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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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Minimal media plus Anti-MUC1* supports the long-term growth of 100% pluripotent hESCs without added bFGF or conditioned media.H9 hESCs were plated at very low density (4×104 cells per chamber slide well) over matrigel-coated wells then grown for 5 weeks under a variety of test conditions. The resulted colonies were stained with antibodies against OCT4 and DAPI to assess pluripotency. Photomicrographs are shown. A,B. The photo shows a single colony that completely filled the well of the chamber slide when hESCs were cultured in minimal stem cell medium to which Anti-MUC1* was added. OCT4 and DAPI staininig showed that 100% of the cells were pluripotent. The addition of bFGF or Anti-MUC1* and bFGF cultured under the same conditions produced no pluripotent cell growth (data not shown). C,D. hESCS grown in media supplemented with Anti-MUC1* and media supplemented with fibroblast (Hs27)-conditioned medium produced partially as well as completely undifferentiated colonies (pictured). E,F. The addition of bFGF and conditioned medium from fibroblasts, which is standard hESC culture medium for feeder-free systems, produced only partially undifferentiated colonies; dotted lines indicate edge of pluripotent portion of the colony. G,H. Cells grown in conditioned media, bFGF and Anti-MUC1* addition resulted only in OCT4-negative, fibroblast-like cells. Scale bars = 100 µm.
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pone-0003312-g008: Minimal media plus Anti-MUC1* supports the long-term growth of 100% pluripotent hESCs without added bFGF or conditioned media.H9 hESCs were plated at very low density (4×104 cells per chamber slide well) over matrigel-coated wells then grown for 5 weeks under a variety of test conditions. The resulted colonies were stained with antibodies against OCT4 and DAPI to assess pluripotency. Photomicrographs are shown. A,B. The photo shows a single colony that completely filled the well of the chamber slide when hESCs were cultured in minimal stem cell medium to which Anti-MUC1* was added. OCT4 and DAPI staininig showed that 100% of the cells were pluripotent. The addition of bFGF or Anti-MUC1* and bFGF cultured under the same conditions produced no pluripotent cell growth (data not shown). C,D. hESCS grown in media supplemented with Anti-MUC1* and media supplemented with fibroblast (Hs27)-conditioned medium produced partially as well as completely undifferentiated colonies (pictured). E,F. The addition of bFGF and conditioned medium from fibroblasts, which is standard hESC culture medium for feeder-free systems, produced only partially undifferentiated colonies; dotted lines indicate edge of pluripotent portion of the colony. G,H. Cells grown in conditioned media, bFGF and Anti-MUC1* addition resulted only in OCT4-negative, fibroblast-like cells. Scale bars = 100 µm.

Mentions: In the absence of conditioned media from fibroblast feeder cells, the only condition that supported the growth of pluripotent stem cells was the addition of Anti-MUC1*, alone (Table 1). The condition that included Anti-MUC1* and bFGF did not produce any pluripotent cells, nor did the addition of bFGF alone. The addition of Anti-MUC1* produced the first colony, the largest colony (completely covered the well) and after five weeks of stimulation with Anti-MUC1* it remained 100% positive for OCT4 (Fig. 8A,B). Stem cells grown under these conditions did not spontaneously differentiate. However, the withdrawal of the Anti-MUC1* antibody, after five weeks, did induce the onset of differentiation. None of the other conditions tested produced any OCT4-positive cells when grown in the absence of conditioned media from fibroblasts. Stem cells grown in minimal media that had been supplemented with conditioned media from fibroblast feeder cells produced a mixture of undifferentiated and differentiated colonies in response to treatment with Anti-MUC1* (Fig. 8C,D) or bFGF (Fig. 8E,F). However, treatment with Anti-MUC1* produced colonies sooner and produced more undifferentiated cells than treatment with bFGF. Long-term growth supplemented with bFGF and Anti-MUC1, together, did not support the growth of undifferentiated colonies in either media (Fig. 8E,F).


MUC1* mediates the growth of human pluripotent stem cells.

