Quantification of regenerative potential in primary human mammary epithelial cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Linnemann JR
PROVIDER: S-EPMC4582177 | biostudies-literature | 2015 Sep
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:We present an organoid regeneration assay in which freshly dissociated human mammary epithelial cells from healthy donors are grown in adherent/rigid or floating/compliant collagen I gels. In both conditions, luminal progenitors (CD49f+EpCAM+) form spheres, whereas basal cells (CD49fhiEpCAM-) generate branched ductal structures. However, in compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Moreover, the membrane metallo-endopeptidase CD10 as an additional surface marker enriches for TDLU-formation and reveals the presence of stromal cells within the CD49fhiEpCAM- population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. Comparison of three cell populations derived from FACS-sorted primary human mammary epithelial cells: luminal progenitors, CD10+ basal cells and CD10- basal cells.
2015-07-02 | E-GEOD-64248 | biostudies-arrayexpress
Project description:We present an organoid regeneration assay in which freshly dissociated human mammary epithelial cells from healthy donors are grown in adherent/rigid or floating/compliant collagen I gels. In both conditions, luminal progenitors (CD49f+EpCAM+) form spheres, whereas basal cells (CD49fhiEpCAM-) generate branched ductal structures. However, in compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Moreover, the membrane metallo-endopeptidase CD10 as an additional surface marker enriches for TDLU-formation and reveals the presence of stromal cells within the CD49fhiEpCAM- population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis.
2015-07-02 | GSE64248 | GEO
Project description:Bio-engineered organs for transplantation may ultimately provide a personalized solution for end-stage organ failure, without the risk of rejection. Building upon the process of whole organ perfusion decellularization, we aimed to develop novel, translational methods for the recellularization and regeneration of transplantable lung constructs. We first isolated a proliferative KRT5(+)TP63(+) basal epithelial stem cell population from human lung tissue and demonstrated expansion capacity in conventional 2D culture. We then repopulated acellular rat scaffolds in ex vivo whole organ culture and observed continued cell proliferation, in combination with primary pulmonary endothelial cells. To show clinical scalability, and to test the regenerative capacity of the basal cell population in a human context, we then recellularized and cultured isolated human lung scaffolds under biomimetic conditions. Analysis of the regenerated tissue constructs confirmed cell viability and sustained metabolic activity over 7 days of culture. Tissue analysis revealed extensive recellularization with organized tissue architecture and morphology, and preserved basal epithelial cell phenotype. The recellularized lung constructs displayed dynamic compliance and rudimentary gas exchange capacity. Our results underline the regenerative potential of patient-derived human airway stem cells in lung tissue engineering. We anticipate these advances to have clinically relevant implications for whole lung bioengineering and ex vivo organ repair.
| S-EPMC5035637 | biostudies-literature
Project description:Many normal adult tissues contain rare stem cells with extensive self-maintaining regenerative potential. During development, the stem cells of the hematopoietic and neural systems undergo intrinsically specified changes in their self-renewal potential. In the mouse, mammary stem cells with transplantable regenerative activity are first detectable a few days before birth. They share some phenotypic properties with their adult counterparts but are enriched in a subpopulation that displays a distinct gene expression profile. Here we show that fetal mammary epithelial cells have a greater direct and inducible growth potential than their adult counterparts. The latter feature is revealed in a novel culture system that enables large numbers of in vitro clonogenic progenitors as well as mammary stem cells with serially transplantable activity to be produced within 7 days from single fetal or adult input cells. We further show that these responses are highly dependent on novel factors produced by fibroblasts. These findings provide new avenues for elucidating mechanisms that regulate normal mammary epithelial stem cell properties at the single-cell level, how these change during development, and how their perturbation may contribute to transformation.
| S-EPMC3742452 | biostudies-literature
Project description:Previously, we showed that some podocytes in juvenile mice are recruited from cells lining Bowman's capsule, suggesting that parietal epithelial cells (PECs) are a progenitor cell population for podocytes. To investigate whether PECs also replenish podocytes in adult mice, PECs were genetically labeled in an irreversible fashion in 5-week-old mice. No significant increase in labeled podocytes was observed, even after 18 months. To accelerate a potential regenerative mechanism, progressive glomerular hypertrophy was induced by progressive partial nephrectomies. Again, no significant podocyte replenishment was observed. Rather, labeled PECs exclusively invaded segments of the tuft affected by glomerulosclerosis, consistent with our previous findings. We next reassessed PEC recruitment in juvenile mice using a different reporter mouse and confirmed significant recruitment of labeled PECs onto the glomerular tuft. Moreover, some labeled cells on Bowman's capsule expressed podocyte markers, and cells on Bowman's capsule were also directly labeled in juvenile podocyte-specific Pod-rtTA transgenic mice. In 6-week-old mice, however, cells on Bowman's capsule no longer expressed podocyte-specific markers. Similarly, in human kidneys, some cells on Bowman's capsule expressed the podocyte marker synaptopodin from 2 weeks to 2 years of age but not at 7 years of age. In summary, podocyte regeneration from PECs could not be detected in aging mice or models of glomerular hypertrophy. We propose that a small fraction of committed podocytes reside on Bowman's capsule close to the vascular stalk and are recruited onto the glomerular tuft during infancy to adolescence in mice and humans.
