Phenotypic and Functional Characterization of Human Mammary Stem/Progenitor Cells in Long Term Culture

Background: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. Methodology: Single cell suspensions derived from human breast `organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres) were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. Principal Findings: We show that primary mammospheres contain a distinct side-population (SP) that displays a CD24(low)/CD44(low) phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high)/CD24(low) cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1) mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. Conclusions: Thus, the self-renewal potential of human breast stem cells is exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance.

Découverte de cellules souches potentielles de l’épithélium thymique

Le thymus subit un vieillissement précoce, appelé involution thymique, qui cause une perte de fonction du thymus avec l’âge. À ce jour, les mécanismes de renouvellement des cellules épithéliales thymiques (TECs) sont encore mal compris, c’est pourquoi nous avons voulu identifier les cellules souches de l’épithélium thymique. Comme les cellules souches sont quiescentes dans plusieurs tissus, les objectifs de notre étude étaient de déterminer si l’épithélium thymique contenait des cellules quiescentes et d’étudier la cinétique de prolifération des TECs chez les souris jeunes et adultes. Pour ce faire, nous avons utilisé une souris transgénique (H2B-GFP Tet-On) nous permettant d’identifier les cellules ne se divisant pas sur une longue période de temps (LRC, label-retaining cells¬). Nous avons d’abord montré que les TECs proliféraient plus rapidement chez les femelles que les mâles. De plus, nous avons trouvé plusieurs différences entre l’épithélium thymique post-natal et adulte : (1) les TECs corticales (cTECs) et médullaires (mTECs) ont un taux de prolifération similaire chez les jeunes souris, mais chez l’adulte, les cTECs prolifèrent plus lentement que les mTECs; (2) les TECs prolifèrent plus rapidement chez les souris jeunes que adultes; (3) des LRC sont détectées chez l’adulte, mais pas chez les jeunes souris. Les LRC, retrouvées dans le compartiment cTEC, sous-expriment des gènes associés à la sénescence et surexpriment des gènes importants pour le développement et le renouvellement des TECs. Ces résultats montrent que ces cellules sont quiescentes et suggèrent qu’elles pourraient bel et bien être les progéniteurs thymiques responsables du renouvellement des TECs adultes.

Regeneration of the Corneal Epithelium after Debridement of its Central Region: An Autoradiographic Study on Rabbits

Purpose: To investigate the proliferative behavior of the corneal and limbal epithelia after debridement on the central region of the rabbit cornea. Methods: After scraping a circular epithelial area, 5 mm in diameter, in the center of the cornea, (3)H-thymidine ((3)H-TdR) was injected intravitreally, and the rabbits killed from 1 to 49 days afterward. The cornea, together with the adjacent conjunctiva, was processed for autoradiography. Results: The regenerating epithelium at the center of the cornea exhibited high frequencies of labeled nuclei when compared to controls. The mitotic indexes for the limbus were comparable in experimental and control eyes. The unique basal stratum of the limbal epithelium exhibited quick proliferation and vertical migration in all eyes. Cells that remained labeled for four weeks or more were observed throughout the corneal epithelium, including its basal stratum, and this did not depend on epithelial damage. Conclusion: Corneal epithelium wounds are healed by sliding and proliferation of cells surrounding the epithelial gap without any evidence for the participation of the limbal epithelium. Daughter cells labeled with (3)H-TdR were visualized in all layers of the corneal epithelium up to 7 weeks after the DNA precursor injection. However, at this long interval, the only labeled cells in the limbus were in the suprabasal layers.

