isolation-culture-dental-epithelial-stem-cells-from-adult-mouse

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

University of California, San Francisco

Overexpression of the epidermal growth factor (EGF) receptor occurs frequently in ovarian cancer and is associated with poor patient prognosis. A constitutively active mutant EGF receptor termed variant III (EGFRvIII) has been detected at a high frequency in many human tumors, including those of the ovary. To identify the consequences of EGFRvIII expression in ovarian tumor cells, we introduced EGFRvIII into the epithelial ovarian cancer cell line (OVCA 433). The EGFRvIII-transfected cells displayed a dissociated, motile phenotype and fibroblastic morphology. The EGFRvIII-dependent phenotype was comparable to that observed in EGF-stimulated parental OVCA 433 cultures and required the catalytic activity of the mutant receptor. Disruption of adherens and desmosomal junctions in EGFRvIII expressing cells was evident by immunofluorescent detection of specific junctional components. In addition, Western blot analysis confirmed decreased levels of cellular plakoglobin and beta-catenin in EGFRvIII-expressing cells, and E-cadherin protein and mRNA were nearly absent. The loss of E-cadherin was accompanied by decreased expression of additional ovarian epithelial markers, including keratins 7, 8, and 18 and mucins 1 and 4. In contrast, the mesenchymal markers N-cadherin and vimentin were elevated in EGFRvIII expressing cells. Overall, the switch in cadherins from E-cadherin to N-cadherin, coupled with gain of vimentin expression and loss of the epithelial keratins and mucins typically expressed in well-differentiated epithelial ovarian carcinomas, are consistent with transition to a mesenchymal phenotype as an outcome of EGFRvIII expression. These findings suggest that EGFRvIII expression may regulate phenotypic plasticity in ovarian cancer and thereby contribute to more aggressive disease.

Mesenchymal transformation in epithelial ovarian tumor cells expressing epidermal growth factor receptor variant III.

Many peptide growth factors, including EGFR ligands, accelerate wound reepithelialization in vivo and in vitro. Furthermore, EGFR expression is transiently increased at wound margins, suggesting an active role for this receptor in wound repair. During reepithelialization of cutaneous wounds, keratinocytes display a phenotypic plasticity resembling aspects of epithelial-mesenchymal transformation. The transcription factor Slug/Snai2 is a regulator of epithelial-mesenchymal transformation during development, and we previously reported that Slug expression is elevated in keratinocytes bordering cutaneous wounds in vivo, ex vivo, and in vitro. In this study we provide evidence that Slug expression is necessary for an EGFR-stimulated reepithelialization response. Epidermal growth factor (EGF) induces Slug expression and the response to EGFR activation is more robust than to other receptor tyrosine kinase ligands. EGFR-stimulated reepithelialization is highly dependent on Slug, as demonstrated by the absence of EGF-stimulated outgrowth in explants derived from Slug null mice. In vitro reepithelialization stimulated by ectopic Slug expression was not impaired by an inhibitor of EGFR catalytic activity, suggesting that Slug is a downstream mediator of this EGFR-stimulated response. Our findings provide evidence that Slug is an essential component of the pathway leading to EGFR-mediated epithelial outgrowth.

Slug/Snai2 is a downstream mediator of epidermal growth factor receptor-stimulated reepithelialization.

Modulation of cell : cell junctions is a key event in cutaneous wound repair. In this study we report that activation of the epidermal growth factor (EGF) receptor disrupts cell : cell adhesion, but with different kinetics and fates for the desmosomal cadherin desmoglein and for E-cadherin. Downregulation of desmoglein preceded that of E-cadherin in vivo and in an EGF-stimulated in vitro wound reepithelialization model. Dual immunofluorescence staining revealed that neither E-cadherin nor desmoglein-2 internalized with the EGF receptor, or with one another. In response to EGF, desmoglein-2 entered a recycling compartment based on predominant colocalization with the recycling marker Rab11. In contrast, E-cadherin downregulation was accompanied by cleavage of the extracellular domain. A broad-spectrum matrix metalloproteinase inhibitor protected E-cadherin but not the desmosomal cadherin, desmoglein-2, from EGF-stimulated disruption. These findings demonstrate that although activation of the EGF receptor regulates adherens junction and desmosomal components, this stimulus downregulates associated cadherins through different mechanisms.

