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

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.

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

Differentiation of human embryonic stem cells to hepatocytes

Noam Beckmann

Many liver diseases involve hepatocyte malfunction. In most cases, transplantation of corrected hepatocytes would correct for disease symptoms. Liver donor shortage and hepatocyte inability to proliferate in vitro accentuate this problem. Embryonic stem and induced pluripotent stem cells proliferate indefinitely and have the capacity to differentiate into every cell type of the body. They could thus represent a solution to this organ donor shortage. In this project, we aim to differentiate ES and iPS cells to hepatocytes. First, we consider an approach to select the ES/iPS cell line showing the best potential to generate liver lineage using teratomas as a selection tool. Then we use lentiviral vectors to transduce this selected cell line with essential liver-‐specific development factors to help differentiation efficiency. Next, we developed an in vitro differentiation protocol based on existing protocols and developmental clues of liver development to generate a pool of hepatocyte precursors. Later on, we injected these differentiated cells in mice liver of Fah-‐, Rag2-‐ and Il-‐2rγ-‐deficient mice to check for engraftment and liver marker expression. Our findings show, after checking for hepatocyte-‐ specific markers in the differentiated cells, that our previous selection for the potentially best cell line seems correct. We also find that the transcription factors involved in liver development appear to have a negative effect in vitro on liver differentiation. Applying our protocol to regular ES cells results on the production of late maturity hepatocyte markers, showing correct differentiation and maturation of newly formed hepatocytes. Finally, mouse in vivo liver transplantation of these differentiated cells gives us human albumin expression detectable in blood serum of said mice

2010

Oligopotent stem cells are distributed throughout the mammalian ocular surface

Georges Abou Jaoudé, Yann Barrandon, Ariane Rochat

The integrity of the cornea, the most anterior part of the eye, is indispensable for vision. Forty- five million individuals worldwide are bilaterally blind and another 135 million have severely impaired vision in both eyes because of loss of corneal transparency(1); treatments range from local medications to corneal transplants, and more recently to stem cell therapy(2). The corneal epithelium is a squamous epithelium that is constantly renewing, with a vertical turnover of 7 to 14 days in many mammals(3). Identification of slow cycling cells ( label- retaining cells) in the limbus of the mouse has led to the notion that the limbus is the niche for the stem cells responsible for the long- term renewal of the cornea(4); hence, the corneal epithelium is supposedly renewed by cells generated at and migrating from the limbus, in marked opposition to other squamous epithelia in which each resident stem cell has in charge a limited area of epithelium(5,6). Here we show that the corneal epithelium of the mouse can be serially transplanted, is self- maintained and contains oligopotent stem cells with the capacity to generate goblet cells if provided with a conjunctival environment. Furthermore, the entire ocular surface of the pig, including the cornea, contains oligopotent stem cells (holoclones)(7,8) with the capacity to generate individual colonies of corneal and conjunctival cells. Therefore, the limbus is not the only niche for corneal stem cells and corneal renewal is not different from other squamous epithelia. We propose a model that unifies our observations with the literature and explains why the limbal region is enriched in stem cells.

2008

Understanding engraftment of cultured epidermal stem cells

Nicolas Grasset

EPFL2008