Perturbation of Fetal Liver Hematopoietic Stem and Progenitor Cell Development by Trisomy 21

Perturbation of fetal liver hematopoietic stem and progenitor cell development by trisomy 21

Anindita Roya,1, Gillian Cowana,1, Adam J. Meadb, Sarah Filippic, Georg Bohna, Aristeidis Chaidosa, Oliver Tunstalla, Jerry K. Y. Chand,e, Mahesh Choolanid,e, Phillip Bennettf, Sailesh Kumarf, Deborah Atkinsonb, Josephine Wyatt-Ashmeadg, Ming Hua, Michael P. H. Stumpfc, Katerina Goudevenoua, David O’Connora, Stella T. Chouh, Mitchell J. Weissh, Anastasios Karadimitrisa, Sten Eirik Jacobsenb, Paresh Vyasi, and Irene Robertsa,2

aCentre for Haematology and fInstitute of Reproductive and Developmental Biology and Centre for Fetal Care, Imperial College London, London W12 0NN, United Kingdom; bHaematopoietic Stem Cell Laboratory, Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, United Kingdom; cCentre for Bioinformatics, Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, United Kingdom; dExperimental Fetal Medicine Group, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119077; eDepartment of Reproductive Medicine, KK Women’s and Children’s Hospital, Singapore 229899; gDepartment of Pathology, Imperial College Healthcare National Health Services Trust, London W2 1NY, United Kingdom; hDivision of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104; and iMedical Research Council Molecular Haematology Unit and Department of Haematology, Weatherall Institute of Molecular Medicine, University of Oxford and Oxford University Hospitals, National Health Services Trust, Oxford OX3 9DS, United Kingdom

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 17, 2012 (received for review July 4, 2012) The 40-fold increase in childhood megakaryocyte-erythroid and GATA1 mutation (11, 12). Specifically, the MK-erythroid pro- B-cell leukemia in Down syndrome implicates trisomy 21 (T21) in genitor (MEP) population is expanded with increased cell-intrinsic + perturbing fetal hematopoiesis. Here, we show that compared MK and erythroid lineage proliferation from CD34 cells. These with primary disomic controls, primary T21 fetal liver (FL) hema- data suggest that T21-mediated developmental alterations to FL topoietic stem cells (HSC) and megakaryocyte-erythroid progeni- myeloid progenitor development provide a cell-specificsubstrate tors are markedly increased, whereas granulocyte-macrophage for selection and expansion of mutant GATA1 clones. This finding progenitors are reduced. Commensurately, HSC and megakaryo- is consistent with results in mice genetically engineered to ex- cyte-erythroid progenitors show higher clonogenicity, with in- press N-terminal truncated GATA1 protein, which develop an creased megakaryocyte, megakaryocyte-erythroid, and replatable altered MK lineage proliferation/differentiation phenotype, re- blast colonies. Biased megakaryocyte-erythroid–primed gene ex- stricted to FL progenitors and not seen in adult mice (13). pression was detected as early as the HSC compartment. In lym- The abnormalities in the myeloid progenitor compartment of phopoiesis, T21 FL lymphoid-primed multipotential progenitors T21 FL would be consistent with one of at least two contrasting and early lymphoid progenitor numbers are maintained, but there scenarios. The first scenario is that effects of T21 are confined to was a 10-fold reduction in committed PreproB-lymphoid progeni- the myeloid progenitor compartment, which through the com- tors and the functional B-cell potential of HSC and early lymphoid bination of T21 and mutant GATA1 acquires increased self-re- progenitor is severely impaired, in tandem with reduced early lym- newal and a selective growth advantage; these cells would be phoid gene expression. The same pattern was seen in all T21 FL predicted to have a relatively short lifespan, which could account samples and no samples had GATA1 mutations. Therefore, T21 itself for frequent spontaneous resolution of TMD (∼80%) within a few causes multiple distinct defects in FL myelo- and lymphopoiesis. months of birth (14). An alternative model, given the increased risk of childhood B-ALL and B-cell immune deficiency (4, 15), is transient myeloproliferative disorder | aneuploidy | human fetus that T21 might perturb hematopoiesis at the hematopoietic stem cell (HSC) or multipotential progenitor level. To distinguish onstitutional trisomy 21 (T21) causes Down syndrome (DS), between these two possibilities, we performed detailed immu- Cthe most common syndrome-associated chromosomal anom- nophenotypic and functional analysis of the HSC/multipotential aly in humans (1). As well as with neurodevelopmental, cardiac, progenitor compartment and committed myeloid and B-lym- and gut anomalies (2), there is a striking increase in childhood phoid compartments of T21 FL without GATA1 mutations and acute leukemia in DS, even though the risk of solid tumors is compared these with normal FL. Here, we are unique in reporting much lower than with the general population (3). Intriguingly, that in human FL, T21 itself increases immunophenotypic HSC this susceptibility to hematopoietic tumors manifests as an in- frequency, clonogenicity and MK-erythroid output with associated creased risk both of acute megakaryocyte (MK)-erythroid leuke- MEP expansion, and severe impairment of B-lymphoid de- mia (known as ML-DS) by 150-fold and of acute B-lymphoblastic velopment. Perturbation of FL HSC/progenitor proliferation and leukemia (B-ALL) by 33-fold (3, 4). lineage commitment may underlie the striking susceptibility of DS leukemias display distinct characteristics that support a T21 hematopoietic cells to both myeloid and lymphoid leukemic crucial role for T21 in their pathogenesis. Hallmarks of ML-DS transformation and, together with human T21 ES and induced are the megakaryoblastic phenotype, clinical presentation confined to the first 5 y of childhood (5, 6), an antecedent clonally linked preleukemic condition (termed transient myeloprolifer- Author contributions: I.R. designed research; A.R., G.C., A.J.M., G.B., A.C., O.T., D.A., J.W.-A., M.H., K.G., and D.O. performed research; J.K.Y.C., M.C., P.B., and S.K. contributed new ative disorder, TMD) in most cases, and acquired N-terminal reagents/analytic tools; A.R., G.C., A.J.M., S.F., M.P.H.S., S.T.C., M.J.W., A.K., S.E.J., P.V.,

