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

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-alpha 6 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 x 10(5) 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.