Human Embryonic Stem Cells Passaged UsingEnzymatic Methods Retain a Normal Karyotype
and Express CD30
ALISON THOMSON, DAVINA WOJTACHA, ZOË HEWITT,* HELEN PRIDDLE,†VIRGINIE SOTTILE,‡ ALEX DI DOMENICO, JUDY FLETCHER,§ MARTIN WATERFALL,
NÉSTOR LÓPEZ CORRALES,¶ RAY ANSELL, and JIM MCWHIR
ABSTRACT
Human embryonic stem cells (hESCs) are thought to be susceptible to chromosomal re-arrangements as a consequence of single cell dissociation. Compared in this study are two meth-ods of dissociation that do not generate single cell suspensions (collagenase and EDTA) withan enzymatic procedure using trypsin combined with the calcium-specific chelator EGTA (TEG),that does generate a single cell suspension, over 10 passages. Cells passaged by single cell dis-sociation using TEG retained a normal karyotype. However, cells passaged using EDTA, with-out trypsin, acquired an isochromosome p7 in three replicates of one experiment. In all of theTEG, collagenase and EDTA-treated cultures, cells retained consistent telomere length and po-tentiality, demonstrating that single cell dissociation can be used to maintain karyotypicallyand phenotypically normal hESCs. However, competitive genomic hybridization revealed thatsubkaryotypic deletions and amplifications could accumulate over time, reinforcing that pres-ent culture regimes remain suboptimal. In all cultures the cell surface marker CD30, reportedlyexpressed on embryonal carcinoma but not karyoptically normal ESCs, was expressed on hESCswith both normal and abnormal karyotype, but was upregulated on the latter.
89
INTRODUCTION
ACRITICAL ASPECT of practical hESC culture ismethod of passage. Most hESC lines require
mechanical disaggregation in early passages, sug-gesting sensitivity at that stage to cell–cell dis-ruption. In the original report of the isolation of
hESCs, collagenase was used to passage the cellsonce established, and supported the long-termgrowth of karyotypically stable, pluripotent cells(Thomson et al., 1998). More recently, this group,in collaboration with others, did report kary-otypic abnormalities in hESCs passaged usingcollagenase (Draper et al., 2004). Aneuploid cells
Division of Gene Function and Development, Roslin Institute, Roslin, Midlothian, Scotland. *Current address: Cen-tre for Stem Cell Biology, The University of Sheffield, Alfred Denny Building, Western Bank, Sheffield, S10 2TN, UK.
†Current address: D Floor East Block, Queen’s Medical Centre, University of Nottingham, Nottingham, NG7 2UH,UK.
‡Current address: Institute of Genetics, Queen’s Medical Centre, University of Nottingham, Nottingham, NG7 2UH,UK.
§Current address: Centre for Regenerative Medicine, University of Edinburgh, 49 Little France Crescent, Edinburgh,EH16 4SB, UK.
¶Current address: Public University of Navarra, Department of Agriculture, Edif. Los Olivos, Campus de Arrosadía31006, Pamplona, Spain.
were reported in H1, H7, and H9 hESCs passagedwith collagenase (Rosler et al., 2004). Others havereported aneuploidy when using enzymatic orchemical, but not mechanical, methods of pas-saging (Mitalipova et al., 2005). These authorsand others (Brimble et al., 2004) have suggestedthat the poor clonal efficiency of hESCs leads toselective pressure for the accumulation of partic-ular chromosomal rearrangements, and that thisoccurs as a consequence of generating a single-cell suspension.
In this paper the karyoptypic stability and po-tentiality of hESCs were evaluated following pas-sage using enzymatic and chemical-based methodsof disaggregation (collagenase, trypsin in combi-nation with EGTA, the Ca�� chelating agent,trypsin in combination with EDTA, a Ca��Mg��
chelator, and EDTA alone). Both collagenase- andtrypsin-based methods supported long-term cul-ture without gross chromosomal changes. Fur-thermore, single-cell suspensions generated withtrypsin could be transfected, and clones isolatedand expanded, that retain normal karyotype. How-ever, gross karyotypic normality does not excludesmall deletions or duplications that can be detectedover as few as 10 passages by competitive genomichybridization. In one experiment, cells passagedwith EDTA did become karyotypically abnormal(duplication of the short arm of chromosome 7).
CD30 expression has been reported to marktransformed hESCs (Herszfeld et al., 2006). CD30is a member of the tumor necrosis factor super-family that is a surface marker for malignant cellsin Hodgkin’s disease (Durkop et al., 1992), and isalso expressed in embryonal carcinoma (EC) cells,that share many of the properties of ESCs but arekaryotypically abnormal (Durkop et al., 2000;Pera et al. 1997).
However, screening of both the normal and ab-normal lines for CD30 expression revealed thatalthough CD30 was upregulated in the karyo-typically abnormal line, it was also detected incultures of karyotypically normal hESCs.
MATERIALS AND METHODS
Cells and media
H1, H7, and H9 hESCs were a gift from GeronCorp. (Menlo Park, CA). Prior to the comparisonof passage regime, hESCs were cultured and pas-saged with collagenase as described previously(McWhir et al., 2006; Xu et al., 2001).
K562 cells, a human erythroleukemia cell line,
were cultured in RPMI 1640 (Invitrogen, Carls-bad, CA) supplemented with 10% fetal bovineserum (FBS) and 2 mM L-glutamine. The humanembryonal carcinoma (hEC) cell line NTera 2 cellswere cultured in DMEM (Invitrogen) supple-mented with 10% FBS, 2 mM L-glutamine, 0.1mM nonessential amino acids and 1 mM sodiumpyruvate. Human trabecular bone (HTB) cellswere isolated as described in (Sottile et al., 2002)and grown in the same medium as the NTera 2cells. HM1 murine ESCs were cultured as de-scribed in McWhir et al. (1996).
