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Embryo |
Division of Stem Cell Biology,3 Medical Research Center, MizMedi Hospital, Kangseo-ku, Seoul 157-280, Korea
Department of Life Science,4 College of Natural Sciences, Hanyang University, Seongdong-gu, Seoul 133-791, Korea
Stem Cell Research Center,5 Seoul, Korea
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cytokines, developmental biology, early development, embryo, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In 1998, human embryonic stem (hES) cells were successfully derived from the ICM of blastocysts produced by in vitro fertilization (IVF) [7, 8]. Unlike mouse embryonic stem (mES) cells and human embryonic germ (hEG) cells, hES cells lost pluripotency and differentiated rapidly when grown on plastic tissue-culture dishes without feeder cells, even in the presence of culture media supplemented with leukemia inhibitory factor (LIF) [9–11]. Furthermore, hES cells had relatively low cloning efficiencies because it was difficult to passage them as a single cell [12].
Several studies suggest that hES cells can differentiate into various cell types, such as pancreatic cells, neural cells, cardiomyocytes, and red blood cells [13–15]. These features of hES cells indicate that they have the potential to provide an unlimited supply of various cell types for regenerative medicine, pharmacokinetic screening, and functional genomics applications [16, 17].
Previous reports have indicated that both ICM culture and undifferentiated hES cell culture require feeder cells, either as a feeder layer or as a source of conditioned medium, and extracellular matrix (ECM) [18]. In mES cell culture, the feeder layer can be replaced by the addition of LIF in the growth medium [11]. However, LIF does not have the same effect on hES cell culture as mES [4]. Therefore, both the derivation and maintenance of hES cells require the use of feeder cells. These feeder cells can be either primary mouse embryonic fibroblasts (PMEFs) or STO as permanently growing cell lines. The STO cell line was derived from mouse SIM embryonic fibroblasts, was resistant to 6-thioguanine and ouabain, and was sensitive to HPRT (hypoxanthine guanine phosphoribosyl transferase) and HAT (hypoxanthine, aminoprotein, and thymidine), as well as negative for mouse poxvirus. It has been used to prepare feeder layers for teratocarcinoma cells, hybridomas, and ES cells [19, 20]. Groups that have established hES cell lines have generally used PMEFs as feeder cells whereas the STO cell line has only been used as a feeder cell line for establishing hEG cells, mouse ES cells, and bovine ES cells [9, 21]. There are some disadvantages of using PMEFs as feeder cells. First, the proliferation of PMEFs is limited, and therefore, it is necessary to isolate PMEFs from mouse fetuses repeatedly to supply PMEFs for ES cell culture. Second, PMEFs present the risk of being contaminated by mycoplasms, animal viruses, and fungi. Finally, PMEFs tend to lose capacity to support proliferation of ES cells with increasing passages. Therefore, improvements are needed in order to supply defined feeder cells to hES cell culture.
In this study, we provide evidence that the STO cell line provides advantages over PMEFs for the establishment and maintenance of hES cell lines.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human pronuclear (PN)-stage embryos were donated for use in this study following Institutional Review Board approval and informed consent by couples undergoing IVF treatment. Frozen and thawed pronuclear-stage human embryos were cultured to blastocysts in G1.2 and G2.2 medium (Vitrolife Inc., Denver, CO) [9].
Human Blastocyst Immunosurgery
Immunosurgery was used to isolate ICM from blastocysts as previously described [22]. Briefly, human blastocysts were exposed to 100% anti-human whole serum antibody (Sigma; St. Louis, MO) for 20 min, followed by a 30 min exposure to guinea pig complement (Life Technologies, Karlsruhe, Germany) in 50-µl droplets at 37°C in 5% CO2. Isolated ICMs were cultured on mitomycin C-treated STO feeder cell layers.
