Immunophenotyping of the human bulge region: the quest to define useful in situ markers for human epithelial hair follicle stem cells and their niche

Abstract

Since the discovery of epithelial hair follicle stem cells (eHFSCs) in the bulge of human hair follicles (HFs) an important quest has started: to define useful markers. In the current study, we contribute to this by critically evaluating corresponding published immunoreactivity (IR) patterns, and by attempting to identify markers for the in situ identification of human eHFSCs and their niche. For this, human scalp skin cryosections of at least five different individuals were examined, employing standard immunohistology as well as increased sensitivity methods. Defined reference areas were compared by quantitative immunohistochemistry for the relative intensity of their specific IR.According to our experience, the most useful positive markers for human bulge cells turned out to be cytokeratin 15, cytokeratin 19 and CD200, but were not exclusive, while b1 integrin and Lhx2 IR were not upregulated by human bulge keratinocytes. Absent IR for CD34, connexin43 and nestin on human bulge cells may be exploited as negative markers. a6 integrin, fibronectin, nidogen, fibrillin-1 and latent transforming growth factor (TGF)-betabinding protein-1 were expressed throughout the connective tissue sheath of human HFs. On the other hand, tenascin-C was upregulated in the bulge and may thus constitute a component of the bulge stem cell niche of human HFs.These immunophenotyping results shed further light on the in situ expression patterns of claimed follicular 'stem cell markers' and suggest that not a single marker alone but only the use of a limited corresponding panel of positive and negative markers may offer a reasonable and pragmatic compromise for identifying human bulge stem cells in situ.

Differential expression of stem-cell-associated markers in human hair follicle epithelial cells

“…Although both K15 and CD200 were expressed in the bulge, as expected, 9,11,23 our results revealed that the areas of expression for the two markers are distinct from each other. CD200 was expressed in a limited range around the bulge, whereas K15 was expressed widely in both the peri-bulge and sub-bulge areas (Figure 2a and b).…”

“…As reported previously, 9,11,17,19,23 K15 (bulge marker), CD200 (bulge marker), CD34, and CD271 were strongly and differentially expressed in the outermost layer of the ORS in cells that adhered to the basement membrane ('basal cells'; Figure 1a). Other cytokeratins (K1 and K14) and integrins Figure 1 Biomarker expression profiles in human epidermis and the anagen hair follicle.…”

“…Thus, unlike rodent CD34, human CD34 could not be regarded as a bulge marker in this study, consistent with previous reports. 17,23 Recent reports suggested that CD34 and CD271 were expressed in human hair follicles, especially in the ORS proximal to the attachment site of the APM. 16,17,19 Our immunohistological assays demonstrated a similar staining pattern; however, we report for the first time that CD271 was expressed proximal to CD34.…”

Eradication of damaged keratinocytes in cutaneous lichen planus forms demonstrated by evaluation of epidermal and follicular expression of CK15, indices of apoptosis and regulatory protein S100

“…The sections were counterstained with Mayer's hematoxylin, washed with tap water, dehydrated, cleared and mounted in PERTEX (mounting medium for light microscopy, Histolab, Gothenburg, Sweden). Initially, reproduction of published immunoreactivity patterns for CK15 was used as an internal positive control as recommended by Kloepper et al (2008). Thereafter, sections from a case of melanoma and human squamous cervical carcinoma were used as positive controls for S100 and CK15 immunohistochemistry, respectively.…”

“…These multipotent stem cells, called epithelial hair follicle stem cells (eHFSCs), are located in the bulge region -specifically, at the insertion site of the arrector pili muscle in the outermost layer of the outer root sheath [14,15] -and contribute to repopulation of the epidermis after injury. According to the results published by Abbas et al (2009Abbas et al ( , 2011, Kloepper et al (2008), and Sabeti et al (2013) [16,17,18,19] cytokeratin 15 (CK15), a stem cell marker, labels the cells in the bulge region of the human hair follicle, the outermost layer of the outer root sheath, the basal layer of the epidermis and eccrine glands, whereas other researchers found that the basal layer of normal epidermis is usually negative for CK15, and the bulge stem cells do not contribute to the interfollicular epidermis reconstitution [20]. A further controversy is added by Cotsarelis' publication reporting about the migration of the bulge cells to the interfollicular epidermis [21].…”