Plating efficiency of hESCs passaged underdifferent regimes
Three passage regimes were compared: (1) col-lagenase IV(200 units/mL) (2) trypsin/EGTA(TEG)—0.25% trypsin in Ca��-Mg��-free phos-phate-buffered saline (PBS) containing 1 mMEGTA detailed in McWhir et al. (1996) and Prid-dle et al. (2004). (3) EDTA—0.2 mg/mL ethyl-enediaminotetra acetate (EDTA) in PBS. H9 hESCswere seeded at 1 � 105 cells/well in six-well platesat passage 40. The collagenase passage regime wasas described in Xu et al. (2001). Prior to dissociationin all treatments, wells were washed with 2 mL KO-DMEM. For the TEG treatment, cells were incu-bated with 0.5 mL TEG at 37°C for approximately2 min. KO-DMEM was then added, cells triturated,and centrifuged at 200 � g for 2 min before resus-pension in conditioned medium (CM). For theEDTA treatment cells were incubated with 0.5 mMEDTA in PBS at 37°C for approximately 3–5 min.After removal of the EDTA the cells were scrapedinto 1 mL CM. Clumps were gently triturated threetimes with a 5-mL pipette. After 10 passages thecells were expanded to generate stocks for cryop-reservation, genomic DNA preparation, isolation,and karyotype analysis.
Each treatment was replicated three times, andthree wells were set up for each replicate. The ini-tial seeding for all replicates of all three treat-ments was from a single collagenase-treated flask(passage 40). One well per replicate was counted16–18 h after plating to allow calculation of theplating efficiency (number of cells plated down/number of cells seeded � 100%). Once the re-maining two wells were confluent one well wastreated with TEG to obtain a single-cell suspen-sion that could be counted. On the basis of thiscell count the sister well was then passaged.Three wells of a six-well plate were seeded withapproximately 1 � 105 cells. It was unavoidable
THOMSON ET AL.90
that those wells set up using collagenase wouldhave greater variability in actual cell number be-cause collagenase tretament results in cell clumpsrather than dissociated cells. The experimentaldesign is summarized schematically in Figure 1A.Replicates from Experiment 1 were designated e1replicate A, B, or C.
The experiment was repeated using earlier pas-
sage (P34) H9 cells as described above but withthe addition of a fourth passage regime similar toTEG, but replacing EGTA with EDTA (TED).Replicates from Experiment 2 were designated e2replicate 1, 2, or 3.
Calculation of doubling time
Cells were seeded at 1 � 105 cells/well in two,six-well plates for each of two replicates. At eachtime point two wells were trypsinized in eachreplicate and the cells were counted using a he-mocytometer. Because collagenase-treated cellsdo not generate a single-cell suspension, addi-tional sacrificial wells were seeded for countingfollowing trypsinization. For each cell type, lin-ear regressions of time were fitted to the loga-rithm of the cell numbers. Doubling time was es-timated as log(2) divided by the slope of theregression. Confidence intervals for doublingtimes were obtained by replacing the slope by itsestimated confidence limits from fit of the re-gression.
DNA preparation
Genomic DNA was prepared from cell culturesby overnight incubation at 55°C in lysis buffer(100 mM Tris–Cl, 5 mM EDTA, 200 mM NaCl,0.2% SDS, 100 �g/mL proteinase K, pH 8.5), fol-lowed by incubation with Rnase A (325 �g/mL)at 37°C for 1 h, and further treatment with pro-teinase K (100 �g/mL) at 55°C for 2 h. DNA wasthen precipitated by addition of isopropanol,washed with 70% ethanol, and resuspended in TEbuffer (10 mM Tris–Cl, 1 mM EDTA pH 8.0). Ge-nomic DNA for comparative genomic hybridiza-tion (CGH) analysis was further purified by phe-nol/chloroform extraction prior to precipitation.
Comparative genomic hybridization (CGH) analysis
The CGH procedure was modified from theoriginal methods of Kallioniemi et al. (1992, 1994).Target human metaphases were obtained fromphytohemagglutinin-stimulated blood lympho-cytes cultures (72 h). Chromosome spreads wereprepared 1 day before hybridization and leftovernight at 43°C. Before CGH analysis the slideswere treated with pepsin (50 �g/mL in 0.01 MHCl), denatured with formamide, and serially de-hydrated in ethanol. hESC genomic DNA was labeled by nick translation with SpectralGreen(Vysis–Abbott Molecular, Des Plaines, IL). Spec-
hESCs PASSAGED ENZYMATICALLY RETAIN A NORMAL KARYOTYPE 91
Collagenase
Collagenase TEG
X3reps
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2 3 4Passage Number
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ting
Effi
cien
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5 106 7 8 9
40
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80EDTA
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Collagenase
FIG. 1. Plating efficiency of H9 hESCs passaged usingdifferent treatments. (A) Schematic representation of ex-perimental design. All treatments originated from a sin-gle flask of H9 cells cultured for 40 passages (P40) usingcollagenase. A sister culture was karyotyped and con-firmed 46XX. Passages within the experiment are num-bered P�1, P�2, etc. Cells were counted 16–18 h afterplating (count 1) and again at time of passage (count 2).(B) Cells were passaged using either collagenase (green),TEG (blue), or EDTA (pink). The plating efficiency valuesare the mean of the three replicates and the error bars in-dicate the standard deviation. (For interpretation of thereferences to color in this figure legend, the reader is re-ferred to the web version of this article at www.liebertonline.com/ars).