Mitotic Inactivation of STO Cells and PMEF Cells
PMEFs were isolated from Day 13.5 postcoitum fetuses of C57BL/6 mice as described [8]. The STO cell line was purchased from the American Type Culture Collection. STO cells and PMEF cells were cultured in Dulbecco modified Eagle medium (DMEM) high-glucose (Life Technologies), supplemented with 2 mM glutamine (Sigma), 0.1 mM ß-mercaptoethanol (Sigma), and 1% nonessential amino acid (Life Technologies). Cells were treated with 10 µg/ml mitomycin C (Sigma) for 1.5 h. The mitomycin C-treated STO cells and PMEF cells were extensively washed in PBS and replated at 75 000 cells/cm2 in gelatin-coated tissue culture dishes.
Estimation of Cell Proliferation and Metabolic Activity of Mitotically Inactivated STO and PMEF Cells
Cell proliferation was assessed by counting cells and by detecting incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA in four independent experiments. For the BrdU incorporation assay, mitomycin C-treated STO cells and PMEF cells were replated at 1 x 103 cells/well in microtiter wells (tissue culture grade, 96 well, flat bottomed) and incubated in humidified atmosphere (37°C, 5% CO2). After its incorporation into DNA, BrdU was detected by immunoassay using a BrdU incorporation assay kit according to the manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, IN). The reaction product was quantified by measuring absorbance using an ELISA reader at 450 nm (reference wavelength 690 nm). The same procedure was performed for negative control samples, which were identical but without BrdU labeling.
Cellular metabolic activity was determined by measuring the conversion of 3 (4,5-dimethythiazozyl-2)-2,5 diphenyl tetrazolium bromide to colored formazan product at the second day after replating, according to the manufacturer's instruction (MTT; Roche Molecular Biochemicals). Briefly, 10 µl of MTT labeling reagent (final concentration 0.5 mg/ml) was added to the cells (at 1 x 103 cell/well) seeded in 96-well plates. The cells were then incubated for 4 h at 37°C. One hundred microliters of solubilization solution was added to each well and the 96-well plates were incubated for 16 h at 37°C. Colored formazan products were quantified by measuring absorbance using an ELISA reader at 550 nm (reference wavelength should be more than 650 nm). The same procedure was performed for a negative control sample without MTT labeling reaction. This experiment was repeated four times.
The Quantification of Collagen, Laminin, and Fibronectin
ELISA assay was performed to determine the amounts of ECM produced by mitotically inactivated STO cells and PMEF cells using antibodies against collagen, laminin, and fibronectin. Mitotically inactivated STO and PMEF cells were replated at 1 x 103 cells/well and incubated for 48 h, then fixed with 4% paraformaldehyde for 1 h at room temperature. The fixed cells were incubated with rabbit anti-mouse fibronectin antibody, rabbit anti-mouse laminin antibody, and rabbit anti-mouse collagen antibody (Chemicon International, Temecula, CA). Antibody binding was detected using goat anti-rabbit IgG conjugated to horseradish peroxidase (Bethy Laboratory, Montgomery, TX). Colorimetric reactions were performed using an ELISA Starter Kit (Bethy Laboratory) according to the manufacturer's instructions. Absorbance was measured using an ELISA reader at 450 nm. The same procedure was performed for a control sample that did not contain the first antibody treatment and for a negative control sample without colorimetric reagents. The experiment was repeated four times.
Human Embryonic Stem Cell Culture
During the early passages of hES cell culture, cells were cultured in DMEM high-glucose medium, without pyruvate, supplemented with 20% fetal bovine serum (FBS), 0.1 mM ß-mercaptoethanol, 1% nonessential amino acids, 1 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2000 units/ml human recombinant leukemia inhibitory factor (hrLIF). After 10 passages, DMEM high-glucose and FBS were replaced with Knockout DMEM and Knockout serum replacement with the addition of basic fibroblast growth factor (bFGF, 4 ng/ml) (Life Technologies). The hES cell colony derived from ICM was dissociated and mechanically disaggregated using a micropipette every 7 days. To induce embryoid body (EB) formation, hES cell colonies were transferred to plastic Petri dishes using 0.1% trypsin and 1 mM EDTA. Human EBs were grown in the same culture medium without LIF and bFGF.
