Stem Cells http://www.stemcellsportal.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Papini, S.
Right arrow Articles by Revoltella, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Papini, S.
Right arrow Articles by Revoltella, R. P.

Stem Cells 2003;21:481-494 www.StemCells.com
© 2003 AlphaMed Press

Meeting Report

Isolation and Clonal Analysis of Human Epidermal Keratinocyte Stem Cells in Long-Term Culture

Sandra Papinia, Denise Cecchettib, Daniela Campanib, Wendy Fitzgeraldc, Jean Charles Grivelc, Silvia Chenc, Leonid Margolisc, Roberto P. Revoltellaa

a Immunobiology and Cell Differentiation Unit, Institute of Biomedical Technologies, Consiglio Nazionale delle Ricerche, Pisa, Italy;
b Department of Oncology, University of Pisa, Pisa, Italy;
c NASA/NIH Center for Three-Dimensional Tissue Culture, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development (NICHD), NIH, Bethesda, Maryland, USA

Key Words. Keratinocyte • Stem cells • Differentiation • Three-dimensional cultures • Cell cloning

Roberto P. Revoltella, M.D., Ph.D., Consiglio Nazionale delle Ricerche, Institute of Biomedical Technologies, Unit of Immunobiology and Cell Differentiation, Via Moruzzi 1, 56100 Pisa, Italy. Telephone: 39-050-315-2772; Fax: 39-050-315-3367; e-mail: r.revoltella{at}imd.pi.cnr.it


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We developed a procedure for growing normal epidermal keratinocyte stem cells isolated from a single punch biopsy of adult human skin in long-term culture. Primary skin epithelial cells were maintained in collagen-coated plates with irradiated human neonatal foreskin fibroblasts (line HPI.1) as a feeder for more than 120 days, approximately 115 population doublings, without signs of replicative senescence. Clonal analysis revealed the presence of holoclones, meroclones, and paraclones. Only emerging colonies with high proliferative potentials and extensive capacities for division (holoclones and meroclones) were subcultured, favoring the expansion of stem cells and progenitors capable of prolonged self-maintenance when subcloned, thus accounting for the prevailing long-term proliferation of the original culture. We found that meroclones included bipotent progenitors capable of generating both keratinocytes and mucin-producing cells. The numbers of these cells were greater after confluence, suggesting that commitment for their differentiation occurred late in the life of a single clone. On a three-dimensional gelatin matrix and on a collagen layer containing the fibroblast feeder, cells isolated from the expansion of holoclones and meroclones formed stratified cohesive layers of keratinocytes that were able to further differentiate, as in normal skin. These results indicate that our procedure will serve as a valuable tool to study expansion of epidermal stem cells as well as the growth mechanisms and cell products associated with their growth and differentiation.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Epidermis, the outermost layer of skin, consists of an external epithelial layer devoid of blood vessels that is separated from a connective tissue, the dermis, by the mesenchymal layer, a specialized basement lamina and a network of fine collagen fibers that extend from the basement membrane to the anchoring plaques in the papillary dermis. This papillary dermis is composed primarily of microvessels, fibroblasts, migrating leukocytes, and collagen fibers [1–10]. Mesenchymal-epithelial interactions regulate the continuous turnover of the epithelial layer. The epidermis normally includes only keratinocytes, which are arranged above the dermis in cohesive multilayers. Three types of keratinocytes have been identified in human epidermis and hair follicles on the basis of their growth, kinetic behaviors, and local distribution. The first, stem cells, are firmly adherent to the basal lamina and include about 10% of the cells in the basal layer at the dermal-epidermal junction. These cells regenerate themselves by division and can also give rise to differentiated progeny. The second type, transient-amplifying cells (approximately 40% of the basal cell layer, including melanocytes), replicate with higher frequencies than stem cells but have far fewer population doublings. They further differentiate, detach from the basement membrane, and migrate toward the epidermis surface, forming multilayered, cohesive sheets of keratinocytes that differentiate into one or more specialized cell types. The third type, terminally differentiated keratinocytes, subsequently die and form external layers of cornified cells [11–19]. These cells obtain their nutrition and growth factors from stromal cells in the dermis [20, 21]. Basal keratinocytes produce and secrete adhesion molecules, which form the anchoring structure of the epidermis [22], and also a variety of nutritive and regulatory molecules, including cytokines, which perform autocrine, paracrine, and endocrine functions, crossing the basement membrane and entering the blood stream [23, 24].

Cultured human keratinocytes are now widely studied in order to understand the basic biology of these cells in vivo and also for in vitro toxicity testing. Moreover, cultured epithelial sheets enriched with proliferative keratinocytes are also used in autografts for skin replacement in full and deep partial-thickness burn injuries, chronic wounds, etc., and have assured, in many cases, the persistence of the regenerated epidermis [25–31]. Furthermore, epidermal stem cells are becoming attractive targets for gene therapy of skin diseases [18, 32–37] as well as for systemic delivery of recombinant proteins in the treatment of a number of genetic disorders [8, 37, 38].

To stimulate self-renewal of skin tissue, current in vitro culture methods include an appropriate matrix surrounding nonreplicating dermal stromal cells as a feeder layer, together with a population of immature keratinocytes grown on top at the air-liquid interface, often with permeable filter inserts as physical supports [20, 39]. By these methods, a normal epidermis can be reconstituted within a few weeks, showing properties of an orderly, structured, self-regenerating epithelium, including the formation of an organized basement membrane, which appears in an orthokerostatic stratum corneum. Because epidermis is renewed monthly in vivo, it is obvious that any attempt to use keratinocytes to take advantage of the persistence of the regenerated epidermis in these grafts depends upon the presence of a sufficiently high proportion of engrafted self-replicating stem cells. It is difficult to maintain this high proportion and to evaluate their numbers in standard monolayer cultures. It was reported that the half-life of a keratinocyte population declines with the age of the donor [13], suggesting an intrinsic difference in the growth potential of stem cells and the rate of generation of terminal differentiation between newborn and elderly donors. Moreover, once progenitor cells are taken from their natural habitat, they might be forced by environmental factors to irreversibly lose their original proliferation capacities, to undergo replicative senescence, and to differentiate. With most current in vitro techniques, the replication of primary keratinocytes is limited, normally to about 15–50 population doublings [17, 40].

