Exploration of the functional hierarchy of the basal layer of human epidermis at the single-cell level using parallel clonal microcultures of keratinocytes

摘要: The basal layer of human epidermis contains both stem cells and keratinocyte progenitors. Because this cellular heterogeneity, the development methods suitable for investigations at a clonal level is dramatically needed. Here, we describe new method that allows multi-parallel cultures keratinocytes. Immediately after extraction from tissue samples, are sorted by flow cytometry based on their high integrin-alpha 6 expression plated individually in microculture wells. This automated cell deposition process enables large-scale characterization primary clonogenic capacities. resulting growth profile provided precise assessment hierarchy, as size distribution 14-day-old clones ranged abortive to highly proliferative containing 1.7 x 10(5) keratinocytes (17.4 doublings). Importantly, these could be used generate three-dimensional reconstructed with progeny single cell. In long-term cultures, fraction sustain extensive expansion >100 population doublings over 14 weeks exhibited reconstruction potency, thus fulfilling candidate functional criteria. summary, parallel microcultures provide relevant model single-cell studies interfollicular keratinocytes, which also other epithelial models, including hair follicle cornea. data obtained using system support hierarchical organization epidermis.