CORRESPONDENCE

transactions are the difficulty in accessing drugs can be manufactured under license for (cell dissociation buffer, CDB) and correct and complete information on local consumption. Such alliances will lead enzymatic (collagenase/trypsin, CT)— potential partners, suppliers or market to a win-win situation for all, both biotech eventually compromised the genetic possibilities and the uncertainty of ensuring companies and the public.” integrity of the hES cell lines that had a partner’s commitment to formal contracts. To sum up, Indian biotech firms basically previously been passaged by manual The latter in particular, requires a judicial have three choices in the short-term as busi- methods. Chromosomal abnormalities system that functions more efficiently and ness innovation strategies2: first, they can dominated the BG01 hES cell cultures after credibly. These conditions have nothing to focus on products that are either off-patent as few as 23 passages after changing over to do with TRIPS. Thus, the impact of TRIPS already or soon to be off-patent (essentially the CDB passaging method. The BG01 cell on either the commercial strategies of foreign the generics market); second, they can col- line maintained a normal karyotype for 42 companies or their strategic alliances with laborate with Western multinationals and manual passages but developed trisomy 12 Indian companies is anyone’s guess, as it is biotech companies (two areas that are likely and 17 in all cells (5 cells out of 20 analyzed only one parameter among many that will be to witness an increase in collaborations are contained an additional X ) used in making foreign investment decisions. clinical trials and R&D outsourcing); or as early as 23 passages after changing from From the perspective of Western firms, third, they can focus on innovations that the manual to the nonenzymatic CDB the implementation of TRIPS in India may the multinationals will not be interested in; passaging method (Supplementary Fig. 1 encourage them to introduce new brand that is, mainly ‘tropical’ or developing world online). The BG02 hES cell line maintained drugs because such products will now enjoy diseases. a normal karyotype for 45 manual passages, patent protection—a situation not possible but trisomy 17 was observed 25 passages since 1970. This will not mean, however, Shyama V. Ramani1, Preeti Pradhan2 & after switching to CDB passaging. The that high-priced, Western-manufactured Mahesh Ravi1 BG02 line, which demonstrated a normal products can be directly shoehorned into 1Department of Economics, Institut National de la karyotype after 12 manual passages, was the Indian market. As K.S.N. Prasad, CEO Recherche Agronomique; UMR INRA-UPMF, BP found to have trisomy 12, 14, 17 and an

http://www.nature.com/naturebiotechnology of Shantha Biotechnics (Hyderabad, India), 47, 38040 Grenoble cedex 9, France. extra copy of the X chromosome in all puts it: “Though TRIPS gives exclusive rights 2Department of Economics, University of cells (3 cells out of 20 analybed contained to Western companies to market their brand California, 543 Evans Hall, c/o Jaeger, Berkeley, CA an extra copy of chromosome 20), when products in India—eliminating competition 94720, USA. studied 56 passages after switching to e-mail: [email protected] from local companies that copy inventions— the enzymatic CT passaging method these multinationals are unlikely to benefit 1. Ames, G. & Sunil, P. Indian pharmaceutical indus- (Supplementary Fig. 2 online). from selling their products at high prices try: market, regulatory, import and investment regime Because manually passaged BG02 hES because Indian consumers simply cannot (Pacific Bridge, Inc., Washington, DC, 2002). http:// cells maintained a normal karyotype www.pacificbridgemedical.com/publications/Indian_ afford the high costs of drugs developed and Pharma_Industry_2002.pdf through 105 passages, we then investigated manufactured abroad. Therefore, it will be 2. Ramani, S.V. & Maria A. TRIPS and its possible impact whether limited CDB or CT passaging of on the biotech based segment of the Indian pharma- necessary for Western and Indian companies ceutical industry. Economic and Political Weekly, in the hES cell colonies could be used to reduce to enter into strategic alliances so that novel press (January 2005). or limit changes in karyotype. Limited disaggregation of BG02 hES cell colonies 2005 Nature Publishing Group Group 2005 Nature Publishing

