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MUC1* mediates the growth of human pluripotent stem cells.

Hikita ST, Kosik KS, Clegg DO, Bamdad C - PLoS ONE (2008)

Bottom Line: Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC1*, or its ligand, NM23.Antibody-induced dimerization of the MUC1* receptor on hESCs stimulated cell growth to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast growth factor (bFGF) as well as factors secreted by fibroblast "feeder cells".These findings suggest that this primal growth mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell Biology and Engineering, University of California Santa Barbara, Santa Barbara, California, USA.

ABSTRACT
The MUC1 protein is aberrantly expressed on an estimated 75% of all human solid tumor cancers. We recently reported that a transmembrane cleavage product, MUC1*, is the predominant form of the protein on cancer cells [1]. Further, our evidence indicated that MUC1* functions as a growth factor receptor on tumor cells, while the full-length protein appeared to have no growth promoting activity. Here, we report that MUC1* acts as a growth factor receptor on undifferentiated human embryonic stem cells (hESCs). Cleavage of the full-length ectodomain to form MUC1*, a membrane receptor, appears to make binding to its ligand, NM23, possible. Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC1*, or its ligand, NM23. Newly differentiated stem cells exclusively present full-length MUC1. Antibody-induced dimerization of the MUC1* receptor on hESCs stimulated cell growth to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast growth factor (bFGF) as well as factors secreted by fibroblast "feeder cells". Further, MUC1* mediated growth was shown to be independent of growth stimulated by bFGF or the milieu of factors secreted by feeder cells. Stimulating the MUC1* receptor with either the cognate antibody or its ligand NM23 enabled hESC growth in a feeder cell-free system and produced pluripotent colonies that resisted spontaneous differentiation. These findings suggest that this primal growth mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions.

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The expression of MUC1 cleavage enzymes, MMP14 and TACE, is high on undifferentiated hESCs but significantly lower on differentiated cells.A–C. Stem cells colonies that were deemed to be undifferentiated by visual inspection and by staining positive for the presence of OCT4 were stained with antibodies against MUC1-FL and the cleavage enzymes. A. Undifferentiated hESC colonies were stained with an antibody that recognizes MMP14. VU4H5, which recognizes MUC1-FL, did not stain this colony (data not shown). B. Undifferentiated hESC colonies were stained with an antibody that recognizes TACE. C. The same undifferentiated colony was also treated with VU4H5 (binds to MUC1-FL) but no immuno-reactivity was detected. D–F. Stem cells that were induced to differentiate by withholding bFGF for 14 days were stained with the same antibodies as in A–C. D. Newly differentiating stem cell colonies co-express MMP14 (red) and MUC-FL (green). E. Similarly, newly differentiating stem cell colonies co-express MUC1 cleavage enzyme TACE (red) and MUC1-FL (green). F. Triple staining of colonies with anti-TACE (red), VU4H5 (green) and DAPI (blue) showed that roughly 50% of the differentiating cells expressed the cleavage enzyme. An experiment using an antibody against MMP14 gave essentially the same result. Scale bars = 100 µm.
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pone-0003312-g005: The expression of MUC1 cleavage enzymes, MMP14 and TACE, is high on undifferentiated hESCs but significantly lower on differentiated cells.A–C. Stem cells colonies that were deemed to be undifferentiated by visual inspection and by staining positive for the presence of OCT4 were stained with antibodies against MUC1-FL and the cleavage enzymes. A. Undifferentiated hESC colonies were stained with an antibody that recognizes MMP14. VU4H5, which recognizes MUC1-FL, did not stain this colony (data not shown). B. Undifferentiated hESC colonies were stained with an antibody that recognizes TACE. C. The same undifferentiated colony was also treated with VU4H5 (binds to MUC1-FL) but no immuno-reactivity was detected. D–F. Stem cells that were induced to differentiate by withholding bFGF for 14 days were stained with the same antibodies as in A–C. D. Newly differentiating stem cell colonies co-express MMP14 (red) and MUC-FL (green). E. Similarly, newly differentiating stem cell colonies co-express MUC1 cleavage enzyme TACE (red) and MUC1-FL (green). F. Triple staining of colonies with anti-TACE (red), VU4H5 (green) and DAPI (blue) showed that roughly 50% of the differentiating cells expressed the cleavage enzyme. An experiment using an antibody against MMP14 gave essentially the same result. Scale bars = 100 µm.

Mentions: MMP14 (MT1-MMP) and TACE (ADAM 17) have been reported to be enzymes that cleave MUC1 on human uterine epithelial cells [31], [32]. If MMP-14 and TACE also cleave MUC1 on embryonic stem cells, then one might expect high expression levels on undifferentiated cells, where MUC1 is cleaved, and lower expression on differentiating cells where it is not. Immunofluorescent imaging revealed that both cleavage enzymes, MMP14 and TACE, are robustly expressed on undifferentiated stem cells that were completely devoid of full-length MUC1 (Fig. 5A–C). However, on newly differentiated stem cells, where MUC1-FL immunoreactivity was present, there was a marked decrease in MMP14 and TACE expression (Fig. 5 D–F). The merged image of a triple staining experiment, which also included DAPI staining, shows that approximately 50% of the cells present stained positive for the cleavage enzymes (Fig. 5F) compared to virtually 100% on undifferentiated colonies (data not shown). These findings support the idea that cleavage enzymes MMP14 and TACE cleave MUC1 on embryonic stem cells.


MUC1* mediates the growth of human pluripotent stem cells.