Differential downregulation of e-cadherin and desmoglein by epidermal growth factor.

Dental epithelial stem cells (DESCs) drive continuous growth in the adult mouse incisors. To date, a robust system for the primary culture of these cells has not been reported, and little is known about the basic molecular architecture of these cells or the minimal extracellular scaffolding that is necessary to maintain the epithelial stem cell population in an undifferentiated state. We report a method of isolating DESCs from the cervical loop of the mouse mandibular incisor. Cells were viable in a two-dimensional culture system and did not demonstrate preferential proliferation when grown on top of various substrates. Characterization of these cells indicated that E-cadherin, integrin alpha-6, and integrin beta-4 mark the DESCs both in vivo and in vitro. We also grew these cells in a three-dimensional microenvironment and obtained spheres with an epithelial morphology and expression patterns. Insights into the mechanisms of stem cell maintenance in vitro will help lay the groundwork for the successful generation of bioengineered teeth from adult DESCs.

Characterization of dental epithelial stem cells from the mouse incisor with two-dimensional and three-dimensional platforms.

This review summarizes approaches used in tissue engineering and regenerative medicine, with a focus on dental applications. Dental caries and periodontal disease are the most common diseases resulting in tissue loss. To replace or regenerate new tissues, various sources of stem cells have been identified such as somatic stem cells from teeth and peridontium. Advances in biomaterial sciences including microfabrication, self-assembled biomimetic peptides, and 3-dimensional printing hold great promise for whole-organ or partial tissue regeneration to replace teeth and periodontium.

Stem cell and biomaterials research in dental tissue engineering and regeneration.

Type 1 diabetes is chronic disease with numerous complications and currently no cure. Tissue engineering strategies have shown promise in providing a therapeutic solution, but maintenance of islet function and survival within these therapies represents a formidable challenge. The islet microenvironment may hold the key for proper islet maintenance. To elucidate the microenvironmental conditions necessary for improved islet function and survival, three-dimensional (3D) polyacrylamide cell scaffolds were fabricated with stiffnesses of 0.1 and 10 kPa to regulate the spatial and mechanical control of biosignals. Specifically, we show a significant increase in insulin mRNA expression of 3D primary mouse islet-derived and Min6-derived β-cell clusters grown on compliant 0.1 kPa scaffolds. Moreover, these compliant 0.1 kPa scaffolds also increase glucose sensitivity in Min6-derived β-cell clusters as demonstrated by the increased glucose stimulation index. Our data suggest that stiffness-specific insulin processing is regulated through the myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK) mechanosensing pathways. Additionally, β-catenin is required for regulation of stiffness-dependent insulin expression. Through activation or inhibition of β-catenin signaling, reversible control of insulin expression is achieved on the compliant 0.1 kPa and overly stiff 10 kPa substrates. Understanding the role of the microenvironment on islet function can enhance the therapeutic approaches necessary to treat diabetes for improving insulin sensitivity and response.

Miquella G. Chavez

Department of Orofacial Sciences and Program in Craniofacial and Mesenchymal Biology,
Department of Bioengineering and Therapeutic Sciences

University of California, San Francisco

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Miquella G. Chavez

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Orofacial Sciences and Program in Craniofacial and Mesenchymal Biology,Department of Bioengineering and Therapeutic Sciences

University of California, San Francisco

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Department of Orofacial Sciences and Program in Craniofacial and Mesenchymal Biology,
Department of Bioengineering and Therapeutic Sciences