The objective of the present study was to optimize cultures of skin keratinocyte stem cells isolated from a single punch biopsy of human skin and to extend their lifespans and expansion potentials while retaining their differentiation capabilities.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Cultures

HPI.1 Cell Line   The human dermal fibroblast HPI.l line was obtained from a neonatal foreskin biopsy. The skin sample was minced into small pieces (l x l mm), treated with type IA collagenase (1 mg/ml) and type IVS hyaluronidase (0.4 mg/ml) (Sigma Chemicals Co.; St. Louis, MO; http://www.sigmaaldrich.com) at 37°C for 45 minutes, washed with phosphate-buffered saline (PBS) at pH 7.4, and transferred to a 35-mm-diameter tissue culture plate (Falcon Plastics, Inc.; London, Ontario; http://www.falconplastics.on.ca). The sample was then incubated in 6.0 ml of high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Euroclone; York, UK; http://www.euroclone.net) supplemented with 20% fetal bovine serum (FBS) (Euroclone) containing gentamicin (Sigma), streptomycin, and penicillin (each 20 µg/ml) in a humidified incubator at 37°C and a 5% CO2-in-air atmosphere for 14 hours. All experiments in this study were performed with a single batch of FBS tested for its ability to support good keratinocyte colony formation (see below). The medium was then removed, and the adherent skin fragments were covered with 10.0 ml of fresh medium that was subsequently changed every 3 days. When abundant fibroblasts began to migrate from the explants and adhere to the plate surface, the skin pieces were removed, and fibroblasts were covered with fresh growth medium (DMEM with 10% FBS) and allowed to grow and reach subconfluence. They were then divided by trypsin-EDTA (0.05% trypsin and 0.02% EDTA, weight/volume), expanded in T25 plastic culture flasks (Corning Inc.; Corning, NY; http://www.corning.com) or six-well culture plates (Falcon), and subcultured at a final seeding density of about 5 x 105 viable cells per ml, with changes of medium every 3 days. Only primary cultures of HPI.1 cells (up to 8–10 passages) were used in all subsequent experiments. Preliminary results indicate that HPI.l cells produced constitutively many growth factors, including fibroblast growth factor, epidermal growth factor (EGF), interleukin-6, stem cell factor, GM-CSF, vascular endothelial growth factor, and insulin-like growth factor-1 (Papini and Revoltella, unpublished). These cells were cultured with base medium until semiconfluence. The conditioned medium was then centrifuged, filtered, diluted 1:3 with fresh base medium, and used.

A5RT1 Cell Line   The high-grade malignant human keratinocyte HaCaT-ras clone A5RT1 is a recultivated tumor cell clone derived after in vivo passage in athymic mice as subcutaneous tumors of a benign clonal line [41]. The established A5RT1 cell line stably maintains the distinct keratinocyte phenotype of the original heterotransplants. A5RT1 cells were adapted to grow in a monolayer in DMEM supplemented with 10% FBS and subcultured at subconfluence every 3 days.

Keratinocyte Cell Culture   Normal human epidermal cell suspensions, which are about 95% keratinocytes, were prepared from human skin biopsies (0.5 x 0.5 cm pieces of full-thickness skin), as described elsewhere [42, 43]. Biopsies isolated from plastic surgery procedures (either from thorax, abdomen, or breast) from l2 unrelated consenting adult (aged 15–58 years) male donors were used in this study. The skin biopsy was maintained for 5 hours at room temperature in DMEM (concentrated 5x), containing penicillin and streptomycin (50 µg/ml each), gentamicin (10 µg/ml), and an antimycotic agent (amphotericin B, 10 µg/ml), and kept in a 35-mm-diameter sterile plastic dish with 0.5% dispase II (Boehringer; Mannheim, Germany; http://www.boeringer.com) for 12–18 hours in a refrigerator (12°C). Each enzyme-treated piece was washed with Ca++- and Mg++-free PBS at pH 7.4 and dissected horizontally through the stroma to separate it into two halves. Connective tissue was removed with sterile surgical tools, and the two halves were further dissected. The dermal surface was brushed gently with curved forceps to release any loosely attached basal keratinocytes into the medium, and this suspension was added to the keratinocytes isolated from the epidermis. Cells were incubated in a solution of 0.3% trypsin and 1% EDTA (both from Sigma) for 3 minutes at room temperature, and the enzyme activity was then blocked with 2 ml of medium containing 20% FBS. Cells were transferred to a 15-ml centrifuge tube and centrifuged for 5 minutes at 110 g. The supernatant was removed, and keratinocytes were gently resuspended in serum-free growth medium and cultured on six-well culture plates (Falcon), either coated with bovine type I collagen (Privates Institut für Biomedizinische Forshung und Beratung GMBH, IBFB; Leipzig, Germany; http://www.ibfb.de) or uncoated, at a final seeding cell density of approximately 3 x 105 viable cells per well. They were cocultured with a 1-day-old feeder layer of irradiated (3,000 rads) HPI.1 cells (3.0 x l05 cells seeded per well) in a keratinocyte growth medium (KGM) containing DMEM with sodium bicarbonate (1.176 g/l) without serum, 20 mM HEPES, 100 IU/ml streptomycin and penicillin, 0.18 nM adenine, 4 mM glutamine, 2 nM triiodothyronine, and 1 nM cholera toxin. Just before use, an additional keratinocyte growth supplement (KGS), containing 0.4 µg/ml hydrocortisone, 50 ng/ml EGF, 5 µg/ml insulin, 5 µg/ml transferrin, 5 µg/ml selenite, and 1.0 µg/ml bovine pituitary extract (Sigma), was added. Initial keratinocyte cultures were grown in the presence of 400 µg/ml geniticin (G-418; GIBCO; Carlsbad, CA; http://www.lifetech.com) to maintain selection pressure. After a 12-hour incubation at 37°C, unattached cells were gently removed by aspiration, and attached keratinocytes were then maintained in culture. Primary keratinocytes that formed colonies were left to grow until they reached about 70% subconfluence in the culture plate. Adherent cells were then harvested, with 0.25% dispase II (for 3 minutes) and then one or two subsequent treatments with trypsin-EDTA (0.3% trypsin and 1% EDTA) at 37°C, gently pipetted to help dislodge the cells, with rapid trypsin neutralization to prevent cell damage, isolated into a single-cell suspension, and subcultured at a final plating density of 0.5–2 x 105 viable cells per well on a 1-day-old feeder layer of irradiated HPI.1 cells in collagen-coated six-well culture plates. Cultures were kept at 37°C in a 5% CO2-in-air atmosphere in a humidified incubator. Plating efficiency, the number of colonies per 100 cells seeded, was determined from plates seeded with 5 x 103 or 5 x 104 cells per well. The cells were then fixed with 3.7% formaldehyde and stained with hematoxylin-eosin. Keratinocytes after the second passage were subsequently serially passaged as above, always at the stage of subconfluence but only with trypsin-EDTA treatment, with KGM supplemented with 10% FBS. At each passage, cells from two wells out of a set of triplicate cultures were detached, pooled, centrifuged, resuspended in fresh growth medium, seeded into three new wells, and expanded until they reached subconfluence again. The growth medium was changed completely every 3 days.

