Madin-Darby canine kidney (MDCK) cells are a model mammalian cell line used in biomedical research. MDCK cells are used for a wide variety of cell biology studies including cell polarity, cell-cell adhesions (termed adherens junctions), collective cell motility, toxicity studies, [1] as well as responses to growth factors. It is one of few cell culture models that is suited for 3D cell culture and multicellular rearrangements known as branching morphogenesis. [2]
Following the initial isolation in 1958 of epithelial cells from the kidney tubule of an adult Cocker Spaniel dog by Stewart H. Madin and Norman B. Darby, Jr., [3] the cell line bearing their name was employed primarily as a model for viral infection of mammalian cells. [4] [5] [6] Indeed, they chose to isolate kidney tubules with precisely this goal in mind, as they had previously succeeded with viral infection of cells derived from kidney tubules from other mammals. [7] Thus the initial goal in isolating and culturing cells from this tissue was not to generate a new model system for epithelial cell biology. It was not until 1970 that the laboratory of Zbynek Brada published work describing MDCK cells as a representative cell line bearing hallmarks of kidney tubule epithelial cells. [8] They based this conclusion on the fluid transport activities of monolayers formed of MDCK cells, the presence of microvilli on their apical (upper) surface, and their ability to self-organize, when grown in 3D, into hollow spheres. In their report, the authors speculated that the "histotypic expression" by which MDCK cells formed structures reminiscent of their tissue of origin might be fruitfully applied to the study of other tissues. The following decades have proved them largely right, although the repertoire for studying the organization and behavior of cells within tissues has vastly expanded. [9]
Through the 1970s, the MDCK cell line found new use as a model for mammalian epithelial tissue. In 1982 Mina Bissell and colleagues showed that MDCK monolayers responded to the addition of a collagen overlay (dubbed a "sandwich culture") by proliferating and forming hollow tubules. [10] This hinted for the first time that the cell line would respond to 3D environments by self-organizing into the appropriate 3D structure reminiscent of kidney tubules. In the following years, the culture of MDCK cells embedded fully in collagen was shown to yield hollow spheres, or acini. [11] These were simple epithelial monolayers with a defined interior and exterior. However, the fact that MDCK cells did not form tubules under these conditions remained unexplained until later.
Over the same period in the 1980s, biologists studying cell motility had hit upon an interesting and reproducible behavior of cells in culture: the scattering response. Epithelial cells in culture grow normally as tight clusters. However, they could be induced to break cell-cell contacts and become elongated and motile after exposure to a "scatter factor" that was secreted by mesenchymal cells such as Swiss 3T3 fibroblasts. [12] This was best described by Julia Gray's group in 1987. [13] During the same period in the mid 1980s, a monoclonal antibody was reported by the group of Walter Birchmeier to disrupt cell-cell contacts and alter the front-rear polarity of cells in culture. [14] [15] The target of this antibody was later identified as a component of cell-cell junctions, E-cadherin. [16] These disparate observations eventually coalesced into a resilient paradigm for cell motility and cell polarity. Epithelial cells are typically nonmotile, but can become motile by inhibiting cell-cell junctions or by addition of growth factors that induce scattering. [17] Both of these are reversible, and both involve the rupture of cell-cell junctions.
In 1991, the response of MDCK acini in 3D culture to the scatter factor was first reported by Lelio Orci and colleagues. [18] They cultured acini of MDCK cells in collagen gels with or without Swiss 3T3 fibroblasts, in which media could exchange but the cell types were not in direct contact. This cell culture strategy, termed coculture, induced MDCK acini to undergo branching morphogenesis, in which cells rearrange into a network of interconnected tubules that resembles the development of many tissues. [19] In the same year, the "scatter factor" was shown to be a previously described protein secreted by fibroblasts, hepatocyte growth factor (HGF). [20] This work solved an outstanding mystery of MDCK culture, as the tissue from which these cells were derived is tubular, yet they had previously only developed into spherical acini in 3D culture. Beyond that immediate paradox, a crucial connection was forged between the acute induction of cell motility in 2D culture by the "scatter factor", and its impact on the spatial organization adopted by tissues in 3D. This connection remains significant as a link between precisely defined mechanisms of cell motility in 2D and complex rearrangements in 3D whose regulation is yet to be understood fully.
In the last 20 years, understanding of MDCK cell biology in 3D culture has been advanced most notably by the laboratory of Keith Mostov. This group has focused on the regulation of cell polarity and its downstream effects on branching morphogenesis. [21] [2] Indeed, the body of work generated by the Mostov group has successfully synthesized decades of knowledge about the spatial segregation of cellular functions, and their molecular markers, into a remarkable model for the generation and homeostasis of cellular polarity in tissues. [22] [23] In 2003 the Mostov group reported the first comprehensive account linking branching morphogenesis with hallmarks of apical-basal polarity. [24] This work established that MDCK cells do not lose contacts with neighbors during the onset of branching morphogenesis, but that canonical markers of cell polarity are transiently lost. One outcome of this shift in polarity is the reorientation of cell division along a newly growing branch of cells, in order to correctly position daughter cells to continue branch extension. Cell motility by which MDCK cells produce and elongate branches was linked with these polarity changes.
