RESEARCH ARTICLE

Molecular Reproduction & Development 79:709–718 (2012)

Evaluation of Unrestricted Somatic Stem Cells as a FeederLayer to Support Undifferentiated Embryonic Stem Cells

SAEED HEIDARI KESHEL,1,2,3* MASOUD SOLEIMANI,4 MOSTAFA REZAEI TAVIRANI,1 MARYAM EBRAHIMI,2

REZA RAEISOSSADATI,1 HEMAD YASAEI,5 DANIAL AFSHARZADEH,6 MAHMOUD JABBARVAND BEHROZ,2

AMIR ATASHI,4 SAEID AMANPOUR,7 AHAD KHOSHZABAN,2 REZA ROOZAFZOON,1,3 AND GHOLAM REZA BEHROUZI1

1 Proteomics Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran2 Eye Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran3 TissueEngineeringDepartment, School of AdvancedMedical Technology, TehranUniversity ofMedical Science, Tehran, Iran4 Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran5 Institute of Cancer Genetics and Pharmacogenomics, Division of Biosciences, School of Health Sciences and Social Care,

Brunel University, Uxbridge, Middlesex, UK6 Universitat Politecnica de Catalunua, Department of Agribusiness, Biotechnology, Barcelona, Spain7 Faculty of Medicine, Cancer Institute, Tehran University of Medical Sciences, Imam Khomeini Hospital, Tehran, Iran

SUMMARY

The use of unrestricted somatic stem cells (USSCs) holds great promise for futureclinical applications. Conventionally, mouse embryonic fibroblasts (MEFs) or otheranimal-based feeder layers are used to support embryonic stem cell (ESC) growth;the use of such feeder cells increases the risk of retroviral and other pathogenicinfection in clinical trials. Implementation of a human-based feeder layer, such ashUSSCs that are isolated from human sources, lowers such risks. Isolated cord bloodUSSCs derived from various donors were used as a novel, supportive feeder layer forgrowth of C4mES cells (Royan C4 ESCs). Complete cellular characterization usingimmunocytochemical and flow cytometric methods were performed on murine ESCs(mESCs) and hUSSCs. mESCs cultured on hUSSCs showed similar cellular mor-phology and presented the same cell markers of undifferentiated mESC as wouldhave been observed in mESCs grown on MEFs. Our data revealed these cells hadnegative expression of Stat3, Sox2, and Fgf4 genes while showing positive expres-sion for Pou5f1, Nanog, Rex1, Brachyury, Lif, Lifr, Tert, B2m, and Bmp4 genes.Moreover, mESCs cultured on hUSSCs exhibited proven differentiation potential togerm cell layers showing normal karyotype. The major advantage of hUSSCs is theirability to be continuously cultured for at least 50 passages. We have also found thathUSSCs have the potential to provide ESC support from the early moments ofisolation. Further study of hUSSC as a novel human feeder layer may lead to theirincorporation into clinical methods, making them a vital part of the application ofhuman ESCs in clinical cell therapy.

Mol. Reprod. Dev. 79: 709–718, 2012. � 2012 Wiley Periodicals, Inc.

Received 18 September 2011; Accepted 25 July 2012

* Corresponding author:Proteomics Research CenterShahid Beheshti University ofMedical Sciences

Tehran, Iran.E-mail: saeed_heidaril@spu.ir

Conflict of interest: none declared.

Grant sponsor: Tarbiat ModaresUniversity

Publishedonline 18September 2012 inWileyOnline Library(wileyonlinelibrary.com).DOI 10.1002/mrd.22079

Abbreviations: C4mESC, Royan C4 mouse embryonic stem cell line; EB,embryoid body; [m]ESC, [mouse] embryonic stem cell; [h]USSC, [human]unrestricted somatic stem cells; LIF, leukemia inhibitory factor; MEF, mouseembryonic fibroblast.

� 2012 WILEY PERIODICALS, INC.

