The long-term objective is to identify markers whose modulation results in epithelial differentiation. This objective has a direct impact on the application of epithelial grafts as therapy for full thickness skin injury and as a delivery system for specific gene products. Connexin-negative cells are found in the basal cell layers that harbor stem cells. Connexin expression or lack thereof will be correlated with markers thought to be indicative of stem cells. For correlation with label retaining cells, which are putative stem cells, thymidine-labeled mouse skin will be used. For correlation with K19 expressing follicular cells, also purported to be stem cells, human skin will be utilized. To accomplish this aim, FACS analysis and immunohistochemistry combined with fluorescent and confocal microscopy will be employed. The functionality of gap junctions within both mouse skin and human hair follicles will also be evaluated. Cell populations able to carry out dye transfer will be compared to those populations containing label retaining cells in mouse skin, and K19 expressing cells in human hair follicles. These experiments will further indicate whether stem cells lack gap junction-mediated cell-to-cell communication. The connexin-negative cells of the epidermis will be isolated and compared in culture with other cells of the basal layer. For these experiments, antibodies against the extracellular domain of connexin molecule will be used as a negative selection factor in isolating cells by FACS. Individual cell populations will be assayed for culture life-span, and colony forming efficiency at each passage. Finally, retroviral vectors will be used to introduce both Cx43 gene and antisense oligonucleotides into keratinocytes. The effects will be assessed in submerged cultures, organotypic cultures and on organotypic cultures grafted onto athymic mice. The proposed research plan, as well as the activities described in the application, are aimed at achieving the career goals.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
5K01AR002079-05
Application #
6795452
Study Section
Arthritis and Musculoskeletal and Skin Diseases Special Grants Review Committee (AMS)
Program Officer
Baker, Carl
Project Start
2000-04-18
Project End
2006-03-31
Budget Start
2004-04-01
Budget End
2006-03-31
Support Year
5
Fiscal Year
2004
Total Cost
$127,710
Indirect Cost
Name
State University New York Stony Brook
Department
Dentistry
Type
Schools of Dentistry
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Kocer, Salih S; Djuric, Petar M; Bugallo, Monica F et al. (2008) Transcriptional profiling of putative human epithelial stem cells. BMC Genomics 9:359
Matic, Maja; Pullis, Christopher; Golightly, Marc G et al. (2005) Analysis of connexin 43 expression on keratinocytes using flow cytometry. Methods Mol Biol 289:193-200
Matic, Maja (2005) A subpopulation of human basal keratinocytes has a low/negative MHC class I expression. Hum Immunol 66:962-8
Matic, Maja; Simon, Marcia (2003) Label-retaining cells (presumptive stem cells) of mice vibrissae do not express gap junction protein connexin 43. J Investig Dermatol Symp Proc 8:91-5
Matic, Maja; Evans, W Howard; Brink, Peter R et al. (2002) Epidermal stem cells do not communicate through gap junctions. J Invest Dermatol 118:110-6