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First published online June 13, 2005
Stem Cells Vol. 23 No. 9 October 2005, pp. 1221 -1227
doi:10.1634/stemcells.2004-0347; www.StemCells.com
© 2005 AlphaMed Press

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EXPERIMENTAL PROTOCOLS FOR EMBRYONIC STEM CELL RESEARCH

Derivation and Growing Human Embryonic Stem Cells on Feeders Derived from Themselves

Qian Wanga, Zhen F. Fangb, Fan Jinc, Yong Lua, Hui Gaia, Hui Z. Shenga,b

a Center for Developmental Biology, Xinhua Hospital, Shanghai Second Medical University, Shanghai, P.R. China;
b Laboratory of Stem Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, P.R. China;
c IVF Center of Women’s Hospital, School of Medicine, Zhejiang University, Zhejiang, P.R. China

Key Words. Human embryonic stem cells • Feeder

Correspondence: Hui Z. Sheng, M.D., Ph.D., Center for Developmental Biology, 1665 Kong Jiang Road, Xinhua Hospital, Shanghai Second Medical University, Shanghai 200092, P.R. China. Telephone: 86-21-55570017; Fax: 86-21-55570017; e-mail: hzsheng{at}sh163.sta.net.cn


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human embryonic stem cells (hESCs) are pluripotent. They have the potential to differentiate into every cell type of an organism. Since many human somatic cell types have the ability to support the growth of hESCs, cells differentiated from hESCs may also be able to support the growth of themselves. We tested this hypothesis by growing hESCs on feeders derived from themselves and demonstrated that such feeders did constitute an environment suitable for the derivation and long-term growth of hESCs. hESCs maintained in this system expressed all the markers indicative of the undifferentiated state and gave rise to cell types representative of all three primary germ layers upon differentiation. By modifying the genome of hESCs, feeders with special features can be derived and mass produced. The system will facilitate large-scale production of hESCs in a standardized animal pathogen-free environment.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human embryonic stem cell (hESC) lines were first isolated by Thomson et al. [1] in 1998. These cells have the potential to produce any type of cells of the body in an unlimited quantity and can be genetically altered [2]. These characteristics make hESCs good candidates for cell-based therapies.

hESCs are in most cases cultured on mouse embryonic fibroblast (MEF). However, concerns arise that contaminations, such as rodent viruses or proteins introduced by MEF, may make hESCs unsuitable for therapeutic purposes. Alternative culture systems have therefore been invented to avoid the use of MEF. Some of these systems use human somatic cells or cell lines to substitute MEF. These include embryonic fibroblasts, adult fallopian tube epithelium [3], bone marrow stromal cells [4], foreskin fibroblasts [5], human cell lines (D551/CCL-10, CCL-2552), adult skin cells [6], and placenta cells [7]. The other systems use a feeder-free environment that cultures hESCs in special media supplemented with Matrigel matrix plus MEF-conditioned medium [8], fibronectin plus transforming growth factor ß1 and basic fibroblast growth factor (bFGF) [9], or Matrigel in combination with activator of WNT pathway [10], respectively.

Although these alternative systems provide solutions for minimizing pathogen contamination, each of them has its own limitations. For example, using human somatic cells as feeder, human materials will be needed frequently and tissues from different sources may bring variations to the culture. Using MEF-conditioned medium may still expose hESCs to animal pathogens. Feeder-free systems, using additional growth factors, will significantly increase the cost of the culture. In addition, until now it has not been reported that current feeder-free systems can be used to derive new hESC lines. Overall, each of these culture systems offers some advantages and disadvantages. Complementary to each other, they provide a variety of choices to meet the needs of different applications.

