Nature Methods
2, 185 - 190 (2005)
Published online: 17 February 2005; | doi:10.1038/nmeth744
Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cellsRen-He Xu1, 2, Ruthann M Peck1, Dong S Li1, Xuezhu Feng2, Tenneille Ludwig2
& James A Thomson1, 21 WiCell Research Institute, Madison, Wisconsin 53707, USA. 2 Department of Anatomy, University of Wisconsin-Madison Medical School, the Wisconsin National Primate Research Center and The Genome Center of Wisconsin, Madison, Wisconsin 53706, USA.
Correspondence should be addressed to Ren-He Xu rxu@wicell.org or James A Thomson thomson@primate.wisc.edu Human embryonic stem cells (hESCs) are routinely cultured on fibroblast feeder layers or in fibroblast-conditioned medium (CM). Bone morphogenetic proteins (BMPs) have previously been shown to induce hESC differentiation, in apparent contrast to mouse embryonic stem (ES) cells, in which BMP4 synergizes with leukemia inhibitory factor (LIF) to maintain self-renewal. Here we demonstrate that hESCs cultured in unconditioned medium (UM) are subjected to high levels of BMP signaling activity, which is reduced in CM. The BMP antagonist noggin synergizes with basic fibroblast growth factor (bFGF) to repress BMP signaling and sustain undifferentiated proliferation of hESCs in the absence of fibroblasts or CM. These findings suggest a basic difference in the self-renewal mechanism between mouse and human ES cells and simplify the culture of hESCs.hESCs were initially isolated on fibroblast feeder layers in medium containing serum1,
2, conditions remarkably similar to those first used to isolate mouse ES cells3,
4. Yet despite these initial culture similarities, the factors that mediate self-renewal of mouse and human ES cells appear to be distinct. LIF is used to culture mouse ES cells5,
6. LIF binds a heterodimer of the LIF receptor and gp130 that activates JAK/Stat3 signaling, and activated Stat3 is sufficient to sustain undifferentiated proliferation of mouse ES cells cultured in serum. It has recently been reported that, in the absence of serum, BMPs can synergize with LIF to maintain self-renewal of mouse ES cells by inducing expression of Id genes7. Addition of LIF to culture medium1 or activation of Stat3 (ref. 8) does not sustain hESCs in conditions that would support mouse ES cells. Of all the growth factors we have tested previously, bFGF has had the greatest effect in promoting hESC self-renewal. Addition of bFGF (also called FGF2) to medium containing a commercially available serum replacement allows the clonal culture of hESCs on fibroblasts9.
We have previously shown that the addition of BMPs to hESCs cultured in CM containing bFGF promotes trophoblast differentiation10. Others have found that blocking BMP activity in serum with the BMP antagonist noggin does not maintain hESC self-renewal, but instead enhances neural differentiation by inhibiting non-neural differentiation11. Here we demonstrate that hESCs cultured in UM show greater BMP signaling activity than those cultured in CM, and that this activity is inhibited by the addition of noggin, bFGF or both. We also demonstrate that noggin combined with high bFGF concentrations supports the long-term undifferentiated proliferation of hESCs in the absence of fibroblasts or CM. Three hESC lines, H1, H9 and H14, were used in these studies.
Results
UM contains BMP-like differentiation-inducing activity
UM (see Methods) contained 20% KNOCKOUT serum replacement (Invitrogen), which includes a proprietary lipid-rich bovine albumin component, ALBUMAX12. UM was conditioned on fibroblasts overnight and then supplemented with 4 ng/ml human bFGF to obtain CM13. We cultured hESCs (H1) in CM, UM, a 1:1 mixture of CM with UM, or a 1:1 mixture of CM with DMEM/F12. The cells in CM or the 1:1 CM-DMEM/F12 mixture remained undifferentiated, and were characterized by typical hESC morphology. However, the cells in UM and in the 1:1 CM-UM mixture both rapidly differentiated within 48 h (Fig. 1). We next substituted purified fetal bovine serum albumin (16.6 g/l, Fisher Scientific) for the serum replacement to determine whether albumin caused the differentiation. This medium allowed hESCs to maintain an undifferentiated morphology for about 7 d; however, the cells had a reduced proliferation rate and eventually differentiated into a mixed population of cells (data not shown). These results suggest that components other than albumin contained in the serum replacement are responsible for the rapid differentiation of UM-cultured cells. CM reduces this differentiation-inducing activity, but also provides positive factors to sustain hESC self-renewal. In addition to albumin, serum replacement also contains other components that are required for hESC culture, so serum replacement rather than albumin was used in all subsequent studies.