Immunocytocheminstry for the Characterization of hES Cells
For immunocytochemistry, hES cells were washed with Ca2+- and Mg2+-PBS and then fixed in 4% paraformaldehyde at 4°C for 30 min. To detect alkaline phosphatase activity, the cells were permeabilized with 0.2% Triton X-100 for 20 min at 20°C and washed three times in Ca2+- and Mg2+-PBS. The cells were then stained with NBT/BCIP (Roche Molecular Biochemicals). Cell surface monoclonal antibodies were supplied by the Developmental Studies Hybridoma Bank (SSEA-1, SSEA-3, and SSEA-4) (University of Iowa, Iowa city, IA), Chemicon (TRA-1-60, and TRA-1-81), and Santa Cruz Biotechnology (anti-laminin receptors [integrin 
6, ß1 dimer] antibody) (Santa Cruz, CA). The antibodies were diluted with Ca2+- and Mg2+-PBS containing 1% BSA to block nonspecific reactivity. Primary monoclonal antibodies were localized using biotinylated secondary antibody, followed by a complex of avidin and horseradish peroxidase (Vectastain ABC system; Vector Laboratory, Burlingame, CA). A Vector NovaRED substrate kit (Vector laboratory) was used to localize the complex.
To identify spontaneously differentiated cells, anti-neurofilament antibody (Chemicon) was used. Anti-neurofilament antibody was localized with goat anti-mouse IgG-FITC conjugated antibody. Stained cells were examined using confocal microscopy.
Cytogenetic Analysis of hES Cells
Cells were incubated in growth medium with 0.1 µg/ml of colcemid for 3–4 h, trypsinized, resuspended in 0.1% sodium citrate, incubated for 15 min at 37°C, and fixed in methanol/acetic acid (3:1, v/v).
Oct4 Expression
To monitor the expression of Oct-4, total RNA was prepared using a QIAGEN RNeasy kit (QIAGEN, Valencia, CA). Standard reverse-transcription reactions were performed with 500 ng of total RNA using random hexamers and AMV reverse transcriptase (Roche Molecular Biochemicals). The PCR was carried out with 2 µl of cDNA template, 1 µl of 10 mM dNTP mixtures, 10 pmol of Oct4 primers (forward, 5'-GGAAAGGCTTCCCCCTCAGGGAAAGG-3'; reverse, 5'-AAGAACATGTGTAAGCTGC GGCCC-3'). The Oct-4 RNA transcripts were amplified using 5 min for denaturation at 94°C, followed by 40 cycles at 94°C for 30 sec, with the final extension at 72°C for 10 min, 62°C for 30 sec, and 72°C for 30 sec in a GeneAmp 9600 (Perkin-Elmer, Irvine, CA). As a loading control, the same amounts of cDNA template were amplified using ß-actin primers: forward, 5'-TTCTTGTGCTGGTTATAGAA-3'; reverse, 5'-GACAACAATGAGAACCTTCA-3'. Products were analyzed on 1.5% agarose gel and visualized by ethidium bromide staining.
Teratoma Formation in SCID Mice
During routine passage, clumps consisting of about 100 cells with an undifferentiated morphology were harvested as described above and injected subcutaneously into 4- to 8-wk-old SCID mice (two or more mice per cell line). Six to seven weeks later, the resulting tumors were fixed in 10% neutral buffered formalin, embedded in paraffin, and examined histologically after hematoxylin and eosin staining.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mitotically inactivated STO cells and PMEF cells did not show any significant changes in numbers by counting (Fig. 1, A and B), BrdU incorporation (Fig. 1C) and MTT assay (Fig. 1D) over 6 days. These results suggest that mitomycin C-treated STO cells and PMEF cells do not proliferate but maintain their metabolic activity.
