SEARCH

SEARCH BY CITATION

To The Editor

  1. Top of page
  2. To The Editor
  3. Acknowledgments
  4. Disclosure of Potential Conflicts of Interest
  5. References
  6. Supporting Information

In human embryonic stem cells (hESC), continuous stimulation with fibroblast growth factor 2 (FGF2) is required for self-renewal. Inhibition of FGF2 signaling results in rapid differentiation, further underscoring the importance of FGF system in maintenance of hESC undifferentiated state [1-3]. In addition to FGF2, human FGF family contains 17 members which presence in hESC remains unclear, with exception of FGF4 [4].

We analyzed the expression of entire FGF family (FGF1–10, 16–23) in a panel of well-characterized hESC lines (CCTL6, CCTL8–10, CCTL12–14) [5]. RT-PCR analysis [6] identified four Fgf genes expressed in hESC, that is, Fgf1, Fgf2, Fgf4, and Fgf19 (Fig. 1A; data not shown). Here, we focus on FGF19 expression. By Western immunoblotting (WB) or immunoprecipitation, FGF19 was found in hESC grown either as colonies on mitotically inactivated mouse embryonal fibroblasts, or feeder-free hESC cultures adapted to growth on Matrigel-coated dishes (Fig. 1B; Supporting Information Fig. S1A, S1B) (Supporting Information Material and Methods). By comparing the FGF19 WB signal to signal obtained from 0.16 to 2.5 ng of recombinant FGF19, we estimated the amount of FGF19 produced by CCTL9, CCTL12, and CCTL14 monolayer cultures between 0.3 and 1.2 ng (0.013–0.05 pmol) per 1 × 105 cells (Supporting Information Fig. S1C; data not shown). By immunocytochemistry (ICC), FGF19 showed both nuclear and membrane localization in hESC grown in colonies, in contrast to hESC grown in monolayer, that showed nuclear and cytoplasmic signal. Similar data were obtained with three commercially available antibodies (Fig. 1C, Supporting Information Fig. S2; data not shown). The amounts of Fgf19 transcripts correlated positively with undifferentiated hESC state (Fig. 1D).

image

Figure 1. Expression of FGF19 and its coreceptor βKLOTHO in human embryonic stem cell (hESC). (A): hESC lines CCTL6, 8–10, 12–14 were cultivated in colonies on mouse embryonal fibroblast (MEF) feeder layer as described in detail in Supporting Information Material and Methods. hESC colonies were manually harvested and analyzed for Fgf19 expression by RT-PCR as described earlier [6]. Note the uniformly high levels of Fgf19 transcript in all tested hESC lines. Human GAPDH expression serves as control for RT-PCR. (B): FGF19 protein expression in 40 µg of total protein harvested from three hESC lines, CCTL9, 12, and 14, grown as feeder-free monolayer. ACTIN serves as loading control. (C): CCTL14 cells were grown in colonies on MEF feeder, fixed, and analyzed for FGF19 or βKLOTHO expression by immunocytochemistry (ICC) as described in Supporting Information Material and Methods. Cell nuclei were visualized by DAPI. Mouse IgM isotype control as a primary antibody was used as negative control for ICC. Note the uniform FGF19 expression throughout the colony, with localization to cell nuclei and membranes (arrows; upper panels). In contrast, βKLOTHO signal (lower panels) is restricted to the centers of hESC colonies and localized both to the cell membranes or cytoplasm (Supporting Information Fig. S3). Scale bars = 200 µm mouse IgM, upper panels FGF19 and βKLOTHO; 20 µm lower panels. (D): CCTL14 cells were subjected to neural differentiation or differentiation into the embryoid bodies as described in Supporting Information Material and Methods, and the amounts of Fgf19 transcripts were determined by quantitative RT-PCR. Note the downregulation of Fgf19 expression upon differentiation compared to expression of known stem cell marker Nanog. Data represent average from two experiments (three biological repeats each), with indicated SEM (n = 6). (E): CCTL12 cells, maintained as MEF-dependent colonies, were treated with FGF19 for 30 minutes, harvested, and activating phosphorylation (p) of FRS2, AKT, and ERK MAP kinase was determined by Western immunoblotting. The total levels of given molecule serve as loading control. (F): CCTL12 cells were grown in colonies, treated with FGF2 (4 ng/ml) or FGF19 (100 ng/ml) for 30 minutes, and analyzed for phosphorylated ERK MAP kinase by ICC. Please note the differences in ERK activation between FGF19 (most signal in the center of the colony) and FGF2 (signal preferentially at the edges of the colony). FGF-naïve cells serve as control for ICC (Supporting Information Fig. S4). Scale bar = 250 µm. (G): βKLOTHO expression in CCTL12 cells grown in hypoxic conditions (5% O2) for 24 hours. Data represent two independent CCTL12 cultures in normoxic and hypoxic conditions. Abbreviations: FGF2, fibroblast growth factor 2; FRS2, fibroblast growth factor receptor substrate 2.

