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 MEDICAL SCIENCES 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. 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 Early Edition | 1of6 Downloaded by guest on September 29, 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 hematopoiesis. 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 T21 and euploid iPSCs produce similar fre- matched by surface-marker expression. quencies of progenitors in the first wave of hematopoiesis to Beginning at days 4–5 of differentiation, a prehematopoietic emerge within EB cultures, and that T21 progenitors exhibit only cell population expressing KDR (VEGF-R) and CD31 (platelet subtle alterations in gene expression. endothelial cell adhesion molecule-1) was detected in EBs (Fig. 1C, Left). By days 7–9, the initial wave of hematopoietic progen- Altered Myelo-Erythroid Differentiation in T21 iPSCs. Between days itors, which coexpress CD43 (leukosialin), CD41 (integrin αIIb), 7 and 14 of EB differentiation, most hematopoietic progenitors and CD235 (glycophorin A) (28, 29), were present in EBs com- were released into the medium and underwent erythroid, mye- prising ∼25% of the total culture (Fig. 1C, Center). At this same loid, and megakaryocytic differentiation, as evidenced by flow time point, EBs released a relatively homogenous population of cytometry and cell morphology (Fig. 2 A and B, and Fig. S2). By + + + CD43 41 235 blood progenitors into the medium (Fig. 1 B and day 12, EB cultures from T21 iPSC lines produced about twofold + – C, Right). These progenitors differentiated into committed ery- increased proportion of erythroid (CD235 41 ) cells with a con- + – + + + + throid (CD235 41 ), myeloid (CD45 18 ), and megakaryocytic comitant decrease in the fraction of myeloid (CD45 18 ) cells,

Fig. 1. T21 iPSCs produce normal levels of early hematopoietic progenitors. (A) Schematic of hematopoietic differentiation protocol via EB formation with the following cytokines: BMP4, VEGF, SCF, TPO, FLT3, bFGF, EPO, IL-3, IL-11, and IGF1. (B) Photograph of iPSC-derived EB culture with hematopoietic cells released into the medium. Original magnification, 10×.(C) Flow + + cytometry analysis showing CD31 KDR hematoendothelial + − + + + precursor cells (Left) and CD34 / 43 235 41 progenitors within EBs (Center), and released into the medium (Right). (D) Meth- ylcellulose colony assays of various purified populations. Cyto- kines include SCF, IL-3, EPO, and GMCSF. Results show mean values ± SEM, n = 3 per group. (E) Globin gene expression in erythroid colonies from iPSCs or fetal liver (FL) determined by quantitative real-time PCR. Charts show fraction of α-(Left)or β-like (Right) genes. Ten to 20 colonies were pooled per sample, + + + n = 3 per group. (F) Frequency of CD43 41 235 progenitor cells in EB cultures on day 7–8 of hematopoietic differentiation (n = 14 and 11 independent experiments for euploid and T21 iPSCs, respectively. (G) Scatter plots of microarray data showing + + + average mRNA expression values in purified CD43 41 235 progenitors from between-group comparison of three euploid and three T21 biological replicate samples.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1211175109 Chou et al. Downloaded by guest on September 29, 2021 compared with controls (Fig. 2 A, C, and D). Morphologies of demonstrate that T21 iPSC-derived hematopoietic progenitors cells released from EBs at days 12 and 20 are shown in Fig. 2B.By exhibit increased propensity for erythroid differentiation, similar day 20, the T21 samples contained a greater proportion of ba- to DS fetal liver hematopoiesis (7, 8). However, in contrast to T21 sophilic erythroblasts and normoblasts but neutrophils were more fetal liver, T21 iPSCs did not generate increased megakaryocyte apparent in the euploid cultures. Of note, T21 fetal liver hema- progenitors or larger erythroid or megakaryocytic colonies com- topoietic