Investigating wound healing characteristics of gingival and skin keratinocytes in organotypic cultures.

OBJECTIVES: The gingiva heals at an accelerated rate with reduced scarring when compared to skin. Potential well-studied factors include immune cell number, angiogenesis disparities and fibroblast gene expression. Differential keratinocyte gene expression, however, remains relatively understudied. This study explored the contrasting healing efficiencies of gingival and skin keratinocytes, alongside their differential gene expression patterns. METHODS: 3D organotypic culture models of human gingiva and skin were developed using temporarily immortalised primary keratinocytes. Models were wounded for visualisation of re-epithelialisation and analysis of keratinocyte migration to close the wound gap. Concurrently, differentially expressed genes between primary gingival and skin keratinocytes were identified, validated, and functionally assessed. RESULTS: Characterisation of the 3D cultures of gingiva and skin showed differentiation markers that recapitulated organisation of the corresponding in vivo tissue. Upon wounding, gingival models displayed a significantly higher efficiency in re-epithelialisation and stratification versus skin, repopulating the wound gap within 24 hours. This difference was likely due to distinct patterns of migration, with gingival cells demonstrating a form of sheet migration, in contrast to skin, where the leading edge was typically 1-2 cells thick. A candidate approach was used to identify several genes that were differentially expressed between gingival and skin keratinocytes. Knockdown of PITX1 resulted in reduced migration capacity of gingival cells. CONCLUSION: Gingival keratinocytes retain in vivo superior wound healing capabilities in in vitro 2D and 3D environments. Intrinsic gene expression differences could result in gingival cells being 'primed' for healing and play a role in faster wound resolution. CLINICAL SIGNIFICANCE STATEMENT: The successful development of organotypic models, that recapitulate re-epithelialisation, will underpin further studies to analyse the oral response to wound stimuli, and potential therapeutic interventions, in an in vitro environment.

Decision-support systems for ambulatory care, including pandemic requirements: using mathematically optimized solutions.

BACKGROUND: The healthcare sector poses many strategic, tactic and operational planning questions. Due to the historically grown structures, planning is often locally confined and much optimization potential is foregone. METHODS: We implemented optimized decision-support systems for ambulatory care for four different real-world case studies that cover a variety of aspects in terms of planning scope and decision support tools. All are based on interactive cartographic representations and are being developed in cooperation with domain experts. The planning problems that we present are the problem of positioning centers for vaccination against Covid-19 (strategical) and emergency doctors (strategical/tactical), the out-of-hours pharmacy planning problem (tactical), and the route planning of patient transport services (operational). For each problem, we describe the planning question, give an overview of the mathematical model and present the implemented decision support application. RESULTS: Mathematical optimization can be used to model and solve these planning problems. However, in order to convince decision-makers of an alternative solution structure, mathematical solutions must be comprehensible and tangible. Appealing and interactive decision-support tools can be used in practice to convince public health experts of the benefits of an alternative solution. The more strategic the problem and the less sensitive the data, the easier it is to put a tool into practice. CONCLUSIONS: Exploring solutions interactively is rarely supported in existing planning tools. However, in order to bring new innovative tools into productive use, many hurdles must be overcome.

Carboxylated-xyloglucan and peptide amphiphile co-assembly in wound healing.

Hydrogel wound dressings can play critical roles in wound healing protecting the wound from trauma or contamination and providing an ideal environment to support the growth of endogenous cells and promote wound closure. This work presents a self-assembling hydrogel dressing that can assist the wound repair process mimicking the hierarchical structure of skin extracellular matrix. To this aim, the co-assembly behaviour of a carboxylated variant of xyloglucan (CXG) with a peptide amphiphile (PA-H3) has been investigated to generate hierarchical constructs with tuneable molecular composition, structure, and properties. Transmission electron microscopy and circular dichroism at a low concentration shows that CXG and PA-H3 co-assemble into nanofibres by hydrophobic and electrostatic interactions and further aggregate into nanofibre bundles and networks. At a higher concentration, CXG and PA-H3 yield hydrogels that have been characterized for their morphology by scanning electron microscopy and for the mechanical properties by small-amplitude oscillatory shear rheological measurements and compression tests at different CXG/PA-H3 ratios. A preliminary biological evaluation has been carried out both in vitro with HaCat cells and in vivo in a mouse model.

Willingness, concerns, incentives and acceptable remuneration regarding an involvement in teaching undergraduates - a cross-sectional questionnaire survey among German GPs.