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To examine whether the differentiation-inducing activity in UM stimulates BMP signaling in hESCs, we assessed by western blotting the abundance of phosphorylated Smad1, an immediate effector downstream of BMP receptors14. Smad1 phosphorylation (the antibody used here could also detect phosphorylation of the other BMP effectors Smad5 and Smad8; refs. 15 and 16, respectively) was low in H1 cells cultured in CM, but was high in cells cultured for 24 h in UM or in CM plus BMP4 (Fig. 2a). The addition of noggin to UM reduced Smad1 phosphorylation, but the addition of 40 ng/ml bFGF to UM did not change Smad1 phosphorylation. BMP signaling can induce expression of BMP ligands, forming a positive feedback loop in cells from various species, including hESCs11. BMP2 and BMP4 proteins were, indeed, detected at an increased level in UM-cultured hESCs compared to cells cultured in CM or in UM plus noggin (Fig. 2a). It is at present unclear whether there are BMPs in UM that directly stimulate BMP signaling in hESCs, or other differentiation-inducing molecules that indirectly stimulate BMP signaling by inducing BMP secretion. Noggin and another BMP antagonist, gremlin17, were both detected in medium conditioned by the fibroblasts (Fig. 2b). These data demonstrate that an elevated, but repressible, BMP signaling activity is present in UM-cultured hESCs, and that both BMP agonists and antagonists are present in fibroblast-supported culture of hESCs.
| | Figure 2. BMP agonistic and antagonistic signals are detected in hESC culture. | | |  | (a) Western blotting of H1 cells cultured in CM, CM + 100 ng/ml BMP4 (CM +BMP4), UM, UM + 40 ng/ml bFGF (UMF), UM + 0.5  g/ml noggin (UMN), or UM + bFGF + noggin (UMFN) for 24 h. Phosphorylated Smad1/5/8 (P-Smad1/5/8), Smad1/5/8, BMP2/4 and  -actin (as a loading control) were examined. (b) BMP antagonists were detected in serum replacement−free medium conditioned by fibroblasts for 24 h. The media were processed for immunoprecipitation (IP) by goat anti-noggin and anti-gremlin antibodies (Ab) or nonspecific goat immunoglobin (IgG). The precipitated proteins were subjected to western blotting with the anti-noggin and anti-gremlin antibodies separately. Recombinant mouse noggin and gremlin proteins were loaded as positive controls. (c) BMP/Smad-luciferase reporter assay on H14 cells cultured for 24 h in CM, DMEM/F12 (D/F12) or UM with or without various concentrations of BMP4, serum replacement (SR), bFGF or noggin. Error bars,  s.d. (d) Quantitative PCR assays for ID1−4 transcripts in H9 cells cultured in various media for 24 h. Results are displayed as relative mRNA level with the level in CM-cultured cells referred to as 1, and shown as mean s.d. *P < 0.05, **P < 0.01 compared to CM condition.
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We further assessed BMP signaling in hESCs (H14) cultured in various media in the presence or absence of protein factors, by using a luciferase reporter plasmid specifically responsive to BMP/Smads18. The reporter activity increased with an increasing concentration of the serum replacement or BMP4, and decreased with an increasing concentration of noggin or bFGF (Fig. 2c). 500 ng/ml noggin and 40 ng/ml bFGF had a synergistic effect in reducing the reporter activity to the level similar to that achieved by CM. Somewhat surprisingly, even higher amounts of bFGF (100 ng/ml) reduced BMP signaling to a level comparable to that found in CM without the addition of noggin. These results suggest that serum replacement indeed contains BMP-like activity, which can be reduced by noggin and/or bFGF.
Both human ID1 (ref. 18) and mouse Id1 (ref. 19) promoters contain BMP-responsive elements18,
19, and ID1 and Id1 were previously shown to be targets of BMP4 signaling in both human10 and mouse7 ES cells, respectively. We therefore examined the expression of ID genes as a second indicator of BMP signaling activity in hESCs cultured in various media. ID1−4 transcripts were more abundant in hESCs (H9) cultured for 24 h in UM or CM plus BMP4 than in cells cultured in CM, and addition of noggin to UM reduced expression of the ID genes (Fig. 2d).
