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Wisconsin National Primate Research Center (B.G.-N., O.V.D., M.D., F.H.W., J.A.T., T.G.G.), University of Wisconsin-Madison; and the Departments of Anatomy (J.A.T.) and Obstetrics and Gynecology (T.G.G.), University of Wisconsin Medical School, Madison, Wisconsin 53715-1299
Address all correspondence and requests for reprints to: Thaddeus G. Golos, Ph.D., Wisconsin National Primate Research Center, University of Wisconsin, 1223 Capitol Court, Madison, Wisconsin 53715-1299. E-mail: golos{at}primate.wisc.edu.
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Abstract |
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Introduction |
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The establishment of human embryonic stem (hES) cells has provided new experimental approaches to studying early human embryonic development. Undifferentiated hES cells can be maintained and propagated on mouse embryonic fetal fibroblast (MEF) feeder layers (4) or on Matrigel (MG), an extracellular matrix substrate primarily composed of laminin, collagen IV, and heparan sulfate proteoglycan, in medium conditioned by MEFs (5). Human ES cells can undergo spontaneous, random differentiation upon withdrawal of conditions that sustain undifferentiated growth. This can occur in adherent culture of hES cell colonies or upon injection into immunocompromised mice and subsequent formation of teratomas. Alternatively, hES cells grown in suspension in the absence of feeder cells or basic fibroblast growth factor (FGF)-2 are capable of forming spheroid aggregates called embryoid bodies (EBs) (6). In the early stages of culture, ES cells form a dense, compact, simple EB; whereas with subsequent culture, cystic EBs demonstrate epithelial and mesenchymal differentiation and formation of a central cavity (7). During maintenance of EBs in suspension culture, hES cells can be induced to enter a program of differentiation in vitro in which there is formation of embryonic germ layers, although these differentiating cells are not organized into embryos.
In the present study, we evaluated trophoblast formation among hES cells allowed to differentiate in vitro. Although trophoblast formation appears to be a relatively inefficient process in adherent ES cell colonies, our results demonstrate that EB differentiation is associated with consistent secretion of placental hormones. In addition, growth of EBs in MG explants appears to promote placental endocrine activity. We suggest that cell-cell or cell-matrix interactions during EB formation are favorable to trophoblast differentiation, and that EB growth in MG may be a useful system to model human embryonic development in the peri-implantation period.
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Materials and Methods |
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-irradiated MEFs after 24 h in culture. A total of 4 ng/ml FGF-2 was added to MEF-CM just before addition to hES cells (5). Approximately 107 hES cells grown on MG (equivalent to six 35-mm cells) were used for making each 100-mm dish of EBs. hES cells were rinsed with 2 ml Dulbecco’s phosphate-buffered saline, (PBS, 1x, without calcium or magnesium, Life Technologies, Inc.) and enzymatically treated with a mixture of 1 ml collagenase, type IV (1 mg/ml in DMEM-F12) and 0.5 ml dispase (10 mg/ml in hES medium) at 37 C, 5% CO2 for 5–7 min, during which the colonies detached from the plate as aggregates, without dispersing into individual cells. Colonies were filtered onto a Cell Strainer (100 µm, BD Falcon, Bedford, MA) and flushed gently onto a 100-mm nontissue culture Petri dish with 15 ml media. Plates were incubated under standard conditions (37 C, 5% CO2) on a Thermolyne shaker (speed setting, 8; angle setting, 6) from 1.5–37 d to allow the ES cells to aggregate into spheroid structures and to prevent adherence to the plate.
To initiate differentiation of EBs, the FGF-2/MEF-CM medium was replaced with medium consisting of 68% DMEM-F12 supplemented with 1% Pen/Strep (Life Technologies, Inc.), 15% KOSR, 15% fetal bovine serum (heat inactivated, FBS, Hyclone, Logan, UT), 1 mM L-glutamine, 0.1 mM ß-Me, and 1% NEAA. After a variable period of suspension culture (see Results and figure legends for details of each experiment), EBs were harvested and counted before plating in 4 ml/35-mm dish or were transferred into MG (see below). Culture medium was collected at 1- to 5-d intervals indicated for each individual experiment by allowing EBs to settle to the center of the dish with gentle swirling. Medium was collected from the periphery of the dishes and replenished with fresh EB media. Hormone levels in EB-conditioned medium were compared with unconditioned medium by ANOVA and Tukey’s honestly-significant-difference test (SPSS Package, Version 11.5.0, Chicago, IL).
