Iterative Three-Dimensional Epidermis Bioengineering and Xenografting to Assess Long-Term Regenerative Potential in Human Keratinocyte Precursor Cells.

The functional definition of somatic adult stem cells is based on their regenerative capacity, which allows tissue regeneration throughout life. Thus, refining methodologies to characterize this capacity is of great importance for progress in the fundamental knowledge of specific keratinocyte subpopulations but also for preclinical and clinical research, considering the high potential of keratinocytes in cell therapy. We present here a methodology which we define as iterative xenografting, which originates in the classical model of human skin substitute xenografts onto immunodeficient recipient mice. The principle of this functional assay is first to perform primary xenografts to assess graft take and the quality of epidermal differentiation. Then, human keratinocytes are extracted from primary graft samples to perform secondary xenografts, to assess the presence and preservation of functional keratinocyte stem cells with long-term regenerative potential. In the example of experiments shown, iterative skin xenografting was used to document the high regenerative potential of epidermal holoclone keratinocytes.

KLF4 inhibition promotes the expansion of keratinocyte precursors from adult human skin and of embryonic-stem-cell-derived keratinocytes.

Expanded autologous skin keratinocytes are currently used in cutaneous cell therapy, and embryonic-stem-cell-derived keratinocytes could become a complementary alternative. Regardless of keratinocyte provenance, for efficient therapy it is necessary to preserve immature keratinocyte precursors during cell expansion and graft processing. Here, we show that stable and transient downregulation of the transcription factor Krüppel-like factor 4 (KLF4) in keratinocyte precursors from adult skin, using anti-KLF4 RNA interference or kenpaullone, promotes keratinocyte immaturity and keratinocyte self-renewal in vitro, and enhances the capacity for epidermal regeneration in mice. Both stable and transient KLF4 downregulation had no impact on the genomic integrity of adult keratinocytes. Moreover, transient KLF4 downregulation in human-embryonic-stem-cell-derived keratinocytes increased the efficiency of skin-orientated differentiation and of keratinocyte immaturity, and was associated with improved generation of epidermis. As a regulator of the cell fate of keratinocyte precursors, KLF4 could be used for promoting the ex vivo expansion and maintenance of functional immature keratinocyte precursors.

Monitoring the cycling activity of cultured human keratinocytes using a CFSE-based dye tracking approach.

The development of methods and tools suitable for functional analysis of keratinocytes placed in an in vitro context is of great importance for characterizing properties associated with their normal state, for detecting abnormalities related to pathological states, or for studying the effects of extrinsic factors. In the present chapter, we describe the use of the intracellular fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) to monitor cell division in mass cultures of normal human keratinocytes. We detail the preparation of CFSE-labeled keratinocyte samples and the identification by flow cytometry of cell subpopulations exhibiting different cycling rates in a mitogenic culture context. In addition, we show that the CFSE-based division-tracking approach enables the monitoring of keratinocyte responsiveness to growth modulators, which is here exemplified by the cell-cycling inhibition mediated by the growth factor TGF-ß1. Finally, we show that keratinocyte subpopulations, separated according to their mitotic history using CFSE fluorescence tracking, can be sorted by flow cytometry and used for further functional characterization, including determination of clone-forming efficiency.

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

The regenerative capacity of human interfollicular epidermis is closely linked to the potential of immature keratinocytes present within its basal layer. The availability of selection methods and culture systems allowing precise assessment of basal keratinocyte characteristics is critical for increasing our knowledge of this cellular compartment. This report presents a multi-parametric comparative study of basal keratinocytes selected according to two different principles: 1) high adhesion capacity on a type-I collagen-coated substrate [Adh⁺⁺⁺], 2) high cell-surface expression of α6-integrin [Itg-α6 (high)]. Importantly, analysis performed at the single-cell level revealed similar primary clone-forming efficiency values of 45.5% ±â€Š6.7% [Itg-α6(high)] and 43.7% ±â€Š7.4% [Adh⁺⁺⁺], which were markedly higher than those previously reported. In addition, both methods selected keratinocytes exhibiting an extensive long-term growth potential exceeding 100 cell doublings and the capacity for generating a pluristratified epidermis. Our study also included a global transcriptome comparison. Genome-wide profiling indicated a strong similarity between [Adh⁺⁺⁺] and [Itg-α6(high)] keratinocytes, and revealed a common basal-associated transcriptional signature. In summary, cross-analysis of [Adh⁺⁺⁺] and [Itg-α6(high)] keratinocyte characteristics showed that these criteria identified highly equivalent cellular populations, both characterized by unexpectedly high growth capacities. These results may have broad impacts in the tissue engineering and cell therapy fields.

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.

Functional investigations of keratinocyte stem cells and progenitors at a single-cell level using multiparallel clonal microcultures.

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.