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

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.

The Cell Differentiation Status Influences the Outcome of Notch-Induced Malignancy

Caroline Poisson

The Notch signaling cascade is a well conserved pathway that regulates many aspects of embryonic development and plays a crucial role in the differentiation process and tissue homeostasis in multiple organs of adult species. Several human pathological disorders, including cancer, arise as a consequence of the deregulation of the Notch signaling pathway. Notch deregulation, up to date, has been best characterized in acute T-cell lymphoblastic leukemia (T-ALL). Indeed, in T-ALL pediatric patients activating Notch1 mutations have been identified in more than 50% of the cases. Increased knowledge of the mechanisms underlying T-ALL development and maintenance is clearly needed as one out of 5 patients with T-ALL has a very poor prognosis. Moreover, the high toxicity of chemotherapeutic regimens has been associated with elevated risks of developing severe secondary health problems. In the present study, we characterized the role of Notch in T-ALL induction and maintenance. Using transgenic mouse models, we could demonstrate that the oncogenic potential of Notch is dependent on RBP-Jκ transcriptional activity. Indeed, the canonical Notch signaling pathway, mediated by the RBP-Jκ transcription factor, is essential for both Notch-mediated T-ALL induction and maintenance, as well as for the induction of a Notch-mediated lymphoproliferative disorder. Moreover, constitutive overexpression of Notch1 at discrete developmental stages within the hematopoietic system, led us to identify a correlation between the differentiation status of a cell, within the hierarchy of the hematopoietic tree, and its sensitivity to Notch1-induced transformation. We demonstrated that forced Notch1 expression in hematopoietic stem cells was not sufficient to induce T-ALL. Nevertheless, aberrant Notch1 expression in the bone marrow resulted in the development of a lethal lymphoproliferative disorder. Overexpression of Notch1 in thmus at the DN2 to DN3 stage of T-cell delvelopment generated an aggressive transplantable T-ALL with important metastatic potential. This suggested that progenitors already committed to the T-cell lineage are highly susceptible to Notch-mediated transformation, which is relevant to the human disease. Nevertheless, forced Notch1 expression after the β-selection checkpoint in the thymys, was no longer sufficient to induce transformation of late DN3 to DN4 thymocytes. We therefore established a murine genetic system that allowed us to dissect the mechanisms of aberrant Notch1 signaling in a lymphoproliferative disorder and a true T-ALL model. We were able to assess the genetic signature that is involved in the transplantability and metastatic potential of leukemic cells and generated a list of candidate genes implicated in these processes. In addition, we correlated genes identified in our murine models with genes associated as well in human T-ALL profiles to identify genes implicated in T-ALL pathogenesis in both mouse and human.

EPFL2011

Thymic epithelial cells

Paola Bonfanti

As primary lymphoid organ, the thymus provides the essential environment for maturation and selection of functional T lymphocytes. Thymic epithelial (TE) cells derive from the endoderm, and more specifically, from the third pharyngeal pouch. The thymic epithelium is organized in a three-dimensional architecture that does not easily fit into standard classifications of simple or stratified epithelia. Moreover, the mechanisms that maintain the epithelial compartment in the post-natal thymus remain unclear. Although they have been postulated to exist as in all epithelia, thymic stem cells have not been clearly identified to date. One crucial property of adult multipotent stem cells (SCs) in the skin and other stratified epithelia is clonogenicity, i.e the ability of a single cell to give rise to a cell population. Epidermal clonogenic keratinocytes can be propagated for several generations in vitro and can permanently engraft when transplanted onto patients. In the absence of reliable molecular markers to identify and purify multipotent SCs, the demonstration that they can selfrenew in vitro and in vivo represents, to date, the best method to assess stemness. Likewise, the thymus contains epithelial cells that express genes usually only expressed in the skin or other stratified epithelia, thus suggesting the existence of a possible relationship. In the work described in this thesis, we investigated the potentialities of rat primary TE cells through clonal analysis and functional assays. We demonstrate that embryonic and postnatal rat primary TE cells have a clonogenic growth pattern in vitro. We also describe the molecular signature of subtypes of thymic keratinocytes and address the postnatal role of developmentally regulated genes which are commonly expressed in skin and thymus. Clones of rat TE cells have been transplanted onto developing skin in order to evaluate their response to skin morphogenetic signals. Similarly, the response of these clones, as well as of clones obtained from hair follicles, to a thymic environment has been investigated. We demonstrate that cultured TE cells share morphological, phenotypic and functional characteristics with skin multipotent stem cells. When challenged in thymic or skin in vivo assays the clonal progeny of a single TE cell is able to integrate into a functional thymus or give rise to all skin derivatives respectively, i.e. epidermis, hair follicle and sebaceous gland. Conversely skin cells cannot participate to thymic reconstitution. Uncovering the relationship of TE cells with stem cells of stratified epithelia may provide important insights into the molecular basis of diseases involving both thymus and skin.