These findings were integrated into a model for branching morphogenesis focused on the transient rearrangement of cell polarity signaling. This model has informally been referred to as the Mostov pathway. This allows normally nonmotile cells to generate protrusions and migrate collectively, followed by redifferentiation and formation of hollow tubules. In support of this model, Mostov and colleagues have identified the effects of HGF on MDCK acini as eliciting a partial transition from epithelial to mesenchymal cell phenotypes. [25] This argument marshals an established signaling program termed the epithelial to mesenchymal transition (EMT), by which sessile epithelial cells become motile and break cell-cell contacts. [17] EMT has been proposed as the transcriptional signaling cascade that drives cell scattering, although previously researchers did not conflate the two. [26] [27] Given the distinction that, for acini in 3D, cell-cell junctions do not rupture, it is unclear how to precisely relate the EMT concept with branching morphogenesis.
The Mostov group has also investigated the means by which HGF activates cell motility during MDCK branching morphogenesis. [28] [29] Their studies have shown that branching morphogenesis requires the Erk transcription factor, downstream of the mitogen activated protein kinase cascade, a well-defined signal transduction pathway implicated in cell motility and proliferation. [30] The precise cell motility machinery responsible for MDCK branching morphogenesis has not been specified by the Mostov group, beyond the requirement for a signaling protein involved in regulating the small GTPase Rho. [29] Moreover, the Gardel lab has shown that invasive motility of MDCK cells in acini requires Dia1, which regulates cell adhesions to individual collagen fibrils. [31] Meanwhile, other groups have demonstrated the requirement for cell-ECM adhesion proteins or their regulators in MDCK branching morphogenesis. [32] [33] Using a modified protocol for MDCK cell culture and branching morphogenesis, Gierke and Wittman established the requirement for microtubule dynamics in regulating the early steps in branching. [34] They observed deficient cell adhesive coupling to the collagen matrix when microtubules were deregulated. This phenotype indicated the importance of trafficking the appropriate cell adhesion and protrusion proteins to the cell front as branching morphogenesis was initiated. Combined with observations from the Mostov group, this work confirmed that cell polarity is indispensable for MDCK acinar homeostasis as well as migratory behaviors during branching morphogenesis.
Morphogenesis is the biological process that causes a cell, tissue or organism to develop its shape. It is one of three fundamental aspects of developmental biology along with the control of tissue growth and patterning of cellular differentiation.
A fibroblast is a type of biological cell that synthesizes the extracellular matrix and collagen, produces the structural framework (stroma) for animal tissues, and plays a critical role in wound healing. Fibroblasts are the most common cells of connective tissue in animals.
With physical trauma or disease suffered by an organism, healing involves the repairing of damaged tissue(s), organs and the biological system as a whole and resumption of (normal) functioning. Medicine includes the process by which the cells in the body regenerate and repair to reduce the size of a damaged or necrotic area and replace it with new living tissue. The replacement can happen in two ways: by regeneration in which the necrotic cells are replaced by new cells that form "like" tissue as was originally there; or by repair in which injured tissue is replaced with scar tissue. Most organs will heal using a mixture of both mechanisms.
A mammary gland is an exocrine gland in humans and other mammals that produces milk to feed young offspring. Mammals get their name from the Latin word mamma, "breast". The mammary glands are arranged in organs such as the breasts in primates, the udder in ruminants, and the dugs of other animals. Lactorrhea, the occasional production of milk by the glands, can occur in any mammal, but in most mammals, lactation, the production of enough milk for nursing, occurs only in phenotypic females who have gestated in recent months or years. It is directed by hormonal guidance from sex steroids. In a few mammalian species, male lactation can occur. With humans, male lactation can occur only under specific circumstances.
Wound healing refers to a living organism's replacement of destroyed or damaged tissue by newly produced tissue.
Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryonic development, wound healing and immune responses all require the orchestrated movement of cells in particular directions to specific locations. Cells often migrate in response to specific external signals, including chemical signals and mechanical signals. Errors during this process have serious consequences, including intellectual disability, vascular disease, tumor formation and metastasis. An understanding of the mechanism by which cells migrate may lead to the development of novel therapeutic strategies for controlling, for example, invasive tumour cells.
The epithelial–mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell–cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells; these are multipotent stromal cells that can differentiate into a variety of cell types. EMT is essential for numerous developmental processes including mesoderm formation and neural tube formation. EMT has also been shown to occur in wound healing, in organ fibrosis and in the initiation of metastasis in cancer progression.
Perlecan (PLC) also known as basement membrane-specific heparan sulfate proteoglycan core protein (HSPG) or heparan sulfate proteoglycan 2 (HSPG2), is a protein that in humans is encoded by the HSPG2 gene. The HSPG2 gene codes for a 4,391 amino acid protein with a molecular weight of 468,829. It is one of the largest known proteins. The name perlecan comes from its appearance as a "string of pearls" in rotary shadowed images.
