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Models of breast morphogenesis based on localization of stem cells in the developing mammary lobule.

Honeth G, Schiavinotto T, Vaggi F, Marlow R, Kanno T, Shinomiya I, Lombardi S, Buchupalli B, Graham R, Gazinska P, Ramalingam V, Burchell J, Purushotham AD, Pinder SE, Csikasz-Nagy A, Dontu G - Stem Cell Reports (2015)

Bottom Line: However, the identity of these cells is a subject of controversy and their localization in the breast epithelium is not known.In this study, we utilized a novel approach to analyze the morphogenesis of mammary lobules, by combining one-dimensional theoretical models and computer-generated 3D fractals.An increased representation of stem cells was found in smaller, less developed lobules compared to larger, more mature lobules, with marked differences in the gland of iparous versus parous women and that of BRCA1/2 mutation carriers versus non-carriers.

View Article: PubMed Central - PubMed

Affiliation: Research Oncology, King's College London School of Medicine, London SE1 9RT, UK. Electronic address: gabriella.honeth@med.lu.se.

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Putative Stem Cell Markers Co-localize in the Normal Breast Epithelium(A) Staining for putative stem cell phenotypes on consecutive sections from normal breast. Pictures show an area with overlap among CD44+, SSEA4+, CK14+CK19+, and ALDH1A1+ phenotypes. CD49f is not expressed in this area. Representative example from six different mammoplasties is shown.(B) Quantification of other marker phenotypes in all ALDH1A1+ cell clusters (n = 61) across six different mammoplasty samples, showing high co-localization of ALDH1A1+, SSEA4+, and CK14+CK19+ phenotypes. EpCAM and CD44 were expressed in about two-thirds and one-third of the ALDH1A1+ cell clusters, respectively, while CD49f and ALDH1A3 were never expressed in these cell clusters. Bars represent mean percentage of each marker presence in the ALDH1A1+ islands, across the six samples, ± SEM.(C and D) Double IF staining for ALDH1A1 together with SSEA4 and CD44, respectively. ALDH1A1 co-localizes with SSEA4 (C) and CD44high (D) in distinct cell clusters. Representative examples from stainings of four (SSEA4) and seven (CD44) different samples, respectively, are shown.(E) Triple IF staining for ALDH1A1 (stained with AlexaFluor647 and detected in the far red filter, shown in red for clarity), CK14 (green), and CK19 (red, shown separate to the far right) illustrating overlap of these three markers in distinct clusters of cells. Representative example from stainings of six different samples is shown.(F and G) Schematic and quantitative overlap of the ALDH1A1+, SSEA4+, CK14+CK19+, and CD44+ phenotypes. The letters in (F) indicate number of additional marker phenotypes co-expressed in ALDH1A1+ areas. Bars in (G) represent mean percentage of areas with indicated number of additional marker phenotypes positive in ALDH1A1+ areas, across six samples, ± SEM.(H) Triple IF staining for ALDH1A1 (stained with AlexaFluor647 and detected in the far red filter, shown in green for clarity), CD49f (red), and EpCAM (green), illustrating the lack of overlap between ALDH1A1+ and CD49fhigh phenotypes. Although ALDH1A1 and CD49f can sometimes be expressed in close proximity, they are not expressed in the same cells (top). EpCAM also was expressed in this area, as illustrated by the far right picture showing EpCAM staining separately. The CD49fhighEpCAMlow cells in the basal layer are negative for ALDH1A1 (bottom, far right). Representative examples from stainings of six different samples are shown.(I) Double immunofluorescent staining for ALDH1A1 and CD10 showing that, in the areas where ALDH1A1 is expressed, there are no, or very few, CD10+ myoepithelial cells (arrow), while high level CD10+ myoepithelial cells can be seen in neighboring acini (arrowhead). Representative example from stainings of four different samples is shown.Scale bar, 100 μm in (A) and 50 μm in (E–I). Blue nuclear staining in fluorescence pictures is DAPI.See also Figures S3 and S4.
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fig2: Putative Stem Cell Markers Co-localize in the Normal Breast Epithelium(A) Staining for putative stem cell phenotypes on consecutive sections from normal breast. Pictures show an area with overlap among CD44+, SSEA4+, CK14+CK19+, and ALDH1A1+ phenotypes. CD49f is not expressed in this area. Representative example from six different mammoplasties is shown.(B) Quantification of other marker phenotypes in all ALDH1A1+ cell clusters (n = 61) across six different mammoplasty samples, showing high co-localization of ALDH1A1+, SSEA4+, and CK14+CK19+ phenotypes. EpCAM and CD44 were expressed in about two-thirds and one-third of the ALDH1A1+ cell clusters, respectively, while CD49f and ALDH1A3 were never expressed in these cell clusters. Bars represent mean percentage of each marker presence in the ALDH1A1+ islands, across the six samples, ± SEM.(C and D) Double IF staining for ALDH1A1 together with SSEA4 and CD44, respectively. ALDH1A1 co-localizes with SSEA4 (C) and CD44high (D) in distinct cell clusters. Representative examples from stainings of four (SSEA4) and seven (CD44) different samples, respectively, are shown.(E) Triple IF staining for ALDH1A1 (stained with AlexaFluor647 and detected in the far red filter, shown in red for clarity), CK14 (green), and CK19 (red, shown separate to the far right) illustrating overlap of these three markers in distinct clusters of cells. Representative example from stainings of six different samples is shown.(F and G) Schematic and quantitative overlap of the ALDH1A1+, SSEA4+, CK14+CK19+, and CD44+ phenotypes. The letters in (F) indicate number of additional marker phenotypes co-expressed in ALDH1A1+ areas. Bars in (G) represent mean percentage of areas with indicated number of additional marker phenotypes positive in ALDH1A1+ areas, across six samples, ± SEM.(H) Triple IF staining for ALDH1A1 (stained with AlexaFluor647 and detected in the far red filter, shown in green for clarity), CD49f (red), and EpCAM (green), illustrating the lack of overlap between ALDH1A1+ and CD49fhigh phenotypes. Although ALDH1A1 and CD49f can sometimes be expressed in close proximity, they are not expressed in the same cells (top). EpCAM also was expressed in this area, as illustrated by the far right picture showing EpCAM staining separately. The CD49fhighEpCAMlow cells in the basal layer are negative for ALDH1A1 (bottom, far right). Representative examples from stainings of six different samples are shown.(I) Double immunofluorescent staining for ALDH1A1 and CD10 showing that, in the areas where ALDH1A1 is expressed, there are no, or very few, CD10+ myoepithelial cells (arrow), while high level CD10+ myoepithelial cells can be seen in neighboring acini (arrowhead). Representative example from stainings of four different samples is shown.Scale bar, 100 μm in (A) and 50 μm in (E–I). Blue nuclear staining in fluorescence pictures is DAPI.See also Figures S3 and S4.

