The Mixed Lineage Kinase Leucine-Zipper Protein Kinase Exhibits a Differentiation-Associated Localization in Normal Human Skin and Induces Keratinocyte Differentiation upon Overexpression

Leucine-zipper protein kinase/dual leucine zipper bearing kinase/mitogen-activated protein kinase-upstream kinase is a recently described protein serine/threonine kinase which belongs to the mixed lineage kinase family. The overall pattern of expression of the leucine-zipper protein kinase/dual leucine zipper bearing kinase/mitogen-activated protein kinase-upstream kinase gene in embryonic and adult mouse tissues suggested that this kinase could be involved in the regulation of epithelial cell proliferation and differentiation. In order to get more insights into the potential role of leucine-zipper protein kinase in these cellular processes, we characterized its expression in normal human skin, both at the mRNA and protein levels. In situ hybridization, western blotting, and indirect immunofluorescence studies were conducted to localize leucine-zipper protein kinase on various human skin tissues. This is one of the first reports that leucine-zipper protein kinase has a very precise pattern of expression in human skin epithelia, as both mRNA and protein are restricted to the granular layer of the epidermis and inner root sheath of hair follicles. Detection of leucine-zipper protein kinase protein on skin from various body sites, donors of different ages as well as on reconstructed human skin always reveals that leucine-zipper protein kinase is present only in the very differentiated keratinocytes of epidermis and hair follicles. To determine directly whether leucine-zipper protein kinase exhibits any effect on cell growth and differentiation, keratinocytes were transfected with an expression vector harboring the leucine-zipper protein kinase cDNA. The presence of this construct in keratinocytes results in growth arrest together with a concomitant increase in filaggrin expression. Collectively, our results indicate that leucine-zipper protein kinase plays an active part in cellular processes related to terminal differentiation of epidermal keratinocytes

Grafting on nude mice of living skinquivalents produced using human collagens

Autologous epidermal transplantation for human burn management is an example of a significant breakthrough in tissue engineering. However, the main drawback with this treatment remains the fragility of these grafts during and after surgery. A new human bilayered skin equivalent (hSE) was produced in our laboratory to overcome this problem. The aim of the present work was to study skin regeneration after hSE grafting onto nude mice. A comparative study was carried out over a period of 90 days, between anchored bovine skin equivalent, hSE and hSE+, the latter containing additional matrix components included at concentrations similar to those in human skin in vivo. The addition of a dermal layer to the epidermal sheet led to successful graft take, enhanced healing, and provided mechanical resistance to the grafts after transplantation. In situ analysis of the grafts showed good ultrastructural organization, including the deposition of a continuous basement membrane 1 week after surgery

Normal Human Merkel Cells are Present in Epidermal Cell Populations Isolated and Cultured from Glabrous and Hairy Skin Sites

The Merkel cell is a highly specialized cell that primarily acts as a slowly adapting mechanoreceptor. Merkel cells are scarce in normal skin but can be identified by the expression of distinct keratin filaments. Merkel cells constitute a very unique population and many questions still remain as to their origin, number, proliferative capacity, and functions in cutaneous biology. The dissociation of epidermal cells from skin is a widely used technique to extract and culture keratinocytes. We took advantage of a two-step extraction method to quantify keratin-20-expressing Merkel cells among total cutaneous cells obtained from either hairy or glabrous skin biopsies. Flow cytometry analysis revealed that keratin-20-labeled Merkel cells represent between 3.6% and 5.7% of freshly dissociated basal epidermal cells. No significant differences were seen between samples derived from glabrous palmar and hairy anatomic sites, from children and adult, respectively. We also report on the presence of Merkel cells in primary and first subcultures of epidermal cells indicating their capacity to remain viable after extraction from skin of various anatomic sites. To our knowledge, this is the first demonstration of nontumorigenic human Merkel cells in culture in vitro. The persistence of a small number of Merkel cells in culture suggests that, with the development of appropriate culture conditions, these cells could be amplified and further studied to unravel long-standing questions relative to their paracrine function or epithelial origin

Production of a bilayered self-assembled skin substitute using a tissue-engineered acellular dermal matrix

