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Following damage, the majority of bone marrow-derived airway cells express an epithelial marker.

MacPherson H, Keir PA, Edwards CJ, Webb S, Dorin JR - Respir. Res. (2006)

Bottom Line: These results were reinforced using deconvolved microscopy and scored by two independent investigators.Using cytokeratin and the universal haematopoietic marker CD45 immunohistochemistry, we find the donor derived cells fall into four phenotypic classes.The cytokeratin positive donor derived cells in the tracheal epithelium are not present in the injected donor cells and must have acquired this novel phenotype in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Human Genetics Unit, Western General Hospital, Crewe Road South, Edinburgh EH4 2XU, UK. heather.macpherson@hgu.mrc.ac.uk

ABSTRACT

Background: Adult-derived bone marrow stem cells are capable of reconstituting the haematopoietic system. However there is ongoing debate in the literature as to whether bone marrow derived cells have the ability to populate other tissues and express tissue specific markers. The airway has been an organ of major interest and was one of the first where this was demonstrated. We have previously demonstrated that the mouse airway can be repopulated by side population bone marrow transplanted cells. Here we investigate the frequency and phenotypic nature of these bone marrow derived cells.

Methods: Female mice were engrafted with male whole bone marrow or side population (SP) cells and subjected to detergent-induced damage after 3 months. Donor cells were identified by Y chromosome fluorescence in situ hybridisation and their phenotype was assessed by immunohistochemistry on the same sections. Slides were visualised by a combination of widefield and deconvolved microscopy and whole cells were analysed on cytospin preparations.

Results: The frequencies of engraftment of male cells in the airway of mice that show this (9/10), range from 1.0-1.6% with whole marrow and 0.6-1.5% with SP cells. Undamaged controls have only between 0.1 and 0.2% male cells in the trachea. By widefield microscopy analysis we find 60.2% (53/88) of male donor derived cells express cytokeratins as a marker of epithelial cells. These results were reinforced using deconvolved microscopy and scored by two independent investigators. In addition cytospin analysis of cells dissociated from the damaged trachea of engrafted mice also reveals donor derived Y chromosome positive cells that are immunopositive for cytokeratin. Using cytokeratin and the universal haematopoietic marker CD45 immunohistochemistry, we find the donor derived cells fall into four phenotypic classes. We do not detect cytokeratin positive cells in whole bone marrow using cytokeratin immunostaining and we do not detect any cytokeratin mRNA in SP or bone marrow samples by RT-PCR.

Conclusion: The appearance of bone marrow derived cells in the tracheal epithelium is enriched by detergent-induced tissue damage and the majority of these cells express an epithelial marker. The cytokeratin positive donor derived cells in the tracheal epithelium are not present in the injected donor cells and must have acquired this novel phenotype in vivo.

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Co-localisation of Y FISH and cytokeratin immunosignal in single cells. Female animals engrafted with male bone marrow were sacrificed after three months and cells dissociated from the tracheas were cytospun onto slides and subjected to Y FISH (red) and cytokeratin immunocytochemistry (green). Y positive (donor derived) cells are arrowed and both Y chromosome positive and cytokeratin negative (panels A,G and I) and Y positive, cytokeratin positive cells (panel B,C and E) were observed. Non-arrowed cells in panels A and B are cytokeratin positive but not Y FISH positive and presumably host female derived. Panels C and G show donor cells (arrow) stained for presence of Y chromosome and then the slides were counterstained with H and E (panel D and H respectively). Panels E and I show the donor Y FISH positive cells (arrowed) with panels F and J showing the same cells stained with H and E. The donor cell (arrowed) in panels I and J is cytokeratin negative and has macrophage morphology.
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Figure 3: Co-localisation of Y FISH and cytokeratin immunosignal in single cells. Female animals engrafted with male bone marrow were sacrificed after three months and cells dissociated from the tracheas were cytospun onto slides and subjected to Y FISH (red) and cytokeratin immunocytochemistry (green). Y positive (donor derived) cells are arrowed and both Y chromosome positive and cytokeratin negative (panels A,G and I) and Y positive, cytokeratin positive cells (panel B,C and E) were observed. Non-arrowed cells in panels A and B are cytokeratin positive but not Y FISH positive and presumably host female derived. Panels C and G show donor cells (arrow) stained for presence of Y chromosome and then the slides were counterstained with H and E (panel D and H respectively). Panels E and I show the donor Y FISH positive cells (arrowed) with panels F and J showing the same cells stained with H and E. The donor cell (arrowed) in panels I and J is cytokeratin negative and has macrophage morphology.

Mentions: The colocalisation of Y chromosome FISH positivity and cytokeratin immunosignal was further confirmed by taking tracheal cell preparations from the 3 month engrafted whole marrow transplanted mice. Following damage all the 5 animals (W1-5) tested showed evidence of Y positive cells by FISH carried out on the cytospin preparations and Figure 3 clearly shows examples of cells that are Y FISH positive and either cytokeratin negative (panels A, G and I) or cytokeratin positive (panel B, C and E). Following H and E staining, it would appear that the donor cell visible in panels G and I is a macrophage (panel J).