BACKGROUND: Worldwide, many undergraduate general practice curricula include community-based courses at general practitioners' (GPs') offices. Usually the academic general practice departments collaborate with networks of affiliated teaching practices. To successfully master the challenge of network development and extension, more information is needed about GPs' willingness to be involved in different teaching formats, important influencing factors, incentives, barriers, and the need for financial compensation. METHODS: In this cross-sectional study a questionnaire survey was conducted among all GPs working in Leipzig and environs (German postal code area 04). In addition to descriptive statistics, group comparisons and logistic regression were performed to reveal differences between GPs with and without an interest in teaching. RESULTS: Response rate was 45.3% with 339 analyzable questionnaires. The average age was 52.0 years and 58.4% were women. Sixty-two participants stated that they were already involved in teaching undergraduates. Altogether 60.1% of all GPs and 53.5% among those who didn't teach yet were basically interested in being involved in undergraduate education. The interested GPs could imagine devoting on average 6.9 h per month to teaching activities. GPs interested in teaching were on average younger, were more actively involved in continuing education and professional associations, and more frequently had pre-existing teaching experiences. The willingness to teach differed substantially among teaching formats. GPs were more willing to teach at their own practices rather than at university venues and they preferred skills-oriented content. Comprehensive organization on the part of the university including long-term scheduling and available teaching materials was rated as most important to increase the attractiveness of teaching. Time restraints and decreased productivity were rated as the most important barriers. Interested GPs appreciated financial compensation, particularly for teaching at university venues, and demanded amounts of money corresponding to German GPs' hourly income. CONCLUSIONS: The GPs' interest in undergraduate teaching is generally high indicating a substantial pool of potential preceptors. Recruitment strategies should consider the collaboration with institutions involved in residency and continuing education as well as with professional associations. Comprehensive organization by the responsible department should be promoted and time restraints and decreased productivity should be overtly addressed and financially compensated.

Efficacy of surveillance ultrasound for venous thromboembolism diagnosis in critically ill children after trauma.

INTRODUCTION: Venous thromboembolism (VTE) is increasingly prevalent in injured children admitted to the intensive care unit (ICU). Few data exist to support VTE pharmacologic prophylaxis or ultrasound (US) surveillance in children with high bleeding risk. After implementation of screening US guidelines, we sought to describe our experience, hypothesizing that screening US of children at highest risk for VTE results in earlier detection and management. STUDY DESIGN: A retrospective analysis was conducted on prospectively collected data of injured children admitted to an American College of Surgeons Verified level 1 Pediatric Trauma Center from 2010 to 2015. In patients at high risk for both VTE and bleeding (HRHR), guidelines recommended deferral of pharmacologic prophylaxis and a screening US at ≥7 ICU days if bleeding risk remained. Outcomes analyzed included VTE rates, guideline compliance, and US timing. The rate of deep vein thrombosis (DVT) detection (number of DVT captured/number of US obtained) was examined. RESULTS: Of 4061 trauma patients, 588 (14.5%) were critically injured including 112 patients who met HRHR criteria. The rate of VTE in the HRHR group ≥7 ICU days was 25% (14/56). Of 23 VTE diagnosed in the ICU, 17 were detected by 49 US performed (34.7%), with the remaining 6 diagnosed by computed tomography. DVT was detected earlier than the US guideline recommended 7 days, independent of symptoms. Guideline compliance was 86%. CONCLUSION: Critically injured children at risk for bleeding frequently develop VTE. Surveillance ultrasound in patients at high risk for both VTE and bleeding allows earlier detection and treatment. LEVEL OF EVIDENCE: Therapeutic study, level II.

Characterization of the Growth of Chlamydia trachomatis in In Vitro-Generated Stratified Epithelium.

