Progress 09/01/12 to 08/31/14
Outputs
Target Audience: The target audiences were mainly students, researchers, and professionals (clinical) who could be benefited with this science knowledge and aslo could positively impact this project. Changes/Problems: Contamination is a major problem of any cell culture, and specially for primary culture, that may have a significant and negative impact on the outcome results. In particular, the contamination by mycoplasm is one of the most challenge, least apparent, and very difficult to treat. It usually impairs and delays cell growth and end up jeopardizing and damaging the cells/culture. Mycoplasma was detected in my culture and it certainly delayed my long-term goals. First, I had to discard ongoing culture plates and I spent a considerable amont of time cleaning the environment (hoods, incubators, pipettes etc), tracking for the contamination source, and tryint to reestablish my cell culture. What opportunities for training and professional development has the project provided? This project provided to me the opportunity for training and professional development, as listed below: - Training on confocal microscopy at the Penn Vet Imaging Core (PVIC), - Advancing my skills in different laboratorial techniques, - Improving research writing, - Active participation in Journal Clubs and Chalk Talks series at New Bolton Center, - Participation in meetings and symposium. How have the results been disseminated to communities of interest? The results obtained in this project have been presented in internal seminars and presentations at the UPENN as well as national and international meetingd and conferences, and published in a peer-reviewed journal: Conferences: IRM and CECR Tissue Engineering Symposium, UPENN IRM, PA, US. September 2012. Annual Meeting of the International Society for Stem Cell Research. Boston, MA, US. June 2013. Penn Vet Faculty Research Retreats. International Equine Conference on Laminitis and Diseases of the Foot. West Palm Beach, FL, US. November, 2013 (Journal of Equine Veterinary Science. Vol. 33, Issue 10, p866-867) American Society for Cell Biology. Philadelphia, PA, US. December, 2014 Peer-reviewed publication: Veterinary Dermatology, 2015 (Epub ahead of press 5-12-15, DOI: 10.1111/vde.12214): Expression and localization of epithelial stem cell and differentiation markers in equine skin, eye and hoof. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
An organotypic culture of the equine lamellar epidermal cell has been developed using a commercial synthetic 3D scaffold (Alvetex® 200 μm thick culture dish inserts, Amsbio) and examined by indirect immunofluorescence (IIF) using the mouse monoclonal anti-K14 antibody (1:500, clone LL002; Abcam, Cambridge, MA; ab7800), against cytokeratin 14, a specific marker for basal keratinocytes. The cytokeratin 14 marker stains less differentiated basal epidermal cells of the secondary epidermal lamellae (Carter et al., 2010) and it has been applied to identify and locate the equine lamellar epidermal cells in the scaffold to validate our organotypic culture system. I investigated differentiation markers to verify the identity of equine lamellar (as well as skin and cornea) epidermal/epithelial cells: cytokeratin (K) 14 (specific marker for basal keratinocytes), K124 (a novel specific keratin for equine lamellar basal and suprabasal epidermal cells identified in the Galantino-Homer laboratory), K10 (skin-specific marker), K3 (cornea-specific marker), and the transcription factor p63 (proliferative potential of epithelial stem cells marker) and phospho-p63 (marker of the stem to transit-amplifying cell transition) in situ (fixed tissues) and in vitro (cell culture/colonies) by IIF. The p63 antibody, previously validated for use on equine tissues, demonstrated that this transcription factor p63, a regulator of epidermal stem cell (SC) proliferative potential, is present throughout the hoof lamellae and highly expressed by holoclones in culture. Phosphorylation of p63 (phospho-p63 or pp63) also served as a biomarker for epidermal SC differentiation. A microscope with epifluorescence illumination (Leica model DM5000B) and a confocal microscope (Leica model DMI6000B) were used to acquire the images. In addition to the retention of basal cell regulation (p63) and epidermal differentiation marker expression (K14, K124, and phospho-p63), the culture was also examined/characterized by proliferation, clone size and shape, cellular morphology, and clonogenic potential. Holoclones, the largest colonies generated from a single ESC, were characterized based on colony size and cobblestone cell morphology, with small cells containing scant cytoplasm at the periphery of the colony, and distinguished from medium-sized, transit-amplifying cell-based meroclones and small paraclones derived from differentiated epidermal cells. Secondary holoclones were generated from cells subcloned from primary holoclones, selected cell colonies (undifferentiated holoclones). The 3D scaffold is a highly porous interconnected scaffold made from cross-linked polystyrene that allows diffusion of gases, nutrients, and waste products, demonstrated that equine lamellar cells grow on the scaffold and tend to form discrete colonies that project from its surface. The use of transmission electron microscopy (TEM) allows the visualization of morphology of these cells in more detail, including abundant cytoskeletal elements, within the scaffold. The junction between the cells and scaffold revealed structures believed to be hemidesmosomes and numerous cell-cell attachments, believed, based on morphology, to be desmosomes and adherens junctions, were also observed by TEM.
Publications
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Progress 09/01/12 to 08/31/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provided to me the opportunity for training and professional development, as listed below: - Training on confocal microscopy at the Penn Vet Imagin Core (PVIC), - Active participation in Journal Clubs and Chalk Talks series at New Bolton Center, - Participation in meetings and symposia. How have the results been disseminated to communities of interest? The results have been presented in internal seminars and presentations at the UPENN as well as national and international meetings and conferences: IRM and CECR Tissue Engineering Symposium, UPENN IRM, PA, US. September 2012. Annual Meeting of the International Society for Stem Cell Research. Boston, MA, US. June 2013. International Equine Conference on Laminitis and Diseases of the Foot. West Palm Beach, FL, US. November, 2013 (Journal of Equine Veterinary Science. Vol. 33, Issue 10, p866-867). Penn Vet Faculty Research Retreats. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The accomplishments for these goals were: a. Isolation of equine hoof lamellar cells: Cells were obtained after mechanical disruption and enzymatic digestion of the lamellar epidermal tissue and underlying dermal tissue which were dissected after the removal of a small section of the dorsal hoof wall. Single cells were cultured on mitomycin-c-arrested 3T3-J2 fibroblasts in serum, growth factor supplemented FAD medium as previously described (Barrandon and Green, 1985). The identity of the equine lamellar culture was verified by cell morphology and expression of differentiation markers or specific markers for keratinocytes such as keratin (K) 14, a basal epidermal marker, and K124, a novel lamellar-specific keratin. b. Development of a tissue-derived scaffold: To create a tissue-derived scaffold, equine peritoneal basement membrane was harvested, mounted on a 9.5-mm diameter cylinder, and submitted to decellularization (chemically stripping the overlying mesothelial cells) with ammonium hydroxide or 0.15% peracetic or in combination with 3% hydrogen peroxide, as per a prococol developed by Hotary et al., 2006 and Padmini et al., 2010, for rat mesentery. c. Comparison of the tissue-derived and a 3D synthetic (Alvetex, Amsbio) scaffolds for the equine epidermal stem cell culture: It was demonstrated that the 3D synthetic scaffold created a more physiological environment to culture lamellar epidermal cells and reproduced better the in vivo conditions and tissue structures. This 3D scaffold allows cells to maintain their normal 3D shape and structure, to have more surface area exposed to other cells or to the matrix, to form complex interactions with adjacent cells, and to receive and transmit signals.
Publications
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