Hum. Reprod. Advance Access originally published online on September 2, 2005
Human Reproduction 2006 21(1):217-222; doi:10.1093/humrep/dei275
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Serum-free medium cultivation to improve efficacy in establishment of human embryonic stem cell lines
1 Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100083, 2 Department of Obstetrics and Gynecology, General Hospital of PLA, Beijing 100853 and 3 Peking University Stem Cell Research Center, Beijing 100083, China
4 To whom correspondence should be addressed at: Peking University Third Hospital, Department of Obstetrics and Gynecology, Beijing 100083, China. E-mail: chenguian{at}bjmu.edu.cn
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Abstract |
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BACKGROUND: Serum-containing and serum-free media were used to derive human embryonic stem (HES) cells from donated oocytes and embryos. METHODS and RESULTS: Inner cell masses (ICM) were isolated by immunosurgery. The HES cells were found to be easily obtained and expanded in a serum-free medium. The efficacy in establishing human embryonic stem cell lines improved in a serum-free medium compared with that in serum-containing media. Four HES cell lines were derived from 13 isolated ICM on mouse embryonic fibroblast feeder layers. All four cell lines possess the same characteristics and differentiating potency: normal 46, XX or 46, XY karyotype; and expressing a series of surface markers such as APase, SSEA-3, SSEA-4, TRA-1–60, TRA-1–81, but not SSEA-1. They can form embryoid bodies in suspension culture and develop teratomas comprising derivatives of three embryonic germ layers when injected into severe combined immunodeficient mice. CONCLUSION: These preliminary results suggest that serum-free cultivation may be superior to serum-containing cultivation for deriving human embryonic stem cells.
Key words: embryonic stem cell/derivation/passage/serum-free cultivation
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Introduction |
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Embryonic stem (ES) cells derived from inner cell masses (ICM) of blastocysts can be cultured indefinitely in an undifferentiated state and kept pluripotent. The first mammalian ES cell lines were derived from mice (Evans and Kaufman, 1981
; Martin, 1981). These ES cells were shown to be able to integrate into blastocysts, participate in normal embryo development and organ formation (Roberson et al., 1986; Stewart et al., 1994), and even form an entire fetus under certain conditions (Nagy et al., 1993; Eggan et al., 2002). Human embryonic stem (HES) cell lines, however, were established just a few years ago (Thomson et al., 1998; Reubinoff et al., 2000) and fulfilled the following criteria: (i) derived from the preimplantation embryo; (ii) prolonged undifferentiated proliferation; and (iii) stable developmental potency after prolonged culture to form derivatives of all three embryonic germ layers (Thomson et al., 1998). They have been shown to be able to differentiate into various cell types in vitro like their mouse counterparts, such as neural precursors (Reubinoff et al., 2001; Zhang et al., 2001), haematopoietic cells (Kaufman et al., 2001), cardiomyocytes (Kehat et al., 2001; C.H.Xu et al., 2002), trophoblast (R.H.Xu, 2002) and insulin-secreting cells (Assady et al., 2001; Segev et al., 2004). Thus, they will have wide usage in treating degenerative diseases such as pubertal diabetes, Parkinson’s disease, cardiac infarcts and cancers.
The general culture conditions for pluripotent stem cells have not changed much since they were used in growing embryonic carcinoma (EC) stem cells, as well as mouse and primate embryonic stem cells. These cells were all grown on mitotically inactivated mouse embryonic fibroblasts (MEF) in a high-glucose formulation medium containing 15–20% fetal bovine serum (FBS). HES cells were cultured under similar conditions. Some problems, however, existed in routine culturing of HES cells under the serum-containing conditions: cells died during routine culturing and passage, colonies underwent natural differentiation (Reubinoff et al., 2000; Amit et al., 2000; Amit and Itskovitz-Eldor, 2002), cells needed to be passed in clumps (Thomson et al., 1998; Reubinoff et al., 2000), and the HES cell derivation rate from different groups varied greatly (2–60%) (Thomson et al., 1998; Reubinoff et al., 2000; Lanzendorf et al., 2001; Amit and Itskovitz-Eldor, 2002). It was shown that a well-defined substitute of chemical serum, supplemented with basic fibroblast growth factor (bFGF), was able to support prolonged HES cell growth in an undifferentiated state, and a higher cloning efficiency was obtained than that in FBS-containing medium (Amit et al., 2000). Serum may be the critical cause.