truncating mutations in the erythroid-MK transcription factor and I.R. analyzed data; and A.R., G.C., A.J.M., S.F., J.K.Y.C., M.C., P.B., S.K., M.P.H.S., S.T.C., MEDICAL SCIENCES GATA1 (7–9). Such mutations in GATA1 are present in both M.J.W., A.K., S.E.J., P.V., and I.R. wrote the paper. ML-DS and TMD (9) but are not found in patients without DS The authors declare no conﬂict of interest. who develop megakaryoblastic leukemia (7) and are not leuke- *This Direct Submission article had a prearranged editor. mogenic in the absence of T21 (10). Freely available online through the PNAS open access option. Molecular, biologic, and clinical data indicate that TMD is 1A.R. and G.C. contributed equally to this work. initiated before birth (9, 11–14). We previously reported that by 2To whom correspondence should be addressed. E-mail: [email protected]. the second trimester, the T21 fetal liver (FL) myeloid progenitor This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. compartment is abnormal and that this occurs in the absence of 1073/pnas.1211405109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1211405109 PNAS | October 23, 2012 | vol. 109 | no. 43 | 17579–17584 Downloaded by guest on September 25, 2021 pluripotent stem cells (iPSCs), T21 FL provides a tractable model progenitor frequency also reﬂect changes in absolute numbers of for dissecting T21-mediated leukemia. these populations in T21 FL.

