Human Milk Protein Production in Xenografts of Genetically Engineered Bovine Mammary Epithelial Stem Cells

Abstract

BackgroundIn 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 FindingsWe 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.ConclusionsThese 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.

Concise Review: Aldehyde Dehydrogenase Bright Stem and Progenitor Cell Populations from Normal Tissues: Characteristics, Activities, and Emerging Uses in Regenerative Medicine

“…The ALDHdim population gave rise only to luminal epithelial cells [9]. In contrast to these results with human cells, ALDHbr cells isolated from bovine mammary epithelium gave rise to cells with luminal phenotype in culture, while ALDHlow cells were myoepithelial progenitors [55].…”

Mammary Stem Cell Research in Veterinary Science: An Update

“…One type of colony had a myoepithelial phenotype based on a CK18-negative and CK14-positive expression profile, whereas the two other colonies had a luminal phenotype characterized by a CK18-positive and CK14-negative phenotype [55]. Moreover, analysis of the ALDH activity showed that ALDH high cells had luminal features, whereas ALDH low cells produced colonies with myoepithelial features [56]. The group of Motyl identified putative MaSC in a suspension of bovine mammary epithelial cells using the fluorescent dye Hoechst 33342 [57].…”

“…1A), (2) differentiation characteristics into epithelial-like, myoepithelial-like, and secretory cells, and (3) their expression of beta 1-integrin and alpha 6-integrin [16]. Martignani et al used CFU assays to study the clonogenic expansion capacities of bovine MaSC and they found three distinct colonies, according to the size and morphology [55,56]. One type of colony had a myoepithelial phenotype based on a CK18-negative and CK14-positive expression profile, whereas the two other colonies had a luminal phenotype characterized by a CK18-positive and CK14-negative phenotype [55].…”

“…Another research group characterized spheres derived from canine mammary gland adenocarcinoma cell lines using flow cytometry and RT-PCR analyses. Their salient findings were that the sphere cell population was predominantly CD44 + CD24 -and had a higher expression of stem cells markers, including CD133, CD34, Notch 3, and the multidrug resistance protein, when compared to the adherent cell population [56]. Moreover, these CD44 + CD24 -populations showed a high ALDH activity and were capable of initiating tumors in NOD/SCD mice [72].…”

Bovine CD49 positive-cell subpopulation remarkably increases in mammary epithelial cells that retain a stem-like phenotype

“…Current methods for detecting bovine mammary progenitors require the preparation of viable single-cell suspensions and their assessment in suitable in vitro or in vivo assays to detect the growth and differentiation properties of the input cells at a clonal density (Stingl et al, 2006). However, the efforts to purify adult stem cells from the bovine mammary gland have been hindered by the lack of cell-surface markers specific for undifferentiated or differentiated mammary cells (Martignani et al, 2009) even if suitable in vitro and in vivo assays for testing stem cell properties have been proposed by our laboratory (Martignani et al, 2010).…”