Characterization and isolation of stem cell–enriched human hair follicle bulge cells
Manabu Ohyama, … , Mark C. Udey, Jonathan C. Vogel
Manabu Ohyama, … , Mark C. Udey, Jonathan C. Vogel
Published January 4, 2006
Citation Information: J Clin Invest. 2006;116(1):249-260. https://doi.org/10.1172/JCI26043.
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Research Article Dermatology

Characterization and isolation of stem cell–enriched human hair follicle bulge cells

Abstract

The human hair follicle bulge is an important niche for keratinocyte stem cells (KSCs). Elucidation of human bulge cell biology could be facilitated by analysis of global gene expression profiles and identification of unique cell-surface markers. The lack of distinctive bulge morphology in human hair follicles has hampered studies of bulge cells and KSCs. In this study, we determined the distribution of label-retaining cells to define the human anagen bulge. Using navigated laser capture microdissection, bulge cells and outer root sheath cells from other follicle regions were obtained and analyzed with cDNA microarrays. Gene transcripts encoding inhibitors of WNT and activin/bone morphogenic protein signaling were overrepresented in the bulge, while genes responsible for cell proliferation were underrepresented, consistent with the existence of quiescent noncycling KSCs in anagen follicles. Positive markers for bulge cells included CD200, PHLDA1, follistatin, and frizzled homolog 1, while CD24, CD34, CD71, and CD146 were preferentially expressed by non-bulge keratinocytes. Importantly, CD200+ cells (CD200hiCD24loCD34loCD71loCD146lo) obtained from hair follicle suspensions demonstrated high colony-forming efficiency in clonogenic assays, indicating successful enrichment of living human bulge stem cells. The stem cell behavior of enriched bulge cells and their utility for gene therapy and hair regeneration will need to be assessed in in vivo assays.

Authors

Manabu Ohyama, Atsushi Terunuma, Christine L. Tock, Michael F. Radonovich, Cynthia A. Pise-Masison, Steven B. Hopping, John N. Brady, Mark C. Udey, Jonathan C. Vogel

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Figure 3

The differential expression of genes identified by microarray analysis was confirmed by immunohistochemistry and FACS analysis of human anagen hair follicle.

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The differential expression of genes identified by microarray analysis w...
(A) The transverse sections of the same individual hair follicle at the bulge and sub-bulge levels are shown for each gene. The antibodies against bulge ORS–upregulated gene products identified in microarray analysis (KRT15, FZD1, FST, DKK3, WIF1, PHLDA1) demonstrated stronger staining in the bulge ORS than sub-bulge ORS, while antibodies against the bulge ORS–downregulated gene products (melanoma adhesion molecule [CD146], EDNR) demonstrated intense staining in the sub-bulge ORS. Scale bar: 50 mm. (B) Mid-follicle single-cell suspension was prepared from the mid-portion of individually isolated human hair follicles. Mid-follicle cells were fixed and stained with anti-FST and anti-KRT15 antibodies and analyzed with FACS. Consistent with microarray and immunohistochemistry data (see Supplemental Figure 2), fewer FST-positive cells were detected in mid-follicle suspensions than KRT15-positive cells. Subsequently, FST was used as the positive marker for human hair follicle bulge cells.