Research Items (9)
RNA synthesis can be detected by 5-ethynyl uridine (EU) incorporation and click chemistry. Despite identifying a fundamental functional process, this technique has yet to be widely applied to complex human tissue systems. By incorporating EU into human hair follicle (HF) organs cultured ex vivo, nascent RNA synthesis was detected in situ. EU differentially incorporated across the HF epithelium. Interestingly, RNA synthesis did not correlate with protein synthesis, proliferation or epithelial progenitor cell marker expression. By treating human HFs with the cytotoxic cell cycle inhibitor (R)-CR8, which inhibits transcriptional regulators CDK7 and CDK9, it was further shown that this technique can be used to sensitively detect changes in global RNA synthesis in situ. Together, this work delineates new insights into nascent RNA synthesis within a human (mini)- organ and describes a novel read-out parameter that will enrich future ex vivo human tissue research studies.
- Apr 2017
Human hair follicle (HF) growth and hair shaft formation require terminal differentiation-associated cell cycle arrest of highly proliferative matrix keratinocytes. However, the regulation of this complex event remains unknown. CIP/KIP family member proteins (p21CIP1, p27KIP1 and p57KIP2) regulate cell cycle progression/arrest, endoreplication, differentiation and apoptosis. Since they have not yet been adequately characterized in the human HF, we asked whether and where CIP/KIP proteins localise in the human hair matrix and pre-cortex in relation to cell cycle activity and HF-specific epithelial cell differentiation that is marked by keratin 85 (K85) protein expression. K85 expression coincided with loss or reduction in cell cycle activity markers, including in situ DNA synthesis (EdU incorporation), Ki-67, phospho-histone H3 and cyclins A and B1, affirming a post-mitotic state of pre-cortical HF keratinocytes. Expression of CIP/KIP proteins was found abundantly within the proliferative hair matrix, concomitant with a role in cell cycle checkpoint control. p21CIP1, p27KIP1 and cyclin E persisted within post-mitotic keratinocytes of the pre-cortex, whereas p57KIP2 protein decreased but became nuclear. These data imply a supportive role for CIP/KIP proteins in maintaining proliferative arrest, differentiation and anti-apoptotic pathways, promoting continuous hair bulb growth and hair shaft formation in anagen VI. Moreover, post-mitotic hair matrix regions contained cells with enlarged nuclei, and DNA in situ hybridisation showed cells that were >2N in the pre-cortex. This suggests that CIP/KIP proteins might counterbalance cyclin E to control further rounds of DNA replication in a cell population that has a propensity to become tetraploid. These data shed new light on the in situ-biography of human hair matrix keratinocytes on their path of active cell cycling, arrest and terminal differentiation, and showcase the human HF as an excellent, clinically relevant model system for cell cycle physiology research of human epithelial cells within their natural tissue habitat.
Human scalp hair follicles (hHF) harbour several epithelial stem (eHFSC) and progenitor cell sub-populations organised into spatially distinct niches. However, the constitutive cell cycle activity of these niches remains to be characterized in situ. Therefore, the current study has studied these characteristics of keratin 15+ (K15), CD200+ or CD34+ cells within anagen VI hHFs by immunohistomorphometry, using Ki-67 and 5-ethynyl-2′-deoxyuridine (EdU). We quantitatively demonstrate in situ the relative cell cycle inactivity of the CD200+/K15+ bulge compared to other non-bulge CD34+ and K15+ progenitor compartments and found that in each recognized eHFSC/progenitor niche, proliferation associates negatively with eHFSC-marker expression. Furthermore, we also show how prostaglandin D2 (PGD2), which is upregulated in balding scalp, differentially impacts on the proliferation of distinct eHFSC populations. Namely, 24 h organ-cultured hHFs treated with PGD2 displayed reduced Ki-67 expression and EdU incorporation in bulge resident K15+ cells, but not in supra/proximal bulb outer root sheath K15+ progenitors. This study emphasises clear differences between the cell cycle behaviour of spatially distinct stem/progenitor cell niches in the hHF, and demonstrates a possible link between PGD2 and perturbed proliferation dynamics in epithelial stem cells.
