Expression and Potential Role of Fibroblast Growth Factor 2 and Its Receptors in Human Embryonic Stem Cells
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《干细胞学杂志》
a Department of Molecular Embryology, Institute of Experimental Medicine AS CR, Brno, Czech Republic;
b Department of Internal Medicine, Hematooncology, University Hospital Brno, Brno, Czech Republic;
c Laboratory of Molecular Embryology, Mendel University Brno, Brno, Czech Republic;
d Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic
Key Words. Growth factor ? Human embryonic stem cells ? Cell signaling ? Differentiation
Correspondence: Petr Dvorak, Ph.D., Laboratory of Molecular Embryology, Mendel University, Brno Zemedelska 1, 613 00 Brno, Czech Republic. Telephone: 420-5-45133298; Fax: 420-5-45133357; e-mail: dvorakp@mendelu.cz
ABSTRACT
Human embryonic stem cells (hESCs) are pluripotent stem cells derived from the human blastocyst-stage embryo. In common with their mouse counterparts (mESCs), hESCs have the capacity to self-renew and, under appropriate conditions, to differentiate into a diverse range of specialized cell types. These two major properties of hESCs, combined with their untransformed character, render them an ideal resource for the study of human development and for transplantation therapy in numerous pathologies. Regarding hESC-based therapies, the current most important challenge in this field is the development of culture systems that are chemically defined and free of animal products and which moreover allow sustained proliferation of undifferentiated cells and/or directed differentiation of hESCs into specific cell types, efficiently and to homogeneity. Here, as with mESCs, it is considered that growth factors represent the key components of such defined culture systems; however, despite great efforts, those growth factors and signaling pathways maintaining pluripotency and regulating self-renewal and/or differentiation of hESCs are largely unknown. Although activation of the JAK/STAT3 pathway by leukemia inhibitory factor (LIF) leads to maintained self-renewal of mESCs, with input also from bone morphogenetic proteins , this particular pathway is dispensable to the self-renewal of hESCs . It has been suggested that Wnt signaling positively regulates expression of transcription factors Oct4, Rex-1, and Nanog, which represent key molecules implicated in the state of hESC stemness and therefore may contribute to undifferentiated growth of hESCs . Nonreceptor tyrosine kinase, cYes, also has been identified as contributing to the maintenance of undifferentiated mESCs and to hESC self-renewal . Although cYes can be activated by LIF or other components present in serum, its inhibition does not interfere with LIF-induced JAK/STAT3 or extracellular signal-regulated kinases (ERK1/2) phosphorylation, suggesting the existence of a new, LIF-independent pathway . It is generally accepted that hESCs in culture depend on the presence of feeder cells to sustain self-renewal and the capacity to differentiate. By convention, hESCs are cultured on feeder layers of mitotically inactivated mouse embryonic fibroblasts (MEFs), using medium supplemented with serum replacement and fibroblast growth factor (FGF)-2. There are several reports in which undifferentiated hESCs are, in addition to FGF-2, grown with LIF and/or transforming growth factor ?1 . However, it is not yet clearly established that such modified culture conditions offer significant improvement for culture of undifferentiated hESCs. Undoubtedly, hESCs maintained under standard conditions may use numerous and as-yet undefined factors produced by the feeder cells. Moreover, hESCs may use several intracrine or autocrine signaling pathways to retain their capacity to proliferate indefinitely in an undifferentiated state or to modify their developmental program. Such autonomous signaling mechanisms might not have relevance in vivo because hESCs represent a greatly expanded cell population that is similar, although not identical, to inner cell mass–derived primitive ectoderm in the developing embryo. The signaling pathways in hESCs may reflect their adaptation to in vitro conditions and therefore may be somewhat simplified compared with signaling processes occurring during normal development. Nevertheless, an understanding of intracrine and autocrine hESC-specific signaling pathways is crucial not only for development of new culture conditions for propagating cells in the undifferentiated state but also for developing efficient differentiation protocols. In this context, using cDNA microarray analysis, several groups have detected high/elevated levels of components of the FGF/fibro-blast growth factor receptor (FGFR) signaling pathway in undifferentiated hESCs compared with their differentiated progeny, human tissue, and mESCs. These components included FGFR-1, -2, -3, and -4, as well as FGF-2, -11, and -13 . However, there is as yet no report addressing the action of endogenous FGFs in undifferentiated hESCs.
In contrast to the current limited understanding of the actions of other FGFs and FGFRs in undifferentiated hESCs, FGF-2 has been shown to induce development of ectodermal and mesodermal cells from predifferentiated hESCs and to support hESC differentiation into neural lineages . However, the effects of exogenous FGF signaling during in vitro differentiation protocols may be peripheral, as studies in mESCs have indicated that for neural specification, autocrine FGF signaling is preferentially involved . Taken together, these data are somewhat contradictory, especially as individual components of the FGF signaling are elevated in undifferentiated hESCs and downregulated during differentiation, in which, on the other hand, the importance of FGF regulatory pathways has been repeatedly proven.
Therefore, as a first step to understanding the role of the FGF/FGFR signaling pathways in hESCs, we determined the expression pattern of molecular isoforms of endogenous FGF-2 in undifferentiated and differentiating hESCs. Then, using a quantitative method, we analyzed and compared the expression of all four FGFRs under the same conditions. The response of hESCs to exogenous FGF-2 was analyzed at both biochemical and cellular levels. Finally, we used an inhibitor of FGFR tyrosine kinases to investigate the autocrine FGF signaling pathway. Our results clearly demonstrate the existence of an operational, autocrine FGF signaling pathway in undifferentiated hESCs, inhibition of which leads to rapid differentiation. We therefore suggest that autocrine FGF signaling through FGFRs, and specifically FGFR1, is unconditionally required for proliferation of hESCs in the undifferentiated state.
MATERIALS AND METHODS
Expression and Release of FGF-2 in hESCs
Human FGF-2 is synthesized in five isoforms that originate from alternative translation start codons within a single mRNA species. Different molecular isoforms of FGF-2 act through distinct pathways: four high-molecular-mass isoforms (22-, 22.5-, 24-, and 34-kDa) are likely to be involved in intracrine regulation of cell proliferation and apoptosis, and the low-molecular-mass isoform (18 kDa), which is released from cells, signals via transmembrane receptors and regulates both proliferation and differentiation in either an autocrine or paracrine manner, depending mainly on the cell type concerned.
An antibody that recognizes all five in vivo FGF-2 isoforms was used here to analyze the FGF-2 expression pattern in hESCs. As this antibody recognizes both human and mouse FGF-2, it was also used to investigate the expression and release of FGF-2 in CF-1 feeder cells. It was revealed that undifferentiated hESCs (maintained in recombinant FGF-2–free medium for 24 hours), unlike mESCs , express 18-, 22-, 22.5-, and 24-kDa isoforms of FGF-2. The 34-kDa FGF-2 molecule was not detected (Fig. 2A). Indirect immunofluorescence with the same antibody demonstrated strong nuclear and nucleolar staining, with less-intensive cytoplasmic staining, which is consistent with the preponderance in hESCs of the high-molecular-mass isoforms of FGF-2, which are nuclear-localized (Fig. 2B). We also demonstrated that hESCs contain nuclear protein FIF (Fig. 2A, lower panel), which interacts with high-molecular-mass FGF-2 and exhibits antiapoptotic properties .