A
B
tralRed (Vysis) labeled normal human genomicDNA was used as reference DNA. Labeled frag-ments were 500–1000 bp. Equal amounts (400ng–1 �g) of hES and reference DNA were mixedand precipitated with 20–30 �g of unlabeledCot-1 DNA (Roche, Indianapolis, IN). DNAsamples were dissolved in hybridization solu-tion and denatured at 75°C. Slides were hy-bridized for 72 h in a humidity chamber at 37°C.After hybridization slides were washed in SSCat high stringency and dehydrated through anethanol series. Slides were counterstained with DAPI in Vectashield (Vector Laboratories,Burlingame, CA). The hybridized metaphasecells were examined using a workstation com-posed of an epifluorescence microscope coupledto a CCD camera (Olympus, Melville, NY) and a CytoVision 2.7 CGH software (AppliedImaging, San Jose, CA). CGH analysis was de-veloped over 10 complete metaphases. For eachmetaphase three fluorochrome images (DAPI,SpectralGreen and SpectralRed) were acquiredand processed using high-resolution CGH soft-ware. Statistical analysis was performed usingthree confidence levels (95, 99.5, and 99.9%).
Karyotype analysis
Exponentially growing cultures were treatedwith Karyomax colcemid solution (Invitrogen,UK) at 100 ng/mL for 2 h at 37°C. Karyotypeswere prepared and analyzed as described inMcWhir et al. (2006), Hewitt et al. (2007), and Gal-limore and Richardson (1973).
Cells were disaggregated using TEG and a sin-gle-cell suspension prepared in FACS buffer(PBS, 0.1% BSA, 0.1% Sodium Azide). Cells werealiquoted at 2–5 � 105 cells/sample, washed, andresuspended in 300 �L FACS buffer. The flow cy-tometer was set up for autofluorescence signalsin FL2 channel using unstained cells. Cells for PIstaining were incubated with 15 �L of stainingsolution (PI at 50 �g/mL in PBS) with gentle mix-ing for 1 min. PI fluorescence data (FL2 channel;575/42 nm) was then acquired for the stainedcells. Data for 5–10,000 ungated events were ac-quired on the FACSAria and analyzed usingFACSDiva software [Beckton Dickinson Im-munocytometry Systems (BD), UK].
Flow cytometry analysis for expression of stemcell surface markers
Single cell suspensions of hESC were stainedas described in Hewitt et al. (2006). Data for40,000 events/sample were acquired and ana-lyzed using CellQuestPro software (BD, UK).Three independent experiments were performed.
Flow cytometry analysis for the cell surfaceCD30 (Ber-H2) epitope
Adherent hESCs washed with PBS were incu-bated with TEG to dissociate cells. Recoveredcells were pelleted at 200 � g and resuspended atbetween 2 � 106 and 1 � 107 cells/mL in PBS(Ca2� and Mg2 free) with 0.2% BSA. Aliquots (100�L) were stained with either CD30-PE (SantaCruz, Santa Cruz, CA; sc19658) or isotype control(mIgG1-PE; BD Pharmingen, San Diego, CA;555749). Cells were incubated in the dark at 4°Cfor 30 min, washed, and finally resuspended inPBS. Flow cytometry was performed using a Bec-ton Dickinson FACSAria and Diva analysis soft-ware. Live cells were gated using forward- andside-scatter profiles. Data were acquired for10,000 live events.
Fixation of cells
Sample fixation was performed immediatelyfollowing CD30 staining. Cells were incubatedwith 0.1% paraformaldehyde for 15 min at roomtemperature, washed, and finally resuspended in100 �L PBS.
Transfection
A linearized plasmid containing a neomycinresistance gene flanked by a PGK promoter andpoly-adenylation signal was used to test stabletransfection efficiencies. Lipofection was carriedout using Lipofectamine 2000 (Invitrogen) ac-cording to the manufacturers instructions. Sub-confluent cells (1 � 106) in three wells of a six-well plate were lipofected with 5 �g of linearplasmid per well. Two different electroporationmethods were compared, using 1 � 106 cells inthe log phase of growth, treated with TEG to givea single-cell suspension. Using a Gene Pulser(BioRad, Hercules, CA), the cell/DNA mix waselectroporated in PBS at room temperature in a0.4-cm electrode cuvette at 940-�F, 200 V. Usinga Multiporator (Eppendorf, Westbury, NY) the
THOMSON ET AL.92
cells were swelled in hypo-osmolar buffer (Ep-pendorf) for 20 min at room temperature, mixedwith DNA, and pulsed in a 0.4-cm electrode cu-vette at 300 V for 100 �S. Following either elec-troporation method, cuvettes were left at roomtemperature for 10 min prior to plating the cellsonto 15-cm matrigel-coated dishes. Selection inG418 at 150 �g/mL was applied 48 h after trans-fection. G418 resistant colonies were fixed withmethanol, stained with 10% Gurrs R66 Giemsa inphosphate-buffered saline pH6.8, and counted(Priddle, 2004).
RT-PCR
RNA was isolated using an RNeasy mini kit(Qiagen, Valencia, CA). One-step reverse tran-scription–polymerase chain reaction (RT-PCR)was performed using Superscript One-Step RT-PCR with Platinum Taq (Invitrogen). Forty-fivecycles were used for each primer pair. Primerswere designed to span exons and distinguish ge-nomic DNA from cDNA products. CD30 mRNAwas amplified using the forward primer 5�-AGC-TAGAGCTTGTGGATTCCA-3� and the reverseprimer 5�-GTCTTCTTTCCCTTCCTCTTCC-3� togive a product of 464 bp. �-Actin mRNA was am-plified using the forward primer 5�-GCCACG-GCTGCTTCCAGC-3� and the reverse primer 5�-CAAGATGAGATTGGCATGGCT-3� to give aproduct of 528 bp.
Cryopreservation
Cells were resuspended in hESC medium andmixed with an equal volume of hESC mediumsupplemented with 10% serum replacement and20% DMSO. Cells were transferred to cryovials,stored at �80°C overnight and transferred to�150°C the next day.
In vitro differentiation of hESCs
Embryoid bodies were prepared by suspensionculture as described in Hewitt et al. (2007). Em-bryoid bodies were plated onto gelatin-coatedplates in differentiation medium [KnockoutDMEM (Invitrogen), 10% FCS (Globepharm, Deer-field, IL), 2 mM L-glutamine (Invitrogen), 1�nonessential amino acids (Invitrogen), and 100 �M�-mercaptoethanol (Invitrogen)] and allowed toreattach to the culture surface. Differentiation wasallowed to proceed for a further 2 weeks.
