SEARCH

SEARCH BY CITATION

Keywords:

  • dermal papilla;
  • hair follicle papilla;
  • hair follicle;
  • cell culture;
  • microdissection

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method and results
  5. Discussion
  6. Acknowledgements
  7. References

Abstract: Study of the involvement of the hair follicle papilla in hair growth regulation was greatly facilitated by the isolation and cultivation of this tiny cluster of fibroblast-like cells in the rat vibrissae and in the human hair follicle. While isolation of the hair follicle papilla from the former is relatively straightforward, the current method to isolate the much smaller human hair follicle requires significant skill. Thus, the routine initiation of primary cultures of human scalp hair follicle papilla cells requires significant training, time, and commitment.

In an attempt to simplify hair follicle papilla cell culture methodology for new laboratory personnel, we have made significant refinements to the current method. Our method requires only two simple manipulations to isolate hair follicle papilla from intact isolated hair follicles. This very rapid and easy method isolates clean and intact hair follicle papillae. Together with their attachment via scratching to the growth surface, the isolation and cultivation of this important hair follicle component can now be achieved easily by the laboratory newcomer. The method relies for its simplicity on the removal of the hair follicle papilla from the outside of the intact hair follicle rather than via internal manipulations from within the hair follicle.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method and results
  5. Discussion
  6. Acknowledgements
  7. References

The current rapid pace of hair biology research owes much to the pioneering studies of Cohen and Oliver (1–3), which demonstrated, via elegant microdissection and implantation experiments, a crucial role for the hair follicle papilla (HFP; aka dermal papilla) in hair growth. This basal lamina-enclosed cluster of specialized fibroblast-like mesenchymal cells is located within the bulb of the anagen hair follicle (HF) and is continuous via its stalk with the connective tissue capsule that encloses the entire growing HF (4). From the earliest stages of HF morphogenesis to the end of adult life, the HFP induces the HF epithelium to produce a functional and cycling HF (5–7).

A major advance in dissecting the contributions of HFP cells in hair growth regulation emerged with successful isolation and cultivation in vitro by Jahoda & Oliver in the rat vibrissae, and Messenger and others in humans (8–11). Despite being mitotically quiescent, apoptosis-resistant and long-lived in vivo(12–14), these fibroblast-like cells proliferate in vitro. Importantly, cultured HFP cells retain their hair growth inductive capacity in vivo(15–17).

Published methodologies of HFP isolation rely on microdissecting manipulation for their successful isolation. While this technique is relatively straightforward for the large vibrissal HFP in the rat (8) or red deer (18), it requires significant skill for the considerably smaller human HF (9). To recap for those not familiar with these established standard methods of HFP isolation, intact anagen HF are removed and bisected above the bulb. Using fine needles and forceps, the lower connective tissue sheath (with attached HFP) is separated from the bulb epithelium. While this first part of the procedure presents little technical challenge, the method thereafter becomes considerably more difficult. The lower connective tissue sheath capsule then needs to be opened and inverted, and the teardrop-shaped HFP (a mere 0.2–0.3 mm across in human scalp HFs) externalized. The papilla is then removed by cutting the stalk attaching it to the now inverted connective tissue sheath (9). The second part of this procedure is particularly difficult for the uninitiated and unpracticed researcher. Moreover, imprecise manipulations at the level of the lower connective tissue sheath may increase the probability of dislodging and subsequently aspirating cells from the lower connective tissue sheath and/or epithelium. These cells may go on to contaminate the HFP culture.

During our attempts to refine and simplify this method, we have developed a significantly less involved method for the routine and rapid isolation of clean and intact HFP. This method relies for its simplicity on the removal of the HFP from the outside of the intact HF rather than via internal manipulations from within the HF.

Method and results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method and results
  5. Discussion
  6. Acknowledgements
  7. References

Isolation of human scalp hair follicles

Normal human scalp specimens were obtained with informed consent from elective face-lift surgeries or from donor and recipient haired scalp during hair restoration surgery for androgenetic alopecia. All cell culture reagents were obtained from Gibco BRL (Paisley, Scotland) unless otherwise indicated. Scalp specimens were transported to the laboratory in William's E serum-free medium supplemented with 2.5-fold concentrated penicillin (250 U/ml), streptomycin (250 μg/ml) and fungazone (12.5 µg/ml amphotericin B) at 4°C. Anagen VI HFs of variable caliber were isolated in fresh transport medium under a binocular dissecting microscope using a method modified from Philpott (19): briefly, small scalp specimens (approximately 1 cm3) were transected at the dermis–subcutis interface by scalpel (Fig. 1a,c) to reveal the mid-to-lower portions of HF embedded in the adipose tissue (Fig. 1b,c). The sides of the fat tissue were pressed carefully with blunt forceps to partially extrude the upper portion of the HFs from the subcutis. This enabled a fine forceps to grip and extract the HFs from the subcutis (Fig. 1c). Upon isolation, the HF were transferred intact into fresh growth medium and washed gently several times (Fig. 1d).

