Author contributions: Y.S.S.: conception and design, collection and assembly of data, and data analysis and interpretation; R.H.S.: administrative support and data analysis and interpretation; C.J.R.: conception and design; Y.S.C. and K.-H.B.: administrative support, data analysis, and financial support; S.J.C.: collection and assembly of data and financial support; B.-H.L.: administrative support and financial support; J.-K.M.: conception and design, collection and assembly of data, data analysis and interpretation, financial support, manuscript writing, and final approval of manuscript; H.J.H.: conception and design, data analysis and interpretation, financial support, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS September 28, 2011.
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.
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 . 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 . 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.
MATERIALS AND METHODS
Detailed Materials and Methods are described in the supporting information.
RESULTS AND DISCUSSION
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).
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.
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.
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.
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).
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 . 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 . 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.
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.
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.).
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors declare no potential conflicts of interest.