In our experiment, aimed at obtaining HES cell lines, we used donated oocytes to obtain high quality blastocysts and chose the well-tested serum-containing medium in which nearly all embryonic stem cell lines were derived. The findings were, however, frustrating even though we changed three batches of sera. Finally, we tried a serum-free medium and obtained great improvements. The results are presented under the two culturing conditions and with four established HES lines.
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Materials and methods |
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Derivation and propagation of ES cells
Sixty-two oocytes were donated by three healthy volunteers. Twelve spare day 3 embryos and semen were donated by couples undergoing infertility treatment. The procedures were approved by the Ethical Committee of Peking University Medical Center and all participants were fully aware of the scope of this study and signed informed consent sheets. Oocytes were inseminated according to conventional clinical IVF procedure. Day 3 embryos were cultured in G-2 medium (Vitrolife Sweden AB; Kungsbacka, Sweden; http://www.vitrolife.com) until expanded blastocyst stage and then graded with a scoring system for human blastocysts (Gardner and Schoolcraft, 1999
). After zona pellucida removal, by digesting with 0.5% pronase E (Merck), the blatocysts were washed three times with Dulbecco’s modified Eagle medium (DMEM) (Gibco/BRL; Carlsbad, CA, USA; http://www.invitrogen.com). The trophectoderms were removed by immunosurgery using a 1:50 dilution of rabbit antiserum to JEG-3 cells in DMEM followed by exposure to a 1:10 dilution of guinea-pig complement as described previously (Solter and Knoeles, 1975). After thorough washing in DMEM, the lysed trophectoderms were removed from the intact ICM by pipetting. ICM were isolated and plated onto mitotic-inactivated (3000 rads gamma irradiation) mouse embryonic fibroblasts prepared as previously reported (Abbondanzo et al., 1993). The serum-containing medium consisted of 80% knock-outTM DMEM (Gibco/BRL), 20% fetal bovine serum (ES-screened, Hyclone; Logan, UT; http://www.hyclone.com), supplemented with 0.1 mmol/l 
-mercaptoethanol (Gibco/BRL), 2 mmol/l glutamine (Gibco/BRL) and 1% non-essential amino acid stock (Gibco/BRL). The serum-free medium contained 80% Knock-outTM DMEM, 20% Knock-outTM Serum Replacer (Gibco/BRL), 0.1 mmol/l -mercaptoethanol, 2 mmol/l glutamine, 1% non-essential amino acid stock and 4 ng/ml bFGF, as described elsewhere (Amit et al., 2000; Assady et al., 2001; Zhang et al., 2001; C.H.Xu et al., 2002). Cells were cultured at 37°C under 6% CO2 in air. After culturing for 7–8 days, the central parts of the masses were manually removed, cut into 2–10 small parts and replated on fresh MEF layer in fresh medium. After three to five passages, colonies were passed every 5–6 days by dissociation after treating with 1 mg/ml collagenase IV (Gibco/BRL), or passed as single cell suspension by 0.05% typsin/0.53 mmol/l EDTA treatment.
In vitro characterization
Surface markers were tested in 35 mm culture dishes. Prior to analysis, cells were fixed by 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature. After three washes with PBS, alkaline phosphatase (APase) activity was determined by histochemical techniques using 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitrotetrazolium blue chloride (NBT) (Vector Laboratories; Burlingame, CA, USA; http://www.vectorlabs.com) as substrates. Antibodies used for detecting surface markers included stage-specific embryonic antigen (SSEA)-1 (MC-480, Developmental Studies Hybridoma Bank (DSHB); Iowa, IA, USA; http://www.uiowa.edu/~dshbwww, 1:100), SSEA-3 (MC-631, DSHB, 1:100), SSEA-4 (MC-813-70, DSHB, 1:200), tumour recognition antigen (TRA)-1–60 and TRA-1–81 (Chemicon; Temecula, CA, USA; www.chemicon.com, 1:100 dilutions for both). Antibodies were detected with goat anti-mouse IgG conjugated to fluorescein isothiocyanate (FITC) (Zhongshan Biotechnology Company; Beijing, www.zsbio.com, 1:50).
