The Control of Epidermal Stem Cells (Holoclones) in the Treatment of Massive Full-Thickness Burns With Autologous Keratinocytes Cultured on Fibrin1

Pellegrini, Graziella; Ranno, Rosario; Stracuzzi, G; Bondanza, Sergio; Guerra, Liliana; Zambruno, Giovanna; Micali, G; Luca, Michele De

doi:10.1097/00007890-199909270-00021

Cited by 337 publications

(289 citation statements)

References 52 publications

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“…Informed consent was obtained from the parents. Keratinocytes were cultivated on a feeder layer of lethally irradiated 3T3-J2 cells as described (28,30,33) and passaged at the stage of subconfluence.…”

Section: Methodsmentioning

confidence: 99%

“…Keratinocytes can generate cohesive sheets of stratified epithelium that maintains virtually the same differentiation features and gene expression pattern of its in vivo counterpart so that it can be routinely transplanted in patients suffering from large skin or mucosal defects (31)(32)(33). When primary keratinocytes are seeded onto dead de-epidermized dermis in organotypic cultures (29), mature HDs are formed in vitro (30).…”

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confidence: 99%

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Gene Correction of Integrin β4-dependent Pyloric Atresia-Junctional Epidermolysis Bullosa Keratinocytes Establishes a Role for β4 Tyrosines 1422 and 1440 in Hemidesmosome Assembly

Dellambra¹,

Prislei²,

Salvati³

et al. 2001

Journal of Biological Chemistry

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The cytoplasmic domain of ␤ 4 integrin contains two pairs of fibronectin-like repeats separated by a connecting segment. The connecting segment harbors a putative tyrosine activation motif in which tyrosines 1422 and 1440 are phosphorylated in response to ␣ 6 ␤ 4 binding to laminin-5. Primary ␤ 4 -null keratinocytes, obtained from a newborn suffering from lethal junctional epidermolysis bullosa, were stably transduced with retroviruses carrying a full-length ␤ 4 cDNA or a ␤ 4 cDNA with phenylalanine substitutions at Tyr-1422 and Tyr-1440. Hemidesmosome assembly was evaluated on organotypic skin cultures. ␤ 4 -corrected keratinocytes were indistinguishable from normal cells in terms of ␣ 6 ␤ 4 expression, the localization of hemidesmosome components, and hemidesmosome structure and density, suggesting full genetic and functional correction of ␤ 4 -null keratinocytes. In cultures generated from ␤ 4 Y1422F/Y1440Fkeratinocytes, ␤ 4 mutants as well as ␣ 6 integrin, HD1/ plectin, and BP180 were not concentrated at the dermalepidermal junction. Furthermore, the number of hemidesmosomes was strikingly reduced as compared with ␤ 4 -corrected keratinocytes. The rare hemidesmosomes detected in ␤ 4 Y1422F/Y1440F cells were devoid of subbasal dense plates and of inner cytoplasmic plaques with keratin filament insertion. Collectively, our data demonstrate that the ␤ 4 tyrosine activation motif is not required for the localization of ␣ 6 ␤ 4 at the keratinocyte plasma membrane but is essential for optimal assembly of bona fide hemidesmosomes.Human epidermis consists of a stratified squamous epithelium composed of keratinocytes organized in distinct cellular layers. Keratinocytes forming the basal layer firmly adhere to the basement membrane by means of hemidesmosomes (HDs), 1 multiprotein complexes linking the epithelial intermediate filament network to the dermal anchoring fibrils (see Refs. 1 and 2 for review). HDs are formed by the clustering of several cytoplasmic and trans-membrane proteins (2). The cytoplasmic HD plaque components, which include HD1/plectin (3) and the bullous pemphigoid antigen 1 (BP230) (4), act as linkers for elements of the cytoskeleton at the cytoplasmic surface of plasma membrane. The trans-membrane constituents of HDs, which include the ␣ 6 ␤ 4 integrin (5, 6) and the bullous pemphigoid antigen 2 (BP180) (7), serve as cell receptors connecting the cell interior to extracellular matrix proteins.In particular, the ␣ 6 ␤ 4 integrin is a receptor for the basement membrane component laminin-5, a heterotrimeric protein composed of three distinct polypeptides, ␣3, ␤3, and ␥2, which are encoded by three different genes known as LAMA3, LAMB3, and LAMC2, respectively (see Ref. 8 for review). Laminin-5 binds to the basal keratinocyte cell surface through the ␣ 6 ␤ 4 integrin and tightens the dermal-epidermal junction by binding also to the N-terminal NC-1 domain of type VII collagen (9). The crucial importance of the interaction between laminin-5 and its ␣ 6 ␤ 4 receptor in maintaining the integrity of the i...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Gene Correction of Integrin β4-dependent Pyloric Atresia-Junctional Epidermolysis Bullosa Keratinocytes Establishes a Role for β4 Tyrosines 1422 and 1440 in Hemidesmosome Assembly

