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EMBRYONIC STEM CELLS

Fibroblast Growth Factor 4 and Its Novel Splice Isoform Have Opposing Effects on the Maintenance of Human Embryonic Stem Cell Self-Renewal

^aDepartment of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel;
^bProChon Biotech Ltd., Nes Ziona, Israel

Key Words. Human embryonic stem cells • Self-renewal • Fibroblast growth factor 4 • Signaling • Erk

Correspondence: Correspondence: Nissim Benvenisty, M.D., Ph.D., Department of Genetics, The Life Sciences Institute, The Hebrew University, Jerusalem 91904, Israel. Telephone: 972-2-6586774; Fax: 972-2-6584972; e-mail: nissimb{at}mail.ls.huji.ac.il

Received on December 18, 2007; accepted for publication on December 27, 2007.

First published online in STEM CELLS EXPRESS  January 10, 2008.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Human embryonic stem cells (HESCs) are unique in their capacity to self-renew while remaining pluripotent. This undifferentiated state must be actively maintained by secreted factors. To identify autocrine factors that may support HESC growth, we have taken a global genetic approach. Microarray analysis identified fibroblast growth factor 4 (FGF4) as a prime candidate for autocrine signaling. Furthermore, the addition of recombinant FGF4 to HESCs supports their proliferation. We show that FGF4 is produced by multiple undifferentiated HESC lines, along with a novel fibroblast growth factor 4 splice isoform (FGF4si) that codes for the amino-terminal half of FGF4. Strikingly, although FGF4 supports the undifferentiated growth of HESCs, FGF4si effectively counters its effect. Furthermore, we show that FGF4si is an antagonist of FGF4, shutting down FGF4-induced Erk1/2 phosphorylation. Expression analysis shows that both isoforms are expressed in HESCs and early differentiated cells. However, whereas FGF4 ceases to be expressed in mature differentiated cells, FGF4si continues to be expressed after cell differentiation. Targeted knockdown of FGF4 using small interfering RNA increased differentiation of HESCs, demonstrating the importance of endogenous FGF4 signaling in maintaining their pluripotency. Taken together, these results suggest a growth-promoting role for FGF4 in HESCs and a putative feedback inhibition mechanism by a novel FGF4 splice isoform that may serve to promote differentiation at later stages of development.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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One of the key issues in human embryonic stem cell (HESC) research involves the unique capacity of HESCs to self-renew while retaining pluripotency [1–3]. Research into the molecular basis of self-renewal has been carried out extensively in mice. However, even though several core molecular features have been shown to be conserved between mouse and human embryonic stem (ES) cells, such as the importance of embryonic stem cell-specific OCT4 and NANOG expression [4–8], the molecular mechanisms governing the maintenance of the undifferentiated state seem to be markedly different. Predominantly, the two key ligands that have been shown to promote mouse ES cell self-renewal, leukemia inhibitory factor (LIF) [9, 10] and bone morphogenetic protein 4 (BMP4) [11], have been shown either to be ineffective or even to induce differentiation in HESCs, respectively [12–16]. Although the mechanisms governing HESC self-renewal remain largely unknown, recent reports have demonstrated that high doses of basic fibroblast growth factor (FGF2) can support feeder-free growth of HESCs [14, 16–18]. Other studies have suggested transforming growth factor β/activin/nodal signaling to be similarly effective [18–21]. In this study, we examined whether growth factors produced by HESCs could be involved in the regulation of their own growth.

Previously, we analyzed the expression of receptors and secreted factors in HESCs using DNA microarrays. We have shown broad expression of fibroblast growth factor receptors (FGFRs), as well as a variety of fibroblast growth factors (FGFs), in HESCs [22]. In this study, we expanded this analysis and showed that among the FGFs expressed in HESCs, FGF2 alone continued to be expressed even after prolonged in vitro differentiation. We propose that another FGF family member, FGF4, may play a more physiologically relevant role in the maintenance of HESC self-renewal.