Chlamydia infection targets the mucosal epithelium, where squamous and columnar epithelia can be found. Research on Chlamydia-epithelia interaction has predominantly focused on columnar epithelia, with very little known on how Chlamydia interacts with the squamous epithelium. The stratification and differentiation processes found in the squamous epithelium might influence chlamydial growth and infection dissemination. For this reason, three-dimensional (3D) organotypic stratified squamous epithelial cultures were adapted to mimic the stratified squamous epithelium and chlamydial infection was characterized. Chlamydia trachomatis infection in monolayers and 3D cultures were monitored by immunofluorescence and transmission electron microscopy to evaluate inclusion growth and chlamydial interconversion between elementary and reticulate body. We observed that the stratified epithelium varied in susceptibility to C. trachomatis serovars L2 and D infection. The undifferentiated basal cells were susceptible to infection by both serovars, while the terminally differentiated upper layers were resistant. The differentiating suprabasal cells exhibited different susceptibilities to serovars L2 and D, with the latter unable to establish a successful infection in this layer. Mature elementary body-containing inclusions were much more prevalent in these permissive basal layers, while the uppermost differentiated layers consistently harbored very few reticulate bodies with no elementary bodies, indicative of severely limited bacterial replication and development. For serovar D, the differentiation state of the host cell was a determining factor, as calcium-induced differentiation of cells in a monolayer negatively affected growth of this serovar, in contrast to serovar L2. The apparent completion of the developmental cycle in the basal layers of the 3D cultures correlated with the greater degree of dissemination within and the level of disruption of the stratified epithelium. Our studies indicate that the squamous epithelium is a suboptimal environment for growth, and thus potentially contributing to the protection of the lower genital tract from infection. The relatively more fastidious serovar D exhibited more limited growth than the faster-growing and more invasive L2 strain. However, if given access to the more hospitable basal cell layer, both strains were able to produce mature inclusions, replicate, and complete their developmental cycle.

Evaluation of guidelines for injured children at high risk for venous thromboembolism: A prospective observational study.

BACKGROUND: Pharmacologic prophylaxis for venous thromboembolism (VTE) is a widely accepted practice in adult trauma patients to prevent associated morbidity and mortality. However, VTE prophylaxis has not been standardized in injured pediatric patients. Our institution identified factors potentially associated with a high risk of VTE in critically injured children that led to prospective implementation of VTE prophylaxis guidelines. We hypothesize that the guidelines are accurate in predicting children at risk for VTE. METHODS: Data were prospectively collected on injured children from August 2010 to August 2015. Pharmacologic prophylaxis was indicated for patients identified by the guidelines as high risk for VTE. Prophylaxis was deferred and a screening ultrasound was performed if the high-risk VTE patients were also at high risk for bleeding. To assess the accuracy of predicting confirmed cases of VTE, stepwise logistic regression analysis was used to measure the association of individual risk factors with VTE controlling for age (≥13 years). A receiver operating characteristic curve measured the accuracy of the final model to predict a VTE. RESULTS: Of 4,061 trauma patients, 588 were admitted to the ICU, with the guidelines identifying 199 as high risk for VTE. VTE occurred in 3.9% (23/588) of the ICU population and 10% (20/199) of the high risk group. The median age of VTE patients in the ICU was 9.7 years. Statistically significant predictors (p < 0.05) of VTE in the multivariate model included presence of a central venous catheter (OR = 5.2), inotropes (OR = 7.7), immobilization (OR = 5.5), and a Glasgow Coma Scale of <9 (OR = 1.3). The area under receiver operating characteristic curve of this model was 0.92, demonstrating its excellent predictive ability. CONCLUSION: Specific clinical factors in critically injured children are associated with a high risk for VTE. Incorporating these risk factors in VTE prophylaxis guidelines facilitates more accurate risk stratification and may allow for improved VTE prevention in pediatric trauma. LEVEL OF EVIDENCE: Prognostic study, level II.

Constitutive Autophagy and Nucleophagy during Epidermal Differentiation.

Epidermal keratinocytes migrate through the epidermis up to the granular layer where, on terminal differentiation, they progressively lose organelles and convert into anucleate cells or corneocytes. Our report explores the role of autophagy in ensuring epidermal function providing the first comprehensive profile of autophagy marker expression in developing epidermis. We show that autophagy is constitutively active in the epidermal granular layer where by electron microscopy we identified double-membrane autophagosomes. We demonstrate that differentiating keratinocytes undergo a selective form of nucleophagy characterized by accumulation of microtubule-associated protein light chain 3/lysosomal-associated membrane protein 2/p62 positive autolysosomes. These perinuclear vesicles displayed positivity for histone interacting protein, heterochromatin protein 1α, and localize in proximity with Lamin A and B1 accumulation, whereas in newborn mice and adult human skin, we report LC3 puncta coincident with misshaped nuclei within the granular layer. This process relies on autophagy integrity as confirmed by lack of nucleophagy in differentiating keratinocytes depleted from WD repeat domain phosphoinositide interacting 1 or Unc-51 like autophagy activating kinase 1. Final validation into a skin disease model showed that impaired autophagy contributes to the pathogenesis of psoriasis. Lack of LC3 expression in psoriatic skin lesions correlates with parakeratosis and deregulated expression or location of most of the autophagic markers. Our findings may have implications and improve treatment options for patients with epidermal barrier defects.