| S-EPMC3968497 | biostudies-literature
Project description:There is an ample epidemiological evidence to support the role of environmental contaminants such as bisphenol A (BPA) in breast cancer development but the molecular mechanisms of their action are still not fully understood. Therefore, we sought to analyze the effects of three common contaminants (BPA; 4-tert-octylphenol, OP; hexabromocyclododecane, HBCD) on mammary epithelial cell (HME1) and MCF7 breast cancer cell line. We also supplied some data on methoxychlor, MXC; 4-nonylphenol, NP; and 2-amino-1-methyl-6-phenylimidazo [4-b] pyridine, PhIP. We focused on testing the prolonged (two months) exposure to low nano-molar concentrations (0.0015-0.0048 nM) presumed to be oncogenic and found that they induced DNA damage (evidenced by upregulation of pH2A.X, pCHK1, pCHK2, p-P53) and disrupted the cell cycle. Some agents induced epigenetic (methylation) changes of tumor suppressor genes TIMP3, CHFR, ESR1, IGSF4, CDH13, and GSTP1. Obviously, the accumulation of these molecular alterations is an essential base for cancer development. Consistent with this, we observed that these agents increased cellular invasiveness through collagen. Cellular abilities to form colonies in soft agar were increased for MCF7. Toxic agents induced phosphorylation of protein kinase such as EGFR, CREB, STAT6, c-Jun, STAT3, HSP6, HSP27, AMPK?1, FAK, p53, GSK-3?/?, and P70S6 in HME1. Most of these proteins are involved in potential oncogenic pathways. Overall, these data clarify the molecular alterations that can be induced by some common environmental contaminants in mammary epithelial cells which could be a foundation to understand environmental carcinogenesis.
| S-EPMC7279350 | biostudies-literature
Project description:Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models.We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ER?, as well as its ability to respond to estrogen stimulation.We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems.The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro. We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.
| S-EPMC5837767 | biostudies-literature
Project description:Primary human mammary epithelial cells (pHMECs) are known to be remarkably difficult to engineer genetically. Here, we present a protocol for efficient transduction of pHMECs using a baboon retroviral envelope glycoprotein for pseudotyping of lentiviral vectors (BaEV-LVs). We describe the preparation of the BaEV-LVs, the isolation of pHMECs from breast samples, and the subsequent transduction of pHMECs. We also detail the use of CRISPRi technology to efficiently silence gene expression in pHMECs, which can then be used for functional assays. For complete details on the use and execution of this protocol, please refer to Richart et al. (2022).1.
| S-EPMC9871349 | biostudies-literature
Project description:Rho family small GTPases serve as molecular switches in the regulation of diverse cellular functions, including actin cytoskeleton remodeling, cell migration, gene transcription, and cell proliferation. Importantly, Rho overexpression is frequently seen in many carcinomas. However, published studies have almost invariably used immortal or tumorigenic cell lines to study Rho GTPase functions and there are no studies on the potential of Rho small GTPase to overcome senescence checkpoints and induce preneoplastic transformation of human mammary epithelial cells (hMEC). We show here that ectopic expression of wild-type (WT) RhoA as well as a constitutively active RhoA mutant (G14V) in two independent primary hMEC strains led to their immortalization and preneoplastic transformation. These cells have continued to grow over 300 population doublings (PD) with no signs of senescence, whereas cells expressing the vector or dominant-negative RhoA mutant (T19N) senesced after 20 PDs. Significantly, RhoA-T37A mutant, known to be incapable of interacting with many well-known Rho effectors including Rho kinase, PKN, mDia1, and mDia2, was also capable of immortalizing hMECs. Notably, similar to parental normal cells, Rho-immortalized cells have WT p53 and intact G(1) cell cycle arrest on Adriamycin treatment. Rho-immortalized cells were anchorage dependent and were unable to form tumors when implanted in nude mice. Lastly, microarray expression profiling of Rho-immortalized versus parental cells showed altered expression of several genes previously implicated in immortalization and breast cancer progression. Taken together, these results show that RhoA can induce the preneoplastic transformation of hMECs by altering multiple pathways linked to cellular transformation and breast cancer.
| S-EPMC2792911 | biostudies-literature
Project description:The TWIST1 embryonic transcription factor displays biphasic functions during the course of carcinogenesis. It facilitates the escape of cells from oncogene-induced fail-safe programs (senescence, apoptosis) and their consequent neoplastic transformation. Additionally, it promotes the epithelial-to-mesenchymal transition and the initiation of the metastatic spread of cancer cells. Interestingly, cancer cells recurrently remain dependent on TWIST1 for their survival and/or proliferation, making TWIST1 their Achilles' heel. TWIST1 has been reported to form either homodimeric or heterodimeric complexes mainly in association with the E bHLH class I proteins. These complexes display distinct, sometimes even antagonistic, functions during development and unequal prometastatic functions in prostate cancer cells. Using a tethered dimer strategy, we successively assessed the ability of TWIST1 dimers to cooperate with an activated version of RAS in human mammary epithelial cell transformation, to provide mice with the ability to spontaneously develop breast tumors, and lastly to maintain a senescence program at a latent state in several breast cancer cell lines. We demonstrate that the TWIST1-E12 complex, unlike the homodimer, is an oncogenic form of TWIST1 in mammary epithelial cells and that efficient binding of both partners is a prerequisite for its activity. The detection of the heterodimer in human premalignant lesions by a proximity ligation assay, at a stage preceding the initiation of the metastatic cascade, is coherent with such an oncogenic function. TWIST1-E protein heterodimeric complexes may thus constitute the main active forms of TWIST1 with regard to senescence inhibition over the time course of breast tumorigenesis.
| S-EPMC4887617 | biostudies-literature