cultures also exhibit enhanced erythroid differentiation pared with controls. Thus, T21 fetal liver and the iPSCs examined + + − (7, 8). The percentage of megakaryocytic (CD41 42 235 ) cells under the in vitro differentiation conditions specified here exhibit was similar in T21 and euploid cultures (Fig. 2 A, C, and D), which common abnormalities in blood formation and also notable dif- contrasts with findings in T21 fetal liver where megakaryocytic ferences. Most likely, the differences reflect distinct effects of T21 progenitors are expanded, suggesting developmental stage- on primitive vs. definitive hematopoiesis (see Discussion). specific hematopoietic patterns (7, 8). We assessed the colony-forming potential of day 7–9 EB-de- mRNA Expression in DS and Control iPSC-Derived Hematopoietic + + + rived CD43 41 235 progenitors in methylcellulose assays and Progenitors. As noted, transcriptome profiling showed remarkably + + + in collagen-based megakaryocyte assays. CD43 41 235 progeni- few differences in gene expression between iPSC-derived T21 + + tors were purified by flow cytometry from the entire culture (dis- and euploid CD41 235 progenitors (Fig. 1G). Applying a para- aggregated EBs plus the cells released into the medium) or ob- metric two-sample Student t test within the entire set of 19,392 + + + tained from the cells released by the EBs (90% CD43 41 235 on genes interrogated failed to identify differentially expressed genes days 7–9) (Fig. 1C, Right). The clonogenic potential varied among beyond the expected number of false positives. Focusing on HSA21, progenitors derived from different iPSC lines (31), but no con- which produces a relatively small proportion of transcripts within sistent differences were noted in the overall efficiency of colony the total dataset (1.2%), we found 71 differentially expressed formation between T21 and control cultures (Fig. 3 A and B). genes that passed a false-discovery rate (FDR) threshold of <0.2 However, T21 iPSC-derived progenitors produced a significantly (Table S2) (32), 13 of which lie within the “Down Syndrome increased proportion of erythroid colonies compared with con- Critical Region,” defined by studies of children with partial tri- trols (Fig. 3 A and C). In contrast, the majority of colonies formed somy 21 (33, 34) (Table 1, top section). Of these genes, DYRK1a, from euploid iPSC progenitors were myeloid (Fig. 3 A and C), in a regulator of calcineurin-nuclear factor of activated T cells sig- agreement with the suspension culture differentiation assays. We naling that has been implicated in DS pathology (35, 36), was up- observed no significant differences in the absolute numbers of regulated 1.3-fold with high statistical significance. Among HSA21 megakaryocyte colonies generated by T21 compared with euploid genes implicated in pathological DS hematopoiesis (reviewed in iPSCs (Fig. 3 B and C). Additionally, there was no consistent refs. 4 and 5), BACH1, GABPA,andSON,inadditiontoDYRK1a, difference in the size of erythroid or megakaryocyte colonies pro- were significantly up-regulated in T21 progenitors (range 1.25– MEDICAL SCIENCES duced by T21 progenitors compared with controls (Fig. 3E). The 1.50, P ≤ 0.05) but RUNX1, ERG, ETS2, and DCSR1 were not cell morphology of iPSC-derived erythroid and myeloid colonies (Table 1, middle section). Similar differences in expression were is shown in Fig. 3F. Similar results were obtained by analyzing observed with semiquantitative RT-PCR validation, with the ex- progenitor populations from whole EBs that were disaggregated ception that DSCR1 was up-regulated 2.4-fold in T21 iPSC-de- into single cells, although the clonogenicity was lower by ∼fivefold rived progenitors (P = 0.01) (Fig. S4). (Fig. S3). Importantly, the total number of colonies generated We separately examined 38 genes outside of HSA21 that play from T21 and euploid total EB cultures was equivalent, suggest- key