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Human milk protein production in xenografts of genetically engineered bovine mammary epithelial stem cells.

Martignani E, Eirew P, Accornero P, Eaves CJ, Baratta M - PLoS ONE (2010)

Bottom Line: Transplantation of the bovine cells transduced with a lentivirus encoding human β-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo.These findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species.These results highlight the potential of novel engineering and transplant strategies for a variety of commercial applications including the production of modified milk components for human consumption.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Morphophysiology, University of Turin, Grugliasco, Italy.

ABSTRACT

Background: In the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show that a mammary stem cell population can be functionally identified and isolated from the bovine mammary gland. We also demonstrate that this stem cell population may be a promising target for manipulating the composition of cow's milk using gene transfer.

Methods and findings: We show that the in vitro colony-forming cell assay for detecting normal primitive bipotent and lineage-restricted human mammary clonogenic progenitors are applicable to bovine mammary cells. Similarly, the ability of normal human mammary stem cells to regenerate functional bilayered structures in collagen gels placed under the kidney capsule of immunodeficient mice is shared by a subset of bovine mammary cells that lack aldehyde dehydrogenase activity. We also find that this activity is a distinguishing feature of luminal-restricted bovine progenitors. The regenerated structures recapitulate the organization of bovine mammary tissue, and milk could be readily detected in these structures when they were assessed by immunohistochemical analysis. Transplantation of the bovine cells transduced with a lentivirus encoding human β-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo.

Conclusions: These findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species. These results highlight the potential of novel engineering and transplant strategies for a variety of commercial applications including the production of modified milk components for human consumption.

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Related in: MedlinePlus

FACS profiles of bovine mammary cells stained for ALDH activity.Cells were stained with ALDEFLUOR with (a) or without (b) diethylamino-benzaldehyde (DEAB), an ALDH inhibitor. Panel c shows the proportional distribution of total clonogenic luminal and myoepithelial progenitors between the Aldehyde Dehydrogenase (ALDH)high and ALDHlow fractions (% of all progenitors of a given type in the fraction shown ± SEM, n = 3).
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pone-0013372-g002: FACS profiles of bovine mammary cells stained for ALDH activity.Cells were stained with ALDEFLUOR with (a) or without (b) diethylamino-benzaldehyde (DEAB), an ALDH inhibitor. Panel c shows the proportional distribution of total clonogenic luminal and myoepithelial progenitors between the Aldehyde Dehydrogenase (ALDH)high and ALDHlow fractions (% of all progenitors of a given type in the fraction shown ± SEM, n = 3).

Mentions: We next sought to determine whether bovine cells with in vitro clonogenic or in vivo regenerative activity could be prospectively enriched within distinct subsets of cells. Unfortunately, an evaluation of antibodies against antigens expressed on primitive human and mouse mammary cells (CD24, CD29, CD49f, EpCAM and MUC1) failed to detect any that showed crossreactivity with bovine cells (data not shown). These findings precluded the adoption of most cell separation strategies previously used to discriminate subsets of primitive mouse and human mammary cells. We therefore turned to an alternative approach that allows cells to be separated based on their aldehyde dehydrogenase (ALDH) activity using a reagent that is not species-restricted. Figure 2a–b shows a representative distribution of viable cells obtained from bovine mammary tissue, in this case from a pubertal virgin cow after staining and FACS analysis of the cells according to their ALDH activity (31% ALDHhigh and 67% ALDH1low). As shown in Figure 2c, assessment of the distribution of clonogenic cells in the sorted ALDHhigh and ALDHlow fractions isolated from 3 different animals showed that almost all of the colonies produced by the ALDHhigh cells had luminal features. Conversely, most of the colonies produced by the ALDHlow fraction had myoepithelial features (progenitor frequencies are shown in Table 1).


Human milk protein production in xenografts of genetically engineered bovine mammary epithelial stem cells.

Martignani E, Eirew P, Accornero P, Eaves CJ, Baratta M - PLoS ONE (2010)

FACS profiles of bovine mammary cells stained for ALDH activity.Cells were stained with ALDEFLUOR with (a) or without (b) diethylamino-benzaldehyde (DEAB), an ALDH inhibitor. Panel c shows the proportional distribution of total clonogenic luminal and myoepithelial progenitors between the Aldehyde Dehydrogenase (ALDH)high and ALDHlow fractions (% of all progenitors of a given type in the fraction shown ± SEM, n = 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2957408&req=5

pone-0013372-g002: FACS profiles of bovine mammary cells stained for ALDH activity.Cells were stained with ALDEFLUOR with (a) or without (b) diethylamino-benzaldehyde (DEAB), an ALDH inhibitor. Panel c shows the proportional distribution of total clonogenic luminal and myoepithelial progenitors between the Aldehyde Dehydrogenase (ALDH)high and ALDHlow fractions (% of all progenitors of a given type in the fraction shown ± SEM, n = 3).
Mentions: We next sought to determine whether bovine cells with in vitro clonogenic or in vivo regenerative activity could be prospectively enriched within distinct subsets of cells. Unfortunately, an evaluation of antibodies against antigens expressed on primitive human and mouse mammary cells (CD24, CD29, CD49f, EpCAM and MUC1) failed to detect any that showed crossreactivity with bovine cells (data not shown). These findings precluded the adoption of most cell separation strategies previously used to discriminate subsets of primitive mouse and human mammary cells. We therefore turned to an alternative approach that allows cells to be separated based on their aldehyde dehydrogenase (ALDH) activity using a reagent that is not species-restricted. Figure 2a–b shows a representative distribution of viable cells obtained from bovine mammary tissue, in this case from a pubertal virgin cow after staining and FACS analysis of the cells according to their ALDH activity (31% ALDHhigh and 67% ALDH1low). As shown in Figure 2c, assessment of the distribution of clonogenic cells in the sorted ALDHhigh and ALDHlow fractions isolated from 3 different animals showed that almost all of the colonies produced by the ALDHhigh cells had luminal features. Conversely, most of the colonies produced by the ALDHlow fraction had myoepithelial features (progenitor frequencies are shown in Table 1).

Bottom Line: Transplantation of the bovine cells transduced with a lentivirus encoding human β-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo.These findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species.These results highlight the potential of novel engineering and transplant strategies for a variety of commercial applications including the production of modified milk components for human consumption.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Morphophysiology, University of Turin, Grugliasco, Italy.

ABSTRACT

Background: In the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show that a mammary stem cell population can be functionally identified and isolated from the bovine mammary gland. We also demonstrate that this stem cell population may be a promising target for manipulating the composition of cow's milk using gene transfer.

Methods and findings: We show that the in vitro colony-forming cell assay for detecting normal primitive bipotent and lineage-restricted human mammary clonogenic progenitors are applicable to bovine mammary cells. Similarly, the ability of normal human mammary stem cells to regenerate functional bilayered structures in collagen gels placed under the kidney capsule of immunodeficient mice is shared by a subset of bovine mammary cells that lack aldehyde dehydrogenase activity. We also find that this activity is a distinguishing feature of luminal-restricted bovine progenitors. The regenerated structures recapitulate the organization of bovine mammary tissue, and milk could be readily detected in these structures when they were assessed by immunohistochemical analysis. Transplantation of the bovine cells transduced with a lentivirus encoding human β-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo.

Conclusions: These findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species. These results highlight the potential of novel engineering and transplant strategies for a variety of commercial applications including the production of modified milk components for human consumption.

Show MeSH
Related in: MedlinePlus