FGF4 has been shown to be transcriptionally regulated by a heterodimer of SOX2 and OCT4 transcription factors [23, 24] and ceases to be expressed following induced differentiation. Initially, FGF4 was identified as an oncogene from stomach cancer and Kaposi sarcoma capable of transforming fibroblasts in vitro [25, 26]. The highly restricted expression pattern of FGF4 in the developing embryo [27], along with its roles in embryonic survival and patterning [28, 29], have established FGF4 as a key player in early development. Two major signaling pathways have recently been found to be crucial for HESC growth and maintenance. Using chemical inhibitors, it has been shown that the phosphoinositide 3-kinase (PI-3K)/Akt pathway is important for survival and growth of HESCs [30]. Similarly, MEK/Erk signaling was shown to be critical in HESCs, as its inhibition causes differentiation of HESC colonies [31, 32]. Given the robust potential of FGFs in general and FGF2 in particular in activating Erk1/2 [31–33], we set out to examine the role of FGF4 in this context.

Here, we show that FGF4 is secreted from undifferentiated HESCs, along with a newly identified fibroblast growth factor 4 splice isoform (FGF4si). Furthermore, whereas FGF4 promotes the undifferentiated growth of HESCs, FGF4si effectively counters its effect. Expression analysis shows that both isoforms are expressed in HESCs and early differentiated cells. Interestingly, whereas FGF4 ceases to be expressed in late-differentiated cells, FGF4si does not. Taken together, these results suggest an autocrine growth-promoting role for FGF4 in HESCs that is restrained by a novel FGF4 splice isoform that may serve to promote differentiation at later stages of development.

	MATERIALS AND METHODS

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Cell Culture
Human ES cells were cultured on mitomycin C-treated mouse embryonic fibroblast (MEF) feeder layer (obtained from 13.5-day embryos) in 85% KnockOut Dulbecco's modified Eagle's medium (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) supplemented with 15% KnockOut SR (a serum-free formulation) (Gibco-BRL), 1 mM glutamine, 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 1% nonessential amino acids stock (Gibco-BRL), penicillin (50 units/ml), streptomycin (50 µg/ml), 100x insulin-transferrin-selenium (ITS) in a 1:300 dilution (Gibco, Grand Island, NY, http://www.invitrogen.com), and 4 ng/ml FGF2 (Gibco). Cells were passaged using trypsin-EDTA (Biological Industries, Beit Haemek, Israel, http://www.bioind.com). To obtain feeder-free cultures, cells were plated on laminin (1 µg/cm²; Sigma-Aldrich) or gelatin (0.1%; Merck & Co., Whitehouse Station, NY, http://www.merck.com)-coated plates and grown with MEF-conditioned medium (MEF-CM). Differentiation in vitro into embryoid bodies (EBs) was performed by trypsinizing the cells to a near single-cell suspension, withdrawing FGF2 from the growth media, and allowing aggregation in Petri dishes.

Western Blot Analysis
HESCs were grown for one passage without feeders on gelatin-coated plates with MEF-conditioned medium. Semiconfluent plates were washed several times with phosphate-buffered saline (PBS), and medium was replaced with a serum replacement-free medium supplemented with 100x ITS at a 1:150 dilution but otherwise similar to the regular growth medium used. Following 24 hours of incubation, medium was collected and mixed with a single wash of PBS. The medium sample was separated on a heparin affinity column using an identical NaCl elution gradient. Resulting fractions were pooled according to the elution step as unbound, low, and high (0.2, 0.5, and 2 M NaCl, respectively). Pooled fractions were precipitated using 80% trichloroacetic acid overnight and resuspended and boiled in sample buffer. Samples were resolved on 12% SDS polyacrylamide gel and stained using a polyclonal anti-FGF4 antibody (Peprotech, Rocky Hill, NJ, http://www.peprotech.com). Specificity was validated using recombinant FGF4si and FGF4 proteins and by preincubation of the antibody with recombinant FGF4. For phosphorylation assays, cells were grown as described above, except that an additional medium change was performed 3 hours prior to induction and after a PBS wash. Phospho-Erk1/2 were detected using Sigma-Aldrich M8159 monoclonal antibody, and total Erk1/2 were detected using M5670 antibody (Sigma-Aldrich).