UM + bFGF + noggin sustains hESC self-renewal
UM supplemented with 0.5 g/ml noggin and 40 ng/ml bFGF sustained undifferentiated proliferation of hESCs (Fig. 3). H1 cells were plated at an equal number and cultured for 7 d in CM, UM, UM plus bFGF, UM plus noggin or UM plus bFGF and noggin. Oct4+ cell numbers were significantly higher after 7 d in CM and in UM plus bFGF and noggin than in UM alone, UM plus bFGF or UM plus noggin (Fig. 3a). Intermediate Oct4+ cell numbers were detected in UM plus bFGF and UM plus noggin, suggesting a synergistic effect between noggin and bFGF (Fig. 3a). hESCs cultured in UM plus bFGF or UM plus noggin could be propagated for multiple passages, but differentiated cells accumulated in either the middle (in UM plus bFGF) or edge (in UM plus noggin) of the hESC colonies (Supplementary Fig. 1 online). Increased differentiation also occurred in cells cultured in UM plus bFGF and noggin if the noggin concentration was reduced to 0.1 g/ml and the bFGF concentration to 10 ng/ml (Supplementary Fig. 1 online). The noggin in UM plus bFGF and noggin could be substituted by gremlin (5 g/ml) or a soluble BMP receptor IA (0.5 g/ml) (data not shown), supporting the idea that the effects of noggin are indeed through the interruption of BMP receptor activation by BMPs.
| | Figure 3. UM containing bFGF and noggin sustains undifferentiated proliferation of hESCs. | | |  | (a) 35,000 H1 cells were added to each of the five media, UM, UMF, UMN, UMFN and CM, and cultured for 7 d. The cells were harvested on day 7, total cell numbers were counted, and Oct4+ cells were detected by FACS analysis. The numbers of Oct4+ and Oct4- cells in each medium are shown. Also shown are H9 and H14 cells cultured in UMFN for 27 and 18 passages, respectively, and then subjected to FACS analysis for Oct4+ and Oct4- cells. *P < 0.05, **P < 0.01 for comparison with UM. No significant difference exists between UMFN and CM or for other comparisons. (b) FACS for Oct4+ cells was performed on H1, H9 and H14 cell lines cultured in UMFN for 7, 6 and 6 passages (each taking more than 40 d), respectively, and on cells derived from the UMFN-cultured cells after subsequent culture in UM for 7 d. Sibling CM-cultured ES cells and their derivatives following 7-d subsequent culture in UM serve as controls. *P < 0.05, **P < 0.01 for comparison with the corresponding subsequent culture in UM. (c) FACS profiles for Oct4+ cells are shown for H9 cells cultured in UMFN for six passages (left) and their derivative cells following subsequent culture in UM for 7 d (right). (d) H1 cells cultured in UMFN for eight passages (about 70 d) remained undifferentiated (i and iii) and Oct4+ (v), whereas they mostly differentiated (ii and iv) and became Oct4- (vi) after subsequent culture in UM for 7 d. The cells were subjected to immunocytochemistry with the anti−human Oct4 antibody and photographed by phase (i−iv) and fluorescent (v and vi) microscopy. Bar, 50 m.
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Three hESC lines (H1, H9 and H14) that had been expanded in UM plus bFGF and noggin for more than 40 d (7, 6 and 6 passages, respectively) remained positive for POU5F1 (also known as Oct4), but differentiated if switched to UM lacking bFGF and noggin (Fig. 3b−d). hESCs cultured in UM plus bFGF and noggin continued to express other ES cell markers, including NANOG and ZFP42 (an ortholog of mouse Rex1; Fig. 4a), and the cell surface markers SSEA4 and TRA-1-60 (data not shown). Even in the best cultures, hESCs are mixed with a small percentage of spontaneously differentiated cells. For example, low levels of the trophoblast marker chorionic gonadotropin -subunit (CGB) can be detectable in CM-cultured ES cells, indicating the presence of small populations of trophoblasts10. This marker, however, was not detectable in cells cultured in UM plus bFGF and noggin (Fig. 4a). Neither the neural progenitor markers PAX6 and NEUROD1; T, a homolog of the mouse mesodermal marker Brachyury; nor the endodermal marker FOXA1 (also known as HNF3 ), were detected in hESCs cultured in CM or UM plus bFGF and noggin (Fig. 4a). Thus, ES cells propagated in UM plus bFGF and noggin maintained characteristic ES cell markers following extended culture.