In all cases differentiated cells were washedwith PBS, fixed with 4% paraformaldehyde (PFA)at room temperature for 20 min, washed twotimes with PBS followed by a 15-min incubationwith PBS and 0.1% Triton X-100, and two washeswith PBS. Nonspecific protein binding wasblocked with 2% bovine serum albumin (BSA) for30 min at room temperature. Primary antibodieswere bound to their antigens in PBS with 2% BSAfor 1 h at room temperature. Antibodies and di-lutions were: monoclonal �-sarcomeric actinin(Sigma, St. Louis, MO) at a 1 in 500, monoclonalanticardiac troponin 1 (Chemicon International,Temecula, CA) at a 1 in 1000, monoclonal anti-cardiac troponinT (Neomarkers, Freemont, CA)at a 1 in 100, monoclonal anti-�-tubulin III(Sigma) 1 in 200, rabbit polyclonal anti-NF200(Sigma) 1 in 100 dilution, monoclonal anti-�-fe-toprotein (Sigma) 1 in 500. Unbound antibodywas removed by two 10-min room temperaturewashes with PBS followed by a 10-min incuba-tion with PBS and 0.05% Tween-20. FITC-conju-gated goat antimouse IgG (Sigma) and FITC con-jugated goat antirabbit (Vector Laboratories)were used as secondary antibodies (1 in 1000) inPBS with 2% BSA by incubation at room temper-ature for 1 h. Coverslips were mounted in Vec-tashield (Vector Laboratories), and viewed witha fluorescence microscope (Zeiss, Thornwood,NY). Control reactions with no primary antibodywere performed to confirm that the secondary an-tibody did not stain cells nonspecifically. Stain-ing on undifferentiated hESCs was also per-formed to assess level of background staining andfound to be negligible.
Formation of tumors in severe combinedimmunodeficiency disease (SCID) mice andhistological analysis
SCID mice were obtained from Harlan UK Ltd.(Bicester, UK) and maintained in a sterile envi-ronment. Human ESCs for injection into SCIDmice were disaggregated by treatment with col-lagenase or TEG, washed once in PBS, and re-suspended in PBS at 1 � 108 cells/mL. For eachpopulation tested, a 100-�L aliquot was injectedinto the leg muscle of each of three SCID mice.
Three to 5 months later, the mice were sacri-ficed and the tumors removed, fixed with 4% PFAfor 20 min at room temperature, embedded inparaffin wax using a Shandon Hypercentre XP
hESCs PASSAGED ENZYMATICALLY RETAIN A NORMAL KARYOTYPE 93
processor (Shandon Scientific UK Ltd), cut into10-�m sections using a Microm HM325 rotary mi-crotome (MICROM international GmbH), andstained using standard hematoxylin and eosin(H&E) protocols.
RESULTS
Plating efficiency
The response of H9 hESCs to three methods ofdisaggregation (collagenase, TEG, and EDTA)was followed over 10 passages. Plating efficien-cies were obtained for each treatment (Fig. 1b). Allthree replicates of cells cultured by passage withcollagenase failed to survive the seventh passage.Although in our experience it is unusual to lose aculture, this exemplifies the erratic nature of theplating efficiencies we have observed with thismethod of passage particulary when plating at therelatively low cell densities used in this experi-ment. The TEG passage regime was less variablethan either EDTA or collagenase, yielding an av-erage plating efficiency of 32%. All three replicatesof the EDTA-treated cells passaged at particularlypoor efficiency at passage 8 (Fig. 1b)
H9 hESCs passaged with collagenase and TEGproliferated with significantly different (p � 001)doubling times of 17.3 and 13.3 h, respectively
(Fig. 2). Human ESCs passaged with TEG hadsimilar doubling time (13.3 h) to mESCs (13.1 h)when measured at subconfluent densities. Dou-bling time increased as TEG-treated H9 cells at-tained confluence. Collagenase-treated H9 cellsdid not attain confluence over the course of theexperiment. When the growth rates of collage-nase-passaged cells and TEG-passaged cells werecompared at lower initial density (Fig. 2b) TEG-passaged cells again reached confluence earlier.
THOMSON ET AL.94
0
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)
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Time in hours
i iiii
20 20 �M 20 20 �M
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FIG. 2. Doubling time of hESCs. (A) Growth curves cal-culated for murine HM1 ESCs and human H9 ESCs byseeding 104 to 105 cells per well of a series of six-wellplates. For each cell type, linear regressions of time werefitted to the logarithm of the cell numbers. Doubling timewas estimated as log(2) divided by the slope of the re-gression. Confidence intervals for doubling times wereobtained by replacing the slope by its estimated confi-dence limits from fit of the regression. The y-axis showslogarithmically transformed cell numbers for: collage-nase-treated H9 cells (black), murine HM1 ESC (blue),and H9 hESCs following long-term passage using TEG(red). TEG treated cells were greater in number at the startof the experiment and reached confluence faster as indi-cated by the nonlinearity of those data. Collagenasetreated cells grew with a constant doubling time of 17.3h while murine ES cells had a doubling time of 13.3 h.When the experiment was repeated for TEG-treated andcollagenase-treated H9 cells at lower seeding density (B)TEG-treated cells again reached confluence more rapidly.(C) The phenotype of H9 cells 90 h after plating at 104 perwell of six-well plate. TEG-treated cells (Ci) showed lessdifferentiation and formed larger colonies than did col-lagenase-treated cells (Cii). (For interpretation of the ref-erences to color in this figure legend, the reader is referredto the web version of this article at www.liebertonline.com/ars).
As initial plating densities were identical, thisconfirms an accelerated growth rate in TEG-treated cells leading to earlier confluence. Figure2C shows the morphology of collagenase- andTEG-passaged cells 90 h postplating. TEG-pas-saged cells typically formed larger colonies, whilecollagenase-passaged cultures, in addition to un-differentiated hESCs, contained a second, differ-entiated cell type of fibroblastic morphology.