Growth Analysis
Cells were seeded in 96-well dishes coated with 0.1% gelatin at a density of 2 x 10⁴ cells per cm² (6,000 cells per well), and medium was changed daily. Recombinant human growth factors, midkine, pleiotrophin, FGF2, and FGF4 were obtained from Peprotech. Cell densities were determined by fixating the cells with 0.5% glutardialdehyde (Sigma-Aldrich) and staining with methylene blue (Sigma-Aldrich) dissolved in 0.1 M boric acid (pH 8.5). Color extraction was performed using 0.1 M hydrochloric acid, and the staining (which is proportional to cell number) was quantitated by measuring absorbance at 650 nM.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction Analysis
RNA was extracted using TRI-reagent for total RNA isolation according to the manufacturer's instructions (Sigma-Aldrich). cDNA was synthesized using random hexamer primers (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com). Amplification was performed on the cDNA using Takara Ex-Taq (Takara Bio, Shiga, Japan, http://www.takara-bio.com). All polymerase chain reactions (PCRs) for FGF4 and FGF4si were performed in the presence of 8% dimethyl sulfoxide. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to evaluate and compare quality of different cDNA samples. Primers for GAPDH (62°C annealing, 25 cycles) were as follows: forward, 5'-AGCCACATCGCTCAGACACC-3'; reverse, 5'-GTACTCAGCGGCCAGCATCG-3'. Primers for OCT4 (64°C annealing, 30 cycles) were as follows: forward, 5'-CTCACCCTGGGGGTTCTATT-3'; reverse, 5'-CTCCAGGTTGCCTCTCACTC-3'. Specific primers for FGF4 (60°C annealing, 40 cycles) were as follows: forward, 5'-TTCTTCGGGCCATGAGCAG-3'; reverse, 5'-CCGAAGAAAGTGCACCAAGG-3'. Specific primers for FGF4si (60°C annealing, 40 cycles) were as follows: forward, 5'-GACACCCTTCTTCACCGATG-3'; reverse, 5'-CTCCAGGTTGCCTCTCACTC-3'. Final products were examined by gel electrophoresis on 2% agarose ethidium bromide-stained gels. For sequencing of the complete FGF4 gene products, nested PCR was performed. Outer primers (60°C annealing, 40 cycles) were as follows: forward, 5'-TCCTCAGAGTCCCAGCTCCA-3'; reverse, 5'-CTCCAGGTTGCCTCTCACTC-3'. Inner primers (60°C annealing, 40 cycles) were as follows: forward, 5'-TCCATGCAGCCGGGGTAGA-3'; reverse, 5'-ACCAAGGTGACCCTCGCACT-3'.

DNA Microarray Analysis
Total RNA was extracted according to the manufacturer's protocol (Affymetrix, Santa Clara, CA, http://www.affymetrix.com). When extracting RNA from undifferentiated HESCs, the cells were grown for one passage on gelatin-coated plates with conditioned medium to avoid contamination by feeder cells. Hybridization to U133A/B DNA microarrays, washing, and scanning were performed according to the manufacturer's protocol, and expression patterns were compared between samples. Microarrays were analyzed using the Affymetrix MAS5 probe condensation algorithm. Results were normalized according to the trimmed mean (95%) of the entire array and multiplied by a factor of 100. Absent calls (nonspecific labeling) as judged by MAS5 were collectively given a value of 10.

Surface-Antigen Expression
Cells were dissociated to a single-cell suspension using trypsin-EDTA. Cells were then incubated for 1 hour with TRA-1–60 antibody (Kind gift of Prof. Peter Andrews and sc-21705 from Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com). As a secondary antibody, Cy3-conjugated rabbit anti-mouse IgM (dilution, 1:200; Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) was used. Fluorescence-activated cell sorting (FACS) analysis was performed using a FACSCalibur flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com).

Cloning, Expression, and Purification of Human FGF4si
The FGF4si open reading frame was amplified by PCR using the following primers and reaction conditions: forward, 5'-CATGCCATGGCATCGGGGCCAGGGACGGC-3'; reverse, 5'-ACGTG-GATCCGATCGGTGAAGAAGGGTGTCGCGGGTGTCCGCGT-G-3'. The PCR product was digested with NcoI and BamHI and cloned in a similarly restricted pET32b (pET32b hFGF4si). BL21 bacteria were transformed with the resulting (sequence-verified) construct. A single colony was picked and cultured overnight. The next day, bacteria were diluted 100 times and cultured to an optical density of 0.6 at 600 nm. Isopropyl β-D-thiogalactoside (0.1 mM) was then added for additional overnight incubation. The overnight culture pellet was resuspended in phosphate buffer and sonicated, and the cleared lysate was loaded on a Ni²⁺ column. Phosphate buffer containing increasing concentrations of imidazol was used for washing and eluting the purified protein. Typically, recombinant human fibroblast growth factor 4 splice isoform (rhFGF4si) eluted at 200 mM imidazol. Prior to use, rhFGF4si was dialyzed against PBS overnight at 4°C.

Small Interfering RNA Knockdown Experiments
HESCs were harvested to single-cell suspension and plated at a low density on gelatin-coated plates in the presence of MEF-CM (5% serum replacer). Transfection of small interfering RNA (siRNA) was carried out the following day using Oligofectamine reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) according to the manufacturer's instructions. Specifically, Oligofectamine reagent was used at a final concentration of 1:350. siRNA oligos were used at a final concentration of 45 nM. siRNA for FGF4 was obtained from Dharmacon, Inc. (Lafayette, CO, http://www.dharmacon.com) (ON-TARGETplus SMARTpool). siRNA for green fluorescent protein (GFP) (Integrated DNA Technologies, Coralville, IA, http://www.idtdna.com) was synthesized according to previously described sequences [34].

	RESULTS

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FGF4 Is a Putative Autocrine Self-Renewal Promoting Factor in HESCs
Previously, we have shown that several growth factors are expressed in HESCs in conjunction with their respective receptors [22]. Here, we expanded this analysis to examine the factors' temporal expression during spontaneous differentiation and maturation as embryoid bodies (EBs). The 18 growth factors shown in our previous study to be expressed in HESCs fall into eight different families. Of these, the fibroblast growth factor (FGF) family displayed the most dramatic downregulation during late differentiation (supplemental online Fig. 1). Notably, FGF4, a known stem cell marker, was among those downregulated in 30-day-old EBs (Fig. 1A), correlating with the reported downregulation of the embryonic transcription factor OCT4, which is among its activators. In addition to the FGF family, another family of growth factors, the secreted pleiotrophin (PTN)/midkine (MK) factors, and their respective receptors are highly expressed in HESCs (Fig. 1C, 1D). In contrast to the FGF family, both members of the PTN/MK heparin-binding factor family (PTN and MK) remained highly expressed throughout differentiation. This suggests that FGF signaling but not PTN/MK signaling would be important for HESC self-renewal.

View larger version (22K): [in this window] [in a new window]   Figure 1. FGF4 is a potential autocrine regulator of human embryonic stem cell (HESC) growth. (A, C): RNA expression profile of the members of the FGF family expressed in HESCs (A) and of the PTN/MK growth factors (C) during in vitro differentiation as embryoid bodies (EBs) for the indicated number of days. In addition, the expression levels of the corresponding receptors in undifferentiated cells are shown ([B] and [D], respectively). Analysis was performed using the Affymetrix U133 DNA microarray set, and each experiment was performed in triplicate. Expression levels were normalized and centered to an average value of 100. Error bars represent SE. (E): Relative cell numbers of treated HESC cultures. Cells were seeded in gelatin-coated 96-well plates (6 x 103 cells per well) and grown in the presence of the indicated factors at two doses for 3 days. Cells were methylene blue-stained, and absorbance (650 nm) was used as an indication of relative cell number. The values shown represent the relative cell number with respect to control wells and are the average of three independent experiments, each performed in triplicate. FGF4 (17 or 50 ng/ml) enhanced the number of cells in the cultures significantly with respect to controls (*, p < .05). Error bars represent SEM. Abbreviations: ES, embryonic stem; FGF, fibroblast growth factor; FGF2, basic fibroblast growth factor; FGFR, fibroblast growth factor receptor; MK, midkine; OD, optical density; PTN, pleiotrophin.

A Novel FGF4 Splice Isoform Is Coexpressed with FGF4 in HESCs
PCR amplification of the entire FGF4 cDNA revealed, in addition to the expected FGF4 PCR fragment, an additional low molecular weight band that was identified by sequence analysis to be a novel FGF4 splice isoform lacking the second exon (Fig. 2A). Isoform-specific reverse transcription (RT)-PCR showed that both FGF4 isoforms were coexpressed in all five HESC lines tested: HES7 and HES9 [35], I3 and I6 [36] (Fig. 2B), and H9 [3] (Fig. 2C). In addition, both isoforms were found to be expressed in mouse ES cells but not in MEF (Fig. 2B). Sequence analysis revealed that following the excision of the second exon, the third exon is decoded at a +1 frame, resulting in the generation of a premature stop codon. To verify the PCR results and test whether this putative splice isoform is translated, we performed Western blot analysis using a polyclonal FGF4 antibody. According to its nucleotide sequence, the putative splice isoform peptide is 118 amino acids (aa) long, with an expected molecular weight (MW) of 12 kDa, compared with the full-length FGF4 protein containing 206 aa (MW, 22 kDa). Examination of the heparin-binding fraction of HESC-conditioned medium revealed two specific bands at 25 and 14 kDa, corresponding to FGF4 and FGF4si, respectively (Fig. 2A). These results demonstrate that both FGF4 and FGF4si are translated and actively secreted by HESCs.

View larger version (39K): [in this window] [in a new window]   Figure 2. A novel splice isoform of FGF4 is expressed and secreted by human embryonic stem cells (HESCs). (A): Reverse transcription (RT)-polymerase chain reaction (PCR) amplification of the entire FGF4 transcript using primers specific for the RNA untranslated regions revealed two distinct products in HESCs. The bands were sequenced and found to correspond to the expected full-length transcript (upper band) and to a novel splice isoform (FGF4si) lacking the second exon (lower band). The cartoon depicts both splice events and illustrates the introduction of a premature stop codon by frameshift, caused by the loss of the second exon in FGF4si (stop codons indicated by flags). HESC-conditioned medium was analyzed by immunoblotting using a polyclonal antibody directed against recombinant human FGF4. Prior to analysis, the sample was run on a heparin affinity column. Shown is the high-salt elution fraction (2 M NaCl). Recombinant human FGF4 (182 amino acids; 19.7 kDa; 2 and 10 ng) served as control. Arrowheads on the left indicate specific labeling. (B): Expression of FGF4 and FGF4si was examined in several HESC lines (I3, I6, HES7, and HES9), in a line of mouse ES cells (E14Tg2a) and MEF. (C): The expression pattern of FGF4 and FGF4si during differentiation was examined by RT-PCR in H9 HESCs. Three stages of differentiation were thus tested: undifferentiated HESCs (ES) and 2- and 30-day-old EBs (EB2 and EB30, respectively). RT-PCR was performed using specific primers for each transcript. The temporal expression of the FGF4 isoforms was compared with the expression pattern of OCT4, a known regulator of FGF4 transcription and a marker of undifferentiated cells. GAPDH was used as a positive control. Abbreviations: ES, embryonic stem; FGF, fibroblast growth factor; FGF4si, fibroblast growth factor 4 splice isoform; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; kb, kilobases; MEF, mouse embryonic fibroblast; mES, mouse embryonic stem; rhFGF4, recombinant human fibroblast growth factor 4.

FGF4 Helps Maintain the Pluripotent State of HESCs and Is Antagonized by FGF4si
To examine the possible effect of FGF4si on the growth of HESCs, we first produced recombinant FGF4si. Parallel experiments were set, in which identical numbers of cells were plated onto laminin-coated plates. The cultures were allowed to grow for 5 days with daily replacement of either basal medium, MEF-CM, media containing either FGF4 with or without FGF4si, or FGF2. HESCs treated with either 50 or 100 ng/ml FGF4 or 100 ng/ml FGF2 grew as large and defined colonies, characteristic of undifferentiated cells (Fig. 3). The addition of 100 ng/ml FGF4si to FGF4-treated cells caused a reduction in colony size, with more differentiated cell morphologies, very similar to the morphology of the untreated control (Fig. 3). To further investigate the nature of the observed effect, the fractions of undifferentiated cells in the different cultures were assessed by FACS using Tra-1–60 antibody. The observed percentage of undifferentiated cells was then multiplied by the total cell count for each culture condition to yield the total number of undifferentiated cells per culture. Dramatic increases in undifferentiated cell numbers in FGF2- and FGF4-treated cultures were observed relative to basal medium control (3.2-fold increase for 100 ng/ml FGF2 and 2.7- and 2-fold increases for 50 and 100 ng/ml FGF4, respectively) (Fig. 4A). The effect of FGF4 was abolished when FGF4si was added to the medium. Thus, FGF4si caused a significant reduction in the number of undifferentiated HESCs in the cultures treated with FGF4 (p < .005) (Fig. 4A). Taken together, these results clearly demonstrate a significant effect of FGF4 in the undifferentiated growth potential of HESCs, an effect countered by FGF4si.

View larger version (157K): [in this window] [in a new window]   Figure 3. FGF4si blocks the self-renewal activity of FGF4. Cells (2 x 104 cells per cm2) were seeded on laminin-coated plates. Cultures were grown in basal medium with the addition of the indicated growth factors. Medium was replaced daily, and representative fields were photographed after 4 days. Abbreviations: FGF2, basic fibroblast growth factor; FGF, fibroblast growth factor; FGF4si, fibroblast growth factor 4 splice isoform; SI, FGF4si 100ng/ml.

View larger version (18K): [in this window] [in a new window]   Figure 4. FGF4 causes an increase in the number of undifferentiated cells in culture and is antagonized by FGF4si. (A): Cells (2 x 104 cells per cm2) were seeded on laminin-coated plates. Cultures were grown in basal medium with the addition of the indicated growth factors, and medium was replaced daily. After 5 days, cells were analyzed by fluorescence-activated cell sorting using TRA-1–60 antibody. Shown are the results of multiplication of the percentage of TRA-1–60-positive cells by the total cell count. FGF4si added together with FGF4 effectively abolished the growth enhancement conferred by FGF4 alone (*, p < .005). Experiments were performed in triplicate. Error bars represent SEM. (B): Percentages of TRA-1–60– cells of successfully transfected HESCs, as judged by GFP reporter knockdown. GFP siRNA alone served as control (*, p < .05). Abbreviations: FGF2, basic fibroblast growth factor; FGF, fibroblast growth factor; FGF4si, fibroblast growth factor 4 splice isoform; GFP, green fluorescent protein; RNAi, RNA interference; SI, fibroblast growth factor 4 splice isoform 100ng/ml.

FGF4si Inhibits FGF4-Induced Erk1/2 Phosphorylation in HESCs
To obtain insight into the underlying mechanism of FGF4si action, we set out to investigate its effect on FGF4-induced signal transduction. It was previously shown that FGF stimulation causes robust phosphorylation of the mitogen-activated protein kinases Erk1 and Erk2 [31, 37]. To test the activated levels of Erk1/2, HESCs were starved and then stimulated with FGF2 or FGF4 with or without FGF4si. Two cell lines were thus tested, H9 (Fig. 5A) and HES9 (Fig. 5B). Both FGFs strongly induced Erk1/2 phosphorylation, as shown by Western blot analysis. The stimulation of Erk1/2 phosphorylation by FGF4 was significantly reduced by FGF4si in both cell lines. However, the effect on Erk1 was more prominent, as its phosphorylation was virtually eliminated in the presence of FGF4si. The specificity of FGF4si antagonistic action was demonstrated by the relatively weak inhibition of FGF2-stimulated MAPK.

View larger version (39K): [in this window] [in a new window]   Figure 5. FGF4si antagonizes FGF4-induced Erk1/2 activation. H9 (A): and HES9 (B): HESCs were treated with either FGF2 or FGF4 (50 ng/ml each) with and without FGF4si (100ng/ml). Cells were incubated for the indicated amounts of time, and phosphorylation levels of Erk1/2 were determined by Western blot using phospho-specific anti-Erk1/2 antibody. Reprobing with anti-total Erk1/2 antibody served as loading control. Abbreviations: FGF2, basic fibroblast growth factor; FGF, fibroblast growth factor; FGF4si, fibroblast growth factor 4 splice isoform; min, minutes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

In contrast to the majority of studies hitherto published, attempting to characterize growth factors important for self-renewal in HESCs, we decided to focus on the identification of endogenous growth factors that could affect HESC fate. Thus, we performed DNA microarray analysis of HESC samples, along with samples of in vitro-differentiated embryoid bodies of successive stages (2-, 10-, and 30-day EBs) [22]. This analysis identified 18 growth factors where both ligand and receptor are expressed in undifferentiated HESCs. Of these, seven showed significant downregulation in their expression upon differentiation. Marked among the downregulated genes are those of the FGF family in which the expression level of three of four expressed members are dramatically reduced upon late differentiation. Given the broad expression of FGFRs in HESCs [22, 31], it seems plausible that FGFs could function as autocrine signals in HESC cultures.

Using a short-term proliferation assay with low-density cultures seeded as single cells, we identified FGF4 as a factor capable of enhancing the growth potential of HESCs under these conditions. Subsequent cloning of the gene revealed a novel alternative splice variant, termed FGF4si. Analysis of additional HESC lines, as well as a mouse ES cell line, showed FGF4si to be expressed in all of them but not in MEF. FGF4si lacks the second exon, as verified by sequence analysis of the RT-PCR products; the absence of this exon results in a ribosomal frameshift leading to premature translation termination. Thus, FGF4si shares with FGF4 the first 113 N-terminal amino acids yet lacks a substantial portion of the protein's C terminus, including several predicted heparin- and receptor-binding sites [42]. Western blot analysis using an FGF4 polyclonal antiserum identified both isoforms of FGF4 in the heparin-bound fraction of HESC-conditioned medium. Thus, we demonstrated that both proteins are actively secreted by HESCs and bind heparin. Indeed at least one heparin-binding site remains intact in this splice isoform [42]. Interestingly, we show that the expression pattern of FGF4si differs from that of FGF4, in that it remains expressed even following late differentiation in vitro.

The continued expression of FGF4si in the absence of FGF4 prompted us to investigate the possible role of serine/arginine-rich (SR) proteins, which are known to modulate alternative splicing [43]. Thus, we examined in HESCs and EBs the expression of four SR proteins with known recognition motifs, namely SC35, SRp40, SRp55, and SF2/ASF. Comparison of DNA microarray data shows that SC35 and SF2/ASF genes are overexpressed in HESCs relative to EBs (Fig. 6A). Subsequent prediction of recognition motifs for the SR proteins [44] identified numerous such sites for SC35 and SF2/ASF along the second exon and flanking introns. However, recognition sites for SRp55 and SRp40 that were expressed at similar levels in HESCs and EBs were less abundant, and only one site displayed the same level of confidence (Fig. 6B). Taken together, these results suggest that a general shift in SR protein expression during differentiation of HESCs may specifically cause the transition from coexpression of both the FGF4 isoforms to the continued expression of FGF4si alone.

View larger version (15K): [in this window] [in a new window]   Figure 6. Changes in SR proteins levels may explain the shift in expression of FGF4 isoforms during differentiation. (A): RNA expression levels of four SR proteins were examined using DNA microarrays. Expression values were normalized to a baseline of averaged normal human adult tissues [46]. (B): Recognition motifs of SR proteins were predicted using the ESEFinder 3.0 online resource [44, 47]. Shown are recognition motifs identified above a threshold of 4, along exon 2 and 200-base pair flanking introns. Abbreviations: FGF, fibroblast growth factor; HESC, human embryonic stem cell; SR, serine/arginine-rich.

Following the results using recombinant proteins, we decided to evaluate the role of FGF4 as a potential autocrine signal. Using an siRNA-mediated knockdown approach, we could show a significant increase in differentiation in cells receiving siRNA for FGF4. This effect was, however, modest and may be an underestimation of the true importance of the FGF4 signal. This may be due to redundancy of FGF signals in HESCs, compensation of FGF4 by untransfected cells, or the knockdown of both FGF4si and FGF4 resulting from sequence identity that precluded us from using isoform-specific siRNA.

In light of the biological effects of FGF4 and FGF4si, we examined their effect on the downstream MAPKs Erk1/2. Previous studies have shown robust phosphorylation of both Erks in response to FGF2 stimulation in HESCs [31, 32]. Interestingly, whereas the activation of Erk signaling has been implicated using chemical inhibitors as necessary for HESC self-renewal [32], studies in the mouse have shown that Erk activity inhibits self-renewal and promotes differentiation [37, 45]. Similar to FGF2 stimulation, we could see robust activation of Erk1/2 by FGF4. Most importantly, however, we could clearly show an almost complete elimination of FGF4-stimulated Erk1/2 phosphorylation with the addition of FGF4si. Moreover, we could show that this antagonistic effect is preferentially directed against FGF4, as FGF2 stimulation of Erk1/2 was less affected by FGF4si. An additional downstream effector of receptor tyrosine kinases is the PI-3K/AKT pathway, which has been shown to promote survival of HESCs in response to neurotrophins [30]. However, Western blot analysis with anti-phospho-AKT antibodies showed no effect of either FGF tested on AKT stimulation (data not shown).

Taken together, these results indicate an important role for FGF4 signaling in HESC growth and differentiation. Furthermore, our study describes a novel endogenous control mechanism that regulates FGFR signaling by inhibition of FGF4-mediated Erk1/2 activation. We suggest a model in which FGF4, expressed in undifferentiated cells in conjunction with FGF4si, is prevalent and actively helps maintain HESCs in their undifferentiated state. During differentiation, with the decline of FGF4 expression and the continued expression of FGF4si, this balance shifts, possibly due to global changes in SR protein expression, shutting down remaining FGF4 signaling and effectively promoting differentiation.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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N.B. has served as a member of the Board for Stem Cell Technologies. A.Y. owns stock in, has served as an officer or member of the Board for, and has a financial interest in Prochon, Ltd. I.C. owns stock in and has a financial interest in Prochon, Ltd. E.R. has a financial interest in Prochon, Ltd.

	ACKNOWLEDGMENTS

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We thank Prof. Peter Andrews for kindly providing us with the TRA-1–60 antibody and Dr. Claudio Basilico for the use of the FGF4 cDNA plasmid. We also thank Dr. Mario Lebendiker for his help with protein purification. This research was partially supported by funds from the Bereshit Consortium, the Israeli Ministry of Trade and Industry, and the European Community (ESTOOLS, Grant number 018739).

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 

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