INTRODUCTIONEmbryonic stem cells (ESCs) are pluripotent cell popula-

tions oftenobtained from the inner cellmassof theblastocyst.ESCs have been used in medical studies since 1981. One ofthe most relevant in vitro studies in this area is the ability tocultureESCsonmouseembryonic fibroblasts (MEFs) whosemitoticactivityhasbeenarrestedby g-irradiationormitomycinC. In culture, ESCs form dense colonies of small cells withscant amounts of cytoplasm and clear nucleoli. They retaintheir normal karyotypes (46XX, 46XY) and their undifferenti-ated state in vitro, although they are capable of differentiatingto a wide range of adult cells under specific conditions. Thedivision time for these cells is between 12 and 18hr, depend-ing on their adaptation to the in vitro microenvironment. Inaddition to MEFs, which are considered to be supporters ofESC growth and proliferation, other feeder layers includeembryonic skin fibroblasts, fallopian tube epithelial cells,mesenchymal stem cells, foreskin fibroblasts, and mouseSIM embryonic fibroblasts (STO) (Amit et al., 2003).

When cultured in vitro without any inhibitors of differen-tiation, ESCs are able to rapidly differentiate into differentcell lineages (Lindvall et al., 2004). Leukemia inhibitoryfactors (LIF), differentiation inhibitory activity (DIA), throm-bopoietin (TPO), stem-cell factor (SCF), and interleukin-6(IL-6) are employed as inhibitors of unwanted ESC differ-entiation. Many supportive layers are able to produce andexpress these inhibitors as secreted or membrane-boundfactors. In the absence of a supportive layer, recombinantLIF, gelatin-coated plates, or extracellular matrices can beused to sustain ESCs in an undifferentiated state (Amitet al., 2004). The results obtained from the effects of LIF onESCs provide a clear example of the regulation that factorshaveon cellular activity. Differentiation of ESCs rarely occurin the presence of LIF, and increasing the amount of LIF hasno effect on the inhibition of the minimal differentiationobserved (Hwang et al., 2004; Jager et al., 2004).

In this present study, unrestricted somatic stem cells(USSCs)wereutilizedasasupportive layer for thegrowthofmurine ESCs (mESCs). In 2004, Jager and his colleaguesfirst isolated USSCs from umbilical cord blood, and evalu-ated the differentiation capacity and cytokine production ofthese cells for transplantation (Jager et al., 2004; Kogleret al., 2004). In fact, USSCs are one of the rare cellpopulations in umbilical cord blood that are consideredpluripotent (Aktas et al., 2010; Zaehres et al., 2010). More-over, USSCs have a high potential of proliferation anddifferentiation. Therefore, USSCs are a valuable sourcefor cell therapy (Kogler et al., 2004; Aktas et al., 2010).These supportive cells are CD45-negative, adherent, andHLA class II-negative stem cells with long telomeres. Addi-tionally, these cells possess a unique profile of cytokinesand a high production rate of self-renewal factors (Kogleret al., 2005; Aktas et al., 2010; Zaehres et al., 2010). Hence,this type of cell would be a suitable option for supportingembryonic stem cells. In this study, we present a noveland inexpensive culture method for expanding RoyanC4 mESCs (C4mESCs) on a supportive layer of humanunrestricted somatic stem cells (hUSSCs).

RESULTS

hUSSC CultureOnly40%of cordbloodsamplesassayedcontainUSSCs.

hUSSCs have a high proliferation capacity, as they neededrepeated and frequent passaging; the cells used in this studywere continuously cultured for 50 passages. The prolifera-tion and morphology of these cells were similar to eachother before and after freezing. hUSSCs are adherent andspindle-shaped cells, 20–25mm in size (Fig. 1).