Type VI Collagen Regulates Dermal Matrix Assembly and Fibroblast Motility.

Type VI collagen is a nonfibrillar collagen expressed in many connective tissues and implicated in extracellular matrix (ECM) organization. We hypothesized that type VI collagen regulates matrix assembly and cell function within the dermis of the skin. In the present study we examined the expression pattern of type VI collagen in normal and wounded skin and investigated its specific function in new matrix deposition by human dermal fibroblasts. Type VI collagen was expressed throughout the dermis of intact human skin, at the expanding margins of human keloid samples, and in the granulation tissue of newly deposited ECM in a mouse model of wound healing. Generation of cell-derived matrices (CDMs) by human dermal fibroblasts with stable knockdown of COL6A1 revealed that type VI collagen-deficient matrices were significantly thinner and contained more aligned, thicker, and widely spaced fibers than CDMs produced by normal fibroblasts. In addition, there was significantly less total collagen and sulfated proteoglycans present in the type VI collagen-depleted matrices. Normal fibroblasts cultured on de-cellularized CDMs lacking type VI collagen displayed increased cell spreading, migration speed, and persistence. Taken together, these findings indicate that type VI collagen is a key regulator of dermal matrix assembly, composition, and fibroblast behavior and may play an important role in wound healing and tissue regeneration.

Meta-regression analyses to explain statistical heterogeneity in a systematic review of strategies for guideline implementation in primary health care.

This study is an in-depth-analysis to explain statistical heterogeneity in a systematic review of implementation strategies to improve guideline adherence of primary care physicians in the treatment of patients with cardiovascular diseases. The systematic review included randomized controlled trials from a systematic search in MEDLINE, EMBASE, CENTRAL, conference proceedings and registers of ongoing studies. Implementation strategies were shown to be effective with substantial heterogeneity of treatment effects across all investigated strategies. Primary aim of this study was to explain different effects of eligible trials and to identify methodological and clinical effect modifiers. Random effects meta-regression models were used to simultaneously assess the influence of multimodal implementation strategies and effect modifiers on physician adherence. Effect modifiers included the staff responsible for implementation, level of prevention and definition pf the primary outcome, unit of randomization, duration of follow-up and risk of bias. Six clinical and methodological factors were investigated as potential effect modifiers of the efficacy of different implementation strategies on guideline adherence in primary care practices on the basis of information from 75 eligible trials. Five effect modifiers were able to explain a substantial amount of statistical heterogeneity. Physician adherence was improved by 62% (95% confidence interval (95% CI) 29 to 104%) or 29% (95% CI 5 to 60%) in trials where other non-medical professionals or nurses were included in the implementation process. Improvement of physician adherence was more successful in primary and secondary prevention of cardiovascular diseases by around 30% (30%; 95% CI -2 to 71% and 31%; 95% CI 9 to 57%, respectively) compared to tertiary prevention. This study aimed to identify effect modifiers of implementation strategies on physician adherence. Especially the cooperation of different health professionals in primary care practices might increase efficacy and guideline implementation seems to be more difficult in tertiary prevention of cardiovascular diseases.

Strategies for guideline implementation in primary care focusing on patients with cardiovascular disease: a systematic review.

BACKGROUND: Guidelines should reduce inappropriate practice and improve the efficiency of treatment. Not only methodological quality but also acceptance and successful implementation in daily practice are crucial for the benefit on patients. Focusing on cardiovascular diseases (CVD), it is still unclear which implementation strategy can improve physician adherence to the recommendations of guidelines in primary care. METHODS: We conducted a systematic review on randomized controlled trials about guideline implementation strategies on CVD. Medline, Embase, CENTRAL, conference proceedings and registers of ongoing studies were searched. RESULTS: Eighty-four trials met our predefined inclusion criteria, of them 54 trials compared unimodal strategies and 30 multimodal strategies to usual care. Concerning unimodal strategies, 15 trials investigated provider reminder systems, 3 audit and feedback, 15 provider education, 4 patient education, 5 promotion of self-management and 14 organizational change. The strongest benefit of a unimodal implementation strategy was found due to organizational change (odds ratio 1.96; 95% CI 1.4 to 2.75), followed by patient education, provider education and provider reminder systems. Trials on the efficacy of audit and feedback and patient self-management showed differing results or small advantages in terms of physician adherence. Multimodal interventions showed almost similar effect measures and ranking of strategies. CONCLUSION: The use of implementation strategies for the distribution of guidelines on CVD can be convincingly effective on physician adherence, regardless whether based on a unimodal or multimodal design. Three distinct strategies should be well considered in such an attempt: organizational changes in the primary care team, patient education and provider education.