Clonal Analysis and Calculation of Population Doublings
At the second cell passage (i.e., about 20 days from the initial plating), keratinocytes from individual large colonies, identified under the microscope in a culture plate containing a primary keratinocyte cultured mass, were detached with a sterile thin Pasteur glass pipette or with top disks (the latter from Sigma, Bel-art Products; Pequannok, NJ; http://www.bel-art.com). They were then transferred (~1 x 103 viable cells per well) into six-well plates (either precoated with type I collagen or uncoated) containing adherent growth-inhibited HPI.1 cells seeded 24 hours previously (2.0 x 104 cells seeded per well) as a feeder layer. At 7–10 days after plating, keratinocyte colonies became evident under phase-contrast inverted microscopy. The area containing colonies was photographed, and the number of cells within each of the clones was counted. Selected individual enlarging colonies were then detached, and each was transferred to a new empty well containing growth medium supplemented with FBS. A single-cell suspension was obtained by gentle pipetting, and individual cells were subcultured into 6- or 12-well plates (Falcon) with feeder irradiated HPI.1 cells. The colonies growing in two of the three wells were used for further serial propagation. Cloning and subcloning were performed by splitting cells at subconfluence (~70% saturation density) at a strict time point, identical for each clone. Each clone was scored according to its size and whether its progeny was progressively growing or aborted. Colony-forming efficiency was evaluated as the ratio of the number of colonies to the number of inoculated cells. The population doubling value (X) of the clones was obtained using the formula: X = 3.322log(N/N0), where N is the total number of cells obtained at each passage and N0 is the number of clonogenic cells, on the assumption that most viable cells seeded were capable of reattachment [19]. The third well of the set was fixed 10 days after cell plating, and the cells were stained for classification of clonal type. Three types of clone were found. The first was the holoclone: enlarging colonies in which the cells were capable of prolonged self-maintenance when subcloned, with a frequency as high as those of the parental cells. The second type was the meroclone: only a limited proportion of cells from a clone could be serially subcloned, and the emerging colonies were of remarkably differing size, reflecting highly variable proliferative potentials. The third type was the paraclone: this formed no colonies or very few small colonies in which the cells terminally differentiated, and their clones rapidly aborted after the first subcloning.

Three-Dimensional Tissue Culture

Cultures on Gelatin Sponge Matrices   Sterile Gelfoam sponges from purified pork skin gelatin (12 x 7 mm, product code NDC 0009-0315-03; Pharmacia & Upjohn; Kalamazoo, MI; http://www.pnu.com) were moisturized with KGM containing 10% FBS and KGS just before use and then transferred to six-well culture plates, on top of a monolayer of metabolically active, irradiated HPI.1 cells as feeders. Keratinocytes were placed on the top surface of each sponge (five injections, each of approximately 1 x l04 cells per 20 µl), as described elsewhere [44]. The cultures were then incubated in a stationary horizontal position in the incubator at 37°C in a 5% CO2-in-air atmosphere. The growth medium was carefully changed every 3 days. Some specimens were then fixed in 3.7% formaldehyde at 14 days, and some at 30 days, at room temperature overnight, kept in PBS at 4°C, and then embedded in paraffin. Sections (≤10 µm) were made by dividing each sponge into four vertical parts (from left to right and from the superficial to the deep surface of the matrix) to achieve a better view of cell diffusion. Observation of cultures within and through sponges was normally unsatisfactory by light microscopy, since very few cell clusters were recognized within the sponge during culture; however, after about 1 week, some epithelial cells began to filter inside and through the sponge and form adherent colonies on the bottom of the culture plate. Individual colonies of 1–2 mm in diameter, growing in a monolayer, were detached from the plate; the cells were dissociated by gentle pipetting, and individual cells were seeded again to study their proliferation and cloning potential, as described above.

Organotypic Cultures   Keratinocytes (1–3 x 105 viable cells) were placed on a cell culture insert (Transwell-COL, 0.4-µm pore size; Corning) coated with bovine type I and type III collagen, and the inserts were transferred into six-well culture plates for organotypic cultures. On the bottom of the plate, adherent, irradiated HPI.1 cells were seeded (5 x 105 cells per well) 24 hours previously in KGM containing l0% FBS and KGS as a feeder layer. A sufficient amount of medium was pipetted into the lower chamber to keep the keratinocytes layered onto the collagen gel of the insert covered. Cultures were incubated at 37°C overnight, after which the growth medium was removed and fresh medium was added, leaving the adherent keratinocytes on top of the gel in the insert at the air-liquid interface. The medium was changed every 2 days.

Histology and Immunohistochemical Analysis
Confluent sheets of epithelial cells generated either in cultured mass or in clonal cultures were detached from the plate with trypsin-EDTA, washed in PBS, dissociated by gentle pipetting, centrifuged on glass slides, fixed and permeabilized for 30 minutes at room temperature with PBS containing 0.5% Nonidet® P40 and 3.7% formaldehyde, incubated with l% glycine and 1% bovine serum albumin in PBS for an additional 10 minutes, and then stained. Alternatively, cytocentrifuged cells were fixed in cold methanol and acetone for 5–10 minutes, rinsed in PBS, and stained. Specimens of cells grown in three-dimensional histotypic cultures were fixed in formaldehyde (3.7% in PBS) overnight at room temperature, kept at 4°C in PBS, and embedded in paraffin. Sections were stained with hematoxylin-eosin or analyzed by immunohistochemical methods. Mouse monoclonal antibodies, AE1 (high molecular weight basal/parabasal cytokeratins) and AE3 (low molecular weight transitional squamous/soprabasal cytokeratins), were from Cell Marque (Austin, TX; http://www.cellmarque.com); CK7 and CK19 (squamous cytokeratins) were from DAKO (Glostruls, Denmark; http://www.us.dakocytomation.com). In some experiments, AE1 and AE3 antibodies were mixed together in order to provide a polyclonal pan-cytokeratin reagent. Monoclonal antibodies to desmin, vimentin, and the nuclear antigen Ki-67 (345 and 395 KDa, Clone MIB-1), expressed throughout the cell cycle, as well as anti-HLA (polyvalent) antibodies, were all from DAKO. An immunoenzymatic method (Vectorstain, avidin-biotin complete kit; Vector Laboratories; Burlingame, CA; http://www.vectorlabs.com) was used for both monoclonal and polyclonal antibody reactions. In those experiments, either the horseradish-peroxidase-dextran-anti-mouse complex (EnVision Plus HRP System; DAKO) that uses 3-3'-diaminobenzidine tetrahydrochloride (Fast DAB; Sigma) as a chromogen or the alkaline phosphatase (AP)-dextran-anti-mouse complex (EnVision AP System; DAKO) that uses Fast Red TR/Naphtol AS-MX (Sigma) as a chromogen was used. After the immunostaining procedures were completed, the sections were lightly counterstained with Mayer hematoxylin. Epithelial cells capable of secreting mucin were detected, after fixation in 3.7% formaldehyde, by Alcian Blue staining.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human Skin Epidermal Keratinocytes in Long-Term Culture

