Download CD34 cell selection CD34 cell separation was carried out on freshly

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.