Karyotypes
The H9 hESCs used to set up the comparisonof different disaggregation treatments had been
cultured for 23 passages with collagenase, andhad a diploid karyotype (46XX) (Fig. 3) at pas-sage 40 in this experiment (Fig. 1A). Cells disag-gregated for 13 passages using TEG retained anormal karyotype. By contrast, cells disaggre-gated using EDTA had acquired an abnormalkaryotype with all three replicates having a du-plication of the short-arm of chromosome 7 anddeletion of the long arm [46,XX,i(7q)]. This resultwas confirmed by comparative genomic hy-bridization (CGH) (Fig. 4). Although TEG-treat-ment led to no gross karyotypic abnormalities,CGH suggested the presence of random deletionsclustered near the telomeres of many chromo-
hESCs PASSAGED ENZYMATICALLY RETAIN A NORMAL KARYOTYPE 95
1 2 3
6 7 8 9 10 11 12
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4 5
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6 7
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13 14 15 16 17 18
4 5
1 2 3
6 7 8 9 10 11 12
19 20 21 22 x
13 14 15 16 17 18
4 5
a. b.
c.
d.
FIG. 3. Karyotypes of H9 cells. (a) Collagenase-treated cells at the start of the experiment (passage 40, 23 passageswith collagenase in our lab); (b) passaged 13 times with TEG (H9-TEGe1repA); (c) passaged 13 times with EDTA(H9-EDTAe1repA). Karyotypes in b and c are representative of 10 spreads fully analyzed for each of three independentreplicates. No abnormalities were apparent in the parental collagenase-treated cells (a) nor in any of three replicatesof the TEG-treated cells(H9-TEGe1reps A, B, and C) (b). However, all three replicates of the EDTA protocol (H9-ED-TAe1reps A, B, and C) carried the same isochromosome isop7 (c and d). (d) Chromosome 7 pairs from three differ-ent EDTA replicates.
A.i.
ii.
B.0.5 1.0 1.5
7 n�23
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n�181
n�196 n�227 n�198 n�219 n�2110 n�2111 n�2012
n�2013 n�1914 n�2115 n�1716 n�2217 n�2218
n�19xn�2319 n�2320 n�2021 n�2122 y
n�182 n�213 n�204 n�195
n�231 n�182 n�193 n�184 n�205
n�2012n�2211n�2010n�229n�218n�187n�196
n�1913 n�2114 n�2115 n�2116 n�2117 n�2118
n�17xyn�1922n�2121n�2220n�2019
? ? ?
FIG. 4. Comparative genome analysis(CGH) of H9 hESCs. (A) Summarizes twoindependent CGH experiments for onereplicate of TEG-passaged H9 cells show-ing a tendency to accumulate small sub-telomeric deletions. When CGH is re-peated with the same DNA samples (Aiand Aii) similar, but not identical sub-telomeric deletions also appear. Circlesare areas showing a tendency for smalldeletions to accumulate in similar sub-telomeric regions. Similar, but not identi-cal subtelomeric deletions were also asso-ciated with the other two TEG replicatesbut not with EDTA or collagenase (datanot shown). (B) Summarizes CGH experi-ments for EDTA-passaged H9 hESCsshowing an abnormality on chromosome7 with duplication of the p arm and lossof the q arm.
hESCs PASSAGED ENZYMATICALLY RETAIN A NORMAL KARYOTYPE 97
Eve
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B) H7 TEG
FIG. 5. Flow cytometric analysis of stem cell markers on H7 hESCs passaged with. (A) collagenase or (B) TEG.Shaded areas are indicative of staining with the stem cell marker antibody (green � FITC conjugated and red � R �PE conjugated secondaries). Data represents 40,000 events; representative plots are shown from three independentexperiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web ver-sion of this article at www.liebertonline.com/ars).
THOMSON ET AL.98
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v) H9 TEGe1repA
012
0
100 101 102
PE-A
57.1% M1
103 104
Eve
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vi) H1 TEG
012
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100 101 102
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35.4% M1
103 104
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vii) H9 EDTA e2rep2
012
0
100 101 102
PE-A
41.1% M1
103 104
Eve
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viii) H9 EDTA e1repB
012
0
100 101 102
PE-A
69.9% M1
103 104
Eve
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ix) H9 EDTA e1repC
012
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100 101 102
PE-A
79.4% M1
103 104
FIG. 6A & B.