Download figure to PowerPoint

FGF19 requires βKLOTHO for FGF-receptor (FGFR) activation. βKLOTHO functions as coreceptor, compensating for poor FGF19's affinity toward “canonical” FGF coreceptors, the heparin sulfate proteoglycans [7-9]. βKLOTHO ICC performed on cells growing in colonies demonstrated both cytoplasmic and membranous signal in hESC. Intriguingly, βKLOTHO expression appeared restricted to hESC growing within the centers of the colonies (Fig. 1C, Supporting Information Fig. S3).

To test whether FGF19 activates FGFR signaling in hESC, we treated hESC with recombinant FGF19 and used WB to detect changes in activating phosphorylation of ERK MAP kinase and protein kinase B (AKT), the two intracellular mediators of FGFR signaling previously shown to be activated by FGF2 in hESC [3]. Figure 1E shows that addition of FGF19 increased AKT and ERK phosphorylation, clearly demonstrating the hESC responsiveness to the FGF19 stimulus. ICC with antibody recognizing phosphorylated ERK demonstrated that FGF19 activates ERK in the center of hESC colonies (Fig. 1F, Supporting Information Fig. S4), that is, within the βKLOTHO expression domain (Fig. 1C, Supporting Information Fig. S3).

Altogether, we show the concomitant expression of FGF19 and its coreceptor βKLOTHO in hESC, and the ability of exogenous FGF19 to activate FGFR signaling therein. One area of FGF19 function may lie in maintenance of hESC undifferentiated phenotype in a fashion similar to the FGF2. Alternatively, FGF19 may have more metabolic actions [10], possibly regulating glucose uptake in βKLOTHO-expressing cells growing in the middle of the colonies, which may suffer from limited glucose and oxygen supply. In support of this speculation, we found upregulation of βKLOTHO expression in hESC growing in hypoxic environment (Fig. 1G), and increased glucose metabolism in hESC exposed to FGF19 (Supporting Information Fig. S5). Although a further research is necessary to clarify the role of FGF19/βKLOTHO in hESC, our data open an attractive possibility for yet another FGF ligand, FGF19, being involved in regulation of hESC physiology.

Acknowledgments

  1. Top of page
  2. To The Editor
  3. Acknowledgments
  4. Disclosure of Potential Conflicts of Interest
  5. References
  6. Supporting Information

We thank to Yuh-Man Sun for providing RNA samples of neural differentiated hESC, and to Pertchoui B. Mekikian for excellent technical support. This study was supported by Ministry of Education, Youth, and Sports of the Czech Republic (MSM0021622430; KONTAKT LH12004), Grant Agency of Masaryk University (0071-2013; www.muni.cz), the Czech Science Foundation (P305/11/0752) (PK), Netherlands Organization for Scientific Research (VIDI Career Development Grant), and European Regional Development Fund (CEITEC) (LT).

Disclosure of Potential Conflicts of Interest

  1. Top of page
  2. To The Editor
  3. Acknowledgments
  4. Disclosure of Potential Conflicts of Interest
  5. References
  6. Supporting Information

The authors indicate no potential conflicts of interest..

References

  1. Top of page
  2. To The Editor
  3. Acknowledgments
  4. Disclosure of Potential Conflicts of Interest
  5. References
  6. Supporting Information

Supporting Information

  1. Top of page
  2. To The Editor
  3. Acknowledgments
  4. Disclosure of Potential Conflicts of Interest
  5. References
  6. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
stem1493-sup-0001-suppinfo1.doc51KSupporting Information 1
stem1493-sup-0002-suppinfo2.doc3322KSupporting Information 2
stem1493-sup-0003-suppinfo3.doc44KSupporting Information 3

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.