Outer root sheath (ORS) cells organize into epidermoid cyst-like spheroids when cultured inside Matrigel: a light-microscopic and immunohistological comparison between human ORS cells and interfollicular keratinocytes.

In organotypic cultures, outer root sheath (ORS) cells of the human hair follicle develop into a stratified epithelium largely reminiscent of the epidermis; this apparently reflects their importance during wound healing. In the present study, ORS cells were grown inside a three-dimensional network of extracellular matrix proteins (Matrigel), together with different mesenchymal cells, in an attempt to mimic their follicular environment. Thus, inside Matrigel, ORS cells formed spheroids differentiating toward the center and showing all the markers of epidermal keratinization. Under identical conditions, normal epidermal keratinocytes developed similar spheroids, but of a significantly smaller size. Human dermal fibroblasts and dermal papilla cells, cocultured in the matrix, had a positive influence on both the proliferation and differentiation within both types of spheroids. Epidermal differentiation markers, such as suprabasal keratins, involucrin, filaggrin, gp80 and pemphigoid antigen, were readily expressed in ORS spheroids, whereas hard (hair) keratins were not detectable by immunostaining. Cells positive for an epithelial membrane antigen, strongly expressed in sebaceous glands, were seen in numerous spheroids. In contrast to organotypic "surface" epithelia, the expression and location of different integrin chains was normalized in ORS spheroids, indicating an enhanced mesenchymal influence in this in vitro system.

Phenotypic modulation of human hair matrix cells (trichocytes) by environmental influence in vitro and in vivo.

Trichocytes, i.e. precursor cells of the hair cortex and medulla, isolated from plucked human scalp hair follicles (HF) were propagated on feeder layers of post-mitotic human dermal fibroblasts (HDF). Cell isolates from five HF routinely yielded about 0.5-1 x 10(5) cells within 3 weeks. When grown as 'surface epithelia' in vitro (on dermal equivalents exposed to air), trichocytes organized into stratified epithelia largely reminiscent of epidermis with regard to both tissue architecture and localization of epidermal differentiation products (keratins K1 and K10, involucrin, filaggrin). However, when HDF in the collagen matrix were replaced by dermal papilla cells (DPC) epidermoid differentiation was largely prevented while still allowing growth and stratification. Epidermal differentiation (keratinization) was virtually complete when trichocytes grown on collagen gels with HDF were transplanted onto nude mice; this was apparent by tissue organization, expression of K1 and K10 and the nearly regular epidermal localization of involucrin. In addition, the deposition of basement membrane components (laminin, type IV collagen, bullous pemphigoid antigen) at the epithelium-collagen interface further increased and was more regular in transplants than in vitro. Cells embedded in Matrigel together with HDF developed large spheroidal structures with inward-directed differentiation and all the epidermal markers found in 'surface' cultures, while only small keratinizing spheroids formed without HDF. In this system co-culture of trichocytes with DPC suppressed almost completely epidermal keratinization. Although typical hair proteins were not detectable, our data clearly demonstrate that: (1) bona fide trichocytes inherit the options for alternative directions of differentiation, and (2) external (in part mesenchymal cell-mediated) influences play a pivotal role in this determination.

Differentiation and polypeptide expression in human papilla and dermal fibroblasts in vitro.

Proliferation and differentiation of human scalp hair follicle papilla fibroblasts (PF) in the anagenic stage of development and of neighboring dermal fibroblasts (DF) of the same donor were studied with stem cell system-specific methods in primary and secondary populations in in vitro systems. Both populations differentiate along the sequence of mitotic fibroblasts (MF) MF I-MF II-MF III and postmitotic fibroblasts (PMF) PMF IV-PMF V-PMF VI-PMF VIIa/PMF VIIb, as described for fibroblast stem cell systems in general elsewhere (Bayreuther et al., Mutat. Res. 256, 233-242 (1991)). PF and DF populations are distinguished by a number of cell-biological parameters, like population dynamics, maximal cumulative population doublings (CPD), changes in cell type frequencies, changes in cloning efficiency as a function of the CPD level (CPDL) of mitotic mass population, in addition by distinct changes of cell type frequencies of postmitotic fibroblasts as a function of the duration of maintenance of postmitotic fibroblast population in stationary culture. Primary and secondary PFs express cell type-specific high molecular weight cellular polypeptides in the M(r) range of 190 to 270 x 10(3) and/or secrete cell type-specific low molecular weight polypeptides in the M(r) range of 27 to 28 x 10(3) as revealed by two-dimensional gel electrophoresis of [35S]methionine-labeled and [14C]proline-labeled polypeptides. These polypeptides are not expressed in the corresponding primary or secondary DF cell types. The expression of specific proteins in primary and secondary PF in vitro may correspond to specific functions of PF in vivo.

Experimental modulation of the differentiated phenotype of keratinocytes from epidermis and hair follicle outer root sheath and matrix cells.

Follicles of human anagen hair were separated into morphologically distinct compartments (by sequential trypsinization and microdissection) for the biochemical and immunological analysis of keratins as differentiation markers to diagnose the type of epithelial differentiation. While outer root sheath contained throughout the "soft" (cyto)keratins K5, 6, 14, 16, and 17, and hair cortex contained exclusively a set of acidic and basic "hard" alpha-keratins (consistent up to the hair tip), in inner root sheath and hair cuticle peptides related or derived from suprabasal epidermal keratins K1 and 10 were detected. These keratin profiles served as in vivo correlates for the evaluation of type and degree of differentiation achieved by the respective isolated epithelial cells, comparing different growth or culture conditions. Cultures of ORS cells and hair matrix cells (PHS cells) as well as normal keratinocytes were initiated using postmitotic human dermal fibroblasts as efficient feeder cells. On lifted collagen gels populated with HDF ("surface" cultures), ORS and PHS cells formed stratified epithelial expressing epidermal differentiation markers such as keratins K1 and 10, involucrin, and filaggrin. Compared with NEK "surface" cultures, balance between growth and differentiation was better maintained by both follicular cell types. In contrast, epidermal tissue homeostasis was largely normalized in transplants on nude mice regardless of the epithelial cell type, apparent from orderly tissue structure, regular distribution of keratin K10, filaggrin, and involucrin, and distinct continuous deposition of basement membrane components at the epithelium-collagen interface. Embedded in Matrigel (on top of HDF collagen gels) ORS cells and NEK formed spheroids exhibiting inward-directed epidermoid differentiation, increasing with time. All epidermal maturation products found in "surface" cultures were likewise expressed, and again differentiation greatly outbalanced proliferation in spheroids of NEK but not of ORS cells. PHS cells embedded together with HDF in Matrigel produced similar spheroids as ORS cells. Size of spheroids and degree of epidermoid differentiation were dramatically reduced when HDF were replaced by follicular DP cells, demonstrating the crucial role of the mesenchymal "companion" cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Restoration of the epidermal phenotype by follicular outer root sheath cells in recombinant culture with dermal fibroblasts.

In order to better understand how outer root sheath (ORS) cells are able to reepithelialize superficial skin wounds, the level of epidermal differentiation achieved by isolated ORS cells in vitro was determined. Using postmitotic human dermal fibroblasts (HDF) as efficient feeder cells, large numbers of ORS cells from individual follicles were generated. Passaged ORS cells were grown exposed to air on HDF-populated collagen gels in the CRD device (Noser and Limat, In vitro 23, 541-545, 1987) which allows histiotypic tissue organization. In such recombinant organotypic cultures, ORS cells developed distinct epidermal strata comparable to interfollicular keratinocytes (NEK). Ultrastructurally, desmosomes and intermediate filaments increased in number toward the epithelial surface and small keratohyalin (KH) granules (but no large irregular KH granules as in NEK) were abundant, adjacent to an electrondense stratum corneum. Also, synthesis of epidermal suprabasal keratins (K1 and 10;2D gels) was lower in ORS cultures, but clearly visible suprabasally by immunofluorescence along with other epidermal markers (involucrin, filaggrin, surface glycoprotein gp80, pemphigus vulgaris antigen). Basement membrane components (laminin, type IV collagen, bullous pemphigoid antigen) were detectable in both ORS and NEK in these assays. Thus, phenotypic expression was largely comparable, but, whereas terminal differentiation (keratinization) was progressing in NEK cultures limiting their lifespan, this seemed to be better controlled in ORS cultures and viable cell layers persisted resulting in longer survival time.

Postmitotic human dermal fibroblasts preserve intact feeder properties for epithelial cell growth after long-term cryopreservation.

In vitro, human dermal fibroblasts (HDF) differentiate through morphologically and biochemically identified compartments. In the course of this spontaneous differentiation through mitotic and postmitotic states, a tremendous increase in cellular and nuclear size occurs. Induction of postmitotic states can be accelerated by chemical (e.g., mitomycin C) or physical (e.g., x-ray) treatments. Such experimentally induced postmitotic HDF cells support very efficiently the growth of cutaneous epithelial cells, i.e. interfollicular keratinocytes and follicular outer root sheath cells, especially in primary cultures starting from very low cell seeding densities. The HDF feeder system provides more fundamental and also practical advantages, i.e. use of initially diploid human fibroblasts from known anatomic locations, easy handling and excellent reproducibility, and the possibility of long-term storage by incubation at 37 degrees C. Conditions for the cryogenic storage of postmitotic HDF cells in liquid nitrogen are presented and related to the feeder capacity for epithelial cell growth. Because postmitotic HDF cells preserve intact feeder properties after long-term storage, the immediate availability of feeder cells and the possibility to repeat experiments with identical materials further substantiate the usefulness of this feeder system.

Post-mitotic human dermal fibroblasts efficiently support the growth of human follicular keratinocytes.

For growth at low seeding densities, keratinocytes isolated from human tissues like epidermis or hair follicles are dependent on mesenchyme-derived feeder cells such as the 3T3-cell employed so far. As an alternative method, the present study describes the use of post-mitotic human dermal fibroblasts sublethally irradiated or mitomycin C-treated. Special emphasis was put on efficient growth of primary keratinocyte cultures plated at very low seeding densities. Thus, outer root sheath cells isolated from two anagen human hair follicles and plated in a 35-mm culture dish (3 - 6 X 10(2) attached cells) grew to confluence within 3 weeks (6 - 8 X 10(5) cells). Similar results were obtained for interfollicular keratinocytes. A crucial point for the function of these fibroblast feeder cells is plating at appropriate densities, considering their tremendous increase in cell size at the post-mitotic state. Plating densities of 4 - 5 X 10(3/cm2 allow full spreading of the feeder cells and do not impede the settling and expansion of the keratinocytes. Major advantages of this system include easier handling and better reproducibility than using 3T3-cells. Moreover, homologous fibroblast feeders mimic more closely the physiologic situation and therefore might provide a valuable tool for studying interactions between human mesenchymal and epithelial cells. Finally, potential hazards of using transformed feeder cells from a different species in keratinocyte cultures raised for wound covering in humans could be thus avoided.

Organotypic culture of outer root sheath cells from human hair follicles using a new culture device.

A new culture vessel for the growth of cells on biological substrata and under organotypic conditions is described. This device, named Combi-ring-dish (CRD), is composed of four concentric rings designed to take up one or several substrata on which cells can be grown either immersed in culture medium or exposed to air and fed from underneath. Using the CRD, outer root sheath cells, isolated from plucked human hair follicles and plated on growth-arrested 3T3 feeder layers, were grown on native collagen lattices populated with living human fibroblasts. After reaching confluence, the immersed cultures were recombined (in vitro) with pieces of freeze-killed dermis and grown further, exposed to air. Thus by mimicking epidermal growth conditions, differentiation was dramatically improved, compared to control cultures on plastic substratum. Virtually all morphologic features of interfollicular epidermis developed. This seems a suitable model to investigate the differentiation potential of human hair follicle cells.

Evaluation of an in vitro percutaneous permeation model with two oxidative hair dyes.

Synopsis The percutaneous permeation of two oxidative hair dyes was measured by means of pig skin in a flow-through diffusion cell system entirely constructed from Teflon. Pig skin membranes were prepared by reducing full thickness skin with a dermatome to a more in vivo-like barrier layer and their integrity was checked by measuring the steady-state permeation of tritiated water. Initially, the inter- and intraindividual variability of percutaneous permeation was determined with an aqueous solution of 1-(2'-hydroxyethyl)-amino-3,4-methylenedioxybenzene-hydrochloride, an oxidative hair dye component. In the same way the proper flow rate of elution fluid through the receptor cell was found to be most favourable at 10 ml h(-1), the thickness of permeation membranes was fixed at 1 mm, and it was shown that storage of the skin at -20 degrees C for up to 35 days did not change the permeability. The percutaneous permeation of the same hair dye component and of 4-amino-2-hydroxymethylphenol-hydrochloride was determined after application to pig skin membranes under practical conditions of hair dyeing. The in vitro skin permeation was in the same order of magnitude as results from comparable in vivo skin absorption studies in rats. Perméation percutanée in vitro de colorants d'oxydation pour cheveux.

Serial cultivation of single keratinocytes from the outer root sheath of human scalp hair follicles.

A method for the isolation of outer root sheath keratinocytes from plucked human hair follicles and for their subsequent cultivation has been developed. The selective trypsinization of outer root sheath keratinocytes provided a single cell suspension of defined origin within the hair follicle. The 3T3 feeder layer technique supports sustained growth of these cells in that as little as one single plucked hair follicle (yielding approximately 1.5 X 10(4) cells) consistently gave rise to a confluent 35-mm culture dish (with approximately 1.5 X 10(6) cells) within about 2 weeks. The outer root sheath keratinocytes can be serially passaged for up to 3 times and also cryopreserved.

Studies on the potential in vivo induction of sister-chromatid exchanges in rat bone marrow by resorcinol.

The hair-dye component resorcinol was tested for potential in vivo induction of sister-chromatid exchanges (SCE) in bone-marrow cells after intraperitoneal, peroral and epicutaneous application to rats. None of the tested drug concentrations produced an increase in SCE frequencies. As a control for our test method we demonstrated dose-dependent SCE increases induced by 2-acetylaminofluorene and by 2-aminoanthracene, cytogenetic response to 2-aminoanthracene being higher in females than in males.