Assessment of replication rates of human keratinocytes in engineered skin substitutes grafted to athymic mice (original) (raw)
Related papers
An alternative approach for traditional clinical mesh grafting in burn wound treatment is the use of expanded autologous keratinocytes in suspension or sheets that are cultured over 2–4 weeks in a remote service facility. While a wound reepithelialization has been described, the functional and aesthetic outcome is under debate. Cell isolation from split-skin donor tissue aims to preserve the valuable stem cell progenitors from the basal epidermal layer and to provide patients with a rapid wound re-epithelialization and a satisfying outcome. While the presence of epidermal progenitors in the cell graft is thought to enable an improved epidermal surface post reepithelialization, we investigated a feasible clinical approach involving cultured versus noncultured epidermal cells comparing the α6int high /K15 high /FSC low /SSC low and α6int high /K5 high / FSC low /SSC low keratinocyte progenitor subpopulations before and after in vitro culture process. Our results show a significant increase of cell size during in vitro passaging and a decrease of progenitor markers linked to a gradual differentiation. A provision of the regenerative epidermal progenitors, isolated from the split-skin biopsy and applied directly onto the wound in an on-site setting of isolation and cell spray grafting in the operation room, could be of interest when choosing options for skin wound care with autologous cells.
Cells
The basal layer of human interfollicular epidermis has been described to harbour both quiescent keratinocyte stem cells and a transit amplifying cell population that maintains the suprabasal epidermal layers. We performed immunofluorescence analyses and revealed that the main proliferative keratinocyte pool in vivo resides suprabasally. We isolated from the human epidermis two distinct cell populations, the basal and the suprabasal keratinocytes, according to the expression of integrin β4 (iβ4). We compared basal iβ4+ or suprabasal iβ4− keratinocytes with respect to their proliferation and colony-forming ability and their Raman spectral properties. In addition, we generated dermo–epidermal substitutes using freshly isolated and sorted basal iβ4+ or suprabasal iβ4− keratinocytes and transplanted them on immuno-compromised rats. We show that suprabasal iβ4− keratinocytes acquire a similar proliferative capacity as basal iβ4+ keratinocytes after two weeks of culture in vitro, with expr...
Reconstituted Skin from Murine Embryonic Stem Cells
Current Biology, 2003
cytokeratin-14 (K14) intermediate filament. Relative quantification was done by careful observation of the 1 INSERM U385 06107 Nice fields under a fluorescent microscope (Table 1). Rare induction was observed when ES cells were cultured on 2 INSERM/UMRS 514 51100 Reims gelatin. A higher but still low number of K14-positive colonies were observed in the presence of matrix de-3 CNRS UMR 6543 06108 Nice rived from SCC25 (epithelial tumor cell line derived from human tongue), 804G (epithelial cell line derived from France a rat bladder carcinoma), and MCF-10A (immortalized murine mammary epithelial) cells (Table 1). However, significant keratinocyte differentiation was obtained Summary when ES cells were seeded on human normal fibroblasts (HNF) (Figures 1Aa and 1Ab) or mouse NIH-3T3 fibro-Embryonic stem (ES) cell lines can be expanded indefinitely in culture while maintaining their potential to blast cell-secreted ECM (Table 1). The molecular basis of keratinocyte differentiation induction by the feeder differentiate into any cell type [1, 2]. During embryonic development, the skin forms as a result of reciprocal matrix remains to be understood, but it must be noted that the efficient matrices were derived from cells of interactions between mesoderm and ectoderm [3].