Results Increased Clonogenicity and MK-Erythroid Potential of T21 FL HSC/ Perturbation of HSC/Progenitor Frequency in T21 FL. We previously Progenitors. Next, we performed in vitro clonogenic assays of found marked MEP expansion, reduced granulocyte-macrophage these immunophenotypic HSC and progenitor populations (sort- progenitors (GMP), and qualitative common myeloid progenitor ing strategy shown in Fig. S2). This process showed increased (CMP) abnormalities in T21 FL in the absence of GATA1 mu- clonogenicity of HSC (fivefold P < 0.01), CMP (2.7-fold P < tation (11, 12). To investigate whether T21 causes wider pertur- 0.05), and MEP (2.5-fold P < 0.05) (Fig. 2A). Lineage output bation of T21 FL hematopoiesis, we used the same scheme to from HSC, MPP, CMP, and MEP showed marked skewing to- define the HSC/progenitor hierarchy as established for cord blood ward the MK lineage with increased MK and MK-erythroid B and adult marrow (16, 17) and compared the HSC/multipotential colonies (Fig. 2 and Table S1). Furthermore immunohisto- fi progenitors (MPP)/lymphoid-primed multipotential progenitors chemistry of FL sections con rmed the marked absolute increase (LMPP) compartment in T21 FL (n = 8; median gestation in MKs (Fig. S1). T21 FL HSC also generated increased absolute 16 wk) with normal FL (n = 13; median gestation 15 wk) by flow numbers of erythroid burst-forming units (BFU-E), given that overall clonogenicity was higher in T21 HSC (Table S1), although cytometry. There was a 3.5-fold increase in immunophenotypic B HSC frequency in T21 compared with normal FL (7.9 ± 0.9% vs. the relative proportion was similar to normal FL (Fig. 2 ). Two types of blast-cell colony (Blast-My and Blast-E) were 2.3 ± 0.4%; P = 0.0025) but MPP and LMPP frequency were identified morphologically in normal and T21 FL HSC, MPP, and preserved and we confirmed the changes in MEP and GMP CMP. Blast-E were also generated by FL MEP (Fig. 2 B–F). Blast-E, frequency we found previously (11, 12) (Fig. 1). The same pat- small compact hemoglobinized colonies (Fig. 2C) appearing after tern was seen in all T21 FL samples and no samples had GATA1 + + day 14 of culture (in contrast to BFU-E, which were easily seen by mutations. There was a marked reduction in CD34 CD19 day 10), contained late and early normoblasts, blast-like cells, and committed B progenitors (CBP) in T21 compared with normal D ± ± P = macrophages (Fig. 2 ), coexpressed CD34 and GlyA in contrast FL (3.1 1.3% vs. 10.7 1.3%; 0.0026), suggesting a block to BFU-E (Fig. 2E) and, unlike BFU-E, had high replating effi- to B-lymphoid differentiation because early lymphoid progenitor + + − − ciency, generating solely erythroid colonies (BFU-E) and no (ELP) (CD34 CD127 CD10 CD19 ) frequency was normal myeloid colonies. Neither Blast-E–derived BFU-E nor BFU-E (Fig. 1). These data show multiple defects in T21 FL hemato- arising directly from plated FL HSC/MPP/CMP or MEP were poiesis, characterized by expansion of immunophenotypic HSC replatable (Fig. 2F), suggesting they lie immediately upstream of and MEP with commensurate reductions in GMP and B-pro- + BFU-E. Absolute numbers of Blast-E colonies were increased in genitors. Because the frequency of CD34 cells was the same in T21 FL HSC, CMP, and MEP because overall clonogenicity was T21 FL as normal FL, both by flow cytometry (3.1 ± 0.8% vs. 4.9 ± higher (Table S1), although the relative proportion was similar to 0.9% of mononuclear cells; T21 vs. normal; P = 0.185) and im- normal FL (Fig. 2B). Blast-My, which appeared on day 10–12 of munohistochemistry of FL sections (15.1 ± 2 vs. 17.7 ± 4.7/100 culture and did not hemoglobinize even after 4 wk (Fig. 2C), high-power fields; P = 0.624) (Fig. S1), these differences in HSC/ contained almost exclusively myeloid blast cells (Fig. 2D), coexpressed CD34 and mature myeloid markers (Fig. 2E), and had high replating efficiency, generating CFU-GM, BFU-E, and BFU- F A NORMAL T21 MK, which had no further replating ability (Fig. 2 ), suggesting 0 3.8% that they lie immediately upstream of CFU-GEMM. Blast-My 0.0310.7% 0 96.2% were increased in T21 FL HSC and CMP compared with normal 77.3% 0.0389.2% 68.4% FL (Fig. 2B). Although Blast-My are detectable in normal cord

10.7% 8 8 13.2% 19 blood and adult bone-marrow HSC and myeloid progenitors, 3 D D CD3 C Blast-E appear to be fetal in origin because we do not detect them CD19 C CD34 CD34 CD34 CD34 in adult bone marrow (Table S2).

HSC 28.3 0.2% 32.1 27.3% HSC 22 14.8% 51.9 19.5% 21.5 19.1% 56.17.1% GMP Impaired B-Lymphoid Differentiation of T21 HSC, LMPP, and ELP. To GMP CMP CMP characterize the B-progenitor defect we performed more de- LMPP + + tailed immunophenotypic analysis of CBP (CD34 CD19 ). Both MEP D90 54.5 1.97% MEP + + − D90 MPP C LMPP MPP 34.4 10.1%

C immature CBP, CD34 CD19 CD10 (PreproB), and mature CD123 CD123 + + + CBP, CD34 CD19 CD10 (ProB), were seen in normal FL (Fig. CD45RA CD45RA CD45RA CD45RA + + 3 A and B), as in cord blood (18), and CD19 /CD20 B cells were easily seen in normal FL (Fig. 3 C and D, and Fig. S1). Normal B ** − + 60 FL HSC, LMPP, and ELP generated CD34 CD19 B cells in Normal T21 MS5 cocultures consistent with FL as a site of normal prenatal B-cell development (Fig. 3E). In T21 FL, both PreproB (0.33 ± 4+ 40 3 ± ±

D 0.1 vs. 3.5 0.5%; T21 vs. normal) and ProB progenitors (3.1

C *

f 0.5 vs. 7.1 ± 1.0) were reduced (Fig. 3 A and B), as were mature ** + 20 ** + C D

%o (CD19 /CD20 ) B cells (Fig. 3 and , and Fig. S1). Moreover, T21 FL HSC demonstrated marked impairment in B-cell dif- − + 0 ferentiation with very few CD34 CD19 cells after 14 or 21 d in P P P P P P E SC P M E M MS5 cocultures (Fig. 3 ). T21 FL LMPP and ELP MS-5 cocul- H MP ELP CB M + LM C G tures also failed to generate CD19 cells apart from transient + occasional CD19 cells from ELP after 7 d but not thereafter Fig. 1. Perturbation of T21 FL HSC/progenitor frequency. (A) Representative + + (Fig. 3E). In contrast, there was no difference in T21 FL T plots from normal FL (n = 13) and T21 FL (n = 8) CD34 cells gated on CD34 + + + − + lo/− progenitor frequency (CD34 CD4 cells: 5.5 ± 1.9 vs. 3.7 ± 0.9% CD38 CD19 cells for CMP, MEP, and GMP, and on CD34 CD38 cells for + P = HSC, MPP, and LMPP. (B) Mean + SEM HSC and progenitor frequencies in of CD34 cells; normal vs. T21; 0.70). These data suggest normal (n = 13; light bars) and T21 (n = 8) FL (dark bars) showing increased speciﬁc impairment of B-cell development in T21 FL which is HSC and MEP frequency and reduced CBP and GMP. **P < 0.01; *P < 0.05. evident at all stages of B-cell differentiation.

