Conditionally Reprogrammed Normal and Transformed Mouse Mammary Epithelial Cells Display a Progenitor-Cell–Like Phenotype

Abstract

Mammary epithelial (ME) cells cultured under conventional conditions senesce after several passages. Here, we demonstrate that mouse ME cells isolated from normal mammary glands or from mouse mammary tumor virus (MMTV)-Neu–induced mammary tumors, can be cultured indefinitely as conditionally reprogrammed cells (CRCs) on irradiated fibroblasts in the presence of the Rho kinase inhibitor Y-27632. Cell surface progenitor-associated markers are rapidly induced in normal mouse ME-CRCs relative to ME cells. However, the expression of certain mammary progenitor subpopulations, such as CD49f+ ESA+ CD44+, drops significantly in later passages. Nevertheless, mouse ME-CRCs grown in a three-dimensional extracellular matrix gave rise to mammary acinar structures. ME-CRCs isolated from MMTV-Neu transgenic mouse mammary tumors express high levels of HER2/neu, as well as tumor-initiating cell markers, such as CD44+, CD49f+, and ESA+ (EpCam). These patterns of expression are sustained in later CRC passages. Early and late passage ME-CRCs from MMTV-Neu tumors that were implanted in the mammary fat pads of syngeneic or nude mice developed vascular tumors that metastasized within 6 weeks of transplantation. Importantly, the histopathology of these tumors was indistinguishable from that of the parental tumors that develop in the MMTV-Neu mice. Application of the CRC system to mouse mammary epithelial cells provides an attractive model system to study the genetics and phenotype of normal and transformed mouse epithelium in a defined culture environment and in vivo transplant studies.

Developing in vitro models of human ductal carcinoma in situ from primary tissue explants

“…Five lines recently developed from a patient in Singapore were genetically manipulated through hTERT transfection and are not commercially available [33]. Herein, we described an adaptation of a recently described protocol for culturing primary epithelial cells that results in rapid expansion of mammary epithelial cells from primary DCIS lesions [19][20][21][22]. Using conditioned medium instead of a feeder layer of irradiated fibroblasts significantly simplifies the primary culture methods and allows more sophisticated in vitro studies without the potentially confounding presence of murine cells.…”

“…Our method is based on a recently reported conditional reprogramming method that involves culturing mechanically and enzymatically dissociated tumor explants in F-medium conditioned by irradiated mouse fibroblasts and supplemented with Y-27632 dihydrochloride rho-associated protein kinase inhibitor (ROCKi), which is thought to function by inhibiting differentiation and inducing proliferation [19][20][21][22]. We have cultured primary DCIS from 19 patients, demonstrated luminal and basal cellular features and the ability of the cultures to maintain heterogeneity, and noted their ability to self-organize.…”

Harnessing TGF-β and BMP signaling for expansion of p63-positive epithelial stem cells

“…This approach is based on the pioneering work by Richard Schlegel of Georgetown University and expands cells through conditional reprogramming (17,18). Using this method, multiple publications have demonstrated expansion of epithelial cells from diverse tissue sources such as airway, cervix, breast and ear hair cells (19,20). One potential downside of this approach is that differentiation may be limited at later passages.…”

Primary cancer cell culture: mammary-optimized vs conditional reprogramming

“…P. Jones et al, 2005; L. Jones et al, 2008; Nakles et al, 2013) and Brca1 replete (Tilli et al, 2003; Díaz-Cruz et al, 2011) mouse models of breast cancer to compare transcriptional consequences and cancer progenitor cell retention of two different primary cancer epithelial cell culture methodologies: the mammary epithelial cell-optimized EpiCult (B) system (Stemcell Technologies, Vancouver, BC, Canada) (Yamaji et al, 2009) and the epithelial-selective conditionally reprogrammed culture (CRC) technology (Liu et al, 2012; Suprynowicz et al, 2012; Palechor-Ceron et al, 2013; Chapman et al, 2014; Saenz et al, 2014; Ligaba et al, 2015). Previously we employed time-lapse microscopy to show that mammary epithelial cells from wild-type (WT) and Brca1 deficient GEMM undergo morphological changes consistent with epithelial-mesenchymal transition (EMT) within four days of initial culture in EpiCult (B) (EpiC) (Nakles et al, 2013).…”

“…Previously we employed time-lapse microscopy to show that mammary epithelial cells from wild-type (WT) and Brca1 deficient GEMM undergo morphological changes consistent with epithelial-mesenchymal transition (EMT) within four days of initial culture in EpiCult (B) (EpiC) (Nakles et al, 2013). The experiments presented here were initiated because CRC was reported to retain mammary epithelial cell cuboidal architecture (Saenz et al, 2014). However mandatory inclusion of the irradiated fibroblast feeder layer or conditioned media burdens the technique and the required Rho kinase (ROCK) inhibitor Y-27632 has the potential to alter a number of cellular processes (Olson, 2008) For that reason we thought it sensible to directly compare biological and transcriptional consequences of these two prominent choices for initial isolation and propagation of primary mammary epithelial cells under 2D adherent conditions to further define the advantages and disadvantages of each approach.…”