Keratinocytes   The proliferation potentials of keratinocytes maintained in long-term cultures with irradiated HPI.1 cells as a feeder layer in KGM containing 10% FBS and KGS are summarized in Table 1Go. Primary epidermal keratinocytes isolated from adult donors consistently formed colonies. Each colony was the progeny of a single cell or a small cell cluster. Measurements of the colonies began at approximately the second cell passage after the processing of the biopsy and the initial cell plating (time 0); the preceding time was used to allow keratinocytes to adapt in vitro and to allow the adherent progenitor cells to grow to a suitably large population. The shapes and general appearances of most colonies in the primary cultured mass, as well as in their subclones, were typical of epithelial cells, forming mosaics of polygonal cells (Fig. 1AGo). Several colonies eventually fused, forming a monolayer of epithelium. The plating efficiency of epithelial cells in the primary mass culture was low (≤0.1%). With successive passages, cells formed large colonies with greater frequency (Fig. 1BGo). Most colonies contained a population of small cells (<15 µm diameter) with the tendency to become cuboidal in monolayer; while at confluence, the cells displayed a more differentiated morphology and stained positively with anticytokeratin antibodies (Fig. 1CGo). With successive passages, they normally reached saturation density within 7 days after plating. Cultures were split each time at subconfluence for approximately 120 days in culture (i.e., about 115 cumulative cell doublings, with an average cumulative doubling time of approximately 24 hours), without showing signs of replicative senescence.


View this table:
[in this window]
[in a new window]
  		Table 1. Proliferative potentials of keratinocytes in cultured mass
 


View larger version (74K):
[in this window]
[in a new window]
  		Figure 1. Microscopic analysis of different keratinocyte colonies. Microscopic analysis of normal human epidermal keratinocytes forming colonies on type-I-collagen-coated six-well culture plates in medium containing 10% FBS, with irradiated HPI.1 fibroblasts as a feeder layer. Cells were plated at a density of 1 x 103 viable cells per well, and colonies were analyzed 5 days later. Colonies were typical of epithelial cells, forming enlarging mosaics of polygonal cells. A) Colonies of different sizes generated by normal skin keratinocytes in a primary cultured mass. B) Colonies after four serial divisions (hematoxylin-eosin stained, original magnification = 100x). C) Colonies immunohistochemically stained with monoclonal antibodies to specific (AE1 and AE3) cytokeratin antigens (original magnification = 400x).

 
Such mass keratinocyte cultures were formed from a single punch biopsy derived from all 12 unrelated donors, irrespective of age, indicating that clonogenic keratinocytes with high self-renewal capacities (typical of stem cells) are widely distributed in the skin.

No differences in cultured keratinocyte doublings were found among cells isolated from skin of different body areas (thorax, breast, abdomen) or from donors of different ages (range, 15–58 years). In the first- or second-passage cultured mass, approximately one-third of the colonies of keratinocytes had apparently been the site of accelerated cell division, reaching a considerably larger size than other colonies in the same culture. The cells of those fast-growing colonies, however, often became terminally differentiated, aborted after a few more doublings, and could not be further serially cultured (data not shown).

Mucin-Producing Cells   When keratinocytes reached a confluent monolayer, they gave rise to large secretory-type cells producing mucin, as revealed with Alcian Blue staining (Fig. 2AGo). Those cells were initially rare but were always present during the entire lifespan of mass-cultured keratinocytes, and their proportions were greater in later cultures. In order to verify that mucin-producing cells originated from bipotential progenitors, keratinocytes of the A5RT1 clone were seeded in parallel culture. Individual A5RT1 cells formed colonies, which eventually enlarged, forming confluent sheets of epithelial cells. Within confluent monolayers, scattered A5RT1 cells spontaneously differentiated into mucin-producing cells (Fig. 2BGo).



View larger version (140K):
[in this window]
[in a new window]
  		Figure 2. Mucin-producing cells. Mucin-producing cells (arrow) observed in 7-day-old confluent epithelial sheets of mass-cultured human epidermal keratinocytes after 20 serial passages (A) and in colonies of the clonal A5RT1 line (B). In all cases, cells were grown on type-I-collagen-coated plates in the absence of a feeder layer. Alcian Blue stain with hematoxylin as contrast, (original magnification = 400x).

 

Other Cells   Approximately 5% of adherent cells revealed positive staining for antidesmin antibodies and, thus, were of the endothelial type (Table 2Go). These cells divided slowly and normally disappeared after successive passages of the cells in culture in collagen-coated plates. In addition, we identified elongated, spindle-shaped stromal cells, classed as fibroblasts on the basis of their reactivity with both anti-HLA and anti-vimentin antibodies. These accounted for a small proportion of the whole epidermal initial population. The growth rate of fibroblasts on collagen was markedly lower than the growth rate of keratinocytes under our culture conditions (not shown).


View this table:
[in this window]
[in a new window]
  		Table 2. Expression of tissue differentiation markers in epidermal cells grown in primary cultured mass
 
Effects of Culture Conditions
Incubation of primary dispersed keratinocytes from all 12 biopsies in KGM supplemented with KGS, together with the use of HPI.1 cells as a feeder layer in the absence of serum for two passages and then with 10% FBS, resulted in consistent formation of colonies whose progeny continued to proliferate well on collagen for approximately 120 days, undergoing, on average, approximately 115 cumulative population doublings without significantly altering their growth rates (Table 3Go; Fig. 3Go, Group 1).


View this table:
[in this window]
[in a new window]
  		Table 3. Culture medium and substrate for human epidermal keratinocyte stem cell production
 


View larger version (16K):
[in this window]
[in a new window]
  		Figure 3. Determination of the number of cell generations. Keratinocytes isolated from the same biopsy were expanded for two passages in serum-free growth medium (KGM with KGS) and subsequently plated on collagen-coated plastic wells in medium containing either 10% FBS (Group 1), 2% FBS (Group 2), or no serum (Group 3). Cells were also plated onto uncoated plastic plates in growth medium containing 10% FBS (Group 4). Irradiated HPI.1 fibroblasts were always added as a feeder layer. In addition, keratinocytes were grown with HPI.1-conditioned medium in the absence of the feeder layer (Group 5). Triplicate cultures in each group were seeded. Cells from two plates at subconfluence (70% saturation) were harvested, and new subcultures were established, while the third culture was analyzed. The number of cumulative population doublings was calculated using the formula X = 3.322log(N/N0), where N is the total number of cells obtained at each passage, and N0 is the number of clonogenic cells (average values from 12 cultures).

 
With a lower concentration of FBS, 2% rather than 10%, in KGM supplemented with KGS, a significantly lower proportion of primary keratinocytes formed colonies, and after transfer, they became senescent within approximately 40 cumulative population doublings (Table 3Go; Fig. 3Go, Group 2). In serum-free medium, the lifespan of the keratinocytes was more markedly affected, with cultures reaching proliferative senescence and generating end-stage cells in about 15 population doublings (Table 3Go; Fig. 3Go, Group 3). When primary epidermal epithelial cells were grown in KGM containing 10% FBS and supplemented with KGS on plastic, in the absence of collagen as a substrate, a significantly lower proportion of keratinocytes formed colonies (about 10 colonies per 1,000 cells seeded), with 25–30 rounds of cell division. However, the cells of those colonies ceased to proliferate after only 30–35 days under the culture conditions described above (Table 3Go; Fig. 3Go, Group 4).

