Early Fate Choice Determines in vivo Engraftment and in vitro Behavior of Epidermal Stem Cells

François Gorostidi
EPFL, 2012
Thèse EPFL

Résumé

Regenerative medicine aims at using stem cells to restore or establish lost, damaged, diseased, or aging tissues and organs function. The transplantation of cultured epidermal autograft (CEA) has saved the lives of many burned patients over the last 30 years, but the resulting epidermis is far from perfect, and clinical outcomes remain unpredictable. The engraftment of cultured epidermal stem cells is poorly understood, and, as a result, suboptimal. Using a large animal model, the pig, we recapitulated the clinical settings and outcomes of human CEA transplantation. With this model, we study the engraftment process, and we demonstrate that cultured epidermal stem cells often, but not always, favor the choice of differentiation over self-renewal when transplanted on full thickness wounds. Differences in this early fate choice are likely to explain the variability of clinical outcomes. We developed a system of in vitro live cell imaging (LCI) to study the fate choices of multiple individual cells in a microenvironment that is much more controlled than the grafting bed of a burned patient. We demonstrate that keratinocytes have a very heterogeneous behavior in vitro. With a fluorescent reporter of cell cycle progression (FUCCI), we show the influence of the cell cycle on early fate choices of cultured keratinocytes. By introducing a ROCK inhibitor in the culture medium, we show that keratinocyte’s early fate choices can be impacted so that they show greater clonogenicity and growth potential. The unique combination of large animal CEA transplantation with individual cell LCI demon- strate the critical role of early fate choice, both for epidermal stem cell engraftment and in vitro behavior. The possibility to influence the early fate choices of stem cells by modifying the microenvironment has a great potential to improve the engraftment and the ex vivo ampli- fication of stem cells, the two limiting steps of cell replacement therapy.

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https://infoscience.epfl.ch/record/180219?ln=fr

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François Gorostidi
EPFL, 2012
Thèse EPFL

Résumé

Official source

https://infoscience.epfl.ch/record/180219?ln=fr

À propos de ce résultat

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Concepts associés

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Publications associées (32)

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Publications associées (32)

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

Chargement

Adult stem cells are characterized by their unique ability to self-renew and give rise to a progeny of specialized/differentiated cells. Therefore, they have a remarkable potential for regenerative medicine. However, the use of adult stem cells is restrained by several obstacles such as limited in vitro amplification and uncontrolled differentiation. Adult stem cells reside in tightly regulated microenvironments termed “niches”. The niche is composed of nursing cells, extracellular matrix and a variety of signalling factors that dynamically regulate stem cell behaviour. Consequently, recapitulating the niche in vitro is fundamental in manipulating adult stem cells. In recent years, the physicochemical microenvironment of the niche (e.g. temperature, pH, oxygen tension) has proven to be as important as classical signalling pathways for the regulation of stem cell fate. Therefore, the aim of this thesis is to understand the influence of temperature variations on human epidermal stem cell behaviour. This effort has been accomplished in three main directions. First, we have contributed to the development of two different telemetric temperature sensors that precisely measure and record temperature in vivo and in vitro. These sensors have allowed us to demonstrate that epidermal keratinocytes are continuously subjected to small physiological temperature variations (3 ± 2oC) in vivo. Then, we have engineered a device that allows a tight and dynamic control of temperature while simultaneously providing real-time imaging of cultured human epidermal stem cells. It is shown that temperature variations of 1oC are not trivial, since they significantly affect human epidermal stem cell growth and proliferation. Second, we have shown that human keratinocytes sense temperature by means of temperature-sensitive calcium channels termed thermoTRPs (Transient Receptor Potential). Consequently, we have monitored intracellular calcium concentrations to evaluate the reaction of epidermal stem cells to thermal or chemical stimulations. The strongest calcium influxes have been observed after temperature drops (from 37 to 32oC) suggesting that human keratinocytes are particularly sensitive to temperature decreases. Furthermore, we have demonstrated the importance of these channels for keratinocyte viability by silencing a thermoTRP channel (i.e. TRPV3) using lentiviral delivery of shRNA. Third, we have demonstrated that thermal signals affecting keratinocytes are integrated through the mTOR (mammalian Target Of Rapamycin) signalling pathway, since cool stimuli (32oC) decreased mTOR complex 1 (mTORC1) kinase activity. Culture at 32oC and 100nm rapamycin treatment affected growth and proliferation of human keratinocyte stem cells similarly, further stressing the inhibitory effects of cool temperatures on mTORC1. We also have observed that inhibition of mTORC1 induced nuclear translocation of mTOR, suggesting a putative transcriptional role for mTOR. To conclude, we have established that prolonged inhibition of mTORC1 favours an apparent maintenance of the stem cell phenotype by reducing clonal conversion, without however stopping stem cell aging. Altogether, our results demonstrate that thermoTRPs are wired to the mTOR signalling in human epidermal stem cells. Furthermore, our work thoroughly supports the hypothesis that temperature is a significant actor of the epidermal stem cell niche as it can directly affect stem cell behaviour.

EPFL2013

Chargement

Capturing epidermal stemness

Andrea Zaffalon

Regenerative medicine aims to replace or regenerate tissues or organs to re-establish their normal function. In 1975, Rheinwald and Green developed a technique to isolate and amplify epidermal stem cells. Their discovery led to the development of cultured epidermal autografts (CEA), the first regenerative therapy using cultured cells. Adult stem cells are the working force behind tissue homeostasis and repair. Through constant division and specialization, they produce enough daughter cells to maintain tissue architecture and function. This process is orchestrated by an elegant cross-talk between the stem cells and their microenvironment. By using irradiated feeder cells (3T3-J2 cells), Rheinwald and Green were able to artificially instruct epidermal cells to grow in vitro. Later on, they found that these cells could regenerate a functional epidermis. Moreover, Barrandon and Green demonstrated that clonogenic keratinocytes lose progressively their growth potential in vitro. This process is called clonal conversion. The 3T3-J2 cells are mouse embryonic fibroblasts. The molecules that they produce are necessary to promote self-renewal of keratinocyte stem cells in vitro. If the quality of the culture system is not monitored, clonal conversion can occur rapidly and the therapeutic potential is lost. Although the system is now used in the clinics for the treatment of large burns and cornea injuries, the regulatory affairs express genuine concerns towards the animal origin of the feeder cells. Ultimately, we would like to replace the current 3T3-J2 culture system by a fully characterized system, devoid of animal products, for the production of CEA. In this thesis, we developed two strategies to work towards this goal. First, we developed a large scale RNAi strategy to investigate the cellular cross-talk between feeder cells and human keratinocytes. We have identified several putative âfeeder genesâ. One of these genes is Furin, a serine protease. Furin is expressed by the 3T3-J2 cells and is required to sustain the proliferation of human keratinocytes. Second, we investigated the impact of ROCK inhibition on the procurement and culture of human keratinocytes. We found that it promoted the adhesion and proliferation of freshly isolated human keratinocytes. In opposition to what was described previously, we did not observe evidences of cellular immortalization or reprogramming when keratinocytes where treated with Y-27632 (ROCK inhibitor). Together, the results of our two approaches provide new leads for the further development of a new culture system for human keratinocyte stem cells.

EPFL2016

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Understanding engraftment of cultured epidermal stem cells

Nicolas Grasset

EPFL2008

EPFL2013

Capturing epidermal stemness

Andrea Zaffalon

EPFL2016