© resulted in normal karyotypes for 13 and 15 passages in CDB and CT treatments, respectively (Table 1). However, trisomy Preserving the genetic integrity of for chromosome 17 was observed by CDB passage 23, indicating that chromosomal human embryonic stem cells abnormalities eventually arose using current enzymatic/nonenzymatic passaging To the editor: methods. The limited number of human embryonic registered hES cell lines BG01 and BG02 Abnormal karyotype was also associated stem (hES) cell lines1–7 heightens the were propagated by manual dissection of with significant changes in expression of need to maintain their genetic integrity. the hES cell colonies3 and have normal candidate implicated in maintaining A report by Draper et al. published in last karyotypes at passages 41, 50, 62, 74 pluripotency9–11 as well as of other genes January’s issue (Nat. Biotechnol., 22, 53–54, and 105. These results confirm previous related to early developmental lineage 2004) and a related correspondence from observations from Buzzard and colleagues, restriction. Differential expression Buzzard and colleagues in the April issue indicating that the difficult and laborious analyses by real-time PCR was conducted (Nat. Biotechnol. 22, 381–382) suggest that manual passaging of hES cells will retain a on four BG02 hES cell groups. We tested hES cell lines propagated in vitro for even stable karyotype, even after 100 passages. the effect of passage number (early versus a few months can develop an abnormal Faster and easier means of passaging hES late) in both manual and enzymatic karyotype. We report here data from our cells are available, but we found that they (CT) passaged cells. laboratory that throw more light on the can promote chromosomal aneuploidy, of the aneuploid late CT was compared genetic stability of hES cell lines and its especially trisomy 12 and/or 17, in to the normal karyotype groups (early relation to how cells are maintained in conjunction with aberrant gene expression. manual, late manual and early CT), and culture. Two means of disaggregating hES cell the late CT exhibited a higher expression In our laboratory, US National Institutes colonies into single cell suspensions for of genes associated with pluripotency, of Health (NIH, Bethesda, MD, USA)- bulk hES cell passaging1,6,8—nonenzymatic including POU5F1, , LEFTY2 (also

NATURE BIOTECHNOLOGY VOLUME 23 NUMBER 1 JANUARY 2005 19 CORRESPONDENCE

Table 1 Results of cytogenetic and fluorescent in situ hybdridization (FISH) analyses of BG01 and BG02 hES cells based on passage number and method of cell dispersal Number of passages using different FISH chromosome 12% signal countb,c FISH chromosome 17% signal countb,c dispersal methods Cell line Mechanical CDB C/TKaryotypea 2 copies 3 copies 2 copies 3 copies BG01 41 46, XY [20] 100% (200) 100% (200) 48, XY, +12, +17 [20]/ BG01 42 23 11% (22) 89% (178) 12% (24) 88% (176) 49, XXY, +12, +17 [5]

BG02 50 46, XY [20] 100% (128) 100% (100) BG02 62 46, XY [9] 100% (145) 100% (200) BG02 74 46, XY[20] 100% (200) 100% (200) BG02 100 46,XY 100% (200) 100% (200) BG02 105 46, XY 100% (200) 100% (200) BG02 45 7 46, XY [20] 100% (200) 100% (200) BG02 45 13 46, XY [20] 100% (200) 100% (200) 47, XY, +17[5]/ BG02 45 25 100% (200) 100% (200) 47, XY, +inv(17) (q11.2q21) [2] BG02 56 3 46, XY [9] 100% (100) 100% (100) BG02 65 15 NA 100% (100) 100% (100) 50, XXY, +12, +14, +17 [17]/ BG02 12 56 51, XXY, +12, +14, +17, +20 7.5% (15) 92.5% (185) 5.5% (11) 94.5% (189) [3] http://www.nature.com/naturebiotechnology

aValues in square brackets [ ] refer to total number of metaphases. b≤5% signal gain or loss is considered background and is not shown. When trisomy is present, apparent ‘losses’ due to overlap- ping signals is increased so that 5.5–12% losses seen above may be due to artifact or may represent low level presence of a normal cell line. NA, not analyzed. cValues in parentheses () refer to total number of nuclei analyzed. Details of cytogenetic methods are presented in Supplementary Methods online.