Figure 2. Expression of FGF-2 in hESCs and release of the low-molecular-mass isoform by undifferentiated hESCs. (A): Western blot analysis to show that undifferentiated, as well as differentiated, hESCs express several molecular isoforms of FGF-2 (upper panels) and synthesize FIF (lower panel). Human recombinant FGF-2 that represents 16.4-kDa band and FGF-2–producing leukemia K562 cells (18-kDa isoform) were used as positive controls for FGF-2; K562 and HL60 cells were used as positive controls for FIF. (B): As determined by indirect immunofluorescence, significant amounts of FGF-2 are localized in nuclei and nucleoli of undifferentiated hESCs as well as intracytoplasmically and at the plasma membrane and intercellular junctions. For Ig control, the primary antibody was omitted. (C): Western blot analysis to show that the 18-kDa isoform of FGF-2 synthesized by hESCs is released into culture medium (CM1, CM2). In contrast, mitomycin C–treated mouse embryonic fibroblasts (feeder cells) derived from strain CF-1 mice neither contain nor release FGF-2. The following samples were probed by Western blotting for the presence of FGF-2: (a) cell extracts from CF-1 fibroblasts and from hESCs CCTL14 (cells); (b) conditioned media (CM1 and CM2) harvested from two independent cultures of mouse fibroblasts and from two high-density cultures of undifferentiated hESCs of the lines CCTL12 and CCTL14 (respectively), subjected to enrichment of heparin-binding growth factors. Data are representative of at least three independent replicates. Abbreviations: CCTL, Center for Cell Therapy Line; EB, embryoid body; FGF, fibroblast growth factor; FIF, FGF-2–interacting factor; hESC, human embryonic stem cell; HMM, high molecular mass; Ig, immunoglobulin; LMM, low molecular mass; PI, propidium iodide–stained DNA.
We next analyzed the expression of endogenous FGF-2 in differentiating/differentiated hESCs (Fig. 2A). The pattern of expression of all four (18-, 22-, 22.5-, and 24-kDa) molecular isoforms of FGF-2 in D8 embryoid bodies, and in adherent cells resulting from the two-step differentiation protocol, is indistinguishable from that in undifferentiated cells. Cells produced by our standard, two-step protocol (D5 + 10) still contain detectable levels of Oct-4, a marker of undifferentiated hESCs; therefore, we prolonged the differentiation period to a total of 24 days, comprising 6 days in suspension and 18 days in adherent culture (D6 + 18), after which time immunoreactivity for Oct-4 is undetectable, and probed these cells for the presence of FGF-2. Even in such highly differentiated cells, there is no observable difference in the gross pattern of FGF-2 expression compared with undifferentiated hESCs.
The capacity of hESCs in culture to release the specific, 18-kDa isoform of FGF-2, mediating autocrine signaling, was examined. Culture medium that had been conditioned for 24 hours by hESCs, maintained on a feeder layer in recombinant FGF-2–free medium, was subjected to enrichment of heparin-binding growth factors. As revealed by Western blot analysis, exportable 18-kDa FGF-2 isoform was recovered from medium conditioned by hESCs on feeder cells but not from medium conditioned by feeder cells alone (Fig. 2C). Crucially, the concentration of 18-kDa FGF-2 in hESC-conditioned medium increased with increasing number of hESCs and reached 80 to 100 pg/ml in high-density cultures. Therefore, it is deduced that hESCs have the capacity, in terms of exported low-molecular-mass isoform of FGF-2, to activate FGFRs in an autocrine manner, and such activity may reach high levels, especially in high-density hESC cultures. It is notable also that feeder layers prepared from CF-1 strain MEFs express no potentially FGFR-stimulatory FGF-2 isoforms in this culture system.
Expression of FGFRs in hESCs
The biological effects of FGFs are mediated by their binding to four FGFR kinases, FGFR1 through FGFR4, each possessing different ligand-binding specificities and cell type–specific and tissue-specific expression. The ligand-binding specificities of FGFR1, FGFR2, and FGFR3 are primarily achieved by alternative splicing of the exons encoding the C-terminal region of extracellular, immunoglobulin-like domain III. Such alternative splicing event results in b and c isoforms of FGFR1, FGFR2, and FGFR3, whereas FGFR4 exists in one sole spliced form.
Quantitative real-time RT-PCR was used to analyze the expression of all four FGFRs in undifferentiated hESCs and in hESCs at various stages of differentiation. The primers were designed to detect human-specific FGF-2–binding, IIIc alternatives of FGFR1, FGFR2, and FGFR3 and the ligand-binding domain of FGFR4. Real-time RT-PCR data were normalized by comparison with expression of the ABL gene that was found to be eligible for quantification of FGFR gene transcripts in the different hESC samples and are summarized in Table 2. To avoid contamination with feeder cells, colonies of hESCs were carefully separated by mechanical scraping with a glass pipette. First, by analyzing four independent hESC lines, it was revealed that undifferentiated cells that are mechanically passaged and cultured at low densities express all four FGFRs in a very stable, relative pattern: in all the lines, FGFR1 was dominant with the other receptors showing lower expression; FGFR1 FGFR3 FGFR4 FGFR2. Notably, this relative expression pattern changed to FGFR1 FGFR4 FGFR3 FGFR2 in two selected hESC lines, CCTL12 and 14, after adaptation to enzymatic passaging, for the purpose of achieving higher cell-culture densities, and this pattern reflected upregulation of FGFR1 and downregulation of FGFR3 expression compared with low-density cultures. A similar trend in expression of FGFRs was observed also in cells that had initiated differentiation, whether by simple 8-day adherent culture without FGF-2 and feeder cells (D8 spontaneous differentiation) or by 8-day culture in aggregates (D8 embryoid bodies). Also for hESCs that had undergone more advanced differentiation by the two-step protocol (D5 + 10), the relative order of expression was FGFR1 FGFR4 FGFR3 FGFR2. Here, compared with undifferentiated hESCs, expression of all four FGFRs was dramatically elevated, with FGFR1 being increased about sixfold, FGFR2 about twofold, and FGFR3 and FGFR4 about sevenfold.
Table 2. Expression of FGFRs in undifferentiated and differentiated human embryonic stem cells
In summary, our data clearly indicate that hESCs are well equipped to accept and transmit FGF-2 signals via all four FGFRs, with FGFR1 potentially representing a dominant target, and that the relative levels of expression of FGFRs are tightly coupled to conditions that direct hESCs to differentiate.
Biological Effects of Exogenous and Endogenous FGF-2 on hESCs
Recombinant FGF-2 added exogenously to culture media may stimulate hESCs only via their cognate receptors, FGFRs. In contrast, FGF-2 produced endogenously by hESCs may function in two ways, depending on the presence or absence of the nuclear localization sequence: high-molecular-mass isoforms may be targeted to the nucleus and operate independently of cell-surface receptors, in an intracrine manner, whereas the low-molecular-mass isoforms may be exported from the cells and act via FGFRs as an autocrine or paracrine factor.
Stimulation of cells by FGF-2 leads to overall tyrosine phosphorylation of various proteins and to activation of extracellular signal-regulated kinases, ERK1/2 in particular, and so we used these two phenomena as criteria for the capacity of hESCs to respond to FGF-2. As evident from Figure 3, a short exposure of hESCs to 5 ng/ml recombinant FGF-2 induces a rapid and significant increase in levels of tyrosine phosphorylation of proteins (Fig. 3A), including ERK1/2 (Fig. 3B). This concentration of recombinant FGF-2 is routinely used for hESC culture and, as shown here, can be further increased by endogenously produced FGF-2. It is also of note that undifferentiated hESCs maintained in FGF-2–free medium possess an unexpectedly high basal level of phosphorylation of ERK1/2, which contrasts with the almost undetectable ERK1/2 phosphorylation typically demonstrated by mESCs D3 cultured under feeder-free conditions with serum- and LIF-supplemented medium (Fig. 3C).
Figure 3. FGF-2–induced phosphorylation of tyrosine residues and activation of ERK1/2 kinases in undifferentiated hESCs. (A): Treatment of hESCs (lines CCTL12 and CCTL14) with 5 ng/ml FGF-2 for 7 minutes results in elevated levels of tyrosine phosphorylation, as determined by Western blotting using antiphosphotyrosine antibody. Amidoblack staining of total proteins is shown in the lower panel, to document equal loading. (B): The phosphorylation of ERKs, as visualized by phospho-specific anti-ERK antibodies (pERK1/2), was strongly increased in hESCs (lines CCTL12 and CCTL14) by treatment with 5 ng/ml FGF-2 for 7 minutes. Membranes were reprobed with anti-ERK antibody to show the total amounts of ERKs (ERK1/2). (C): The basal level of ERK1/2 phosphorylation in serum-starved and FGF-2–starved hESCs (line CCTL12) is high relative to that in undifferentiated mESCs (feeder-independent line D3) maintained in serum- and LIF-supplemented medium. Data are representative of two independent replicates. Abbreviations: CCTL, Center for Cell Therapy Line; ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; hESC, human embryonic stem cell; LIF, leukemia inhibitory factor; mESC, mouse embryonic stem cell.