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

Minimal media plus Anti-MUC1* supports the long-term growth of 100% pluripotent hESCs without added bFGF or conditioned media.H9 hESCs were plated at very low density (4×104 cells per chamber slide well) over matrigel-coated wells then grown for 5 weeks under a variety of test conditions. The resulted colonies were stained with antibodies against OCT4 and DAPI to assess pluripotency. Photomicrographs are shown. A,B. The photo shows a single colony that completely filled the well of the chamber slide when hESCs were cultured in minimal stem cell medium to which Anti-MUC1* was added. OCT4 and DAPI staininig showed that 100% of the cells were pluripotent. The addition of bFGF or Anti-MUC1* and bFGF cultured under the same conditions produced no pluripotent cell growth (data not shown). C,D. hESCS grown in media supplemented with Anti-MUC1* and media supplemented with fibroblast (Hs27)-conditioned medium produced partially as well as completely undifferentiated colonies (pictured). E,F. The addition of bFGF and conditioned medium from fibroblasts, which is standard hESC culture medium for feeder-free systems, produced only partially undifferentiated colonies; dotted lines indicate edge of pluripotent portion of the colony. G,H. Cells grown in conditioned media, bFGF and Anti-MUC1* addition resulted only in OCT4-negative, fibroblast-like cells. Scale bars = 100 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553196&req=5

pone-0003312-g008: Minimal media plus Anti-MUC1* supports the long-term growth of 100% pluripotent hESCs without added bFGF or conditioned media.H9 hESCs were plated at very low density (4×104 cells per chamber slide well) over matrigel-coated wells then grown for 5 weeks under a variety of test conditions. The resulted colonies were stained with antibodies against OCT4 and DAPI to assess pluripotency. Photomicrographs are shown. A,B. The photo shows a single colony that completely filled the well of the chamber slide when hESCs were cultured in minimal stem cell medium to which Anti-MUC1* was added. OCT4 and DAPI staininig showed that 100% of the cells were pluripotent. The addition of bFGF or Anti-MUC1* and bFGF cultured under the same conditions produced no pluripotent cell growth (data not shown). C,D. hESCS grown in media supplemented with Anti-MUC1* and media supplemented with fibroblast (Hs27)-conditioned medium produced partially as well as completely undifferentiated colonies (pictured). E,F. The addition of bFGF and conditioned medium from fibroblasts, which is standard hESC culture medium for feeder-free systems, produced only partially undifferentiated colonies; dotted lines indicate edge of pluripotent portion of the colony. G,H. Cells grown in conditioned media, bFGF and Anti-MUC1* addition resulted only in OCT4-negative, fibroblast-like cells. Scale bars = 100 µm.
Mentions: In the absence of conditioned media from fibroblast feeder cells, the only condition that supported the growth of pluripotent stem cells was the addition of Anti-MUC1*, alone (Table 1). The condition that included Anti-MUC1* and bFGF did not produce any pluripotent cells, nor did the addition of bFGF alone. The addition of Anti-MUC1* produced the first colony, the largest colony (completely covered the well) and after five weeks of stimulation with Anti-MUC1* it remained 100% positive for OCT4 (Fig. 8A,B). Stem cells grown under these conditions did not spontaneously differentiate. However, the withdrawal of the Anti-MUC1* antibody, after five weeks, did induce the onset of differentiation. None of the other conditions tested produced any OCT4-positive cells when grown in the absence of conditioned media from fibroblasts. Stem cells grown in minimal media that had been supplemented with conditioned media from fibroblast feeder cells produced a mixture of undifferentiated and differentiated colonies in response to treatment with Anti-MUC1* (Fig. 8C,D) or bFGF (Fig. 8E,F). However, treatment with Anti-MUC1* produced colonies sooner and produced more undifferentiated cells than treatment with bFGF. Long-term growth supplemented with bFGF and Anti-MUC1, together, did not support the growth of undifferentiated colonies in either media (Fig. 8E,F).

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