Christine Baldeschi: Assistant Professor (UEVE)
Gilles Lemaître: Research engineer (UEVE)
Nathalie Holic-Barlet : Assistant Professor (UEVE)
Manoubia Saidani: Qualified research technician (CECS)
Sophie Domingues: Engineer (CECS)
Annabelle Darle : Engineer (CECS)
Aims and background:
Genodermatoses are characterized by genetic disorders of the skin affecting mainly either keratinocytes or melanocytes the two majors cell types of the epidermis.
Human epidermis has been produced in vitro for decades using adult epidermal stem cells from donors to provide cell therapy. However, its use is hampered by the difficulty in supplying the amount of epidermal substitute needed in order to extend the application to all patients who may benefit from it. Derivation of pluripotent stem cells, either of embryonic origin or following genetic reprogramming, has opened the path for an alternative source for epidermal cell therapy as these cells are both immortal and pluripotent, theoretically capable of providing any requested number of cells of any desired phenotype. For this purpose, we developed protocols allowing to generate homogenous and functional populations of keratinocytes and melanocytes derived from pluripotent stem cells.
In parallel to this cell therapy application, keratynocytes and melanocytes derived from pluripotent stem cells will be used for pathological modelling of genodermatoses allowing identification of new disease-specific pharmacological treatments
Strategy, means and methods:
Homogenous and functional population of keratinocytes and melanocytes derived from pluripotent stem cells were generated by designing a multi-step sequential protocol respecting the chronobiology of epidermis formation during human ontogenesis.
For keratinocytes derivation, pluripotent stem cells were seeded onto a feeder layer in a medium supplemented by a high concentration of BMP4 during 40 days. After this period of induction, keratinocytes derived from pluripotent stem cells were isolated and amplified to obtain a homogenous and pure population of keratinocytes presenting all the phenotypic characteristic of adult keratinocytes. Once seeded onto a proper support, these keratinocytes appeared capable of forming a pluristratified epidermis both in vitro and in vivo as xenografts in immunodeficient mice. Human epidermis was shown to persist for at least three months, i.e. three full renewal cycles, displaying normal human skin characteristics.
In parallel, derivation of melanocytes from pluripotent stem cells was performed by modulating concentration of BMP4 in the same experimental scheme. Once isolated and amplified melanocytes derived from pluripotent stem cells exhibited all characteristic features of their adult counterpart. This includes the enzymatic machinery required for production and functional delivery of melanosomes to keratinocytes. In addition, melanocytes integrated appropriately in organotypic epidermis reconstructed in vitro. Treatment of melanised epidermis with alpha-melanocyte stimulating hormone (αMSH), a physiological agent that controls skin pigmentation, activated the production of melanin in the tissue.
Results and future prospects:
Our ability to obtain functional pluristratified epidermis from pluripotent stem cells opens the way to epidermis cell therapy in the treatment of chronic and acute wounds. This project is presented in the Pioneer Advanced Cell therapy of the Epidermis (PACE) booklet. These technical developments will allow also the establishment of pathological modelling of the epidermolysis bullosa disorders.
The availability of human pluripotent stem cells committed to the melanocytic lineage in vitro, at all stages of differentiation, will be used to decipher the mechanisms that guide developmental processes. In parallel, the generation of functional melanocytes from pluripotent stem cells, capable of producing melanosomes and transferring them to keratinocytes, will facilitate in vitro analysis of molecular mechanisms underlying melanocytic defects observed in genetic diseases such as neurofibromatosis or albinism.
Guenou H et al., Human embryonic stem-cell derivatives for full reconstruction of the pluristratified epidermis: a preclinical study. Lancet. 2009 Nov 21;374(9703):1745-53.
Nissan X et al., Pluristratified epidermis from human embryonic stem cells., Med Sci. 2010 Jan;1(26):5-8.
Peschanski M et al., Epidermis grafting from adult to embryonic stem cells. Regen Med. 2010 Mar;5(2):157-9.
Dr. Fernando Larcher, Ciemat, Epithelial Biomedecine-Madrid, Spain, (mice skin graft)
Dr. Guerrino Meneguzzi, Inserm U 634, Biologie et Physopathologie de la peau, Nice, France, (epidermolysis bullosa modelling)
Dr. Robert Ballotti, Inserm U597, Biologie et Pathologie des cellules mélanocytaires : de la pigmentation cutanée aux mélanomes malin, Nice, France, (melanocytes)
Dr. Dominique Vidaud, Inserm UMR745, Génétique et Biothérapie des Maladies Dégénératives et Prolifératives du SystèmeNerveux, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France, (neurofibromatosis modelling)