known as EBAF), GABRB3, GBX2 and gene expression profile of undifferentiated National Science Foundation under award no. EEC- FGF13 (Supplementary Table 1a online). hES cells. 9731643. This work was also supported in part by Eleven of 17 pluripotent genes were more In summary, our results suggest that funding from the National Institutes of Health (NIH- HL074303). We thank Bresagen Inc., for providing highly expressed in late CT hES cells bulk passage methods (CT and CDB) can the hES cells used in this correspondence, and Roger compared with the three chromosomally perpetuate aneuploid cell populations Nielsen for assistance with real-time PCR analysis. normal hES cell groups (highlighted in after extended passage in culture, but may 1,3 1,3 Supplementary Table 1a online). Analysis be used for shorter periods (up to at least Maisam M. Mitalipova , Raj R. Rao , Deborah M. Hoyer1, Julie A. Johnson2, of early embryonic development genes 15 passages) without compromising the 2005 Nature Publishing Group Group 2005 Nature Publishing Lorraine F. Meisner2, Karen L. Jones1,

© (Supplementary Tables 1b–e online) karyotypes. Together with the findings of Stephen Dalton1 & Steven L. Stice1 also revealed a higher expression in hES Draper et al. and Buzzard and colleagues 1 cells with an abnormal karyotype (late on karyotypic instability in hES cells, our Rhodes Animal Science Center, The University CT) compared with the other three data indicate that bulk passaging can lead of Georgia, Athens, Georgia 30602, USA. 2Cytogenetic Laboratory at Wisconsin State karyotypically normal groups. Overall, not only to abnormal karyotypes but also to Laboratory of Hygiene, 465 Henry Mall, more than 70% of the genes analyzed were quantitative differences in gene expression. Madison, Wisconsin 53706, USA. found to be significantly different between Even so, it may be possible to maintain 3These authors contributed equally to this work. the abnormal karyotype late CT group and a normal karyotype in hES cells under e-mail [email protected] the normal early manual, late manual and long-term manual propagation conditions 1. Thomson, J.A. et al. Science 282, 1145–1147 early CT groups (P < 0.05; Supplementary followed by limited bulk passaging in (1998). Table 1 online). experiments requiring greater quantities 2. Reubinoff, B.E. et al. Nat. Biotechnol. 18, 399–404 (2000). Increased expression of pluripotent genes of hES cells than manual passage methods, 3. Mitalipova, M. et al. Stem Cells 21, 521–526 may be a consequence of in vitro selection alone, can provide. In addition, our data (2003). for specific aneuploid hES cell populations underscore the need for simultaneous 4. Stojkovic, M. et al. Stem Cells 22, 790–797 (2004). 5. http://stemcells.nih.gov/research/registry. with enhanced proliferative ability under karyotypic and quantitative gene expression 6. Cowan, C.A. et al. N. Engl. J. Med. 350, 1353–1356 extended bulk passaging. Our data also analyses to preserve and monitor the genetic (2004). indicate that, in addition to increased integrity of hES cells in continuous culture. 7. Amit, M. & Itskovitz-Eldor, J. J. Anat. 200, 225–232 (2002). expression of pluripotent genes, abnormal 8. Carpenter, M.K. et al. Cloning Stem Cells 5, 79–88 cell lines also exhibit increased expression ACKNOWLEDGEMENTS (2003). This work was supported in part by funding provided 9. Rao, R.R. & Stice, S.L. Biol Reprod (2004). of early differentiation genes, implying that by the Georgia Tech/Emory Center (GTEC) for the 10. Sato, N. et al. Dev Biol 260, 404–413 (2003). bulk passaging compromises the normal Engineering of Living Tissues, an ERC Program of the 11. Bhattacharya, B. et al. Blood (2003).

20 VOLUME 23 NUMBER 1 JANUARY 2005 NATURE BIOTECHNOLOGY

Supplementary Figure 1 G-banded karyotype of BG01 hES cells at p42 (mechanical) + p23

(non-enzymatic): 48,XY,+12,+17

Supplementary Figure 2 Fluorescence in situ hybridization signal patterns for cell line BG02 p12 (mechanical) + p56 (enzymatic): (a) Trisomy 12: 12p13 TEL gene (green) and 21q22 AML1 gene normal control probe (red) (b) Trisomy 17: 17 centromere probe (green) and 9 centromere control probe (red).