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Furthermore, no significant differences were found between STO and PMEFs with respect to collagen, laminin, or fibronectin content (Fig. 1E).
Derivation of hES Cell Lines (Miz-hES1, Miz-hES2, and Miz-hES3)
Thirty inner cell masses were isolated and cultured on mitomycin C-treated STO cells. After 8 days of culture, groups of small, tightly packed cells had proliferated from the ICM (Fig. 2A). These ICM-derived colonies were separated and replated every 7 days. The replated clumps gave rise to flat colonies of cells that morphologically resembled human ES or primate ES cells (Fig. 2B). Under high magnification (200x), these hES cells were found to have a high ratio of nucleus to cytoplasm and prominent nucleoli (Fig. 2C). Moreover, these hES cells maintained the undifferentiated state when cultured in the presence of the STO feeder layer, both with and without human LIF (data not shown). Each of the Miz-hES1, 2- and 3-cell lines has now been passaged continuously for more than 110 passages, 75 passages, and 66 passages, respectively. When Miz-hES cell lines were cultured over 14 days in a feeder-free culture, these cells spontaneously differentiated into various cell types. Neurofilament was expressed in some of these differentiated cells (Fig. 2D). When our hES cells were cultured on plastic Petri dishes, human EB formation was induced (data not shown).
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Marker Expression and Karyotypes of the hES Cells
In common with pluripotent stem cells, our hES cells showed a high level of AP activity (Fig. 3A). Immunophenotyping of the hES cells was performed using several antibodies against cell-surface carbohydrates, which are specifically expressed on undifferentiated hES and hEC (embryonic carcinoma) cells. While SSEA-1 was not detected on undifferentiated hES cell colonies (Fig. 3B), SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 were strongly stained in undifferentiated hES cells. None of these proteins were detected in differentiated hES cells (Fig. 3, C through F). Similar results were obtained from two other cell lines (Miz-hES 2 and Miz-hES 3) (data not shown). Oct-4, expressed in hES, is essential for the pluripotent stem cell population and its expression is downregulated when ES cells differentiate. Thus, we compared Oct-4 expression in undifferentiated and differentiated hES cells by reverse transcription-polymerase chain reaction. All of the three hES cell lines expressed the Oct-4 in undifferentiated cells whereas Oct-4 expression was not detected in differentiated cells (Fig. 4). Karyotype analyses were performed at 10–15 passages of hES cells and all three hES cell lines were normal. In this assay, two cell lines (Miz-hES1 and Miz-hES2) had normal 46-XY karyotypes (Fig. 5, A and B), and the other cell line (Miz-hES3) had a normal 46-XX karyotype (Fig. 5C).
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In Vivo Differentiation and In Vitro Spontaneous Differentiation
The hES cells were injected into severe combined immunodeficiency (SCID) mice to confirm the formation of teratomas. All three hES cell lines produced teratomas in each injected SCID mouse, which were found to contain tissues of the three embryonic germ layers: endoderm (endothelial cell types), mesoderm (bone, smooth muscle, and striated muscle), and ectoderm (neural epithelium, embryonic ganglia, and stratified squamous epithelium) (Fig. 6).
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In some colonies cultured over 10 days, hES cells observed in the center of the colonies spontaneously differentiated into fibroblast-like cells. However, hES cells at the edges of colonies still maintained their morphologically undifferentiated state. These cells also were strongly stained with AP, SSEA-3, and laminin receptor (integrin 6, ß1) (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been suggested that coculture with PMEFs exposes hES cells to mouse retroviruses, which may prevent the future use of these cells in cell-based therapy. Therefore, self-made hEF and human adult marrow cells have been recently used to support the proliferation of hES cells instead of PMEFs [23, 24]. However, the need to recreate lines may raise technical and ethical problems, because the derivation of self-made human embryonic fibroblasts (hEFs) involves abortion. Furthermore, it is difficult to justify the use of human adult marrow cells as feeder cells because human adult marrow cells can be directly used for cell therapy and research for guided differentiation.
