Isolating a pure population of epidermal stem cells for use in tissue engineering.
Continuously renewing tissues, such as the epidermis, are maintained by stem cells that slowly proliferate and remain in the tissue for life. Although it has been known for decades that epithelial stem cells can be identified as label-retaining cells (LRCs) by long term retention of a nuclear label, isolating a pure population of stem cells has been problematic. Using a Hoechst and propidium iodide dye combination and specifically defined gating, we sorted mouse epidermal basal cells into three fractions, which we have now identified as stem, transient amplifying (TA), and non-proliferative basal cells. More than 90% of freshly isolated stem cells showed a G0/G1 cell cycle profile, while greater than 20% of the TA cells were actively dividing. Both stem and TA cells retained proliferative capacity, but the stem cells formed larger, more expandable colonies in culture. Both populations could be transduced with a retroviral vector and used to bioengineer an epidermis. However, only the epidermis from the stem cell population continued to grow and express the reporter gene for 6 months in organotypic culture. The epidermis from the transient amplifying cell fraction completely differentiated by 2 months. This novel sorting method yields pure viable epithelial stem cells that can be used to bioengineer a tissue and to test permanent recombinant gene expression.
Dunnwald
M
M
Department of Anatomy and Cell Biology, The University of Iowa, Iowa City 52242, USA.
Tomanek-Chalkley
A
A
Alexandrunas
D
D
Fishbaugh
J
J
Bickenbach
J R
JR
D000818:Animals; D000831:Animals, Newborn; D001562:Benzimidazoles / Q000378:metabolism; D001973:Bromodeoxyuridine / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D000078404:Epidermal Cells; D004817:Epidermis / Q000502:physiology; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D005818:Genetic Engineering; D015316:Genetic Therapy; D007763:Lac Operon; D051379:Mice; D011419:Propidium / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology; D014162:Transfection
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D011419:Propidium; D001973:Bromodeoxyuridine; C017807:bisbenzimide ethoxide trihydrochloride