Mentions: To determine possible overlaps among these phenotypes, we stained a series of consecutive sections from six different reduction mammoplasties (Figures 2A and S4). Furthermore, analysis of pairs of markers was done on samples from additional patients. Overall, a total of 18 samples were used; each combination of markers was analyzed in at least nine different samples. Clinical characteristics of the patients are presented in the Supplemental Experimental Procedures. We detected a strikingly clear overlap among staining patterns for ALDH1A1+, SSEA4+, and CK14+CK19+ in all the samples analyzed (Figures 2A and S4). To quantify this co-localization, we identified ALDH1A1+ areas across sections from the consecutively stained samples and analyzed the rest of the markers in the same areas on consecutive sections (Figure 2B). SSEA4+ and CK14+CK19+ staining was detected in 83% and 69% of the ALDH1A1+ areas, respectively. CD44 was present in 38% of these areas, at a higher level compared to the surrounding epithelium and in a luminal position. In the rest of the mammary epithelium tree, CD44 was expressed ubiquitously in the basal layer. SSEA4 was detected in distinct clusters of cells in cytoplasmic location. These cells also expressed ALDH1A1. Additionally, SSEA4 was detected as an apical luminal staining in a minority of the samples (Figure S3C). This apical staining did not overlap with the expression of ALDH1A1.