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We further examined hESCs after long-term culture in UM plus bFGF and noggin. H9 cells were continuously cultured in UM plus bFGF and noggin for 32 passages. H1 and H14 cells cultured in UM plus bFGF and noggin were frozen after passages 20 and 16, respectively. H14 cells were subsequently thawed directly into UM plus bFGF and noggin and cultured to passage 18. The population doubling time (Supplementary Table 1 online) and percentage of Oct4+ cells (Fig. 3a) of both H9 and H14 cells cultured in UM plus bFGF and noggin for 27 and 18 passages, respectively, were similar to those for CM-cultured control hESCs.
UM + bFGF + noggin maintains hESC developmental potential
When treated with BMP4 in CM for 3−7 d, hESCs that had been previously cultured in UM plus bFGF and noggin for 10 passages differentiated into a flattened epithelium and secreted human chorionic gonadotropin (HCG) into the medium, indicating trophoblast differentiation10 (Fig. 4b). Embryoid bodies derived from H1 cells cultured in UM plus bFGF and noggin for 5 passages, and from control CM-cultured cells, expressed the trophoblast marker CGB and markers of the three germ layers, including PAX6, NEUROD1, T and FOXA1. Embryoid body cells also had reduced expression of the ES cell markers Oct4, NANOG and ZFP42 (Fig. 4a). H1 and H9 cells cultured in UM plus bFGF and noggin for 7 and 6 passages, respectively, were injected into SCID-beige mice. Teratomas exhibiting complex differentiation developed in the mice 5−6 weeks after inoculation (Supplementary Fig. 2 online).
ES cells in UM + bFGF + noggin have normal karyotype
H1 cells cultured in UM plus bFGF and noggin for 5 passages, H9 for 33 passages and H14 for 19 passages were karyotyped by standard G-banding, and chromosomes 12 and 17 (ref. 20) were examined by fluorescence in situ hybridization. The cells retained normal karyotypes (Fig. 5).
Discussion
The evolutionary conservation of important developmental pathways across divergent model organisms is a central concept in modern developmental biology. There is, therefore, a high expectation that mechanisms controlling self-renewal and pluripotency in various mammalian ES cells should share essential features. Recent expression analyses of human and mouse ES cells confirm that there is a substantial overlap in the expression of key genes21,
22,
23,
24, and this overlap has already been important in directing studies of self-renewal. For example, elements of the TGF and Wnt signaling pathways are well represented in both mouse and human ES cells21,
22,
23,
24, which led to the testing of TGF1 (ref. 25) and Wnt3a (ref. 26) on hESC self-renewal.
Yet there appear to be important differences in the growth factor requirements of mouse and human ES cells. BMP signaling through Id genes and LIF signaling through Stat3 are sufficient to promote the clonal growth of mouse ES cells in serum-free medium7. Activation of the LIF/Stat3 signaling pathway1,
8 does not sustain hESCs in conditions that would support mouse ES cells, and we have been unable to culture undifferentiated hESCs in serum-free medium supplemented only with LIF and BMP4 (unpublished data). BMPs cause hESC differentiation either to trophoblast10 or primitive endoderm11 in conditions that otherwise support their undifferentiated proliferation. The BMP/Smad reporter assay indicated the presence of BMP-like activity in UM (containing 20% serum replacement) that was roughly comparable to that in CM supplemented with 10 ng/ml BMP4 (Fig. 2c), a concentration we have previously shown causes differentiation10. There remain low but detectable levels of Smad1 phosphorylation and BMP2 and BMP4 production in current conditions that maintain undifferentiated hESC proliferation, including culture on fibroblasts11, in CM and in UM plus noggin and bFGF (Fig. 2a). Although it is possible that this low level of Smad1 phosphorylation is from contaminating differentiated cells rather than from the hESCs themselves, a role for BMP signaling in hESC self-renewal cannot be completely ruled out yet. In every condition we have tested to date, however, suppression of BMP signaling has been beneficial and not deleterious to hESC culture.
bFGF allows the clonal growth of hESCs on fibroblasts in medium containing serum replacement9, and the same medium supports hESCs after conditioning on fibroblasts13. Both the fibroblasts (unpublished data) and hESCs express multiple FGF genes, and ES cells themselves express multiple FGF receptors22. The increased concentration of bFGF (40 ng/ml) used in the current studies allowed hESCs to be expanded through multiple passages even in unconditioned medium without noggin. During the first several days after plating, cultures in UM plus bFGF and in UM plus bFGF and noggin seemed similar, but the proportion of differentiated cells consistently increased, especially in the middle of the cell colonies, with each culture passage in UM plus bFGF. By contrast, in the UM cultures containing bFGF and noggin, the differentiated cells were largely eliminated (Supplementary Fig. 1 online). Although 500 ng/ml noggin (a median effective dose (ED50) recommended by the manufacturer) was used throughout this study, we found that 100 ng/ml noggin was also sufficient to demonstrate the same effect as 500 ng/ml when used together with 40 ng/ml bFGF (data not shown). The noggin doses we used were far higher than the detected level in CM (Fig. 2b), so it is unlikely that noggin alone is the cause of reduced BMP signaling activity in CM-cultured cells.
