BJD British Journal of Dermatology. Summary DERMATOPATHOLOGY

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1 DERMATOPATHOLOGY BJD British Journal of Dermatology CD10 and CD34 in fetal and adult human hair follicles: dynamic changes in their immunohistochemical expression during embryogenesis and hair cycling E. Poblet and F. Jiménez* Department of Pathology and AECC Research Unit, Hospital General Universitario de Albacete, Albacete, Spain *Dermatological private practice, Angel Guimerá 2, Las Palmas de Gran Canaria, Spain Summary Correspondence Francisco Jiménez. Accepted for publication 7 May 2008 Key words CD10, CD34, hair follicle, immunohistochemistry, placode Conflicts of interest None declared. DOI /j x Background CD10 and CD34 have been detected in both epithelial and mesenchymal components of anagen human hair follicles. Objectives To analyse the expression of CD10 and CD34 in human hair follicle development as well as in different phases of the hair cycle. Methods Fetal and adult hair follicles at different stages of the hair cycle were examined by immunohistochemistry for CD10 and CD34. Results In fetal follicles, CD10 is expressed by the cells of the placodes, and CD34 by the mesenchymal cells of the dermal condensate. As the follicle matures, CD10 can be seen in the matrix cells, inner root sheath and dermal sheath. In adult follicles, the expression of CD10 in the follicular epithelium is present in anagen follicles, but tends to disappear in catagen, and is not detected in telogen. The CD10 positivity of the dermal sheath is more intense in catagen than in anagen follicles. CD34 immunostaining of the external root sheath was seen in adult anagen follicles but not in fetal follicles. This staining of the anagen outer sheath tends to disappear in catagen and is not detected in telogen. Conclusions CD10 and CD34 are not proteins constantly present in a specific cell type of the hair follicle, but are proteins that can be expressed by both epithelial and mesenchymal cells depending on the stage of development and hair cycle. The distribution of the immunoreactivity to CD10 in the placode and CD34 in the dermal condensate suggests a role of these proteins in initial stages of hair formation. CD10, also known as neutral endopeptidase or as common acute lymphoblastic leukaemia antigen, is a cell surface zincdependent metalloproteinase. It has a well known function as a cell surface enzyme that degrades several neuropeptides including substance P, bradykinin, enkephalins, bombesin and calcitonin gene-related peptide. 1 Because CD10 controls the local concentrations of peptide substrates, its role in each tissue is different depending on the availability of the substrate. Based on immunohistochemical findings, the CD10 antigen was originally believed to be a tumour-specific marker of acute lymphoblastic leukaemia, but expression of CD10 was later demonstrated in numerous normal and neoplastic lymphoid and nonlymphoid tissues. CD10 is expressed in normal cells and tissues including lymphoid precursor cells, enterocytes, glomeruli, and myoepithelial cells of the breast among others. 2 The first study of the cutaneous immunohistochemical expression of CD10 was reported by Orelund et al., 3 who described in frozen sections the CD10 staining of the epidermal keratinocytes, epithelial and myoepithelial cells of eccrine glands, and perifollicular cells of hair follicles. However, numerous and more recent studies using standard immunohistochemical methods on formalin-fixed, paraffin-embedded tissue specimens failed to detect the epidermal expression of CD10, showing instead that CD10 expression is more restricted to the pilosebaceous structure. 4 6 Specifically, CD10 immunoreactivity has been reported in the epithelial cells of the inner root sheath, hair matrix, and in the perifollicular dermal sheath, that is, the spindle-shaped cells that surround the inferior segment of the follicular epithelium. All these findings have been described in adult anagen human follicles. To our knowledge the expression of CD10 in catagen and telogen follicles has not been studied. Besides normal skin, CD10 has been found in a variety of cutaneous tumours including basal cell carcinomas, 6,7 in follicular derived tumours such as pilomatricomas and trichoblastomas, 7 646

2 CD10 and CD34 in fetal and adult human hair follicles, E. Poblet and F. Jiménez 647 and in mesenchymal skin tumours such as dermatofibromas, 8 dermatofibrosarcoma protuberans, neurofibromas and atypical fibroxanthomas. 9 CD10 has been expressed with some frequency in xanthomatous neoplasms of the skin, 10 and in approximately one-third of primary and metastatic melanomas. 11 CD34 is a heavily glycosylated cell surface protein that is expressed in immature haematopoietic cells and in endothelial cells. In mice hair follicles, CD34 identifies a subset of bulge keratinocytes with characteristics of stem cells. 12 However, this is not the case in human hair follicles. Immunohistochemical studies have shown positive immunostaining for CD34 in the epithelial cells of the outer root sheath below the isthmus level, 13,14 as well as perifollicular spindle-shaped cells. 