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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 cleaved form, MUC1*, is expressed on undifferentiated human embryonic stem cells while the full-length, uncleaved protein (MUC1-FL) is expressed on differentiated stem cells.A–C. Undifferentiated H9 hESC colonies were triple stained with antibodies against: A. MUC1*, B. OCT4, and C. MUC1-FL. The dotted line indicates the border of the undifferentiated colony. D–F. H9 hESC colonies were induced to differentiate by withholding bFGF for 14 days. Visual inspection indicated that the colonies had differentiated. The hESC colonies were triple stained with antibodies against: D. MUC1*, E. OCT4, and F. MUC1-FL. Scale bars = 100 µm.
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pone-0003312-g003: The cleaved form, MUC1*, is expressed on undifferentiated human embryonic stem cells while the full-length, uncleaved protein (MUC1-FL) is expressed on differentiated stem cells.A–C. Undifferentiated H9 hESC colonies were triple stained with antibodies against: A. MUC1*, B. OCT4, and C. MUC1-FL. The dotted line indicates the border of the undifferentiated colony. D–F. H9 hESC colonies were induced to differentiate by withholding bFGF for 14 days. Visual inspection indicated that the colonies had differentiated. The hESC colonies were triple stained with antibodies against: D. MUC1*, E. OCT4, and F. MUC1-FL. Scale bars = 100 µm.

Mentions: Unexpectedly, we found that although the cleaved form, MUC1*, is highly expressed on undifferentiated hESCs, no full-length MUC1 was detectable. Undifferentiated stem cells stained positive for MUC1* and OCT4 but negative for full-length MUC1 (Fig. 3A–C). In stark contrast, newly differentiated embryonic stem cells exclusively expressed full-length MUC1 but not the cleaved form, MUC1*. Differentiating stem cells tested positive for full-length MUC1 and negative for both MUC1* and OCT4 (Fig. 3D–F). No full-length MUC1 was detectable on undifferentiated H9 or H14 hESCs, whether probed with the VU4H5 antibody or HMPV; after differentiation, both cell lines stained positive for MUC1-FL using either antibody (Fig. S2 A–H; Fig. S3 A–F). Both cell lines stained positive for the presence of MUC1* in the undifferentiated state, but not in the differentiated state (data not shown). We cannot rule out the possibility that Anti-MUC1* is also staining an alternative splice isoform such as MUC1/X, MUC1/Y or MUC1/Z. However, Western blot analysis of H9 hESCs revealed a 20 kD Anti-MUC1* reactive species that co-migrated with the MUC1 cleavage product from T47D breast cancer cells and with transfected MUC1*1110, that contains only forty-five (45) amino acids of the extracellular domain (Fig. S4). It follows that this low molecular weight species is the cleavage product of full-length MUC1, since it runs with an apparent molecular weight that is roughly half the apparent molecular weight of MUC1/Y and MUC1/Y is not cleaved.


MUC1* mediates the growth of human pluripotent stem cells.

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

The cleaved form, MUC1*, is expressed on undifferentiated human embryonic stem cells while the full-length, uncleaved protein (MUC1-FL) is expressed on differentiated stem cells.A–C. Undifferentiated H9 hESC colonies were triple stained with antibodies against: A. MUC1*, B. OCT4, and C. MUC1-FL. The dotted line indicates the border of the undifferentiated colony. D–F. H9 hESC colonies were induced to differentiate by withholding bFGF for 14 days. Visual inspection indicated that the colonies had differentiated. The hESC colonies were triple stained with antibodies against: D. MUC1*, E. OCT4, and F. MUC1-FL. Scale bars = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2553196&req=5

pone-0003312-g003: The cleaved form, MUC1*, is expressed on undifferentiated human embryonic stem cells while the full-length, uncleaved protein (MUC1-FL) is expressed on differentiated stem cells.A–C. Undifferentiated H9 hESC colonies were triple stained with antibodies against: A. MUC1*, B. OCT4, and C. MUC1-FL. The dotted line indicates the border of the undifferentiated colony. D–F. H9 hESC colonies were induced to differentiate by withholding bFGF for 14 days. Visual inspection indicated that the colonies had differentiated. The hESC colonies were triple stained with antibodies against: D. MUC1*, E. OCT4, and F. MUC1-FL. Scale bars = 100 µm.
Mentions: Unexpectedly, we found that although the cleaved form, MUC1*, is highly expressed on undifferentiated hESCs, no full-length MUC1 was detectable. Undifferentiated stem cells stained positive for MUC1* and OCT4 but negative for full-length MUC1 (Fig. 3A–C). In stark contrast, newly differentiated embryonic stem cells exclusively expressed full-length MUC1 but not the cleaved form, MUC1*. Differentiating stem cells tested positive for full-length MUC1 and negative for both MUC1* and OCT4 (Fig. 3D–F). No full-length MUC1 was detectable on undifferentiated H9 or H14 hESCs, whether probed with the VU4H5 antibody or HMPV; after differentiation, both cell lines stained positive for MUC1-FL using either antibody (Fig. S2 A–H; Fig. S3 A–F). Both cell lines stained positive for the presence of MUC1* in the undifferentiated state, but not in the differentiated state (data not shown). We cannot rule out the possibility that Anti-MUC1* is also staining an alternative splice isoform such as MUC1/X, MUC1/Y or MUC1/Z. However, Western blot analysis of H9 hESCs revealed a 20 kD Anti-MUC1* reactive species that co-migrated with the MUC1 cleavage product from T47D breast cancer cells and with transfected MUC1*1110, that contains only forty-five (45) amino acids of the extracellular domain (Fig. S4). It follows that this low molecular weight species is the cleavage product of full-length MUC1, since it runs with an apparent molecular weight that is roughly half the apparent molecular weight of MUC1/Y and MUC1/Y is not cleaved.

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