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Keywords:

  • L1 cell adhesion molecule;
  • Cell-surface marker;
  • Human embryonic stem cells;
  • Monoclonal antibody;
  • Fibroblast growth factor receptor 1

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Despite the recent identification of surface markers of undifferentiated human embryonic stem cells (hESCs), the crucial cell-surface molecules that regulate the self-renewal capacity of hESCs remain largely undefined. Here, we generated monoclonal antibodies (MAbs) that specifically bind to undifferentiated hESCs but not to mouse embryonic stem cells. Among these antibodies, we selected a novel MAb, 4-63, and identified its target antigen as the L1 cell adhesion molecule (L1CAM) isoform 2. Notably, L1CAM expressed in hESCs lacked the neuron-specific YEGHH and RSLE peptides encoded by exons 2 and 27, respectively. L1CAM colocalized with hESC-specific cell-surface markers, and its expression was markedly downregulated on differentiation. Stable L1CAM depletion markedly decreased hESC proliferation, whereas L1CAM overexpression increased proliferation. In addition, the expression of octamer-binding transcription factor 4, Nanog, sex-determining region Y–box 2, and stage-specific embryonic antigen (SSEA)-3 was markedly downregulated, whereas lineage-specific markers and SSEA-1 were upregulated in L1CAM-depleted hESCs. Interestingly, the actions of L1CAM in regulating the proliferation and differentiation of hESCs were exerted predominantly through the fibroblast growth factor receptor 1 signaling pathway. Taken together, our results suggest that L1CAM is a novel cell-surface molecule that plays an important role in the maintenance of self-renewal and pluripotency in hESCs. Stem Cells 2011;29:2094–2099.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Human embryonic stem cells (hESCs), which are derived from the inner cell mass of blastocysts, have the ability to self-renew and give rise to differentiated progeny of all three germ layers while maintaining pluripotency [1–3]. Therefore, hESCs can serve as an unlimited source of cells for regenerative medicine, which aims to replace or restore tissue damaged by disease or injury through the transplantation of differentiated and functional hESCs [4–6]. For these purposes, pure populations of selected cell types will be a likely prerequisite. However, recent studies have revealed that hESCs both within the pluripotent stem cell compartment and within cultures that are termed hESC lines are morphologically and phenotypically heterogeneous [7]. It is therefore critical to develop specific and efficient tools to detect and isolate pluripotent cells.

The systematic identification and characterization of the cell-surface molecules of hESCs provide simple tools to identify and analyze specific cell populations and has practical applications in the purification of cells for cell transplantation therapy [8]. Moreover, the identification of novel hESC-specific cell-surface molecules is essential for understanding the relationship between the phenotype of hESCs and their pluripotency and the mechanisms involved in hESC differentiation and self-renewal. In addition, the development of monoclonal antibodies (MAbs) against these surface molecules will facilitate efficient purification of pluripotent cells from mixed populations of cultured hESCs.

L1 cell adhesion molecule (L1CAM) was originally described as a neural cell adhesion molecule and has been shown to initiate a variety of dynamic motile processes, including cerebellar cell migration and neurite extension in the central nervous system [9–11]. L1CAM is also expressed in other cell types [12–15]. However, its expression and role in hESCs have not been previously studied.

In the present study, we generated hybridomas by immunizing mice with hESC clumps and selected MAbs that bind to hESCs but not to mouse embryonic stem cells (mESCs). Among these, we selected and characterized a new MAb, 4-63 (IgG1, κ), that binds specifically to human L1CAM. Our results demonstrated that L1CAM is a novel surface molecule for undifferentiated hESCs and plays an important role in the maintenance of self-renewal and pluripotency.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Identification and Characterization of L1CAM in hESCs as a Novel Surface Molecule

To discover novel surface molecules of hESCs, we generated MAbs that bind to cell-surface proteins on hESCs. Among these, we selected a MAb, 4-63, and characterized its activity. Four-63 bound to the surface of three hESC lines but did not bind to mouse embryo fibroblasts (MEFs) or the mESC line J1 (Fig. 1A, 1B). Consistently, immunofluorescence analysis showed that the 4-63 antigen colocalized with TRA-1-60 in undifferentiated hESCs (Fig. 1C). To identify the target antigen of 4-63, mass spectrometry of immunoprecipitates using 4-63 was performed. Peptide sequencing revealed that the antigen was the L1CAM isoform two precursor, a non-neuronal isoform lacking the neuron-specific exons 2 (YEGHH) and 27 (RSLE; Supporting Information Fig. 1A, 1B), implying that its activities in hESCs may be different from those in neural cells. The specificity of 4-63 for L1CAM was also confirmed using 5G3, a commercial antibody against L1CAM (Fig. 1D).

