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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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Related in: MedlinePlus

MUC1* mediates growth of pluripotent human embryonic stem cells.Undifferentiated H9 hESCs were grown for 41 hours in the presence of bivalent Anti-MUC1* (Bivalent Ab), which can dimerize the receptor, the monovalent Fab of Anti-MUC1* (Monovalent Ab), that blocks receptor dimerization, and/or basic fibroblast growth factor (bFGF). The results were quantified as follows. A–F. A live/dead (green/red) calcein assay was performed 41 hours post treatment. Photos record the results. A. Treatment with bivalent Anti-MUC1* (Bivalent Ab) and bFGF produced mostly viable cells (green) and very few dead cells (red). B. Treatment with the monovalent Fab of Anti-MUC1* (Monovalent Ab) and bFGF resulted in essentially total cell death within 12 hours. C. Treatment with bFGF alone produced mostly viable cells. D. Treatment with bivalent Anti-MUC1* alone produced more viable cells than with the addition of bFGF. E. Treatment with monovalent Anti-MUC1* killed essentially all cells. F. Treatment without antibodies or bFGF resulted in more dead cells and less viable cells. G. A bar graph shows that after 41 hours of treatment with Anti-MUC1* (Bivalent Antibody) there were more than 2-times the number of live cells than without the antibody (No Ab). Treatment with the monovalent Fab (Monovalent Ab) killed all the cells. Fluorescence of live cells in a calcein assay is plotted. Data are represented as mean fluorescence units±SEM. H. The percentage hESC growth is plotted for undifferentiated cells grown in the presence or absence of bFGF and with bivalent or monovalent Anti-MUC1* (Bivalent Ab, Monovalent Ab). Student's two-tailed test was used for statistical analysis. The graph shows that Anti-MUC1* with or without bFGF stimulated growth roughly twice as well as with bFGF.
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pone-0003312-g007: MUC1* mediates growth of pluripotent human embryonic stem cells.Undifferentiated H9 hESCs were grown for 41 hours in the presence of bivalent Anti-MUC1* (Bivalent Ab), which can dimerize the receptor, the monovalent Fab of Anti-MUC1* (Monovalent Ab), that blocks receptor dimerization, and/or basic fibroblast growth factor (bFGF). The results were quantified as follows. A–F. A live/dead (green/red) calcein assay was performed 41 hours post treatment. Photos record the results. A. Treatment with bivalent Anti-MUC1* (Bivalent Ab) and bFGF produced mostly viable cells (green) and very few dead cells (red). B. Treatment with the monovalent Fab of Anti-MUC1* (Monovalent Ab) and bFGF resulted in essentially total cell death within 12 hours. C. Treatment with bFGF alone produced mostly viable cells. D. Treatment with bivalent Anti-MUC1* alone produced more viable cells than with the addition of bFGF. E. Treatment with monovalent Anti-MUC1* killed essentially all cells. F. Treatment without antibodies or bFGF resulted in more dead cells and less viable cells. G. A bar graph shows that after 41 hours of treatment with Anti-MUC1* (Bivalent Antibody) there were more than 2-times the number of live cells than without the antibody (No Ab). Treatment with the monovalent Fab (Monovalent Ab) killed all the cells. Fluorescence of live cells in a calcein assay is plotted. Data are represented as mean fluorescence units±SEM. H. The percentage hESC growth is plotted for undifferentiated cells grown in the presence or absence of bFGF and with bivalent or monovalent Anti-MUC1* (Bivalent Ab, Monovalent Ab). Student's two-tailed test was used for statistical analysis. The graph shows that Anti-MUC1* with or without bFGF stimulated growth roughly twice as well as with bFGF.

Mentions: We performed similar experiments to determine whether ligands of MUC1* could mediate the growth of pluripotent hESCs. Undifferentiated stem cells were grown on matrigel-coated wells and cultured according to current methods which included feeding with minimal stem cell media that had been supplemented with 30% conditioned media from Hs27 fibroblast feeder cells. Cells were treated with bivalent Anti-MUC1* or the monovalent Fab, in the presence or absence of exogenous bFGF. The addition of Anti-MUC1* to undifferentiated hESCs had a dramatic, stimulatory effect on cell growth. Treating hESCs with bivalent Anti-MUC1* for forty-one (41) hours, in the presence or absence of added bFGF, resulted in cells that were more viable and abundant than control cells that were cultured according to standard methods, which included adding bFGF. In stark contrast, the addition of the monovalent Fab fragment of Anti-MUC1* resulted in nearly total cell death within 12 hours of treatment (Fig. 7A–F). Presumably the monovalent Fab competed with MUC1*'s native ligand, NM23, for binding and blocked receptor dimerization. The growth effects of the MUC1*-targeting antibodies were quantified by measuring the fluorescence at 530 nm for live cells grown under each test condition. In the absence of added exogenous bFGF, the addition of bivalent Anti-MUC1* resulted in a greater than 2-fold enhancement of cell growth compared to the control (Fig. 7G). A plot of normalized cell growth with added bFGF defined as 100%, is shown in Figure 7H. Bivalent Anti-MUC1* greatly enhances the growth of undifferentiated stem cells and does not require the addition of exogenous bFGF. Notably, the addition of bFGF cannot rescue stem cells when treated with monovalent Anti-MUC1*. A similar experiment was performed in which the effects of bivalent Anti-MUC1*, its monovalent Fab, and a control Fab, on H9 and on H14 hESCs were measured (Fig. S5 A,B). The results were essentially the same as those depicted in Figure 7.