We compared whether the greater expansion was due to cell-to-cell contact or due primarily to supportive growth factors secreted by the neonatal foreskin fibroblasts. Primary keratinocytes were grown in KGM containing 10% FBS supplemented with KGS on collagen-coated plates in the absence of HPI.1 cells as a feeder layer but with medium supplemented with conditioned medium (1:3) harvested from 4-day-old HPI.1 cell cultures. Without HPI.1 cells as a feeder layer, a significantly lower proportion of keratinocytes formed colonies. These colonies generated progeny forming holoclones and meroclones for at least 60 population doublings before reaching proliferative senescence (Fig. 3Go, Group 5).

These results indicate the advantage of collagen over uncoated plastic in enhancing the growth potential of keratinocyte progenitors. Moreover, keratinocytes maintained with serum in the culture medium depended on the fibroblast feeder layer as well as on growth factors secreted by those cells for their establishment both in early cultures and in subcultures.

Additionally, we found cells producing mucin in keratinocyte colonies. In all five groups, mucin-producing cells were rare in primary keratinocyte mass cultures, and their numbers were difficult to evaluate. However, in confluent monolayers, they were always present (Fig. 4AGo). The numbers of Alcian-Blue-positive cells were progressively greater late in the life of the cultures (Fig. 4BGo).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 4. Mucin-producing cells. A) Keratinocytes from an exponentially growing mass culture after 20 serial passages were seeded in triplicate on collagen-coated glass coverslips kept in six-well plates (105 cells per well) with irradiated 1-day-old HPI.1 fibroblasts (2 x 104 cells) as a feeder layer, reaching confluence about 6 days after plating. At each time interval, three coverslips were removed, and the cells were fixed in 3.7% formaldehyde and stained. Cells producing mucin were identified and counted. Means and standard deviations are shown. Note that their numbers were several-fold greater after the keratinocytes had reached confluence. B) Keratinocytes generated from a single secondary clone were serially propagated on collagen-coated six-well plates by division at subconfluence every 5 days. At each indicated time interval, cells were detached and pooled: two-thirds of the cells were used for further cell expansion, while one-third was seeded on collagen-coated coverslips maintained for 7 days to reach confluence, fixed, and stained. Cells producing mucin were present during the entire lifespan of the culture. They were rare in early cultures, but their numbers were several-fold greater with successive population doublings.

 
Clonogenic Ability and Proliferative Potential of Keratinocyte Stem Cells
A total of 729 clones from 12 individual colonies isolated from biopsies derived from different body sites (thorax, breast, and abdomen) were analyzed. All clones isolated consisted only of replicating keratinocytes. On average, 1,000 cells plated in the initial mass culture formed approximately 80 colonies. However, the proportions of cells forming colonies were gradually greater with successive cell passages. Furthermore, a large proportion of recloned cells collected from a single primary colony was able to generate new colonies. Individual clones generated from single colonies after five passages in a serially propagated clonal subculture were photographed, and their areas were measured. The cells of six individual clones were collected, dispersed, and transferred to sets of three separate wells coated with collagen (100 viable cells per well) on top of irradiated HPI.1 cells as a feeder layer. Two wells (containing three-fourths of the clone) were used for serial propagation, and the third well (containing one-fourth of the clone) was fixed 10 or 12 days later and used to classify the cell clonal type on the basis of the size of clones and the relative number of aborted colonies. Holoclones, meroclones, and paraclones, were detected in all six cases (Table 4Go).


View this table:
[in this window]
[in a new window]
  		Table 4. The proliferation and clonogenic potential of epidermal keratinocytes from a primary cultured mass
 
In a typical representative experiment, 31 clones generated from a single large colony of keratinocytes, isolated from a 10-day primary cultured mass of epithelial cells from one of the thorax skin biopsies, were analyzed. In agreement with the number of doublings found for the original cultured mass (i.e., mean ± standard error, 115 ± 7 cumulative population doublings), the five holoclones scored in this experiment accounted for almost the entire proliferative potential of the primary cultured mass, producing 108 (± 12) population doublings before senescence. Eighteen clones were classed as meroclones, displaying a wide variability in self-renewal potential, with clones undergoing 25–66 population doublings. Eight paraclones consisted primarily of mature keratinocyte progenitors: they were capable only of a few divisions (5 ± 2), eventually leading to aborted colonies. Generation of large mucin-producing cells was observed mainly among meroclones; their numbers were greater after confluence.

The high replicative potentials of holoclones were confirmed by a large proportion of MIB-1+ staining; however, they were only in part AE1+ and were consistently AE3-, CK7-, and CK19-. Meroclones had variable replicative potentials; they were MIB-1+, AE1+ and AE3+, and, less frequently, also CK7+ and CK19+. Paraclones included rare MIB-1+ cells; most of the cells were strongly stained either with anti-AE1 and anti-AE3 or with anti-CK7 and anti-CKl9 cytokeratin antibodies.

Our present studies demonstrate that keratinocyte stem cells isolated from the skin of normal adult donors can be successfully expanded in our culture system with high frequencies. However, consistent with previous observations, we demonstrated that the relative percentage of epidermal keratinocyte stem cells declines in long-term culture, suggesting that these cells are programmed to undergo a limited number of cell divisions before differentiating, generating transient-amplifying keratinocytes that eventually irreversibly undergo replicative senescence and cease to proliferate [13–15, 45–47].

Clonal Analysis of Keratinocytes in Three-Dimensional Tissue Culture

Cultures on Gelatin Gelfoam Sponge Matrixes   Clones of keratinocytes forming colonies of a similarly large size (possibly holoclones and meroclones) were harvested from a cultured mass. These cells were dispersed, pooled, and then seeded on top of gelatin gelfoam sponges. About 20 days after cell seeding, the sponges were fixed and processed for paraffin embedding. Sections were then analyzed after hematoxylin-eosin and immunohistochemical staining. After removal of the sponges, several enlarged colonies of keratinocytes were found adherent to the bottom of the collagen-coated plates. These colonies apparently derived from keratinocytes that had filtered through the sponge gel. The cells showed high proliferative potentials and long-lasting self-maintenance. They included a homogeneous population of poorly differentiated keratinocytes (AE1+, AE3+/-, CK7-, and CK19-). After these colonies were subcloned for several passages, the cells formed primarily meroclones, so classed on the basis of the variable proliferative potentials of their progeny.

Immunohistological staining with anti-AE1 or anti-AE3 antibodies of sections of paraffin-embedded sponges revealed the frequent presence of large clusters of keratinocytes tightly bound to the top region of the gel (Fig. 5AGo). The frequencies of these clusters from the top toward the bottom of the sponge were gradually greater with greater culture durations, and each cluster included differentiated keratinocytes, predominantly adherent to gelatin (Fig. 5BGo), as well as cycling keratinocytes (Fig. 5CGo). After 20 days of culture, mucin-producing cells were also detected within each cell cluster, particularly those of larger dimensions (Fig. 5DGo), in greater frequencies.



View larger version (144K):
[in this window]
[in a new window]
  		Figure 5. Three-dimensional organotypic cultures. A) Clusters of tightly adherent keratinocytes growing on and inside the matrix of a gelatin sponge after 20 days in culture (hematoxylin-eosin stained). B) Cells positively stained with anti-AE1 monoclonal antibody (original magnification = 400x). C) Cycling keratinocytes stained positively with anti-MIB-1 monoclonal antibody. D) Cells producing mucin (arrows) detected particularly inside large cell clusters (Alcian Blue stained, original magnification = 400x). E) Three-dimensional organotypic culture of keratinocytes growing in ordered multilayers on collagen gel. HPI.1 fibroblasts were used as a feeder layer in the lower chamber of the culture plate. Keratinocyte progenitors, pooled from six different large primary colonies, were plated onto a type-I- and type-III-collagen-coated insert and grown at the air-liquid interface. After 10 days in culture, cultures were fixed and paraffin embedded, and sections were subsequently stained with hematoxylin-eosin (original magnification = 400x).

 
These findings demonstrate that keratinocytes migrated through sponges, leading to an initially rapid increase in the proportion of the most primitive keratinocyte progenitors generating early progeny within the sponge. After gradual migration through the sponge, keratinocytes were capable of originating clones with variable replicative potentials, typical of meroclones, when replated on type-I-collagen-coated plates. Additionally, we found that meroclones could include mucin-producing cells that clearly differentiated from a common bipotent progenitor late in the life of a single clone.

Development of Keratinocyte Competence in Organotypic Cultures   After keratinocytes were seeded in the inserts, they became adherent to collagen and formed an adherent basal cell layer (Fig. 5EGo). Subsequently, cells of this layer began to proliferate and migrate toward the outer surface, developing cohesive multilayers of further differentiated keratinocytes, which stained positively with the anticytokeratin antibodies AE1 (basal level) and AE3 (parabasal level) (not shown). In 30-day-old cultures, cells secreting mucin (Alcian Blue positive) were constantly detected, primarily in the epibasal differentiated levels (not shown). These findings confirm that colonies with high replicative potentials isolated from human adult skin biopsies contained keratinocyte stem cells able to self-replicate on collagen-coated plates with fibroblasts as a feeder layer and also to further differentiate and reconstitute an organized epithelium in three-dimensional organotypic cultures.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we report the development of a new procedure for the long-term growth of human epidermal keratinocyte stem cells derived from a single punch biopsy from normal adult skin. The system uses isolated epidermal keratinocytes seeded on an artificial dermis matrix formed by a collagen layer and irradiated human neonatal foreskin fibroblasts (line HPI.1). To support plating and long-term proliferation, keratinocytes are maintained in a growth medium (initially in the absence of FBS and, after two passages, supplemented with FBS) containing a supplement of several chemically defined compounds that enhance the keratinocyte lifespan. This system has been successfully applied to grow keratinocyte progenitors for at least 115 population doublings, which corresponds to an approximately 1018- to 1020-fold increase from original cell numbers. This system greatly extends the lifespans of cultured keratinocytes isolated from normal human adult donors beyond that obtained with most other traditional methods (typically 15–50 population doublings after the primary culture stage) [13, 17, 40]. By clonal analysis of the primary epidermal proliferating population, we recognized the previously described [11–19] three main types of keratinocytes, which can all initiate colonies, but which do so with different cloning efficiencies and proliferative capacities. These cells have been identified as keratinocyte progenitors giving rise to holoclones, meroclones, and paraclones, based both on the sizes of the growing colonies and the frequencies of terminal colonies produced after serial cell transfers [13]. Holoclones contain cells with extensive capacities for cell division and are probably representative of in vivo keratinocyte stem cells. During early cultures, these clones, in fact, represented a very small fraction of the initial primary cultured mass. However, the proportion of self-maintaining cells was significantly greater with successive cell passages, accounting for almost the entire population of the mass culture, and these cells retained normal differentiating capabilities. On a collagen-coated plate, with irradiated HPI.1 cells as a feeder layer, cells of these clones underwent rapid proliferation. Moreover, in support of the "stemness" of these cells, we demonstrated that, in three-dimensional cultures, they generated progeny that subsequently differentiated, forming committed basal and epibasal keratinocytes, adjusting their proliferative and differentiation potentials to the requirements of the tissue of origin.

The discrete location of replicating keratinocyte progenitors in the basal layer of skin epidermis suggests that most of the early-passage replicating cells in the cultures were formed by meroclones, containing committed, transient-amplifying cells. These cells represented a source of immature keratinocytes, able to develop progeny with marked asymmetries in development and apparently capable of undergoing several rounds of cell division but eventually losing self-maintenance capability with later passages.

Additionally, replicating keratinocyte progenitors included keratinocytes that ultimately generated paraclones, representing the end of a unidirectional process of maturation and differentiation that occurs during prolonged keratinocyte culture and probably occurs in vivo during natural aging.

It is conceivable that, in normal skin, cells forming meroclones and paraclones are slow cycling but may become stimulated to enter the S phase by environmental signals (e.g., physical stimulation, skin infection, or wounding), to initiate rapid rounds of division followed by rapid terminal differentiation and cessation of replication. This resembles human hair follicles, in which a subpopulation of transient-amplifying keratinocytes is capable of initiating only a few replication cycles before undergoing terminal differentiation and ceasing to proliferate [17, 47]. Another comparable situation has been described for corneal and conjunctival epithelial stem cells [19].

It has been postulated that early epidermal keratinocyte progenitors are multipotent, capable of self-replication and also of developing committed keratinocytes at different levels of differentiation, which migrate from the basal to epibasal layers. Here, we provide evidence for the first time, to the best of our knowledge, for the existence of mucin-secreting cells during epidermal development in culture. These cells seem to derive from "old" meroclones and perhaps from paraclones. They are located primarily in the epibasal differentiated layers of the epidermal epithelium. The presence of mucin-containing cells within the upper layers of epithelium has been reported, particularly in pathologic conditions [48–51]. The results of our present study strongly suggest that both these mucin-producing cells and the other keratinocytes in the developing epidermis derived from a common bipotent keratinocyte progenitor representing the final progeny of individual keratinocyte clones. This suggestion is based on the finding that Alcian-Blue-positive cells spontaneously originated in well-established human keratinocyte A5RT1 clones after reaching confluence.