hESCs PASSAGED ENZYMATICALLY RETAIN A NORMAL KARYOTYPE 99
Eve
nts
i) NTera2
012
0
100 101 102
PE-A103 104
C
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PE-A
75.2% M1
103 104
Eve
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overlay without fixation with fixation
012
0
100 101 102
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45.8% M1
103 104
Eve
nts
ii) H9 TEGe1repA
012
0
100 101 102
PE-A103 104
Eve
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0
100 101 102
PE-A
63.3% M1
103 104
Eve
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100 101 102
PE-A
61.1% M1
103 104
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iii) H9 EDTAe1repC
012
0
100 101 102
PE-A103 104
Eve
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62.8% M1
103 104
Eve
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PE-A
57.9% M1
103 104
FIG. 6. Expression of CD30 in hESCs. (A) RT-PCR analysis of expression of CD30 transcript. Cells with normal kary-otype as well as those with an abnormal karyotype express CD30 mRNA. Detection of beta-actin product as a load-ing control. Lanes 1, no RNA control; lane 2, positive control NTera2; lane 3, positive control K562; lane 4, H9 hESCspassaged with collagenase (H9-Collagenase e2rep1); lane 5, H1 hESCs passaged with TEG; lane 6, H9 hESCs pas-saged with TEG, replicate A from the first passaging experiment (H9-TEGe1repA); lane 7, H9 hESCs passaged withTEG, replicate C from first passaging experiment (H9-TEGe1repC); lane 8, H9 hESCs passaged with EDTA, replicate2 from the second passaging experiment (H9-EDTAe2rep2); lane 9, H9 hESCs passaged with EDTA, replicate B fromthe first passaging experiment (H9-EDTAe1repB, abnormal karyotype); lane 10, H9 hESCs passaged with EDTA, repli-cate C from the first passaging experiment (H9-EDTAe1repC, abnormal karyotype); lane 11, negative control HTBcells. Position of molecular weight markers are shown on the left. (B) Flow cytometric analysis of CD30 expression.Cells with normal karyotype as well as those with an abnormal karyotype express the CD30 epitope. (i) K562, (ii)NTera 2, (iii) HTB, (iv) H9 hESCs passaged with collagenase, (v) H9 hESCs passaged with TEG (H9 TEGe1repA), (vi)H1 hESCs passaged with TEG, (vii) H9 hESCs passaged with EDTA; these cells were from replicate 2 of the secondpassaging experiment (H9-EDTAe2rep2), (viii) H9 hESCs passaged with EDTA; these cells were from replicate B ofthe first passaging experiment (H9-EDTAe1repB, abnormal karyotype), (ix) H9 hESCs passaged with EDTA; thesecells were from replicate C of the first passaging experiment (H9-EDTAe1repC, abnormal karyotype). The analysiswas performed on at least three separate occassions; representative plots are shown. (C) CD30 staining is affected byfixation treatment of the cells. CD30 staining was analyzed by flow cytometry. (i) N-Tera2; (ii) H9 TEGe1repA, (iii)H9 EDTAe1repC. The analysis was performed on at least two separate occasions; representative plots are shown.
somes. No similar pattern was observed with col-lagenase-treated cells or with EDTA-treated cells(data not shown). No specific deletions achievedstatistical significance; however, the clusteredpattern of subtelomeric deletion was consistentacross three independent replicates, and suggestsa mixed population containing cells with manydifferent small deletions. The different passageregimes were repeated using earlier passage cells(P34), and extended to include TED. Cells pas-saged with TEG, TED, trypsin in the absence ofa chelating agent, or collagenase were found tobe karyotypically normal (data not shown). Bycontrast to the first experiment, cells passagedwith EDTA were also found to be karyotypicallynormal (data not shown). H1 and H7 cells previ-ously passaged with collagenase have also beenswitched to the TEG-based protocol and shownto retain a normal karyotype (data not shown).
Analysis of telomere length showed that cellssubjected to all four treatments showed similartelomere lengths, suggesting that the variousmethods of passage had no differential effect ontelomere length, and that the telomeres were be-ing maintained (data not shown).
Stem cell markers
The expression of surface markers characteris-tic of undifferentiated (SSEA3, SSEA- 4,TRA-1-60and TRA-1-81) and differentiated (SSEA-1) hESCsby TEG-passaged H7 hESCs (Fig. 5) was com-pared by flow cytometry with that of collagenasepassaged cells. Cells passaged using TEG re-tained a high level of expression of markers char-acteristic of undifferentiated hESCs and a reduc-tion in expression of SSEA-1, a marker ofdifferentiation. This was consistent with the mor-phological observation that TEG-passaged cellsare predominantly of undifferentiated pheno-type. Similar results were obtained for H1 and H9cells, and H9 TEG-passaged cells were also pos-itive for expression of Oct-4 and alkaline phos-phatase (data not shown).
As concerns have been raised about the viabil-ity of hESCs once in a single cell suspension, cell
viability was assessed by propidium iodide stain-ing after passaging with TEG. The average cell vi-ability, was 93.1 2.0% (SD) (n � 8).
CD30 expression
CD30 expression has been reported in trans-formed hESCs, but not in karyotypically normalsister cultures (Herszfeld et al., 2006), suggestingthat its expression may be diagnostic for karyo-typic abnormality. hESC lines were screened forexpression of CD30 transcripts by RT-PCR (Fig.6A). Because CD30 expression is characteristic ofEC cells and of human erythroid progenitors, thehuman EC line NTera 2 and the human eryth-roblastoid leukemic cell line K562 were includedas positive controls and human trabecular bonecells (HTBs), as a negative control. Expressionwas detected in the positive control cell linesNTera 2 and K562 but not in HTBs. The lines H9-EDTA e1repB and C (EDTA passaged cells fromthe first experiment, lanes 9 and 10), identified asbeing karyotypically abnormal, also showed ex-pression of CD30 mRNA. Surprisingly, otherhESC lines that had been shown to have a nor-mal diploid karyotype at a similar passage alsoexpressed the transcript. This included cells pas-saged with collagenase, EDTA, and TEG (lanes4–8). Screening these cell lines for expression ofCD30 protein by flow cytometry using the samemonoclonal antibody used by Herszfeld et al.(2006), showed, as expected, that a high propor-tion of cells in both K562 and N-Tera 2 cultureswere CD30 positive (Fig. 6B), and that HTBslacked the CD30 epitope. Consistent with the RT-PCR data, CD30 expression was detected in allthe hESC lines tested. The abnormal cell lines H9-EDTA e1repB and C (viii and ix) did expresshigher levels than the “normal” hESCs (iv–vii).Herszfeld et al. (2006) had reported CD30 flowcytometry data using fixed cells. To test whetherfixation steps affected the level of CD30 stainingseveral cell lines were examined using differentstaining protocols either with or without fixation.Postfixation of samples reduced the number ofcells that stained positive for CD30 expression
THOMSON ET AL.100
FIG. 7. Differentiation of H9 cells in vivo and in vitro. (A) Sections from tumors generated from, collagenase- (a–f)or TEG-treated (g–n) H9 cells. Cartilage (a, g), mucus-secreting epithelium (b, m), pigmented epithelium (c), neuroglia(d), sebaceous gland (e, n), adipose tissue (h), smooth muscle (i), primitive neural tissue (j), ganglia (k), sweat glands(l). Bar � 50 �m. (B) Immunostaining of in vitro differentiated cultures from TEG-passaged H9 cells using primaryantibodies against �-sarcomeric Actinin (a), Troponin 1 (b), cardiac Troponin T (c), �3-Tubulin (d), Neurofilament 200(e), �-Fetoprotein (f).
hESCs PASSAGED ENZYMATICALLY RETAIN A NORMAL KARYOTYPE 101
FIG. 7.