15 Ohyama et al. 16 have characterized the gene expression profile and cell surface markers of the bulge region of human follicles, confirming that CD34 expression is low or absent in the bulge cells with stem cell characteristics. The purpose of this article is to analyse the expression of CD10 and CD34 in what we have termed a dynamic view over the lifetime of the hair follicle. The strategy used was the following: (i) to study the immunohistochemical expression of CD10 and CD34 during hair follicle embryogenesis using human fetal samples; and (ii) to analyse whether CD10 expression varies according to different phases of the hair cycle. Materials and methods Tissue samples Fetal human skin was collected from the scalp of 11 spontaneous abortions received at the Department of Pathology of the Hospital General Universitario de Albacete. The fetuses selected for this study were only those that did not present signs of maceration macroscopically, and that presented well-preserved follicular structures microscopically. The fetuses selected for the study had a gestational age of 10, 11, 13, 14 (three fetuses), 19 (two fetuses), 21, 28 and 32 weeks. Adult human hair follicles were selected from surgical pathology specimens received at the Department of Pathology of the Hospital General Universitario de Albacete, as well as from human scalp of volunteer patients treated in a hair restoration surgery clinic (Las Palmas de Gran Canaria). All specimens were formalin fixed (using buffered formalin) and paraffin embedded. From the material obtained at the Department of Pathology, 200 hair follicles in different stages of the hair cycle were examined. Most were in the anagen phase, while four hairs were in catagen and 18 in telogen. Each hair follicle selected for the study had its entire length represented on the slide. Paraffin blocks of the selected hair follicles were retrieved from the pathology files. Serial sections of these blocks were obtained following a protocol in order to examine the same hair follicle stained with haematoxylin and eosin (HE), anti-cd10 and anti-cd34. The protocol was as follows: sections were between 3 and 5 lm thick, and three consecutive single sections were retrieved and mounted on separate slides for HE, anti-cd10 and anti-cd34. In addition, 11 follicular units from human scalp were also harvested using a 1-mm circular punch. Manipulation of these follicular units was very meticulous when it came to making the paraffin blocks and the microtome cutting, as the goal was to obtain parallel sections that included the entire length of the hair follicles. To obtain consecutive sections of the same hair follicles the same protocol described above was used. (a) (b) Fig 1. Fetus of 11th week of gestational age. (a) Immunohistochemical staining with antibody to CD10. The first evidence of CD10 expression in fetal skin is the staining of the epithelial cells of the placode. (b) Immunohistochemical staining with antibody to CD34. The first evidence of CD34 expression in fetal skin is the staining of mesenchymal cells of the dermal condensate, located just below the CD10-positive cells of the placode.

3 648 CD10 and CD34 in fetal and adult human hair follicles, E. Poblet and F. Jiménez Immunohistochemistry Primary antibodies used were an anti-cd10 monoclonal antibody (clone 56C6, dilution 1 10; Novocastra, Newcastle upon Tyne, U.K.) and an anti-cd34 monoclonal antibody (clone QBEND 10; Dako, Glostrup, Denmark). Briefly, the immunohistochemical method used was as follows: paraffin sections were first deparaffinized, and then steamed for 40 min in citrate buffer, ph 7, at 95 C. Human hair follicles were immunostained using the standard avidin streptavidin staining method in an automated platform (Dako- Cytomation Autostainer, Glostrup, Denmark). The incubation time with the primary antibodies was 30 min. Diaminobenzidine reaction was used for visualization, followed by haematoxylin counterstain. Results Fetal hair follicles Expression of CD10 The first microscopic sign of hair follicle development, the foci of basal epidermal cells that bud into the dermis, known as the placode, shows a strong CD10 positivity (Fig. 1). In a later stage of development, CD10 staining is detected in (a) (b) Fig 2. (a) CD34 in fetal follicle of 14 weeks of gestational age. The CD34-positive mesenchymal cells of the dermal condensate form a primitive follicular papilla immediately below the follicular epithelium. (b) CD10 in fetal follicles of 17 weeks of gestational age showing strong positive staining of the epithelial matrix cells of the bulb and weak staining of perifollicular spindle-shaped cells. (a) (b) (c) Fig 3. Serial longitudinal sections of fetal skin of 21 weeks of gestational age stained with haematoxylin and eosin (a), antibody to CD10 (b), and antibody to CD34 (c). Section (b) shows that CD10 clearly delineates the perifollicular dermal sheath of the inferior portion of the anagen follicle, and is expressed by matrix cells and internal root sheath. Section (c) shows that CD34 is present mainly in perifollicular spindle-shaped cells (original magnification 10).