To determine whether the observed increased tyrosine phosphorylation and concomitant activation of growth-related kinases result in accelerated proliferation of hESCs, we used two complementary assays: measurement of the number of viable/metabolically active cells by the cleavage of tetrazolium salt WST-1 and analysis of the proportion of cells in S-phase by incorporation of BrdU into newly synthesized DNA. Although overall metabolic activity as determined by the cleavage of WST-1 was increased slightly in FGF-2–treated hESC line CCTL12 compared with untreated controls, such effect was not observed in CCTL14. We also determined that the number of cells in S-phase remained unchanged at any tested concentration of FGF-2 (Fig. 4A). From this data, metabolic activity of hESCs rather than progression of their cell cycle is influenced by exogenously added FGF-2. The absence or presence of recombinant FGF-2 also had no effect on the expression of Oct-4 (Fig. 4B). We therefore conclude that exogenously added FGF-2 at concentrations up to 20 ng/ml is dispensable for growth and self-renewal of hESCs maintained under standard conditions with feeder cells.
Figure 4. Effect of FGF-2 concentration on the proliferation and differentiation of hESCs. (A): Cells were maintained for 2, 3, and 5 days in culture medium supplemented with increasing concentrations of FGF-2, and growth was measured using the WST-1 proliferation reagent (upper panels) and by metabolic labeling of DNA with BrdU followed by ELISA (lower panels). Nontreated cultures (0 ng/ml FGF-2) served as a control. For both assays, growth is expressed as the absorbance quantified spectrophotometrically at indicated wavelengths, at the given time points. Values obtained by measurement of either metabolic activity or BrdU incorporation by feeder cells alone were subtracted. For hESC line CCTL12, metabolic activity was slightly augmented upon culture with FGF-2; however, treatment with FGF-2 had no effect on the proportion of BrdU-labeled, i.e., proliferating, hESCs. Data are representative of three independent experiments. Each value represents the mean of 24 replicates (wells) with the calculated standard deviation. (B): Western blot analysis illustrates that for hESCs cultured for 5 days without or with exogenous FGF-2 at indicated concentrations, the undifferentiated state was not affected, as documented by relatively constant levels of Oct-4 expression. Amidoblack staining of total proteins is shown in the lower panel, to document equal loading. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; CCTL, Center for Cell Therapy Line; ELISA, enzyme-linked immunosorbent assay; FGF, fibroblast growth factor; hESC, human embryonic stem cell.
Although no effect of FGF-2 on the proliferation of hESCs was found, there are still observable differences in hESC cultures depending on the concentration of exogenous FGF-2, which was manifest by the ability of cells to spread on the culture substratum. To quantify this effect, we used a simple strategy based on determining the change with time in size of hESC colonies in cultures that were exposed to various concentration of exogenous FGF-2 for 4, 5, and 6 days. Interestingly, the outgrowth of hESC colonies grown in medium containing 10 and 20 ng/ml FGF-2 increased at day 5 of culture and then was much less than the outgrowth in medium without or with only 5 ng/ml FGF-2 at day 6 of culture. This reduction in colony size at day 6 of culture cannot be attributed to reduced proliferation (see above) but instead reflected a morphological change to more compacted colonies, with well-defined borders and without the habitual, flattened cells on their periphery (Figs. 5A, 5B).
Figure 5. Effect of FGF-2 at various concentrations on the outgrowth of undifferentiated hESC colonies. (A): Cells were grown on feeder layers in 24-well dishes in medium supplemented with FGF-2 (at 5, 10, and 20 ng/ml) or without FGF-2. At 4, 5, and 6 days of culture, individual cultures were photographed and then sizes of colonies were determined from the photomicrographs. Representative hESC cultures are shown. (B): A minimum of 80 colonies was measured for each culture condition and time point, and average colony size was calculated; the increase in size (colony outgrowth) at days 5 and 6 was expressed as a percentage of the initial measurement at day 4, which is represented by a value of 0 on the Y-axis. At day 6 of culture, when the cells are routinely passaged, FGF-2 in the medium at concentrations of 10 and 20 ng/ml caused hESCs to grow in more compact/less-spread colonies compared with conditions without FGF-2 or with FGF-2 at only 5 ng/ml. Comparable results were obtained for both lines, CCTL12 and CCTL14, and therefore were port representative data for CCTL14. Abbreviations: CCTL, Center for Cell Therapy Line; FGF, fibroblast growth factor; hESC, human embryonic stem cell.
To test whether autocrine FGF signaling is essential for proliferation of undifferentiated hESCs, cultures of hESCs in FGF-2–free medium were treated with the pharmacological inhibitor of FGFR tyrosine kinases, SU5402, which specifically interacts with intra-cellular catalytic domain of FGFRs . After 2 days of continuous exposure to SU5402 at a concentration of 10–30 μM, cells in the centers of colonies acquired flattened morphology that resembled those spontaneously differentiating hESCs that occasionally occur in standard cultures, or the crater cells obtained during neuronal differentiation using HepG2-conditioned medium . Upon further culture totaling 5 days in the presence of SU5402, such morphology (Fig. 6A, upper panel), accompanied by loss of alkaline phosphatase activity (Fig. 6A, lower panel), finally developed in all hESC colonies . Furthermore, decreased phosphorylation of MEK1/2 and its substrate ERK1/2 were both observed in cells maintained in medium with inhibitor (Fig. 6B, – FGF-2). A similar effect was observed also in hESCs that were treated with 10 ng/ml recombinant FGF-2 (Fig. 6B, +FGF-2). Crucially, no signs of decreased cell viability were observed in SU5402-treated cultures (determined by cleavage of lamin B; Fig. 6C, upper left panel); instead, the presence of flattened cells was accompanied by downregulation of Oct-4 and upregulation of cyclin-dependent kinase inhibitor p27 (Fig. 6C, upper right panel), a common event that characterizes differentiation of cells of early embryonic origin and slower proliferation (Fig. 6C, lower panel). Moreover, differentiation process was manifested by upregulation of TROMA-1, a marker for primitive endoderm, and of nestin, a marker for developing neuroepithelium and for epithelial precursors in the embryonic pancreas (Fig. 6D, Western blots and immunofluorescence). Recombinant FGF-2 at concentrations up to 20 ng/ml did not reverse the phenotype produced by the noncompetitive inhibitor, SU5402 (results not shown), thus supporting our hypothesis of a critical role for autocrine FGF signaling in maintaining the pluripotent state of hESCs.
Figure 6. Inhibition of autocrine FGF signaling in undifferentiated hESCs. (A): hESCs were cultured on feeder layers in medium with 4 ng/ml FGF-2 (+FGF-2) or without FGF-2 (–FGF-2) and supplemented with the indicated concentration of SU5402. Cells were cultured for 5 days with daily change of medium, fixed, stained for alkaline phosphatase, and photographed at low (upper panel showing entire culture wells) and high (lower panel) magnifications. For cells cultured with 10 μM SU5402, differentiation starts at the centers of some colonies (data not shown); for cells cultured with 20 and/or 30 μM SU5402, there is more homogenous differentiation throughout all colonies (upper panel), which is characterized by a central area of flattened cells (asterisk in lower panel) surrounded by a ring of alkaline phosphatase–positive cells (arrow in lower panel); meanwhile, cells in SU5402-untreated control colonies showed homogeneous staining for alkaline phosphatase. Cells maintained under standard conditions, i.e., in hESC medium supplemented with 4 ng/ml FGF-2 alone (control 1), or with the diluent, dimethylsulfoxide (control 2) served as controls. (B): hESCs maintained without recombinant FGF-2 in the presence of 20 μM SU5402 for 4 days show decreased phosphorylation of MEK1/2 and ERK1/2. A similar decay of phosphorylated MEK1/2 and ERK1/2 was obtained in SU5402-treated hESCs after stimulation with 10 ng/ml FGF-2 for 7 minutes. (C): hESCs maintained in the presence of 20 μM SU5402 for 5 days were not apoptotic (as demonstrated by lack of cleavage of lamin B, upper left panel). In addition, cells cultured with inhibitor were negative for Oct-4 (upper right panel), although most of the hESC colonies still contained at their peripheries a surrounding ring of alkaline phosphatase–positive cells (A), upregulate p27 (upper right panel), and showed significantly retarded proliferation (p < .05), as documented by 5-bromo-2'-deoxy-uridine staining (lower panel). (D): SU5402-treated cells demonstrated strongly upregulated expression of the differentiation markers, TROMA-1 (approximately 55 kDa) and nestin (220–240 kDa), as documented by Western blotting (upper panels) and immunofluorescence (lower panels). Identical results were obtained for CCTL9 and CCTL14, and therefore we present data for CCTL14. Data are representatives of atleast three independent replicates. Abbreviations: CCTL, Center for Cell Therapy Line; ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; FL, feeder layer; hESC, human embryonic stem cell; MEK, mitogen-activated protein kinase kinase.