Morphological and autoradiographic studies on the corneal and limbal epithelium of rabbits

The investigation was centered on the morphological features of the conjunctiva-cornea transition (limbus) of the rabbit eye and the proliferative behavior of its epithelium. The eyes were processed for examination with light and electron microscopy, as well as for autoradiography after intravitreal injection of [H-3]thymidine ([H-3]TdR). At the sites of extraocular muscle insertion, the vascularization of the stroma extended to the peripheral cornea, and the limbal epithelium was thin with its basal stratum made up by clear cuboidal cells. In between the muscle insertions, the cuboidal clear cells, as well as the stroma blood vessels; were scarce. At the light microscope level, the basement membrane was distinct in the cornea but not in the limbus or the conjunctiva. Autoradiographs demonstrated that, at the limbus, the basal cells migrated very quickly to the suprabasal region and remained there up to the 28-day interval. Labeled cells were identified in all epithelial layers of the cornea, including the basal one, at 21 and 28 days but not in the limbal basal clear cells. The rate of renewal of conjunctival epithelium was similar to that observed for the transition with scarce clear cells. The high-resolution autoradiographs demonstrated that the basal cuboidal clear limbal cells exhibit a quick renewal and that they are not label-retaining cells. These latter ones were detected all over the corneal epithelium and in the suprabasal layers of the limbus up to 28 days, in physiological conditions, without the need of stimulation by damage to the corneal epithelium.

Nicho espermatogonial em Cyprinus carpio e papel do Amh na espermaogênese

Pós-graduação em Aquicultura - FCAV

Stem cells in embryonic skin development

The skin is a complex stratified organ which acts not only as a permeability barrier and defense against external agents, but also has essential thermoregulatory, sensory and metabolic functions. Due to its high versatility and activity, the skin undergoes continuous self-renewal to repair damaged tissue and replace old cells. Consequently, the skin is a reservoir for adult stem cells of different embryonic origins. Skin stem cell populations reside in the adult hair follicle, sebaceous gland, dermis and epidermis. However, the origin of most of the stem cell populations found in the adult epidermis is still unknown. Far more unknown is the embryonic origin of other stem cells that populate the other layers of this tissue. In this review we attempt to clarify the emergence, structure, markers and embryonic development of diverse populations of stem cells from the epidermis, dermis and related appendages such as the sebaceous gland and hair follicle.

Enrichment for murine keratinocyte stem cells based on cell surface phenotype

The identification and physical isolation of epithelial stem cells is critical to our understanding of their growth regulation during homeostasis, wound healing, and carcinogenesis. These stem cells remain poorly characterized because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying (TA) cells, which have a more restricted proliferative potential. Cell kinetic analyses have permitted the identification of murine keratinocyte stem cells (KSCs) as slowly cycling cells that retain [3H]thymidine ([3H]Tdr) label, termed label-retaining cells (LRCs), whereas TA cells are visualized as rapidly cycling cells after a single pulse of [3H]Tdr, termed pulse-labeled cells (PLCs). Here, we report on the successful separation of KSCs from TA cells through the combined use of in vivo cell kinetic analysis and fluorescence-activated cell sorting. Specifically, we demonstrate that murine dorsal keratinocytes characterized by their high levels of α6 integrin and low to undetectable expression of the transferrin receptor (CD71) termed α6briCD71dim cells, are enriched for epithelial stem cells because they represent a minor (≈8%) and quiescent subpopulation of small blast-like cells, with a high nuclear:cytoplasmic ratio, containing ≈70% of label-retaining cells, the latter being a well documented characteristic of stem cells. Conversely, TA cells could be enriched in a phenotypically distinct subpopulation termed α6briCD71bri, representing the majority (≈60%) of basal keratinocytes that are actively cycling, and importantly contain ≈70% of [3H]Tdr pulse-labeled cells. Importantly, immunostaining of dorsal skin revealed the presence of CD71dim cells in the hair follicle bulge region, a well documented location for KSCs.