In this study, we successfully established cultures of hES cells on mitomycin C-treated STO cells. The STO cell line, a permanently growing cell line, has been used in the past to establish hEG cells [9]. The hEG cells are less successfully generated and passaged when PMEFs and human fetal fibroblasts are substituted for STO cells. Although STO cells have some advantages over PMEF cells and hEF cells (they are easier to handle, may be passaged repeatedly over long periods, and are negative in the ectromelia virus test), these cells have never been used as feeder cells to establish and cultivate hES cell lines.
In order to use STO cells as feeder cells and take advantage of the properties listed above, it is essential to stop their proliferation so that they do not overgrow in any ES cell cultures but yet they continue to support the growth of ES cells. This can be achieved in two ways—
-irradiation and mitomycin C treatment. While -irradiation leads to DNA strand breaks, mitomycin C has the extraordinary ability to crosslink DNA with high efficiency and is absolutely specific for the CpG sequence [25]. The choice of -irradiation or mitomycin C treatment is often guided by the availability of -irradiation equipment. In most laboratories, mitomycin C has been used to prepare feeder cells because of availability and cost. In the present study, mitomycin C-treated STO cells were evaluated for their proliferation and metabolic activity for 6 days because the efficiency of mitomycin C treatment for the preparation of feeder cells is dependent on the feeder cell type. Mitomycin C treatment did not significantly change the proliferation and metabolic activity of STO cells in comparison with PMEF cells.
Most normal cells require adhesion to the ECM for survival, migration, and growth. Collagen type IV is an ECM protein that promotes the proliferation and differentiation of rat cortical progenitor cells during corticogenesis [26]. Laminin, the first ECM protein to be expressed in two- to four-cell-stage mouse embryos, is a major component of the extracellular matrix of all basal laminae in vertebrates. In particular, previous reports have indicated that soluble factors and ECM produced by feeder cells might be important for proliferation and maintenance of undifferentiated hES cells [18]. In this study, ECM (laminin, collagen type IV, and fibronectin) was not significantly different between STO and PMEF cells treated with mitomycin C.
These results suggest that mitotically inactivated STO cells, like PMEFs, are able to produce the conditions that support the establishment and maintenance of hES cells.
Three hES cell lines were derived from 30 ICMs on STO cells. These hES cell lines had features in common with human pluripotent stem cells, including a similar morphology, expression of surface markers (SSEA-3, SSEA-4, TRA-1-61, and TRA-1-80), pluripotency, a lack of response to LIF, and the formation of embryoid bodies in suspension culture. The differentiation of hES cells occurred spontaneously in feeder-free culture conditions and was independent of exogenous hLIF. Both ES and EC cells of murine and human species express the transcription factor, Oct4. In mice, Oct4 expression is limited to the ICM of blastocyst and pluripotent populations and has been shown to be essential for the establishment of pluripotent cell lineages [27]. Also, teratoma formation in SCID mice, arising from injection of our cell lines, was similar to that obtained by injecting the mES cell line, and induced various cell types of the three embryonic germ layers in mice. Our results suggest that Miz-hES cell lines established on STO cells are a hES cell type, as previously described [4].
Recently, it was reported that hES cells could be maintained in conditioned media from PMEF culture but that they may undergo spontaneous differentiation in conditioned media from STO cell culture [18]. This result implies that the soluble factors secreted into conditioned media from STO cell culture may not be sufficient to maintain the undifferentiated state of hES cells [28]. In this study, we observed that hES cells contacting feeder cells did not show any evidence of differentiation but hES cells located in the middle of colonies (no direct contact with feeder cells) differentiated spontaneously. These results also suggest that, whereas STO cells might produce sufficient amounts of cell surface factors for the renewal and maintenance of the undifferentiated state of hES cells, STO cells might not produce sufficient amounts of soluble factors for self-renewal of hES cells growing without direct contact with STO feeder cells.