Supplementary Table 1 Fold change values for individual genes across three populations. (BG02-p45: Early Manual [EM]; BG02- p93: Late Manual [LM]; BG02-p65/15: Early Trypsin [ECT]. Genes have been classified based on (a) Pluripotency (b) Ectoderm- related (c) Mesoderm-related (d) Endoderm-related and (e) Trophoblast and Others.

Table 1a. Pluripotency

Gene Chromosome LOG10(Fold Change) Symbol Locus Gene Name EM LM ECT BUB1 2p11-q21 Budding uninhibited by benzimidazoles 1, S. cerevisiae, homolog of -0.57* -0.53* -0.49* CCNA2 4q25-q31 Cyclin A2 -0.39* -0.32* -0.38* CD9 12p13 CD9 antigen -0.93* -0.91* -0.81* DNMT3B 20q11.2 DNA methyltransferase 3B +0.09 +0.25* +0.08 LEFTY2 1q42.1 Endometrial bleeding associated factor -0.32* -1.10* -0.50* FGF13 Xq26.3 Fibroblast growth factor 13 -0.71* -0.75* -0.73* FGF2 4q26 Fibroblast growth factor 2 -0.24* +0.00 -0.27* FGF4 11q13.3 Fibroblast growth factor 4 -0.09 +0.13 +0.06 FGFR4 5q33-qter Fibroblast growth factor 4 +0.06 +0.39* +0.12 FOXH1 8q24 Forkhead box H1 -0.04 +0.09 -0.09 GABRB3 15q11.2-q12 Gamma-aminobutyric acid receptor, beta3 -0.53* -0.44* -0.43* GBX2 2q37 Gastrulation brain 2 -2.26* -1.28* -2.36* LDB2 4p16 Lim domain-binding 2 -0.29* -0.29* -0.22* SALL2 14q11.1-q12.1 Sal-like 2 +0.76* +0.93* +0.74* POU5F1 6p21.31 POU domain, class 5, 1 -0.69* -0.68* -0.75* SOX2 3q26.3-q27 SRY-box 2 -0.40* -0.26 -0.48* ZNF42 19q13.2-q13.4 42 +0.39* +0.40* +0.36*

Table 1b. Ectoderm-related

Gene Chromosome LOG10(Fold Change) Symbol Locus Gene Name EM LM ECT FGF5 4q21 Fibroblast growth factor 5 -1.48* -1.12* -1.39* FN1 2q34-q36 Fibronectin 1 -0.63* -1.10* -0.31* HOXB4 17q21-q22 Homeobox B4 +0.73* -1.52* +0.68* LMX1B 9q34 Lim homeobox transcription factor 1, beta +0.42 -0.09 -0.30 MCAM 11q23.3 Melanoma adhesion molecule -0.64* -0.76* -0.77* MSI1 12q24 Musashi, drosophila, homolog of, 1 -0.46* -0.43* -0.59* MSI2 17q23.2 Musashi, drosophila, homolog of, 2 -0.44* -0.52* -0.50* NCAM1 11q23-q24 Neural cell adhesion molecule 1 -0.47* -0.75* -0.68* 12q22 Neural precursor cell expressed, developmentally downregulated NEDD1 1 -0.77* -1.06* -0.66* NEDD4L 18q21 Ubiquitin protein ligase NEDD4-like -0.58* -0.53* -0.52* 2q37 Neural precursor cell expressed, developmentally downregulated NEDD5 5 -0.39* -0.41* -0.49* 14q11.2 Neural precursor cell expressed, developmentally downregulated NEDD8 8 -0.48* -0.48* -0.46* NEFH 22q12.1-q13.1 Neurofilament protein, heavy polypeptide -0.33* -0.04 -0.46* NEFL 8p21 Neurofilament protein, light polypeptide -0.42* -0.55* -0.33* NPDC1 9q34.3 Neural proliferation, differentiation and control protein 1 +0.41* +0.85* +0.62* NR4A2 2q22-q23 subfamily 4, group A, member 2 +0.28 +0.30 -1.16* 15q Neural precursor cell expressed, developmentally downregulated NEDD4 4 -0.14 -0.96* -0.37* OTX2 14q21-q22 Orthodenticle, drosophila, homolog of 2 -0.16* -0.29* -0.37* PAX6 11p13 Paired box gene 6 +2.09* +0.54* +1.34* MCFD2 2p21 Multiple coagulation factor deficiency 2 -0.53* -0.57* -0.53* SOX3 Xq26-q27 SRY-box 3 -0.78* -0.35* -0.57*