roles in hematopoietic lineage determination and differen- ing that the primitive hematopoietic progenitor compartment is tiation (37, 38) (Table 1, bottom section, and Table S2). Thirty- not expanded with T21 (Fig. S3). Semiquantitative RT-PCR (Fig. five of these genes, including GATA1, GATA2, FOG1 (ZFPM1), 1E) of globin chains from iPSC-generated erythrocytes confirmed KLF1, PU.1 (SFPI1), CEBPa, IKZF1 (Ikaros), and GFI1B were primarily embryonic globin gene expression. Overall, our findings not differentially expressed between T21 and controls (P > 0.05). Among the cohort of 38 genes queried, the microarrays detected three with statistically significantly different expression: down- regulated CD34 (1.75×)andCSF1R (1.19×), and up-regulated MYB (1.18×). However, semiquantitative RT-PCR demonstrated no significant difference (Fig. S4). Thus, the T21 hematopoietic progenitors exhibit relatively small changes in the expression of numerous hematopoietic and HSA21 genes relative to euploid controls. Discussion iPSCs provide new opportunities to model human diseases and investigate early embryonic development. We used iPSCs to assess the effects of T21 at the onset of human hematopoiesis, which is difficult to study in equivalent 4- to 8-wk-gestation embryos. Based on stringent controls and consistent effects in multiple independent iPSC lines, our findings reflect the developmental influence of T21 and are not caused by artifacts of iPSC deriva- tion, culture, or clonal variation. The present findings are unique in indicating that T21 hematopoietic abnormalities begin in the embryonic yolk sac. Specifically, we show that T21 yolk sac-type hematopoiesis is skewed toward the production of erythroid cells, similar to what occurs in T21 fetal liver hematopoiesis (7, 8). Thus, increased erythroid mass may occur in early human T21 embryos, Fig. 2. Propensity for erythroid differentiation by T21 iPSCs. (A) Flow cytometry analysis of suspension cells in day 12 differentiation cultures showing which could potentially impair viability by increasing blood vis- − + mature hematopoietic lineages: erythroid (Ery, CD41 235 ), megakaryocytic cosity and contribute to the high rate of early spontaneous abor- + + + + (Meg, CD41 42 ), and myeloid (CD45 18 ). (B) May–Grunwald Giemsa- tion observed for this chromosomal aneuploidy (1). stained cells from EB suspension cultures at days 12 and 20. (Scale bars, In contrast to what was observed in studies of human fetal liver, 50 μm.) (C) Distribution of lineage-committed cells in EB suspension cultures T21 did not enhance megakaryopoiesis or increase the proliferative at days 12–14 of differentiation. n = 3–5 independent experiments per iPSC capacity of erythro-megakaryocytic precursors in the first wave of line. (D) Summary of data with all iPSC lines combined according to geno- hematopoiesis to emerge from iPSCs. Fetal liver contains de- type (n = 15 per group). finitive hematopoietic progenitors, but the iPSC-derived progenitors

Chou et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 + + + Fig. 3. Increased erythroid progenitors in T21 iPSC differentiation cultures. CD43 41 235 hematopoietic progenitors derived from three euploid and four T21 iPSC lines were analyzed. (A) Methylcellulose colony assays of CD43+41+235+ progenitors containing SCF, IL-3, EPO, GM-CSF, and (B) Colony-forming megakaryocyte (CFU-Mk) assays that include TPO, IL-3, and IL-6. Results show mean values ± SEM for three independent experiments per iPSC line. (C) Summary of data with all iPSC lines combined according to genotype for methylcellulose colony assays and (D) CFU-Mk assays. Results show mean values ± SEM, n = 9 for WT, n = 12 for T21. (E) Representative hematopoietic colonies from iPSC-derived progenitors. (Scale bars, 200 μm.) Meg, megakaryocyte. (F) Morphology of cells from iPSC-derived erythroid and myeloid colonies. May–Grunwald Giemsa stain. (Scale bars, 20 μm.)