Colony-Forming Unit AssayColony-forming unit assays provide a convenient

means of assessing the clonogenic capacity of the hUSSCexpanded in culture. The results showed that 88.0�3.2,

Figure 1. Themorphologies of feeder cell. A: hUSSCs at passage 50.B: MEFs at passage 6. The two feeder cell populations show differentmorphologies. Scale bar, 50mm. [Color figure can be seen in theonline version of this article, available at http://wileyonlinelibrary.com/journal/mrd]

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89.0� 2.3, 88.0�3.5, 88.0� 3.2, and 89.0� 3.6 colonieswere formed per 100 cells at passages 2, 10, 20, 35, and46, respectively. In comparison, the clonogenic capacityof MEFs were 81, 83, 70, 35, and 22 colonies formedper 100 cells at passages 2, 4, 6, 8, and 10, respectively.

C4mESC MorphologyThe morphology of C4mESCs on hUSSCs was similar

to those grown on MEFs. The C4mESCs maintained on aMEF feeder layer exhibited a typical morphology, includinga low cytoplasm-to-nucleus ratio, a clear nucleus, anda little space between the cells. Continuous proliferationof C4mESCs on the human feeder layer was normallymaintained for more than 30 passages with preservationin their undifferentiated state (Fig. 2).

Karyotype Analysis of C4mESCs and hUSSCsKaryotype analysis was performed on hUSSCs at pas-

sages 2 and 48; all lines analyzed have a normal chromo-some karyotype of 44XX. After 20 continuous passages,the karyotype of C4mESCs that had been cultured ona hUSSC feeder layer was normal, 38XX as previouslyreported (Fig. 3).

Figure 2. A: Morphology of C4mESC colonies on hUSSCs; mESCswere continuously cultured (for 30 passages). Individual spindle-shaped fibroblastic hUSSCs are also visible. B: Morphology ofC4mESC colonies cultured on MEFs. Scale bar, 50mm. [Color figurecan be seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/mrd]

Figure 3. Karyotype analysis of feeder cells and C4mESCs. A: Kar-yotype analysis of hUSSCs at passage 2 represented a normal 46XXkaryotype. B: Karyotype analysis of hUSSCs at passage 48 repre-sented a normal 46XX karyotype. C: mESCs expanded on humanfeeder cells. mESC culture on hUSSC feeder cells exhibit the normal38XX karyotype after 20 continuous passages.

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Immunophenotypic Characterization of hUSSCsThe immunophenotype of the hUSSC cultures was in-

vestigated via flow cytometry. All cells were highly positivefor the surface antigens CD166, CD73, CD90, and CD105.On the contrary, cells were negative for CD34 and CD45(Fig. 4).

Expression of Pluripotent Cell-Specific MarkersC4mESCs cultured on the hUSSCs versus MEFs were

analyzed for pluripotent genes.Reverse transcriptase-PCR(RT-PCR) was used to assess C4mESCs at passages 30,37, 40, and 53. The expression of Pou5f1 (Oct4), Nanog,Tert, Lifr, Lif, Brachyury, Rex1, Bmp4, and B2m wasassessed; b-globin was used as a positive control. It wasconfirmed that the expression of these pluripotency geneshad been preserved in all of the mESC colonies assayed.Expression of these genes signified that the C4mESCs onthe hUSSCs feeder layer were in an undifferentiated stateand that the hUSSCs were effectively serving as a feederlayer (Fig. 5). An immunocytochemistry assay was alsocarried out for POU5F1. All of the C4mESCs on hUSSCsand MEFs were positive for this specific marker of mESCs(Fig. 6).

Embryoid Body Formation and Their Capacity toDifferentiate

Seven to 10 days after formation, embryoid bodies (EBs)formed from the C4mESCs were subjected to RT-PCR todetect the expression of tissue-specific markers. The EBs

gradually formed a heterogeneous cell populationin vitro. The expression of NF-68 (ectoderm), kallikrein(mesoderm), and albumin (endoderm) was detected indifferentiated EBs. The results showed that after 7 days,NF-68 was strongly expressed during EB differentiation,but albumin and kallikrein were not (Fig. 7).

In Vivo Differentiation AssayAfter extended culture on the hUSSC feeder, the devel-

opmental potential of the C4mESCs was examined usingthe in vivo teratoma model. The teratomas contained rep-resentative tissues of the three embryonic germ layers,including adipose tissue, cartilage tissue, gastrointestinalepithelium, primitive neuroepitheliom, connective tissue,and vein and muscle cells. Representative tissues formedin these teratomas are shown in Figure 8.

DISCUSSION

USSCs are CD45-negative, adherent cells isolated fromthe umbilical cord blood. These cells have unrestrictedproliferation and are classified as pluripotent (Kogleret al., 2005; Aktas et al., 2010). USSCs have the abilityto differentiate into osteoblasts, chondrocytes, blood cells,neurons, hepatocytes, and heart tissue under ex vivo con-ditions (Kogler et al., 2004; Aktas et al., 2010) whereasothermesenchymal cord blood cells are able to differentiateonly to osteoblasts, chondrocytes, adipocytes, and neuronsunder the same condition (Heins et al., 2004; Suss-Toby

Figure 4. Flow cytometry analysis of hUSSCs for surface markers CD34, CD45, CD166, CD73,CD105, CD90. Shaded histogram indicates background signal; open histogram, positive reactivity withthe indicated antibody.

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et al., 2004). USSCs express different factors, includingadheresion molecules, growth factors, and various cyto-kines suchasSCF,VEGF,GM-CSF,M-CSF, TGF-1b, IL-6,G-CSF, LIF, Flt3 ligand, TPO, HGF, SDF-1a, IL-15, IL-12,IL-8, and IL-1b (Kogler et al., 2005). The study presentedhere revealed that cytokine secretion of the hUSSC as afeeder layer allows for the maintenance of mESCs in anundifferentiated state.

Most studiesmaintain mESCs and hESCs onMEFs, butusing MEFs increases the risk of cross-species viral andpathogenic infections, which is the main limiting factorbehind the application of ESC for clinical purposes(Richards et al., 2002; Amit et al., 2003; Hovatta et al.,2003). Other recent studies have ruled out potential feederlayers in which the extracellular matrix, conditioned media,and/or other factors endogenous to the cells negativelyaffect the growth and maintenance of ESCs in an undiffer-entiated state (Xu et al., 2001; Amit et al., 2004; Carpenteret al., 2004; Rosler et al., 2004). Other studies have

reported that embryonic skin cells and muscle cellsobtained from a 14-week-old embryo had the capacity tosupport the growth and continuous proliferation of ESCs(Richards et al., 2002). Immortalized, human artificial fal-lopian tubes have also been employed as a feeder layerto support ESCs, although this was not very successfulas they had to be expanded several million times beforesuccessful co-culture with ESCs. In the present study, cordblood-derived hUSSCs were applied to support the contin-uous proliferation of mESCs. hUSSCs collected from dif-ferent donors are able to maintain and support proliferationof undifferentiatedmESCs from2 to50passages.Basedonthe expression of molecular markers and cell morphology,the proliferation of mESCs on hUSSCs is very similar tothose cultured on MEFs.

One of the benefits of hUSSCs, when compared to otherfeeder layers in the expansion of C4mESCs or humanESCs, is that utilization of foreign hUSSCs involves nostimulation of T-lymphocytes in the immune system. It

Figure 5. Analysis of mESC gene markers after 30, 37, 40, and 53 passages on (A) hUSSCs and(B) MEFs as undifferentiated ESCs. Gene expressions of Stat3, Sox2, B2m, Bmp4, Rex1, Nanog,Brachyury, Tert, Lif, Lifr, and Fgf4 were evaluated by RT-PCR. B2m: positive control, �RT: negativecontrol.

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was reported that hUSSCs have considerable potential todown-regulate the host immune responses during threetransplantations (Kogler et al., 2004). Another advantageof hUSSCs is their ability to undergo more than 50 continu-ous passages without any deficiency in their proliferativepotential or negative effects on karyotype. This is in contrastto MEF feeder layers, which are limited to seven passagesbefore entering cellular senescence, which then increasesthe rate of autonomous ESC differentiation. Additionally,hUSSCs can be used to maintain ESCs soon after theirisolation, whereas ESCs have to be placed onMEFs beforebeing transferred to other feeder layers (Amit et al., 2003).hUSSCs are also durable as they can support andmaintainESC growth after freeze-down at �80�C or cryopreserva-tion in liquid nitrogen (�196�C), both of which furtherincrease the longevity of the hUSSC populations. One ofthemost interesting observationsmade is that the quality ofC4mES support by higher-passage hUSSCs is enhancedcompared to lower-passage populations. This is likely dueto the more developed and balanced cytokines profile thatmay be established during the higher passages. This maybe further aided by longer telomeres, compared to bonemarrow mesenchymal cells, thereby allowing many morepassages of hUSSCs for maturation to occur (Kogler et al.,2004).

In this study, we presented the gene expression ofC4mESCs in a co-culture condition with hUSSCs. Theimportant roles of several down-regulated factors, suchas Stat3, Rex1, Sox2, and Bmp4, in the maintenance ofundifferentiated ESCs have previously been demonstrated(Rao and Stice, 2004; Palmqvist et al., 2005; Masui et al.,2007).Our results showed thatStat3,Sox2, andFgf4geneswerenot expressed inC4mESCsco-culturedwith hUSSCs,butBmp4andPou5f1werepositively expressed.Accordingto some recent studies, Pou5f1 and Sox2 are able to assistin the maintenance of undifferentiated ESCs (Masui et al.,2007). Two possibilities are proposed here: The first is thatPou5f1 maintains an undifferentiated state alone or bycooperating with other factors while the second possibilityis that the C4mESC microenvironment maintains thisstate. Although the ability of hUSSCs to support thehESC lines was not tested, hUSSCs would be an appropri-ate candidate feeder layer. Further experiments will benecessary, such as comparative microarray analysis, tounderstand the characterization of our novel, human-basedfeeder layer.

MATERIALS AND METHODS

Culture and Isolation of hUSSC From FreshUmbilical Cord Blood

Cord blood was collected from the umbilical cord vein of30 mothers who gave informed consent. The mean age ofdonors was 28 years. After sample collection, red bloodcells were lysed using ammonium chloride (NH4Cl) (SigmaChemical Co, St. Louis) and the isolation procedurewas continued via Ficoll-Hypaque (Amersham). A cellsolution was gently overlaid onto the Ficoll-Hypaque and

Figure 6. Immunostaining of mESCs after culturing for 30 passages.Panels on the left column show DAPI stained C4mESCs on hUSSCs(A andC) andMEFs (E andG). Arrows point tomESC colonies. Panelson the right showPOU5F1 expression in hUSCCs (B andD) andMEFs(F andH) alone. NomESCswere observed in control plates (D andH).Scale bar, 200mm.

Figure 7. Expression of tissue-specific markers in differentiatedEBs. Various marker genes, including NF-68 (ectoderm), kallikrein(mesoderm), and albumin (endoderm), were detected in differentiat-ed EBs derived from mESCs cultured on hUSSCs. The resultsshowed that after 7 days, NF-68 was strongly expressed duringEB differentiation.

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centrifuged at 400g for 30min at room temperature. Aftercentrifugation, the white cell layer between the Ficoll andplasmawas collected and diluted with Hank’s balanced saltsolution (HBSS). It was then centrifuged at 400g for 10minat room temperature. The isolated mononuclear cell layerwas washed in PBS, then resuspended in growth mediumcontaining Dulbecco’s modified Eagle’s medium (DMEM)containing low glucose supplementedwith 10% fetal bovineserum (FBS), 2mMglutamine, 100mg/ml streptomycin, and100U/ml penicillin (all provided fromGibco�). The isolatedmononuclear cell layer was then plated onto polystyreneplastic of 75 cm2 tissue-culture flasks (Nunc). The cellsbeing cultured were maintained at 37�C in a humidified,5% CO2 incubator (Binder). When the cells reached80% confluence, the cultures were harvested with 0.25%Trypsin–EDTA solution (Gibco�). Non-adherent cellswere removed after 48 hr. The medium was replaced every3–4 days.

Flow Cytometric Analysis of hUSSCshUSSCs were first trypsinized and counted. Tubes con-

taining �106 cells were centrifuged at 300g for 6min, andthen the cell pellets were resuspended in a solution of3% human serum in phosphate-buffere saline (PBS).This mixture was incubated at room temperature for30min, and then centrifuged again as above. The secondresuspensionwasonly inPBS. Thecellmixturewas passedthrough a nylon mesh; 100ml of the mixture was added toeach tube along with the following antibodies: anti-CD73,anti-CD105, anti-CD166, anti-CD45, anti-CD90, and anti-CD34 (all products from Abcam). These tubes were incu-bated at 4�C in a dark room for 45min. After the washingprocess, the cells were fixed in 100ml of 1% paraformalde-hyde in PBS before flow cytometric analysis was carriedout. The ratios of fluorescence versus scattered signalwere calculated by the flow cytometer (Partec). Histogramswere generated using the WinDmi 2.9 software.

Figure 8. In vivo differentiation (teratoma formation) ofmESCs cultured on hUSSCs. (A) Adipose tissue,(B) cartilage tissue, (C) gastrointestinal epithelium, (D) primitive neuroepitheliom, (E) vein, (F) muscle.Hematoxylin and eosin stain. Scale bar, 50mm. [Color figure can be seen in the online version of thisarticle, available at http://wileyonlinelibrary.com/journal/mrd]

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APPLICATION OF USSCS AS A FEEDER LAYER FOR MES CELLS

Preparation of Feeder Layer Containing hUSSCshUSSC passages 2–55 were utilized as a supportive

layer forC4mESCs (RoyanC4embryonic stemcell line). Tostop mitosis, hUSSCs were first treated using 10mg/ml ofmitomycin C (Sigma Chemical Co, St. Louis) for 90min.Theywere thenwashedwith sterilePBS three times in orderto remove the mitomycin C. Cells were detached from theflask using trypsin–EDTA 0.25% (Gibco�), and collectedby centrifugation. The cell pellet was resuspended withDMEM (Gibco�) and 10% FBS (Gibco�), counted, andtransferred to a culture flask. After adhesion of hUSSCs tothe bottom of the flasks, their culture medium was ex-changed for KnockOut� D-MEM (Gibco�) supplementedwith 2.0mM L-glutamine (Gibco�), 0.1mM non-essentialamino acids (Gibco�), 0.1mM 2-mercaptoethanol (SigmaChemical Co), 5mg/ml insulin, 5mg/ml transferrin, 5mg/mlselenium ITS (Gibco�), 100U/ml mouse leukemia inhibi-tory factor (LIF) (Chemicon International), and 10% ESC-qualified fetal bovine serum (Gibco�). The inactivatedhUSSCs were adapted to ESC culture medium for 24 hr.

Preparation of MEF Feeder LayerMEFs were prepared from 13-day-old mouse embryos.

Each embryo was detached from the placenta and sur-rounding membranes. With mouse brain and dark-redorgans snipped off beforehand, each embryo was washedwith fresh PBS and its cells/tissues were suspended intrypsin–EDTA (about 1–2ml per embryo). Cells werethen incubated at 37�C for 15min with gentle shaking.The suspension was transferred to a 50-ml Falcon tube,and about two volumes of fresh media were added. TheMEF media contained DMEM (high glucose), 10% (v/v)FCS (Gibco�), 200mM L-glutamine (Gibco�), 100mg/mlstreptomycin and 100U/ml penicillin (Gibco�). For prepa-ration of the feeder layer, mitomycin C was diluted in MEFmedium to a final concentration of 10mg/ml. Cells wereexposed to the drug for 2 hr at 37�C, and then washed withsterile PBS three times in order to remove themitomycin C.Finally, cells were trypsinized, centrifuged, resuspended,and replated.

Culture of C4mESCs on Human orMEF Feeder Layer

After preparation and adaptation of the feeder layer,C4mESCs frozen at �80�C were retrieved and transferredonto the feeder layer. Cultures were maintained at 37�Cwith 5%CO2, and culturemediumwas replacedevery 24 hr.Themost suitable stage for the passage of the cells is whenthe diameter of the C4mESC colonies is 200–400mm with3� 106 cells/T25. The feeder layer culture medium wasreplaced with the ESC culture medium approximately 2 hrbefore the passage of C4mESCs.

Colony-Forming Unit AssayThe clonogenic potential of hUSSC (passages 2, 10, 20,

35, and46) fromall donors (n¼ 13) orMEFs (passages 2, 4,

6, 8, and10)was testedbyacolony-formingunit assay.Onehundred cells were plated on a 60-mm2 dish in completemedium. Samples were incubated for 7 days in completemedium, at which time the plates were stained with 3%crystal violet in methanol at room temperature for 10min.All visible colonies were counted. This step was repeated10 times, and the data were presented as a mean�standard deviation.

Immunocytochemistry AnalysisThe cultured C4mESCs on the hUSSC feeder layer was

used for immunocytochemical analysis. The cells were firstfixed in 4%paraformaldehyde. Theywere incubated initiallyat 4�C for 30min, and then at room temperature for 30min.After washing twice with cold PBS, 0.4% Triton X-100 wasadded to the cells. After incubation at room temperature for40min, the cells were rinsed with 0.1% PBS-Tween andplaced in a blocking solution containing 1% human serumalbumin in PBS for 30min. After washing twice with PBS,the primary antibody of the rabbit antimouse-POU5F1(Chemicon International) was added to the cells and thenincubated at 4�C for 12 hr in a dark room. After washingthree times with 1% PBS-Tween, the secondary antibody(PE-conjugated) (Sigma-Aldrich Chemie GM, Steinheim,Germany) was added to the cells at a 1:30 dilution.After 3 hr, the cells were washed with PBS-Tween. DAPI(Sigma-Aldrich Chemie GM, Steinheim) was used to coun-terstaining the nuclei.

Karyotype Analysis for C4mESC and hUSSCsKaryotype analysis was performed onmESC cells main-

tained on both feeder layers, and on the first and lastpassages of the hUSSC feeder layer the C4mESCs werecultured on. The cells were first placed in an incubator with0.1mg/ml colcemid for 3–4 hr. Next, they were trypsinizedand 0.075MKCl solution was added to them. Then the cellswere incubated with 5% CO2 at 37

�C for 20min. In the nextstep, methanol and acetic acid in 1:3 ratios were added tofix the samples. Finally, the cells scattered over the slidesurface and chromosomes were subjected to karyotypeanalysis. At least 20 metaphase spreads were analyzedper cell line, and the final karyotype was stated if it waspresent in at least 90% of the metaphases.

Embryoid Body (EB) Formation and TheirDifferentiation Capacity

In order to induce EB formation, C4mESCs were firstseparated into a single-cell suspension in ESCmedia. Aftersuspension, EB formation was induced by exchanging theESCmedia for inductive media (DMEMwith 5% ESC-FBS,5% normal bovine serum, 2mM L-glutamine, 0.1mMof 2-mercaptoethanol, 50U/ml penicillin and 50mg/mlstreptomycin). About 800 undifferentiated ESCs were in-cluded in a hanging drop. After 3–4 days, ESCs aggregatedinto EBs. In the next step, EBs were transferred to culturemedium. A period of 7–10 days was considered sufficient

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for spontaneous differentiation of EBs. After this period,the EBs were characterized to detect gene markers of allthree germ layers, including kallikrin (mesoderm), NF-68(ectoderm), and albumin (endoderm) using reversetranscriptase-PCR (RT-PCR).

