Exploration of the functional hierarchy of the basal layer of human epidermis at the single-cell level using parallel clonal microcultures of keratinocytes
| Autor: | Nicolas O. Fortunel, Stephan Bouet, Emmanuelle Cadio, Paul-Henri Romeo, Loubna Chadli, Pierre Vaigot, Michèle T. Martin, Sandra Moratille |
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| Přispěvatelé: | Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de radiobiologie et d'étude du génome (LREG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA), This work was supported by research grants from the Comité de Radioprotection d’Electricité de France (EDF) and from ANR (BIRAD, n° 05961)., Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA) |
| Rok vydání: | 2010 |
| Předmět: |
Keratinocytes
[SDV]Life Sciences [q-bio] Cellular differentiation Population Cell Culture Techniques Cell Count Dermatology Integrin alpha6 Biology Biochemistry 03 medical and health sciences 0302 clinical medicine Chromosomal Instability medicine Chromosomes Human Humans Progenitor cell education Clonogenic assay Molecular Biology Cell Proliferation 030304 developmental biology Comparative Genomic Hybridization 0303 health sciences education.field_of_study Tissue Engineering human keratinocyte Epidermis (botany) Integrin beta1 Stem Cells flow cytometry Cell Differentiation progenitor α6 integrin Hair follicle Clone Cells Cell biology stem cell parallel clonal culture medicine.anatomical_structure Epidermal Cells tissue reconstruction 030220 oncology & carcinogenesis Immunology Stem cell Keratinocyte |
| Zdroj: | Experimental Dermatology Experimental Dermatology, Wiley, 2010, 19 (4), pp.387-392. ⟨10.1111/j.1600-0625.2009.01046.x⟩ Experimental Dermatology, 2010, 19 (4), pp.387-392. ⟨10.1111/j.1600-0625.2009.01046.x⟩ |
| ISSN: | 1600-0625 0906-6705 |
| DOI: | 10.1111/j.1600-0625.2009.01046.x |
| Popis: | Chantier qualité GA; International audience; The basal layer of human epidermis contains both stem cells and keratinocyte progenitors. Because of this cellular heterogeneity, the development of methods suitable for investigations at a clonal level is dramatically needed. Here, we describe a new method that allows multi-parallel clonal cultures of basal keratinocytes. Immediately after extraction from tissue samples, cells are sorted by flow cytometry based on their high integrin-α6 expression and plated individually in microculture wells. This automated cell deposition process enables large-scale characterization of primary clonogenic capacities. The resulting clonal growth profile provided a precise assessment of basal keratinocyte hierarchy, as the size distribution of 14-day-old clones ranged from abortive to highly proliferative clones containing 1.7 × 105 keratinocytes (17.4 cell doublings). Importantly, these 14-day-old primary clones could be used to generate three-dimensional reconstructed epidermis with the progeny of a single cell. In long-term cultures, a fraction of highly proliferative clones could sustain extensive expansion of >100 population doublings over 14 weeks and exhibited long-term epidermis reconstruction potency, thus fulfilling candidate stem cell functional criteria. In summary, parallel clonal microcultures provide a relevant model for single-cell studies on interfollicular keratinocytes, which could be also used in other epithelial models, including hair follicle and cornea. The data obtained using this system support the hierarchical model of basal keratinocyte organization in human interfollicular epidermis. |
| Databáze: | OpenAIRE |
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