The function of e-cadherin in stem cell pluripotency and self-renewal

Abstract

E-cadherin; pluripotency; embryonic stem cell; induced pluripotent stem cell; iPS; ES; signaling pathways; Activin; Nodal.

Figure 1

A generalized view of the derivation of stem cells from pre- and post-implantation stages. (A) Pre-implantation blastocysts (left) are formed by two different cell types; the external trophectoderm surrounds a cavity where the inner cell mass (ICM) is found. In post-implantation stages, the ICM differentiates into the epiblast (internal) and the extra-embryonic endoderm (in contact with the cavity); (B) List of cell types found in pre- and post-implantation stages and the relative stem cell lines that have been isolated and maintained in vitro (green). ES = Embryonic Stem cells from the ICM, TS = Trophoblast Stem cells from the trophectoderm, XEN = Extraembryonic endoderm-derived cells, EpiS = Epiblast Stem cells. The black arrows indicate the tissues that each cell type will develop into during embryogenesis.

Figure 2

Diagrammatic representation of the pathways associated with leukemia inhibitory factor (LIF)-dependent pluripotency in mouse ES cells.

Figure 3

Diagrammatic representation of Activin, Nodal, TGFβ and BMP signaling pathways.

Figure 4

Diagrammatic representation of the E-cadherin promoter and protein. (A) Promoter region of the Cdh1 gene encoding for E-cadherin. The figure shows both positive (GC boxes and CCAAT boxes) and negative (E boxes) regulatory elements. Note the location of E-box 4 after the transcription initiation site (orange arrow) and the presence of an epithelial specific enhancer (ESE) located within an unusually large intron 2. (B) Diagrammatic representation of the E-cadherin/catenin complex anchored to the actin cytoskeleton. The extracellular region of E-cadherin contains four extracellular cadherin domains (ECD) and an atypical membrane proximal domain (MPED). Calcium ion-binding sites are located between ECDs and are necessary for cell adhesion mediation. β-Catenin and p120-catenin bind specific regions within the cytoplasmic domain of E-cadherin. α-E-catenin might directly anchor the complex to the actin cytoskeleton by binding with F-actin and β-catenin or indirectly through an additional bridging molecule (e.g., EPLIN). ECD = extracellular cadherin domain; MPED = Membrane Proximal Extracellular Domain.

Figure 5

Diagrammatic representation of the transcriptional and translational events associated with epithelial-mesenchymal transition during mouse and human ES cell differentiation. Undifferentiated ES cells exhibit E-cadherin-mediated cell-cell contact and this is associated with low levels of N-cadherin, E-cadherin repressors (Slug, Snail and SIP1) and matrix metalloproteinases (MMPs) [112,113]. Upon induction of ES cell differentiation, E-cadherin protein is rapidly lost from the cell surface and this is associated with increased N-cadherin, E-cadherin repressor (Slug, Snail and SIP1) and MMP expression [112,113]. Green denotes changes in both transcripts and protein; red denotes changes in transcripts only.

Figure 6

Culture of hES cells in the presence of E-cadherin neutralizing antibody SHE78.7 allows their culture in the absence of FGF2. HES4 and H1 human ES cell lines were cultured in the presence of a minimal fibroblast feeder layer (approximately 1000 cells/dish) in the absence of FGF2 in serum replacement medium in the presence of cAb or nAb (0.5 μL/mL of media of 0.5 mg/mL stock solution). (a) Phase contrast microscopy of HES4 ES cells cultured in control antibody (cAb) or E-cadherin neutralizing antibody SHE78.7 (nAb) after 2 passages in the absence of FGF-2. Note that nAb cells exhibited normal colony morphology whereas cAb treated cells differentiated; (b) Phase contrast microscopy of H1 ES cells cultured in control antibody (cAb) or E-cadherin neutralizing antibody SHE78.7 (nAb) after 2 passages in the absence of FGF-2. Note that nAb cells exhibited normal colony morphology whereas cAb treated cells differentiated; (c) (i) HES4 ES cell colonies were cultured in nAb (0.5 μL/mL of media of 0.5 mg/mL stock solution) in the presence of a minimal fibroblast feeder layer in the absence of FGF2 in serum replacement medium for 10 passages (approximately 90 days) and assessed for expression of transcripts associated with pluripotency and various lineage markers as previously described [142]. Note that the transcript profile expression is consistent with that observed for undifferentiated HES4 ES cells (as described in Ward et al. [142]). (ii) HES4 ES cell colonies described above were allowed to overgrow in the culture plates (i.e., no passaging) in normal ES cell culture medium (i.e., +FGF2) for 20 days to induce differentiation of the cells and assessed for expression of transcripts associated with pluripotency and various lineage markers (as described in [142]). Note that markers of differentiation expressed following differentiation of the cells included all three germ layers (endoderm-HNF, TF, AMY; mesoderm-FLK, CD34, AC133; ectoderm-NES, NFM, NSE, PAX and PLP) and extra-embryonic visceral endoderm (AFP); (d) RT-PCR analysis of (i) undifferentiated and (ii) differentiated H1 ES cells as described in (c). Oct-4 (OCT); α-fetoprotein (AFP); hepatocyte nuclear factor (HNF); nestin (NES); neurofilament middle chain (NFM); neuron-specific enolase (NSE); Pax-6 (PAX); proteolipid protein (PLP); amylase (AMY); α1-antitrypsin (TRP); Flk-1 (Flk); CD34 (CD); AC133 (AC); Transferrin (Tf); β-tubulin (BT); alpha-fetal protein (AFP); (e) Cell surface expression of the pluripotent marker Tra-1-60 was assessed on cAb and nAb treated HES4 ES cells (HES4) after 3 passages in the absence of FGF2 and HES4 ES cells cultured under normal conditions (HES4) on a fibroblast feeder layer containing FGF2 by fluorescent flow cytometry. Note that nAb-treated cells exhibited similar expression of Tra-1-60 compared to HES4 cells cultured under normal conditions; (f) Cell surface expression of the pluripotent marker Tra-1-60 was assessed on nAb treated HES4 ES cells (all cAb treated cells died) after 5 passages in the absence of FGF2. Note that >99% of the nAb treated cells exhibited Tra-1-60 expression.

Figure 6

Culture of hES cells in the presence of E-cadherin neutralizing antibody SHE78.7 allows their culture in the absence of FGF2. HES4 and H1 human ES cell lines were cultured in the presence of a minimal fibroblast feeder layer (approximately 1000 cells/dish) in the absence of FGF2 in serum replacement medium in the presence of cAb or nAb (0.5 μL/mL of media of 0.5 mg/mL stock solution). (a) Phase contrast microscopy of HES4 ES cells cultured in control antibody (cAb) or E-cadherin neutralizing antibody SHE78.7 (nAb) after 2 passages in the absence of FGF-2. Note that nAb cells exhibited normal colony morphology whereas cAb treated cells differentiated; (b) Phase contrast microscopy of H1 ES cells cultured in control antibody (cAb) or E-cadherin neutralizing antibody SHE78.7 (nAb) after 2 passages in the absence of FGF-2. Note that nAb cells exhibited normal colony morphology whereas cAb treated cells differentiated; (c) (i) HES4 ES cell colonies were cultured in nAb (0.5 μL/mL of media of 0.5 mg/mL stock solution) in the presence of a minimal fibroblast feeder layer in the absence of FGF2 in serum replacement medium for 10 passages (approximately 90 days) and assessed for expression of transcripts associated with pluripotency and various lineage markers as previously described [142]. Note that the transcript profile expression is consistent with that observed for undifferentiated HES4 ES cells (as described in Ward et al. [142]). (ii) HES4 ES cell colonies described above were allowed to overgrow in the culture plates (i.e., no passaging) in normal ES cell culture medium (i.e., +FGF2) for 20 days to induce differentiation of the cells and assessed for expression of transcripts associated with pluripotency and various lineage markers (as described in [142]). Note that markers of differentiation expressed following differentiation of the cells included all three germ layers (endoderm-HNF, TF, AMY; mesoderm-FLK, CD34, AC133; ectoderm-NES, NFM, NSE, PAX and PLP) and extra-embryonic visceral endoderm (AFP); (d) RT-PCR analysis of (i) undifferentiated and (ii) differentiated H1 ES cells as described in (c). Oct-4 (OCT); α-fetoprotein (AFP); hepatocyte nuclear factor (HNF); nestin (NES); neurofilament middle chain (NFM); neuron-specific enolase (NSE); Pax-6 (PAX); proteolipid protein (PLP); amylase (AMY); α1-antitrypsin (TRP); Flk-1 (Flk); CD34 (CD); AC133 (AC); Transferrin (Tf); β-tubulin (BT); alpha-fetal protein (AFP); (e) Cell surface expression of the pluripotent marker Tra-1-60 was assessed on cAb and nAb treated HES4 ES cells (HES4) after 3 passages in the absence of FGF2 and HES4 ES cells cultured under normal conditions (HES4) on a fibroblast feeder layer containing FGF2 by fluorescent flow cytometry. Note that nAb-treated cells exhibited similar expression of Tra-1-60 compared to HES4 cells cultured under normal conditions; (f) Cell surface expression of the pluripotent marker Tra-1-60 was assessed on nAb treated HES4 ES cells (all cAb treated cells died) after 5 passages in the absence of FGF2. Note that >99% of the nAb treated cells exhibited Tra-1-60 expression.