17580 | www.pnas.org/cgi/doi/10.1073/pnas.1211405109 Roy et al. Downloaded by guest on September 25, 2021 Normal A B HSC MPP LMPP CMP MEP GMP 100 T21 * BFU-E ** * 80 NORMAL Blast - E CFU-GEMM 60 CFU- GM M H M Blast-My 40 MK T21 20 MkE colonies/100 cells

HSC MPP MEP LMPP CMP GMP C NORMAL T21 CFU-GM Blast-My BFU-E Blast -E MK MkE CFU-GM Blast-My BFU-E Blast -E MK MkE

D 6 6 1 1 / E / 5 5 1 1 / 4/ 14 1 GLYA D GLYA C GLYA CD GLYA CD34 CD34 CD41a CD34 CD34 CD41a

F BFU-E 100

100 ng ng CFU-GM a l 50 MK

p 50 epla r re

0 % 0 % CFU-GM Blast-My BFU-E Blast -E MK MkE CFU-GM Blast-My BFU-E Blast -E MK MkE

Fig. 2. Increased clonogenicity and megakaryocyte/erythroid potential of normal and T21 FL HSC and progenitors. (A) Clonogenicity of ﬂow-sorted HSC and progenitors from normal (n = 8) and T21 (n = 5) FL (mean + SEM). Cells (100 cells/mL) were plated in Methocult H4230 with IL-3, IL-6, IL-11, SCF, FLT3, GM-CSF, TPO, and EPO. Clonogenicity of T21 HSC, CMP, and MEP was increased compared to normal FL. **P < 0.01; *P < 0.05. (B) Lineage read out of clonogenic data in A showing increased MK and MK-erythroid (MkE) colonies from T21 FL HSC, MPP, CMP, and MEP, and Blast-My colonies from T21 FL LMPP compared to normal FL. T21 HSC generated increased absolute numbers of CFU-MK, MkE, BFU-E, Blast-E, and Blast-My compared to normal FL HSC and T21 CMP and MEP increased CFU-MK, MkE, and Blast-E (for quantitation see Table S1). (C) Representative colonies (Scale bars, 100 μm.) and (D) colony cytospins (Scale bars, 10 μm.) from normal (Left) and T21 (Right) FL clonogenic assays. (E) Only Blast-My and Blast-E had secondary replating activity. No tertiary replating was seen. There was no difference between replating activity of normal and T21 FL Blast-My or Blast-E.

Expression of MK-Erythroid and B-Lineage Genes in T21 FL HSC and compared with normal FL HSC: NOTCH1, FLT3, ETS1, MEF2C, Progenitors. To determine whether changes in HSC/progenitors HES1,andDYRK1A (Fig. 4B). Increased MK-erythroid com- in T21 FL reflected alterations in lineage-affiliated gene- mitment by T21 FL HSC did not appear to be a result of of in- expression signatures, we flow-sorted normal and T21 HSC, CMP, creased MPL or EPOR or to the key MK-erythroid regulators SCL MEP, LMPP, ELP, and PreproB progenitors, as shown (Fig. S2), or KLF1, because their levels of expression were normal or re- and measured expression of key lineage-associated genes by duced in T21 FL HSC (Fig. 4C). Expression of several early stem quantitative RT-PCR. Both normal and T21 FL HSC-expressed cell MK-erythroid genes, including GATA2, GATA1, VWF (Fig. genes highly expressed in human adult bone-marrow HSC, in- 4C), RUNX1, and ERG (Fig. 4A), was higher in T21 FL HSC and, cluding MPL, IL3RA, IKZF1, RUNX1, ERG (17), and inter- NOTCH1 although the increase for each gene individually was not statisti- estingly also expressed high levels of compared with cally significant, collectively these changes may contribute to the differentiated progenitors, consistent with murine fetal HSC (19) fi A EPOR increased MK-erythroid commitment. In line with this nding, (Fig. 4 ). Intriguingly, high-level expression of was seen GATA2 GATA1 C and expression were increased in T21 FL CMP in normal FL HSC, equivalent to FL MEP (Fig. 4 ), in contrast C to low EPOR expression in adult marrow HSC (17), suggesting (Fig. 4 ). Taken together, differences in T21 FL HSC gene ex- FL HSC may be erythroid-primed to meet the fetal demand for pression are consistent with their increased MK-erythroid and red cells. FL HSC also expressed genes important in early lym- impaired B-lymphoid potential.

