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Identification of Stem Cell Units in the Terminal End Bud and Duct of the Mouse Mammary Gland.

Kenney NJ, Smith GH, Lawrence E, Barrett JC, Salomon DS - J. Biomed. Biotechnol. (2001)

Bottom Line: Additionally, TUs are located every 250 +/- 75 &mgr;m in ducts or in the terminal end bud 200-600 &mgr;m in diameter.Molecules expressed in TUs were Zonula Occludens-1 and alpha-catenin proteins which were significantly detected in 75%-91% (P <0.001)of the LRCs cells that make up the TU.These data suggest that transitional units may be a group of label-retaining stem cells and maybe involved in the developmental or cancer process.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
The mouse mammary gland may undergo cycles of proliferation, terminal differentiation, tissue remodeling, and more importantly malignant transformation.Mammary epithelial stem cells and their progeny participate in these processes.Mammary epithelial stem cells are multipotent, exhibit properties of self renewal (up to 7 divisions)and may exist either as long-lived nondividing cells or as proliferating-differentiating cells. The focus of this study was to locate stem cells by identifying them as long-lived, label-retaining mammary epithelial cells (LRCs)in growth active (developing)or growth static (aged)mammary ducts. Initially, primary epithelial cells were pulse labeled with either fluorescent tracker dye and/or BrdU. Cells were then transplanted into cleared juvenile syngeneic mammary fat pads and held for 5 weeks or 8 weeks. In this study, we demonstrate that LRCs are stem cells and their progeny (transitional cells)are arranged as transitional units (TUs). Additionally, TUs are located every 250 +/- 75 &mgr;m in ducts or in the terminal end bud 200-600 &mgr;m in diameter. Molecules expressed in TUs were Zonula Occludens-1 and alpha-catenin proteins which were significantly detected in 75%-91% (P <0.001)of the LRCs cells that make up the TU. These data suggest that transitional units may be a group of label-retaining stem cells and maybe involved in the developmental or cancer process.

No MeSH data available.


Related in: MedlinePlus

Graph depicting the number of divisions HC-11 cells undergo while losing TLC label. In cells that reached 5 ± 1 generations, over 80% of TLC label was lost. HC-11 cell morphologies (□) and C57BL/6N mouse primary epithelial cultures (□) that were scanned for fluorescent intensities and grown according to materials and methods. Inset, bright TLC labeled stem cell surrounded by differentiated progeny using IF (A), phase contrast (B) and bright field microscopy (C). Arrow in (C) represents pale counterstained stem cell. (D), Differentiated cell and its nearby differentiated daughter cell. (E), One of 4 differentiated HC-11 morphologies that can be labeled using the TRITC cell linker. Scale bars (A–E) 15 μm.
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Figure 1: Graph depicting the number of divisions HC-11 cells undergo while losing TLC label. In cells that reached 5 ± 1 generations, over 80% of TLC label was lost. HC-11 cell morphologies (□) and C57BL/6N mouse primary epithelial cultures (□) that were scanned for fluorescent intensities and grown according to materials and methods. Inset, bright TLC labeled stem cell surrounded by differentiated progeny using IF (A), phase contrast (B) and bright field microscopy (C). Arrow in (C) represents pale counterstained stem cell. (D), Differentiated cell and its nearby differentiated daughter cell. (E), One of 4 differentiated HC-11 morphologies that can be labeled using the TRITC cell linker. Scale bars (A–E) 15 μm.

Mentions: In order to identify the location of stem cells as long-lived label-retaining mammary epithelial cells (LRCs) in the mammary duct in vivo, BrdU [29] and BKH26-GL [26], were utilized as tracking markers. PKH26-GL is a fluorescent cell linker compound (Excitation max = 551 nm, Emission max = 587) that incorporates aliphatic reporter molecules into the cell membrane by selective partitioning [26]. The cells can be visualized under the TRITC channel during immunofluorescence of confocal microscopy. The technology has been limited to a number of model systems in vitro and in vivo. The half-life of elution of PKH26 from rabbit red blood cells is greater than 100 days in vivo. Labeled cells usually vary from bright and uniform labeling to a punctuate or patchy appearance. In addition, the labeling is not a saturation reaction but a function of both dye and cellconcentration, therefore 2x10-6M of linker was used in the experiment. Over-labeling of the cells would result in loss of membrane integrity and cell recovery. We have also evaluated the uniformity of staining, cell viability (by propidium staining), and the coefficient of variation of fluorescent peaks to maximize the optimum dye/cell kinetics [26, 29]. Initially, TRITC-cell linker labeled cells (TLC) were examined for labeling potential in vitro with the nontransformed immortalized mouse mammary epithelial HC-11 cell line. Cells were pulsed for3 min with TLC and grown as single cells in 96 well plates for 5 days. Following cell fixation and histological counterstaining, the number of cell divisions which is inversely proportional to fluorescent signal was calculated using the image-based format developed by NIH Image. The data suggests that following the first 24 h of growth, cells lose 40% ±10% of TLC signal after their first cell division (Figure 1). Following the second cell division, HC-11 cells lose an additional 20% of label until reaching basal levels (6–7 cell divisions) (P > 0.05). Similar results were also observed in pulse-labeled mammary epithelial primary cultures established from female FVB mice (Figure 1). The results suggest that the TLC method can be used to track the number of cell divisions in both mammary cell lines and mammary cell cultures in vitro.