Although bFGF did not seem to inhibit Smad1 phosphorylation (Fig. 2a), the BMP/Smad reporter assay indicated that downstream BMP signaling events were inhibited by bFGF (Fig. 2c). Previous studies have shown an ability of bFGF to interrupt BMP signaling either by preventing the nuclear translocation of phosphorylated Smad1 (ref. 27) or by repressing Smad1 activity in the nucleus28. Notably, at 100 ng/ml bFGF, BMP signaling activity was repressed to levels observed in CM, even without the addition of noggin (Fig. 2c). Indeed, we have found recently that at 100 ng/ml bFGF, an effect of noggin can no longer be demonstrated, and hESCs can be maintained through multiple passages with minimal background differentiation (unpublished data). Clearly, other effects of these high concentrations of bFGF are also important to hESC self-renewal, yet suppressed BMP signaling remains a consistent hallmark of current methods of hESC culture.
Undifferentiated hESCs can be sustainably expanded in the described culture system, yet it is important to examine the limitations and the challenges that remain. First, although the elimination of fibroblasts is an important step in the development of a defined medium, UM containing bFGF and noggin is not yet a defined medium. Both the serum replacement and the cell matrix Matrigel, which was used to coat plates for hESC culture, are complex mixtures of animal origin. Although we have had some success replacing Matrigel with laminin, we have observed marked differences in effectiveness between different commercial sources of both mouse and human laminins, and even between batches of laminin from the same source. Second, UM containing bFGF and noggin supports the clonal growth of hESCs extremely poorly, if at all. Cells need to be plated in clumps, and in general, higher-density cultures grow better. Finally, although this medium did support each of the hESC lines we tested, the true test of a new medium will be whether it supports growth from initial derivation. In spite of these limitations, our results define some of the signals important in the self-renewal of hESCs, eliminate the need for some animal-derived materials, and should further facilitate the use of hESCs as an experimental model.
Methods
Medium and cell culture.
UM contained 80% DMEM/F12 and 20% KNOCKOUT serum replacement, and was supplemented with 1 mM L-glutamine, 1% nonessential amino acids (all from Invitrogen) and 0.1 mM 2-mercaptoethanol (Sigma). CM was prepared as described13. hESCs were cultured on plates coated with Matrigel (BD Scientific) in CM or UM with or without either 0.5 g/ml mouse noggin (R&D Systems), or 40 ng/ml human bFGF (Invitrogen), or both, and propagated by using 2 mg/ml dispase (Invitrogen) to loosen the cell colonies. For evaluation of Oct4+ cell number, suspended colonies containing 35,000 cells were added to each medium in multiple wells and cultured for 7 d. Cells were harvested and counted on days 1 and 7, and Oct4+ cells on day 7 were detected by fluorescence-activated cell sorting (FACS, see below). Embryoid bodies were formed by suspending hESCs that had been cultured in CM or in UM containing bFGF and noggin (UMFN) as cell clumps in UM on a noncoated plate and culturing them on a rocker for 7 d. The embryoid body cells were then replated in DMEM medium supplemented with 10% fetal bovine serum on a gelatin-coated plate and cultured for 5 d followed by harvesting and RT-PCR analysis. Experiments were repeated multiple times and ANOVA was used for statistic analysis throughout the studies.
Immunoprecipitation and western blotting.
15 ml of DMEM/F12 medium was conditioned by 3.8  106 irradiated mouse embryonic fibroblast cells in a T75 flask overnight. The medium was collected and concentrated to about 0.7 ml with a 5 kDa molecular weight cutoff filter (Millipore) and immunoprecipitated with goat anti-mouse noggin and gremlin antibodies (R&D Systems; 5 g each) or 10 g goat IgG as a negative control. The precipitated proteins (Fig. 2b) or cell lysates (Fig. 2a) were electrophoresed on a 4%−20% linear gradient polyacrylamide Tris-HCl precast gel (Bio-Rad) for western blotting. The antibodies against mouse noggin and gremlin were used for the immunoprecipitated proteins, and antibodies against human Smad1/5/8, phosphorylated Smad1/5/8 (Cell Signaling Technology), BMP2/4 (R&D Systems) and -actin (Abcam) were used for the cell lysates. The blots were treated with the ECL substrate solutions 1 and 2 (Amersham Biosciences) and exposed in a Fuji Imager (Fuji Medical Systems) for chemiluminescence.