analyzed in our study likely represent the yolk sac primitive progenitors. These authors demonstrate that T21 impacts both lineage. Of note, the iPSC-derived hematopoietic progenitors myeloid and lymphoid lineages. Taken together, these studies produced neutrophils, which are not generated during primitive better define T21 hematopoiesis throughout ontogeny, illustrating hematopoiesis in mice (39). This finding may reflect interspecies similarities and differences in three distinct, successive stages of differences in developmental potential of the primitive lineage or embryonic/fetal hematopoiesis. low-level definitive hematopoiesis occurring in our cultures. How- Notably, our group and Maclean et al. used similar protocols ever, colony and cell morphology, as well as globin analysis, indi- for differentiation of ESC/iPSCs (42), but produced blood line- cates that most of the hematopoietic progenitors examined in our ages representing distinct stages of ontogeny. Specifically, we study represent the primitive lineage, a transient population with derived primitive yolk sac-type hematopoietic progenitors and limited proliferative capacity compared with definitive progeni- Maclean et al. generated definitive fetal liver-type progenitors. tors (13). Failure of T21 to enhance the expansion of primitive Although our combined findings define T21 hematopoietic ab- progenitors indicates that this lineage does not likely give rise to normalities more comprehensively than either study could alone, DS-associated TMD or AMKL. More likely, DS-associated TMD/ the reason for the observed differences in iPSC developmental AMKL derives from definitive hematopoietic progenitors that output is unknown. Numerous properties of iPSCs, including originate from either late-stage yolk sac or the AGM region and gene-expression patterns, global DNA methylation, developmental migrate to the fetal liver, which provides an essential microen- capacity, and X chromosome inactivation state can vary, at least in vironment for expansion of TMD blasts (40, 41). part, from differences in culture methods (45–47). Indeed, prior Murine models are useful for the study of DS, but do not studies indicate that relatively small differences in ES/iPSC dif- recapitulate the developmental hematopoietic abnormalities of ferentiation protocols can affect the developmental stage of eryth- human T21. Alford et al. (14) and our own data (Fig. S5) show rocytes generated (23, 24, 48). These findings, along with the that fetal liver hematopoiesis is normal in the Tc1 and Ts65Dn present work, suggest that when patient iPSCs are used to model murine DS models, respectively. Ts65Dn mice, which are tri- human blood disorders, it is essential to stage the hematopoietic somic for 104 HSA21 genes and display some postnatal hema- progeny. Long term, it will be important to better define culture topoietic abnormalities that occur in DS (16), also exhibit normal conditions that reproducibly drive differentiation of iPSCs into yolk sac-primitive hematopoiesis (Fig. S5), contrasting with our primitive vs. definitive hematopoiesis. findings using human T21 iPSCs. Taken together, these studies We generated multiple human T21 iPSC lines that all exhibited indicate that murine models for DS do not recapitulate human enhanced erythropoiesis, an abnormality that appears to be T21 defects in developmental hematopoiesis within the yolk sac a consistent property throughout development in DS. Most likely, or fetal liver, highlighting the need for alternative models to multipotent progenitors are directed preferentially toward ery- study human T21 hematopoietic disorders. throid or erythro-megakaryocytic fates in T21-associated primi- Controlling developmental shifts in human iPSC hematopoi- tive and definitive hematopoiesis, respectively (7, 8, 42, 44, and etic differentiation is challenging and highly dependent on cul- the present study). This process must be initiated through altered ture methods. Our results show that iPSCs can be used to define HSA21-encoded genes that control hematopoietic fate directly the effects of T21 on the earliest stage of human hematopoiesis and through intermediary factors. In comparing the transcript- + + + (i.e., yolk sac-derived primitive). In an accompanying article, omes of CD43 41 235 progenitors generated from T21 and MacLean et al. (42) generated definitive, but not primitive pro- euploid iPSCs, only a few differentially expressed genes stand out genitors from T21 ES and iPSCs (42). This study likely recapit- as obvious candidates to explain the lineage-preference phe- ulates late yolk sac or fetal liver-derived definitive hematopoiesis, notypes (Table 1). These candidates include HSA21 genes reflecting a later wave of blood formation in embryos (43). A third BACH1, SON, GABPA, and DYRK1A, all of which exhibited accompanying article by Roy et al. analyzes second trimester fetal relatively small (≤ 1.5-fold) but significant up-regulation in T21 liver hematopoiesis (44), which represents AGM-derived definitive progenitors. The small differences in expression of numerous