image

Figure 1. Transection of a normal human scalp specimen at the connective tissue-subcutis interface ([RIGHTWARDS ARROW]). Bright field microscopy (a) . Removal of the upper portion of the tissue transected in (a) leaving the lower half of the anagen VI hair follicles in the subcutis. Dark field microscopy (b) . Schematic of (a) and (b) with the method of isolation of hair follicles from the subcutis (c) . Collection of anagen VI hair follicles isolated as in (c). Phase contrast microscopy (d) . The hair follicle is gripped gently with a forceps at the suprabulbar region (e,e′). With gentle closing pressure applied to the forceps, the bulb becomes slightly compressed and globe-shaped (f,f′). With a beveled scalpel blade, the connective tissue sheath capsule is transected at the level of the stalk to open up the proximal capsule just underneath the hair follicle papilla (HFP; g,g′). Release of the gentle pressure imparted by the gripped forceps induces the HFP to emerge freely and intact from the bulb (h,h′). Note that this method generates little debris, thereby facilitating easy aspiration of very clean HFPs.

In the case of a incomplete transection of the HFP stalk, the distal edge of the HFP remains partially attached to the epithelium, but can be released easily with the scalpel blade (i,i′). Individual isolated HFP from anagen VI hair follicle: phase-contrast microscopy (j). The isolated HFP are attached to the plastic substratum by a needle scratch (k). Explantation of cells from a single HFP attached to the plastic substratum after 10 days. Note the mitotic cells in the insets: phase-contrast microscopy (l). HFP cells after subculture with their characteristic flattened morphology: phase-contrast microscopy (m). High-resolution light microscopy of an isolated HFP. Note that the HFP is isolated intact and with clean borders using this method. No stalk is retained. Toluidine blue stain (n). Transmission electron microscopy of the border of an isolated HFP showing the intact basal lamina with multiplication internally. Uranyl acetate and lead citrate stain (o).

Download figure to PowerPoint

Isolation of human scalp hair follicle papillae

Hair follicle papillae were individually isolated from intact isolated HF under bright-field stereoscopic illumination: the HF was gripped gently with a forceps at the suprabulbar region (Fig. 1e,e′ and stabilized to the base of the Petri dish. By gently closing the pressure applied to the forceps, the bulb became slightly compressed and globe-shaped (Fig. 1f,f′). Using a fresh beveled scalpel blade (size 15, Swan-Morton, Sheffield, UK), the connective tissure sheath capsule was transsected at the level of the stalk, thereby ′opening up′ the proximal capsule just underneath the HFP (Fig. 1g,g′). The result of transecting this portion of the capsule was to release the gentle pressure imparted by the gripped forceps and to induce the HFP to emerge freely and intact from the bulb (Fig. 1h,h′). In the case of incomplete transection of the HFP stalk, the distal edge of the HFP remained partially attached to the epithelium (Fig. 1i,i′), but could easily be released with a scalpel blade. Importantly, this method generates little debris, thereby facilitating easy aspiration of very clean HFP (Fig. 1j). This is an important consideration, as the collection/transfer of cells originating from the HF capsule or epithelium may contaminate the HFP culture.

Establishment of human scalp hair follicle papilla cell cultures

After HFP isolation it is our routine practice to transfer from the same donor, using an automatic micropipette set to 30–50 µl, up to four HFP to a single sterile 35-mm plastic Petri dish. The HFP were incubated in 2 ml RPMI 1640 supplemented with 2 mM L-glutamine, 20% fetal calf serum (FCS) and 1x concentrated penicillin and streptomycin without amphotericin B. The HFPs were anchored to the substratum by a single scratch through their center using a fine needle (Fig. 1k). This step, in addition to adhering the HFP to the growth surface, also breaks the HFP basal lamina case to facilitate the emigration of cells during the subsequent cell explantation phase. The dishes were then moved to a 37°C incubator (humidified atmosphere of 5% CO2) and left unmoved for 7 days. The growth medium was refreshed with RPMI 1640 supplemented with 10% FCS when the first cell migration was apparent, i.e. after approximately 7–10 days for the majority of isolated HFP (Fig. 1l).

The HFP cells were permitted to expand until all four explants began to merge and were then subcultured by detachment with trypsin/EDTA and transferal to a T25 cell culture flask, i.e. passage 1 (Fig. 1m). This expansion was complete approximately 3 weeks after the initiation of the primary culture. Cells were cryopreserved in liquid nitrogen at passage 3 and used for routine experimentation at passages 3–6.