DISCUSSION
This research was supported in part by the Grant Agency of the Czech Republic (301/03/1122, 305/05/0434), Ministry of Education, Youth, and Sports(1M0021620803), Ministry of Health (MZ 00065269705), and Academy of Sciences of the Czech Republic (AV0Z50390512). We are very grateful to Dr. Elena Notarianni for valuable comments on the manuscript and for having revised the English of this manuscript.
REFERENCES
Niwa H, Burdon T, Chambers I et al. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998;12:2048–2060.
Matsuda T, Nakamura T, Nakao K et al. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J 1999;18:4261–4269.
Ying QL, Nichols J, Chambers I et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281–292.
Humphrey RK, Beattie GM, Lopez AD et al. Maintenance of pluripotency in human embryonic stem cells is STAT3 independent. STEM CELLS 2004;22:522–530.
Sato N, Meijer L. Skaltsounis L at al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004;10:55–63.
Annerén C, Cowan CA, Melton DA. The Src family of tyrosine kinases is important for embryonic stem cell self-renewal. J Biol Chem 2004;279:31590–31598.
Amit M, Shariki C, Margulets V et al. Feeder and serum-free culture of human embryonic stem cells. Biol Reprod 2004;70:837–845.
Mitalipova M, Calhoun J, Shin SJ et al. Human embryonic stem cell lines derived from discarded embryos. STEM CELLS 2003;21:521–526.
Cowan CA, Klimanskaya I, McMahon J et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 2004;350:1354–1356.
Sperger JM, Chen X, Draper JS et al. Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc Natl Acad Sci U S A 2003;100:13350–13355.
Sato N, Sanjuan IM, Heke M et al. Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev Biol 2003;260:404–413.
Ginis I, Luo Y, Miura T et al. Differences between human and mouse embryonic stem cells. Dev Biol 2004;269:360–380.
Schuldiner M, Yanuka O, Itskovitz-Eldor J et al. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2000;97:11307–11312.
Carpenter MK, Inokuma MS, Denham J et al. Enrichment of neurons and neural precursors from human embryonic stem cells. Exp Neurol 2001;172:383–397.
Zhang SC, Wernig M, Duncan ID et al. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 2001;19:1129–1133.
Reubinoff BE, Itsykson P, Turetsky T et al. Neural progenitors from human embryonic stem cells. Nat Biotechnol 2001;19:1134–1140.
Schulz TC, Palmarini GM, Noggle SA et al. Directed neuronal differentiation of human embryonic stem cells. BMC Neurosci 2003;4:27–39.
Park S, Lee KS, Lee YJ et al. Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors. Neurosci Lett 2004;359:99–103.
Ying Q, Stavridis M, Griffiths D et al. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 2003;21:183–186.
Gabert J, Beillard E, van der Velden VHJ et al. Standardization and quality control studies of "real-time" quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) of fusion gene transcripts for minimal residual disease detection in leukemia: a Europe Against Cancer Program. Leukemia 2003;17:2318–2357.
Jirmanova L, Pacholikova J, Krejci P et al. O-linked carbohydrates are required for FGF-2-mediated proliferation of mouse embryonic cells. Int J Dev Biol 1999;43:555–562.
Van den Berghe L, Laurell H, Huez I et al. FIF , a nuclear putatively antiapoptotic factor, interacts specifically with FGF-2. Mol Endocrinol 2000;14:1709–1724.
Mohammadi M, McMahon G, Sun L et al. Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science 1997;276:955–960.
Bryja V, Pachernik J, Soucek K et al. Increased apoptosis in differentiating p27-deficient mouse embryonic stem cells. Cell Mol Life Sci 2004;61:1384–1400.
Arnaud E, Touriol C, Boutonnet C et al. A new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-AUG start codon and behaves as a survival factor. Mol Cell Biol 1999;19:505–514.
Stachowiak MK, Fang XH, Myers JM et al. Integrative nuclear FGFR1 signaling (INFS) as a part of a universal "feed-forward-and-gate" signaling module that controls cell growth and differentiation. J Cell Biochem 2003;90:662–691.
Arese M, Chen Y, Florkiewicz RZ et al. Nuclear activities of basic fibroblast growth factor: potentiation of low-serum growth mediated by natural or chimeric nuclear localization signals. Mol Biol Cell 1999;10:1429–1444.
Sheng Z, Lewis JA, Chirico WJ. Nuclear and nucleolar localization of 18-kDa fibroblast growth factor-2 is controlled by C-terminal signals. J Biol Chem 2004;279:40153–40160.
Gaubert F, Escaffit F, Bertrand C et al. Expression of the high molecular weight fibroblast growth factor-2 isoform of 210 amino acids is associated with modulation of protein kinases C and and ERK activation. J Biol Chem 2001;276:1545–1554.
Burdon T, Stracey C, Chambers I et al. Suppression of SHP-2 and ERK signaling promotes self-renewal of mouse embryonic stem cells. Dev Biol 1999;210:30–43.
Amit M, Carpenter MK, Inokuma MS et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 2000;227:271–278.
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974.
Rosler ES, Fisk GJ, Ares X et al. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn 2004;229:259–274.
Debiais F, Lemonnier J, Hay E et al. Fibroblast growth factor-2 (FGF-2) increases N-cadherin expression through protein kinase C and Src-kinase pathways in human calvaria osteoblasts. J Cell Biochem 2001;81:68–81.
El-Hariry I, Pignatelli M, Lemoine NR. FGF-1 and FGF-2 regulate the expression of E-cadherin and catenins in pancreatic adenocarcinoma. Int J Cancer 2001;94:652–661.
Jang JH, Ku Y, Chung CP et al. Enhanced fibronectin-mediated cell adhesion of human osteoblast by fibroblast growth factor, FGF-2. Biotechnol Lett 2002;24:1659–1663.
Richard C, Roghani M, Moscatelli D. Fibroblast growth factor (FGF)-2 mediates cell attachment through interactions with two FGF receptor-1 isoforms and extracellular matrix or cell-associated heparan sulfate proteoglycans. Biochem Biophys Res Commun 2000;276:399–405.
Cha S, Hwang Y, Chae J et al. Inhibition of FGF signaling causes expansion of the endoderm in Xenopus. Biochem Biophys Res Commun 2004;315:100–106.
Esni F, Stoffers DA, Takeuchi T et al. Origin of exocrine pancreatic cells from nestin-positive precursors in developing mouse pancreas. Mech Dev 2004;121:15–25.
Bursdal CA, Flannery ML, Pedersen RA. FGF-2 alters the fate of mouse epiblast from ectoderm to mesoderm in vitro. Dev Biol 1998;198:231–244.
Dvorak P, Flechon JE, Thompson EM et al. Embryoglycans regulate FGF-2-mediated mesoderm induction in the rabbit embryo. J Cell Sci 1997;110:1–10.