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1211175109 Chou et al. Downloaded by guest on September 29, 2021 Table 1. Transcriptome analysis of T21 vs. euploid iPSC-derived progenitors Gene symbol Chromosome Fold-change P value FDR

Differentially expressed genes located in DS critical region SH3BGR chr21 1.66 (+) 0.0050 0.07 BRWD1 chr21 1.64 (+) 0.0287 0.14 DSCR3 chr21 1.60 (+) 0.0579 0.19 MORC3 chr21 1.55 (+) 0.0024 0.06 BACE2 chr21 1.54 (+) 0.0136 0.09 HLCS chr21 1.54 (+) 0.0141 0.09 WRB chr21 1.52 (+) 0.0041 0.07 TTC3 chr21 1.39 (+) 0.0091 0.08 PIGP chr21 1.35 (+) 0.0288 0.14 DYRK1A chr21 1.34 (+) 0.0027 0.06 PSMG1 chr21 1.27 (+) 0.0066 0.07 HMGN1 chr21 1.22 (+) 0.0223 0.12 SETD4 chr21 1.22 (+) 0.0045 0.07 Chromosome 21-encoded hematopoietic regulators BACH1 chr21 1.50 (+) 0.0313 0.15 RUNX1 chr21 1.38 (+) 0.1074 0.30 ETS2 chr21 1.36 (+) 0.1605 0.37 GABPA chr21 1.29 (+) 0.0023 0.06 SON chr21 1.25 (+) 0.0141 0.09 RCAN1 (DSCR1) chr21 1.16 (+) 0.1455 0.35 ERG chr21 1.68 (−) 0.2175 0.42 Genes involved in hematopoietic lineage determination and differentiation GATA2 chr3 1.20 (+) 0.1230 0.78 MYB chr6 1.18 (+) 0.0003 0.01 IKZF1 (Ikaros) chr7 1.07 (+) 0.6739 0.95 MEDICAL SCIENCES SPI1 (PU.1) chr11 1.02 (+) 0.9308 0.95 CEBPA chr19 1.02 (−) 0.8777 0.95 KLF1 chr19 1.04 (−) 0.7328 0.95 GFI1B chr9 1.05 (−) 0.5540 0.95 ZFPM1 (FOG1) chr16 1.06 (−) 0.3857 0.95 GATA1 chrX 1.08 (−) 0.2710 0.95 CSF1R (C-FMS) chr5 1.19 (−) 0.0150 0.29 CD34 chr1 1.75 (−) 0.0443 0.56

Differential gene expression between T21 and euploid iPSC-derived progenitors is analyzed for two separate comparisons: genes located on chromosome 21 (FDR < 0.2; top two portions of table) and 38 selected genes outside of HSA21 that play key roles in hematopoietic lineage determination and differentiation (bottom portion of table). “Fold-change” is the level of up-regulation (+) or down-regulation (−) of mean gene expression in T21 with respect to euploid cells. P values were obtained from a parametric two-sample Student t test comparing differences in mean expression between T21 and euploid cells (n = 3 distinct iPSC lines per group). FDR values were obtained using the Benjamini–Hochberg-FDR method. Genes encoding regulators of hematopoiesis with signiﬁcant differences in expression are in boldface.

genes likely produce an aggregate effect. Our finding that only previously described (17). Mononuclear cells were infected with pHage2- a subset of HSA21 genes are overexpressed in T21 hematopoi- CMV-RTTA-W and pHage-Tet-hSTEMMCA-loxP virus, as previously described etic progenitors is consistent with prior studies demonstrating (25). Reprogramming details and pluripotency characterization methods are that in DS models, increased dosages of HSA21 are complex and provided in SI Materials and Methods. tissue type-specific (30, 49). Although gain- and loss-of-function studies are required to identify the HSA21 genes that contribute Hematopoietic Differentiation. iPSCS were feeder-depleted and EBs generated to erythroid expansion in T21, standard approaches using by standard methods as described in ref. 20. EBs were cultured in StemPro-34 siRNA knockdown or cDNA overexpression may create artifacts (Invitrogen) media supplemented with 2 mM glutamine, 50 mcg/mL ascorbic by altering expression beyond physiologic ranges or at in- acid, 150 mcg/mL transferrin, 0.4 mM monothioglycerol, and with bone morphogenic protein 4 (BMP4) 25 ng/mL, VEGF 50 ng/mL (day 0–2); BMP4 25 appropriate times during development. Genetic manipulation of fi ng/mL, VEGF 50 ng/mL, stem cell factor (SCF) 50 ng/mL, thrombopoietin (TPO) iPSCs using zinc- nger and transcription activator-like effector 50 ng/mL, FLT3-ligand (FLT3) 50 ng/mL, bFGF 20 ng/mL (day 2–4); VEGF 50 ng/ nucleases should provide more physiological approaches to pre- mL, SCF 50 ng/mL, TPO 50 ng/mL, FLT3 50 ng/mL, bFGF 20 ng/mL (day 4–8); SCF cisely manipulate copy numbers of candidate HSA21 genes 50 ng/mL, TPO 50 ng/mL, IL-3 10 ng/mL, IL-11 5 ng/mL, erythropoietin (EPO) 2 suspected to alter hematopoiesis (50, 51). Similar approaches U/mL, and insulin growth factor-1 (IGF1) 25 ng/mL (day 8+). All cytokines ex- using directed differentiation of T21 iPSCs into other lineages, cept EPO (Amgen) and bFGF (Invitrogen) were purchased from R&D Systems.

