Clinical application of autologous epithelial stem cells in disorders of squamous epithelia

Epidermis and epithelia lining the ocular surface, the oral cavity, the pharynx, the oesophagus, the larynx, and the vagina, called stratified squamous epithelia (SSE), contain stem/progenitor cells that support renewal and repair. Under appropriate conditions, these cells can be massively expanded in culture. Restoration of the integrity and the function of SSE is obtained by transplantation and engraftment of the autologous cultivated stem cells in case of several severe clinical conditions (e.g., extensive third-degree burns, limbal deficiency). Successful gene therapy for hereditary SSE disorders has also been achieved by means of correction of the genetic defect in cultivated autologous keratinocyte stem cells. However, the mechanisms controlling engraftment of the transplanted stem cells remain poorly understood, leading to unpredictable clinical results. Further fundamental investigations to explore the behaviour of the transplanted stem cells and their plasticity, anticipating the regulatory affairs main concerns, are needed for successful cell and gene therapy

c-Myc is required for the formation of intestinal crypts but dispensable for homeostasis of the adult intestinal epithelium

Christelle Dubey

In self-renewing tissues such as the skin epidermis and the bone marrow, Myc proteins control differentiation of stem cells and proliferation of progenitor cell types. In the epithelium of the small intestine, we show that c-Myc and N-Myc are expressed in a differential manner. Whereas c-Myc is expressed in the proliferating transient-amplifying compartment of the crypts, N-Myc is restricted to the differentiated villus epithelium and a single cell located near the crypt base. c-Myc has been implicated as a critical target of the canonical Wnt pathway, which is essential for formation and maintenance of the intestinal mucosa. To genetically assess the role of c-Myc during development and homeostasis of the mammalian intestine we induced deletion of the c-myc(flox) allele in the villi and intestinal stem cell-bearing crypts of juvenile and adult mice, via tamoxifen-induced activation of the CreER(T2) recombinase, driven by the villin promoter. Absence of c-Myc activity in the juvenile mucosa at the onset of crypt morphogenesis leads to a failure to form normal numbers of crypts in the small intestine. However, all mice recover from this insult to form and maintain a normal epithelium in the absence of c-Myc activity and without apparent compensation by N-Myc or L-Myc. This study provides genetic and molecular evidence that proliferation and expansion of progenitors necessary to maintain the adult intestinal epithelium can unexpectedly occur in a Myc-independent manner.

2005

A single epidermal stem cell strategy for safe ex vivo gene therapy

Yann Barrandon, Sara Benmohammed, Stéphanie Danièle Hélène Droz-Georget, Nicolas Grasset, Graham Knott, Alessandra Recchia, Ariane Rochat, Jacques Rougemont, Emmanuelle Savioz-Dayer, Andrea Zaffalon

There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance, particularly for ex vivo autologous gene therapy. Yet current technology makes it difficult to thoroughly evaluate the behaviour of genetically corrected stem cells before they are transplanted. To address this, we have developed a strategy that permits transplantation of a clonal population of genetically corrected autologous stem cells that meet stringent selection criteria and the principle of precaution. As a proof of concept, we have stably transduced epidermal stem cells (holoclones) obtained from a patient suffering from recessive dystrophic epidermolysis bullosa. Holoclones were infected with self-inactivating retroviruses bearing a COL7A1 cDNA and cloned before the progeny of individual stem cells were characterised using a number of criteria. Clonal analysis revealed a great deal of heterogeneity among transduced stem cells in their capacity to produce functional type VII collagen (COLVII). Selected transduced stem cells transplanted onto immunodeficient mice regenerated a non-blistering epidermis for months and produced a functional COLVII. Safety was assessed by determining the sites of proviral integration, rearrangements and hit genes and by whole-genome sequencing. The progeny of the selected stem cells also had a diploid karyotype, was not tumorigenic and did not disseminate after long-term transplantation onto immunodeficient mice. In conclusion, a clonal strategy is a powerful and efficient means of by-passing the heterogeneity of a transduced stem cell population. It guarantees a safe and homogenous medicinal product, fulfilling the principle of precaution and the requirements of regulatory affairs. Furthermore, a clonal strategy makes it possible to envision exciting gene-editing technologies like zinc finger nucleases, TALENs and homologous recombination for next-generation gene therapy.

Wiley Open Access2015

Innovative strategy of corneal epithelium regeneration using fabricated soft contact lens containing cavities for stem cells transplantation

Julien Graber

Transplantation of donor cornea is widely performed with a high success rate to treat patients with impaired corneal transparency. However the limited number of donors has led scientists to search for new technologies and treatments, including stem cell therapies. We have developed a device that permits the transplantation of stem cells onto the cornea in order to regenerate a functional epithelium. It is shaped as a contact lens and it possesses cavities in its internal portion that are filled with a cell suspension. The device is used as a carrier to transfer the cells onto the cornea. To test and validate our approach we created an ex vivo system to perform long-term organotypic culture on the cornea of enucleated pig eyes. We seeded corneal epithelial cells onto pig stromas. We demonstrate that 30'000 donor cells can regenerate an entire epithelium in organotypic culture conditions. By using our culture system no living animals were required during the conception and evaluation of the device. We performed preliminary in vivo trials on rabbits, which showed promising results. We aim to create a user-friendly and low cost ocular surface reconstruction procedure that will be an alternative to current treatments. The advantages of our strategy are that it will not require cell culture or tissue preparation, as is the case for usual tissue-based grafts. Hopefully this technique could extend to clinical studies and could one day be used as an ocular surface reconstruction procedure.

EPFL2014

A single epidermal stem cell strategy for safe ex vivo gene therapy

Wiley Open Access2015