4 CD10 and CD34 in fetal and adult human hair follicles, E. Poblet and F. Jiménez 649 epithelial matrix cells of the bulb and in spindle-shaped perifollicular cells (Fig. 2). At week 21 the hair follicle showed a mature appearance, with presence of inner and outer root sheaths and dermal papilla, and CD10 was strongly expressed by the perifollicular dermal spindle-shaped cells and weakly by cells of the internal root sheath (Fig. 3). Expression of CD34 CD34 appears initially in the mesenchymal portion of the hair germ, the so called dermal condensate, located below the CD10-positive placode (Fig. 1). Later on, the cells of the dermal condensate form a primitive follicular papilla that is also CD34 positive (Fig. 2). When the hair follicle is more mature, CD34 staining is found only in the perifollicular spindleshaped cells but not in the dermal papilla (Fig. 3). In fetal follicles, we have not seen the typical CD34 staining of the outer root sheath that is always present in anagen adult follicles. Adult hair follicles Expression of CD10 In anagen follicles, CD10 is expressed by the epithelial cells of the inner root sheath, and by the perifollicular dermal sheath in the inferior portion of the hair follicle (below the level of the isthmus) (Fig. 4). We have observed that follicles in the early (a) (b) Fig 5. Adult catagen follicle immunostained with antibody to CD10, showing intense expression in perifollicular dermal sheath cells (original magnification 35). Fig 4. (a) Adult human hair follicle in early anagen phase stained with antibody to CD10, showing CD10 positivity in epithelial cells that form the bulb. (b) Adult human hair follicle in late anagen phase stained with antibody to CD10, showing positive staining of the inner root sheath and perifollicular dermal sheath cells. anagen phase also show CD10 positivity in epithelial cells that form the bulb; however, this CD10 epithelial staining of the hair bulb disappears in fully developed anagen follicles (Fig. 4). In catagen follicles, CD10 does not show any significant staining of the epithelial cells, but shows an intense staining in the perifollicular dermal sheath surrounding the epithelium of the lower follicle. This perifollicular network appears to be remarkably more intense in catagen follicles than in anagen follicles (Fig. 5). In telogen follicles the dermal sheath surrounding the telogen bulb shows a strong CD10 staining. CD10 also delineates the residual fibrous tract that corresponds to the remnant of the dermal sheath that surrounds the epithelium of the inferior portion of the anagen follicle, which disintegrates during catagen. The follicular epithelium at the telogen stage is CD10 negative (Fig. 6).

5 650 CD10 and CD34 in fetal and adult human hair follicles, E. Poblet and F. Jiménez (a) (b) (c) Fig 6. Serial section of an adult telogen follicle stained with haematoxylin and eosin (a), antibody to CD34 (b), and antibody to CD10 (c). The follicular epithelium at this stage is CD34 and CD10 negative. However, an intense CD10 expression is noted in the follicular dermal sheath that surrounds the telogen bulb and in the residual fibrous tract (original magnification 20). Expression of CD34 The findings in the hair follicles analysed coincide with those reported in our previous work. 14 In short, typical mature anagen human follicle shows CD34 expression in the outer root sheath, in the inferior segment of the follicle, below the isthmus level. CD34 immunoreactivity tends to disappear in catagen hair follicles, and is not detected in the telogen phase. As expected, perifollicular spindle-shaped cells are also CD34 positive, as well as endothelial cells and dendritic spindleshaped dermal cells (Fig. 6). Discussion Hair follicles are complex biological structures composed of epithelial cells (the matrix and root sheaths) and mesenchymal cells (the perifollicular dermal sheath and the dermal papillae). The first morphological sign of hair follicle formation is the appearance of focal, regularly spaced, thickenings of the fetal epidermis known as the placodes at about the 10th week of gestational age. In turn, signals from the placodes induce the clustering of primitive mesenchymal cells, which can be microscopically identified, and are known as the dermal condensate. 17 The interaction between the placodes with these subjacent mesenchymal cells is thought to play a major role in hair development. 