Table 1c. Mesoderm-related

Gene Chromosome LOG10(Fold Change) Symbol Locus Gene Name EM LM ECT BMP4 14q22-q23 morphogenetic protein 4 -0.51* -0.91* -0.63* CD34 1q32 Hematopoietic progenitor cell antigen CD34 -0.32 -0.84 +0.22 CDH5 16q22.1 Cadherin 5 -1.24* -1.84* -1.15* EOMES 3p21.3-21.2 , xenopus, homolog of +0.13* -0.37* -0.42* FLT1 13q12 FMS-related tyrosine kinase 1 +0.36* +0.55* +0.45* GATA2 3q21 GATA-binding protein 2 -0.68* -0.98* -0.54* GSC 14q32.1 Goosecoid +0.56* -0.09 +0.26* HLA-C 6p21.3 Major histocompatibility complex, class 1C -0.57* -0.64* -0.51* HLA-A 6p21.3 Major histocompatibility complex, class 1A -0.39* -0.52* -0.72* HLA-B 6p21.3 Major histocompatibility complex, class 1B -0.90* -0.99* -0.63* ITGA2B 17q21.32 Integrin, alpha2B -0.80* -0.52* -0.56* KDR 4q11-q12 Kinase insert domain receptor +0.34* +0.26* -0.09 LMO2 11p13 Lim domain only 2 -1.15 -1.11 -0.88 MYL4 17q21-qter Myosin, light chain 4, alkali, atrial, embryonic +0.32 -1.34* +0.51 NKX2.5 5q34 NK2, drosophila, homolog of, E -0.85 -0.81 -1.17 PECAM1 17q23 Platelet endothelial cell adhesion molecule 1 -1.28* -1.28* -1.89* PITX2 4q25-q27 Paired-like homeodomain transcription factor 2 -0.46* -0.66* -0.29 RUNX1 21q22.3 RUNT-related transcription factor 1 -0.65* -0.90* -0.73* T 6q27 T , mouse homolog of -0.65* -0.58* -0.76* TBX1 22q11.21 T-box 1 -0.79 -0.91 -1.59* TBX5 12q24.1 T-box 5 -0.62 -0.78 -1.07 TBX6 16p11.2 T-box 6 -0.66* -0.69* -0.73* TEK 9p21 TEK tyrosine kinase, endothelial -0.64* -0.89* -0.26* THBS1 15q15 Thrombospondin 1 -0.85* -0.67* -0.85* THBS2 6q27 Thrombospondin 2 -0.62* -0.12 -0.27* 1p34-p33 Tyrosine kinase with immunoglobin and EGF factor homology TIE domains -1.22* -0.43 -0.30 VCAM1 1p32-p31 Vascular cell adhesion molecule 1 +0.63* -1.33* -0.23 VWF 12p13.3-p13.2 Von willebrand factor +0.64* -0.06 +0.22

Table 1d. Endoderm-related

Gene Chromosome LOG10(Fold Change) Symbol Locus Gene Name EM LM ECT AFP 4q11-q13 Alpha-feto protein +0.48 +0.33 +2.27* CER1 9p23-p22 Cerberus 1, xenopus, homolog of +0.43* -0.44* +0.15* EXO1 1q42-q43 Exonuclease 1, S. cerevisiae, homolog of -0.51* -0.57* -0.52* FOXA2 20p11 Forkhead box A2 +0.40* +0.04 -0.07 GATA4 8p23.1-p22 GATA-binding protein 4 +0.45* -0.63* -0.02 GATA6 18q11-q12 GATA-binding protein 6 +0.39* -0.68* +0.03 HNF4α 20q12-20q13.1 Hepatocyte nuclear factor F4 alpha +0.61 -1.41* +0.23

Table 1e. Trophoblast and others

Gene Chromosome LOG10(Fold Change) Symbol Locus Gene Name EM LM ECT GATA3 10p15 GATA-binding protein 3 +0.88* -0.04 +0.27* HEY1 8q21 Hairy/enhancer of split related with YRPW motif 1 -0.27 +0.27 +0.44* MSX2 5q34-q35 Muscle segment homeobox, drosophila, homolog of 2 -0.24 -1.46* -0.41 CASP-2 7q34-q35 Caspase 2, apoptosis-related cysteine protease -0.13 -0.11 -0.19 SMAD2 18q21 Mothers against decapentaplegic, drosophila, homolog of, 2 -0.28* -0.36* -0.40* PTCH 9q22.1-q31 Patched, drosophila, homolog of -0.30* -0.03 -0.19* WNT5A 3p21-p14 Wingless-type MMTV integration site family, member 5A -0.79 -1.53* -0.66 NODAL 10q22.1 Nodal, mouse, homolog of -0.47* -0.58* -0.62*

FOOTNOTE: For each gene, the fold changes values have been calculated keeping the BG02-p12/54: Late Trypsin [LCT] as the baseline. Genes that exhibited highest expression in LCT group have been highlighted in red. (*)-Individual Comparisons with LCT group were significantly different at a p-value <0.05. SUPPLEMENTARY METHODS

Human ES cell culture. Human ES cell lines BG01 and BG02 were cultured as described previously. Briefly, they were cultured on mitotically inactivated mouse embryonic fibroblast

(MEF) feeder layers in (hES medium) DMEM/F12 medium supplemented with 15% fetal bovine serum (FBS, HyClone) , 5% Knock-out serum replacer (KSR, Gibco), 2mM L-glutamine

(Gibco), 1% nonessential amino acids, 50 U/ml penicillin, 50 µg/ml streptomycin, 1,000 U/ml recombinant human leukemia inhibitory factor (LIF, Chemicon), 0.1 mM β-mercaptoethanol, 4 ng/ml basic fibroblast growth factor (bFGF, Sigma).

Manual passaging of human ES cells. Human ES cells were passaged by using pulled firepolished Pasteur Pipette with colonies of hES cells cut into pieces of 10 to 100 cells and transferred to fresh MEF layer.

Enzymatic passaging of human ES cells. Cells were incubated in collagenase (1mg/ml, Gibco) solution for 2-5 min, followed by treatment with 0.05%trypsin/EDTA solution (Gibco).

Dislodged cells were centrifuged at 200g for 4min at RT and resuspended in hES medium and replated onto fresh MEF layer.

Non-enzymatic passaging of human ES cells. Human ES cells were incubated in Cell dissociation Buffer (CDB, Gibco) for 1 min and dislodged cells were centrifuged at 200g for

4min at RT. Cells were resuspended in hES medium and replated onto fresh MEF layer.

Preparation of hES cells for karyotype analysis. Human ES cells were incubated with

Ethidium Bromide (12 µg/ml) for 40 min at 37ºC, 5% CO2 , followed by 120ng/ml of colcemid

(Gibco) treatment for 20 min. The cells were treated with 0.25% trypsin, and dislodged cells were centrifuged at 200g for 8 min at RT. The cell pellet was gently resuspended in 0.075 M

KCL solution, and fixed in a solution containing 3:1 of methyl alcohol and glacial acetic acid. Fixed cells were dropped on wet slides, air dried and baked at 90°C for 1 hour. G banding was performed using Trypsin-EDTA and Lieschman Stain (GTL) by immersing slides in 1X Trypsin-

EDTA with 2 drops of 0.4N Na2HPO4 for 20 to 30 seconds, rinsed in distilled water and stained with Lieschman Stain (Sigma)for 1.5 minutes, rinsed in distilled water, and air dried.

GTL Banding Analysis. 20 metaphases were fully karyotyped at the microscope using an

Olympus BX40 microscope equipped with 10X and 100X plan-apo objectives. Images were captured and 2-5 cells were karyotyped using the CytoVysion® digital imaging system (Applied

Imaging, Pittsburgh, PA).

