Clonal derivation and characterization of humanembryonic stem cell lines
Nico Heins a, Anders Lindahl b, Ulrika Karlsson a, Marie Rehnstrom a,Gunilla Caisander a, Katarina Emanuelsson a, Charles Hanson c,
Henrik Semb d, Petter Bjorquist a, Peter Sartipy a, Johan Hyllner a,∗
a Cellartis AB, Arvid Wallgrens Backe 20, 413 46 Goteborg, Swedenb Department of Clinical Chemistry/Transfusion Medicine, Sahgrenska University Hospital, 413 45 Goteborg, Sweden
c Department of Obstetrics and Gynaecology, Sahgrenska University Hospital, 413 45 Goteborg, Swedend Section of Endocrinology, Lund University, 221 84 Lund, Sweden
Received 30 June 2005; received in revised form 19 September 2005; accepted 10 October 2005
Abstract
Human embryonic stem cells (hESC) are isolated as clusters of cells from the inner cell mass of blastocysts and thus shouldformally be considered as heterogeneous cell populations. Homogenous hESC cultures can be obtained through subcloning.Here, we report the clonal derivation and characterization of two new hESC lines from the parental cell line SA002 and the
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blastocysts and they have the capacity for indefinite,undifferentiated proliferation in vitro (Thomson et al.,1998; Reubinoff et al., 2000). Based on these uniqueproperties, hESC are extremely useful in basic researchand exciting future applications of hESC and theirderivatives include cell replacement therapies (Jonesand Thomson, 2000). In addition, the hESC technologyplatform holds enormous potential for the developmentof novel approaches for improving processes in drugdiscovery and in vitro toxicology (Rohwedel et al.,2001; Davila et al., 2004; Klemm and Schrattenholz,2004; McNeish, 2004).
The use of hESC in various high throughput appli-cations in drug discovery requires homogeneous cellpopulations. The general procedure for establishmentof hESC lines is based on the isolation and subsequentin vitro culture of the inner cell mass cells from humanblastocysts recovered at days 5–7 after fertilization(Thomson et al., 1998; Reubinoff et al., 2000; Cowanet al., 2004; Heins et al., 2004). Based on morpho-logical evaluation and specific hESC marker analysis,most of the previously described hESC lines appear tobe homogenous populations of cells (Carpenter et al.,2003). However, since most hESC lines available todayare not clonally derived, it cannot be excluded that thesepopulations are mixtures of multiple precursors cellsalready committed to certain lineages. These popula-tions can be further refined by the clonal derivation ofsublines from the existing parental hESC lines (Amitet al., 2000; Heins et al., 2004). The clonal character isorttdIabll
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these cultures a small subpopulation of cells with adiploid karyotype existed. The frequency of diploidcells varied slightly between passages and was in therange of 5–15%. Based on this observation, an effortto derive stable diploid subclones from SA002 wasundertaken. In addition, the clonally derived hESC lineAS034.1 (Heins et al., 2004) was subjected to recloningin order to further refine this cell population. We suc-cessfully established subclones, designated SA002.5and AS034.1.1, respectively, with diploid karyotypesfrom both of these hESC lines and demonstrate thatthey retain their developmental potential and replica-tive capacity. Importantly, the availability of hESC linesSA002 (trisomy 13) and SA002.5 (diploid) provide theunique opportunity to use these cell lines as modelsystems for example when studying diseases and syn-dromes associated with chromosome 13.
2. Material and methods
2.1. Culture of hESC
The hESC line SA002 and the clonally derivedhESC line AS034.1 were established and character-ized as described previously (Heins et al., 2004).The cell lines were maintained at Cellartis AB usingmitomycin-C inactivated mouse embryonic fibrob-last (MEF) feeder layers and VitroHESTM medium(Vitrolife AB, Goteborg, Sweden) supplemented with4Csb(
2
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f importance when using hESC in applications whichely on cell populations with identical genetic consti-ution. In addition, recent reports have demonstratedhat, under certain culture condition, hESC are prone toevelop karyotypic abnormalities (Draper et al., 2004;nzunza et al., 2004; Rosler et al., 2004; Mitalipova etl., 2005). Thus, based on these observations, it woulde necessary to periodically subclone parental hESCines in order to maintain a homogenous diploid popu-ation of undifferentiated cells.