18 In our immunohistochemical study, a result that clearly stands out is that the first microscopic structures of hair development, the placodes and the dermal condensates, show a strong CD10 and CD34 expression, respectively. To our knowledge, only Yada et al. 6 and Schirren et al. 19 have reported the expression of CD10 and CD34 in fetal follicles, but neither mentioned the immunoreactivity of the placode and the dermal condensate, probably because they studied fetal skin samples of later stages of development. Although our study entails a merely descriptive analysis of the immunohistochemical expression of CD10 and CD34 in hair follicles, the anatomical location of CD10 immunoreactivity in the epithelial cells of the placode and CD34 in the mesenchymal cells of the dermal condensate suggests that these two proteins might play a role in the early stage of hair morphogenesis. Moreover, the peculiar arrangement of the CD10 and CD34 proteins, facing each other in a complementary fashion, gives rise to speculation of a possible role of these proteins in the process of communication that takes place between the epithelial cells of the placode and the mesenchymal cells of the dermal condensate. This hypothesis is supported by the fact that CD10 has also been found in specialized mesenchymal cells (onychofibroblasts) in the nail unit, which appears to have an influence on the nail matrix for nail formation through epithelial mesenchymal interactions. 20 However, in contrast to the nail unit, the CD10- positive cells of the placode are epithelial in origin and not mesenchymal. The expression of CD10 and CD34 in hair follicle embryogenesis could be of interest in other skin hair-related fields such as follicular regeneration. Different strategies to achieve hair neoformation are currently under investigation that look promising as a useful therapy for hair loss. The idea would be to isolate hair follicle epithelial stem cells and combine them

6 CD10 and CD34 in fetal and adult human hair follicles, E. Poblet and F. Jiménez 651 with inductive dermal cells to produce new hair follicles. 21 The positive CD10 and CD34 staining in the very primitive epithelial and mesenchymal cells that give rise to the hair follicle indicates that these two proteins might be markers of follicular cells with inductive capacity of hair follicle formation. In addition, Reynolds et al. 22 demonstrated the inductive capacity of the follicular dermal sheath when the dermal sheath from the scalp of a man was transplanted to the forearm of a woman and 3 5 weeks later new hair follicles were found at the site of the implantation. It would be interesting to reproduce this experiment and investigate whether the cells responsible for this inductive capacity of the dermal sheath express the protein CD10. Another interesting aspect of our work is that the immunohistochemical expression of CD10 and CD34 appears to change according to the degree of hair follicle development and to the stage of hair follicle cycle. This brings into the discussion a concept that we have named the dynamic view of the hair follicle. As a hair follicle is a unique structure that experiences cycles of growth (anagen), regression (categen) and rest (telogen), it is reasonable to envision that the immunohistochemical expressions of these proteins might change according to the phase of the hair follicle cycle. Most immunohistochemical studies performed in human hair follicles describe the staining pattern from a static viewpoint, normally using only anagen follicles. Contrary to this fixed picture, the results of this article demonstrate that CD10 and CD34 can be expressed by cells of the hair follicle depending on the degree of maturation or the functional activity that these cells acquire. This dynamic expression can be observed as well with other molecules expressed by cells of the hair follicles such as the epithelial cell adhesion molecule (detected by the antibody Ber-EP4). 23 As examples of this changing expression, we have observed that CD10, which is initially expressed in fetal skin by the epithelial placode cells, is later found in the hair matrix and mesenchymal perifollicular cells and, when the hair follicle is more mature, only remnants of CD10 staining can be observed at the inner root sheath. Likewise, adult hair follicles also show changing CD10 immunoreactive patterns according to the hair cycle. For example, in early anagen follicles CD10 is expressed in epithelial cells of the bulb, in a very similar pattern to that seen in fetal follicles (compare Figs 2b and 4a). However, when the anagen follicle is fully mature, the epithelial CD10 staining of the bulbs is lost and the CD10 staining remains limited to the internal root sheath and to the dermal sheath. Regarding the protein CD34, in a previous study we had reported that the expression of CD34 in the follicular epithelium differs according to the hair cycle, being present in the external root sheath of the anagen but not in the telogen hair follicles, with a tendency to disappear when catagen begins. 