phoid speciﬁcation, including FLT3, CRLF2, E2A, ETS1, MEF2C, T21 FL LMPP also failed to up-regulate the early lymphoid MEDICAL SCIENCES IKZF1 RUNX1 NOTCH1 FLT3 GATA3,andHES1 (20–22) (Fig. 4B), as well as early stem MK- gene program ( , , ,and ) seen in erythroid genes, including SCL, GATA2, low levels of GATA1 and normal FL LMPP and several key B-lymphoid genes, including VWF (Fig. 4C), and early myeloid lineage-afﬁliated genes, such EBF, IGH@, CRLF2, and IL7RA (Fig. 4 A and B). In contrast, as CEBPA, PU1, CSF1R, CSF2R,andCSF3R (Fig. 4D). These E2A and PU1 expression in T21 FL was the same as normal FL, data are consistent with multilineage priming of human FL HSC, consistent with their wider roles in hematopoiesis (21). Similar analagous to murine and adult HSC (17, 22, 23). patterns of reduced expression of early lymphoid and B-lym- T21 FL HSC also expressed a multilineage program but with phoid genes, including EBF1, IGH@, and PAX5, but not CD19, marked reductions in expression of early lymphoid genes were seen in T21 ELP and PreproB progenitors (Fig. 4B).

Roy et al. PNAS | October 23, 2012 | vol. 109 | no. 43 | 17581 Downloaded by guest on September 25, 2021 A NORMAL T21 0 48.9% 0 19.5% 0 80.9% 0 44% 0 4.3% 0 96.6% 0 51.1% 0 96.1% 0 19.1% 0 56% 0 96.1% 0 3.6% CD45RA

CD10 Fig. 3. Impaired lymphoid differentiation of T21 CD19 CD45RA CD19 CD10 FL HSC, LMPP, and ELP. (A) Representative plots CD34 CD34 CD19 CD34 CD34 CD19 from normal (Left; n = 11) and T21 (Right; n = 6) FL + + − showing reduced Pre-proB (CD34 CD19 CD10 ) B Normal CDNORMAL T21 * + + + 10 * 12 100 T21 and ProB progenitors (CD34 CD19 CD10 ). (B) 8 7.7 0% 0.7 0% 80 Mean B-lymphoid progenitor frequencies in nor- 92 0% 95.5 0% 8 6 *** 60 mal (light bars; n = 11) and T21 (n = 6) FL (dark 4 40 4 bars) showing reduced PreProB and ProB progeni- 2 MNC % of 20 % of CD34+ % of CD19

CD19 < < 0 tors in T21 FL. ***P 0.001; *P 0.02. (C)Repre- 0 no of CD20 pos cells/100 HPF cells/100 CD20 pos no of 0 − + CD34 CD34 CD34-CD19+ l S sentative plots showing reduced CD34 CD19 cells D ELP pro B orma Pro B n in T21 compared to normal FL and summary data Pre = HSC LMPP ELP in normal (light bars, n 5) and T21 FL (dark bars, = < + E Day 0 Day 14 Day 0 Day 14 Day 0 Day 7 Day 14 Normal n 8). *P 0.05. (D) Mean number of CD20 cells/ 3.6% 5.5 0.3% T21 2.3 3.1% 0 0.0% 81.3 2.1% 13.1 100000 ﬁ = 0 0.0% 12.4% high-power elds from normal (n 4; light bars) 0 100% 65.8 28.9% 0 100% 12.4 4.5% 0 0.0% 70.8 86.18.15% NORMAL 0 100% = 10000 and T21 FL (n 4; dark bars). (E) Differentiation of ﬂow-sorted 100 HSC, LMPP, and ELP on MS5 1000 stroma showing representative results on day 0 0.0% 0.2 0.3% 0 0.0% 0 0.0% 0 0.0% 3.7 0.2% 0 0% 100 97.72.3% 0 and day 14 (day 7 also shown for ELP) from 0 100% 14.4 85.0% 0 100% 30 70% 0 100% 94 2.2% T21 = = 10 normal (n 3) and T21 FL (n 4) and (Right)mean + CD19 absolute number of CD19 cells on day 14 from 1 normal FL HSC, LMPP, and ELP (light bars) or T21 No of CD19 pos cells/100 HSC/LMPP/ELP cells/100 CD19 pos No of CD34 HSC, LMPP or ELP (dark bars).