Our bilayered self-assembled skin substitutes (SASS) are skin substitutes showing a structure and functionality very similar to native human skin. These constructs are used, in life-threatening burn wounds, as permanent autologous grafts for the treatment of such affected patients even though their production is exacting. We thus intended to shorten their current production time to improve their clinical applicability. A self-assembled decellularized dermal matrix (DM) was used. It allowed the production of an autologous skin substitute from patient's cells. The characterization of SASS reconstructed using a decellularized dermal matrix (SASS-DM) was performed by histology, immunofluorescence, transmission electron microscopy, and uniaxial tensile analysis. Using the SASS-DM, it was possible to reduce the standard production time from about 8 to 4 and a half weeks. The structure, cell differentiation, and mechanical properties of the new skin substitutes were shown to be similar to the SASS. The decellularization process had no influence on the final microstructure and mechanical properties of the DM. This model, by enabling the production of a skin substitute in a shorter time frame without compromising its intrinsic tissue properties, represents a promising addition to the currently available burn and wound treatments

Improved methods to produce tissue-engineered skin substitutes suitable for the permanent closure of full-thickness skin injuries

There is a clinical need for skin substitutes to replace full-thickness skin loss. Our group has developed a bilayered skin substitute produced from the patient’s own fibroblasts and keratinocytes referred to as Self-Assembled Skin Substitute (SASS). After cell isolation and expansion, the current time required to produce SASS is 45 days. We aimed to optimize the manufacturing process to standardize the production of SASS and to reduce production time. The new approach consisted in seeding keratinocytes on a fibroblast-derived tissue sheet before its detachment from the culture plate. Four days following keratinocyte seeding, the resulting tissue was stacked on two fibroblast-derived tissue sheets and cultured at the air–liquid interface for 10 days. The resulting total production time was 31 days. An alternative method adapted to more contractile fibroblasts was also developed. It consisted in adding a peripheral frame before seeding fibroblasts in the culture plate. SASSs produced by both new methods shared similar histology, contractile behavior in vitro and in vivo evolution after grafting onto mice when compared with SASSs produced by the 45-day standard method. In conclusion, the new approach for the production of high-quality human skin substitutes should allow an earlier autologous grafting for the treatment of severely burned patients

Improvement of human keratinocyte isolation and culture using thermolysin

We propose a modification of the conventional keratinocyte isolation method which has shown a significant improvement in the purity, colony forming efficiency (c.f.e.) and growth capacity of the isolated epidermal cell population. This method utilized thermolysin since it selectively digests the dermo-epidermal junction. Following separation from the dermis, the epidermis was digested with trypsin to obtain a single cell suspension. Compared with the conventional procedure, this isolation method was shorter and resulted in (i) cells displaying a higher colony forming efficiency, (ii) cells reaching confluence 1–3 days earlier, (iii) cells not contaminated by fibroblasts, (iv) a cell population containing all the basal layer keratinocytes. These cells were suitable for the establishment of primary cultures and could be subcultured. Such cell populations should be advantageous in studies of epithelial-mesenchymal interactions in which keratinocyte populations, free of fibroblasts, are desirable. In the treatment of extensively burned patients using cultured epidermal sheets, the main problem remains the time required for their production. Thus, the absence of fibroblast overgrowth of the keratinocyte cultures and the significantly reduced time to obtain confluent cultures and epidermal sheets with our method have very important implications for the treatment of large burn wounds

Early basement membrane formation following the grafting of cultured epidermal sheets detached with thermolysin or Dispase

The basement membrane zone is important for graft adhesion and stability. The aim of the present study was to visualize the regeneration of the basement membrane and determine the sequential appearance of its constituents in the early postgrafting period of cultured human epidermal sheets. A keratinocyte single cell suspension, devoid of dermal fibroblast contamination, was obtained from human skin by a two-step tissue digestion method with thermolysin and trypsin. After culturing, epidermal sheets were generated, detached enzymatically by incubating with thermolysin (for 20–30 min) or Dispase (for 45–60 min), and deposited on a muscular graft bed of athymic mice. Immunohistochemistry and ultrastructural analyses were performed on biopsies harvested 2, 4 and 21 days postgrafting. Bullous pemphigoid antigens and laminin were detected at the dermo—epidermal junction, showing an almost continuous line 2 days postgrafting. Type IV collagen was generally absent at this time, but it was detected 4 days postgrafting. Type VII collagen was labelled as a discontinuous line of increasing intensity from 2 to 21 days postgrafting. Ultrastructural analysis revealed hemidesmosomes and a discontinuous lamina densa 2 days postgrafting, and a complete basement membrane with a continuous lamina densa, hemidesmosomes and anchoring fibrils 21 days postgrafting. The sequence of appearance of major basement membrane components was similar for cultured sheets detached with thermolysin or Dispase. However, it differed from that of other wound healing models. Results are discussed in terms of the variable keratinocyte migration requirement between various wound healing models

Optimization of murine keratinocyte culture for the production of graftable epidermal sheets

The aim of the present study was to optimize murine epidermal cell cultures in order to obtain graftable sheets. Newborn (1–3 days old) Balb/c mice skin were used to optimize culture media and plating cell concentration, then epidermal sheet production, and grafting. Epidermal cells were plated at various concentrations in different culture media containing low (0.1 mM) or high (>1 mM) Ca2+ levels. After a 3 day culture at the 104 cells/cm2 plating cell concentration, the percentage of differentiated cells was more than 80% in the high Ca2+ culture medium and less than 50% with bulky cells in the low Ca2+ culture medium. Under these conditions confluence was not obtained. At the 105 cells/cm2 seeding inoculum, the percentage of confluence increased to 95–100% during the first 72 h of culture in both high and low Ca2+ culture media. Three-day-old culture showed stratified multilayer epidermal sheets in the high calcium medium, and monolayer epidermal sheets were present in the low calcium medium after seeding keratinocytes in fibronectin precoated flasks. Seven days after plating, post confluent cultures were composed of a high percentage of differentiated cells (90%) with an increase in shedding cells in the medium. Considering the above morphological observations, sheets obtained with 105 cells/cm2 in MCDB-153 (A), DME-HAM (B) or GMEM (C) media after 3 days in culture were grafted. Twenty days after grafting, histological analysis of biopsies showed an epidermal structure and organization comparable to normal murine epidermis without hair follicles. Epidermal transplants showed a complete basement membrane, hemidesmosomes, and tonofilament bundles. Sheets obtained after seven day culture in all media showed lower coverage of the wound bed. These studies point out the importance of the plating cell and Ca2+ concentrations, and the culture time for murine keratinocyte confluence and differentiation to obtain graftable epidermal sheets

Role and innocuity of Tisseel ®, a tissue glue, in the grafting process and in vivo evolution of human cultured epidermis

Cultured epidermal sheets are currently used for burn wound treatment but reported results on graft take are variable. This study was designed to evaluate the role and influence of Tisseel®, a fibrin glue, in the take of cultured human epidermal sheets in an athymic mouse model. On days 4, 10 and 21 post-grafting, histology, electron microscopy and immunofluorescence staining confirmed the presence of a human epithelium and the development of a basement membrane. Tisseel® was detectable on day 4 only, but overall treated and untreated grafts were similar. The use of Tisseel® enhanced the mechanical stability of these fragile grafts, increased the percentage of graft take, and its innocuity on thein vivo evolution of cultured epidermal sheets was demonstrated. For these reasons, we think that Tisseel® may be advantageous in a clinical setting

Are the Effects of the Cholera Toxin and Isoproterenol on Human Keratinocytes’ Proliferative Potential Dependent on Whether They Are Co-Cultured with Human or Murine Fibroblast Feeder Layers?

Human keratinocyte culture has provided the means to treat burns, wounds and skin pathologies. To date, to efficiently culture keratinocytes, cells are cultured on an irradiated feeder layer (iFL), either comprising human (iHFL) or murine (i3T3FL) fibroblasts, and the culture medium is supplemented with a cyclic adenosine monophosphate (cAMP) accumulation inducing agent such as isoproterenol (ISO) or cholera toxin (CT). Previous studies have characterized how the feeder layer type and the cAMP inducer type influence epithelial cells’ phenotype independently from one another, but it is still unknown if an optimal combination of feeder layer and cAMP inducer types exists. We used sophisticated statistical models to search for a synergetic effect of feeder layer and cAMP inducer types on human keratinocytes’ proliferative potential. Our data suggests that, when culturing human keratinocytes, using iHFL over i3T3FL increases population doublings and colony-forming efficiency through signaling pathways involving Ak mouse strain thymoma (Akt, also known as protein kinase B) isoforms 1 to 3, signal transducer and activator of transcription 5 (STAT5), p53, and adenosine monophosphate activated protein kinase α1 (AMPKα1). Both tested cAMP inducers ISO and CT yielded comparable outcomes. However, no significant synergy between feeder layer and cAMP inducer types was detected. We conclude that, to promote human keratinocyte growth in the early passages of culture, co-culturing them with a human feeder layer is preferable to a murine feeder layer