Mathieu J, Griffin K, Herbomel P et al. Nodal and Fgf pathways interact through a positive regulatory loop and synergize to maintain mesodermal cell populations. Development 2004;131:629–641.(Petr Dvoraka,c,d, Dana Dv)
b Department of Internal Medicine, Hematooncology, University Hospital Brno, Brno, Czech Republic;
c Laboratory of Molecular Embryology, Mendel University Brno, Brno, Czech Republic;
d Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic
Key Words. Growth factor ? Human embryonic stem cells ? Cell signaling ? Differentiation
Correspondence: Petr Dvorak, Ph.D., Laboratory of Molecular Embryology, Mendel University, Brno Zemedelska 1, 613 00 Brno, Czech Republic. Telephone: 420-5-45133298; Fax: 420-5-45133357; e-mail: dvorakp@mendelu.cz
ABSTRACT
Human embryonic stem cells (hESCs) are pluripotent stem cells derived from the human blastocyst-stage embryo. In common with their mouse counterparts (mESCs), hESCs have the capacity to self-renew and, under appropriate conditions, to differentiate into a diverse range of specialized cell types. These two major properties of hESCs, combined with their untransformed character, render them an ideal resource for the study of human development and for transplantation therapy in numerous pathologies. Regarding hESC-based therapies, the current most important challenge in this field is the development of culture systems that are chemically defined and free of animal products and which moreover allow sustained proliferation of undifferentiated cells and/or directed differentiation of hESCs into specific cell types, efficiently and to homogeneity. Here, as with mESCs, it is considered that growth factors represent the key components of such defined culture systems; however, despite great efforts, those growth factors and signaling pathways maintaining pluripotency and regulating self-renewal and/or differentiation of hESCs are largely unknown. Although activation of the JAK/STAT3 pathway by leukemia inhibitory factor (LIF) leads to maintained self-renewal of mESCs, with input also from bone morphogenetic proteins , this particular pathway is dispensable to the self-renewal of hESCs . It has been suggested that Wnt signaling positively regulates expression of transcription factors Oct4, Rex-1, and Nanog, which represent key molecules implicated in the state of hESC stemness and therefore may contribute to undifferentiated growth of hESCs . Nonreceptor tyrosine kinase, cYes, also has been identified as contributing to the maintenance of undifferentiated mESCs and to hESC self-renewal . Although cYes can be activated by LIF or other components present in serum, its inhibition does not interfere with LIF-induced JAK/STAT3 or extracellular signal-regulated kinases (ERK1/2) phosphorylation, suggesting the existence of a new, LIF-independent pathway . It is generally accepted that hESCs in culture depend on the presence of feeder cells to sustain self-renewal and the capacity to differentiate. By convention, hESCs are cultured on feeder layers of mitotically inactivated mouse embryonic fibroblasts (MEFs), using medium supplemented with serum replacement and fibroblast growth factor (FGF)-2. There are several reports in which undifferentiated hESCs are, in addition to FGF-2, grown with LIF and/or transforming growth factor ?1 . However, it is not yet clearly established that such modified culture conditions offer significant improvement for culture of undifferentiated hESCs. Undoubtedly, hESCs maintained under standard conditions may use numerous and as-yet undefined factors produced by the feeder cells. Moreover, hESCs may use several intracrine or autocrine signaling pathways to retain their capacity to proliferate indefinitely in an undifferentiated state or to modify their developmental program. Such autonomous signaling mechanisms might not have relevance in vivo because hESCs represent a greatly expanded cell population that is similar, although not identical, to inner cell mass–derived primitive ectoderm in the developing embryo. The signaling pathways in hESCs may reflect their adaptation to in vitro conditions and therefore may be somewhat simplified compared with signaling processes occurring during normal development. Nevertheless, an understanding of intracrine and autocrine hESC-specific signaling pathways is crucial not only for development of new culture conditions for propagating cells in the undifferentiated state but also for developing efficient differentiation protocols. In this context, using cDNA microarray analysis, several groups have detected high/elevated levels of components of the FGF/fibro-blast growth factor receptor (FGFR) signaling pathway in undifferentiated hESCs compared with their differentiated progeny, human tissue, and mESCs. These components included FGFR-1, -2, -3, and -4, as well as FGF-2, -11, and -13 . However, there is as yet no report addressing the action of endogenous FGFs in undifferentiated hESCs.
In contrast to the current limited understanding of the actions of other FGFs and FGFRs in undifferentiated hESCs, FGF-2 has been shown to induce development of ectodermal and mesodermal cells from predifferentiated hESCs and to support hESC differentiation into neural lineages . However, the effects of exogenous FGF signaling during in vitro differentiation protocols may be peripheral, as studies in mESCs have indicated that for neural specification, autocrine FGF signaling is preferentially involved . Taken together, these data are somewhat contradictory, especially as individual components of the FGF signaling are elevated in undifferentiated hESCs and downregulated during differentiation, in which, on the other hand, the importance of FGF regulatory pathways has been repeatedly proven.
Therefore, as a first step to understanding the role of the FGF/FGFR signaling pathways in hESCs, we determined the expression pattern of molecular isoforms of endogenous FGF-2 in undifferentiated and differentiating hESCs. Then, using a quantitative method, we analyzed and compared the expression of all four FGFRs under the same conditions. The response of hESCs to exogenous FGF-2 was analyzed at both biochemical and cellular levels. Finally, we used an inhibitor of FGFR tyrosine kinases to investigate the autocrine FGF signaling pathway. Our results clearly demonstrate the existence of an operational, autocrine FGF signaling pathway in undifferentiated hESCs, inhibition of which leads to rapid differentiation. We therefore suggest that autocrine FGF signaling through FGFRs, and specifically FGFR1, is unconditionally required for proliferation of hESCs in the undifferentiated state.
MATERIALS AND METHODS
Expression and Release of FGF-2 in hESCs
Human FGF-2 is synthesized in five isoforms that originate from alternative translation start codons within a single mRNA species. Different molecular isoforms of FGF-2 act through distinct pathways: four high-molecular-mass isoforms (22-, 22.5-, 24-, and 34-kDa) are likely to be involved in intracrine regulation of cell proliferation and apoptosis, and the low-molecular-mass isoform (18 kDa), which is released from cells, signals via transmembrane receptors and regulates both proliferation and differentiation in either an autocrine or paracrine manner, depending mainly on the cell type concerned.
An antibody that recognizes all five in vivo FGF-2 isoforms was used here to analyze the FGF-2 expression pattern in hESCs. As this antibody recognizes both human and mouse FGF-2, it was also used to investigate the expression and release of FGF-2 in CF-1 feeder cells. It was revealed that undifferentiated hESCs (maintained in recombinant FGF-2–free medium for 24 hours), unlike mESCs , express 18-, 22-, 22.5-, and 24-kDa isoforms of FGF-2. The 34-kDa FGF-2 molecule was not detected (Fig. 2A). Indirect immunofluorescence with the same antibody demonstrated strong nuclear and nucleolar staining, with less-intensive cytoplasmic staining, which is consistent with the preponderance in hESCs of the high-molecular-mass isoforms of FGF-2, which are nuclear-localized (Fig. 2B). We also demonstrated that hESCs contain nuclear protein FIF (Fig. 2A, lower panel), which interacts with high-molecular-mass FGF-2 and exhibits antiapoptotic properties .
