Cellular adhesion on collagen: a simple method to select human basal keratinocytes which preserves their high growth capacity

Fortunel, Nicolas O.; Chadli, Loubna; Bourreau, Emilie; Cadio, Emmanuelle; Vaigot, Pierre; Marie, Mélanie; Deshayes, Nathalie; Rathman-Josserand, Michelle; Leclaire, Jacques; Martin, Michèle

doi:10.1684/ejd.2011.1268

Cited by 8 publications

(10 citation statements)

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“…However, despite optimization of the rapid adhesion method, CSFC leads to a better enrichment of KSC since the CFE is five times increased versus total cell suspension, whereas it is only 1.4 times for the adhesion method. These results are consistent with those observed by Kaur, who showed that cells adhered regardless of the coating (collagen I, IV and keratinocyte-secreted extra cellular matrix) and the adhesion time (from 5 to 20 min) actually corresponds to a whole α6 high population [30], as confirmed latter by Fortunel et al, who demonstrated that cells having high adhesion capacity on a type I collagen and cells with high expression of α6 integrin showed high convergence at the functional and molecular levels [21]. Our results also confirm that several markers are necessary for qualifying isolated cells as stem cells.…”

Section: Discussionmentioning

confidence: 81%

“…However, techniques using one or several basal cell markers have been proposed to enrich keratinocyte population in basal cells (KSC and TA). For example, integrin α6-antibody coupled to fluorochrome or to magnetic beads allows the isolation of positive cells by flow cytometry [21] or under a magnetic field, respectively. Combination of β1-integrin and Rhodamine 123A [22], as well as β1-integrin and Desmoglein 3 [10], or β1-integrin and K1/K10 [23], would allow isolation of epidermal stem cells candidate.…”

Section: Introductionmentioning

confidence: 99%

“…However, even if several authors have used this technique for studying KSC [14,25,26,27,28,29], the stem cell characteristics of these rapid adherent (RA) cells is still not proved. Currently, it has only been shown that RA cells are similar to α6+ [21] and β1+ [14] cells.…”

Section: Introductionmentioning

confidence: 99%

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α6 Integrin (α6high)/Transferrin Receptor (CD71)low Keratinocyte Stem Cells Are More Potent for Generating Reconstructed Skin Epidermis Than Rapid Adherent Cells

Metral

Bêchetoille²,

Demarne³

et al. 2017

IJMS

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The epidermis basal layer is composed of two keratinocyte populations: Keratinocyte Stem cells (KSC) and Transitory Amplifying (TA) cells that arise from KSC division. Unfortunately, no specific marker exists to differ between KSC and TA cells. Here, we aimed at comparing two different methods that pretended to isolate these two populations: (i) the rapid adhesion method on coated substrate and (ii) the flow cytometry method, which is based on the difference in cell surface expressions of the α6 integrin and transferrin receptor (CD71). Then, we compared different parameters that are known to discriminate KSC and TA populations. Interestingly, we showed that both methods allow enrichment in stem cells. However, cell sorting by flow cytometry (α6high/CD71low) phenotype leads to a better enrichment of KSC since the colony forming efficiency is five times increased versus total cell suspension, whereas it is only 1.4 times for the adhesion method. Moreover, α6high/CD71low cells give rise to a thicker pluristratified epithelium with lower seeding density and display a low Ki67 positive cells number, showing that they have reached the balance between proliferation and differentiation. We clearly demonstrated that cells isolated by a rapid adherent method are not the same population as KSC isolated by flow cytometry following α6high/CD71low phenotype.

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Section: Discussionmentioning

confidence: 81%

Section: Introductionmentioning

confidence: 99%

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α6 Integrin (α6high)/Transferrin Receptor (CD71)low Keratinocyte Stem Cells Are More Potent for Generating Reconstructed Skin Epidermis Than Rapid Adherent Cells

Metral

Bêchetoille²,

Demarne³

et al. 2017

IJMS

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“…This method, although not specific for stem cells, has been previously been demonstrated to reproducibly isolate a keratinocyte population with high clonogenic potential, extensive long term growth capacity (exceeding 100 cell doublings) and the ability to generate a pluristratified epidermis. Transcriptomic profiling studies have additionally shown a strong similarity of this population with the cell population selected for high surface expression of integrin α6, a marker often associated with epithelial stem cells 14. The cells were then resuspended in a DMEM Hams F12 medium (3:1 mixture) containing 2 mM L-glutamine and 1 mM sodium pyruvate (all from Gibco-Life Technologies SAS) and supplemented with 10 fetal calf serum (HyClone-Fisher Scientific SAS, Illkirch, France), nonessential amino acids (Gibco-Life Technologies SAS, Saint Aubin, France), 5 mg/ml insulin, 0.18 mM adenine, 0.4 mg/mL hydrocortisone, 2 nm tri-iodothyronine (all from Sigma-Aldrich Chimie S.a.r.l, Lyon, France), 10 ng/mL epidermal growth factor (Chemicon, Temecula, CA), 1 mM isoproterenol (Sigma-Aldrich Chimie S.a.r.l), 5 mg/mL transferin (Sigma-Aldrich Chimie S.a.r.l), 4 mM glutamine (Gibco-Life Technologies SAS), and 50 U/mL penicillin/streptomycin (Gibco-Life Technologies SAS) and seeded at very low densities (1000 cells per Petri dish) on a feeder-layer of irradiated 3T3 fibroblasts.…”

Section: Methods and Resultsmentioning

confidence: 98%

“…After extraction of the total keratinocyte population, a cell fraction highly enriched in stem and progenitor cells was selected based on the capacity of such cells to adhere very quickly to an extracellular matrix 12–14. This method, although not specific for stem cells, has been previously been demonstrated to reproducibly isolate a keratinocyte population with high clonogenic potential, extensive long term growth capacity (exceeding 100 cell doublings) and the ability to generate a pluristratified epidermis.…”

Section: Methods and Resultsmentioning

confidence: 99%

Evolution of the clonogenic potential of human epidermal stem/progenitor cells with age

Zobiri

Deshayes²,

Rathman-Josserand³

2012

SCCAA

Self Cite

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A number of clinical observations have indicated that the regenerative potential and overall function of the epidermis is modified with age. The epidermis becomes thinner, repairs itself less efficiently after wounding, and presents modified barrier function recovery. In addition, the dermal papillae flatten out with increasing age, suggesting a modification in the interaction between epidermal and dermal compartments. As the epidermal regenerative capacity is dependent upon stem and progenitor cell function, it is naturally of interest to identify and understand age-related changes in these particular keratinocyte populations. Previous studies have indicated that the number of stem cells does not decrease with age in mouse models but little solid evidence is currently available concerning human skin. The objective of this study was to evaluate the clonogenic potential of keratinocyte populations isolated from the epidermis of over 50 human donors ranging from 18 to 71 years old. The data indicate that the number of epidermal cells presenting high regenerative potential does not dramatically decline with age in human skin. The authors believe that changes in the microenvironment controlling epidermal basal cell activity are more likely to explain the differences in epidermal function observed with increasing age.

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Influence of fibrin matrices and their released factors on epidermal substitute phenotype and engraftment

Alexaline

Magne

Rodríguez

et al. 2019

J Tissue Eng Regen Med

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SUMMARY Cultured epithelial autografts (CEAs) represent a life‐saving surgical technique for full‐thickness skin burns covering more than 60% total body surface area. However, CEAs present numerous drawbacks leading to heavy cosmetic and functional sequelae. In our previous study, we showed that human plasma‐based fibrin matrices (hPBM) could improve the reparative potential of CEAs. Therefore, in the present work, we sought to investigate the role of hPBM compared with fibrin from purified fibrinogen (FPF) or plastic support on epidermal substitute formation and engraftment. The use of hPBM for epidermal substitute culture improved keratinocyte migration, proliferation, and epidermal substitute organization to a better extent than FPF in vitro. Both fibrin matrices favored greater dermal–epidermal junction protein deposition and prevented their degradation. Keratinocyte differentiation was also decreased using both fibrin matrices. Basement membrane protein deposition was mainly influenced by matrix whereas growth factors released from fibrin especially by hPBM were shown to enhance in vitro keratinocyte migration, proliferation, and epidermal substitute organization. Ultimately, epidermal substitutes grown on hPBM displayed better engraftment rates than those cultured on FPF or on plastic support in a NOD‐SCID model of acute wound with the formation of a functional dermal–epidermal junction. Together, these results show the positive impact of fibrin matrices and their released growth factor on epidermal substitute phenotype and grafting efficiency. Fibrin matrices, and especially hPBM, may therefore be of interest to favor the treatment of full‐thickness burn patients.

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Cellular adhesion on collagen: a simple method to select human basal keratinocytes which preserves their high growth capacity

Cited by 8 publications

References 0 publications

α6 Integrin (α6high)/Transferrin Receptor (CD71)low Keratinocyte Stem Cells Are More Potent for Generating Reconstructed Skin Epidermis Than Rapid Adherent Cells

α6 Integrin (α6high)/Transferrin Receptor (CD71)low Keratinocyte Stem Cells Are More Potent for Generating Reconstructed Skin Epidermis Than Rapid Adherent Cells

Evolution of the clonogenic potential of human epidermal stem/progenitor cells with age

Influence of fibrin matrices and their released factors on epidermal substitute phenotype and engraftment

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