Optimization of a transplant model to assess skin reconstitution from stem cell-enriched primary human keratinocyte populations

Given that an important functional attribute of stem cells in vivo is their ability to sustain tissue regeneration, we set out to establish a simple and easy technique to assess this property from candidate populations of human keratinocyte stem cells in an in vivo setting. Keratinocytes were inoculated into devitalized rat tracheas and transplanted subcutaneously into SCID mice, and the epithelial lining regenerated characterized to establish the validity of this heterotypic model. Furthermore, the rate and quality of epidermal tissue reconstitution obtained from freshly isolated unfractionated vs. keratinocyte stem cell-enriched populations was tested as a function of (a) cell numbers inoculated; and (b) the inclusion of irradiated support keratinocytes and dermal cells. Rapid and sustained epidermal tissue regeneration from small numbers of freshly isolated human keratinocyte stem cells validates the utilization of this simple and reliable model system to assay for enrichment of epidermal tissue-reconstituting cells.

Interoperable Standards Development : Final Report

Since 1995 the buildingSMART International Alliance for Interoperability (buildingSMART)has developed a robust standard called the Industry Foundation Classes (IFC). IFC is an object oriented data model with related file format that has facilitated the efficient exchange of data in the development of building information models (BIM). The Cooperative Research Centre for Construction Innovation has contributed to the international effort in the development of the IFC standard and specifically the reinforced concrete part of the latest IFC 2x3 release. Industry Foundation Classes have been endorsed by the International Standards Organisation as a Publicly Available Specification (PAS) under the ISO label ISO/PAS 16739. For more details, go to http://www.tc184- sc4.org/About_TC184-SC4/About_SC4_Standards/ The current IFC model covers the building itself to a useful level of detail. The next stage of development for the IFC standard is where the building meets the ground (terrain) and with civil and external works like pavements, retaining walls, bridges, tunnels etc. With the current focus in Australia on infrastructure projects over the next 20 years a logical extension to this standard was in the area of site and civil works. This proposal recognises that there is an existing body of work on the specification of road representation data. In particular, LandXML is recognised as also is TransXML in the broader context of transportation and CityGML in the common interfacing of city maps, buildings and roads. Examination of interfaces between IFC and these specifications is therefore within the scope of this project. That such interfaces can be developed has already been demonstrated in principle within the IFC for Geographic Information Systems (GIS) project. National road standards that are already in use should be carefully analysed and contacts established in order to gain from this knowledge. The Object Catalogue for the Road Transport Sector (OKSTRA) should be noted as an example. It is also noted that buildingSMART Norway has submitted a proposal

Teachers' and education students' perceptions of and reactions to children with and without the diagnostic label 'ADHD'

Thirty-four elementary school teachers and 32 education students from Canada rated their reactions towards vignettes describing children who met attention-deficit/hyperactivity disorder (ADHD) symptom criteria that included or did not include the label “ADHD.” “ADHD”-labeled vignettes elicited greater perceptions of the child's impairment as well as more negative emotions and less confidence in the participants, although it also increased participants' willingness to implement treatment interventions. Ratings were similar to vignettes of boys versus girls; however, important differences in ratings between teachers and education students emerged and are discussed. Finally, we investigated the degree to which teachers' professional backgrounds influenced bias based on the label “ADHD.” Training specific to ADHD consistently predicted label bias, whereas teachers' experience working with children with ADHD did not.

'Any body is better than nobody?' Ethical questions around recruiting and/or retaining health professionals in rural areas

The literature on recruiting and/or retaining health professionals in rural areas focuses primarily on the development of recruitment and retention strategies and assessing whether such strategies are effective. The objective of this article is to argue that it is important for all stakeholders involved in rural recruitment and/or retention processes to consider their decisions and actions from an ethics perspective. Recruitment and/or retention processes are not value neutral and it is important to understand their ethical dimensions. Methods: From the literature, elements of the recruitment and/or retention strategies that have been employed were identified and organised in respect of levels of governance (namely, the levels of health system/government, community, and individual health professionals). The elements identified in these levels were subjected to analysis to identify their ethical dimensions and to determine whether a clash or complement of values arose at each level of governance or between governance levels. Results: There is very little literature in this area that considers the ethical dimensions of rural recruitment and/or retention processes. However, all policies and practices have ethical dimensions that need to be identified and understood as they may have significant implications for recruitment and/or retention processes. Conclusion: This article recommends the application of an ethics perspective when reflecting on rural recruitment and/or retention strategies. The collective decisions of all involved in rural recruitment and/or retention processes may fundamentally influence the 'health' (broadly understood) of rural communities.