Figure 2. Expression of FGF-2 in hESCs and release of the low-molecular-mass isoform by undifferentiated hESCs. (A): Western blot analysis to show that undifferentiated, as well as differentiated, hESCs express several molecular isoforms of FGF-2 (upper panels) and synthesize FIF (lower panel). Human recombinant FGF-2 that represents 16.4-kDa band and FGF-2–producing leukemia K562 cells (18-kDa isoform) were used as positive controls for FGF-2; K562 and HL60 cells were used as positive controls for FIF. (B): As determined by indirect immunofluorescence, significant amounts of FGF-2 are localized in nuclei and nucleoli of undifferentiated hESCs as well as intracytoplasmically and at the plasma membrane and intercellular junctions. For Ig control, the primary antibody was omitted. (C): Western blot analysis to show that the 18-kDa isoform of FGF-2 synthesized by hESCs is released into culture medium (CM1, CM2). In contrast, mitomycin C–treated mouse embryonic fibroblasts (feeder cells) derived from strain CF-1 mice neither contain nor release FGF-2. The following samples were probed by Western blotting for the presence of FGF-2: (a) cell extracts from CF-1 fibroblasts and from hESCs CCTL14 (cells); (b) conditioned media (CM1 and CM2) harvested from two independent cultures of mouse fibroblasts and from two high-density cultures of undifferentiated hESCs of the lines CCTL12 and CCTL14 (respectively), subjected to enrichment of heparin-binding growth factors. Data are representative of at least three independent replicates. Abbreviations: CCTL, Center for Cell Therapy Line; EB, embryoid body; FGF, fibroblast growth factor; FIF, FGF-2–interacting factor; hESC, human embryonic stem cell; HMM, high molecular mass; Ig, immunoglobulin; LMM, low molecular mass; PI, propidium iodide–stained DNA.
We next analyzed the expression of endogenous FGF-2 in differentiating/differentiated hESCs (Fig. 2A). The pattern of expression of all four (18-, 22-, 22.5-, and 24-kDa) molecular isoforms of FGF-2 in D8 embryoid bodies, and in adherent cells resulting from the two-step differentiation protocol, is indistinguishable from that in undifferentiated cells. Cells produced by our standard, two-step protocol (D5 + 10) still contain detectable levels of Oct-4, a marker of undifferentiated hESCs; therefore, we prolonged the differentiation period to a total of 24 days, comprising 6 days in suspension and 18 days in adherent culture (D6 + 18), after which time immunoreactivity for Oct-4 is undetectable, and probed these cells for the presence of FGF-2. Even in such highly differentiated cells, there is no observable difference in the gross pattern of FGF-2 expression compared with undifferentiated hESCs.
The capacity of hESCs in culture to release the specific, 18-kDa isoform of FGF-2, mediating autocrine signaling, was examined. Culture medium that had been conditioned for 24 hours by hESCs, maintained on a feeder layer in recombinant FGF-2–free medium, was subjected to enrichment of heparin-binding growth factors. As revealed by Western blot analysis, exportable 18-kDa FGF-2 isoform was recovered from medium conditioned by hESCs on feeder cells but not from medium conditioned by feeder cells alone (Fig. 2C). Crucially, the concentration of 18-kDa FGF-2 in hESC-conditioned medium increased with increasing number of hESCs and reached 80 to 100 pg/ml in high-density cultures. Therefore, it is deduced that hESCs have the capacity, in terms of exported low-molecular-mass isoform of FGF-2, to activate FGFRs in an autocrine manner, and such activity may reach high levels, especially in high-density hESC cultures. It is notable also that feeder layers prepared from CF-1 strain MEFs express no potentially FGFR-stimulatory FGF-2 isoforms in this culture system.
Expression of FGFRs in hESCs
The biological effects of FGFs are mediated by their binding to four FGFR kinases, FGFR1 through FGFR4, each possessing different ligand-binding specificities and cell type–specific and tissue-specific expression. The ligand-binding specificities of FGFR1, FGFR2, and FGFR3 are primarily achieved by alternative splicing of the exons encoding the C-terminal region of extracellular, immunoglobulin-like domain III. Such alternative splicing event results in b and c isoforms of FGFR1, FGFR2, and FGFR3, whereas FGFR4 exists in one sole spliced form.
Quantitative real-time RT-PCR was used to analyze the expression of all four FGFRs in undifferentiated hESCs and in hESCs at various stages of differentiation. The primers were designed to detect human-specific FGF-2–binding, IIIc alternatives of FGFR1, FGFR2, and FGFR3 and the ligand-binding domain of FGFR4. Real-time RT-PCR data were normalized by comparison with expression of the ABL gene that was found to be eligible for quantification of FGFR gene transcripts in the different hESC samples and are summarized in Table 2. To avoid contamination with feeder cells, colonies of hESCs were carefully separated by mechanical scraping with a glass pipette. First, by analyzing four independent hESC lines, it was revealed that undifferentiated cells that are mechanically passaged and cultured at low densities express all four FGFRs in a very stable, relative pattern: in all the lines, FGFR1 was dominant with the other receptors showing lower expression; FGFR1 FGFR3 FGFR4 FGFR2. Notably, this relative expression pattern changed to FGFR1 FGFR4 FGFR3 FGFR2 in two selected hESC lines, CCTL12 and 14, after adaptation to enzymatic passaging, for the purpose of achieving higher cell-culture densities, and this pattern reflected upregulation of FGFR1 and downregulation of FGFR3 expression compared with low-density cultures. A similar trend in expression of FGFRs was observed also in cells that had initiated differentiation, whether by simple 8-day adherent culture without FGF-2 and feeder cells (D8 spontaneous differentiation) or by 8-day culture in aggregates (D8 embryoid bodies). Also for hESCs that had undergone more advanced differentiation by the two-step protocol (D5 + 10), the relative order of expression was FGFR1 FGFR4 FGFR3 FGFR2. Here, compared with undifferentiated hESCs, expression of all four FGFRs was dramatically elevated, with FGFR1 being increased about sixfold, FGFR2 about twofold, and FGFR3 and FGFR4 about sevenfold.
Table 2. Expression of FGFRs in undifferentiated and differentiated human embryonic stem cells
In summary, our data clearly indicate that hESCs are well equipped to accept and transmit FGF-2 signals via all four FGFRs, with FGFR1 potentially representing a dominant target, and that the relative levels of expression of FGFRs are tightly coupled to conditions that direct hESCs to differentiate.
Biological Effects of Exogenous and Endogenous FGF-2 on hESCs
Recombinant FGF-2 added exogenously to culture media may stimulate hESCs only via their cognate receptors, FGFRs. In contrast, FGF-2 produced endogenously by hESCs may function in two ways, depending on the presence or absence of the nuclear localization sequence: high-molecular-mass isoforms may be targeted to the nucleus and operate independently of cell-surface receptors, in an intracrine manner, whereas the low-molecular-mass isoforms may be exported from the cells and act via FGFRs as an autocrine or paracrine factor.
Stimulation of cells by FGF-2 leads to overall tyrosine phosphorylation of various proteins and to activation of extracellular signal-regulated kinases, ERK1/2 in particular, and so we used these two phenomena as criteria for the capacity of hESCs to respond to FGF-2. As evident from Figure 3, a short exposure of hESCs to 5 ng/ml recombinant FGF-2 induces a rapid and significant increase in levels of tyrosine phosphorylation of proteins (Fig. 3A), including ERK1/2 (Fig. 3B). This concentration of recombinant FGF-2 is routinely used for hESC culture and, as shown here, can be further increased by endogenously produced FGF-2. It is also of note that undifferentiated hESCs maintained in FGF-2–free medium possess an unexpectedly high basal level of phosphorylation of ERK1/2, which contrasts with the almost undetectable ERK1/2 phosphorylation typically demonstrated by mESCs D3 cultured under feeder-free conditions with serum- and LIF-supplemented medium (Fig. 3C).
Figure 3. FGF-2–induced phosphorylation of tyrosine residues and activation of ERK1/2 kinases in undifferentiated hESCs. (A): Treatment of hESCs (lines CCTL12 and CCTL14) with 5 ng/ml FGF-2 for 7 minutes results in elevated levels of tyrosine phosphorylation, as determined by Western blotting using antiphosphotyrosine antibody. Amidoblack staining of total proteins is shown in the lower panel, to document equal loading. (B): The phosphorylation of ERKs, as visualized by phospho-specific anti-ERK antibodies (pERK1/2), was strongly increased in hESCs (lines CCTL12 and CCTL14) by treatment with 5 ng/ml FGF-2 for 7 minutes. Membranes were reprobed with anti-ERK antibody to show the total amounts of ERKs (ERK1/2). (C): The basal level of ERK1/2 phosphorylation in serum-starved and FGF-2–starved hESCs (line CCTL12) is high relative to that in undifferentiated mESCs (feeder-independent line D3) maintained in serum- and LIF-supplemented medium. Data are representative of two independent replicates. Abbreviations: CCTL, Center for Cell Therapy Line; ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; hESC, human embryonic stem cell; LIF, leukemia inhibitory factor; mESC, mouse embryonic stem cell.