Basal and epibasal keratinocytes are filament-rich cells forming stratified layers, conferring resistance and the capability of defense on the epithelium. By contrast, mucin-producing cells are typical terminally differentiated secretory cells, which have a different architectural structure and whose main function is likely to be the production and release of mucin for epithelial protection. The commitment of keratinocytes to a secretory function apparently reaches a peak in frequency at a particularly late time in cell culture, when keratinocytes form a confluent epithelium. We have shown that regeneration of mucin-secreting cells occurs also in serum-free medium and in the absence of HPI.1 fibroblasts as a feeder layer, suggesting that the onset of their differentiation is controlled by an autonomous mechanism. Instructive or selective stimuli of external factors (e.g., cell-cell contacts, regulatory soluble factors, etc.) could trigger a bipotent transient-amplifying keratinocyte progenitor to replicate or to differentiate along one or the other functional pathway.

The molecular mechanisms of these differentiation events occurring in normal skin keratinocytes are still unknown and require further investigation. Bipotent and multipotent progenitors, capable of differentiating along different cell lineages of a tissue, have been well studied [52]. The differentiation and clonal characterization of stem cells, of transient-amplifying cells, and of terminally differentiated cells assuming different and alternative functions have been shown to occur also in cultured ocular epithelium [53]. Corneal and conjunctival stem cells are selectively located, generating a gradient of cells with different multiplicative abilities. In the developing human cornea, a very similar differentiation process, leading to the formation of mucin-producing cells, has been discovered [19].

In conclusion, all this evidence supports our present findings that, in epidermal tissue, normal keratinocyte progenitor cells can generate clones with variable growth and differentiation potentials. The results of our present work have major implications in two different fields. First, we have demonstrated that, with our improved culturing procedure, keratinocyte progenitor cells can be isolated directly from a normal adult skin biopsy and expanded with high efficiency, providing evidence that keratinocyte "stemness" can be preserved in culture for a prolonged time. Second, we have provided evidence that transient-amplifying keratinocytes forming meroclones may contain bipotent keratinocyte progenitors capable of generating either differentiated keratinocytes or mucin-producing cells.

The molecular mechanisms of these differentiation events occurring in normal skin keratinocytes are still unclear. To further investigate the observed mechanistic phenomena, work is now in progress attempting to identify the phenotype of skin stem cells, as well as the growth factors produced by HPI.1 cells required for the growth and expansion of the stem cells (cells able to form holoclones). The possibility of identifying the molecular factors that help to bypass the replicative senescence of keratinocyte stem cells and those that stimulate transient-amplifying cells to differentiate along two different pathways is of great interest and of practical importance for the clinical use of epithelial cultures and also for the study of epidermal differentiation.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The work of Sandra Papini, Denise Cecchetti, Daniela Campani, and Roberto P. Revoltella was supported in part by grants from the Consiglio Nazionale delle Ricerche (CNR, P.F., MADESS II), the Ministero Italiano dell’Università e della Ricerca Scientifica e Tecnologica for projects on "Technologies in Oncology" and "Stem Cells 2001," and the Ministero Italiano Istruzione, Università e Ricerca for Strategic Projects on Quality of Life ("Post-Genome" and "Neo-Organs"). The work of Wendy Fitzgerald, Jean Charles Grivel, Silvia Chen, and Leonid Margolis was supported in part by the NASA/NIH Center for Three-Dimensional Tissue Culture.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Green H. The keratinocyte as differentiated cell type. Harvey Lect 1980;74:101–139.[Medline]

  2. Watt FM. Terminal differentiation of epidermal keratinocytes. Curr Opin Cell Biol 1989;6:1107–1115.

  3. De Luca M, Tamura RN, Kajiji S et al. Polarized integrin mediates human keratinocyte adhesion to basal lamina. Proc Natl Acad Sci USA 1990;87:6888–6892.[Abstract/Free Full Text]

  4. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992;69:11–25.[CrossRef][Medline]

  5. Fuchs E, Byrne C. The epidermis: rising to the surface. Curr Opin Genet Dev 1994;4:725–736.[CrossRef][Medline]

  6. Jones JC, Asmuth J, Baker SE et al. Hemidesmosomes: extracellular matrix/intermediate filament connectors. Exp Cell Res 1994;213:1–11.[CrossRef][Medline]

  7. Roop D. Defects in the barrier. Science 1995;267:474–475.[Free Full Text]

  8. Christiano AM, Uitto J. Molecular complexity of the cutaneous basement membrane zone. Revelations from the paradigms of epidermolysis bullosa. Exp Dermatol 1996;5:1–11.[CrossRef][Medline]

  9. Borradori L, Sonnenberg A. Hemidesmosomes: roles in adhesion, signaling and human diseases. Curr Opin Cell Biol 1996;8:647–656.[CrossRef][Medline]

  10. Dowling J, Yu QC, Fuchs E. Beta4 integrin is required for hemidesmosome formation, cell adhesion and cell survival. J Cell Biol 1996;134:559–572.[Abstract/Free Full Text]

  11. Lavker RM, Sun TT. Epidermal stem cells. J Invest Dermatol 1983;81(suppl 1):121–127.[CrossRef]

  12. Miller SJ, Lavker RM, Sun TT. Keratinocyte stem cells of cornea skin and hair follicles. In: Potten CS, ed. Stem Cells. San Diego, CA: Academic Press Inc., 1997:331–362.

  13. Barrandon Y, Green H. Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA 1987;84:2302–2306.[Abstract/Free Full Text]

  14. Barrandon Y, Green H. Three clonal types of keratinocytes with different capacities of differentiation. Proc Natl Acad Sci USA 1993;84:209–215.[CrossRef]

  15. Barrandon Y. The epidermal stem cell: an overview. Dev Biol 1993;4:209–214.

  16. Jones PH, Watt FM. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 1993;73:713–724.[CrossRef][Medline]

  17. Rochat A, Kobayashi K, Barrandon Y. Location of stem cells of human hair follicles by clonal analysis. Cell 1994;76:1063–1073.[CrossRef][Medline]

  18. Mathor MB, Ferrari G, Dellambra E et al. Clonal analysis of stably transduced human epidermal stem cells in culture. Proc Natl Acad Sci USA 1996;93:10371–10376.[Abstract/Free Full Text]

  19. Pellegrini G, Golisano O, Paterna P et al. Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J Cell Biol 1999;145:769–782.[Abstract/Free Full Text]

  20. Fusenig NE. Epithelial-mesenchymal interactions regulate keratinocyte growth and differentiation in vitro. In: Leigh IM, Lane EB, Watt FM, eds. The Keratinocytes Handbook. Cambridge: Cambridge University Press, 1994:71–94.

