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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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Anti-MUC1* stimulates the growth of pluripotent stem cells in a dose-dependent manner.H9 hESCs at passage 67 were plated at very low density (1.9×104 per well, in triplicate) over matrigel-coated 96-well plates. Cells were cultured in minimal stem cell media, supplemented with Anti-MUC1* antibody to a final concentration of 0, 80 ng/ml, 0.25 ug/ml, 0.5 ug/ml, 1 ug/ml, or 2 ug/ml and cultured for 10 days. Media plus antibody was replaced every other day. After 10 days of growth, cells were stained with Amido Black according to manufacturer's instructions and absorbance at 570 nm was measured on a microplate reader. Stem cell growth was plotted as a function of Anti-MUC1* concentration. The dashed line marks the amount of cell growth that was measured for the control, in which cells were grown in 30% conditioned media from Hs27 feeder cells, supplemented with 4 ng/ml bFGF.
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pone-0003312-g009: Anti-MUC1* stimulates the growth of pluripotent stem cells in a dose-dependent manner.H9 hESCs at passage 67 were plated at very low density (1.9×104 per well, in triplicate) over matrigel-coated 96-well plates. Cells were cultured in minimal stem cell media, supplemented with Anti-MUC1* antibody to a final concentration of 0, 80 ng/ml, 0.25 ug/ml, 0.5 ug/ml, 1 ug/ml, or 2 ug/ml and cultured for 10 days. Media plus antibody was replaced every other day. After 10 days of growth, cells were stained with Amido Black according to manufacturer's instructions and absorbance at 570 nm was measured on a microplate reader. Stem cell growth was plotted as a function of Anti-MUC1* concentration. The dashed line marks the amount of cell growth that was measured for the control, in which cells were grown in 30% conditioned media from Hs27 feeder cells, supplemented with 4 ng/ml bFGF.

Mentions: To verify that the stimulation of stem cell growth that we observed was in fact due to the activation of the MUC1* receptor, we measured the stimulatory effect of Anti-MUC1* as a function of antibody concentration. H9 hESCs were plated at 1.9×104 cells/well (in triplicate) on matrigel-coated 96-well plates. Cells were cultured in minimal media without any added fibroblast extracts or growth factors. Anti-MUC1* was added at concentrations that ranged from 0 to 2 ug/ml. Media plus antibody was changed every other day. After ten (10) days most wells had reached 75% confluency. Cell numbers were measured by staining with Amido Black and measuring absorbance at 570 nm. A plot of cell growth as a function of antibody concentration indicates that the bivalent antibody stimulates stem cell growth in a dose-dependent manner (Fig. 9). A control experiment performed in parallel, wherein stem cells were plated at the same density but grown according to standard protocol which included the addition of 30% conditioned media from fibroblast feeder cells and exogenous bFGF. The degree of cell growth that was achieved using the state of the art conditions is denoted on the graph.


MUC1* mediates the growth of human pluripotent stem cells.

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

Anti-MUC1* stimulates the growth of pluripotent stem cells in a dose-dependent manner.H9 hESCs at passage 67 were plated at very low density (1.9×104 per well, in triplicate) over matrigel-coated 96-well plates. Cells were cultured in minimal stem cell media, supplemented with Anti-MUC1* antibody to a final concentration of 0, 80 ng/ml, 0.25 ug/ml, 0.5 ug/ml, 1 ug/ml, or 2 ug/ml and cultured for 10 days. Media plus antibody was replaced every other day. After 10 days of growth, cells were stained with Amido Black according to manufacturer's instructions and absorbance at 570 nm was measured on a microplate reader. Stem cell growth was plotted as a function of Anti-MUC1* concentration. The dashed line marks the amount of cell growth that was measured for the control, in which cells were grown in 30% conditioned media from Hs27 feeder cells, supplemented with 4 ng/ml bFGF.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553196&req=5

pone-0003312-g009: Anti-MUC1* stimulates the growth of pluripotent stem cells in a dose-dependent manner.H9 hESCs at passage 67 were plated at very low density (1.9×104 per well, in triplicate) over matrigel-coated 96-well plates. Cells were cultured in minimal stem cell media, supplemented with Anti-MUC1* antibody to a final concentration of 0, 80 ng/ml, 0.25 ug/ml, 0.5 ug/ml, 1 ug/ml, or 2 ug/ml and cultured for 10 days. Media plus antibody was replaced every other day. After 10 days of growth, cells were stained with Amido Black according to manufacturer's instructions and absorbance at 570 nm was measured on a microplate reader. Stem cell growth was plotted as a function of Anti-MUC1* concentration. The dashed line marks the amount of cell growth that was measured for the control, in which cells were grown in 30% conditioned media from Hs27 feeder cells, supplemented with 4 ng/ml bFGF.
Mentions: To verify that the stimulation of stem cell growth that we observed was in fact due to the activation of the MUC1* receptor, we measured the stimulatory effect of Anti-MUC1* as a function of antibody concentration. H9 hESCs were plated at 1.9×104 cells/well (in triplicate) on matrigel-coated 96-well plates. Cells were cultured in minimal media without any added fibroblast extracts or growth factors. Anti-MUC1* was added at concentrations that ranged from 0 to 2 ug/ml. Media plus antibody was changed every other day. After ten (10) days most wells had reached 75% confluency. Cell numbers were measured by staining with Amido Black and measuring absorbance at 570 nm. A plot of cell growth as a function of antibody concentration indicates that the bivalent antibody stimulates stem cell growth in a dose-dependent manner (Fig. 9). A control experiment performed in parallel, wherein stem cells were plated at the same density but grown according to standard protocol which included the addition of 30% conditioned media from fibroblast feeder cells and exogenous bFGF. The degree of cell growth that was achieved using the state of the art conditions is denoted on the graph.

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