MG island formation and EB injection
An aliquot of 100 µl (
1 mg) cold MG stock was pipetted onto a sterilized coverslip in three increments at 5-min intervals, allowing matrix to gel between aliquots to ensure three-dimensional structures rather than flat discs. MG "islands" were then allowed to gel for 1 h, covered with a sterile Petri dish to prevent desiccation. EBs were recovered from suspension culture after 1.5 d (experiment MG 3) or 8 d (MG 4, MG 5), washed, and counted. Approximately 50–70 EBs were injected into each MG island. Islands were removed from coverslips and transferred to 35-mm dishes, for a total of three islands per dish containing EB medium. EBs were then cultured for 53 d (experiment MG 3) or 47 d (MG 4, MG 5), and medium was changed at 1-, 3-, or 5-d intervals for hormone assays.
In two further experiments, EBs were maintained in suspension for 36 h (MG 6) or 18 d (MG 7), then transferred to MG-coated 35-mm culture dishes and allowed to adhere. One half the medium was changed daily and assayed for human chorionic gonadotropin (hCG) secretion. In a final experiment (MG 8), EBs were cultured in suspension for 37 d, and culture medium was collected daily. On d 37, some of the EBs were injected to MG as described, and a similar number were maintained in parallel suspension cultures for an additional 31 d. Culture medium was assayed for hCG and steroid hormones.
Immunoassays
Media samples from hES and EB cultures were collected at specified intervals, and levels of hCG and estradiol-17ß were determined by RIA; levels of progesterone were determined by enzyme immunoassay as previously described (9, 10, 11). All hCG determinations were made on duplicate samples; steroid hormone determinations were with either single or duplicate determinations, depending on sample availability. The hCG assay used monoclonal antibody 518B7 (9), and assay of culture medium samples with an human LH RIA (12) demonstrated identical parallelism of EB culture medium samples with hCG but lack of parallelism with human LH (not shown). The interassay coefficient of variation for the hCG assay was 13.69%, and the intraassay coefficient of variation was 6.31%. The sensitivity of the assay was 0.074 ± 0.013 ng hCG/tube (n = 18).
Immunohistochemistry (IHC)
EBs grown in suspension culture or in MG islands were fixed for 2 h in 2% paraformaldehyde, washed twice with PBS, placed in warmed (48 C) 1% agarose, which was then gelled on ice, and subsequently embedded in paraffin. For hCG IHC, 5-µm paraffin sections were boiled in a microwave oven (800 W, Samsung, Model MW5536) using sodium citrate buffer (pH 6.0) for 7 min at full power, followed by 6 min at power 6, and immunostained with polyclonal (1:300 or 1:600, No. A0231, Dako, Carpinteria, CA) or monoclonal (1:200 anti-CGß Ab-5, NeoMarkers, Inc., Freemont, CA) hCG antibodies. Sections were also immunostained without boiling with monoclonal antibodies against cytokeratins 7 and 8 (25 µg/ml, CAM5.2, B-D/PharMingen, San Diego, CA) or vimentin (5.1 µg/ml, V6630, Sigma), using IHC protocols as previously described (13). The negative controls for polyclonal or monoclonal antibodies were rabbit or mouse IgG 1 
(Sigma), at the same concentration as the specific primary antibodies, and were included in each IHC experiment. Endogenous peroxidase activity was quenched using 5% hydrogen peroxide in methanol for 20 min, and sections were blocked by incubation for 30 min with 20% horse serum in 4% TBS/Triton X-100. Positive immunostaining was visualized using an ABC Peroxidase Kit (Vector Labs, Burlingame, CA) and freshly prepared Nova Red substrate (Vector Labs). Human first-trimester placental tissue, obtained as unidentified discarded tissues under approval of the University of Wisconsin IRB, was used in each staining experiment as a positive control for hCG immunostaining. Sections were counterstained with hematoxylin, mounted in organic mount Cytoseal XYL (both from Richard-Allan Scientific, Kalamazoo, MI), and analyzed by light microscopy. Photomicrographic images were captured using a Leica DMIRB microscope and a MagnaFire digital camera and software (W. Nuhsbaum, Inc., McHenry, IL), or with a Nikon TMS microscope and a Nikon N6000 film camera (Fryer Co., Inc., Huntley, IL).
