Reconstruction of a Human Skin Equivalent Using a Spontaneously Transformed Keratinocyte Cell Line (HaCaT)

Boelsma, Esther; Verhoeven, Mary C.H.; Ponec, Maria

doi:10.1046/j.1523-1747.1999.00545.x

Cited by 121 publications

(103 citation statements)

References 48 publications

(58 reference statements)

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“…In organotypic co-cultures with fibroblasts, conditions that induce normal keratinocytes to form stratified epithelia with all features of a normal epidermis (Smola et al, 1998;Stark et al, 1999), HaCaT cells have been described to be deficient in this complex function of tissue regeneration (Haake and Polakowska, 1993;Syrjänen et al, 1996;Steinsträsser et al, 1997;Boelsma et al, 1999). We have been able to improve their epidermal tissue formation and differentiation in this model by intensifying stromal influences by elevating the number of supporting fibroblasts (Schoop et al, 1999).…”

Section: Discussionmentioning

confidence: 79%

“…This is best demonstrated with the HaCaT cell line, a non-tumorigenic keratinocyte population derived from adult trunk skin exhibiting a rather normal differentiation capacity despite multiple chromosomal alterations (Boukamp et al, 1988;Boukamp et al, 1997;Breitkreutz et al, 1997;Breitkreutz et al, 1998;Ryle et al, 1989). However, organotypic co-cultures of HaCaT cells with skin fibroblasts exhibit some stratification but usually lack typical criteria of an ordered structure and regular keratinization (Haake and Polakowska, 1993;Syrjänen et al, 1996;Steinsträsser et al, 1997;Boelsma et al, 1999). This deficiency, however, was not due to the permanent loss of essential differentiation functions.…”

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confidence: 99%

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Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGF-α

Maas‐Szabowski

Stärker

Fusenig

2003

Journal of Cell Science

117

114

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The human keratinocyte cell line HaCaT expresses essentially all epidermal differentiation markers but exhibits deficiencies in tissue organization as surface transplants in nude mice and even more so in organotypic co-cultures with fibroblasts. Whereas tissue differentiation by normal keratinocytes(NEKs) is regulated by stromal interactions, this mechanism is impaired in HaCaT cells. This regulatory process is initiated by interleukin-1 (IL-1)release in keratinocytes, which induces expression of keratinocyte growth factor (KGF/FGF-7) and granulocyte macrophage-colony stimulating factor(GM-CSF) in fibroblasts. Production and release of IL-1 is very low and,consequently, expression of the fibroblast-derived growth factors KGF/FGF-7 and GM-CSF is absent in HaCaT-fibroblast co-cultures. However, addition of KGF and GMCSF, respectively, is inefficient to improve stratification and differentiation by HaCaT cells due to the low expression of their cognate receptors. More importantly, expression and release of the autocrine keratinocyte growth factor TGF-α is dramatically decreased in HaCaT cells. Addition of TGF- α or EGF stimulated HaCaT cell proliferation but, even more effectively, suppressed apoptosis, thus facilitating the formation of a regularly stratified epithelium. Furthermore, TGF-αenhanced the expression of the receptors for KGF and GM-CSF so that addition of these growth factors, or of their inducer IL-1, further improved epidermal tissue differentiation leading to in vitro skin equivalents comparable with cultures of NEKs. Thus, supplementing TGF-α normalized epidermal tissue regeneration by immortal HaCaT keratinocytes and their interaction with stromal cells so that regular skin equivalents are produced as standardized in vitro models.