Stem cells of the skin epithelium
Tissue stem cells form the cellular base for organ homeostasis and repair. Stem cells have the unusual ability to renew themselves over the lifetime of the organ while producing daughter cells that differentiate into one or multiple lineages. Difficult to identify and characterize in any tissue, these cells are nonetheless hotly pursued because they hold the potential promise of therapeutic reprogramming to grow human tissue in vitro, for the treatment of human disease. The mammalian skin epithelium exhibits remarkable turnover, punctuated by periods of even more rapid production after injury due to burn or wounding. The stem cells responsible for supplying this tissue with cellular substrate are not yet easily distinguishable from neighboring cells. However, in recent years a significant body of work has begun to characterize the skin epithelial stem cells, both in tissue culture and in mouse and human skin. Some epithelial cells cultured from skin exhibit prodigious proliferative potential; in fact, for >20 years now, cultured human skin has been used as a source of new skin to engraft onto damaged areas of burn patients, representing one of the first therapeutic uses of stem cells. Cell fate choices, including both self-renewal and differentiation, are crucial biological features of stem cells that are still poorly understood. Skin epithelial stem cells represent a ripe target for research into the fundamental mechanisms underlying these important processes.
Differentiation, 1998
Cultured epithelial autografts (CEA) derived from sole skin were transplanted to full-thickness wounds excised to muscle fascia over a variety of diverse body sites in 12 pediatric patients treated for acute burns or giant congenital nevi. The skin regenerated from the grafts was biopsied from 7 days to 6 years after grafting. The resultant epidermal phenotype was analyzed histologically and by immunohistochemical localization of keratin 9 (K9) as objective evidence of sole-type site-specific differentiation. Expression of K9 was also verified by onedimensional gel electrophoresis of epidermal cytoskeletal extracts and K9 immunoblot analysis. Grafts prepared from epidermis of axilla, groin or foreskin and transplanted to wounds of comparable depth in an identical manner in the same patients served as controls of postgrafting differentiation. Biopsies of sole skin from amputation specimens from patients of comparable age served as normal positive controls, and biopsies of nonsole skin from patients of comparable age served as normal negative controls. As early as 2 weeks postgrafting, the histologic appearance of sole-derived CEA differed substantively from that of axilla-or groin-derived CEA controls and displayed a phenotype characteristic of sole skin with a thick compact stratum corneum, a thick stratum granulosum, and a distinct stratum lucidum. In sole-derived grafts rete ridges regenerated within 2 months postgrafting, whereas nonsole-derived grafts required 4-6 months for rete ridge regeneration. Once acquired, the sole skin phenotype was maintained long-term by all sole-derived CEA. In vitro, sole-derived keratinocytes synthesized little, if any, K9. However, within 7 days after grafting, K9 synthesis by multiple suprabasal keratinocytes was seen within the epidermis regenerated from sole-derived CEA. Protein of K9 appeared progressively more diffuse throughout the suprabasal layers, attaining a confluent pattern of expression comparable to normal controls of sole skin by 6 to 12 months postgrafting, and the confluent pattern of suprabasal K9 synthesis was maintained long-term. The results demonstrate that site-specific differentiation is an intrinsic property of postnatal human keratinocytes and can be expressed and maintained in a permissive environment in the absence of dermal tissue.& b d y :
Experimental Dermatology, 2005
Given that an important functional attribute of stem cells in vivo is their ability to sustain tissue regeneration, we set out to establish a simple and easy technique to assess this property from candidate populations of human keratinocyte stem cells in an in vivo setting. Keratinocytes were inoculated into devitalized rat tracheas and transplanted subcutaneously into SCID mice, and the epithelial lining regenerated characterized to establish the validity of this heterotypic model. Furthermore, the rate and quality of epidermal tissue reconstitution obtained from freshly isolated unfractionated vs. keratinocyte stem cell-enriched populations was tested as a function of (a) cell numbers inoculated; and (b) the inclusion of irradiated support keratinocytes and dermal cells. Rapid and sustained epidermal tissue regeneration from small numbers of freshly isolated human keratinocyte stem cells validates the utilization of this simple and reliable model system to assay for enrichment of epidermal tissue-reconstituting cells.