14 However, in the present study we unexpectedly observed that the typical CD34-positive staining of the external root sheath seen in adult anagen follicles was not present in any of the fetal hair follicles analysed. This observation again emphasizes that CD10 and CD34 are not structural proteins linked to specific cell types, but rather proteins that can be expressed by follicular cells of epithelial and mesenchymal lineages depending on the stage of development and hair cycle. Acknowledgments We thank Dr Jose M. Godinez and Pedro J. Benito Castellanos for their technical assistance in immunohistochemistry, and Dr Fernando Larcher for his critical review of the manuscript. This work has been supported by grants JCCM and FIS PI , IN056. References 1 Roques B, Noble F, Dauge V et al. Neutral endopeptidase 24.11: structure, inhibition and experimental and clinical pharmacology. Pharmacol Rev 1993; 45: McIntosh GG, Lodge AJ, Watson P et al. NCL-CD10-270: a new monoclonal antibody recognizing CD10 in paraffin-embedded tissue. Am J Pathol 1999; 154: Orelund JE, Usui ML, Seckin D et al. Neutral endopeptidase expression and distribution in human skin and wounds. J Invest Dermatol 1999; 112: Kanitakis J, Bourchany D, Claudy A. Expression of the CD10 antigen (neutral endopeptidase) by mesenchymal tumors of the skin. Anticancer Res 2000; 20: Lee KJ, Choi YL, Kim WS et al. CD10 is expressed in dermal sheath cells of the hair follicles in human scalp. Br J Dermatol 2006; 155: Yada K, Kashima K, Daa T et al. Expression of CD10 in basal cell carcinoma. Am J Dermatopathol 2004; 26: Pham TTN, Selim MA, Burchette JL Jr et al. CD10 expression in trichoepithelioma and basal cell carcinoma. J Cutan Pathol 2006; 33: Lee KJ, Yang JM, Lee ES et al. CD10 is expressed in dermatofibromas. Br J Dermatol 2006; 155: Hultgren TL, DiMaio DJ. Immunohistochemical staining of CD10 in atypical fibroxanthoma. J Cutan Pathol 2007; 34: Perna AG, Smith MJ, Krishnan B, Reed JA. CD10 is expressed in cutaneous clear cell lesions of different histogenesis. J Cutan Pathol 2005; 32: Kanitakis J, Narvaez D, Claudy A. Differential expression of the CD10 antigen (neutral endopeptidase) in primary versus metastatic malignant melanomas of the skin. Melanoma Res 2002; 12: Trempus CS, Morris RJ, Bortner CD et al. Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. J Invest Dermatol 2003; 120: Poblet E, Jiménez F, Rocamora A. Qbend 10 (anti CD34 antibody) in external root sheath cells and follicular tumors. J Cutan Pathol 1994; 21: Poblet E, Jiménez F, Godínez JM et al. The immunohistochemical expression of CD34 in human hair follicles: a comparative study with the bulge marker CK15. Clin Exp Dermatol 2006; 31: Nickoloff BJ. The human progenitor cell antigen (CD34) is localized on endothelial cells, dermal dendritic cells, and perifollicular cells in formalin-fixed normal skin, and on proliferating endothelial cells and stromal spindle-shaped cells in Kaposi s sarcoma. Arch Dermatol 1991; 127: Ohyama M, Terunuma A, Tock CL. Characterization and isolation of stem cell-enriched human hair follicle bulge cells. J Clin Invest 2006; 116:

7 652 CD10 and CD34 in fetal and adult human hair follicles, E. Poblet and F. Jiménez 17 Holbrook KA. Minami SI. Hair follicle embryogenesis in the human. Characterization of events in vivo and in vitro. Ann NY Acad Sci 1991; 642: Millar SE. Molecular mechanisms regulating hair follicle development. J Invest Dermatol 2002; 118: Schirren CG, Burgdorf WHC, Sander CA, Plewig G. Fetal and adult hair follicle: an immunohistochemical study of anticytokeratin antibodies in formalin-fixed and paraffin-embedded tissue. Am J Dermatopathol 1997; 19: Lee KJ, Kim WS, Lee JH et al. CD10, a marker for specialized mesenchymal cells (onychofibroblasts) in the nail unit. J Dermatol Sci 2006; 42: Stenn KS, Cotsarelis G. Bioengineering the hair follicle: fringe benefits of stem cell technology. Curr Opin Biotechnol 2005; 16: Reynolds AJ, Lawrence C, Cserhalmi-Freidman PB et al. Transgender induction of hair follicles. Nature 1999; 402: Sellheyer K, Krahl D. Basal cell (trichoblastic) carcinoma. Common expression pattern for epithelial cell adhesion molecule links basal cell carcinoma to early follicular embryogenesis, secondary hair germ, and outer root sheath of the vellus hair follicle: a clue to the adnexal nature of basal cell carcinoma? J Am Acad Dermatol 2008; 58: