Abstract
We developed protocols for isolation and characterization of mesenchymal progenitors from murine dermis. Our protocols are part of a more general isolation procedure starting with neonatal murine skin, which has been described in detail by U. Lichti and coauthors (Nat Protoc 3(5):799–810, 2008). We list Lichti’s procedures in an abbreviated form as part of this methods section. Our methods to isolate mesenchymal stem cells are presented as a continuous workflow of isolation and characterization, including flow cytometry, cell survival assays, colony formation assays, immunoblotting, immunostaining, multipotential differentiation assays, and in vivo engraftment. In most cases, the protocols are standard; in others, they were adapted to our particular purpose. We made special emphasis on the use of in vitro three-dimensional cultures to cue mesenchymal progenitors into epidermal cells.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Weinberg WC, Goodman LV, George C, Morgan DL, Ledbetter S, Yuspa SH, Lichti U (1993) Reconstitution of hair follicle development in vivo: determination of follicle formation, hair growth, and hair quality by dermal cells. J Invest Dermatol 100(3):229–236
Lichti U, Weinberg WC, Goodman L, Ledbetter S, Dooley T, Morgan D, Yuspa SH (1993) In vivo regulation of murine hair growth: insights from grafting defined cell populations onto nude mice. J Invest Dermatol 101(1 Suppl):124S–129S
Crigler L, Kazhanie A, Yoon TJ, Zakhari J, Anders J, Taylor B, Virador VM (2007) Isolation of a mesenchymal cell population from murine dermis that contains progenitors of multiple cell lineages. FASEB J 21(9):2050–2063. doi:fj.06-5880com [pii], 10.1096/fj.06-5880com
Dlugosz AA, Cheng C, Williams EK, Dharia AG, Denning MF, Yuspa SH (1994) Alterations in murine keratinocyte differentiation induced by activated rasHa genes are mediated by protein kinase C-alpha. Cancer Res 54(24):6413–6420
Dlugosz AA, Yuspa SH (1994) Protein kinase C regulates keratinocyte transglutaminase (TGK) gene expression in cultured primary mouse epidermal keratinocytes induced to terminally differentiate by calcium. J Invest Dermatol 102(4):409–414
Hennings H, Lowry DT, Robinson VA, Morgan DL, Fujiki H, Yuspa SH (1992) Activity of diverse tumor promoters in a keratinocyte co-culture model of initiated epidermis. Carcinogenesis 13(11):2145–2151
Li L, Tennenbaum T, Yuspa SH (1996) Suspension-induced murine keratinocyte differentiation is mediated by calcium. J Invest Dermatol 106(2):254–260
Tennenbaum T, Li L, Belanger AJ, De Luca LM, Yuspa SH (1996) Selective changes in laminin adhesion and alpha 6 beta 4 integrin regulation are associated with the initial steps in keratinocyte maturation. Cell Growth Differ 7(5):615–628
Al-Batran SE, Astner ST, Supthut M, Gamarra F, Brueckner K, Welsch U, Knuechel R, Huber RM (1999) Three-dimensional in vitro cocultivation of lung carcinoma cells with human bronchial organ culture as a model for bronchial carcinoma. Am J Respir Cell Mol Biol 21(2):200–208
Fuchs E (1990) Epidermal differentiation: the bare essentials. J Cell Biol 111(6 Pt 2):2807–2814
Lichti U, Anders J, Yuspa SH (2008) Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice. Nat Protoc 3(5):799–810. doi:nprot.2008.50 [pii], 10.1038/nprot.2008.50
Acknowledgments
This work was supported by the Intramural Research Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health.
The authors have no conflicting financial interests to declare.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Kimlin, L., Virador, V. (2013). Cellular Populations Isolated from Newborn Mouse Skin Including Mesenchymal Stem Cells. In: Turksen, K. (eds) Skin Stem Cells. Methods in Molecular Biology, vol 989. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-330-5_17
Download citation
DOI: https://doi.org/10.1007/978-1-62703-330-5_17
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-329-9
Online ISBN: 978-1-62703-330-5
eBook Packages: Springer Protocols