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Figure 1. Identification and characterization of L1CAM in human embryonic stem cells (hESCs) as a novel surface marker. Undifferentiated hESCs (A), mouse embryonic stem cells (B), and MEFs (B) were stained with anti-SSEA-1, anti-SSEA-3, anti-SSEA-4, 4-63, or 5G3 antibodies. Uncolored populations indicate antibody staining, whereas red-colored populations indicate control staining in each panel. (C): Confocal microscopy demonstrates membranous staining with 4-63 and the anti-TRA-1-60 antibody in hESCs. (D): Extracts of unbiotinylated hESCs (H9, HSF6, and H1) were immunoprecipitated with 4-63, 5G3, or no antibody (bead), followed by Western blot analysis using biotinylated 4-63 and streptavidin-HRP. Four-63 and 5G3 in the non-IP section indicate that either 4-63 or 5G3 antibody was loaded in each lane on the same gel. Four-63 reacted with identical protein bands in the cell lysates immunoprecipitated with 5G3, demonstrating that its target antigen is L1CAM. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; FL1-anti-m IgG FITC, first fluorescence detector 1–anti-mouse immunoglobulin fluorescein isothiocyanate; IP, immunoprecipitation; L1CAM, L1 cell adhesion molecule; MEF, mouse embryo fibroblast; SSEA, stage-specific embryonic antigen.

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Next to determine whether L1CAM could be used as a surface marker to identify, isolate, and characterize undifferentiated hESCs and whether or not L1CAM expression is downregulated on differentiation, L1CAM expression was examined in undifferentiated hESCs and hESC-derived embryoid bodies (EBs). Surface expression as well as mRNA level of L1CAM was markedly downregulated in EBs, similar to the stage-specific embryonic antigen (SSEA)-3, SSEA-4, and hESC markers (Supporting Information Fig. 2A, 2B). Likewise, L1CAM expression was detected in mESCs and also downregulated in mouse EBs (Supporting Information Fig. 2C). These results suggest that L1CAM could be a novel surface marker of undifferentiated hESCs and mESCs.

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Figure 2. L1CAM contributes to the proliferation of human embryonic stem cells. (A): Relative levels of L1CAM expression in L1CAM-overexpressing or -depleted H9 cell lines were analyzed by quantitative RT-PCR (qRT-PCR) (left top), Western blotting with β-actin as a loading control (left bottom), and flow cytometry (right panel). CTL indicates cells transfected with control short hairpin RNA. (B): Each stable cell line was labeled with BrdU for 45 minutes. Cells were incubated with FITC-labeled anti-BrdU antibody and PI and then analyzed with flow cytometry. The relative percentages of cells are shown. Three independent experiments were performed in duplicate. Data are the means ± SD (**p < .01). (C): Analysis of cell cycle distribution in each stable cell line as assessed with flow cytometry. The relative percentages of cells in G0-G1, G2-M, and S phases are shown. This experiment was performed twice in duplicate. (D): The expression of cyclin A in H9-derived stable cell lines was examined with Western blot analysis. Abbreviations: FITC, fluorescein isothiocyanate; BrdU, bromodeoxyuridine; CTL, control; L1CAM, L1 cell adhesion molecule.

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L1CAM is Essential for Self-Renewal and Pluripotency

To understand the role of L1CAM in hESCs, L1CAM was stably depleted or overexpressed (Fig. 2A), and its effect on the proliferation of hESCs was analyzed. Basal levels of bromodeoxyuridine (BrdU)-positive cells in control short hairpin RNA– or mock-transfected hESC lines accounted for ∼70% of the total cell population, similar to the levels in normal H9 cells (Fig. 2B). Remarkably, stable depletion of L1CAM significantly decreased the BrdU-positive cell population, whereas overexpression of L1CAM increased. In addition, cell cycle analysis showed that L1CAM depletion induced accumulation of the cells in G1 phase and subsequently led to reduced S-phase entry, whereas L1CAM-overexpressing hESCs displayed an increase in the number of S-phase cells (Fig. 2C). Consistently, cyclin A expression was markedly reduced in L1CAM-depleted hESCs, whereas it was significantly increased in the L1CAM-overexpressing hESCs (Fig. 2D).