Microscopic evaluation of isolated human scalp hair follicle papillae

The quality of the HFP isolated using this method was assessed by high-resolution light microscopy (HRLM) and transmission electron microscopy, as previously described (20). Hair follicle papillae were placed immediately upon isolation into half-strength Karnovsky's fixative (21), and postfixed in 2% osmium tetroxide. After dehydration in graded ethanols, the HFP were infiltrated with araldite resin and polymerized at 60°C. Semi-thin and ultra-thin sections were cut with a Reichart-Jung microtome; the former were stained with the metachromatic stain and toluidine blue/borax, examined by light microscopy and photographed (Leitz, Wetzlar, Germany). Ultra-thin sections were stained with uranyl acetate and lead citrate, and examined and photographed using a Jeol 1200X transmission electron microscope (Jeol, Tokyo, Japan). By HRLM, the isolated HFPs were seen to be full and intact (Fig. 1n). An intact basal membrane was seen to encase many of the HFP cells that were widely dispersed in copious extracellular matrix. Furthermore, the base of the HFP lacked mesenchymal material from the stalk or lower connective tissue sheath. Transmission electron microscopy of the isolated HFP confirmed the retention of an intact basal lamina that was multilaminated in places (Fig. 1o). Hair follicle papilla cells with active cytoplasm were present in HFP that were isolated from these anagen HF.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method and results
  5. Discussion
  6. Acknowledgements
  7. References

We describe here a novel method for the rapid and easy isolation of HFP from human scalp anagen HF. This method has several significant advantages over current methodologies. Primary amongst these is the greatly increased ease of HFP isolation, as only a single and external transection/manipulation is required. Second, this increased ease results in a considerably more rapid isolation when several HFP are required. After a short period of habituation, users in the author's laboratory and in cooperating laboratories obtained strikingly higher yields with this new technique. Once the HF is isolated, the process of HFP isolation should not take longer than approximately 3 min. Third, the increased ease of HFP isolation increases the availability of this method to those researchers not skilled in microdissection. Thus, HFP culture no longer need be restricted in its use by workers in the field. Fourth, the lack of manipulation of the HF internally removes any potential contamination with debris from the lower connective tissue sheath and/or hair bulb epithelium.

Cultures of HFP are primarily obtained from terminal HFs. However, the much-reduced manipulation of the HF using the current method will allow easy isolation of HFP from intermediate HFs in a balding scalp. The isolation of vellus HFs in toto is technically very demanding because of their inherent lack of pigmentation and size. We have not yet tested our new technique for the isolation of these HF, but the point made for intermediate balding HF may indeed be transferable.

Cultured HFP cells have several applications in hair biology research. In addition to their obvious application as a model for hair growth, their expression of steroid receptors enables their use in dissecting the mechanisms of steroid action in hair growth. Moreover, cultured HFP cells are also likely to be useful in hair follicle disorders where the HFP may be a target (22,23). Cultured HFP cells also lend themselves to investigating the effects of various pharmacologic agents e.g. including K+ channel openers on hair growth (24,25) or for the identification of secreted factors (26). Perhaps the most significant potential of these cells is the further exploration of dermal–epidermal interactions between DP cells and follicular epithelial cell populations (4,27,28), which may reveal specific regulatory molecules in hair growth. The availability of a method that greatly increases the ease and speed of isolating this tiny, but critically important, component of the HF should facilitate wider exploitation of these cells in our long-term goal of understanding the intricacies of hair growth and cycling.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method and results
  5. Discussion
  6. Acknowledgements
  7. References

This study was supported in part by a grant from Pharmacia and from the European Union (Brite-Euram 3: BE97-4301).

The authors thank Drs Nilofer and Bessam Farjo (Farjo Medical Center, Manchester, UK) for their constant support, and Mrs Joanna Carder for her excellent technical assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method and results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Cohen J. The transplantation of individual rat and guinea-pig whisker papilla. J Embryol Exp Morphol 1961: 9: 117127.
  • 2
    Oliver RF. Whisker growth after the removal of the dermal papilla and lengths of the follicle in the hooded rat. J Embryol Exp Morphol 1966: 15: 331347.
  • 3
    Oliver RF. The experimental induction of whisker growth in the hooded rat by implantation of dermal papillae. J Embryol Exp Morphol 1967: 18: 4351.
  • 4
    Jahoda CAB, Oliver R F. The dermal papilla and the growth of hair. In: OrfanosCE, HappleR, eds. Hair and hair diseases. Berlin: Springer-Verlag, 1990: 1944.
  • 5
    Hardy MH. The secret life of the hair follicle. Trends Genet 1992: 8: 5560.
  • 6
    Matsuzaki T, Yoshizato K. Role of hair papilla cells on induction and regeneration processes of hair follicles. Wound Repair Regen 1998: 6: 524530.
  • 7
    Kishimoto J, Burgeson R E, Morgan B A. Wnt signaling maintains the hair-inducing activity of the dermal papilla. Genes Dev 2000: 14: 11811185.
  • 8
    Jahoda CAB, Oliver RF. The growth of vibrissa dermal papilla cells in vitro. Br J Dermatol 1981: 105: 623627.
  • 9
    Messenger AG. The culture of dermal papilla cells from human hair follicles. Br J Dermatol 1984: 110: 685689.
  • 10
    Messenger AG, Senior HJ, Bleehen SS. The in vitro properties of dermal papilla cell lines established from human hair follicles. Br J Dermatol 1986: 114: 425430.
  • 11
    Warren R, Chestnut MH, Wong TK, Otte TE, Lammers KM, Meili ML. Improved method for the isolation and cultivation of human scalp dermal papilla cells. J Invest Dermatol 1992: 98: 693699.
  • 12
    Silver AF, Chase HB. The incorporation of tritiated uridine in hair germ and dermal papilla during dormancy (telogen) and activation (early anagen). J Invest Dermatol 1977: 68: 201205.
  • 13
    Tezuka M, Ito M, Ito K, Sato Y. Cell kinetic study of human and mouse hair tissues using anti-bromodeoxyuridine monoclonal antibody. J Dermatol Sci 1990: 1: 335346.
  • 14
    Bassukas ID, Kiesewetter F, Schell H, Hornstein OP. In situ [3H]thymidine labeling of human hair papilla: an in vitro autoradiographic study. J Dermatol Sci 1992: 3: 7881.
  • 15
    Jahoda CA, Horne KA, Oliver RF. Induction of hair growth by implantation of cultured dermal papilla cells. Nature 1984: 311: 560562.
  • 16
    Watson SA, Pisansarakit P, Moore GP. Sheep vibrissa dermal papillae induce hair follicle formation in heterotypic skin equivalents. Br J Dermatol 1994: 131: 827835.
  • 17
    Inamatsu M, Matsuzaki T, Iwanari H, Yoshizato K. Establishment of rat dermal papilla cell lines that sustain the potency to induce hair follicles from afollicular skin. J Invest Dermatol 1998: 111: 767775.DOI: 10.1046/j.1523-1747.1998.00382.x
  • 18
    Thornton MJ, Kata S, Hibberts NA, Brinklow BR, Loudon SI, Randall VA. Ability to culture dermal papilla cells from red deer (Cervus elaphus) hair follicles with differing hormonal responses in vivo offers a new model for studying the control of hair follicle biology. J Exp Zool 1996: 275: 452458.DOI: 10.1002/(sici)1097-010x(19960815)275:6<452::aid-jez7>3.3.co;2-1
  • 19
    Philpott MP, Green MR, Kealey T. Human hair growth in vitro. J Cell Sci 1990: 3: 463471.
  • 20
    Tobin DJ, Mandir N, Dover R. Morphological analysis of in vitro human hair growth. Arch Dermatol Res 1993: 285 (3): 158164.
  • 21
    Karnovsky MJ. Formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Biol 1965: 27: 137138.
  • 22
    McDonagh AJ, Elliott KR, Messenger AG. Cytokines and dermal papilla function in alopecia areata. J Invest Dermatol 1995: 104: 9S10S.
  • 23
    Hoffmann R, Happle R. Current understanding of androgenetic alopecia. Part I: Etiopathogenesis. Eur J Dermatol 2000: 10 (4): 319327.
  • 24
    Hamaoka H, Minakuchi K, Miyoshi H, Arase S, Chen CH, Nakaya Y. Effect of K+ channel openers on K+ channel in cultured human dermal papilla cells. J Med Invest 1997: 44: 7377.
  • 25
    Sato T, Tadokoro T, Sonoda T, Asada Y, Itami S, Takayasu S. Minoxidil increases 17 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase activity of cultured human dermal papilla cells from balding scalp. J Dermatol Sci 1999: 19: 123125.
  • 26
    Lachgar S, Moukadiri H, Jonca F et al. Vascular endothelial growth factor is an autocrine growth factor for hair dermal papilla cells. J Invest Dermatol 1996: 106: 1723.
  • 27
    Arase S, Sadamoto Y, Katoh S, Urano Y, Takeda K. Co-culture of human hair follicle and dermal papillae in a collagen matrix. J Dermatol 1990: 17: 667676.
  • 28
    Reynolds AJ, Jahoda CAB. Hair follicle stem cells ? A distinct germinative epidermal cell population is activated in vitro by the presence of hair dermal papilla cells. J Cell Sci 1991: 99: 373385.
  • 29
    Itami S, Kurata S, Sonoda T, Takayasu S. Interaction between dermal papilla cells and follicular epithelial cells in vitro: effect of androgen. Br J Dermatol 1995: 132: 527532.