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Results |
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). The presence of trophoblasts in these short-term suspension cultures was additionally suggested by the secretion of progesterone and estradiol-17ß (Fig. 1), because together these three hormones define a differentiated primate trophoblast phenotype. In contrast to EB cultures, these hormones were not secreted by hES cells allowed to spontaneously differentiate in adherent culture for 8 d (not shown).
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). Consistent with the secretion of hCG into the culture medium by these EBs, a significant population of cells within most EBs was identified as hCG-positive by IHC (Fig. 2). The majority of these hCG-positive cells were seen to be located on the surface of EBs, cells which were also cytokeratin-positive.
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Trophoblast differentiation in MG-embedded EB cultures
We reasoned that trophoblast differentiation in EBs might be promoted by the presence of extracellular matrix, as has been shown for trophoblast outgrowth and differentiation with human placental villous explants (14). We thus transplanted EBs to MG drops and maintained them for up to 8 wk in culture. We first determined hCG secretion in EBs after transfer to MG, evaluating the effects of changing culture medium every 1, 3, or 5 d (Fig. 3). Although there were low levels of hCG for approximately 3 wk, secretion began to dramatically increase at approximately 3 wk of culture, with daily medium change promoting the highest levels of hormone production. Elevated hCG secretion was sustained for nearly 4 additional weeks. In parallel with the secretion of hCG, progesterone and estradiol-17ß levels also increased in experiments MG3, MG4, and MG5 (Fig. 4). In these experiments, the EBs were prepared in three different culture medium regimens before injecting into MG [1.5-d EB medium (MG3), 3-d hES medium and 5-d EB media (MG4), or 8-d EB medium only (MG5)]. The highest levels of hCG, progesterone, and estradiol-17ß are seen in MG5; although in all three experiments, hormone production started increasing on approximately d 20.
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). Thorn-like projections (Fig. 5B) may be adhesion points of individual cells on the surface of EBs with the extracellular matrix. In some cases, these projections were seen across the entire surface of an EB; whereas in others, they were more localized. In the case of branch-like outgrowths, columns or groups of cells project away from EBs, into the surrounding matrix (Fig. 5, A, C, and D).
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). Both simple and stratified epithelial cell populations were present, as well as extensive nonepithelial (cytokeratin-negative) components (Fig. 6). We detected hCG-positive cells in many, although not in all, MG-EBs. The hCG-positive cells within EBs were of heterogeneous morphology: in some cases, they appeared to be interspersed with other nonepithelial cells (Fig 6G); whereas in others, hCG was expressed in simple (Fig. 6K) or stratified (Fig. 6, G and K) epithelia. In general, hCG-positive cells were also positive for cytokeratins, a marker of the trophoblast lineage (e.g. compare Fig. 6, C and D). In addition, the intensity of hCG staining in these EBs, after prolonged culture, tended to be higher than the early suspension culture EBs, consistent with the level of hormone secretion.
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). EBs remaining in suspension had essentially undetectable levels of hCG secretion (<1.8 ng/ml).
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), regardless of whether EBs were transferred 1.5 d after formation, or after 18 d of growth in suspension. Cellular outgrowths on the MG-coated plates were also seen within this timeframe. However, the period of elevated hCG secretion in these experiments was limited to approximately 2 wk, in contrast with that seen in EBs embedded in MG drops.
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Discussion |
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The enhancement of hormone secretion and, presumably, trophoblast differentiation by introduction to extracellular matrix is a novel finding of these studies, but one whose mechanism remains to be explored. It is possible that the three-dimensional interaction of EBs cultured in MG explants could mimic trophectoderm differentiation during the early stages of embryo implantation. Embryo implantation into the endometrium during early pregnancy initiates specific patterns in cell proliferation, differentiation, and function. Mechanochemical signals generated by interactions between cells growing out from the EBs into the extracellular matrix could provide a mechanism to promote cell differentiation. The combinatorial signals provided by cell-cell as well as cell-matrix interaction may play synergistic roles in hES cell proliferation and differentiation, specifically to the trophoblast lineage. Recently, Hubner et al. (15) reported the expression of cytokeratin 8 (troma-1) in blastocyst-like structures derived from cultured mouse ES cells, and several trophoblast-related mRNAs (Hand1, hash2, placental lactogen-I) were detected by RT-PCR in these cultures. This may indicate that under specific (although not yet well-understood) conditions, mouse ES cells may also move toward the trophoblast lineage.