Finally, to investigate chromosome 21 (HSA21) genes im- previously (11, 12) and here, we have been unable to demon- portant in hematopoiesis, we compared levels of RUNX1, ERG, strate self-renewal of T21 MEP (replating activity instead resided DYRK1A, and GABPA (Fig. 4 A and B) in T21 and normal FL further up the differentiation hierarchy), although T21 MEP HSC/progenitors. Importantly, none of these genes showed the clonogenicity is increased, indicating a proliferative advantage in predicted 1.5-fold increase in expression across all HSC/pro- MEP, as well as HSC and MKs, were increased in vivo in T21 FL genitor populations expected for cells trisomic for HSA21. In- (on FL sections). Because all samples were screened (and were stead, a significant increase in expression, compared with normal negative) for the presence of the disease-causing exon 2/3 muta- FL, was found only for GABPA (1.3- to 1.4-fold) and this was tions in GATA1 characteristic of TMD (7–9), this argues strongly specific to the LMPP and PreproB populations, because GABPA that T21 is responsible for these effects. This theory is supported expression in HSC and other progenitors was the same in normal by data from human iPSCs reported by MacLean et al. (25), which and T21 samples. RUNX1 and ERG expression were comparable recapitulated many of the abnormalities in fetal myelopoiesis we in T21 and normal FL across all populations apart from signifi- observed in primary fetal cells. cantly reduced expression in PreproB, although modest, poten- Impaired B-progenitor development in T21 FL manifest both tially relevant, increases in RUNX1 (1.4-fold) and ERG (1.2-fold) as a marked selective reduction in committed B-progenitor (Pre- were seen specifically in HSC (Fig. 4A). Interestingly, DYRK1A proB and ProB) frequency (T-progenitor frequency was normal) expression was lower in all T21 HSC/progenitors compared with and as reduced ability of HSC, LMPP, and ELP to generate mature normal FL (Fig. 4A). B cells. These abnormalities were underpinned by an equally marked reduction in T21 FL HSC of expression of FLT3, one of Discussion the earliest regulatory events triggering B-lymphoid development The striking association of T21 with myeloid and B-cell malig- (20). Furthermore, in T21 LMPP, FLT3 expression failed to un- nancies in young children (3) led us to investigate whether T21 dergo the up-regulation needed to activate normal B-progenitor itself alters HSC/progenitor biology using detailed immunophe- development (22). Consistent with this finding, levels of expression notypic and functional analysis of GATA1 mutation-negative, of other transcriptional activators that specify B-cell fate, including second trimester T21 FL compared with normal FL. Here, we IL7RA, CRLF2, ETS1, MEF2C,andEBF, were reduced in T21 are unique in reporting evidence in primary human FL cells that LMPP and downstream B-progenitors and PAX5 expression was T21 itself causes multiple distinct defects in FL myelo- and reduced in ELP. These data show extensive dysregulation of B-cell lymphopoiesis, altering not only the myeloid progenitor com- development from HSC to mature B cells. Whether similar dysre- partment (MEP/CMP/GMP), but also causing perturbation of gulation of B-progenitor development persists beyond birth is un- immunophenotypically defined HSC, MPP, LMPP, and of early known, but we hypothesize that “molecular resetting” of the fetal B- and committed B-lymphoid progenitors. Gene-expression studies lymphoid differentiation program contributes to B-cell immune demonstrate the molecular complexity underlying these changes deficiency (15) and B-ALL in children with DS (26). and argue that effects of T21 are cell context-dependent and It is important to note that the differences between T21 and influenced not only by cell lineage, but also by maturational stage. normal FL hematopoiesis are not only marked, but the pattern Expansion of HSC was accompanied by functional and mo- of hematologic abnormalities seen in every T21 fetal sample is lecular evidence of MK-erythroid bias, although confirmation the same over the gestation range investigated (14–22 wk). This that there is a true increase in MK-erythroid–biased HSC would finding supports our earlier observations of consistent FL ab- require experiments at a single-cell level and appropriate xeno- normalities despite the absence of GATA1 mutations, which was graft models (24). Increased MEP frequency in T21 FL may independently confirmed in two laboratories (11, 12). The pre- therefore be a downstream consequence of this, because more cise mechanisms by which T21 causes the consistent pattern of profound differences in functional assays and gene expression perturbed FL HSC/progenitor specification and function were were seen in HSC rather than MEP in T21 FL. Indeed, both not directly addressed in this study. However, by characterizing

17582 | www.pnas.org/cgi/doi/10.1073/pnas.1211405109 Roy et al. Downloaded by guest on September 25, 2021 AB Stem cell Lymphoid CD Megakaryocyte-erythroid Myeloid 0.045 0.009 0.006 0.008 0.0014 0.04 0.008 0.007 MPL CRLF2 EBF1 0.005 EPO R CEBPA 0.035 0.0012 0.007 0.006 0.03 * 0.001 0.006 0.004 0.005 0.025 * * 0.005 0.0008 0.003 0.004 0.02 * 0.004 * 0.0006 0.003 0.015 * 0.003 0.002 0.0004 0.002 0.01 0.002 * * 0.001 0.001 0.005 0.001 0.0002

0 0 0 0 0

0.0035 0.045 0.04 0.12 0.08 0.04 0.035 0.003 IL3Ra E2A IGH@ SCL 0.07 0.1 PU1 0.035 0.03 0.0025 * 0.06 0.03 0.08 0.025 * 0.05 0.002 0.025 0.06 0.02 0.04 0.0015 0.02 * * * 0.015 * 0.03 0.015 0.001 0.04 * * * 0.01 0.01 * 0.02 0.0005 0.02 * * 0.005 * 0.005 0.01