It was observed that in the feeder-free culture system, there were some stroma-like cells at the peripheral of the hESC colonies [8]. It is possible that those differentiated cells may function as feeders to help maintain the rest of hESCs in an undifferentiated status. This observation has brought up the question of whether feeders can be derived from hESCs and used to support their own growth. This system would provide a growth environment for a broad range of hESC lines without increasing the possibility of heterogeneous contaminations. In this study, we show that hESCs can indeed produce feeder cells that are capable of supporting derivation and long-term growth of themselves. Potential applications of the method are discussed.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of hESC-Derived Feeder
hESC lines H1 (WiCell Research Institute, Madison, WI, http://www.wicell.org), SH1, and SH2 (derived by Center for Developmental Biology, Xinhua Hospital, Shanghai, P.R. China) were initially cultured on irradiated MEF (55 Gy). A serum-free culture medium for hESCs was adopted that consisted of 79% Dulbecco’s modified Eagle’s medium (DMEM)/F-12 supplemented with 20% knockout serum replacement (SR), 2 mM L-glutamine, 1% minimal essential medium (MEM)–nonessential amino acid solution, 0.1 mM ß-mercaptoethanol, and 4 ng/ml human recombinant bFGF (hrbFGF) (all from Gibco BRL, Gaithersburg, MD, http://www.gibcobrl.com). hESCs were split every 5–7 days using 1 mg/ml collagenase IV (Gibco BRL). Embryoid bodies (EBs) were obtained by culturing hESCs in suspension for 4 days using a previously published protocol [11]. To prepare hESC-derived feeders (EDFs), EBs were plated in a 25-cm2 tissue culture flask coated with 0.1% gelatin. After 10–14 days, differentiated cells were digested with 0.05% trypsin/0.53 mM EDTA (Gibco BRL) and split into two flasks (passage 1 [P1]). After 3–5 days, when cells reached 90% confluence, cells were again split to obtain P2 cells. Cells of P5 and after were used as feeders and were named EDF. Alternatively, hESCs were kept at high density for 12–14 days to induce differentiation. The differentiated cells were then digested and plated to obtain P1 EDF cells. The medium used for EDF is DMEM (Gibco BRL) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, http://www.hyclone.com) or 10% human serum (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 1 x 10–8 M dexamethasone (Sigma-Aldrich), x1 insulin-transferrin-selenium (Gibco BRL), and 10 ng/ml epidermal growth factor (Sigma-Aldrich). The EDFs were cryopreserved using a solution containing 10% dimethyl sulfoxide (Sigma-Aldrich), 10% FBS, and 80% DMEM. To support derivation and long-term culture of hESCs, EDFs were irradiated (55 Gy) and plated at 10,000 to 12,500 cells per cm [2].

Derivation and Culture of hESCs on EDF
Human embryos were produced by the IVF Center of Women’s Hospital, School of Medicine, Zhejiang University (Zhejiang, P.R. China) for clinical purposes. Surplus embryos were used for hESC derivation with informed consent. The procedure to derive hESCs from surplus embryos was in accordance with the Guidelines for Research on Human Embryonic Stem Cells issued by the Ministry of Science and Technology and Ministry of Health of P.R. China and approved by the Ethics Committee of Xinhua Hospital.

Zona pellucida of the blastocyst was removed with 0.1% pronase (Sigma-Aldrich). Inner cell mass was isolated manually and cultured on irradiated EDFs prepared as described above. The culture medium consisted of 78% DMEM/F-12, 20% knockout SR, 2 mM L-glutamine, 2% nonessential amino acids, 0.1 mM ß-mercaptoethanol, and 8 ng/ml hrbFGF. The medium was changed every 3 days. Ten to 14 days after initial plating, colonies with typical hESC morphology appeared. These colonies were dissociated either mechanically or with 1 mg/ml collagenase IV and transferred onto a fresh dish with EDFs. After 10 passages, the medium was changed to 79% DMEM/F-12 supplemented with 20% knockout SR, 2 mM L-glutamine, 1% MEM-nonessential amino acids solution, 0.1 mM ß-mercaptoethanol, and 4 ng/ml hrbFGF.

To transfer hESCs previously grown on MEFs to EDFs, colonies were digested with 1 mg/ml collagenase IV for 5–10 minutes at 37°C, dissociated, and transferred to culture dishes preplated with EDFs. Long-term culture of hESCs was performed by passaging hESCs every 5–6 days using collagenase digestion in combination with manual dissociation. hESCs were cryopreserved in freezing media consisting of 45% SR, 45% DMEM/F-12, and 10% dimethylsulfoxide.

