Nicolas O. Fortunel

Fibroblast Growth Factor Type 2 Signaling Is Critical for DNA Repair in Human Keratinocyte Stem Cells

Tissue stem cells must be endowed with superior maintenance and repair systems to ensure genomic stability over multiple generations, which would be less necessary in more differentiated cells. We previously reported that human keratinocyte stem cells were more resistant to ionizing radiation toxicity than their direct progeny, the keratinocyte progenitor cells. In the present study we addressed the mechanisms underlying this difference. Investigations of DNA repair showed that both single and double DNA strand breaks were repaired more rapidly and more efficiently in stem cells than in progenitors. As cell signaling is a key regulatory step in the management of DNA damage, a gene profiling study was performed. Data revealed that several genes of the fibroblast growth factor type 2 (FGF2) signaling pathway were induced by DNA damage in stem cells and not in progenitors. Furthermore, an increased content of the FGF2 protein was found in irradiated stem cells, both for the secreted and the cellular forms of the protein. To examine the role of endogenous FGF2 in DNA repair, stem cells were exposed to FGF2 pathway inhibitors. Blocking the FGF2 receptor (FGF receptor 1) or the kinase (Ras‐mitogen‐activated protein kinase 1) resulted in a inhibition of single and double DNA strand‐break repair in the keratinocyte stem cells. Moreover, supplementing the progenitor cells with exogenous FGF2 activated their DNA repair. We propose that, apart from its well‐known role as a strong mitogen and prosurvival factor, FGF2 helps to maintain genomic integrity in stem cells by activating stress‐induced DNA repair. STEM CELLS 2010; 28:1639–1648.

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

Please 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. Experimental Dermatology 2010.

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% ± 6.7% [Itg-α6(high)] and 43.7% ± 7.4% [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.

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.

Bioengineering a Human Plasma-Based Epidermal Substitute With Efficient Grafting Capacity and High Content in Clonogenic Cells

Cultured epithelial autografts (CEAs) produced from a small, healthy skin biopsy represent a lifesaving surgical technique in cases of full‐thickness skin burn covering >50% of total body surface area. CEAs also present numerous drawbacks, among them the use of animal proteins and cells, the high fragility of keratinocyte sheets, and the immaturity of the dermal‐epidermal junction, leading to heavy cosmetic and functional sequelae. To overcome these weaknesses, we developed a human plasma‐based epidermal substitute (hPBES) for epidermal coverage in cases of massive burn, as an alternative to traditional CEA, and set up critical quality controls for preclinical and clinical studies. In this study, phenotypical analyses in conjunction with functional assays (clonal analysis, long‐term culture, or in vivo graft) showed that our new substitute fulfills the biological requirements for epidermal regeneration. hPBES keratinocytes showed high potential for cell proliferation and subsequent differentiation similar to healthy skin compared with a well‐known reference material, as ascertained by a combination of quality controls. This work highlights the importance of integrating relevant multiparameter quality controls into the bioengineering of new skin substitutes before they reach clinical development.

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-β1. 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 organization of the human epidermal basal layer: Clues sustaining a hierarchical model

Abstract Purpose: The basal layer of adult interfollicular epidermis is a highly dynamic cellular system, ensuring the continuous physiological renewal of this tissue, as well as regenerative processes in the context of wound healing. In human skin, despite its major importance for the maintenance of epidermal homeostasis and regenerative processes, the functional organization of basal keratinocytes is still debated today. Progress in this understanding is closely linked to the development of research models enabling investigations of the different coexisting basal keratinocyte subpopulations, to address their specific functional and molecular characteristics, particularly through clonal analyses. We review here different strategies that have led to significant advances in the knowledge of human basal keratinocyte properties, at both phenotypic and functional levels. Conclusions: Convincing clues supporting a hierarchical organization of the keratinocyte basal layer in humans have emerged from the different functional studies. In particular, the hierarchical model constitutes a straight forward interpretation of the clearly non-equivalent potentialities observed when basal keratinocytes were studied individually in a cell culture context.

Quantitative Detection of Low-Abundance Transcripts at Single-Cell Level in Human Epidermal Keratinocytes by Digital Droplet Reverse Transcription-Polymerase Chain Reaction

Genetic and epigenetic characterization of the large cellular diversity observed within tissues is essential to understanding the molecular networks that ensure the regulation of homeostasis, repair, and regeneration, but also pathophysiological processes. Skin is composed of multiple cell lineages and is therefore fully concerned by this complexity. Even within one particular lineage, such as epidermal keratinocytes, different immaturity statuses or differentiation stages are represented, which are still incompletely characterized. Accordingly, there is presently great demand for methods and technologies enabling molecular investigation at single-cell level. Also, most current methods used to analyze gene expression at RNA level, such as RT-qPCR, do not directly provide quantitative data, but rather comparative ratios between two conditions. A second important need in skin biology is thus to determine the number of RNA molecules in a given cell sample. Here, we describe a workflow that we have set up to meet these specific needs, by means of transcript quantification in cellular micro-samples using flow cytometry sorting and reverse transcription-digital droplet polymerase chain reaction. As a proof-of-principle, the workflow was tested for the detection of transcription factor transcripts expressed at low levels in keratinocyte precursor cells. A linear correlation was found between quantification values and keratinocyte input numbers in a low quantity range from 40 cells to 1 cell. Interpretable signals were repeatedly obtained from single-cell samples corresponding to estimated expression levels as low as 10-20 transcript copies per keratinocyte or less. The present workflow may have broad applications for the detection and quantification of low-abundance nucleic acid species in single cells, opening up perspectives for the study of cell-to-cell genetic and molecular heterogeneity. Interestingly, the process described here does not require internal references such as house-keeping gene expression, as it is initiated with defined cell numbers, precisely sorted by flow cytometry.

Skin physiology: crossroads for a multidisciplinary science

ejd.2011.1305 Auteur(s) : Nicolas O FORTUNEL nicolas.fortunel@cea.fr Alternative Energies and Atomic Energy Commission (CEA), Institute of Cellular and Molecular Radiobiology (IRCM), Laboratory of Genomics and Radiobiology of Keratinopoiesis (LGRK), 2 rue Gaston Cremieux, CP 5722, 91057 Evry Cedex, France Skin homeostasis results from a complex network of interactions, which involves different tissue compartments with specific cell lineage contents and three-dimensional architectures. Consequently, the [...]