In addition, laminin-specific receptors (integrin 6, ß1 dimer) were highly expressed in the region of hES cells contacting STO cells [29, 30]. Therefore, laminin-specific receptor(s) may be important cell surface factors for the maintenance of undifferentiated hES cells.
In conclusion, our study strongly suggests that STO cells are a valuable candidate as feeder cells for the establishment and cultivation of hES cell lines.
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FOOTNOTES |
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2 Correspondence: Hyun Soo Yoon, Division of Stem Cell Biology, Medical Research Center, MizMedi Hospital, 701-4 Naebalsan-dong, Kangseo-ku, Seoul, Korea. FAX: 82 2 2007 1285; yoon{at}mizmedi.net
Received: 25 March 2003.
First decision: 11 April 2003.
Accepted: 31 July 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6ß1 integrin. J Cell Biol 1991 115:843-850This article has been cited by other articles:
|
|
 |
|
  S.-W. Cho, S.-H. Moon, S.-H. Lee, S.-W. Kang, J. Kim, J. M. Lim, H.-S. Kim, B.-S. Kim, and H. M. Chung Improvement of Postnatal Neovascularization by Human Embryonic Stem Cell-Derived Endothelial-Like Cell Transplantation in a Mouse Model of Hindlimb Ischemia Circulation, November 20, 2007; 116(21): 2409 - 2419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Mannello and G. A. Tonti Concise Review: No Breakthroughs for Human Mesenchymal and Embryonic Stem Cell Culture: Conditioned Medium, Feeder Layer, or Feeder-Free; Medium with Fetal Calf Serum, Human Serum, or Enriched Plasma; Serum-Free, Serum Replacement Nonconditioned Medium, or Ad Hoc Formula? All That Glitters Is Not Gold! Stem Cells, July 1, 2007; 25(7): 1603 - 1609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Lee, H. K. Kim, J.-Y. Rho, Y.-M. Han, and J. Kim The Human OCT-4 Isoforms Differ in Their Ability to Confer Self-renewal J. Biol. Chem., November 3, 2006; 281(44): 33554 - 33565. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Trounson The Production and Directed Differentiation of Human Embryonic Stem Cells Endocr. Rev., April 1, 2006; 27(2): 208 - 219. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Cai, J. Chen, Y. Liu, T. Miura, Y. Luo, J. F. Loring, W. J. Freed, M. S. Rao, and X. Zeng Assessing Self-Renewal and Differentiation in Human Embryonic Stem Cell Lines Stem Cells, March 1, 2006; 24(3): 516 - 530. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
|
|
Y. S. Son, J. H. Park, Y. K. Kang, J.-S. Park, H. S. Choi, J. Y. Lim, J. E. Lee, J. B. Lee, M. S. Ko, Y.-S. Kim, et al. Heat Shock 70-kDa Protein 8 Isoform 1 Is Expressed on the Surface of Human Embryonic Stem Cells and Downregulated upon Differentiation Stem Cells, October 1, 2005; 23(10): 1502 - 1513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Li, S. Wang, Y. Xie, Y. Lu, X. Zhang, L. Wang, S. Yang, D. Wolf, Q. Zhou, and W. Ji Homologous Feeder Cells Support Undifferentiated Growth and Pluripotency in Monkey Embryonic Stem Cells Stem Cells, September 1, 2005; 23(8): 1192 - 1199. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. S. Kim, S. K. Oh, Y. B. Park, H. J. Ahn, K. C. Sung, M. J. Kang, L. A. Lee, C. S. Suh, S. H. Kim, D.-W. Kim, et al. Methods for Derivation of Human Embryonic Stem Cells Stem Cells, September 1, 2005; 23(9): 1228 - 1233. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Chen, K. Qian, J. Hu, D. Liu, W. Lu, Y. Yang, D. Wang, H. Yan, S. Zhang, and G. Zhu The derivation of two additional human embryonic stem cell lines from day 3 embryos with low morphological scores Hum. Reprod., August 1, 2005; 20(8): 2201 - 2206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Stojkovic, M. Lako, S. Przyborski, R. Stewart, L. Armstrong, J. Evans, X. Zhang, and M. Stojkovic Human-Serum Matrix Supports Undifferentiated Growth of Human Embryonic Stem Cells Stem Cells, August 1, 2005; 23(7): 895 - 902. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Lee, B. K. Rhee, G.-Y. Bae, Y.-M. Han, and J. Kim Stimulation of Oct-4 Activity by Ewing's Sarcoma Protein Stem Cells, June 1, 2005; 23(6): 738 - 751. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Oh, H. S. Kim, Y. B. Park, H. W. Seol, Y. Y. Kim, M. S. Cho, S. Y. Ku, Y. M. Choi, D.-W. Kim, and S. Y. Moon Methods for Expansion of Human Embryonic Stem Cells Stem Cells, May 1, 2005; 23(5): 605 - 609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Woolf, W. Prudhomme, L. Daheron, G. Q. Daley, and D. A. Lauffenburger Bayesian analysis of signaling networks governing embryonic stem cell fate decisions Bioinformatics, March 15, 2005; 21(6): 741 - 753. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Stojkovic, M. Lako, R. Stewart, S. Przyborski, L. Armstrong, J. Evans, A. Murdoch, T. Strachan, and M. Stojkovic An Autogeneic Feeder Cell System That Efficiently Supports Growth of Undifferentiated Human Embryonic Stem Cells Stem Cells, March 1, 2005; 23(3): 306 - 314. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Oh, H. S. Kim, H. J. Ahn, H. W. Seol, Y. Y. Kim, Y. B. Park, C. J. Yoon, D.-W. Kim, S. H. Kim, and S. Y. Moon Derivation and Characterization of New Human Embryonic Stem Cell Lines: SNUhES1, SNUhES2, and SNUhES3 Stem Cells, February 1, 2005; 23(2): 211 - 219. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. B. Lee, J. E. Lee, J. H. Park, S. J. Kim, M. K. Kim, S. I. Roh, and H. S. Yoon Establishment and Maintenance of Human Embryonic Stem Cell Lines on Human Feeder Cells Derived from Uterine Endometrium under Serum-Free Condition Biol Reprod, January 1, 2005; 72(1): 42 - 49. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. B. Lee, J. M. Song, J. E. Lee, J. H. Park, S. J. Kim, S. M. Kang, J. N. Kwon, M. K. Kim, S. I. Roh, and H. S. Yoon Available human feeder cells for the maintenance of human embryonic stem cells Reproduction, December 1, 2004; 128(6): 727 - 735. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Pera and A. O. Trounson Human embryonic stem cells: prospects for development Development, November 15, 2004; 131(22): 5515 - 5525. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Kim, J. H. Park, J. E. Lee, J. M. Kim, J. B. Lee, S. Y. Moon, S. I. Roh, C. G. Kim, and H. S. Yoon Effects of Type IV Collagen and Laminin on the Cryopreservation of Human Embryonic Stem Cells Stem Cells, November 1, 2004; 22(6): 950 - 961. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Stojkovic, M. Lako, T. Strachan, and A. Murdoch Derivation, growth and applications of human embryonic stem cells Reproduction, September 1, 2004; 128(3): 259 - 267. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Stojkovic, M. Lako, P. Stojkovic, R. Stewart, S. Przyborski, L. Armstrong, J. Evans, M. Herbert, L. Hyslop, S. Ahmad, et al. Derivation of Human Embryonic Stem Cells from Day-8 Blastocysts Recovered after Three-Step In Vitro Culture Stem Cells, September 1, 2004; 22(5): 790 - 797. [Abstract] [Full Text] [PDF]
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