To determine whether the observed increased tyrosine phosphorylation and concomitant activation of growth-related kinases result in accelerated proliferation of hESCs, we used two complementary assays: measurement of the number of viable/metabolically active cells by the cleavage of tetrazolium salt WST-1 and analysis of the proportion of cells in S-phase by incorporation of BrdU into newly synthesized DNA. Although overall metabolic activity as determined by the cleavage of WST-1 was increased slightly in FGF-2–treated hESC line CCTL12 compared with untreated controls, such effect was not observed in CCTL14. We also determined that the number of cells in S-phase remained unchanged at any tested concentration of FGF-2 (Fig. 4A). From this data, metabolic activity of hESCs rather than progression of their cell cycle is influenced by exogenously added FGF-2. The absence or presence of recombinant FGF-2 also had no effect on the expression of Oct-4 (Fig. 4B). We therefore conclude that exogenously added FGF-2 at concentrations up to 20 ng/ml is dispensable for growth and self-renewal of hESCs maintained under standard conditions with feeder cells.
Figure 4. Effect of FGF-2 concentration on the proliferation and differentiation of hESCs. (A): Cells were maintained for 2, 3, and 5 days in culture medium supplemented with increasing concentrations of FGF-2, and growth was measured using the WST-1 proliferation reagent (upper panels) and by metabolic labeling of DNA with BrdU followed by ELISA (lower panels). Nontreated cultures (0 ng/ml FGF-2) served as a control. For both assays, growth is expressed as the absorbance quantified spectrophotometrically at indicated wavelengths, at the given time points. Values obtained by measurement of either metabolic activity or BrdU incorporation by feeder cells alone were subtracted. For hESC line CCTL12, metabolic activity was slightly augmented upon culture with FGF-2; however, treatment with FGF-2 had no effect on the proportion of BrdU-labeled, i.e., proliferating, hESCs. Data are representative of three independent experiments. Each value represents the mean of 24 replicates (wells) with the calculated standard deviation. (B): Western blot analysis illustrates that for hESCs cultured for 5 days without or with exogenous FGF-2 at indicated concentrations, the undifferentiated state was not affected, as documented by relatively constant levels of Oct-4 expression. Amidoblack staining of total proteins is shown in the lower panel, to document equal loading. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; CCTL, Center for Cell Therapy Line; ELISA, enzyme-linked immunosorbent assay; FGF, fibroblast growth factor; hESC, human embryonic stem cell.
Although no effect of FGF-2 on the proliferation of hESCs was found, there are still observable differences in hESC cultures depending on the concentration of exogenous FGF-2, which was manifest by the ability of cells to spread on the culture substratum. To quantify this effect, we used a simple strategy based on determining the change with time in size of hESC colonies in cultures that were exposed to various concentration of exogenous FGF-2 for 4, 5, and 6 days. Interestingly, the outgrowth of hESC colonies grown in medium containing 10 and 20 ng/ml FGF-2 increased at day 5 of culture and then was much less than the outgrowth in medium without or with only 5 ng/ml FGF-2 at day 6 of culture. This reduction in colony size at day 6 of culture cannot be attributed to reduced proliferation (see above) but instead reflected a morphological change to more compacted colonies, with well-defined borders and without the habitual, flattened cells on their periphery (Figs. 5A, 5B).
Figure 5. Effect of FGF-2 at various concentrations on the outgrowth of undifferentiated hESC colonies. (A): Cells were grown on feeder layers in 24-well dishes in medium supplemented with FGF-2 (at 5, 10, and 20 ng/ml) or without FGF-2. At 4, 5, and 6 days of culture, individual cultures were photographed and then sizes of colonies were determined from the photomicrographs. Representative hESC cultures are shown. (B): A minimum of 80 colonies was measured for each culture condition and time point, and average colony size was calculated; the increase in size (colony outgrowth) at days 5 and 6 was expressed as a percentage of the initial measurement at day 4, which is represented by a value of 0 on the Y-axis. At day 6 of culture, when the cells are routinely passaged, FGF-2 in the medium at concentrations of 10 and 20 ng/ml caused hESCs to grow in more compact/less-spread colonies compared with conditions without FGF-2 or with FGF-2 at only 5 ng/ml. Comparable results were obtained for both lines, CCTL12 and CCTL14, and therefore were port representative data for CCTL14. Abbreviations: CCTL, Center for Cell Therapy Line; FGF, fibroblast growth factor; hESC, human embryonic stem cell.
To test whether autocrine FGF signaling is essential for proliferation of undifferentiated hESCs, cultures of hESCs in FGF-2–free medium were treated with the pharmacological inhibitor of FGFR tyrosine kinases, SU5402, which specifically interacts with intra-cellular catalytic domain of FGFRs . After 2 days of continuous exposure to SU5402 at a concentration of 10–30 μM, cells in the centers of colonies acquired flattened morphology that resembled those spontaneously differentiating hESCs that occasionally occur in standard cultures, or the crater cells obtained during neuronal differentiation using HepG2-conditioned medium . Upon further culture totaling 5 days in the presence of SU5402, such morphology (Fig. 6A, upper panel), accompanied by loss of alkaline phosphatase activity (Fig. 6A, lower panel), finally developed in all hESC colonies . Furthermore, decreased phosphorylation of MEK1/2 and its substrate ERK1/2 were both observed in cells maintained in medium with inhibitor (Fig. 6B, – FGF-2). A similar effect was observed also in hESCs that were treated with 10 ng/ml recombinant FGF-2 (Fig. 6B, +FGF-2). Crucially, no signs of decreased cell viability were observed in SU5402-treated cultures (determined by cleavage of lamin B; Fig. 6C, upper left panel); instead, the presence of flattened cells was accompanied by downregulation of Oct-4 and upregulation of cyclin-dependent kinase inhibitor p27 (Fig. 6C, upper right panel), a common event that characterizes differentiation of cells of early embryonic origin and slower proliferation (Fig. 6C, lower panel). Moreover, differentiation process was manifested by upregulation of TROMA-1, a marker for primitive endoderm, and of nestin, a marker for developing neuroepithelium and for epithelial precursors in the embryonic pancreas (Fig. 6D, Western blots and immunofluorescence). Recombinant FGF-2 at concentrations up to 20 ng/ml did not reverse the phenotype produced by the noncompetitive inhibitor, SU5402 (results not shown), thus supporting our hypothesis of a critical role for autocrine FGF signaling in maintaining the pluripotent state of hESCs.
Figure 6. Inhibition of autocrine FGF signaling in undifferentiated hESCs. (A): hESCs were cultured on feeder layers in medium with 4 ng/ml FGF-2 (+FGF-2) or without FGF-2 (–FGF-2) and supplemented with the indicated concentration of SU5402. Cells were cultured for 5 days with daily change of medium, fixed, stained for alkaline phosphatase, and photographed at low (upper panel showing entire culture wells) and high (lower panel) magnifications. For cells cultured with 10 μM SU5402, differentiation starts at the centers of some colonies (data not shown); for cells cultured with 20 and/or 30 μM SU5402, there is more homogenous differentiation throughout all colonies (upper panel), which is characterized by a central area of flattened cells (asterisk in lower panel) surrounded by a ring of alkaline phosphatase–positive cells (arrow in lower panel); meanwhile, cells in SU5402-untreated control colonies showed homogeneous staining for alkaline phosphatase. Cells maintained under standard conditions, i.e., in hESC medium supplemented with 4 ng/ml FGF-2 alone (control 1), or with the diluent, dimethylsulfoxide (control 2) served as controls. (B): hESCs maintained without recombinant FGF-2 in the presence of 20 μM SU5402 for 4 days show decreased phosphorylation of MEK1/2 and ERK1/2. A similar decay of phosphorylated MEK1/2 and ERK1/2 was obtained in SU5402-treated hESCs after stimulation with 10 ng/ml FGF-2 for 7 minutes. (C): hESCs maintained in the presence of 20 μM SU5402 for 5 days were not apoptotic (as demonstrated by lack of cleavage of lamin B, upper left panel). In addition, cells cultured with inhibitor were negative for Oct-4 (upper right panel), although most of the hESC colonies still contained at their peripheries a surrounding ring of alkaline phosphatase–positive cells (A), upregulate p27 (upper right panel), and showed significantly retarded proliferation (p < .05), as documented by 5-bromo-2'-deoxy-uridine staining (lower panel). (D): SU5402-treated cells demonstrated strongly upregulated expression of the differentiation markers, TROMA-1 (approximately 55 kDa) and nestin (220–240 kDa), as documented by Western blotting (upper panels) and immunofluorescence (lower panels). Identical results were obtained for CCTL9 and CCTL14, and therefore we present data for CCTL14. Data are representatives of atleast three independent replicates. Abbreviations: CCTL, Center for Cell Therapy Line; ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; FL, feeder layer; hESC, human embryonic stem cell; MEK, mitogen-activated protein kinase kinase.