Characterizing the phenotype of murine epidermal progenitor cells: complementary whole-mount visualization and flow cytometry strategies.

The epidermis and its appendages, the hair follicle and sebaceous gland, have the capacity to constantly regenerate throughout adult life. Postnatal hair follicles undergo a cyclic mode of tissue homeostasis, defined by periods of growth, degeneration, and rest. A multipotent population of stem cells residing within the hair follicle bulge not only generates the hair lineages during each hair cycle, but also transiently contributes to the repair of epidermis following wounding. In this chapter, we provide methods for identifying epidermal stem cells and investigating their proliferative and apoptotic characteristics. We introduce whole-mount and flow cytometry techniques, which complement each other by permitting visualization of the epidermal stem cell compartment in situ and assessment of the phenotype of purified cells. These techniques can easily be adapted to characterize novel putative epidermal stem or progenitor cell populations. By applying whole-mount and flow cytometry techniques to characterize normal and genetically modified mice with skin defects, we expect to learn more about the factors that regulate stem cell self-renewal and differentiation.

Effectiveness of clinical guidelines for deep vein thrombosis prophylaxis in reducing the incidence of venous thromboembolism in critically ill children after trauma.

BACKGROUND: Historically, 6% of critically ill children developed clinically apparent venous thromboembolism (VTE) after trauma at our Level I pediatric trauma center. We hypothesized that implementation of clinical guidelines for thrombosis prophylaxis incorporating both VTE risk and bleeding risk would reduce VTE incidence without increased bleeding. METHODS: VTE, both clinically apparent and those only detected by guideline-directed screening, were prospectively identified for all children admitted to the intensive care unit after trauma during three time periods: preimplementation of guidelines for VTE thromboprophylaxis (PRE; April 1, 2006-June 30, 2007), the intervening period (ROLL OUT; July 1, 2007-November 4, 2008), and postguideline implementation (POST; November 5, 2008-June 1, 2010). For patients classified as high risk for VTE, anticoagulation was recommended. For those patients at high risk of VTE with high risk of bleeding, anticoagulation was deferred and screening ultrasound performed. RESULTS: Fourteen of 546 subjects developed VTE. There was a decrease in total VTE (p = 0.041) and clinical VTE (p = 0.001) after guideline implementation. The nine VTE PRE (5.2%) were clinically symptomatic, while the three VTE POST (1.8%) were detected by guideline-directed screening ultrasound. Implementation of guidelines did not increase overall thromboprophylaxis, with decreased anticoagulation in patients at low risk of VTE. No bleeding complications occurred. No patients classified by the guidelines as low risk for VTE developed VTE. CONCLUSION: The incidence of clinical VTE and total VTE decreased after implementation of clinical guidelines for thromboprophylaxis in critically ill children after trauma. This decrease in VTE was not associated with increased prophylactic anticoagulation nor increased bleeding. The guidelines were predictive in identifying patients at low risk for VTE. LEVEL OF EVIDENCE: II, therapeutic study.

iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia.

iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR-574-3p and miR-720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63-mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis.

Rac1 deletion causes thymic atrophy.

The thymic stroma supports T lymphocyte development and consists of an epithelium maintained by thymic epithelial progenitors. The molecular pathways that govern epithelial homeostasis are poorly understood. Here we demonstrate that deletion of Rac1 in Keratin 5/Keratin 14 expressing embryonic and adult thymic epithelial cells leads to loss of the thymic epithelial compartment. Rac1 deletion led to an increase in c-Myc expression and a generalized increase in apoptosis associated with a decrease in thymic epithelial proliferation. Our results suggest Rac1 maintains the epithelial population, and equilibrium between Rac1 and c-Myc may control proliferation, apoptosis and maturation of the thymic epithelial compartment. Understanding thymic epithelial maintenance is a step toward the dual goals of in vitro thymic epithelial cell culture and T cell differentiation, and the clinical repair of thymic damage from graft-versus-host-disease, chemotherapy or irradiation.