0 0 0 0 0

0.05 0.02 0.08 0.0018 0.04 0.045 0.018 0.07 0.0016 0.035 GATA2 CSF1R 0.04 IKZF1 0.016 ETS1 PAX5 0.06 0.0014 * 0.03 0.035 0.014 0.0012 0.05 * 0.03 * 0.012 0.025 0.001 0.025 0.01 0.04 0.02 * 0.0008 0.02 * 0.008 0.03 0.015 0.0006 0.015 0.006 * * * 0.02 0.01 0.004 * * 0.01 0.0004 * * * 0.01 0.005 0.002 * 0.005 0.0002 0 0 0 0 0

0.25 0.04 0.08 0.06 0.007 RUNX1 0.035 MEF2C 0.07 IL7Ra CSF2R 0.2 0.05 GATA1 0.006 0.03 * 0.06 * * 0.005 0.025 * 0.05 0.04 0.15 * * 0.02 0.04 0.004 * 0.03 * 0.1 0.015 * 0.03 * 0.003 0.02 0.01 0.02 * * 0.002 0.05 0.005 * 0.01 0.01 0.001 0 0 0 0 0

0.05 0.012 0.35 0.03 0.00018 0.045 ERG GATA3 CD19 0.00016 0.3 CSF3R 0.04 0.01 0.025 VWF 0.00014 0.035 * 0.25 0.008 0.02 0.03 0.00012 0.2 0.0001 0.025 0.006 0.015 0.15 0.02 * 0.00008 0.004 0.015 0.01 0.00006 0.1 0.01 0.00004 0.002 0.005 0.05 0.005 0.00002 0 0 0 0

0.01

B P 0.0025 PPM PL 0.03 C 0.009 0.05

NOTCH1 o

HES1 S 0.008 0.045 KLF1 MC * 0.002 GABPA 0.025 r

0.007 0.04 E MEP

H * p * 0.035 * 0.02 0.006 0.0015

* * L 0.005 0.03 * * e 0.015 0.004 * * 0.025 rP * 0.001 0.003 * * 0.02 0.01 0.002 0.015 * 0.0005 0.001 0.01 0.005 0 0.005 0 0 0 0.03 0.014

B PM

PLE 0.025 FLT3 B 0.012 DYRK1A orp

0.02 0.01 r

ELP

HSC * C MEP HSC CMP MEP

pe 0.015 0.008 * LMPP

* LMPP * * * e * 0.006 * 0.01 * rP * * * * * * rP

* T21 * 0.004 0.005 * * Normal 0.002 0 0 HSC/LMPP

r orp Lymphoid

ELP

HSC CMP MEP

ELP

HSC CMP MEP progenitors

LMPP

r P Myeloid

P progenitors

Fig. 4. (A–D) Altered gene expression in T21 FL HSC/progenitors Mean gene expression levels by quantitative RT-PCR from ﬂow-sorted HSC/progenitors (50 cells in triplicate for each population) from normal (n = 5; light bars) and T21 FL (n = 3; dark bars) shown relative to GAPDH. Signiﬁcant differences between T21 and normal FL are shown as *P < 0.05; **P < 0.01, and ***P < 0.001, using Bayesian analysis of differences in mean (see SI Experimental Procedures).

the effects of T21 on HSC and each progenitor population in- of gene expression (34, 35). For example, small changes in mRNA dividually, our data reveal the molecular complexity underlying expression (1.5-fold) of both HSA21 genes DYRK1A and DSCR1 the effects of T21 and suggest that a single molecular event is caused profound nuclear factor of activated T cells dysregulation unlikely to explain all of these abnormalities and that the impact and cardiac defects (36). Similarly, our data may provide impor- of changes in expression of HSA21 genes is not just tissue-specific, tant clues in primary human hematopoietic cells to possible but also varies according to lineage, differentiation stage, and the mechanisms through which T21 may perturb their growth and regulatory machinery of individual genes, as reported for other differentiation and a model with which to investigate these. These T21 tissues in DS (2) and in DS leukemias (27). data help direct specific questions about how lineage-restricted The marked phenotypic differences in T21 FL hematopoiesis changes in expression of HSA21 genes (such as GABPA and contrast with the relative modest effects on gene expression. This possibly RUNX1, ERG, and DYRK1A) or the five HSA21 miRs finding is not surprising given the very modest differences in contribute to HSC/progenitor dysregulation, as suggested from global gene expression reported in the accompanying article by ML-DS and TMD (35, 37, 38), or the role of altered HSC/pro- Chou et al. (28) and in other primary T21 tissues (average 1.5 genitor interactions with microenvironmental regulators, as sug- – fold) (29 31), which also show variation in the level of increase gested for insulin-like growth factor signaling (39) and CRLF2/ MEDICAL SCIENCES both between genes and within different tissues, leading to thresh- IL7RA (26) in T21 leukemias. olds for differences in expression between T21 and disomic tis- Finally, the impact of T21 on HSC/progenitors is likely to vary sues being set at ∼1.2-fold in some studies (32, 33). In support of with age given the different patterns of hematopoietic abnor- this approach, there is good evidence that small changes in gene malities during fetal and postnatal life (11, 13, 15). Because we expression can account for phenotypic/functional differences. In found that by 14-wk gestation, FL hematopoiesis is already mouse models with different combinations of HSA21, orthologs perturbed, this raises the question of whether T21 also affects also show phenotype attenuation when fewer genes are tripli- HSC/progenitor behavior during early embryonic development cated, demonstrating the importance of multiple small changes and in primitive hematopoietic cells, which is clearly extremely