Unsupervised multi-label text classification using a world knowledge ontology

The development of text classification techniques has been largely promoted in the past decade due to the increasing availability and widespread use of digital documents. Usually, the performance of text classification relies on the quality of categories and the accuracy of classifiers learned from samples. When training samples are unavailable or categories are unqualified, text classification performance would be degraded. In this paper, we propose an unsupervised multi-label text classification method to classify documents using a large set of categories stored in a world ontology. The approach has been promisingly evaluated by compared with typical text classification methods, using a real-world document collection and based on the ground truth encoded by human experts.

Akin Collection : curation of a collaboration between Indigenous artists and fashion designers to create a retail-ready luxury fashion label

The Akin collection is the outcome of a project to lead, guide and curate a luxury, retail-ready fashion collection from a collaboration between five emerging fashion designers and five established Indigenous artists. Research background There is a history of Indigenous artists in Australia being treated unethically; by misappropriation and misrepresentation of their work, inequity of payment for their creativity and little acknowledgement of their cultural contribution to collaborative fashion product sold globally. This has created an atmosphere of bad press for fashion, as well as a fear for emerging designers to include/collaborate with Indigenous artists for textile prints. This fear has been so intense that many emerging Australian designers are now seeking collaboration with other countries’ Indigenous communities, thus missing out on a rich cultural and diverse aesthetic that could brand a truly unique Australian label in the international marketplace. The fashion brands that have collaborated with Indigenous Australian artists have traditionally been a ONE designer label incorporating Indigenous prints, for collections that have little acknowledgement of the artist’s contribution and strong branding for the label and/or fashion designer. This collection seeks to create an equitable and profitable fashion collection under one brand where all artists and designers receive equal payment, equal promotion/credibility, as well as equal royalties for any garments ordered by retailers. Research question Is it possible to curate an ethical, luxury, retail-ready, international fashion brand with a collaboration of five (5) emerging designers and five (5) Indigenous artists? Research contribution In the fashion industry, existing collaborations for Australian Indigenous artists have been with ONE fashion designer or one existing fashion label. This is the first fashion collection created under one brand name with equal credibility and profits for both artists and designers. The process involved presenting workshops ranging from understanding the logistics and timing of the fashion supply chain, costing of garments, the process of ‘ranging’ fashion product for a collection and creating repeat prints from a specific artwork, ready for digital printing. A workshop was also facilitated so both designer and artist could work together to create (and co-own) unique t shirt prints. Lawyers were consulted and ethical contracts were drawn up to cover all participants in this innovative collaboration. While the collaboration of artist and designer was important, the collection required curation of all elements so that the final collection came together as a professional and cohesive, quality, retail- ready product. This could only be created by experienced practitioners. Research significance The Akin Collection is the first Australian fashion brand to be created as a collaboration between five equally recognised Indigenous artists and five emerging fashion designers. It has familiarized the Indigenous artists to the logistics and culture of the fashion industry and the emerging fashion designers have been familiarized to the logistics and culture of how to collaborate with the unique Indigenous artwork that exists in Australia. After only three months, this culminated in a fashion parade showcasing the Akin collection to over 400 members of the public, government, media and retail. Feedback has been strong from the media and the industry, and a lookbook and photoshoot has been organised to promote and sell the collection both nationally and internationally. These concepts plus the curation outlined, has created a successful, luxury, quality collection ready for the international runways. This project has devised an ethical template for other Indigenous artists and emerging designers to create fashion collections that offer a unique aesthetic that could position and brand Australian fashion in the international marketplace. Key Words Indigenous artists, emerging fashion designers, Australian fashion design, ethical fashion, luxury Australian brand