DISCUSSION
This research was supported in part by the Grant Agency of the Czech Republic (301/03/1122, 305/05/0434), Ministry of Education, Youth, and Sports(1M0021620803), Ministry of Health (MZ 00065269705), and Academy of Sciences of the Czech Republic (AV0Z50390512). We are very grateful to Dr. Elena Notarianni for valuable comments on the manuscript and for having revised the English of this manuscript.
REFERENCES
Niwa H, Burdon T, Chambers I et al. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998;12:2048–2060.
Matsuda T, Nakamura T, Nakao K et al. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J 1999;18:4261–4269.
Ying QL, Nichols J, Chambers I et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281–292.
Humphrey RK, Beattie GM, Lopez AD et al. Maintenance of pluripotency in human embryonic stem cells is STAT3 independent. STEM CELLS 2004;22:522–530.
Sato N, Meijer L. Skaltsounis L at al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004;10:55–63.
Annerén C, Cowan CA, Melton DA. The Src family of tyrosine kinases is important for embryonic stem cell self-renewal. J Biol Chem 2004;279:31590–31598.
Amit M, Shariki C, Margulets V et al. Feeder and serum-free culture of human embryonic stem cells. Biol Reprod 2004;70:837–845.
Mitalipova M, Calhoun J, Shin SJ et al. Human embryonic stem cell lines derived from discarded embryos. STEM CELLS 2003;21:521–526.
Cowan CA, Klimanskaya I, McMahon J et al. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 2004;350:1354–1356.
Sperger JM, Chen X, Draper JS et al. Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc Natl Acad Sci U S A 2003;100:13350–13355.
Sato N, Sanjuan IM, Heke M et al. Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev Biol 2003;260:404–413.
Ginis I, Luo Y, Miura T et al. Differences between human and mouse embryonic stem cells. Dev Biol 2004;269:360–380.
Schuldiner M, Yanuka O, Itskovitz-Eldor J et al. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2000;97:11307–11312.
Carpenter MK, Inokuma MS, Denham J et al. Enrichment of neurons and neural precursors from human embryonic stem cells. Exp Neurol 2001;172:383–397.
Zhang SC, Wernig M, Duncan ID et al. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 2001;19:1129–1133.
Reubinoff BE, Itsykson P, Turetsky T et al. Neural progenitors from human embryonic stem cells. Nat Biotechnol 2001;19:1134–1140.
Schulz TC, Palmarini GM, Noggle SA et al. Directed neuronal differentiation of human embryonic stem cells. BMC Neurosci 2003;4:27–39.
Park S, Lee KS, Lee YJ et al. Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors. Neurosci Lett 2004;359:99–103.
Ying Q, Stavridis M, Griffiths D et al. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 2003;21:183–186.
Gabert J, Beillard E, van der Velden VHJ et al. Standardization and quality control studies of "real-time" quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) of fusion gene transcripts for minimal residual disease detection in leukemia: a Europe Against Cancer Program. Leukemia 2003;17:2318–2357.
Jirmanova L, Pacholikova J, Krejci P et al. O-linked carbohydrates are required for FGF-2-mediated proliferation of mouse embryonic cells. Int J Dev Biol 1999;43:555–562.
Van den Berghe L, Laurell H, Huez I et al. FIF , a nuclear putatively antiapoptotic factor, interacts specifically with FGF-2. Mol Endocrinol 2000;14:1709–1724.
Mohammadi M, McMahon G, Sun L et al. Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science 1997;276:955–960.
Bryja V, Pachernik J, Soucek K et al. Increased apoptosis in differentiating p27-deficient mouse embryonic stem cells. Cell Mol Life Sci 2004;61:1384–1400.
Arnaud E, Touriol C, Boutonnet C et al. A new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-AUG start codon and behaves as a survival factor. Mol Cell Biol 1999;19:505–514.
Stachowiak MK, Fang XH, Myers JM et al. Integrative nuclear FGFR1 signaling (INFS) as a part of a universal "feed-forward-and-gate" signaling module that controls cell growth and differentiation. J Cell Biochem 2003;90:662–691.
Arese M, Chen Y, Florkiewicz RZ et al. Nuclear activities of basic fibroblast growth factor: potentiation of low-serum growth mediated by natural or chimeric nuclear localization signals. Mol Biol Cell 1999;10:1429–1444.
Sheng Z, Lewis JA, Chirico WJ. Nuclear and nucleolar localization of 18-kDa fibroblast growth factor-2 is controlled by C-terminal signals. J Biol Chem 2004;279:40153–40160.
Gaubert F, Escaffit F, Bertrand C et al. Expression of the high molecular weight fibroblast growth factor-2 isoform of 210 amino acids is associated with modulation of protein kinases C and and ERK activation. J Biol Chem 2001;276:1545–1554.
Burdon T, Stracey C, Chambers I et al. Suppression of SHP-2 and ERK signaling promotes self-renewal of mouse embryonic stem cells. Dev Biol 1999;210:30–43.
Amit M, Carpenter MK, Inokuma MS et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 2000;227:271–278.
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974.
Rosler ES, Fisk GJ, Ares X et al. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn 2004;229:259–274.
Debiais F, Lemonnier J, Hay E et al. Fibroblast growth factor-2 (FGF-2) increases N-cadherin expression through protein kinase C and Src-kinase pathways in human calvaria osteoblasts. J Cell Biochem 2001;81:68–81.
El-Hariry I, Pignatelli M, Lemoine NR. FGF-1 and FGF-2 regulate the expression of E-cadherin and catenins in pancreatic adenocarcinoma. Int J Cancer 2001;94:652–661.
Jang JH, Ku Y, Chung CP et al. Enhanced fibronectin-mediated cell adhesion of human osteoblast by fibroblast growth factor, FGF-2. Biotechnol Lett 2002;24:1659–1663.
Richard C, Roghani M, Moscatelli D. Fibroblast growth factor (FGF)-2 mediates cell attachment through interactions with two FGF receptor-1 isoforms and extracellular matrix or cell-associated heparan sulfate proteoglycans. Biochem Biophys Res Commun 2000;276:399–405.
Cha S, Hwang Y, Chae J et al. Inhibition of FGF signaling causes expansion of the endoderm in Xenopus. Biochem Biophys Res Commun 2004;315:100–106.
Esni F, Stoffers DA, Takeuchi T et al. Origin of exocrine pancreatic cells from nestin-positive precursors in developing mouse pancreas. Mech Dev 2004;121:15–25.
Bursdal CA, Flannery ML, Pedersen RA. FGF-2 alters the fate of mouse epiblast from ectoderm to mesoderm in vitro. Dev Biol 1998;198:231–244.
Dvorak P, Flechon JE, Thompson EM et al. Embryoglycans regulate FGF-2-mediated mesoderm induction in the rabbit embryo. J Cell Sci 1997;110:1–10.
Mathieu J, Griffin K, Herbomel P et al. Nodal and Fgf pathways interact through a positive regulatory loop and synergize to maintain mesodermal cell populations. Development 2004;131:629–641.(Petr Dvoraka,c,d, Dana Dv)