BMP/Smad luciferase reporter assay.
hESCs cultured in CM were transfected with a BMP/Smad-responsive firefly luciferase reporter plasmid, pID120-Lux18, together with trace amount of pRL-tk plasmid (Promega) to express Renilla luciferase as an internal control. One day after transfection, the cells were treated variously for 24 h. Cell lysates were extracted and both the firefly and Renilla luciferase activities tested by using the Dual-Luciferase Reporter Assay System (Promega) on a 3010 Luminometer (BD Biosciences). Results are shown as the firefly luciferase activity normalized to the Renilla luciferase activity.
Quantitative PCR and RT-PCR.
Total cellular RNA was extracted using a RNeasy kit (Qiagen) and treated with RNase-free DNase according to the manufacturer's instructions. Then, 1 g RNA was reverse transcribed to cDNA with Improm-II Reverse Transcription System (Promega). Quantitative PCR was performed using the SYBR green Q-PCR mastermix (Stratagene) on the AB 7500 Real Time PCR System (Applied Biosystems) under the following conditions: 10 min at 95 °C; 40 cycles of 30 s at 95 °C, 1 min at 60 °C and 1 min at 72 °C; and 3 min extension at 72 °C. GAPDH transcript was tested as an endogenous reference to calculate the relative expression levels of target genes according to Applied Biosystems' instructions. For RT-PCR, the following conditions were used: 3 min at 94 °C, and then various cycles (see below) of 20 s at 94 °C, 30 s at 55 °C and 1 min at 72 °C. The PCR reactions were separated by gel electrophoresis and the DNA bands were visualized under ultraviolet light for photography. The primer sequences and PCR cycle numbers are listed in Supplementary Table 2 online.
FACS and immunocytochemistry.
hESCs cultured in various media were processed for FACS analysis10 to detect Oct4+ cells. Mouse anti-human Oct4 antibody (Santa Cruz Biotechnology) at 2 g/ml and fluorescent isothiocyanate−labeled rabbit anti-mouse secondary antibody (Molecular Probes) at 1:1,000 dilution were used. Statistical analysis was performed on Arcsine values converted from the percentages of Oct4+ cells. For immunocytochemistry10, the mouse anti-Oct4 (at 2 g/ml) antibody was used and followed by Alexa Fluor 488-labeled anti-mouse IgG secondary antibody (Molecular Probes) at 1:1,000 dilution.
Immunoassay of HCG in the culture medium.
hESCs cultured in UMFN for multiple passages were subsequently cultured in CM plus 100 ng/ml BMP4 up to 7 d with daily refreshment of the medium and BMP4. The spent media were collected on days 3, 5 and 7, and assayed for HCG as described10.
G-banding and fluorescence in situ hybridization.
hESCs cultured in UMFN for various passages were processed for G-banding and fluorescence in situ hybridization as described20,
29,
30. From all the dispersed and fixed cells, 20 cells at metaphase were analyzed for G-banding, and 100−200 nuclei were assayed for fluorescence in situ hybridization using probes to detect marker genes in chromosomes of interest. Representative images captured by the CytoVysion digital imaging system (Applied Imaging) are shown.
Note: Supplementary information is available on the Nature Methods website.
Received 13 January 2005; Accepted 26 January 2005; Published online: 17 February 2005.
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Acknowledgments
We thank J. Johnson for karyotyping, D. Manning and E. Mitchen for technical support, M. Piekarczyk for the teratoma assays, T. López-Rovira for pID120-Lux plasmid, W. Hu for statistical analyses, B. Becker for image editing, and M. Levenstein and T. Berggren for critical reading of the manuscript. This work was supported by WiCell Research Institute, a nonprofit subsidiary of the Wisconsin Alumni Research Foundation, National Institutes of Health grants # P20 GM069981-01 to J.A.T., and # 5P51 RR000167 to Wisconsin National Primate Research Center.
Competing interests statement:
The authors declare competing financial interests.
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