(Fig. 6C), as did prefixation (data not shown).Monitoring expression of CD30 on fixed cells,however, still showed that cultures of hESCs pas-saged with collagenase, TEG, and EDTA all con-tained CD30-positive cells. Although the propor-tion of positive cells was similar, the abnormalhESC lines H9-EDTAe1rep B and C displayedhigher levels of CD30 expression than the normalhESC lines. We have also detected expression ofthe CD30 epitope on another hESC line, RH1, de-rived at Roslin Institute (Fletcher et al., 2006) andpassaged with collagenase.
Potentiality in vivo and in vitro
To test the potentiality of H9 hESCs disaggre-gated by TEG or collagenase, cells were injectedinto SCID mice. Four animals were injected witheach cell type, giving rise to tumors in 1/4 of therecipients of collagenase-treated cells and all fourrecipients of the TEG-treated cells. Tumors gen-erated from TEG- and collagenase-dissociatedcells gave rise in each case to a variety of tissuesderived from the three germ layers (Fig. 7A).There was no apparent accumulation of undif-ferentiated cells in recipients of cells from eithertreatment. When submitted to in vitro differenti-ation protocols, the TEG-treated cells gave rise tocell types arising from all three germ layers (Fig.7B).
TEG-treated H9 cells were tested for stabletransfection efficiency using electroporation,multiporation, and lipofection procedures. A setof conditions was defined using a multiporator(Eppendorf), which gave transfection efficienciessome fivefold higher (4 � 10�4) than wereachieved by lipofection or with a Gene Pulserelectroporator (Table 1).
The TEG-passaged cells have subsequentlybeen used successfully in several transfection ex-
periments. The TEG, EDTA, and collagenasemethods of disaggregation were compared fortheir effects on the proportion of frozen cells sur-viving thaw and replating. TEG treatment led toa significant improvement in freeze/thaw sur-vival over collagenase (76.2 vs. 15.8%, n � 3).
DISCUSSION
In this study hESCs passaged by single-cell dissociation using trypsin/EGTA (TEG) weremaintained and retained stable morphology,karyotype, and growth rate over 10 passages. Single-cell dissociation of hESCs did not lead tokaryotypic instability. However, subkaryotypicanalysis by CGH suggests that TEG passage maybe associated with small subtelomeric deletionsand amplifications.
Plating efficiency and doubling time
A doubling time of 17.3 h was calculated forcollagenase-passaged H9 hESCs, comparedwith 13.3 h for TEG-passaged cells. A previousreport calculated the doubling time of H9hESCs to be 35.3 h (Amit et al., 2000). This dis-crepancy may reflect the differences in cultureconditions between labs; notably the growth ofcells on matrigel versus a fibroblast feederlayer. The shorter doubling time of TEG pas-saged cells may be a consequence of the re-duction in differentiated companion cells whencompared with sister cultures passaged withcollagenase.
The high plating efficieny and short doublingtime of the TEG-treated cells contrasts with re-ports that hESCs are sensitive to single-cell dis-sociation, for example (Amit et al., 2000; Draperet al., 2004; Hasegawa et al., 2006). However,Hasegawa and colleagues were able to subclonehESCs that they then showed were adapted tosingle-cell dissociation (Hasegawa et al., 2006).Furthermore, the ability to maintain and re-cover cells as single-cell suspensions affords theopportunity for cell sorting by flow cytometry(Hewitt et al., 2006). The efficiency of clonal iso-lation can also be increased by culturing thecells in 2% oxygen (Forsyth et al., 2006; Hewittet al., 2006). Hence, it appears that hESCs canadapt in culture to overcome sensitivity to sin-gle-cell dissociation without gross karyotypicabnormality.
THOMSON ET AL.102
TABLE 1. COMPARISON OF STABLE
TRANSFECTION EFFICIENCIES
TransfectionMethod Number of clones efficiency
Lipofection 41 4.1 � 10�5
Gene Pulser 36 3.6 � 10�5
ElectroporatorMultiporator 231 4.1 � 10�4
Transfection efficiencies were obtained for H9 hESCsusing lipofection, a gene pulser electroporator and a mul-tiporator.
Single-cell dissociation using TEG supportsstable karyotype
In the original report of the isolation of hESCs,long-term karyotypic stability was demonstratedwhen collagenase was used for routine passageof established lines (Thomson et al., 1998). Sincethen, several reports have described the detectionof aneuploid hESCs using similar, collagenase-based, passage regimes (Amit et al., 2000; Draperet al., 2004; Inzunza et al., 2004; Mitalipova et al.,2005). Of particular note, the abnormalities inchromsomes 17q and 12 have been shown to oc-cur on several independent occasions (Draper etal., 2004; Ludwig et al., 2006). Mitalipova et al.(2005) reported abnormal karyotypes after ex-tended passage in culture using enzymatic- orchemical dissociation-based methods, but notwhen physical methods of disaggregation wereused. Buzzard et al. (2004) hypothesized that theuse of mechanical disaggregation may preventthe types of chromosomal abnormalities previ-ously reported.
H9 hESCs were passaged using two enzymaticmethods (collagenase and TEG) for similar num-bers of passages to the Mitalipova et al. (2005) re-port, and maintained a normal gross karyotypewith both protocols. Of the three methods com-pared in this study only trypsin-based protocolsgenerated a single-cell suspension, and the karyo-typic stability of these trypsin-passaged cells doesnot support the hypothesis that single-cellcloning gives rise to karyotypic instability.Notwithstanding concerns about subtelomericstability, this study indicates that the use of theTEG regime for rapid cell amplification, electro-poration, and freezing offers important advan-tages. Regardless of the method of passage, cau-tion is advisable when cells are cultured at lowdensity, as this may lead to the selection of cellsharboring abnormal karyotypes.