Roy et al. PNAS | October 23, 2012 | vol. 109 | no. 43 | 17583 Downloaded by guest on September 25, 2021 difficult to investigate in detail in human embryos. Indeed, data 50 ng/mL (all Peprotech) and erythropoietin (EPO) 4 U/mL (R&D Systems)] (see from human T21 iPSCs recapitulated many, but not all, of the SI Experimental Procedures). abnormalities we observed in second trimester primary FL cells and show that T21 can also perturb hematopoiesis in the em- MS5 Stromal Cocultures for B-Lymphoid Differentiation. MS5 stromal cells bryonic yolk sac (25, 28). Thus, integration of studies in primary were cocultured with 100 sorted HSC/progenitors in MS5 medium (a-MEM; human fetal tissue with the iPSC model systems of fetal and Invitrogen) with 10% (vol/vol) FCS, Flt-3L (10 ng/mL), SCF (20 ng/mL), IL-2 (10 ng/mL), IL-7 (5 ng/mL), GM-CSF (20 ng/mL), and G-CSF (10 ng/mL) (Peprotech). embryonic hematopoiesis characterized by MacLean et al. (25) – and Chou et al. (28) will be necessary to properly elucidate the B-lymphoid differentiation was assessed weekly by FACS from day 7 21 (see SI Experimental Procedures). molecular complexity underlying the impact of T21 on human hematopoiesis and leukemogenesis. Gene Expression Analysis by Dynamic Arrays. Gene expression was assessed Experimental Procedures using the BioMark real-time PCR (qPCR) system (Fluidigm). Fifty HSC or progenitors from five normal FL and three T21 FL were sorted into 200-μLPCR Samples. Second trimester FL collected during elective surgical termination of tubes with 10 μL RT-STA mix. Each population was tested in triplicate. Sorted pregnancy were processed immediately. GATA1 analysis, CD34+ separation, populations were analyzed for relative level of expression of ≤48 genes si- and immunohistochemistry were performed as previously described (11) (SI multaneously, as previously described (17). Gene expression was normalized Experimental Procedures). FISH was used to confirm T21 in T21 FL and the to GAPDH expression using the 2 -ΔCq method. Data are presented as lack of chromosomal abnormalities in normal FL. The study was approved by ^ ± the Hammersmith and Queen Charlotte’s Hospitals Research Ethics Com- mean SEM. For list of assays (ABI) used for qPCR, see Table S3. mittee (ref 04/Q0406/145). Statistics. The difference in means for two sample groups was tested fi Flow Cytometric Analysis and Sorting. Cells were stained with ≤8 fluorophore- for signi cance using a Bayesian analysis of differences in mean and Wilcoxon ± conjugated monoclonal antibodies (see SI Experimental Procedures) and test. Data are expressed as mean SEM unless otherwise indicated. analyzed using a BD LSR Fortessa or FACSAria II (Becton Dickinson). Gates were set with unstained controls gating on viable cells using DAPI. Data ACKNOWLEDGMENTS. We thank P. May, A. Reid, and V. Melo for FISH were analyzed on FlowJo software (Tree Star). analysis. This study was supported in part by Leukemia and Lymphoma Re- search Specialist Programme Award 08030 (to G.C., I.R., and P.V.); the Impe- fl rial College Biomedical Research Centre (I.R.); Oxford Partnership Biomedical Clonogenic Assays. Clonogenic assays were performed on ow-sorted HSC/ Research Centre National Institute for Health Research Biomedical Research progenitors (100 cells/mL) using Methocult H4230 (Stem Cell Technologies) Centre scheme (P.V., A.J.M., and S.E.J.); Leukemia and Lymphoma Research with cytokines [IL-3 20 ng/mL, IL-6 10 ng/mL, IL-11 10 ng/mL, stem cell factor (A.R., G.B., A.J.M., D.A., and A.K.); the Medical Research Council (S.F., M.P.H.S., (SCF) 10 ng/mL, FLT3 10 ng/mL, GM-CSF 50 ng/mL, thrombopoietin (TPO) and S.E.J.); and the Kay Kendall Leukaemia Fund (to O.T.).

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