The hESC line SA002 was established prior to 9ugust 2001 and is listed on the National Institutes ofealth Human Embryonic Stem Cell Registry (http://
temcells.nih.gov/research/registry/index.asp) and ishus eligible for US Federal funding. It was recentlyeported that SA002 displays an abnormal karyotypetrisomy 13) (Heins et al., 2004). After subsequent cul-ure and further analysis, it became apparent that within
ng/ml human recombinant bFGF (Invitrogen Co.,arlsbad, CA, USA). Undifferentiated hESC were pas-
aged every 4–5 days with fresh medium and MEFy mechanical dissociation using a “Stem Cell Tool”Swemed Lab International AB, Billdal, Sweden).
.2. Clonal derivation of hESC
The procedure used for clonal derivation of hESCines in the present study has previously been describedn detail (Heins et al., 2004). Briefly, hESC coloniesere mechanically dissociated and treated with.5 mM EDTA for 20 min at 37 ◦C and resuspendedn culture medium. To promote cell survival afterissociation, concentrated conditioned medium fromESC grown in the presence of 15% FCS was used.he conditioned medium was made of KO-DMEMupplemented with 15% FCS, 3.5 mM glucose, 1%
N. Heins et al. / Journal of Biotechnology 122 (2006) 511–520 513
PEST, 1% Glutamax, 1% NEAA, and 4 ng/ml bFGF.This medium was concentrated by a factor 4 usinga Centriprep concentration column WM50. Thecloning medium was subsequently prepared at a finalcomposition of 82% KO-DMEM, 15% concentratedconditioned medium, 3.5 mM d-glucose, 4 ng/mlbFGF, 1% PEST, 1% Glutamax and 1% NEAA. Singlecells were picked under direct microscopic obser-vation and transferred into individual MEF-coatedwells in 96-well plates. The cloning efficiency wascalculated as the number of single cells plated thatsurvived and gave rise to new proliferating hESCcolonies in relation to the total number of single cellsplated.
2.3. Fluorescent in situ hybridization (FISH)
A commercially available kit containing probes forchromosomes 13, 18, 21 and the sex chromosomes (Xand Y) was used following the instructions from themanufacturer (Vysis Inc, Downers Grove, IL, USA)with minor modifications. The slides were analyzedin a fluorescence microscope equipped with appropri-ate filters and software (CytoVision, Applied Imaging,Santa Clara, CA, USA).
2.4. Karyotyping
For preparation of metaphase spreads, the hESCwere washed in cell culture medium, incubated in thepcgai
2
sA1moa1Cs
son ImmunoResearch Laboratories Inc., West Grove,PA, USA). The nuclei were visualized by DAPI stain-ing (Sigma Diagnostics, Stockholm, Sweden). Alka-line phosphatase (ALP) activity was determined usinga commercially available kit following the instructionsindicated by the manufacturer (Sigma Diagnostics).
2.6. Telomerase activity
For analyzing the telomerase activity the TeloTAGGG Telomerase PCR ELISAPLUS kit (Roche,Basel, Switzerland) was employed according to themanufacturer’s instructions. The assay uses the internalactivity of telomerase, amplifying the product by PCRand detecting it with an enzyme linked immunosorbentassay.
