Keratinocyte stem cells are best identified by their ability to retain a BrdU label, and we have successfully FACS-sorted pure populations of these label-retaining cells (LRC). Unfortunately, the detection of BrdU requires that cells be fixed and permeablized, making them unsuitable for biological studies requiring living cells. In order to obtain living cells for biological studies, we have tried to identify unique cell surface markers on keratinocyte LRC, using both microarray analysis of global gene expression and proteomic mass spectrometry analysis of membrane proteins. For microarray analysis, we have developed """"""""navigated"""""""" laser capture microdissection to isolate enriched populations of LRC in the human hair follicle and have identified panels of genes, including genes that encode membrane proteins, that are differentially expressed in the LRC population of the human hair follicle bulge area (CD200, Frizzled1 receptor, and follistatin) as compared to non-LRC keratinocytes (CD24, CD34, CD71, CD146). In collaboration with the Biomedical Proteomics Program at FCRF, we have developed high-throughput mass spectrometry methods able to identify membrane proteins on LRC, and we are now using semi-quantitative mass spectrometry to compare the relative levels of the plasma membrane proteins prepared from LRC keratinocytes to control keratinocyte populations (transit amplifying basal keratinocytes). We have also assessed stem cell behavior in other putative KSC populations including side population or SP cells. These cells can be identified by unique fluorescent emission characteristics due to their ability to exclude HO33342 nuclear dye. We have recently described a SP population of keratinocytes and have completed characterization of SP keratinocytes long-term repopulating ability, using our recently described in vivo competitive repopulation stem cell assay.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010542-04
Application #
7338617
Study Section
(DB)
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2006
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Terunuma, Atsushi; Cross, Justin W; Dyke, Michelle et al. (2008) Behavior of human foreskin keratinocytes expressing a hair follicle stem cell marker CD200. J Invest Dermatol 128:1332-4
Terunuma, Atsushi; Kapoor, Veena; Yee, Carole et al. (2007) Stem cell activity of human side population and alpha6 integrin-bright keratinocytes defined by a quantitative in vivo assay. Stem Cells 25:664-9
Ohyama, Manabu; Vogel, Jonathan C; Amagai, Masayuki (2007) Gene ontology analysis of human hair follicle bulge molecular signature. J Dermatol Sci 45:147-50
Fan, Qingyuan; Yee, Carole Lee; Ohyama, Manabu et al. (2006) Bone marrow-derived keratinocytes are not detected in normal skin and only rarely detected in wounded skin in two different murine models. Exp Hematol 34:672-9
Ohyama, Manabu; Terunuma, Atsushi; Tock, Christine L et al. (2006) Characterization and isolation of stem cell-enriched human hair follicle bulge cells. J Clin Invest 116:249-60
Blonder, Josip; Terunuma, Atsushi; Conrads, Thomas P et al. (2004) A proteomic characterization of the plasma membrane of human epidermis by high-throughput mass spectrometry. J Invest Dermatol 123:691-9
Blonder, Josip; Conrads, Thomas P; Yu, Li-Rong et al. (2004) A detergent- and cyanogen bromide-free method for integral membrane proteomics: application to Halobacterium purple membranes and the human epidermal membrane proteome. Proteomics 4:31-45
Terunuma, A; Ye, J; Emmert, S et al. (2004) Ultraviolet light selection assay to optimize oligonucleotide correction of mutations in endogenous xeroderma pigmentosum genes. Gene Ther 11:1729-34
Terunuma, Atsushi; Shaya, Melissa B; Udey, Mark C et al. (2004) An in vivo competitive repopulation assay system to evaluate human keratinocyte stem cells. J Invest Dermatol 123:993-5
Terunuma, Atsushi; Jackson, Kimberly L; Kapoor, Veena et al. (2003) Side population keratinocytes resembling bone marrow side population stem cells are distinct from label-retaining keratinocyte stem cells. J Invest Dermatol 121:1095-103

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