MUC1* mediates the growth of human pluripotent stem cells.

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

MUC1* mediates growth of pluripotent human embryonic stem cells.Undifferentiated H9 hESCs were grown for 41 hours in the presence of bivalent Anti-MUC1* (Bivalent Ab), which can dimerize the receptor, the monovalent Fab of Anti-MUC1* (Monovalent Ab), that blocks receptor dimerization, and/or basic fibroblast growth factor (bFGF). The results were quantified as follows. A–F. A live/dead (green/red) calcein assay was performed 41 hours post treatment. Photos record the results. A. Treatment with bivalent Anti-MUC1* (Bivalent Ab) and bFGF produced mostly viable cells (green) and very few dead cells (red). B. Treatment with the monovalent Fab of Anti-MUC1* (Monovalent Ab) and bFGF resulted in essentially total cell death within 12 hours. C. Treatment with bFGF alone produced mostly viable cells. D. Treatment with bivalent Anti-MUC1* alone produced more viable cells than with the addition of bFGF. E. Treatment with monovalent Anti-MUC1* killed essentially all cells. F. Treatment without antibodies or bFGF resulted in more dead cells and less viable cells. G. A bar graph shows that after 41 hours of treatment with Anti-MUC1* (Bivalent Antibody) there were more than 2-times the number of live cells than without the antibody (No Ab). Treatment with the monovalent Fab (Monovalent Ab) killed all the cells. Fluorescence of live cells in a calcein assay is plotted. Data are represented as mean fluorescence units±SEM. H. The percentage hESC growth is plotted for undifferentiated cells grown in the presence or absence of bFGF and with bivalent or monovalent Anti-MUC1* (Bivalent Ab, Monovalent Ab). Student's two-tailed test was used for statistical analysis. The graph shows that Anti-MUC1* with or without bFGF stimulated growth roughly twice as well as with bFGF.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003312-g007: MUC1* mediates growth of pluripotent human embryonic stem cells.Undifferentiated H9 hESCs were grown for 41 hours in the presence of bivalent Anti-MUC1* (Bivalent Ab), which can dimerize the receptor, the monovalent Fab of Anti-MUC1* (Monovalent Ab), that blocks receptor dimerization, and/or basic fibroblast growth factor (bFGF). The results were quantified as follows. A–F. A live/dead (green/red) calcein assay was performed 41 hours post treatment. Photos record the results. A. Treatment with bivalent Anti-MUC1* (Bivalent Ab) and bFGF produced mostly viable cells (green) and very few dead cells (red). B. Treatment with the monovalent Fab of Anti-MUC1* (Monovalent Ab) and bFGF resulted in essentially total cell death within 12 hours. C. Treatment with bFGF alone produced mostly viable cells. D. Treatment with bivalent Anti-MUC1* alone produced more viable cells than with the addition of bFGF. E. Treatment with monovalent Anti-MUC1* killed essentially all cells. F. Treatment without antibodies or bFGF resulted in more dead cells and less viable cells. G. A bar graph shows that after 41 hours of treatment with Anti-MUC1* (Bivalent Antibody) there were more than 2-times the number of live cells than without the antibody (No Ab). Treatment with the monovalent Fab (Monovalent Ab) killed all the cells. Fluorescence of live cells in a calcein assay is plotted. Data are represented as mean fluorescence units±SEM. H. The percentage hESC growth is plotted for undifferentiated cells grown in the presence or absence of bFGF and with bivalent or monovalent Anti-MUC1* (Bivalent Ab, Monovalent Ab). Student's two-tailed test was used for statistical analysis. The graph shows that Anti-MUC1* with or without bFGF stimulated growth roughly twice as well as with bFGF.
Mentions: We performed similar experiments to determine whether ligands of MUC1* could mediate the growth of pluripotent hESCs. Undifferentiated stem cells were grown on matrigel-coated wells and cultured according to current methods which included feeding with minimal stem cell media that had been supplemented with 30% conditioned media from Hs27 fibroblast feeder cells. Cells were treated with bivalent Anti-MUC1* or the monovalent Fab, in the presence or absence of exogenous bFGF. The addition of Anti-MUC1* to undifferentiated hESCs had a dramatic, stimulatory effect on cell growth. Treating hESCs with bivalent Anti-MUC1* for forty-one (41) hours, in the presence or absence of added bFGF, resulted in cells that were more viable and abundant than control cells that were cultured according to standard methods, which included adding bFGF. In stark contrast, the addition of the monovalent Fab fragment of Anti-MUC1* resulted in nearly total cell death within 12 hours of treatment (Fig. 7A–F). Presumably the monovalent Fab competed with MUC1*'s native ligand, NM23, for binding and blocked receptor dimerization. The growth effects of the MUC1*-targeting antibodies were quantified by measuring the fluorescence at 530 nm for live cells grown under each test condition. In the absence of added exogenous bFGF, the addition of bivalent Anti-MUC1* resulted in a greater than 2-fold enhancement of cell growth compared to the control (Fig. 7G). A plot of normalized cell growth with added bFGF defined as 100%, is shown in Figure 7H. Bivalent Anti-MUC1* greatly enhances the growth of undifferentiated stem cells and does not require the addition of exogenous bFGF. Notably, the addition of bFGF cannot rescue stem cells when treated with monovalent Anti-MUC1*. A similar experiment was performed in which the effects of bivalent Anti-MUC1*, its monovalent Fab, and a control Fab, on H9 and on H14 hESCs were measured (Fig. S5 A,B). The results were essentially the same as those depicted in Figure 7.

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