Hikita ST, Kosik KS, Clegg DO, Bamdad C - PLoS ONE (2008)

The expression of MUC1 cleavage enzymes, MMP14 and TACE, is high on undifferentiated hESCs but significantly lower on differentiated cells.A–C. Stem cells colonies that were deemed to be undifferentiated by visual inspection and by staining positive for the presence of OCT4 were stained with antibodies against MUC1-FL and the cleavage enzymes. A. Undifferentiated hESC colonies were stained with an antibody that recognizes MMP14. VU4H5, which recognizes MUC1-FL, did not stain this colony (data not shown). B. Undifferentiated hESC colonies were stained with an antibody that recognizes TACE. C. The same undifferentiated colony was also treated with VU4H5 (binds to MUC1-FL) but no immuno-reactivity was detected. D–F. Stem cells that were induced to differentiate by withholding bFGF for 14 days were stained with the same antibodies as in A–C. D. Newly differentiating stem cell colonies co-express MMP14 (red) and MUC-FL (green). E. Similarly, newly differentiating stem cell colonies co-express MUC1 cleavage enzyme TACE (red) and MUC1-FL (green). F. Triple staining of colonies with anti-TACE (red), VU4H5 (green) and DAPI (blue) showed that roughly 50% of the differentiating cells expressed the cleavage enzyme. An experiment using an antibody against MMP14 gave essentially the same result. Scale bars = 100 µm.
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Related In: Results  -  Collection

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pone-0003312-g005: The expression of MUC1 cleavage enzymes, MMP14 and TACE, is high on undifferentiated hESCs but significantly lower on differentiated cells.A–C. Stem cells colonies that were deemed to be undifferentiated by visual inspection and by staining positive for the presence of OCT4 were stained with antibodies against MUC1-FL and the cleavage enzymes. A. Undifferentiated hESC colonies were stained with an antibody that recognizes MMP14. VU4H5, which recognizes MUC1-FL, did not stain this colony (data not shown). B. Undifferentiated hESC colonies were stained with an antibody that recognizes TACE. C. The same undifferentiated colony was also treated with VU4H5 (binds to MUC1-FL) but no immuno-reactivity was detected. D–F. Stem cells that were induced to differentiate by withholding bFGF for 14 days were stained with the same antibodies as in A–C. D. Newly differentiating stem cell colonies co-express MMP14 (red) and MUC-FL (green). E. Similarly, newly differentiating stem cell colonies co-express MUC1 cleavage enzyme TACE (red) and MUC1-FL (green). F. Triple staining of colonies with anti-TACE (red), VU4H5 (green) and DAPI (blue) showed that roughly 50% of the differentiating cells expressed the cleavage enzyme. An experiment using an antibody against MMP14 gave essentially the same result. Scale bars = 100 µm.
Mentions: MMP14 (MT1-MMP) and TACE (ADAM 17) have been reported to be enzymes that cleave MUC1 on human uterine epithelial cells [31], [32]. If MMP-14 and TACE also cleave MUC1 on embryonic stem cells, then one might expect high expression levels on undifferentiated cells, where MUC1 is cleaved, and lower expression on differentiating cells where it is not. Immunofluorescent imaging revealed that both cleavage enzymes, MMP14 and TACE, are robustly expressed on undifferentiated stem cells that were completely devoid of full-length MUC1 (Fig. 5A–C). However, on newly differentiated stem cells, where MUC1-FL immunoreactivity was present, there was a marked decrease in MMP14 and TACE expression (Fig. 5 D–F). The merged image of a triple staining experiment, which also included DAPI staining, shows that approximately 50% of the cells present stained positive for the cleavage enzymes (Fig. 5F) compared to virtually 100% on undifferentiated colonies (data not shown). These findings support the idea that cleavage enzymes MMP14 and TACE cleave MUC1 on embryonic stem cells.

Bottom Line: Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC1*, or its ligand, NM23.Antibody-induced dimerization of the MUC1* receptor on hESCs stimulated cell growth to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast growth factor (bFGF) as well as factors secreted by fibroblast "feeder cells".These findings suggest that this primal growth mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell Biology and Engineering, University of California Santa Barbara, Santa Barbara, California, USA.

ABSTRACT
The MUC1 protein is aberrantly expressed on an estimated 75% of all human solid tumor cancers. We recently reported that a transmembrane cleavage product, MUC1*, is the predominant form of the protein on cancer cells [1]. Further, our evidence indicated that MUC1* functions as a growth factor receptor on tumor cells, while the full-length protein appeared to have no growth promoting activity. Here, we report that MUC1* acts as a growth factor receptor on undifferentiated human embryonic stem cells (hESCs). Cleavage of the full-length ectodomain to form MUC1*, a membrane receptor, appears to make binding to its ligand, NM23, possible. Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC1*, or its ligand, NM23. Newly differentiated stem cells exclusively present full-length MUC1. Antibody-induced dimerization of the MUC1* receptor on hESCs stimulated cell growth to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast growth factor (bFGF) as well as factors secreted by fibroblast "feeder cells". Further, MUC1* mediated growth was shown to be independent of growth stimulated by bFGF or the milieu of factors secreted by feeder cells. Stimulating the MUC1* receptor with either the cognate antibody or its ligand NM23 enabled hESC growth in a feeder cell-free system and produced pluripotent colonies that resisted spontaneous differentiation. These findings suggest that this primal growth mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions.

Show MeSH
Related in: MedlinePlus