Identification of Stem Cell Units in the Terminal End Bud and Duct of the Mouse Mammary Gland.

Kenney NJ, Smith GH, Lawrence E, Barrett JC, Salomon DS - J. Biomed. Biotechnol. (2001)

Graph depicting the number of divisions HC-11 cells undergo while losing TLC label. In cells that reached 5 ± 1 generations, over 80% of TLC label was lost. HC-11 cell morphologies (□) and C57BL/6N mouse primary epithelial cultures (□) that were scanned for fluorescent intensities and grown according to materials and methods. Inset, bright TLC labeled stem cell surrounded by differentiated progeny using IF (A), phase contrast (B) and bright field microscopy (C). Arrow in (C) represents pale counterstained stem cell. (D), Differentiated cell and its nearby differentiated daughter cell. (E), One of 4 differentiated HC-11 morphologies that can be labeled using the TRITC cell linker. Scale bars (A–E) 15 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC129060&req=5

Figure 1: Graph depicting the number of divisions HC-11 cells undergo while losing TLC label. In cells that reached 5 ± 1 generations, over 80% of TLC label was lost. HC-11 cell morphologies (□) and C57BL/6N mouse primary epithelial cultures (□) that were scanned for fluorescent intensities and grown according to materials and methods. Inset, bright TLC labeled stem cell surrounded by differentiated progeny using IF (A), phase contrast (B) and bright field microscopy (C). Arrow in (C) represents pale counterstained stem cell. (D), Differentiated cell and its nearby differentiated daughter cell. (E), One of 4 differentiated HC-11 morphologies that can be labeled using the TRITC cell linker. Scale bars (A–E) 15 μm.
Mentions: In order to identify the location of stem cells as long-lived label-retaining mammary epithelial cells (LRCs) in the mammary duct in vivo, BrdU [29] and BKH26-GL [26], were utilized as tracking markers. PKH26-GL is a fluorescent cell linker compound (Excitation max = 551 nm, Emission max = 587) that incorporates aliphatic reporter molecules into the cell membrane by selective partitioning [26]. The cells can be visualized under the TRITC channel during immunofluorescence of confocal microscopy. The technology has been limited to a number of model systems in vitro and in vivo. The half-life of elution of PKH26 from rabbit red blood cells is greater than 100 days in vivo. Labeled cells usually vary from bright and uniform labeling to a punctuate or patchy appearance. In addition, the labeling is not a saturation reaction but a function of both dye and cellconcentration, therefore 2x10-6M of linker was used in the experiment. Over-labeling of the cells would result in loss of membrane integrity and cell recovery. We have also evaluated the uniformity of staining, cell viability (by propidium staining), and the coefficient of variation of fluorescent peaks to maximize the optimum dye/cell kinetics [26, 29]. Initially, TRITC-cell linker labeled cells (TLC) were examined for labeling potential in vitro with the nontransformed immortalized mouse mammary epithelial HC-11 cell line. Cells were pulsed for3 min with TLC and grown as single cells in 96 well plates for 5 days. Following cell fixation and histological counterstaining, the number of cell divisions which is inversely proportional to fluorescent signal was calculated using the image-based format developed by NIH Image. The data suggests that following the first 24 h of growth, cells lose 40% ±10% of TLC signal after their first cell division (Figure 1). Following the second cell division, HC-11 cells lose an additional 20% of label until reaching basal levels (6–7 cell divisions) (P > 0.05). Similar results were also observed in pulse-labeled mammary epithelial primary cultures established from female FVB mice (Figure 1). The results suggest that the TLC method can be used to track the number of cell divisions in both mammary cell lines and mammary cell cultures in vitro.

Bottom Line: Additionally, TUs are located every 250 +/- 75 &mgr;m in ducts or in the terminal end bud 200-600 &mgr;m in diameter.Molecules expressed in TUs were Zonula Occludens-1 and alpha-catenin proteins which were significantly detected in 75%-91% (P <0.001)of the LRCs cells that make up the TU.These data suggest that transitional units may be a group of label-retaining stem cells and maybe involved in the developmental or cancer process.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
The mouse mammary gland may undergo cycles of proliferation, terminal differentiation, tissue remodeling, and more importantly malignant transformation.Mammary epithelial stem cells and their progeny participate in these processes.Mammary epithelial stem cells are multipotent, exhibit properties of self renewal (up to 7 divisions)and may exist either as long-lived nondividing cells or as proliferating-differentiating cells. The focus of this study was to locate stem cells by identifying them as long-lived, label-retaining mammary epithelial cells (LRCs)in growth active (developing)or growth static (aged)mammary ducts. Initially, primary epithelial cells were pulse labeled with either fluorescent tracker dye and/or BrdU. Cells were then transplanted into cleared juvenile syngeneic mammary fat pads and held for 5 weeks or 8 weeks. In this study, we demonstrate that LRCs are stem cells and their progeny (transitional cells)are arranged as transitional units (TUs). Additionally, TUs are located every 250 +/- 75 &mgr;m in ducts or in the terminal end bud 200-600 &mgr;m in diameter. Molecules expressed in TUs were Zonula Occludens-1 and alpha-catenin proteins which were significantly detected in 75%-91% (P <0.001)of the LRCs cells that make up the TU. These data suggest that transitional units may be a group of label-retaining stem cells and maybe involved in the developmental or cancer process.

No MeSH data available.


Related in: MedlinePlus