Next, we examined whether L1CAM expression is associated with maintenance of an undifferentiated state in hESCs. Expression of octamer-binding transcription factor 4 (Oct4), Nanog, sex-determining region Y–box 2 (Sox2), forkhead box protein D3 (FoxD3), and SSEA-3 was markedly downregulated in L1CAM-depleted hESCs, whereas it was upregulated in L1CAM-overexpressing hESCs (Fig. 3A). In addition, surface expression of SSEA-1 was upregulated in the L1CAM-depleted hESCs, whereas that of SSEA-3 was downregulated (Fig. 3B). Moreover, L1CAM-depleted hESCs displayed a flattened morphology with defined separate cell to colony border and increased expression of lineage markers (Fig. 3C, 3D). Furthermore, L1CAM depletion significantly reduced alkaline phosphatase staining (Supporting Information Fig. 3A, 3B), suggesting that spontaneous differentiation had occurred in the L1CAM-depleted hESCs.

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Figure 3. L1CAM is essential for the maintenance of an undifferentiated state in human embryonic stem cells (hESCs). (A): Total mRNA was isolated from H9-derived stable cell lines, and expression of Oct4, Nanog, Sox2, and FoxD3 was compared using qRT-PCR. Gene expression was normalized to that of GAPDH and presented as the fold change over control cells, which was set at 1. Three independent experiments were performed in duplicate. Data are the means ± SD (**p < .01). (B): Histogram showing expression levels of hESC surface markers in the H9-derived stable cell lines. (C): Morphology of the H9-derived stable cell lines. L1CAM-depleted hESCs display a flattened morphology with defined separate cell to cell borders (arrow and circle region), in contrast to control short hairpin RNA–transfected hESCs, which exhibit the typical densely packed colony morphology of undifferentiated hESCs with clearly defined colony borders. However, no significant difference in morphology was detected between L1CAM-overexpressing cells and mock-transfected cells. (D): Comparison of expression levels of three germ layer (ectoderm, mesoderm, and endoderm) markers between L1CAM-depleted and control hESCs. L1CAM-depleted cells exhibited increased expression of the lineage markers compared to the control cells. Three independent experiments were performed in duplicate. Data are the means ± SD (**p < .01). Abbreviations: AFP, α-fetoprotein; CTL, control; FITC, fluorescein isothiocyanate; FoxD3, forkhead box protein D3; FOXA2, forkhead box protein A2; GATA6 and A4, GATA-binding factor 6 and 4; GFAP, glial fibrillary acidic protein; LEF1, lymphoid enhancer-binding factor 1; L1CAM, L1 cell adhesion molecule; Oct4, octamer-binding transcription factor 4; Sox2, sex-determining region Y–box 2; SOX17, sex-determining region Y–box 17; SSEA, stage-specific embryonic antigen.

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To investigate the role of L1CAM in the pluripotency of hESCs, EBs were derived from each cell line. Most of the tested lineage markers were not significantly increased in L1CAM-depleted cells, whereas they were upregulated in control cells, indicating that pluripotency is not maintained in the L1CAM-depleted cells (Fig. 4A). Furthermore, teratoma analysis showed that the L1CAM-depleted cells produced fewer teratomas with longer latencies compared with control cells (Fig. 4B), while the teratomas from both cell lines exhibited all three germ layers (data not shown). However, gene expression analysis of the teratoma samples revealed a significant decrease in the expression of some ectoderm and mesoderm markers (Fig. 4C). Thus, L1CAM is required for the maintenance of self-renewal and pluripotency in hESCs.

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Figure 4. Expression levels of lineage markers in EBs and teratomas. (A): Normal H9, control short hairpin RNA–transfected, or shL1-transfected cells were differentiated into EBs for 3 and 6 days, and the relative expression levels of ectoderm, mesoderm, and endoderm markers were assessed with qRT-PCR. Three independent experiments were performed in duplicate. Data are the means ± SD. **p < .01 versus CTL EBs on 0 day; ##p < .01 versus untransfected EBs on 0 day. (B): Table summarizing the efficiency, latency, and histological analysis of teratomas differentiated from H9-derived stable cell lines. (C): The relative expression levels of ectoderm, mesoderm, and endoderm markers in teratomas were assessed with qRT-PCR. Transcript levels are presented as fold changes over control, which was set at 1. Three independent experiments were performed in triplicate. Data are the means ± SD (**p < .01, *p < .05). Abbreviations: CTL, control; EB, embryoid body; FOXA2, forkhead box protein A2; GATA4, GATA-binding factor 4; GFAP, glial fibrillary acidic protein; LEF1, lymphoid enhancer-binding factor 1; Pax6, paired box gene 6; SOX17, sex-determining region Y–box 17.

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L1CAM Activates Fibroblast Growth Factor Receptor 1 Signaling that Supports Self-Renewal

Some reports have shown that the neuronal type of L1CAM mediates growth factor signaling events via interaction of its extracellular domain with a CAM homology domain in the fibroblast growth factor (FGF) family of receptors, thereby potentiating receptor phosphorylation and neuronal migration [16–18]. As fibroblast growth factor receptor (FGFR) signaling plays a key role in the maintenance of an undifferentiated state in proliferating hESCs [19–21], we examined whether L1CAM interacts with FGFRs in hESCs, resulting in the activation of FGFR signaling. The interaction of L1CAM with FGFR1 was prominently increased in L1CAM-overexpressing hESCs (Fig. 5A), whereas it was not detected in L1CAM-depleted hESCs (Fig. 5B). Concomitantly, the activities of FGFR1 and downstream signaling molecules were significantly increased in the L1CAM-overexpressing cells, whereas they were significantly reduced in the L1CAM-depleted cells (Fig. 5B). Interestingly, the expression of FGFR1 was markedly downregulated at the protein and mRNA levels in L1CAM-depleted hESCs (Fig. 5A-5C).

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Figure 5. L1CAM supports the self-renewal capacity of human embryonic stem cells by activating FGFR1 signaling. (A): Coimmunoprecipitation of L1CAM with anti-FGFR1. Lysates were prepared from H9-derived stable cell lines, immunoprecipitated with anti-FGFR1 antibody, followed by Western blot analysis using 4-63 or anti-FGFR1 antibody. HC indicates the heavy chain. Blots are representative of three independent experiments (B) H9-derived stable cell lines were grown for 5 days, and then FGFR, AKT, and ERK phosphorylation was analyzed. Blots are representative of three independent experiments. (C): FGFR1 expression in H9-derived stable cell lines was assessed with qRT-PCR. Three independent experiments were performed in duplicate. Data are the means ± SD (**p < .01). (D–F): H9-derived mock- or L1CAM short hairpin RNA–transfected cells were incubated with or without 10 μM SU5402 for 72 hours. (D) FGFR1 phosphorylation and Oct4 expression were analyzed with Western blotting with β-actin as a loading control. Blots are representative of two independent experiments. (E) Each stable cell line was labeled with BrdU as described in Figure 2. This experiment was performed twice in triplicate. Data are the means ± SD (**p < .01). (F) Oct4, Nanog, Sox2, and FoxD3 expression was compared with qRT-PCR. This experiment was performed twice in triplicate. Data are the means ± SD (**p < .01). Abbreviations: AKT, v-akt murine thymoma viral oncogene homolog 1; BrdU, bromodeoxyuridine; CTL, control; ERK, extracellular signal-regulated kinase 1/2; FGFR1, fibroblast growth factor receptor 1; FoxD3, forkhead box protein D3; IP, immunoprecipitation; L1CAM, L1 cell adhesion molecule; Oct4, octamer-binding transcription factor 4; P-AKT, phospho-AKT; P-ERK, phospho-ERK; P-FGFR1, phospho-FGFR1; Sox2, sex-determining region Y–box 2; WB, western blot.

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Finally, to investigate that L1CAM-mediated FGFR1 signaling supports self-renewal in hESCs, cells were incubated in a conditioned medium without FGF-2 then treated with an inhibitor of FGFR kinases, SU5402. Treatment with SU5402 reduced the activation of FGFR1 in L1CAM-overexpressing cells (Fig. 5D) and significantly reduced the cell proliferation and the expression of Oct4, Nanog, Sox2, and FoxD3 (Fig. 5E, 5F), demonstrating that L1CAM supports the self-renewal of hESCs through the activation of FGFR1 signaling in a ligand-independent manner.