2.7. In vitro and in vivo differentiation of hESC
For in vitro studies, undifferentiated hESC colonieswere transferred to suspension cultures, using a “StemCell Tool” (Swemed Lab International AB), and cul-tured in EB-medium (KnockOut-DMEM, 20% FCS,1% PEST, 1% Glutamax, 1% NEAA, and 0.1 mM�-merkaptoetanol). Embryoid bodies (EB) were cul-tured for 6 days in suspension cultures. Subsequently,the EB were plated in culture dishes coated withgelatin and grown in the presence of EB-medium.The medium was changed every 2–3 days and after14 days the cells were fixed in 4% PFA. The cellswumtBaOu
tso1iw(aC
resence of Calyculin A, and then again washed in cellulture medium. The cells were collected by centrifu-ation, hypotonically treated, and fixed using ethanolnd glacial acetic acid. The chromosomes were visual-zed using trypsin-Giemsa- or DAPI staining.
.5. Immunohistochemical analysis
Undifferentiated hESC were fixed in 4% PFA andubsequently permeabilized using 0.5% Triton X-100.fter consecutive washing and blocking steps (using0% dry milk), the cells were incubated with the pri-ary antibody (as indicated) at a final concentration
f 1 �g/ml. The primary antibodies used were directedgainst SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA--81, and Oct-4 (Santa Cruz Biotechnology, Santaruz, CA, USA). FITC-, Cy3-, or TRITC-conjugated
econdary antibodies were used for detection (Jack-
ere subjected to immunohistochemical evaluationsing primary antibodies directed against �-smoothuscle actin (ASMA) (Sigma Diagnostics), �-III-
ubulin (Sigma Diagnostics) and HNF3-� (Santa Cruziotechnology). The secondary antibodies were Goatnti-mouse IgG-Alexa (Molecular Probes, Eugene,R, USA) and Donkey anti-goat IgG-Alexa (Molec-lar Probes).
For assessment of the in vivo differentiation poten-ial of the hESC, 1 × 105 undifferentiated cells wereurgically placed under the kidney capsule of 5 weeksld severe combined immuno-deficient mice (C.B-7/lcrCrl-ScidBR) (Charles River Laboratories, Wilm-ngton, MA, USA). The mice were sacrificed after 8eeks and the teratomas were evaluated as described
Heins et al., 2004). All animal studies were reviewednd approved by the Institutional Animal Care and Useommittee at Goteborg University in accordance with
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the policy regarding the use and care of laboratory ani-mals.
3. Results
3.1. Clonal derivation of hESC lines
Based on the observation that rare diploid cells werepresent in cultures of hESC line SA002 we set out toderive clonal hESC populations from the parental cellline. The cloning efficiency of SA002 was 5% and, intotal, 33 subclones were established from SA002 inpassages 137, 138, 144, 150, and 155. A representativeillustration of a proliferating hESC colony originatingfrom a single hESC plated in one individual well isdepicted in Fig. 1A.
In order to further refine the previously clonallyderived hESC line AS034.1 we recloned this cell pop-ulation. The cloning efficiency was substantially lowerfor AS034.1 compared to SA002 and only 0.1% of thesingle cells plated survived and resulted in proliferat-ing hESC colonies. However, this result was similarto the cloning efficiency previously reported for theparental line AS034 (Heins et al., 2004). One sublinewas established from AS034.1 in passage 74 + 71 andit was designated AS034.1.1.
Subsequently, for expansion, colonies of the clon-ally derived hESC were cultured on MEF in the pres-ence of VitroHES medium supplemented with bFGFacuTdo
3
aosicds1
Fig. 1. (A) Representative illustration of a hESC colony derivedfrom a single hESC (SA002) manually transferred and seeded ontop of a MEF layer in an individual well of a 96-well plate, (B)typical morphology of hESC line SA002.5 maintained on MEFusing VitroHES-medium supplemented with 4 ng/ml bFGF in pas-sage 155 + 27, and (C) in passage 155 + 76. Magnification 100×.
nd passaged every 4–5 days using mechanical disso-iation. The morphology of representative colonies ofndifferentiated hESC is presented in Fig. 1B and C.he colonies appear as tightly packed monolayers withistinct borders and individual cells have a high nucle-cytoplasmic ratio and prominent nucleoli.
.2. Cytogenetic analysis
Fig. 2 shows representative illustrations from a FISHnalysis of SA002 in passage 23 in which about 7%f the cells were disomic for chromosome 13. Theubclones established from hESC line SA002 werenitially screened using FISH and probes specific forhromosome 13 in order to determine if the cells wereisomic or trisomic for chromosome 13. Out of the 33ubclones derived, one was disomic for chromosome3. This apparent diploid subclone was derived from
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Fig. 2. FISH analysis of hESC line SA002 in passage 23. Among the trisomic population of cells (A), rare (approximately 7%) individualcells carrying two copies of chromosome 13 were observed (B) (red, chromosome 13; aqua, chromosome 18; green, chromosome 21; blue,chromosome X).
hESC SA002 in passage 155 and it was designatedSA002.5.
The clonally derived hESC lines SA002.5 andAS034.1.1 were subjected to extended cytogeneticanalysis. Traditional karyotyping and G-bandingdemonstrated that the cells had normal karyotypesand that SA002.5 was 46, XX and AS034.1.1 was46, XY (Fig. 7). Sixteen karyotypes were obtainedin passage 155 + 19 and 10 karyotypes were obtainedin passage 155 + 41 for SA002.5. Nineteen karyotypeswere obtained in passage 74 + 71 + 19 and 6 karyotypeswere obtained in passage 74 + 71 + 22 for AS034.1.1.Furthermore, FISH analysis, using probes directedagainst chromosomes 13, 18, 21, X, and Y, was carriedout as a complement to the karyotype analysis. Cellline SA002.5 was analyzed in passage 155 + 31 andAS034.1.1 was analyzed in passage 74 + 71 + 50. Foreach cell line, approximately 100 nuclei were screenedand the FISH analysis confirmed the data obtained fromkaryotyping and demonstrated that the cell lines werediploid (Table 1).
3.3. Human ESC marker expression analysis
Stem cell marker expression analysis was per-formed using SA002.5 and AS034.1.1 cells. Rep-resentative illustrations are presented in Fig. 3 andshows that the undifferentiated cells expressed SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4, whichah
also expressed by the undifferentiated cells (data notshown). Importantly, SSEA-1 was not expressed by thecells further confirming their undifferentiated state.
3.4. Telomerase activity
The telomerase is responsible for the maintenanceof chromosome length and a significant telomeraseactivity in the cells is instrumental for the indefi-nite replication capacity of hESC (Thomson et al.,1998; Amit et al., 2000). The telomerase activitywas analyzed using undifferentiated SA002.5 andAS034.1.1 cells in passage 155 + 31 and 74 + 71 + 20,respectively (Fig. 4). The cells displayed high telom-erase activity as measured using the Telo TAGGGTelomerase PCR ELISAPLUS kit suggesting that these
Table 1Summary of characteristics of SA002.5 and AS034.1.1
Parameter SA002.5 AS034.1.1
Karyotype 46, XX 46, XYFISH (X, Y, 13, 18, 21) Diploid, XX Diploid, XYSSEA-3 Positive PositiveSSEA-4 Positive PositiveTRA-1-60 Positive PositiveTRA-1-81 Positive PositiveALP Positive PositiveOct-4 Positive PositiveSSEA-1 Negative NegativeTelomerase activity Positive PositivePluripotency in vitro Endo, ecto, meso Endo, ecto, mesoP
re all markers commonly used to identify pluripotentESC (Carpenter et al., 2003). In addition, ALP was
luripotency in vivo Endo, ecto, meso Endo, ecto, meso
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Fig. 3. Undifferentiated hESC colonies (SA002.5) maintained onMEF were analyzed using immunohistochemistry as described inSection 2. The panels show representative DAPI stainings and thecorresponding specific antibody staining for SSEA-3 (A), SSEA-4(B), TRA-1-60 (C), TRA-1-81 (D), Oct-4 (E), and SSEA-1 (F). Thecells were fixed and analyzed in passage 155 + 40. Magnification100×.