- Apr 2016
The cell cycle is of major importance to human hair follicle (HF) biology. Not only is continuously active cell cycling required to facilitate healthy hair growth in anagen VI HFs, but perturbations in the cell cycle are likely to be of significance in HF pathology (i.e. in scarring, non-scarring, chemotherapy induced and androgenic alopecias). However cell cycle dynamics of the human hair follicle (HF) are poorly understood in contrast to what is known in mouse. The aims of this method review are two fold; principally to address this gap we present a primer to introduce histological techniques to study the cell cycle in the human HF whilst concurrently encouraging the exploitation of the human HF as a powerful and clinically relevant tool to investigate mammalian cell cycle biology in situ. To achieve this we describe methods to study general 'proliferation' (nuclei count, Ki-67 expression), apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labelling, cleaved caspase 3), mitosis (phospho- histone H3, 'pS780'), DNA synthesis (5-ethynyl-2'-deoxyuridine) and cell cycle regulation (Cyclins) in the human HF, whilst also providing specific examples of dual labelling to further provide instructive cell cycle analyses or to investigate the cell cycle behaviours of specific HF keratinocyte sub-populations (i.e. Keratin 15+ stem / progenitor cells). This article is protected by copyright. All rights reserved.
- Mar 2015
In the murine hair follicle (HF) the transcription factors LHX2 and SOX9 are involved with epithelial hair follicle stem cell (eHFSC) self-renewal and the maintenance of eHFSC niche characteristics. However, the exact expression patterns of LHX2 and SOX9 in human HF are unclear. We have quantitatively mapped the localisation of known human eHFSC markers keratin 15 (K15) and keratin 19 (K19) in the ORS of male occipital scalp anagen HFs, and related this to the localisation of LHX2 and SOX9 protein expression. As expected, K15(+) and K19(+) cells represented two distinct progenitor cell populations in the bulge and in the proximal bulb ORS (pbORS). Interestingly, cell fluorescence for K19 was significantly stronger within the pbORS versus the bulge, and vice versa for K15, describing a hitherto unrecognised differential expression pattern. LHX2 and SOX9 expressing cells were distributed throughout the ORS, including the bulge, but were not restricted to it. SOX9 expression was most prominent in the ORS immediately below the human bulge whereas LHX2(+) cells were similarly distributed between the sub-bulge and pbORS, i.e. compartments not enriched with quiescent eHFSCs. During catagen development, the intensity of LHX2 and SOX9 protein expression increased in the proximal HF epithelium. Double-immunostaining showed that SOX9(+) cells in human anagen HF epithelium generally do not express K15, K19 or Lhx2. This data suggests that LHX2 and SOX9 may highlight distinct epithelial progenitor cell populations, in addition to K15(+) or K19(+) cells, that could play an important role in the maintenance of human HF epithelium. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
- May 2014
Epithelial hair follicle stem cells (eHFSCs) are required to generate, maintain and renew the continuously cycling hair follicle (HF), supply cells that produce the keratinized hair shaft and aid in the reepithelialization of injured skin. Therefore, their study is biologically and clinically important, from alopecia to carcinogenesis and regenerative medicine. However, human eHFSCs remain ill defined compared to their murine counterparts, and it is unclear which murine eHFSC markers really apply to the human HF. We address this by reviewing current concepts on human eHFSC biology, their immediate progeny and their molecular markers, focusing on Keratin 15 and 19, CD200, CD34, PHLDA1, and EpCAM/Ber-EP4. After delineating how human eHFSCs may be selectively targeted experimentally, we close by defining as yet unmet key challenges in human eHFSC research. The ultimate goal is to transfer emerging concepts from murine epithelial stem cell biology to human HF physiology and pathology.