Models of breast morphogenesis based on localization of stem cells in the developing mammary lobule.

Honeth G, Schiavinotto T, Vaggi F, Marlow R, Kanno T, Shinomiya I, Lombardi S, Buchupalli B, Graham R, Gazinska P, Ramalingam V, Burchell J, Purushotham AD, Pinder SE, Csikasz-Nagy A, Dontu G - Stem Cell Reports (2015)

Putative Stem Cell Markers Co-localize in the Normal Breast Epithelium(A) Staining for putative stem cell phenotypes on consecutive sections from normal breast. Pictures show an area with overlap among CD44+, SSEA4+, CK14+CK19+, and ALDH1A1+ phenotypes. CD49f is not expressed in this area. Representative example from six different mammoplasties is shown.(B) Quantification of other marker phenotypes in all ALDH1A1+ cell clusters (n = 61) across six different mammoplasty samples, showing high co-localization of ALDH1A1+, SSEA4+, and CK14+CK19+ phenotypes. EpCAM and CD44 were expressed in about two-thirds and one-third of the ALDH1A1+ cell clusters, respectively, while CD49f and ALDH1A3 were never expressed in these cell clusters. Bars represent mean percentage of each marker presence in the ALDH1A1+ islands, across the six samples, ± SEM.(C and D) Double IF staining for ALDH1A1 together with SSEA4 and CD44, respectively. ALDH1A1 co-localizes with SSEA4 (C) and CD44high (D) in distinct cell clusters. Representative examples from stainings of four (SSEA4) and seven (CD44) different samples, respectively, are shown.(E) Triple IF staining for ALDH1A1 (stained with AlexaFluor647 and detected in the far red filter, shown in red for clarity), CK14 (green), and CK19 (red, shown separate to the far right) illustrating overlap of these three markers in distinct clusters of cells. Representative example from stainings of six different samples is shown.(F and G) Schematic and quantitative overlap of the ALDH1A1+, SSEA4+, CK14+CK19+, and CD44+ phenotypes. The letters in (F) indicate number of additional marker phenotypes co-expressed in ALDH1A1+ areas. Bars in (G) represent mean percentage of areas with indicated number of additional marker phenotypes positive in ALDH1A1+ areas, across six samples, ± SEM.(H) Triple IF staining for ALDH1A1 (stained with AlexaFluor647 and detected in the far red filter, shown in green for clarity), CD49f (red), and EpCAM (green), illustrating the lack of overlap between ALDH1A1+ and CD49fhigh phenotypes. Although ALDH1A1 and CD49f can sometimes be expressed in close proximity, they are not expressed in the same cells (top). EpCAM also was expressed in this area, as illustrated by the far right picture showing EpCAM staining separately. The CD49fhighEpCAMlow cells in the basal layer are negative for ALDH1A1 (bottom, far right). Representative examples from stainings of six different samples are shown.(I) Double immunofluorescent staining for ALDH1A1 and CD10 showing that, in the areas where ALDH1A1 is expressed, there are no, or very few, CD10+ myoepithelial cells (arrow), while high level CD10+ myoepithelial cells can be seen in neighboring acini (arrowhead). Representative example from stainings of four different samples is shown.Scale bar, 100 μm in (A) and 50 μm in (E–I). Blue nuclear staining in fluorescence pictures is DAPI.See also Figures S3 and S4.
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fig2: Putative Stem Cell Markers Co-localize in the Normal Breast Epithelium(A) Staining for putative stem cell phenotypes on consecutive sections from normal breast. Pictures show an area with overlap among CD44+, SSEA4+, CK14+CK19+, and ALDH1A1+ phenotypes. CD49f is not expressed in this area. Representative example from six different mammoplasties is shown.(B) Quantification of other marker phenotypes in all ALDH1A1+ cell clusters (n = 61) across six different mammoplasty samples, showing high co-localization of ALDH1A1+, SSEA4+, and CK14+CK19+ phenotypes. EpCAM and CD44 were expressed in about two-thirds and one-third of the ALDH1A1+ cell clusters, respectively, while CD49f and ALDH1A3 were never expressed in these cell clusters. Bars represent mean percentage of each marker presence in the ALDH1A1+ islands, across the six samples, ± SEM.