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 × 104 cells) consistently gave rise to a confluent 35-mm culture dish (with approximately 1.5 × 106 cells) within about 2 weeks. The outer root sheath keratinocytes can be serially passaged for up to 3 times and also cryopreserved

Characterization of an 80-kD Membrane Glycoprotein (GP80) of Human Keratinocytes: A Marker for Commitment to Terminal Differentiation In Vivo and In Vitro

We have characterized an 80-kD cell-surface glycoprotein (gp80) identified by monoclonal antibody BT 15, the expression of which is closely associated with a commitment to terminal squamous or follicular differentiation of keratinocytes in normal adult and fetal human epidermis. Maximum expression was found in the suprabasal layers, but basal cells located at the epidermal sulci were also clearly positive, in contrast to the virtually negative basal cells at the epidermal ridges. This protein was also present in benign hyperproliferative disorders of the epidermis (i.e., common warts, keratoacanthoma, psoriasis, and seborrhoic keratoses) with monoclonal antibody BT 15 preferentially staining suprabasal cells and some basal cells at the epidermal sulci. Gp80 was completely lacking in most basal cell carcinomas; the only exceptions were two cases of partially cornifying tumors that were strongly stained around keratotic pearls. In squamous cell carcinomas, gp80 was expressed in keratinized areas of the tumors. In organotypic keratinocyte cultures that resemble the in vivo situation, gp80 was strongly expressed in the suprabasal layers. However, unlike known markers for tenainal differentiation, gp80 was weakly expressed by basal cells. Synthesis rates of gp80 were high in keratinocyte cell suspensions freshly prepared from skin, and decreased in primary cultures and first and second subcultures (ratio 10:4:2:1). Elevated concentrations of the Ca++ that increased stratification of cultured keratinocytes resulted in a two- to threefold increase of gp80 synthesis. Gp80 was not synthesized at detectable levels by the immortal keratinocyte cell line HaCaT; however, it was expressed in HaCaT cultures treated with mitomycin C, indicating an association with cessation of growth. Pulse-chase experiments revealed that gp80 is synthesized from a 55-kD precursor molecule, the maturation of which was prevented by treating cells with tunicamycin. Glycosidase digestion of BT 15 immunoprecipitates from untreated cells indicated that the predominant post-translational modification of the protein is N-linked glycosylation. Our data indicate that gp80 is a glycoprotein that is expressed by growth-arrested human keratinocytes or as part of the terminal differentiation program

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

AbstractFor 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 × 102 attached cells) grew to confluence within 3 weeks (6–8 × 105 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 × 103/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

[Cost of hospital-based management of acute myeloid leukemia: from analytical to procedure-based tarification]

International audienceThe confrontation of the macro- and micro-economic approaches of hospital costs is a recurrent question, in particular for pathologies where length of stay is highly variable, like acute myeloid leukemias (AML). This monocentric and retrospective study compares direct hospital medical costs of induction and relapse treatment sequences for AML, valued according to four different approaches: the analytic accounting system of our hospital, the French Diagnosis Related Group (DRG) cost databases of hospital discharges (readjusted, or not, to actual hospital stay duration), and official tariffs from the new French DRG prospective payment system. The average cost of hospital AML care valued by the analytic accounting system of our hospital is 61,248 euros for the induction phase and 91,702 euros for the relapse phase. All other national valuation methods result in a two- to four-fold underestimation of these costs. Even though AMLs are now individualized in the 10th version of the French diagnosis related group (DRG) classification, the impact of this issue in other pathologies is going to increase with the gradual implementation of the French DRG prospective payment system. That is why it must be assessed before the progressive extension of this financing system