RNA Preparation and Gene Expression AnalysisRT-PCR was performed to evaluate the expression of

Pou5f1 (Oct4),Nanog,Rex1,Brachyury,Lif,Lifr,Tert,B2m,Stat3, Sox2, Bmp4, and Fgf4 in C4mESCs that had beencultured on MEFs or hUSSCs, or kallikrin, NF-68, andalbumin in differentiated EBs. Total RNA was isolatedfrom the cells using an RNA extraction kit (Fermentas,Ontario, Canada). RNA samples were treated with DNaseI (Fermentas) to remove contamination of genomic DNA.The primer sequences used are shown in Table 1. Reversetranscription was performed using the RevertAid� firststrand cDNA synthesis kit (Fermentas) and 2mg of totalRNA per reaction, according to the manufacturer’s instruc-tions. The PCR thermal cycling program was as follows:initial denaturation at 95�C for 1min, followed by 40 cyclesof 95�C for 40 sec, annealing temperature for 40 sec, andextension at 72�C for 1min, with a final extension at 72�Cfor 5min. The PCR products were run on 2% agarosegel electrophoresis and stained with ethidium bromide.

Formation of TeratomasAfter 20 passages on hUSSC feeder cells, 2� 106

C4mESCs cells were inoculated into the hind leg of severecombined immunodeficient (SCID) mice, according to NIHanimal treatment protocols. Teratomas were recovered10 weeks later. Tumors were embedded in paraffin andhistologically examined after hematoxylin and eosin stain-ing. hUSSC feeder cells (106) that were inoculated into thesame sites of the SCID mice, as the control, never inducedany tumors.

ACKNOWLEDGMENTS

We are grateful to the hematology department ofTarbiat Modares University for supporting this work and

also to Dr. Said Kaviani, Naser Ahmadbigi, Ehsan Arefian.This project is part of the PhD thesis of Saeed HeidariKeshel.

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TABLE 1. Primer Sequences

Gene Sense Antisense

Pou5F1 50-TGTGGACCTCAGGTTGGACT 50-CTTCTGCAGGGCTTTCATGTNanog 50-GGGAACGCCTCATCAATGCCTG 50-TGCGCATGGCTTTCCCTAGTGGTERT 50-TGGTTGCCCAATGCCTAGTGTG 50-GGACTTGGCACCCATGATTTGCB2M 50-TTC AGTCGCGGTCGCTTCAGTC 50-CAATGTGAGGCGGGTGGAACTGBMP4 50-AATGGAGCCATTCGGTAGTGCC 50-GGGATGCTGCTGAGGTTGAAGREX1 50-ACATGCGGGCCTGACCACTTTG 50-TTCTCGATGGCCTCCTGGTTGGBrachyury 50-TTCTTGCTGGACTTCGTGACGG 50-TGCAGCATGGACAGACAAGCAGLIF 50-AAGGTGTTGGCCGCAGGGATTG 50-TGGCCCACACGGTACTTGTTGCLIFR 50-TGTCGCGCCAAATTTCACCG 50-CGCCAACAATGACAGCCACAGCSTAT3 50-AGCATCGAGCAGCTGACAACGC 50-TGCCTCCTCCTTGGGATTGTCGSOX2 50-TGATGGAGACGGAGCTGAAGCC 50-AGCTGTCCATGCGCTGGTTCAGFGF4 50-AGGGTGTGCTTCCGAGGCTGAG 50-GCAGCACTCACCGCCCTCACGG

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Molecular Reproduction & Development KESHEL ET AL.