  21. Eady RA, Dunnill MG. Epidermolysis bullosa: hereditary skin fragility diseases as paradigms in cell biology. Arch Dermatol Res 1994;287:2–9.[CrossRef][Medline]

  22. Boyce ST. Epidermis as a secretory tissue. J Invest Dermatol 1994;102:8–10.[CrossRef][Medline]

  23. Fenjves ES, Gordon DA, Pershing LK et al. Systemic distribution of apolipoprotein E secreted by grafts of epidermal keratinocytes: implications for epidermal function and gene therapy. Proc Natl Acad Sci USA 1989;86:8803–8807.[Abstract/Free Full Text]

  24. Alexander MY, Bidichandani SI, Cousins FM et al. Circulating human factor IX produced in keratin-promoter transgenic mice: a feasibility study for gene therapy of haemophilia B. Hum Mol Genet 1995;4:993–999.[Abstract/Free Full Text]

  25. Cuono C, Langdon R, McGuire J. Use of cultured epidermal autografts and dermal allografts as skin replacement after burn injury. Lancet 1986;1:1123–1124.[CrossRef][Medline]

  26. De Luca M, D’Anna F, Bondanza S et al. Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis. J Cell Biol 1988;107:1919–1926.[Abstract/Free Full Text]

  27. Compton CC, Gill JM, Bradford DA et al. Skin regenerated from cultured epithelial autografts on full-thickness burn wounds from 6 days to 5 years after grafting. A light electron microscopic and immunohistochemical study. Lab Invest 1989;60:600–612.[Medline]

  28. De Luca M, Albanese E, Bondanza S et al. Multicentre experience in the treatment of burns with autologous and allogenic cultured epithelium fresh or preserved in a frozen state. Burns 1989;15:303–309.[CrossRef][Medline]

  29. Gallico GG 3rd, O’Connor NE, Compton CC et al. Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 1984;311:448–451.[Medline]

  30. Matousková E, Vesely’ P, Königová R. Modified method of in vitro cultivation of human keratinocytes suitable for grafting. Folia Biol (Praha) 1989;35:267–271.

  31. Matousková E, Vogtová D, Königová R. A recombined skin composed of human keratinocytes cultured on cell-free pig dermis. Burns 1993;19:118–123.[CrossRef][Medline]

  32. Teumer J, Lindhal A, Green H. Human growth hormone in the blood of athymic mice grafted with cultures of hormone-secreting human keratinocytes. FASEB J 1990;4:3245–3250.[Abstract]

  33. Fenjves ES. Approaches to gene transfer in keratinocytes. J Invest Dermatol 1994;103(suppl 5):70–75.[CrossRef]

  34. Krueger GG, Morgan JR, Jorgensen CM et al. Genetically modified skin to treat disease: potential and limitations. J Invest Dermatol 1994;103(suppl 5):76S–84S.[CrossRef][Medline]

  35. Greenhalgh DA, Rothnagel JA, Roop DR. Epidermis: an attractive target tissue for gene therapy. J Invest Dermatol 1994;103(suppl 5):63S–69S.[CrossRef][Medline]

  36. Vogt PM, Thompson S, Andree C et al. Genetically modified keratinocytes transplanted to wounds reconstitute the epidermis. Proc Natl Acad Sci USA 1994;91:9307–9311.[Abstract/Free Full Text]

  37. Dellambra E, Vailly J, Pellegrini G et al. Corrective transduction of human epidermal stem cells in laminin-5-dependent junctional epidermolysis bullosa. Hum Gene Ther 1998;9:1359–1370.[Medline]

  38. Pellegrini G, Bondanza S, Guerra L et al. Cultivation of human keratinocyte stem cells: current and future clinical applications. Cell Eng 1998;36:1–13.

  39. Parenteau NL, Bilbo P, Nolte CJM et al. The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function. Cytotechnology 1992;9:163–171.[CrossRef][Medline]

  40. Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975;6:331–344.[CrossRef][Medline]

  41. Fusenig NE, Boukamp P. Multiple stages and genetic alterations in immortalization malignant transformation and tumor progression of human skin keratinocytes. Mol Carcinog 1998;23:144–158.[CrossRef][Medline]

  42. Toda K, Grinnell F. Activation of human keratinocyte fibronectin receptor function in relation to other ligand-receptor interactions. J Invest Dermatol 1987;88:412–417.[CrossRef][Medline]

  43. Toda K, Tuan TL, Brown PJ et al. Fibronectin receptors of human keratinocytes and their expression during cell culture. J Cell Biol 1987;105:3097–3104.[Abstract/Free Full Text]

  44. Leighton J. Epithelial tissue formation in matrix culture and in histophysiologic gradient culture. J Tissue Cult Methods 1992;14:201–208.

  45. Lajtha LG. Stem cell concepts. Differentiation 1979;14:23–34.[CrossRef][Medline]

  46. Morrison SJ, Shah NM, Anderson DJ. Regulatory mechanisms in stem cell biology. Cell 1997;88:287–298.[CrossRef][Medline]

  47. Lavker RM, Miller S, Wilson C et al. Hair follicle stem cells: their location role in hair cycle, and involvement in skin tumor formation. J Invest Dermatol 1993;101(suppl 1):16S–26S.[CrossRef][Medline]

  48. Rongioletti F, Rebora A. Cutaneous mucinoses: microscopic criteria for diagnosis. Am J Dermatopathol 2001;23:257–267.[CrossRef][Medline]

  49. Walker AN, Morton BD. Acral mucinous syringometaplasia. A benign cutaneous lesion associated with verrucous hyperplasia. Arch Pathol Lab Med 1986;110:248–249.[Medline]

  50. Val-Bernal JF, Hernandez-Nieto E. Benign mucinous metaplasia of the penis. A lesion resembling extramammary Paget’s disease. J Cutan Pathol 2000;27:76–79.[CrossRef][Medline]

  51. Friedman KJ, Hood AF, Farmer ER. Cutaneous squamous cell carcinoma with mucinous metaplasia. J Cutan Pathol 1988;15:176–182.[CrossRef][Medline]

  52. Potten CS, ed. Stem Cells. San Diego, CA: Academic Press Inc., 1997:1–474.

  53. Sun TT, Green H. Cultured epithelial cells of cornea, conjunctiva and skin: absence of marked intrinsic divergence of their differentiated states. Nature 1977;269:489–493.[CrossRef][Medline]

Received September 16, 2002; accepted for publication February 26, 2003.



This article has been cited by other articles:


Home page
 J. Cell Sci.Home page
M. Lee and V. Vasioukhin
Cell polarity and cancer - cell and tissue polarity as a non-canonical tumor suppressor
J. Cell Sci., April 15, 2008; 121(8): 1141 - 1150.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
F. G. Chen, W. J. Zhang, D. Bi, W. Liu, X. Wei, F. F. Chen, L. Zhu, L. Cui, and Y. Cao
Clonal analysis of nestin vimentin+ multipotent fibroblasts isolated from human dermis
J. Cell Sci., August 15, 2007; 120(16): 2875 - 2883.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
M. T. Budak, O. S. Alpdogan, M. Zhou, R. M. Lavker, M.A. M. Akinci, and J. M. Wolosin
Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells
J. Cell Sci., April 15, 2005; 118(8): 1715 - 1724.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow