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

Soncin F, Ward CM - Genes (Basel) (2011)

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

View Article: PubMed Central - PubMed

Affiliation: Core Technology Facility, Faculty of Medical and Human Sciences, The University of Manchester, 46 Grafton Street, M13 9NT, UK. fsoncin@sanfordburnham.org.

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

No MeSH data available.


Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3924836&req=5

f6-genes-02-00229: 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.

Mentions: We have previously demonstrated that inhibition of E-cadherin-mediated cell-cell contact in hES cells using a neutralizing antibody (nAb) does not induce differentiation of these cells [112], although it does decrease their proliferative capacity. Titration of nAb onto hES cells such that cell-cell contact and proliferation are unaffected allowed the prolonged culture of the cells in the absence of FGF2 (Figure 6). After two passages in the presence of nAb and absence of FGF2, both HES4 (Figure 6a) and H1 (Figure 6b) ES cells exhibited characteristic colony morphology whereas cAb treated colonies were mostly differentiated. Cell surface expression of the pluripotent marker Tra-1-60 was assessed on cAb and nAb treated HES4 hES cells after three passages in the absence of FGF2 (Figure 6e). nAb-treated HES4 ES cells exhibited similar expression of Tra-1-60 compared to HES4 cells cultured under normal conditions. By contrast, cAb-treated HES4 hES cells exhibited significantly decreased expression of Tra-1-60. Similarly, H1 hES cells treated with nAb for five passages in the absence of FGF2 exhibited high levels of Tra-1-60 expression (Figure 6f) whereas all cAb treated cells had died. Following culture of H1 and HES4 ES cells for 10 passages (approximately 90 days) in the presence of nAb and absence of FGF2, RT-PCR analysis was performed to assess expression of transcripts associated with pluripotency (Oct4) and various lineage markers (Figure 6c(i) and 6d(i)). Both cell lines exhibited an undifferentiated transcript profile consistent with our previous observations [142]. To determine the differentative potential of hES cells cultured for 10 passages in the presence of nAb and absence of FGF2, we allowed the cells 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 and assessed the cells for expression of lineage specific transcript markers (Figure 6c(ii) and 6d(ii)). Both hES cell lines expressed markers of differentiation, demonstrating that the cells maintained the ability to differentiate into cells representative of the three primary germ layers. Whilst the exact mechanism for self-renewal of human ES cells in the absence of FGF2 is unclear (for example, it may be due to exogenous FGF2 expression induced by the nAb) it does illustrate that E-cadherin functions in both mouse and human ES cells to positively regulate pluripotent signaling pathways.


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

Soncin F, Ward CM - Genes (Basel) (2011)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924836&req=5

f6-genes-02-00229: 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.
Mentions: We have previously demonstrated that inhibition of E-cadherin-mediated cell-cell contact in hES cells using a neutralizing antibody (nAb) does not induce differentiation of these cells [112], although it does decrease their proliferative capacity. Titration of nAb onto hES cells such that cell-cell contact and proliferation are unaffected allowed the prolonged culture of the cells in the absence of FGF2 (Figure 6). After two passages in the presence of nAb and absence of FGF2, both HES4 (Figure 6a) and H1 (Figure 6b) ES cells exhibited characteristic colony morphology whereas cAb treated colonies were mostly differentiated. Cell surface expression of the pluripotent marker Tra-1-60 was assessed on cAb and nAb treated HES4 hES cells after three passages in the absence of FGF2 (Figure 6e). nAb-treated HES4 ES cells exhibited similar expression of Tra-1-60 compared to HES4 cells cultured under normal conditions. By contrast, cAb-treated HES4 hES cells exhibited significantly decreased expression of Tra-1-60. Similarly, H1 hES cells treated with nAb for five passages in the absence of FGF2 exhibited high levels of Tra-1-60 expression (Figure 6f) whereas all cAb treated cells had died. Following culture of H1 and HES4 ES cells for 10 passages (approximately 90 days) in the presence of nAb and absence of FGF2, RT-PCR analysis was performed to assess expression of transcripts associated with pluripotency (Oct4) and various lineage markers (Figure 6c(i) and 6d(i)). Both cell lines exhibited an undifferentiated transcript profile consistent with our previous observations [142]. To determine the differentative potential of hES cells cultured for 10 passages in the presence of nAb and absence of FGF2, we allowed the cells 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 and assessed the cells for expression of lineage specific transcript markers (Figure 6c(ii) and 6d(ii)). Both hES cell lines expressed markers of differentiation, demonstrating that the cells maintained the ability to differentiate into cells representative of the three primary germ layers. Whilst the exact mechanism for self-renewal of human ES cells in the absence of FGF2 is unclear (for example, it may be due to exogenous FGF2 expression induced by the nAb) it does illustrate that E-cadherin functions in both mouse and human ES cells to positively regulate pluripotent signaling pathways.

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

View Article: PubMed Central - PubMed

Affiliation: Core Technology Facility, Faculty of Medical and Human Sciences, The University of Manchester, 46 Grafton Street, M13 9NT, UK. fsoncin@sanfordburnham.org.

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

No MeSH data available.


Related in: MedlinePlus