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Section: Discussionmentioning

confidence: 79%

mentioning

confidence: 99%

Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGF-α

Maas‐Szabowski

Stärker

Fusenig

2003

Journal of Cell Science

117

114

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“…Involucrin is a marker of the epidermis and is only expressed in the suprabasal layers of stratified squamous epithelium [29]. K6 is an epidermal marker of hyperproliferation and is expressed in the suprabasal layers of non-keratinized squamous epithelium [30].…”

Section: Characterization and Differentiation Of Hfscsmentioning

confidence: 99%

A simple culture method for epithelial stem cells derived from human hair follicle

et al. 2013

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The challenge arises among researchers when hair follicle stem cells (HFSCs) derived from a human hair follicle remain poorly expanded in defined culture medium. In this study, we isolated the HFSCs and they became confluent after 10 days of cultivation. Comparing the viability of HFSCs cultured in defined keratinocytes serum free medium (KSFM) in a coated plate and CnT07 medium in an uncoated plate, the number of HFSCs cultured in CnT07 was significantly higher at days 2, 4, 6 and 8 (P=0.004). The population doubling time of HFSCs was 21.48±0.44 hours in non-coated plates with CnT07 and 30.73±0.75 hours in coated plates with KSFM. Our primary HFSC cultures were positive for CD200 and K15 with brownish color. Flow cytometry analysis showed that the percentage of HFSCs expressing CD200 and K15 were 65.20±3.16 and 72.07±6.62 respectively. After reaching 100% confluence, the HFSCs were differentiated into an epidermal layer in vitro using CnT02-3D defined media. HFSCs were differentiated into an epidermal layer after 2 weeks of induction. Involucrin- and K6-positive cells were detected in the differentiated epidermal layer. This method is a simple technique for HFSC isolation and has a lower cost of processing and labor, and it represents a promising tool for skin tissue engineering.

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“…Keratinocytes can be obtained from different sites of the oral cavity, such as the hard palate [9], gingiva [10] or buccal mucosa [11]. Normal human keratinocytes should also be used at very early passages, but immortalized human keratinocytes, such as HaCaT cells [12] or TR146 cells [13], can be used at extended passages in the reconstruction of oral mucosal test models. However, epidermal differentiation of transformed keratinocytes is not perfect, and tumor derived cells are abnormal and not suitable for clinical use [1].…”

Section: Discussionmentioning

confidence: 99%

Medium-term Culture of Normal Human Oral Mucosa: A Novel Three-dimensional Model to Study the Effectiveness of Drugs Administration

Bucchieri

Fucarino²,

Gammazza³

et al. 2012

CPD

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Tissue-engineered oral mucosal equivalents have been developed for in vitro studies for a few years now. However, the usefulness of currently available models is still limited by many factors, mainly the lack of a physiological extracellular matrix (ECM) and the use of cell populations that do not reflect the properly differentiated cytotypes of the mucosa of the oral cavity. For this reason, we have developed a novel three-dimensional culture model reflecting the normal architecture of the human oral mucosa, with the main aim of creating a better in vitro model where to test cellular responses to drugs administration. This novel 3D cell culture model (3D outgrowth) was set up using an artificial extracellular matrix (Matrigel TM ), allowing the interactions required for proper differentiation of the various citotypes which form the mucosal layer. Biopsies of human oral mucosa, in fragments of about 0.5 mm 3 , were placed onto 6.5mm Transwells, covered with Matrigel TM and grown in a specific culture medium. A gradual formation of an architectural structure similar to that of the in vivo oral mucosa was observed. Transmission electron and confocal microscopy were employed to characterize the newly developed model: the cell components (keratinocytes and fibroblasts) differentiated properly within the outgrowth and reconstituted, in vitro, the physiological structure of the human oral mucosa, including a stratified non-keratinized squamous layer composed of four different layers, a proper basal membrane and a lamina propria where fibroblasts produce ECM. Moreover, keratinocytes expressed CK5, CK13, CK19 and E-cadherin, whereas fibroblasts expressed collagen type I and IV, laminin and fibronectin. 3D outgrowths could be considered a valid alternative to animal models, and provide useful information for researchers interested in studying the responses of the human oral mucosa to locally delivered drugs or other exogenous treatments.

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Reconstruction of a Human Skin Equivalent Using a Spontaneously Transformed Keratinocyte Cell Line (HaCaT)

Cited by 121 publications

References 48 publications

Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGF-α

Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGF-α

A simple culture method for epithelial stem cells derived from human hair follicle

Medium-term Culture of Normal Human Oral Mucosa: A Novel Three-dimensional Model to Study the Effectiveness of Drugs Administration

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