Dellambra¹,

Prislei²,

Salvati³

et al. 2001

Journal of Biological Chemistry

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“…Several studies have shown that progenitor epidermal cells are better than differentiated ones in the generation of successful tissue-engineered skin [17,18].…”

Section: Introductionmentioning

confidence: 99%

Dermal Papilla Cells Improve the Wound Healing Process and Generate Hair Bud-Like Structures in Grafted Skin Substitutes Using Hair Follicle Stem Cells

Leirós

Kusinsky

Drago³

et al. 2014

Stem Cells Translational Medicine

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Tissue-engineered skin represents a useful strategy for the treatment of deep skin injuries and might contribute to the understanding of skin regeneration. The use of dermal papilla cells (DPCs) as a dermal component in a permanent composite skin with human hair follicle stem cells (HFSCs) was evaluated by studying the tissue-engineered skin architecture, stem cell persistence, hair regeneration, and graft-take in nude mice. A porcine acellular dermal matrix was seeded with HFSCs alone and with HFSCs plus human DPCs or dermal fibroblasts (DFs). In vitro, the presence of DPCs induced a more regular and multilayered stratified epidermis with more basal p63-positive cells and invaginations. The DPC-containing constructs more accurately mimicked the skin architecture by properly stratifying the differentiating HFSCs and developing a well-ordered epithelia that contributed to more closely recapitulate an artificial human skin. This acellular dermal matrix previously repopulated in vitro with HFSCs and DFs or DPCs as the dermal component was grafted in nude mice. The presence of DPCs in the composite substitute not only favored early neovascularization, good assimilation and remodeling after grafting but also contributed to the neovascular network maturation, which might reduce the inflammation process, resulting in a better healing process, with less scarring and wound contraction. Interestingly, only DPC-containing constructs showed embryonic hair bud-like structures with cells of human origin, presence of precursor epithelial cells, and expression of a hair differentiation marker. Although preliminary, these findings have demonstrated the importance of the presence of DPCs for proper skin repair.

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“…The use of fibrin sealant improves the percentage of skin graft take, and evidence also suggests improved hemostasis and a protective effect resulting in reduced bacterial infection 17 . Cell culture studies have shown that the relative percentage of holoclones, meroclones and paraclones of basal keratinocytes is maintained when keratinocytes are cultivated on fibrin, proving that fibrin does not induce clonal conversion and consequent loss of epidermal stem cells 18 . When they are applied on massive full-thickness burns, the take of keratinocytes is high, reproducible, and permanent 14 .…”

Section: Discussionmentioning

confidence: 99%

“…When they are applied on massive full-thickness burns, the take of keratinocytes is high, reproducible, and permanent 14 . Furthermore, it allows a significant reduction of the costs of cultured autografts and eliminates problems related to handling and transportation 14,18 . However, to obtain these results, it has been emphasized that the presence of a "feeder layer" of lethally irradiated mouse fibroblasts is an absolute requirement 14 ; and because fibrinolysis occurs rapidly as soon as epidermal cells or irradiated 3T3 cells are added 13 , until now there are no long-term evaluations of the in vitro differentiation process in the epidermis reconstituted under this condition.…”

Section: Discussionmentioning

confidence: 99%

Ultrastructural evaluation of human keratinocyte growth and differentiation on a fibrin substrate

Tanikawa¹,

Alonso²,

Herson³

et al. 2010

Acta Cir. Bras.