Fig. 4. Telomerase activity in undifferentiated hESC was analyzedusing a Telo TAGGG Telomerase PCR ELISAPLUS kit. The bars showthe results obtained from the internal control and the clonally derivedcell lines SA002.5 and AS034.1.1 analyzed in passage 155 + 31 and74 + 71 + 20, respectively. The error bars represent the positive valueof the S.D. (n = 3).
cells have the capacity to proliferate extensivelyin vitro.
3.5. In vitro and in vivo differentiation of hESC
Spontaneous differentiation in vitro was inducedby mechanically dissecting whole colonies of undif-ferentiated hESC and transferring these to suspensioncultures resulting in formation of EB with similar fea-tures as reported before (Itskovitz-Eldor et al., 2000).After 6 days, the EB were plated in gelatin coated tissueculture dishes and the cells were left to proliferate anddifferentiate over a period of 14 days. Differentiatedcells were identified based on their expression of mark-ers specific for cells of the three embryonic germ layers.Fig. 5 shows representative illustrations of differenti-ated cells from SA002.5 expressing �-III-tubulin (ecto-derm), ASMA (mesoderm), and HNF-3� (endoderm).Areas of spontaneously contracting cells, resemblingcardiomyocytes (mesoderm), were also observed (notshown). Similar results were obtained for AS034.1.1(Table 1).
To examine the in vivo differentiation potentialof the cells, undifferentiated hESC colonies fromSA002.5 and AS034.1.1 were manually dissociatedinto smaller clusters and transplanted under the kidneycapsule of SCID mice. After 8 weeks the mice were sac-rificed and the teratomas dissected and analyzed. ThehESC generated complex teratomas comprised of dif-
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Fig. 5. In vitro differentiation of hESC was performed as described in Section 2 using SA002.5 cells in passage 155 + 29. Differentiated cellswere identified using immunohistochemistry. The panels show representative DAPI stainings and the corresponding specific antibody stainingfor �-III-tubulin (A), ASMA (B), and HNF-3� (C). Magnification 400×.
ferentiated cells representing the three embryonic germlayers as identified by histological analysis (Fig. 6). Inaddition, at the site of transplantation, we also observedcystic masses filled with fluid, similar to what has beendescribed before (Reubinoff et al., 2000; Heins et al.,2004).
Taken together, these results demonstrate that theclonally derived hESC lines SA002.5 and AS034.1.1are pluripotent and differentiated cells representing thethree embryonic germ layers can be generated in vitroand in vivo.
The characteristics of SA002.5 and AS034.1.1described herein are summarized in Table 1.
4. Discussion
In the present study, we have demonstrated thesuccessful clonal derivation and characterization oftwo new sublines originating from existing hESClines (Heins et al., 2004). One was a diploid subline(SA002.5) derived from the parental hESC line SA002in which cells with trisomy 13 dominate. Hence, thisis the first report showing that hESC with an identicalgenomic constitution can be purified from a heteroge-neous population using subcloning. The other subline(AS034.1.1) was a reclone from a previously derivedsubclone (AS034.1). Following cytogenetic analysis
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Fig. 6. Undifferentiated hESC (SA002.5 in passage 155 + 39) were surgically placed under the kidney capsule of SCID mice. The mice weresacrificed after 8 weeks and the tumors were fixed in PFA. Histological evaluation of hematoxylin–eosin stained paraffin sections demonstratedthe presence of tissues derived from endo- (A, secretory epithelium), meso- (B, cartilage), and ectoderm (C, neuroectoderm). Magnification400×.
Fig. 7. Karyotype analysis was performed on undifferentiated hESC. The chromosomes were visualized using trypsin-Giemsa staining. Thefigure shows representative karyotypes from SA002.5 (passage 155 + 41) (A) and from AS034.1.1 (passage 74 + 71 + 19) (B).