including neurons, endothelial, and cardiac cells, should further Cultures were maintained at 37 °C, 5% CO2,5%O2, and 90% (vol/vol) N2. elucidate how altered HSA21 gene dosages disrupt tissue formation and function in DS. Hematopoietic Colony-Forming Assays. CD43+41+235+ cells were seeded into H4230 methylcellulose (Stem Cell Technologies) with EPO 5 U/mL, IL-3 10 ng/ Materials and Methods mL, SCF 5 ng/mL, and GMCSF 5 ng/mL, at 2,000–5,000 cells/mL (or 10,000 Generation of iPSCs. Fibroblasts/stromal cells were transduced with pMXs- cells/mL for total EB cells). Colonies were scored at 12 d. 2,000–5,000 cells/mL based retroviral supernatant with human OCT4, SOX2, KLF4,orMYC,as (or 10,000 cells/mL for total EB cells) were seeded into Megacult-C (Stem Cell

Chou et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 Technologies) with TPO 50 ng/mL, IL-6 10 ng/mL, and IL-3 10 ng/mL. After 12 by the Bioconductor R “oligo” package (53, 54). Robust Multichip Average d, cultures were dehydrated, fixed, and stained with anti-GPIIb/IIIa antibody. processing was applied to the whole dataset, which resulted in 33,297 pro- Large CFU-Mks were defined as having >50 positively stained cells. besets that were then mapped to 19,392 RefSeq genes. In cases where several probe sets mapped to one RefSeq gene, the expression values were Semiquantitative Real-Time PCR. RNA was isolated with the RNeasy kit averaged to obtain one value per gene. Additional methods are provided in (Qiagen), cDNA prepared by the oligo(dT) method (Invitrogen), and PCR- SI Materials and Methods. quantified using SYBR green (Roche) on a Light Cycler 480II System (Roche). For all genes except globin, expression was normalized to cyclophilin, and ACKNOWLEDGMENTS. We thank Darrell Kotton and Gustavo Mostoslavsky fi relative quanti cation determined by comparative CT method. For globin for the reprogramming vectors; Nancy Spinner for the DS1 fibroblast cell quantification, expression was determined by standard-curve quantification line; Danielle Crawford and Sandra Ryeom for assistance in mouse studies; using genomic DNA, and expressed as a fraction of α-orβ-like genes. Primers and the Children’s Hospital of Philadelphia human embryonic and induced sequences are provided in SI Materials and Methods. pluripotent stem cells core facility, located in the Center for Cellular and Molecular Therapeutics, for technical and scientific advice. This work was supported by National Institutes of Health Grants K08 HL093290 (to S.T.C.), Microarray and Bioinformatic Analysis. RNA was isolated using an RNeasy kit RC2 HL10166 (to M.J.W.), P30 DK090969 (to M.J.W. and S.T.C.), R01 (Qiagen) and hybridized to Affymetrix HuGene 1.0 ST microarrays. To obtain HL091724 (to N.A.S.), and R01 DK065806 (to R.C.H.). S.T.C. is an American gene-expression values, raw intensity values from Affymetrix CEL data files Society of Hematology Scholar. J.M.T. is a Special Fellow of the Leukemia were processed using the Robust Multichip Average method (52), implemented and Lymphoma Society.

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