Following damage, the majority of bone marrow-derived airway cells express an epithelial marker.

MacPherson H, Keir PA, Edwards CJ, Webb S, Dorin JR - Respir. Res. (2006)

Co-localisation of Y FISH and cytokeratin immunosignal in single cells. Female animals engrafted with male bone marrow were sacrificed after three months and cells dissociated from the tracheas were cytospun onto slides and subjected to Y FISH (red) and cytokeratin immunocytochemistry (green). Y positive (donor derived) cells are arrowed and both Y chromosome positive and cytokeratin negative (panels A,G and I) and Y positive, cytokeratin positive cells (panel B,C and E) were observed. Non-arrowed cells in panels A and B are cytokeratin positive but not Y FISH positive and presumably host female derived. Panels C and G show donor cells (arrow) stained for presence of Y chromosome and then the slides were counterstained with H and E (panel D and H respectively). Panels E and I show the donor Y FISH positive cells (arrowed) with panels F and J showing the same cells stained with H and E. The donor cell (arrowed) in panels I and J is cytokeratin negative and has macrophage morphology.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1764737&req=5

Figure 3: Co-localisation of Y FISH and cytokeratin immunosignal in single cells. Female animals engrafted with male bone marrow were sacrificed after three months and cells dissociated from the tracheas were cytospun onto slides and subjected to Y FISH (red) and cytokeratin immunocytochemistry (green). Y positive (donor derived) cells are arrowed and both Y chromosome positive and cytokeratin negative (panels A,G and I) and Y positive, cytokeratin positive cells (panel B,C and E) were observed. Non-arrowed cells in panels A and B are cytokeratin positive but not Y FISH positive and presumably host female derived. Panels C and G show donor cells (arrow) stained for presence of Y chromosome and then the slides were counterstained with H and E (panel D and H respectively). Panels E and I show the donor Y FISH positive cells (arrowed) with panels F and J showing the same cells stained with H and E. The donor cell (arrowed) in panels I and J is cytokeratin negative and has macrophage morphology.
Mentions: The colocalisation of Y chromosome FISH positivity and cytokeratin immunosignal was further confirmed by taking tracheal cell preparations from the 3 month engrafted whole marrow transplanted mice. Following damage all the 5 animals (W1-5) tested showed evidence of Y positive cells by FISH carried out on the cytospin preparations and Figure 3 clearly shows examples of cells that are Y FISH positive and either cytokeratin negative (panels A, G and I) or cytokeratin positive (panel B, C and E). Following H and E staining, it would appear that the donor cell visible in panels G and I is a macrophage (panel J).

Bottom Line: These results were reinforced using deconvolved microscopy and scored by two independent investigators.Using cytokeratin and the universal haematopoietic marker CD45 immunohistochemistry, we find the donor derived cells fall into four phenotypic classes.The cytokeratin positive donor derived cells in the tracheal epithelium are not present in the injected donor cells and must have acquired this novel phenotype in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Human Genetics Unit, Western General Hospital, Crewe Road South, Edinburgh EH4 2XU, UK. heather.macpherson@hgu.mrc.ac.uk

ABSTRACT

Background: Adult-derived bone marrow stem cells are capable of reconstituting the haematopoietic system. However there is ongoing debate in the literature as to whether bone marrow derived cells have the ability to populate other tissues and express tissue specific markers. The airway has been an organ of major interest and was one of the first where this was demonstrated. We have previously demonstrated that the mouse airway can be repopulated by side population bone marrow transplanted cells. Here we investigate the frequency and phenotypic nature of these bone marrow derived cells.

Methods: Female mice were engrafted with male whole bone marrow or side population (SP) cells and subjected to detergent-induced damage after 3 months. Donor cells were identified by Y chromosome fluorescence in situ hybridisation and their phenotype was assessed by immunohistochemistry on the same sections. Slides were visualised by a combination of widefield and deconvolved microscopy and whole cells were analysed on cytospin preparations.

Results: The frequencies of engraftment of male cells in the airway of mice that show this (9/10), range from 1.0-1.6% with whole marrow and 0.6-1.5% with SP cells. Undamaged controls have only between 0.1 and 0.2% male cells in the trachea. By widefield microscopy analysis we find 60.2% (53/88) of male donor derived cells express cytokeratins as a marker of epithelial cells. These results were reinforced using deconvolved microscopy and scored by two independent investigators. In addition cytospin analysis of cells dissociated from the damaged trachea of engrafted mice also reveals donor derived Y chromosome positive cells that are immunopositive for cytokeratin. Using cytokeratin and the universal haematopoietic marker CD45 immunohistochemistry, we find the donor derived cells fall into four phenotypic classes. We do not detect cytokeratin positive cells in whole bone marrow using cytokeratin immunostaining and we do not detect any cytokeratin mRNA in SP or bone marrow samples by RT-PCR.

Conclusion: The appearance of bone marrow derived cells in the tracheal epithelium is enriched by detergent-induced tissue damage and the majority of these cells express an epithelial marker. The cytokeratin positive donor derived cells in the tracheal epithelium are not present in the injected donor cells and must have acquired this novel phenotype in vivo.

Show MeSH
Related in: MedlinePlus