (11–13). GSE35561). T21 alters hematopoiesis during embryonic development. 1To whom correspondence may be addressed. E-mail: [email protected] or weissmi@ Human T21 fetal livers with normal GATA1 alleles contain ex- email.chop.edu. panded megakaryocyte-erythroid progenitors, the progeny of which This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. exhibit enhanced proliferation (7, 8). How T21 impacts yolk sac 1073/pnas.1211175109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1211175109 PNAS | October 23, 2012 | vol. 109 | no. 43 | 17573–17578 Downloaded by guest on September 25, 2021 + + – silencing of viral encoded reprogramming genes, and production of (CD41 42 235 ) lineages by day 12 of differentiation (Fig. S2). three embryonic germ layers in teratomas (Fig. S1). Karyotype Colony-forming assays of flow cytometry-sorted cells from whole analysis of iPSCs (passages 11–21) showed no acquired abnormal- EBs performed on days 7–8 of differentiation showed that all + ities and confirmed T21 in the appropriate lines. Cells were pas- hematopoietic progenitors reside in the CD43 population (Fig. saged at least 20 times to avoid “memory effects” that could exert 1D). Erythroid colonies produced from iPSC progenitors ex- lineage bias to iPSC lines derived from different tissues (26, 27). press mainly ζ-andε-globin genes, indicative of primitive hema- topoiesis. In contrast, fetal liver-derived colonies express mostly Trisomy 21 iPSCs Produce Primitive Hematopoietic Progenitors at α-andγ-globin genes, reflecting definitive hematopoiesis (Fig. 1E). + + + Normal Frequency. We compared the blood-forming capacities of The CD43 41 235 progenitors were present at similar proportions T21 and euploid iPSC lines by generating embryoid bodies (EBs) within day 7–8 EB cultures generated from T21 and euploid iPSCs in defined media containing sequential combinations of cytokines (Fig. 1 C and F). Transcriptome analysis of flow cytometry-sorted + + + that support primitive streak/mesoderm formation, transition to CD43 41 235 progenitors showed remarkable similarity be- hematoendothelial progenitors, and terminal hematopoietic dif- tween T21 and euploid cells. Only 21 of 236 HSA21 genes were ferentiation (Fig. 1A), using modifications of a published protocol up-regulated ≥1.5-fold in T21 progenitors (Fig. 1G), consistent by Kennedy et al. (20). The kinetics for emergence of hemato- with complex lineage-specific patterns of gene-dosage imbalance poiesis varied slightly among different iPSC lines. We controlled in human and murine T21 tissues (30). Taken together, our for these differences by examining progenitors that were stage- results suggest that
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