(C and D) Double IF staining for ALDH1A1 together with SSEA4 and CD44, respectively. ALDH1A1 co-localizes with SSEA4 (C) and CD44high (D) in distinct cell clusters. Representative examples from stainings of four (SSEA4) and seven (CD44) different samples, respectively, are shown.(E) Triple IF staining for ALDH1A1 (stained with AlexaFluor647 and detected in the far red filter, shown in red for clarity), CK14 (green), and CK19 (red, shown separate to the far right) illustrating overlap of these three markers in distinct clusters of cells. Representative example from stainings of six different samples is shown.(F and G) Schematic and quantitative overlap of the ALDH1A1+, SSEA4+, CK14+CK19+, and CD44+ phenotypes. The letters in (F) indicate number of additional marker phenotypes co-expressed in ALDH1A1+ areas. Bars in (G) represent mean percentage of areas with indicated number of additional marker phenotypes positive in ALDH1A1+ areas, across six samples, ± SEM.(H) Triple IF staining for ALDH1A1 (stained with AlexaFluor647 and detected in the far red filter, shown in green for clarity), CD49f (red), and EpCAM (green), illustrating the lack of overlap between ALDH1A1+ and CD49fhigh phenotypes. Although ALDH1A1 and CD49f can sometimes be expressed in close proximity, they are not expressed in the same cells (top). EpCAM also was expressed in this area, as illustrated by the far right picture showing EpCAM staining separately. The CD49fhighEpCAMlow cells in the basal layer are negative for ALDH1A1 (bottom, far right). Representative examples from stainings of six different samples are shown.(I) Double immunofluorescent staining for ALDH1A1 and CD10 showing that, in the areas where ALDH1A1 is expressed, there are no, or very few, CD10+ myoepithelial cells (arrow), while high level CD10+ myoepithelial cells can be seen in neighboring acini (arrowhead). Representative example from stainings of four different samples is shown.Scale bar, 100 μm in (A) and 50 μm in (E–I). Blue nuclear staining in fluorescence pictures is DAPI.See also Figures S3 and S4.
Mentions: To determine possible overlaps among these phenotypes, we stained a series of consecutive sections from six different reduction mammoplasties (Figures 2A and S4). Furthermore, analysis of pairs of markers was done on samples from additional patients. Overall, a total of 18 samples were used; each combination of markers was analyzed in at least nine different samples. Clinical characteristics of the patients are presented in the Supplemental Experimental Procedures. We detected a strikingly clear overlap among staining patterns for ALDH1A1+, SSEA4+, and CK14+CK19+ in all the samples analyzed (Figures 2A and S4). To quantify this co-localization, we identified ALDH1A1+ areas across sections from the consecutively stained samples and analyzed the rest of the markers in the same areas on consecutive sections (Figure 2B). SSEA4+ and CK14+CK19+ staining was detected in 83% and 69% of the ALDH1A1+ areas, respectively. CD44 was present in 38% of these areas, at a higher level compared to the surrounding epithelium and in a luminal position. In the rest of the mammary epithelium tree, CD44 was expressed ubiquitously in the basal layer. SSEA4 was detected in distinct clusters of cells in cytoplasmic location. These cells also expressed ALDH1A1. Additionally, SSEA4 was detected as an apical luminal staining in a minority of the samples (Figure S3C). This apical staining did not overlap with the expression of ALDH1A1.

Bottom Line: However, the identity of these cells is a subject of controversy and their localization in the breast epithelium is not known.In this study, we utilized a novel approach to analyze the morphogenesis of mammary lobules, by combining one-dimensional theoretical models and computer-generated 3D fractals.An increased representation of stem cells was found in smaller, less developed lobules compared to larger, more mature lobules, with marked differences in the gland of iparous versus parous women and that of BRCA1/2 mutation carriers versus non-carriers.

View Article: PubMed Central - PubMed

Affiliation: Research Oncology, King's College London School of Medicine, London SE1 9RT, UK. Electronic address: gabriella.honeth@med.lu.se.

Show MeSH
Related in: MedlinePlus