An organoid is a miniaturised and simplified version of an organ produced in vitro in three dimensions that mimics the key functional, structural and biological complexity of that organ. They are derived from one or a few cells from a tissue, embryonic stem cells or induced pluripotent stem cells, which can self-organize in three-dimensional culture owing to their self-renewal and differentiation capacities. The technique for growing organoids has rapidly improved since the early 2010s, and The Scientist names it as one of the biggest scientific advancements of 2013. Scientists and engineers use organoids to study development and disease in the laboratory, drug discovery and development in industry, personalized diagnostics and medicine, gene and cell therapies, tissue engineering and regenerative medicine.
A myofibroblast is a cell phenotype that was first described as being in a state between a fibroblast and a smooth muscle cell.
Hepatocyte growth factor (HGF) or scatter factor (SF) is a paracrine cellular growth, motility and morphogenic factor. It is secreted by mesenchymal cells and targets and acts primarily upon epithelial cells and endothelial cells, but also acts on haemopoietic progenitor cells and T cells. It has been shown to have a major role in embryonic organ development, specifically in myogenesis, in adult organ regeneration, and in wound healing.
Fibroblast growth factor 10 is a protein that in humans is encoded by the FGF10 gene.
SCRIB, also known as Scribble, SCRIBL, or Scribbled homolog (Drosophila), is a scaffold protein which in humans is encoded by the SCRIB gene. It was originally isolated in Drosophila melanogaster in a pathway (also known as the Scribble complex) with DLGAP5 (Discs large) and LLGL1 (Lethal giant larvae) as a tumor suppressor. In humans, SCRIB is found as a membrane protein and is involved in cell migration, cell polarity, and cell proliferation in epithelial cells. There is also strong evidence that SCRIB may play a role in cancer progression because of its strong homology to the Drosophila protein.
Glia-activating factor is a protein that in humans is encoded by the FGF9 gene.
Transcription factor 21 (TCF21), also known as pod-1, capsuling, or epicardin, is a protein that in humans is encoded by the TCF21 gene on chromosome 6. It is ubiquitously expressed in many tissues and cell types and highly significantly expressed in lung and placenta. TCF21 is crucial for the development of a number of cell types during embryogenesis of the heart, lung, kidney, and spleen. TCF21 is also deregulated in several types of cancers, and thus known to function as a tumor suppressor. The TCF21 gene also contains one of 27 SNPs associated with increased risk of coronary artery disease.
Cadherin-1 or Epithelial cadherin(E-cadherin), is a protein that in humans is encoded by the CDH1 gene. Mutations are correlated with gastric, breast, colorectal, thyroid, and ovarian cancers. CDH1 has also been designated as CD324. It is a tumor suppressor gene.
Cell polarity refers to spatial differences in shape, structure, and function within a cell. Almost all cell types exhibit some form of polarity, which enables them to carry out specialized functions. Classical examples of polarized cells are described below, including epithelial cells with apical-basal polarity, neurons in which signals propagate in one direction from dendrites to axons, and migrating cells. Furthermore, cell polarity is important during many types of asymmetric cell division to set up functional asymmetries between daughter cells.
An organ-on-a-chip (OOC) is a multi-channel 3-D microfluidic cell culture, integrated circuit (chip) that simulates the activities, mechanics and physiological response of an entire organ or an organ system. It constitutes the subject matter of significant biomedical engineering research, more precisely in bio-MEMS. The convergence of labs-on-chips (LOCs) and cell biology has permitted the study of human physiology in an organ-specific context. By acting as a more sophisticated in vitro approximation of complex tissues than standard cell culture, they provide the potential as an alternative to animal models for drug development and toxin testing.
Protein Wnt-9b is a protein that in humans is encoded by the WNT9B gene.
Keith E. Mostov is an American cell biologist. He received a BA from University of Chicago in 1976 and during 1976–77 he was a Rhodes Scholar at New College, Oxford. Mostov received a PhD in Biological Science from the Rockefeller University in the laboratory of Nobel laureate Günter Blobel in 1983, and an MD from Weill Cornell Medicine in 1984. He was a Whitehead Fellow at the Whitehead Institute of MIT from 1984 to 1989. In 1989, Mostov joined the faculty of the University of California, San Francisco, School of Medicine, where he is currently Professor. Mostov and colleagues discovered and sequenced the Polymeric Immunoglobulin Receptor (pIgR) and proposed the generally accepted model of its pathway and function. Neil E. Simister and Mostov cloned and sequenced the Neonatal Fc Receptor (FcRn). Mostov and colleagues showed how signals in the pIgR direct its polarized trafficking and how polarized MDCK epithelial cells form three-dimensional structures with lumens and tubules. Mostov and colleagues further found how simple rules cause different branching patterns in kidney as compared to other branching tubular organs