Recently, Ding et al. revealed that the removal of FGF-2 from culture medium resulted in a significant downregulation of FGFR1, 3, and 4 and Oct4 at the protein level, and SU5402 treatment also decreased the expression of Oct3/4 in hESCs and induced pluripotent stem cells [21]. These results strongly suggest that FGFRs-mediated signaling is crucially involved in the regulation of FGFR1 and Oct4. We also observed that L1CAM regulates their expression in a ligand-independent fashion through the activation of FGFR1. Although we did not elucidate the exact mechanism by which L1CAM regulates the gene transcription of FGFR1, a recent study has suggested that the C-terminal fragment of L1CAM is translocated to the nucleus and is involved in L1CAM-dependent gene regulation [22]. However, the mechanism by which endocytosed L1CAM is transported to the nucleus and is involved in the regulation of gene transcription remains to be elucidated.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

This study shows for the first time that L1CAM is a new cell-surface molecule of undifferentiated hESCs and functions to maintain self-renewal and pluripotency. The anti-L1CAM antibody may be useful for identifying and isolating undifferentiated hESCs.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

This work was supported by a Korea Research Institute of Bioscience & Biotechnology grant (to K.-H.B., S.J.C., and J.-K.M.), a grant from Korea Research Council of Fundamental Science and Technology (Stem Cell Research Program; to Y.S.C. and J.-K.M.), a Korea Science and Engineering Foundation grant (no. M1AN41-2006-04440), and a grant (SC12023) from the Stem Cell Research Center of the 21st Century Frontier Research Program, funded by the Ministry of Science and Technology of Korea (to H.J.H.).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. REFERENCES
  10. Supporting Information

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

FilenameFormatSizeDescription
STEM_754_sm_supplFigure1.pdf643KFigure S1. Identification of MAb 4-63 antigen. (A) Extracts of biotinylated H9 hESCs were immunoprecipitated with 4-63 or no antibody (bead), followed by Western blot analysis using streptavidin-HRP. 4-63 in the non-IP section indicates that only 4-63 antibody was loaded. (B) Sequence analysis of L1CAM expressed in hESCs. The MS/MS spectrum of L1CAM obtained after trypsin digestion was analyzed by Q-TOF mass spectrometry. The precursor ion shown in the figure is m/z 673.93; resultant peaks were searched against the NCBInr database. Four tryptic peptides that matched the L1CAM isoform 2 are underlined in the sequence presented in B. The hESC-expressed L1CAM was the non-neuronal isoform because RSLE was not present in the peptide (N-DETFGEYSDNEEK-C) sequenced from the precursor ion of m/z 781.88. To confirm this result, we cloned the full-length cDNA encoding L1CAM from H9 cells and determined its entire nucleotide sequence. The L1CAM sequence lacks the YEGHH and RSLE peptides found in neural L1CAM.
STEM_754_sm_supplFigure2.tif2492KFigure S2. Specificity of L1CAM expression in undifferentiated hESCs and mESCs. (A) H9 cells and day 12-derived EBs were incubated with anti-SSEA-3, anti-SSEA-4, or L1CAM, followed by FITC-conjugated anti-rat IgM or anti-mouse IgG. Fluorescence is compared with control in each panel. (B-C) Total mRNAs were isolated from the hESCs (B) or msESC (C) cultured for 6 or 12 days, or the EBs derived at indicated days. The expression level of transcription factors essential for the maintenance of an undifferentiated state in ESCs (Nanog, Oct4, and Sox2) and three germ layer markers (ectoderm, Pax6; mesoderm, CD34; and endoderm, AFP) was assessed by RT-PCR.
STEM_754_sm_supplFigure3.tif1922KFigure S3. AP staining in H9-derived stable cell lines. (A) AP staining of H9-derived stable cell lines. (B) The graph shows the data of AP-positive cells. L1CAM depletion in hESCs significantly reduced AP staining. In contrast, L1CAM-overexpressing cells strongly expressed AP. Three independent experiments were performed in duplicate. Data are presented as means ± SD (**p < 0.01).
STEM_754_sm_supplTable1.pdf113KSupplementary Table 1.
STEM_754_sm_supplTable2.pdf76KSupplementary Table 2.
STEM_754_sm_suppldata.pdf160KSupplementary Data

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