Subkaryotypic abnormalities can accumulate inTEG-passaged hESCs
Comparative genomic analysis confirmed thediploid karyotypes of cells passaged with eitherTEG or collagenase. Pluripotency of TEG-treated cells was not obviously different fromthat of collagenase-treated cells when assayedboth in vivo (tumors in SCID mice) and in vitro(response to embryoid body formation and re-moval of bFGF and conditioned medium). This
study has not addressed the possible accumu-lation of point mutations as reported for late-passage hESCs (Maitra et al., 2005). Analysis ofsubkaryotypic abnormalities at the level ofCGH have demonstrated small subtelomericdeletions and duplications when using the TEGpassage regime, although these were statisti-cally significant only up to 95% confidence lev-els. This technique detects only those aberra-tions that are shared by a large proportion ofthe population. Hence, random changes that arenot fixed in the population would not be de-tected. The tendency for accumulation of sub-telomeric deletions must give rise to concernabout using the TEG passage regime for thepreparation of cells for therapeutic use.
EDTA-treated cells can acquire abnormal karyotype
In the first experiment (Fig. 1a), EDTA treat-ment was associated with the appearance, inthree separate replicates, of the same duplicationof the short arm of chromosome 7. The completeabsence of cells with normal karyotype in allthree replicates argues strongly for a competitiveadvantage of this rearrangement when cells arepassaged with EDTA. As the same abnormalitywas detected in all three replicates it is likely thatthe duplication was present in the starting pop-ulation. It was noted that during the culture ofthe cells that acquired the karyotypic abnormal-ity there was one exceptionally poor passage af-fecting all three replicates (Fig. 1, plating effi-ciency of 3% compared with an average of 24%).Those cells harboring the duplication of the shortarm of chromosome 7 may have had a selectiveadvantage at this point. Repeating the passageregime using EDTA with an earlier passage (p34)of collagenase-passaged cells, no karyotypic ab-normalities were detected. Draper et al. (2004) re-ported that the gain of chromosome 12 and am-plification of chromosome 17q in H1 and H14hESCs was observed after the cells had beenthrough clonal selection or switched from afeeder-layer to a feeder-free culture system. Bothof these treatments could result in low cell den-sities that may allow a competitive advantage forcells of abnormal karyotypes. Observation of aparticularly poor recovery among the three repli-cates of EDTA-treated cells at p8, followed by fix-ation of the isoP7 rearrangement, would be con-sistent with the presence of a minority population
hESCs PASSAGED ENZYMATICALLY RETAIN A NORMAL KARYOTYPE 103
of isoP7 cells that preferentially survived underthe conditions of that particular passage regime.
SUMMARY
The TEG protocol offers advantages of conve-nience and repeatability, leading to reduction inthe time required to amplify cells for experimen-tal purposes. TEG-treated cells are amenable toelectroporation, leading to stable transfection ef-ficiencies approximately one order of magnitudehigher than those previously reported (Siemen etal., 2005; Zwaka and Thomson, 2003). TEG treat-ment provides fivefold freeze/thaw efficiencygains over the collagenase protocol. Cells switchedfrom collagenase- to TEG-passage have now beenused for several projects. A clonal line, M2, de-rived from H9 hESCs that had been passaged us-ing collagenase for 23 passages, and then TEG for57 passages, also retained a diploid karyotype(Hewitt et al., 2006). The TEG protocol has beenused successfully to passage cells for single-cellsorting (Hewitt et al., 2007), and clones from theseexperiments have been expanded using TEG-pas-sage and retained a normal karyotype.
Spontaneous chromosomal aberrations (SCA)were previously shown to be reduced by cultureof TEG-passaged hESCs at physiologicaly rele-vant oxygen partial pressure (Forsyth et al., 2006).SCAs are precursors to karyotype abnormalities.Of concern was the observation that the rate ofSCAs was higher in later passage cells. Othershave reported an increase in mutation rate withpassage of hESCs (Maitra et al., 2005). These datareinforce the need to thoroughly characterizecells prior to therapeutic use, and to provide fail-safe protection for cancerous phenotypes by suchmeans as conditional suicide genes (Hewitt et al.,2007; Schuldiner et al., 2003). Comparative geneexpression profiling of early- and late-passagehESCs reveals differences in gene expression dueto both karyotypic and epigenetic adaptation toculture (Enver et al., 2005). Together with thepresent observation that culture regime can leadto detectable levels of gene amplification anddeletion, these observations also underline theneed to establish a definitive assay of “normal-ity” that does not rely solely on karyotype.
CD30 was found to be expressed by all hESCsexamined in this study, but its level of expressionwas greater in the karyotypically abnormalhESCs. Hence, the use of CD30 as a diagnostic
tool for normality of hESCs as suggested by Herszfeld et al. (2006) may require quantitativeanalysis and will need to be supported by exam-ination of additional markers.
All models of the application of hESCs in re-generative medicine are based upon massive exvivo expansion of a few or possibly even a singlecell(s) from the inner cell mass into very large cellnumbers in vitro. To ensure the safety of the re-sulting graft a great deal remains to be done tooptimize culture conditions, define and identify“normal cells,” and to provide failsafe featuresthat protect against cellular overgrowth posten-graftment (Hewitt et al., 2007) .
ACKNOWLEDGMENTS
We thank Nick Forsyth for the primers used inthe RT-PCR analysis and discussions on the CD30work, Laura Dick for tumor embedding, Dr. Ed-ward Duvall for the tumor analysis, and DaveWaddington for statistical advice. This work wassupported by the Biotechnology and BiologicalSciences Research Council (BBSRC) and Geron.
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Address reprint requests to:Dr. Alison Thomson
Division of Gene Function and DevelopmentRoslin Institute