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we characterized these two new sublines extensivelyusing markers for undifferentiated hESC and assays toinvestigate their developmental potential. The resultspresented here show that the cells have normal kary-otypes (Fig. 7) and that they express the standard panelof hESC markers (Fig. 3) and importantly, they remainpluripotent and can differentiate to cells representingthe three embryonic germ layers in vitro and in vivo(Figs. 5 and 6). At the time of writing, SA002.5 hasbeen cultured for 155 + 85 passages and AS034.1.1 hasbeen cultured for 74 + 71 + 58 passages. The cloningefficiency was remarkably different between the twoparental cell lines (5% for SA002 versus 0.1% forAS034.1) and the reason for this observation remainsto be investigated but most likely represents eitheradaptation to cell dissociation or inherent differences,such as expression of cell–cell adhesion molecules orreceptors for cell survival pathways, between the celllines.
The most common method of subcloning cells isto dilute the cell culture out, so that when plated ina microtiter plate, there is only one cell deposited ineach well. This cell then divides and gives rise to aculture where all cells originate from one single cell.Here we used a slightly different approach in whichthe hESC were initially dissociated into single cellsuspensions using EDTA. Subsequently, single cellswere picked and transferred to individual culture wellsusing a micropipette under direct visual examinationin a light microscope. This approach was also used inpcactmerowucn
odts
studies have highlighted the possibility that karyotypicabnormalities may occur in hESC during in vitroculture, although the frequency of these events appearsto be mainly dependent on the culture conditions used(Draper et al., 2004; Inzunza et al., 2004; Rosler et al.,2004; Mitalipova et al., 2005). In any case, subcloningis a useful approach for maintaining a homogenousdiploid cell population. On the other hand, subcloningcan also be preferentially used when establishinghESC lines with a uniform abnormal karyotype.Cell lines with various abnormal karyotypes mayprove very valuable as investigative tools for researchconcerning specific genetic diseases and syndromes.In this regard, using the unique combination of hESClines SA002 and SA002.5 the molecular mechanismscausing the phenotype of Patau syndrome (trisomy13) (Cox, 1999) can be interrogated. It is likelythat there can be direct effects due to primary upregulation of genes on chromosome 13 but even moreimportantly a secondary generalized transcriptionalmisregulation can contribute significantly to theclinical manifestations of trisomy 13 (FitzPatricket al., 2002). Since hESC are pluripotent and candifferentiate into virtually any cell type it would alsobe possible to investigate the tissue specific effects oftrisomy 13.
Note added in proof
tBhOcs(
A
(Ahog
revious studies and appears to work relatively well forlonal derivation of hESC (Amit et al., 2000; Heins etl., 2004). However, using either of the methods indi-ated above there is potential risk for transferring morehan one cell into an individual well. In order to mini-
ize this formal possibility we recloned the previouslystablished AS034.1. The same line of reasoning is alsoelevant for the subcloning of SA002. However, sincenly a small fraction of cells with a diploid karyotypeas present within the trisomic parental hESC pop-lation the risk of transferring two diploid cells wasonsidered to be negligible. Therefore, SA002.5 wasot subjected to recloning.
There are several advantages with clonal derivationf hESC lines. For instance, in order to undoubtedlyemonstrate the pluripotency of hESC it is necessaryo assay a population of hESC originating from aingle cell (Amit et al., 2000). Furthermore, recent
During the revision of this manuscript, new impor-ant data was obtained on the clonally derived SA002.5.ased on SNP analysis of the hESC using Affymetrixigh-density oligonucleotide arrays (Maitra et al., 2005ct. Nat. Genet. 37(10), 1099–1103) it could be con-
luded that SA002.5 is a true heterozygote for chromo-ome 13 and it does not display uniparental disomy 13Dr. A. Maitra, personal communication).
cknowledgments
This work was supported by Cellartis ABGoteborg, Sweden) and by a grant from the Swedishnimal Welfare Agency (to AL). The work related toESC line SA002 (listed on the NIH Human Embry-nic Stem Cell Registry) was partly supported by NIHrant R24RR019514-01 awarded to Cellartis AB.
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