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CD34+ cell selection CD34 cell separation was carried out on freshly isolated mononuclear cells (MNC) using StemSep (Stem Cell Technologies, Vancouver, Canada) and Miltenyi MS magnetic columns, as previously described.1,2 Post- separation CD34+ purity was >95% in all cases. Flow cytometry Freshly collected MNC were stained with up to four fluorochrome-conjugated mAb(s) as previously described.1,2 Conjugated antibodies used were: CD61-FITC, CD34-FITC, CD45FITC, CD45RA-FITC, CD123-PE, Glycophorin A-PE, CD34-PE, CD45-PerCP, CD42a-PerCP, CD34-PerCP, CD38-APC, CD34-APC, CD19-APC, CD10-FITC, CD14-FITC, CD2-FITC; all antibodies were from Becton-Dickinson (Oxford, UK). Data were acquired using a dual-laser FACSCalibur (Becton-Dickinson). For each sample stained with antigen-specific mAb, an isotypic control conjugated with the appropriate fluorochrome was used. Compensation settings for 4-colour analysis were set using single-stained samples. Data were analysed on CellQuest (BD) and FlowJo software (Tree Star, Oregon, USA). MEP were defined as described by Manz et al3: CD34+CD38+CD45RA-CD123-; CMP as CD34+CD38+CD45RA-CD123lo; GMP as CD34+CD38+CD45RA+CD123lo, however since lineage depletion was not performed because of the extremely small sample size, cells were stained where possible with mature erythroid, lymphoid and monocyte markers to confirm specificity (<1% of CD34+CD38+ DS and normal FL and bone marrow cells expressed CD19, CD10, CD14, CD2 or GPA as expected). Clonogenic assays Methylcellulose hemopoietic progenitor assays were performed using Methocult H4230 (Stem Cell Technologies, Vancouver, Canada) in the presence of IL-3 (20ng/ml), IL-6 (10 ng/ml), IL11 (10 ng/ml), SCF 10 ng/ml, Flt3L (10 ng/ml), GM-CSF (50 ng/ml), thrombopoietin (Tpo; 50 ng/ml) and erythropoietin (Epo; 4 u/ml) as described by Manz et al.3 All cytokines were from Peprotech (Peprotech Inc, Rocky Hill, NJ, USA) except Epo which was from R&D (R&D Systems Europe, Abingdon, UK). Cell cultures (in duplicate or triplicate depending on cell numbers) were incubated at 37°C, 100% humidity and 5% CO2 for 10-14 days and colonies identified on the basis of morphology. Liquid cultures Erythroid cultures: MACS-purified FL CD34+ cells were cultured for 10 days at 105/ml in liquid culture in IMDM with 10% FCS, L-glutamine (Invitrogen, Carlsbad, CA), penicillin/streptomycin (Cambrex) containing SCF 50ng/ml, Flt-3 Ligand 50ng/ml, IL-3 10ng/ml (Peprotech) and Epo 10u/ml (R&D). Megakaryocyte cultures: MACS-purified FL CD34+ cells were cultured for 10 days at 105/ml in serum-free liquid culture (Stemspan, Stem Cell Technologies, Vancouver, Canada) in 50ng/ml Tpo. Both liquid culture systems were modified from Sato et al.4 Proliferation was assessed by cell counts on days 7 and 10 and differentiation by flow cytometry at day 10 using CD34/GlyA/CD45 and CD34/CD61/CD42 for erythroid and megakaryocyte maturation respectively. GATA1 mutation analysis DNA was extracted using QiAmp DNA Mini Kit (Qiagen, Germany) as per the manufacturer’s instructions. GATA1 mutations were identified by WAVE dHPLC (Transgenomics Inc, Omaha, Nebraska, USA) and PCR/cloning followed by sequencing. Exons 2 and 3 of GATA1 were 1 amplified using previously published primers5 and primers designed for the study (available on request). Each amplicon was directly sequenced. Amplicons were also ligated into pGEM–T Easy vector, cloned and the DNA extracted from individual colonies was sequenced. Finally, all amplicons were analysed for heterodimer formation using dHPLC. Typical GATA1 mutations were identified using all three techniques. The accuracy and sensitivity of the techniques used was verified by analysis of DS-AMKL samples with known GATA1 mutations (dilution experiments mixing normal DNA with DS-AMKL samples with GATA1 clones of known sizes showed that clones of >5% were consistently detected using dHPLC). RT-PCR Primary cells were initially suspended in RNAlater medium (Qiagen) and stored at -80°C. RNA extraction was then carried out with Qiagen RNeasy kits, following the manufacturer’s protocol. An in-column DNase treatment (Qiagen) was carried out to avoid genomic DNA contamination. Directly after RNA extraction a 40µl reverse transcriptase reaction was performed using random primers (Invitrogen, Paisley, UK) and SuperScript II reverse transcriptase (Invitrogen) following the manufacturer’s protocol. cDNA samples were used with cDNA specific primers. Taqman assays for GATA1 splice variants were designed using Primer 3 software. Primer and probe sequences are available on request. Assays for all other genes including 18s were purchased from Applied Biosystems (Foster City, California). Taqman quantitative RT-PCR reactions were carried out on an Applied Biosystems 7500 Fast Real-Time PCR System and analysed on the proprietary software. Expression levels were corrected for 18s levels. Statistics Sample distributions were tested for normality by the Shapiro-Wilk test and the difference in means tested for significance using Student’s T test. Data are expressed as the mean + SEM. References 1. Campagnoli C, Fisk N, Overton T, Bennett P, Watts T, Roberts I: Circulating hemopoietic cells in first trimester fetal blood. Blood 2000; 95: 1967-72. 2. Kotsianidis I, Silk JD, Patterson S, Almeida A, Tsatalas C, Bourikas G, Cerundolo V, Roberts IAG, Karadimitris A: Regulation of hematopoiesis in vitro and in vivo by invariant NKT. Blood 2006; 107: 3138-3144. 3. Manz MG, Miyamoto T, Akashi K, Weissman IL: Prospective isolation of human clonogenic common myeloid progenitors. Proc Natl Acad Sci USA 2002; 99: 11872-2. 4. Sato T, Ono M, Fujita H et al: Development of a liquid culture system to megakaryocyte terminal differentiation: fibrinogen promotes megakaryocytopoiesis but not thrombopoiesis. Brit J Haematol 2003; 121: 315-323. 5. Ahmed M, Sternberg A, Hall G et al. Natural history of GATA1 mutations in Down syndrome. Blood 2004; 103:2480-9.