Functional characterization of highly adherent CD34+ keratinocytes isolated from human skin.

Compared to murine models, data on cells responsible for the homeostasis of human epidermis are scarce and often contradictory. Given the conflicting results and the availability of clinical grade protocols to purify CD34 cells from a given tissue, we pursued to phenotypically characterize human epidermal CD34+ population. After magnetic separation of whole skin CD34+ and CD34- cell fractions and selection for cells highly adherent to extracellular matrix, both CD34+/- fractions retained the ability to form a stratified epidermis in organotypic cultures and presented similar in vitro migratory phenotypes. However CD34- cells showed higher clonogenic potential and in vitro proliferative capacity. These results indicated that CD34- cell fraction contains stem/early progenitor cells, while CD34+ cells might be a transit-amplifying precursor for hair follicle (HF) sheath cells. The ability to isolate living cells using differential cell adhesion and surface markers provides an opportunity to study cells from different morphological regions of the HF.

Cutaneous cancer stem cells: beta-catenin strikes again.

Cancer stem cells (CSCs) are a subpopulation of tumor cells that retain properties of tissue-specific stem cells, including the ability to self-renew. In a recent article in Nature, Malanchi et al. (2008) identified a population of CD34(+) cells in epidermal tumors that require beta-catenin signaling to maintain a CSC phenotype.

Characterization of bipotential epidermal progenitors derived from human sebaceous gland: contrasting roles of c-Myc and beta-catenin.

The current belief is that the epidermal sebaceous gland (SG) is maintained by unipotent stem cells that are replenished by multipotent stem cells in the hair follicle (HF) bulge. However, sebocytes can be induced by c-Myc (Myc) activation in interfollicular epidermis (IFE), suggesting the existence of bipotential stem cells. We found that every SZ95 immortalized human sebocyte that underwent clonal growth in culture generated progeny that differentiated into both sebocytes and cells expressing involucrin and cornifin, markers of IFE and HF inner root sheath differentiation. The ability to generate involucrin positive cells was also observed in a new human sebocyte line, Seb-E6E7. SZ95 xenografts differentiated into SG and IFE but not HF. SZ95 cells that expressed involucrin had reduced Myc levels; however, this did not correlate with increased expression of the Myc repressor Blimp1, and Blimp1 expression did not distinguish cells undergoing SG, IFE, or HF differentiation in vivo. Overexpression of Myc stimulated sebocyte differentiation, whereas overexpression of beta-catenin stimulated involucrin and cornifin expression. In transgenic mice simultaneous activation of Myc and beta-catenin revealed mutual antagonism: Myc blocked ectopic HF formation and beta-catenin reduced SG differentiation. Overexpression of the Myc target gene Indian hedgehog did not promote sebocyte differentiation in culture and cyclopamine treatment, while reducing proliferation, did not block Myc induced sebocyte differentiation in vivo. Our studies provide evidence for a bipotential epidermal stem cell population in an in vitro model of human epidermal lineage selection and highlight the importance of Myc as a regulator of sebocyte differentiation.

The cell-surface marker MTS24 identifies a novel population of follicular keratinocytes with characteristics of progenitor cells.

We describe a novel murine progenitor cell population localised to a previously uncharacterised region between sebaceous glands and the hair follicle bulge, defined by its reactivity to the thymic epithelial progenitor cell marker MTS24. MTS24 labels a membrane-bound antigen present during the early stages of hair follicle development and in adult mice. MTS24 co-localises with expression of alpha6-integrin and keratin 14, indicating that these cells include basal keratinocytes. This novel population does not express the bulge-specific stem cell markers CD34 or keratin 15, and is infrequently BrdU label retaining. MTS24-positive and -negative keratinocyte populations were isolated by flow cytometry and assessed for colony-forming efficiency. MTS24-positive keratinocytes exhibited a two-fold increase in colony formation and colony size compared to MTS24-negative basal keratinocytes. In addition, both the MTS24-positive and CD34-positive subpopulations were capable of producing secondary colonies after serial passage of individual cell clones. Finally, gene expression profiles of MTS24 and CD34 subpopulations were compared. These results showed that the overall gene expression profile of MTS24-positive cells resembles the pattern previously reported in bulge stem cells. Taken together, these data suggest that the cell-surface marker MTS24 identifies a new reservoir of hair follicle keratinocytes with a proliferative capacity and gene expression profile suggestive of progenitor or stem cells.