Journal of Investigative Dermatology, 1993
This study compares two techniques for making cultured skin substitutes: a composite graft made of human fibroblasts and keratinocytes on a collagen-glycosaminoglycan membrane (CG) and a cultured epidermal cell sheet graft (CEG), without a dermal component. The "take" and quality of these cultured skin substitutes were evaluated by placing them on full-thickness, excised wounds of athymic mice. These cultured skin substitutes were placed onto 2-X-2em wounds created on athymic mice. Mice were sacrificed at days 10, 20, and 42 with histologic sections obtained for light, electron, immunofluorescent, a?d immunohistoche~ ical microscopy. "Take" was determmed separately by a dIrect immunofluorescent stain for human leukocyte ABC antigens. There were ten mice of each graft type with at least two animals sacrificed at each time point. Results showed positive "take" for all animals. Grossly, T he use of cultured skin substitutes in burn care and reconstructive surgery is expanding. Two key factors have shaped the design of these cultured grafts: the ability to grow keratinocytes in vitro and the inc~eas ing practice of early wound excision in the extensively burned patient. Medawar reported the first successful isolation of buman keratinocytes in 1941 [1], but it was not until 1975 that Rheinwald and Green reported the growth of human keratinocytes at clonal densities on a feeder layer [2]. This achievement, the culturing of a great number of epidermal cells with dividing times under 24 h, meant that enough keratinocytes could be grown in 3-4 weeks to cover a whole body surface area. Further work in this field over the last 15 years has led to decreased culture times and the formation of serum-free systems [3-6] that can match the growth of the previous serum-based systems. The second major step leading to more experimentation with permanent biologic wound coverings was the increased use of early excision of the burn wound with major burn victims. Several centers Manuscript
Journal of Investigative Dermatology, 2009
We screened a series of antibodies for their exclusive binding to the human hair follicle bulge. In a second step these antibodies were to be used to identify basal keratinocytes and potential epithelial stem cells in the human epidermis and in engineered skin substitutes. Of all the antibodies screened, we identified only one, designated C8/144B, that exclusively recognized the hair follicle bulge. However, C8/144B-binding cells were never detected in the human epidermal stratum basale. In the bulge C8/144B-binding cells gave rise to cytokeratin 19-positive cells, which were also tracked in the outer root sheath between bulge and the hair follicle matrix. Remarkably, cytokeratin 19-expressing cells were never detected in the hair follicle infundibulum. Yet, cytokeratin 19-expressing keratinocytes were found in the epidermal stratum basale of normal skin as a subpopulation of cytokeratin 15-positive (not C8/144B-positive) basal keratinocytes. Cytokeratin 19/cytokeratin 15-positive keratinocytes decreased significantly with age. We suggest that cytokeratin 19-expressing cells represent a subpopulation of basal keratinocytes in neonates and young children (up to 1.5 years) that is particularly adapted to the lateral expansion of growing skin. Our data show that cytokeratin 19 in combination with cytokeratin 15 is an important marker to routinely monitor epidermal homeostasis and (at least indirectly) the self-renewing potential of engineered skin.
Cell proliferation and differentiation in a model of human skin equivalent
The Anatomical Record, 2001
Recent advances in culturing technology has permitted the production of organotypic models that may be referred to as human skin equivalents (HSE). We have studied histochemical, ultrastructural, and kinetic aspects of an HSE composed by an epidermal equivalent and a dermal equivalent separated by a basement membrane. Only keratinocytes and fibroblasts were present in the epidermal and dermal equivalents, respectively; cells of other lineages were lacking. Keratinocyte stratification and differentiation seemed similar to natural skin. Evidence is shown that such an HSE may also release growth factors such as vascular endothelial growth factor that are believed to play a role in skin grafting. The distribution of cycling cells as well as the values of the growth fraction are comparable to those observed in natural skin. Although the absence of several cells populations that reside in natural skin is a remarkable feature of this HSE, the high levels of tissue organization and cell differentiation lead us to believe that such an HSE may be considered a candidate substitute of human skin in biological, pharmacologic, and clinical applications. Anat Rec 264: [261][262][263][264][265][266][267][268][269][270][271][272] 2001.