Karyotype analysis
Karyotype analyses of human ES cells were carried out during passage 9 to passage 30, using G-banding techniques. Briefly, cells were incubated with 50 ng/ml of colcemid for 2 h, trypsinized, re-suspended and incubated in 0.075 mol/l KCl solution for 8 min at 37°C, then fixed in 3:1 methanol/acetic acid and dropped onto cold slides. After being dried, spreads were treated with 0.02% trypsin for 
10 s, stained with Giemsa solution and analysed with Cytoktype 6.2 Software.
Teratoma formation
Human ES cells were detached with 1 mg/ml collagenase type IV, pelleted, re-suspended in PBS and injected into the rear leg muscles of 6–8 week old SCID–beige mice (106 cells per injection). Tumours were removed after 12–16 weeks, fixed overnight in 4% paraformaldehyde, embedded in paraffin, sectioned and examined histologically after being stained with eosin and haematoxylin.
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Results |
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Establishment of HES cell lines
A total of 50 day 3 embryos were cultured in G-2 medium with 30 being grown to the blastocyst stage. Fifteen ICM were isolated by immunosurgery. However, one was cultured on a human endometrial fibroblast feeder layer but died 3 days after plating (data not shown) and one was damaged; thus, 13 ICM were cultured on MEF feeder layers under the different culturing conditions as shown in Table I. The first four ICM obtained from the first oocyte donor were cultured in a serum-containing medium (serum batch 1, S1). All four ICM attached overnight, but gradually flattened out and died, resulting in no outgrowth at all. From the second volunteer who donated 28 oocytes, only two embryos developed to the blastocyst stage and one ICM was isolated. This ICM was again cultured in a serum-containing medium in which serum batch was changed (serum batch 2, S2). The result was the same as that in S1-medium, with no outgrowth at all. Then five ICM were isolated from 10 spare embryos. They were cultured in a medium containing the third batch of serum (serum batch 3, S3). All five ICM grew at different rates. The faster four ICM outgrowths were passed manually (one cut into two) 6–9 days after plating, but only one developed. The other three died or differentiated in passage 1. From that one ICM outgrowth, two clumps were replated, where the bigger clump grew and the smaller one differentiated. It was passed again 4 days later mechanically (one into two), the same as passage 1, the smaller one expanded and the bigger one grew slowly. Then the medium was changed for the cells of passage 3 when the serum was replaced by Knock-out SRTM at the same concentration of FBS. The results were exciting in that the cells of passage 3 proliferated rapidly and 6 days later they were passed mechanically and resulted in >10 ES colonies in passage 4. No clumps died or differentiated. They were passed mechanically again 6 days later and hundreds of colonies appeared. After passage 5, the cells were passed by treating them with 1 mg/ml collagenase type IV every 5–6 days routinely. Thus, the first HES cell line (B4) was established. Meanwhile, the slowest ICM was first cultured in the S3 medium for 13 days, then changed to the medium containing SR for 3 days, and thereafter, passed and cultured in the same serum-free medium. Cells propagated rapidly and the second cell line (B7) was established. Later on, two ICM were isolated from five blastocysts in the third oocyte donor. One was cultured in S3 medium and the other in SR-containing medium to see whether any difference existed between these two culturing conditions in obtaining HES cells from the very beginning. One week later the outgrowths were passed mechanically without changing the culturing conditions. Cells in SR-medium multiplied vigorously, but those in serum-containing medium died in passage 1. Line PKU-1 was established in solely SR-containing medium. The last line (PKU-2) was derived from one spare frozen–thawed embryo in the same SR-containing medium. Compared to the results in serum-containing media, derivation efficacy was greatly improved in the serum-free SR medium.
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Several methods were used to pass HES cells. Initially, they were all passed manually. After three to five passages, there were so many colonies that it was impossible to pass them manually any more. Then digestion with either 1 mg/ml collagenase IV (Gibco/BRL) or 0.05% typsin/0.53 mmol/l EDTA was tried separately. We were able to pass HES cells both in clumps and in single cell suspension in SR-medium; thus, either of these two methods was equally effective in passing HES cells. However, collagenase was chosen to treat HES cells during routing passage in order to reduce the harmful effect of FBS which was used to stop digestion of trypsin. To date, lines B4 and B7 have both been passed for 6 months and are stored frozen. Lines PKU-1 and PKU-2 have been passed continuously for >1 year.
Characterization in vitro and karyotype analysis
All four cell lines showed the typical morphology of human embryonic stem cells as described by others (Thomson et al., 1998; Reubinoff et al., 2000): flat and compact colonies with small cells having a high nucleus/cytoplasm ratio and prominent nucleoli (Figure 1). They all expressed a series of surface markers such as alkaline phosphatase (Figure 2A), OCT-4 (Figure 2B), SSEA-3 (Figure 2C), SSEA-4 (Figure 2D), high molecular weight glycoproteins TRA-1–60 (Figure 2E) and TRA-1–81 (Figure. 2F), but no SSEA-1 (data not shown). They have normal karyotypes: B4 46,XX; B7 46,XY; PKU-1 46,XX; PKU-2 46,XY.
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Differentiation in vivo
Four SCID–beige mice were given injections in their rear legs. Every injection resulted in a teratoma. Tumours were analysed by histology. Many types of cells were identified to belong to three germ layers. Among these, squamous epithelial tissue, neural tissue, columnar gland and cartilage were demonstrated (Figure 3A–D respectively).
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Discussion |
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The four human ES cell lines have similar properties as described previously (Thomson et al., 1998
; Reubinoff et al., 2000). Several features were similar in non-human and human ES cells: (i) a high nuclear/cytoplasmic ratio, prominent nucleoli, and flat colony morphology with distinct cells different from mouse ES and human EG colonies which showed piled-up morphology; (ii) expression of a series of surface markers that characterize undifferentiated human EC and ES cells, including alkaline phosphatase, SSEA-3, SSEA-4, TRA-1–60 and TRA-1–81; (iii) stable, normal karyotypes even after prolonged culture in vitro; (iv) pluripotency: all four cell lines were induced to form teratomas in vivo comprising derivatives of three germ layers. Therefore, these cells are considered typical pluripotent HES cells. We tried three different batches of sera, all of which were tested to be able to support mES cells, but unable to support hES cell derivation. HES cell culturing seems to need a higher quality of serum than mouse ES cell culturing—that may be the main difficulty in HES cell derivation and maintenance. The different results from the different culturing conditions indicate that serum quality is the most critical factor for HES cell derivation and maintenance. Although FBS can provide nutritive supplementation to ES cells, serum is a complex mixture containing compounds both beneficial and detrimental to human ES cells. Different batches vary widely in their ability to support vigorous undifferentiated proliferation of human ES cells, so each lot should be screened prior to use.
Unlike mouse ES cells, human ES cells were difficult to pass in single cell suspension in serum-containing media (Thomson et al., 1998; Reubinoff et al., 2000). In our experiments, several methods have been tried to pass HES cells: (i) manually; (ii) in small cell clumps with mechanical dissociation following 1 mg/ml collagenase IV treatment; and (iii) in single cell suspension with 0.05% typsin/0.53 mmol/l EDTA treatment. The same difficulties were experienced as with others in passing HES cells in that some of the colonies (especially the small ones) flattened out or died in serum-containing media (Reubinoff et al., 2000). However, HES cells could be grown more easily in the serum-free medium and rarely differentiated during routine culturing no matter whether being passed in clumps or in single cell suspensions. It was shown also that HES cloning efficiency was several-fold increased in SR-containing medium as compared to that in FBS-containing medium (Amit et al., 2000). All these data indicated that the serum-free cultivation system is more effective in establishing and maintaining human ES cells, and, furthermore, makes ES cells free from contamination with heterogeneous proteins and latent infectious agents from animal sera.
In order to culture any cell successfully it is important to understand the nutrient requirements and physiology of the cell in question (Gardner and Lane, 2003). The serum-free culture system would successfully support HES cells, not only catering for the requirement of HES cell proliferation, but also minimizing the harmful impact; thereby keeping HES cells in an undifferentiated state. Little is known about the physiology of HES cells and their nutrient requirements. The culturing systems had been chosen empirically for mouse ES cells, non-human primate ES cells and human ES cells. For mouse ES cells, the first line was established in DMEM medium supplemented with 10% fetal calf serum and 10% newborn calf serum on mitomycin C-inactivated STO fibroblast feeder layer (Evans and Kaufman, 1981). Several years later, leukaemia inhibitory factor (LIF) was shown to be essential for effectively maintaining mouse ES cells in an undifferentiated state (Smith et al., 1988; Williams et al., 1988). Then the general culture conditions were established: mouse ES cells were usually grown on a mitotically inactivated fibroblast feeder layer; the general medium was a DMEM formulation supplemented with FBS, L-glutamine, non-essential amino acids, -mercaptoethanol and antibiotics; LIF was often used to keep ES cells in an undifferentiated state (Abbondanzo et al., 1993; Smith, 2001). The first primate embryonic stem cell line was derived using the same culture conditions as that for mES cells (Thomson et al., 1995). Later, several pluripotent cell lines were derived from common marmoset blastocysts under similar conditions, but LIF was shown to be unnecessary to derive and maintain non-human primate ES cells (Thomson et al., 1996). Then five human embryonic stem cell lines were derived from preimplantation blastocysts under the same conditions as used for general culturing of mouse ES cells and primate ES cells (Thomson et al., 1998). However, some difficulties seemed to lie in routine passage. HES cells were found to be prone to dying or differentiating naturally under such conditions (Thomson et al., 1998; Reubinoff et al., 2000). Thereafter, Knock-out SR, a serum replacer, supplemented with bFGF was introduced into the culture system of HES cells. The authors compared the results of cloning efficiency in media supplemented with either serum or serum replacer. It was shown that a several-fold increase in cloning efficiency of HES cells was consistently observed when SR-containing medium was used instead of serum-containing medium (Amit et al., 2000). After that, many investigations demonstrated that SR-containing medium could effectively support proliferation of HES cells (Assady et al., 2001; Kaufman et al., 2001; Zhang et al., 2001; R.H.Xu et al., 2002; Segev et al., 2004). Although the components of this defined chemical material are yet unknown, Knock-out SR seemed to contain less harmful factors than sera.
One of the most important parameters surrounding ES cell culture is the maintenance of ES cells in an undifferentiated state. SR medium was shown to be effective in retaining the pluripotency of mouse ES cells. Mouse ES cells cultured for multiple passages in Knock-out SR or FBS-containing media yielded similar results in chimeric formation and germline transmission after receipt blastocysts were injected with those two mES cells (manufacturer’s data). In HES cell culture, although many results had shown that SR medium was effective in supporting proliferation of HES cells, the pluripotency of HES cells should be questioned after prolonged culture under such serum free conditions. Our data demonstrated that human ES cells derived and maintained in SR medium for >1 year maintain a pluripotency similar to those in serum-containing media.
Our data provide some evidence suggesting that serum-free cultivation of human ES cells is superior to the conventional serum-dependent system, and may provide a better method for establishing human ES cell lines in order to avoid the possible contamination from animal sera that will be important in clinical usage of ES cells in the future. Hopefully, it can be used to establish a better protocol for culturing human ES cells in stem cell research and also clinical usage. However, these results must be considered as being preliminary, due to the lack of carefully designed and controlled experiments.
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Acknowledgements |
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The authors are grateful for help from Professor Shin Yong-Moon and his colleagues Department of Obstetrics and Gynaecology, Seoul National University College of Medicine, Seoul, Republic of Korea) during establishment of the HES cell lines, and for the technical support provided by all members in the IVF laboratory of Reproductive Medical Center, Peking University Third Hospital. This work was supported by grants from the Chinese National 973 Project (2002CB510100), 863 Project (2003AA205070).
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References |
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Abbondanzo SJ, Gadi I and Stewart CL (1993) Derivation of embryonic stem cell lines. Meth Enzymol 225,803–823.[ISI][Medline]
Amit M and Itskovitz-Eldor J (2002) Derivation and spontaneous differentiation of human embryonic stem cells. J Anat 200,225–232.[CrossRef][ISI][Medline]
Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, Itskovitz-Eldor J and Thomson JA (2000) Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 227,271–278.[CrossRef][ISI][Medline]
Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL and Tzukerman M (2001) Insulin production by human embryonic stem cells. Diabetes 50,1691–1697.
Eggan K, Rode A, Jentsch I, Samuel C, Hennek T, Tintrup H, Zevnik B, Erwin J, Loring J, Jackson-Grusby L et al (2002) Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation. Nat Biotechnol 20,455–459.[CrossRef][ISI][Medline]
Evans MJ and Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292,154–156.[CrossRef][Medline]
Gardner DK and Lane M (2003) Culture media for the human embryo. In Revelli A, Tur-Kaspa I, Holte GJ and Massobrio M (eds) Biotechnology of Human Reproduction. Carnforth: Parthenon Press, USA, pp 181–199.
Gardner DK and Schoolcraft WB. In vitro culture of human blastocysts. In: Jansen R, Mortimer D, Towards Reproductive Certainty: Fertility and Genetics Beyond 1999: Carnforth: Parthenon Press; 1999:378–388.
Kaufman DS, Hanson ET, Lewis RL, Auerbach R and Thomson JA (2001) Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 98,10716–10721.
Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, Livne E, Binah O, Itskovitz-Eldor J and Gepstein L (2001) Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 108,407–414.[CrossRef][ISI][Medline]
Lanzendorf SE, Boyd CA, Wright DL, Muasher S, Oehninger S and Hodgen GD (2001) Use of human gametes obtained from anonymous donors for the production of human embryonic stem cell lines. Fertil Steril 76,132–137.[CrossRef][ISI][Medline]
Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78,7634–7638.
Nagy A, Rossant J, Nagy R, Ambramow-Newerly W and Roder JC (1993) Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci USA 90,8421–8428.
Reubinoff BE, Pera MF, Fong CY, Trounson A and Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18,399–404.[CrossRef][ISI][Medline]
Reubinoff BE, Irsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A and Ben-Hur T (2001) Neural progenitors from human embryonic stem cells. Nat Biotechnol 19,1134–1140.[CrossRef][ISI][Medline]
Robertson E, Bradley A, Kuehn M, Evans M (1986) Germ-line transmission of gene introduced into cultured pluripotential cells by retroviral vector. Nature 323,445–448.[CrossRef][Medline]
Segev H, Fishman B, Ziskind A, Shulman M and Itskovitz-Eldor J (2004) Differentiation of human embryonic stem cells into insulin-producing clusters. Stem Cells 22,265–274.
Smith AG (2001) Embryonic stem cells. In Marshak DR, Gardner RL and Gottlieb D (eds) Stem Cell Biology. Cold Spring Harbor Laboratory Press, New York, USA, pp 205–230.
Smith AG, Health JK, Donaldson DD, Wong GG, Moreau J, Stahl M and Rogers D (1988) Inhibition of pluropotential embryonic stem cell differentiation by purified polypeptides. Nature 336,688–690.[CrossRef][Medline]
Solter D and Knoeles BB (1975) Immunosurgery of mouse blastocyst. Proc Natl Acad Sci USA 72,5099–5102.
Stewart CL, Gadi I and Bhatt H (1994) Stem cells from primordial germ cells can reenter the germ line. Dev Biol 161,626–628.[CrossRef][ISI][Medline]
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS and Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282,1145–1147.
Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, and Hearn JP (1996) Pluripotent cell lines derived from common marmoset blastocysts. Biol Reprod 55,254–259.[Abstract]
Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, and Becker RA and Hearn JP (1995) Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci USA 92,7844–7848.
Xu CH, Police S, Rao N and Carpenter MK (2002) Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circln Res 91,501–508.
Xu RH, Chen X, Li DS, Li R, Addicks GC, Glennon C, and Thomson JA (2002) BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotechnol 20,1261–1264.[CrossRef][ISI][Medline]
Zhang S-C, Wernig M, Duncan ID, Brustle O and Thomson JA (2001) In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 19,1129–1133.[CrossRef][ISI][Medline]
Williams RL, Hilton DJ, Pease S, Wilson TA, Stewart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA and Gough NM (1988) Myeloid leukemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336,684–687.[CrossRef][Medline]
Submitted on December 5, 2005; resubmitted on May 24, 2005; accepted on July 26, 2005.
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