EPFL2008

Understanding engraftment of cultured epidermal stem cells

Nicolas Grasset

The epidermis and its appendages protect our body from environmental hazards. Cells generated in the basal layer continuously replace the terminally differentiated keratinocytes that are shed off the epidermal surface. Long-term renewal depends on specific tissue cells, called stem cells. The epidermis can efficiently repair itself when wounded, thanks to the proliferation, migration and differentiation of the stem cells and their progeny. In extensive full-thickness burns, restoration of the epidermal barrier can only be achieved by means of autologous skin grafts. Cell therapy using cultured epithelium autografts (CEA) has been part of the therapeutic arsenal since the early eighties (Gallico et al., 1984; O'Connor et al., 1981) and it has saved the life of many patients worldwide. Paradoxically, little is known on the behavior of the transplanted stem cells and engraftment has remained variable. Unpublished data from our laboratory have shown that the number of stem cells decrease rapidly in transplanted CEA in humans. This further highlights the necessity to thoroughly comprehend the cellular and molecular mechanisms of stem cell engraftment. Several fundamental questions must be addressed, for example: 1) By which mechanisms do stem cells adjust to the stress of transplantation? 2) How many stem cells are required for long-term renewal of the regenerated epidermis? 3) Can one manipulate the niche? To thoroughly investigate the mechanisms of engraftment, it is critical to develop a predictable animal model since it is difficult to experiment on human. We have chosen the pig as a large animal model because clinical procedures used for CEA transplantation cannot be recapitulated in laboratory animals. Firstly, we have demonstrated that pig and human skin share many features. In particular, the skin of the pig contains keratinocyte stem cells that can be characterized by clonal analysis using the same criteria as in the human (holoclone, meroclone, paraclone). Secondly, we have reproducibly produced CEA, including some made from the progeny of a single EGFP-labeled keratinocyte stem cell. Thirdly, we have recapitulated in the pig all surgical procedures used to transplanting CEA in humans. Fourthly, CEA engraftment has been systematically investigated by histology, immunochemistry and clonal analysis. Fifthly, we have demonstrated that the number of stem cells rapidly decreases following CEA transplantation and that there is little apoptosis in transplanted CEA, which suggests that stem cells are rather lost through terminal differentiation. Collectively our results demonstrate that the pig is the best available model system to investigate the mechanisms that govern stem cell engraftment. Superior engraftment may be achieved by improving the grafting bed, by designing smart matrices to better mimic the niche, or by manipulating stem cell behavior. A better comprehension of stem cell engraftment will certainly benefit patients suffering from large burns and those with disabling skin diseases (e.g. dystrophic epidermolysis bullosa). It will also profit stem cell therapy at large since optimal engraftment is also needed for hematopoïetic, muscle and neural stem cells.

EPFL2008

The Cell Differentiation Status Influences the Outcome of Notch-Induced Malignancy

Caroline Poisson

EPFL2011

Understanding engraftment of cultured epidermal stem cells

Nicolas Grasset

EPFL2008

Thymic epithelial cells

Paola Bonfanti

EPFL2008