To determine population doubling (PD) time, cell numbers in five selected independent colonies were counted under an inverted microscope (CKX41; Olympus, Tokyo, http://www.olympus-global.com). Data collected on days 1 and 2 (with 36 hours apart) were used to calculate PD values: PD = log2, in which N1 and N2 are the cell numbers of selected colonies counted on day 1 and day 2, respectively.

Immunohistochemistry and Alkaline Phosphatase Staining
For immunocytochemistry and alkaline phosphatase staining, hESCs or EDF cells were fixed in 4% paraformaldehyde in phosphate buffered saline, 0.05% Triton X-100 for 30 minutes at room temperature and incubated with primary antibodies overnight at 4°C. Fluorescein isothiocyanate (FITC)–conjugated secondary antibodies (1:100) were from Jackson Immunoresearch (West Grove, PA; http//www.jacksonimmuno.com). Antibodies against SSEA-1 (1:15), SSEA-3 (1:40), SSEA-4 (1:40), TRA-1-60 (1:100), and TRA-1-81 (1:100) were from Chemicon International Inc. (Temecula, CA; http://www.chemicon.com). Anti–OCT-3A/4 antibody (1:200) was from Santa Cruz Biotechnology (SantaCruz, CA; http://www.scbt.com). Anti-vimentin antibody (1:50) was from DAKO (Glostrup, Denmark, http://www.dakocytomation.com). The specificity of each antibody was verified by negative controls included in each experiment. The slides were analyzed using a confocal microscope (FV500; Olympus). Alkaline phosphatase staining was performed using Nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate stock solution(Roche Diagnostics, Basel Switzerland, http://www.roche-applied-science.com) according to the manufacturer’s recommendations.

Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted from cells using Trizol-Reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and treated with RNase-free DNase (Promega, Madison, WI, http://www.promega.com). cDNA was synthesized from total RNA using Moloney murine leukemia virus (M-MLV) reverse transcription (RT) (Promega). Polymerase chain reaction (PCR) was carried out using the following parameters: denature at 94°C for 4 minutes, 35 cycles at 94°C for 30 seconds, 55°C (60°C for NANOG) for 30 seconds, and 72°C for 30 seconds, final extension at 72°C for 10 minutes. PCR primers used are listed in Table 1Go. Negative control RT-PCR reactions were carried out under the same conditions, except that the cDNA synthesis was carried out without the M-MLV RT.


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  		Table 1. Polymerase chain reaction primers used in the present study
 
Karyotype Analysis
To eliminate EDF before karyotyping, H1 ESCs grown on EDF for 18 passages were transferred onto irradiated MEF for another three to four passages. For karyotyping, hESCs were treated with 0.1 µg/ml colcemid (Gibco BRL) for 3 hours, incubated in hypo-tonic solution at 37°C for 30 minutes in a 5% CO2 atmosphere, fixed in carnoy’s solution (methanol:glacial acetic acid at 3:1) three times, and dropped onto chilled glass slides. Chromosome spreads were Giemsa-banded and photographed. More than 30 metaphase spreads and six banded karyotypes were analyzed for a cell line.

EB Formation and In Vitro Differentiation
hESCs were digested with 1 mg/ml collagenase IV and scrubbed into small clumps. The clumps of hESCs were cultured in suspension in a medium consisting of 79% DMEM, 20% FBS, 2 mM L-glutamine, 1% MEM nonessential amino acids, and 0.1 mM ß-mercaptoethanol for 4–6 days. For further differentiation, EBs were plated onto a 100-mm tissue culture dish (no. 3005; Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, http://www.bd.com) for another 30 days before RT-PCR analysis.

Formation of Teratomas
After 11 passages on EDF, 1 x 106 H1 ESCs were injected into the rear leg muscle of NOD/LtSz-scid mice. Teratomas were collected 10–11 weeks later, embedded in paraffin, and examined histologically after staining with hematoxylin-eosin.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of EDFs
Early passages of differentiated hESCs were composed of various cells of different shapes as well as many cellular aggregates. As culture went on, cellular aggregates gradually disappeared and cells with a fibroblast-like morphology prevailed, which expressed at least some fibroblast cell markers, such as proline 4-hydroxylase [12–14] and vimentin (Fig. 1Go) [15, 16]. These cells at the fifth or higher passages were used as feeders. EDF cells reached confluence and were split every 2–3 days in the early stages of culture. The interval between passages was gradually prolonged to 4–5 days. Using the current protocol, these cells can be passaged at least 15 times (approximately 20 PDs) and still maintain uniform fibroblast-like morphology. When EDF cells were transferred to the same culture medium supplemented with human serum instead of FBS, they maintained the same morphology and were passaged at the same interval (data not shown).



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  		Figure 1. Expression of fibroblast markers by human embryonic stem cell–derived feeder (passage 13) as detected by (A) immunofluorescent staining using a vimentin-specific antibody and (B) reverse transcription–polymerase chain reaction analysis. Lanes 1, 3, 5: expression of P4HB, Vimentin, and ß-actin by EDF cells, respectively. Lanes 2, 4, 6: negative controls of those genes (see Materials and Methods). M, 100-bp ladder.

 
Three hESC lines, H1, SH1, and SH2, have been used to derive EDFs. Eleven EDF subcultures were established from these lines, and all of them were able to support sustained growth of hESCs. We have also compared EDF from early passage (passage 5) to later passage (passage 15) for supporting the growth of hESCs. Both of them were effective. EDF cells can also be cryopreserved without losing their ability to proliferate and to support the growth of hESCs (data not shown). These results indicate that the growth-promoting property of EDFs is very stable and not influenced by either the number of passages or freeze-thaw cycles.

Derivation and Growth of hESC Lines on EDF
To examine whether new hESC lines could be derived on EDF, inner cell mass of the blastocyst was dissected and plated on EDFs. Cells with morphology typical of hESCs grew out of the cell cluster on the second week after plating. Once dissociated and passaged to new EDFs, these cells continued to proliferate to form a new hESC line, SH7. The SH7 cells appeared as round, monolayer colonies with clearly defined edges. Under high magnification, individual ESCs showed a high nucleus-to-cytoplasm ratio and prominent nucleoli (Fig. 2Go). They are positive for SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and OCT-4, as shown by immunofluorescent analysis (Fig. 3Go). At present, they have been passaged 30 times (>180 days).



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  		Figure 2. Morphology of human embryonic stem cell (SH7) colonies, which were derived on human embryonic stem cell–derived feeder. (A): Outgrowth of inner cell mass 9 days after plating. SH7 cell line at passage 5 (B), passage 24 (C), and passage 32 (D). Pictures are taken in different magnifications (see bars) to show various aspects of the colonies. Scale bar = 100 µm.

 


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  		Figure 3. Fluorescent immunocytochemistry of H1 cells on mouse embryonic fibroblast (MEF) (passage 67), H1 cells on human embryonic stem cell–derived feeder (EDF) (passage 30), and SH7 cells on EDF (passage 28). Lane 1, OCT-4; lane 2, SSEA-3; lane 3, SSEA-4; lane 4, TRA-1-60; lane 5, TRA-1-81. Scale bar = 100 µm.

 
hESCs previously grown on MEF can be easily adapted to EDFs. Usually at the first one or two passages, some colonies tended to differentiate, but the percentage of differentiated colonies never exceeded 20% of all the colonies in the culture. Most colonies maintained an undifferentiated morphology after transferring from MEF to EDF. Similarly, when they were first transferred from EDFs to MEFs, a certain percentage of colonies also differentiated spontaneously. Such spontaneous differentiation seen during transition soon reduced to a normal range in subsequent passages using the same type of feeder, either MEFs or EDFs. hESCs on EDF were usually round, flat colonies with a large surface area, maintained the same morphology as they grow on MEFs (Fig. 4Go). Under high magnification, individual ESCs on EDFs remained round and small, with a high nucleus:cytoplasm ratio and prominent nucleoli (Fig. 4EGo). The H1, SH1, and SH2 lines were plated on either EDF or MEF, and their growth rates were compared. The three lines grown on EDF had a PD time of approximately 38, 35, and 37 hours, respectively, and on MEF, of approximately 37, 33, and 38 hours, respectively. At the time of writing this article, the H1 line had been maintained on EDF for 52 passages (>300 days), the SH1 line for 31 passages (>150 days), and the SH2 line for 34 passages (>200 days).



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  		Figure 4. Phase-contrast micrographs of feeders, human embryonic stem cells (hESCs), and embryoid bodies (EBs). (A): Mouse embryonic fibroblast (MEF) feeder cells. (B): Colony of H1 ESCs on MEF layers at passage 50. (C): Human embryonic stem cell–derived feeder (EDF) cells. (D): Colony of H1 ESCs on EDF layers at passage 24. (E): Colonies of H1 ESCs on EDF at high magnification. (F): Day-3 EBs derived from H1 cells, which were cultured on EDF for 20 passages. Scale bar = 100 µm.

 
Marker Expression of hESCs Grown on EDFs
To test whether EDFs were able to support proliferation of hESCs in an undifferentiated status, we examined expression of undifferentiated markers of the hESC lines: the SH7 line, which was derived and maintained on EDF (passage 28), and the H1 line grown on either MEF (passage 67) or EDF (passage 30). All three lines were positive for OCT-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 (Fig. 3Go) and negative for SSEA-1 (data not shown). These cells also displayed strong alkaline phosphatase activity (data not shown). To ensure undifferentiated status of hESCs maintained on EDF, we also examined differentiated markers of the H1 cell line grown on either MEF or EDF. Although EBs formed from H1 cells (grown on EDF for 20 passages) expressed markers representative of all three germ layers, the H1 cell line grown on either MEF or EDF expressed OCT-4 and NANOG but none of the differentiated markers (Fig. 5Go). These data indicated that hESCs remained undifferentiated after long-term culture on EDFs.



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  		Figure 5. Reverse transcription–polymerase chain reaction analysis of gene expression of H1 cells cultured on human embryonic stem cell–derived feeder (EDF) (passage 20) (lane 2) and on mouse embryonic fibroblast (passage 60) (lane 3). Human embryonic stem cells cultured on both feeders expressed OCT4 and Nanog but not differentiated markers. After embryoid body formation, H1 cells grown on EDF expressed differentiated markers representative of the three germ layers (lane 1).

 
Pluripotency of hESCs on EDF
hESCs that had been cultured on EDF long term were tested for their potential to form EBs and teratomas. These cells formed EBs when cultured insuspension (Fig.4FGo). After 1 month, the EBs were examined for their gene expression using an RT-PCR assay. Those EBs expressed genes representative of all three primary germ layers, including nestin, neurofilament L (ectoderm), alpha-fetoprotein, albumin (endoderm), and BMP4 (mesoderm) (Fig. 5Go).

hESCs grown on EDF for 11 passages formed teratomas when injected into NOD/LtSz-scid mice. The teratomas contained a variety of tissue types, including epithelial cells containing melanin (ectoderm, Fig. 6AGo), muscle and cartilage (mesoderm, Figs. 6A, 6DGo), and columnar epithelium with goblet cells (endoderm, Figs. 6B, 6CGo). Therefore, hESCs grown on EDFs retained pluripotency, as demonstrated by both in vivo and in vitro assays.



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  		Figure 6. Histology of differentiated elements in teratomas formed by H1 embryonic stem cells after 11 passages on human embryonic stem cell–derived feeder. (A): Epithelium with cells containing melanin and striated muscle. (B): Gut-like epithelium with mucous-containing cells and glandular epithelium. (C): Columnar epithelium with goblet cells. (D): Cartilage. Scale bar = 100 µm.

 
Karyotype of hESCs on EDF
We counted 30 karyotypes for the H1 cells after 18 passages on EDF. Each of them contained 46 chromosomes. Of the six samples on which we performed G-band analysis, all of them showed a normal 46,XY karyotype (Fig. 7Go).



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  		Figure 7. Normal 46,XY karyotype of H1 cells cultured on human embryonic stem cell–derived feeder for 18 passages.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Grown on EDFs, hESCs have retained all of the known characteristics of pluripotent cells, even after prolonged culture for more than 10 months (>50 passages). They are positive for SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT-4, and NANOG. They are negative for SSEA-1, displaying a gene expression pattern typical of undifferentiated primate ESCs [17–22]. These cells formed both EBs and teratomas that contained cell and tissue types of all three germ layers.

hESCs grown on EDF were able to proliferate for a long time in a pluripotent status, indicating that EDF provided an environment that fulfills the growth requirements of hESCs. Recently, Xu et al. [23] demonstrated that conditional medium of fibroblast-like cells differentiated from hESCs had the ability to support hESC growth under feeder-free conditions. These results provide some insight to understand the function of EDF. It is possible that EDF support the growth of hESCs partially through secreted factors. Because it is not reported that the condition medium derived from feeder supports the derivation of hESC lines using current protocols, feeder cells (including EDF) may also function through additional molecules that are not released to the medium.

Recently, while this paper was being revised, Stojkovic et al. [24] showed that autogenetic feeders support the growth of hESCs, which is consistent with our results. In our report, we demonstrate that the EDF culture system supports not only sustained growth of established cell lines but also the derivation of new hESC lines. The hESC line derived on EDF behaves the same as that derived on MEF in morphology and gene expression.

To avoid potential pathogens brought into the culture by mouse feeders, hESC lines can be derived using human embryonic fibroblast feeders [3] or human foreskin fibroblasts [25] as reported. Once the first hESC line derived on human feeders is established, it can be expanded and used to produce EDF to support the derivation and growth of new hESC lines. In this way, mouse feeder can be excluded completely from the culture system. In addition, we showed that EDF cells also grow well in medium supplemented with human serum instead of FBS. This provides an additional means to minimize contamination of animal pathogen.

The genome of hESCs can be readily altered [26–28]. A variety of phenotypically different EDFs could be derived from genetically modified ESCs. By introducing an antibiotic-resistance gene, antibiotic-resistant feeders can be produced to facilitate the selection of hESCs with special traits after gene modification. It is also possible to introduce growth-promoting factors, such as bFGF, so that one can grow hESCs without the addition of exogenous growth factors.

hESCs can be expanded infinitely, providing an inexhaustible source for a large quantity of feeders with a uniform genetic background. Also, both hESCs and EDF can be cultured in simple media at relatively low cost. These characteristics make the system particularly suitable for bulk production of standardized feeders to support the large-scale production of hESCs. EDFs from well-defined hESC lines will ensure tight quality control.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Q.W., Z.F.F., and F.J. contributed equally to this article. The authors would like to thank Jianxin Chu for analysis of the teratomas; Tianlong Gao for assistance with the confocal microscopy, and Youming Zhu and Ailian Liu for technical assistance. We thank Drs. Michal Amit and J. Itskovitz-Eldor for sharing their protocol for hESC karyotyping. The authors would also like to thank Drs. Ray and Gayla Sessions for valuable suggestions on this manuscript. This work was funded by the National Basic Research Program (001CB5099), Projects of Shanghai Science, and Technology Development Foundation (03DJ14017).

DISCLOSURES
The authors indicate no potential conflicts of interest.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.[Abstract/Free Full Text]

  2. Brivanlou AH, Gage FH, Jaenisch R et al. Stem cells: setting standards for human embryonic stem cells. Science 2003;300:913–916.[Abstract/Free Full Text]

  3. Richards M, Fong CY, Chan WK et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 2002;20:933–936.[CrossRef][Medline]

  4. Cheng L, Hammond H, Ye Z et al. Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. STEM CELLS 2003;21:131–142.[Abstract/Free Full Text]

  5. Amit M, Margulets V, Segev H et al. Human feeder layers for human embryonic stem cells. Biol Reprod 2003;68:2150–2156.[Abstract/Free Full Text]

  6. Richards M, Tan S, Fong CY et al. Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. STEM CELLS 2003;21:546–556.[Abstract/Free Full Text]

  7. Miyamoto K, Hayashi K, Suzuki T et al. Human placenta feeder layers support undifferentiated growth of primate embryonic stem cells. STEM CELLS 2004;22:433–440.[Abstract/Free Full Text]

  8. Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974.[CrossRef][Medline]

  9. Amit M, Shariki C, Margulets V et al. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 2004;70:837–845.[Abstract/Free Full Text]

  10. Sato N, Meijer L, Skaltsounis L et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004;10:55–63.[CrossRef][Medline]

  11. Itskovitz-Eldor J, Schuldiner M, Karsenti D et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 2000;6:88–95.[Medline]

  12. Bosseloir A, Heinen E, Defrance T et al. Moabs MAS516 and 5B5, two fibroblast markers, recognize human follicular dendritic cells. Immunol Lett 1994;42:49–54.[CrossRef][Medline]

  13. Kivirikko KI, Myllyla R, Pihlajaniemi T. Protein hydroxylation: prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit. FASEB J 1989;3:1609–1617.[Abstract]

  14. Vanderwinden JM, Rumessen JJ, De Laet MH et al. CD34+ cells in human intestine are fibroblasts adjacent to, but distinct from, interstitial cells of Cajal. Lab Invest 1999;79:59–65.[Medline]

  15. Koumas L, King AE, Critchley HO et al. Fibroblast heterogeneity: existence of functionally distinct Thy 1(+) and Thy 1(–) human female reproductive tract fibroblasts. Am J Pathol 2001;159:925–935.[Abstract/Free Full Text]

  16. Strutz F, Okada H, Lo CW et al. Identification and characterization of a fibroblast marker: FSP1. J Cell Biol 1995;130:393–405.[Abstract/Free Full Text]

  17. Thomson JA, Marshall VS. Primate embryonic stem cells. Curr Top Dev Biol 1998;38:133–165.[Medline]

  18. Thomson JA, Kalishman J, Golos TG et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A 1995;92:7844–7848.[Abstract/Free Full Text]

  19. Andrews PW, Oosterhuis JW, Damjanov I. Cell lines from human germ cell tumours. In: Robertson EJ, ed. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford, U.K.: IRL Press, 1987:207–248.

  20. Yeom YI, Fuhrmann G, Ovitt CE et al. Germline regulatory element of OCT-4 specific for the totipotent cycle of embryonal cells. Development 1996;122:881–894.[Abstract]

  21. Mitsui K, Tokuzawa Y, Itoh H et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003;113:631–642.[CrossRef][Medline]

  22. Chambers I, Colby D, Robertson M et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113:643–655.[CrossRef][Medline]

  23. Xu C, Jiang J, Sottile V et al. Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth. STEM CELLS 2004;22:972–980.[Abstract/Free Full Text]

  24. Stojkovic P, Lako M, Stewart R et al. An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. STEM CELLS 2005;23:306–314.[Abstract/Free Full Text]

  25. Inzunza J, Gertow K, Stromberg MA et al. Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cell. STEM CELLS 2005;23:544–549.[Abstract/Free Full Text]

  26. Pfeifer A, Ikawa M, Dayn Y et al. Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci U S A 2002;99:2140–2145.[Abstract/Free Full Text]

  27. Ma Y, Ramezani A, Lewis R et al. High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors. STEM CELLS 2003;21:111–117.[Abstract/Free Full Text]

  28. Zwaka TP, Thomson JA. Homologous recombination in human embryonic stem cells. Nat Biotechnol 2003;21:319–321.[CrossRef][Medline]

Received December 7, 2004; accepted for publication May 31, 2005.



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