It is not readily understood why there is a delay of approximately 20 d before hCG secretion begins to increase in MG-embedded EBs. On the other hand, when EBs grown in suspension culture are allowed to adhere to the culture surface, hCG secretion begins to increase within 9–10 d. Contrasting these situations, there is an approximate association between the appearance of cellular outgrowths and placental hormone secretion. During embryo implantation, it is believed that hCG secretion is detectable in the peripheral blood within 8–10 d (16); in nonhuman primates (rhesus monkey), an increase in circulating CG can be detected 6–7 d after implantation is initiated (17). Within the definitive placenta, hCG is primarily secreted by the syncytiotrophoblasts, although the specific cells secreting hCG in the first days after implantation cannot be directly demonstrated in humans. With regard to EBs in MG explants, outgrowths were typically seen 10–13 d before an elevation of hCG secretion. It is not confirmed that the outgrowths seen in MG explants are trophoblasts, although this is strongly suggested by immunohistochemical evaluation of paraffin specimens. In MG-embedded EBs, there were significant hCG-positive cells located on the outer surface of the EBs. With regard to the EBs maintained as adherent cultures, outgrowths were seen within 2–3 d, and it is possible that the outgrowths seen are trophoblast-like cells. In appearance they are similar to the trophoblast cells that differentiate directly from hES cells upon treatment with BMP-4 and secrete hCG, progesterone, and estradiol-17ß (18). Further studies will be needed to define the phenotype of the EB-derived outgrowths.
In an effort to optimize culture conditions for trophoblast differentiation in EBs, we changed half the media every day, every 3 d, or every 5 d; and hCG secretion was seen to be maximal with more frequent changes. This may indicate that metabolic needs of the EBs were not adequately met by the 3–5 d regimen, compromising hormone secretion; alternatively, it may be that an inhibitor of trophoblast differentiation or hormone secretion is being more efficiently removed with daily medium changes. Higher hormone secretion corresponded to the groups receiving somewhat longer suspension culture in EB medium before transfer to MG; however, the mechanisms by which this promotes higher hCG secretion remain to be investigated. Autocrine and paracrine control of trophoblast differentiation can be readily studied with the MG-EB paradigm.
It is interesting that hCG secretion by adherent EBs is sustained for only several weeks, whereas hCG secretion with MG-embedded EBs persists for at least 4 wk. Perhaps continued exposure to matrix is available in the MG explants, whereas in MG-coated dishes, migrating cells deplete the matrix and the extracellular stimulus that sustains hCG secretion is lost. Although this is speculative, there is significant information available on the important instructive role of the extracellular matrix, not simply providing cells with a passive anchoring scaffold but providing active instructive/inductive signaling input. This input influences cell adhesion and migration (19) as well as proliferation and DNA and protein synthesis related to cell morphology and shape (20). In the human placenta, integrins expressed on endometrial, decidual, and extravillous cytotrophoblast cells (21) present ample receptors for the extracellular matrix to influence early differentiation events. We propose that the MG-embedded EB model can provide novel insights and a new experimental approach to study human trophoblast differentiation and placental morphogenesis.
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Acknowledgments |
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Footnotes |
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B.G.-N. and O.V.D. contributed equally to this work.
This manuscript is publication no. 43-014 of the Wisconsin National Primate Research Center.
Abbreviations: EB, Embryoid body; FGF, fibroblast growth factor; hCG, human chorionic gonadotropin; hES, human embryonic stem; IHC, immunohistochemistry; MEF, mouse embryonic fetal fibroblast; MEF-CM, MEF-conditioned medium; MG, Matrigel.
Received September 18, 2003.
Accepted for publication December 9, 2003.
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