FISH Slide Preparation. The fixed cell suspensions were dropped on wet slides and air dried.

Slides were immersed in 2XSSC for 2 minutes at 73°C, digested in Pepsin (Sigma, P-7012) for

10 minutes at 37°C, rinsed in distilled water, dehydrated through an alcohol series for 2 minutes each, and air dried using parameters set on the VP2000 slide processing instrument (Vysis,

Downers Grove, IL). 3µL of TEL/AML1 probe (Vysis) and 3µL of chromosome 17 specific probe (PathVysion® Her-2 neu/17CEP, or chromosome 17/9 CEP cocktail probes,(Vysis) were placed on separate scribed hybridization areas, covered with coverslips and sealed with rubber cement. Slides were co-denatured for 2 minutes at 73°C and allowed to hybridize over night at

37°C using programmed parameters set up on the Hybrite instrument (Vysis). The following morning the coverslips and rubber cement were removed and the slides were placed in a 0.4X

SSC stringency wash with 0.3% NP-40 (Roche) for 2 minutes at 73°C, and placed in 1XPBS

(Invitrogen) with 0.1% NP-40. Slides were counterstained with 1 drop of 20% DAPI Vectashield in Vecashield Anti-Fade (Vector, Burlington, CA) coverslipped, and stored in the dark at -20°C until microscopic examination was performed. FISH Analysis. 100-200 nuclei were analyzed by 2 technologists using a BX41 fluorescence microscope equipped with a 100W mercury lamp, 20X and 100X objectives and Spectrum

Orange/Spectrum Green dual filter cube (Vysis). Signal enumeration was performed according to prescribed guidelines (Vysis). Representative nuclei were imaged using the CytoVysion® digital imaging system.

DNA Fingerprinting. DNA was isolated from 1XPBS (Invitrogen) washed cell pellets using the

DNA IQ isolation system (Promega, Madison, WI). 16 STR sequences were amplified using the

PowerPlex® 16 DNA fingerprint kit (Promega) and the amplified product was sequenced on the

PRISM 3100® genetic analyzer, (Applied Biosystems, Foster City, CA). Electropherograms were generated using GeneScan®v3.7.1 analysis software (Applied Biosystems) and alleles were determined by manual comparison to an allelic ladder.

Real time PCR analysis. In preparation for real time PCR analysis, total RNA was isolated from

4 samples for which karyotype analysis had been carried out (EM, LM, ECT, LCT), resuspended in 1 ml Trizol (Invitrogen) and triturated until homogenized. RNA was isolated from the crude homogenate according to the manufacturer’s protocols (Trizol, Molecular Research

Corporation). The integrity of the RNA produced from all samples used was verified and quantified using a RNA 600 Nano Assay (Agilent Technologies) and the Agilent 2100

Bioanalyzer. 5 µg total RNA was reverse transcribed using the cDNA Archive Kit (Applied

Biosystems Inc.) according to manufacturer’s protocols using the MultiScribeTM Reverse

Transcriptase. Reactions were incubated initially at 25oC for 10 min and subsequently at 37oC for 120 min. Quantitative PCR (TaqmanTM) assays were chosen for the transcripts to be evaluated from Assays-On-DemandTM (ABI) a pre-validated library of QPCR assays and incorporated into 384-well Micro-Fluidic CardsTM. Two (2.0) µl of the cDNA samples (diluted to 50µl) along with 50µl of 2X PCR master mix were loaded into respective channels on the micro- fluidic card followed by a brief centrifugation. The card was then sealed and real-time PCR and relative quantitation was carried out on the ABI PRISM 7900 Sequence Detection System

(Applied Biosystems Inc.). For calculation of relative fold change values, initial normalization was achieved against endogenous 18S ribosomal RNA using the ∆∆CT method of quantification

(ABI). Average fold changes from three independent runs were calculated as 2–CT. Data analysis for differential expression between the different populations was conducted in triplicate. Proc

GLM (SAS) was used to estimate differences between populations. The p-values attached to the estimated differences were used in the assessment of significance.