International audienc

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-a6 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 · 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.Key words: a6-integrin -flow cytometry -human keratinocytesparallel clonal cultures -progenitors -stem cells -tissue reconstructionPlease cite this paper as: Exploration of the functional hierarchy of the basal layer of human epidermis at the single-cell level using parallel clonal microcultures of keratinocytes.

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The basal layer of human interfollicular epidermis is thought to contain a minor compartment of quiescent or slowly cycling epithelial stem cells. These primitive keratinocytes give rise to the progenitors, which are the proliferating keratinocytes and which can be defined as early to late progenitors, according to their differentiation status. Because of the intrinsic heterogeneity of the basal layer, the development of new methods suitable for functional analysis of basal keratinocytes directly isolated from skin samples is greatly needed. We describe here a new method that allows a rapid and multiparallel deposition of single keratinocytes into 96-well plates, using flow cytometry. The first step of the process allows the clonal analysis of the growth potential of freshly isolated epithelial cells in primary cultures. In a second step, various techniques of functional characterization can be performed on the progeny of the cloned cell, including the generation of reconstructed epidermis, colony assays, and secondary cloning. In a third step, a long-term characterization of the progeny of the cloned keratinocytes can be performed, either by successive subclonings or mass expansion cultures.