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Purpose:In order to circumvent several difficulties that have been met in the routine use of the in vitro keratinocyte cultures using the standard procedure described by Rheinwald and Green, and obtain a more resilient and the least possible immunogeneic skin substitute for a future clinical application, this work studied a new keratinocyte culture system, which envisages the utilization of a fibrin substrate in association with high densities of human keratinocytes. Methods: Through light and transmission electron microscopy and immunohistochemical assays, long-term proliferative and differentiative characteristics of keratinocytes cultured onto a fibrin gel under immerse and air-liquid interface culture conditions were evaluated. Results: Despite the absence of a dermal substitute, the results demonstrated that the proposed composite was constituted of a transparent and elastic fibrin film covered by a well-attached, multistratified epithelium with morphological characteristics that resemble human epidermis, including the neoformation, albeit incomplete, of the basement membrane. Conclusions: Increased mechanical resistance due to the presence of an easy handling substrate, the delivery of nonclonfluent keratinocytes as well as the removal of animal-derived cells from the culture system suggest its potential use for future transplantation purposes. Key words: Fibrin tissue adhesive. Keratinocytes. Immunohistochemistry. Microscopy, Electron. RESUMO Objetivo:Com o intuito de contornar diversas dificuldades encontradas no uso rotineiro de queratinócitos cultivados in vitro pela técnica descrita por Rheinwald e Green, e obter um substituto cutâneo mais resistente e o menos imunogênico possível para futuras aplicações clínicas, este trabalho avaliou um novo sistema de cultura de queratinócitos que prevê a utilização de um substrato de fibrina em associação com queratinócitos humanos em alta densidade. Métodos: Através de microscopia óptica e eletrônica e análise imunohistoquímica, foram avaliadas as características proliferativas e de diferenciação em longo prazo de queratinócitos cultivados em condição imersa e na interface ar-líquido. Resultados: Apesar da ausência de um substituto dérmico, foi demonstrado que o composto proposto constituiu-se de um substrato de fibrina transparente e elástico coberto por epitélio multi-estratificado, bem aderido, com características morfológicas semelhantes à epiderme humana, incluindo a neo-formação, embora incompleta, da membrana basal. Conclusões: A maior resistência mecânica com a presença de um substrato de fácil manuseio, a possível liberação de queratinócitos não-confluentes, e a remoção de células com origem animal dos sistemas de cultura sugerem que o composto proposto neste estudo apresenta grande potencial para uso clínico futuro. Descritores: Adesivo tecidual de fibrina. Queratinócitos. Imunoistoquímica. Microscopia Eletrônica.

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The Control of Epidermal Stem Cells (Holoclones) in the Treatment of Massive Full-Thickness Burns With Autologous Keratinocytes Cultured on Fibrin1

Cited by 337 publications

References 52 publications

Gene Correction of Integrin β4-dependent Pyloric Atresia-Junctional Epidermolysis Bullosa Keratinocytes Establishes a Role for β4 Tyrosines 1422 and 1440 in Hemidesmosome Assembly

Gene Correction of Integrin β4-dependent Pyloric Atresia-Junctional Epidermolysis Bullosa Keratinocytes Establishes a Role for β4 Tyrosines 1422 and 1440 in Hemidesmosome Assembly

Dermal Papilla Cells Improve the Wound Healing Process and Generate Hair Bud-Like Structures in Grafted Skin Substitutes Using Hair Follicle Stem Cells

Ultrastructural evaluation of human keratinocyte growth and differentiation on a fibrin substrate

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