Les cellules souches de l’épiderme interfolliculaire humain, appelées cellules souches kératinocytaires (CSK), assurent l’homéostasie et le renouvellement du tissu durant toute la vie d’un individu grâce à leur importante capacité d’autorenouvellement. Ma thèse de doctorat a porté sur l’étude des effecteurs moléculaires impliqués dans la balance entre prolifération et quiescence dans un modèle in vitro de CSK, isolées de manière clonale et définies par le terme holoclone. Je me suis tout d’abord intéressée à la réponse des holoclones à l’effet d’un régulateur important de la prolifération et de la quiescence des cellules souches adultes : le facteur de croissance TGF β1. Mon travail s’est ensuite focalisé sur l’étude d’un gène agissant en aval de la voie de signalisation TGF β, le facteur de transcription Klf4, et dont le rôle dans la biologie des cellules souches adultes reste largement méconnu. Klf4 est en effet surtout décrit pour son rôle dans la reprogrammation des cellules somatiques en cellules iPS. Le maintien de la sensibilité des holoclones aux signaux inhibiteurs de la croissance constitue un gage de la normalité des CSK. Notre étude de la réponse des holoclones à l’effet antiprolifératif du TGF β1 montre que les holoclones, dotés d’un fort potentiel de croissance, caractéristique des CSK, conservent leur sensibilité à l’effet du TGF β1. Ces résultats nous ont permis de valider les holoclones comme constituant un modèle pertinent pour caractériser la biologie normale des CSK et décrypter les contrôles moléculaires de l’état souche. Le modèle des holoclones a été exploité dans le cadre d’une approche de génomique fonctionnelle visant à déterminer le rôle du facteur de transcription Klf4 dans les CSK. L’utilisation de vecteurs lentiviraux exprimant un shARN dirigé contre l’ARNm de Klf4 nous a permis d’étudier l’impact d’une modulation fine du niveau d’expression de Klf4 sur les propriétés des holoclones. La répression transcriptionelle de Klf4, d’environ un facteur 2, favorise de manière significative l’expansion du compartiment clonogénique au sein des holoclones. Ce gain de fonction concerne à la fois les potentiels de croissance et de reconstruction épidermique des holoclones. Un aspect important de ce travail a concerné la recherche des réseaux moléculaires régulés par Klf4 dans les holoclones. Une analyse du transcriptome nous a permis de montrer que Klf4 participe au contrôle des mécanismes de cycle cellulaire et de différenciation. Klf4 interviendrait également dans la régulation des voies de signalisation TGF β/BMP et Wnt, connues pour exercer des rôles clés dans la biologie des cellules souches. Klf4 constituerait donc un censeur de l’activité du compartiment immature dans l’épiderme interfolliculaire. Il participerait aux mécanismes de régulation du cycle cellulaire et serait susceptible d’intervenir dans le contrôle de l’autorenouvellement du compartiment souche. / Stem cells present within the human interfollicular epidermis, which are defined as keratinocytes stem cells (KSC), ensure the homeostasis and renewal of the tissue throughout the whole individual life. These functions are related to their important self-renewal capacity. My PhD project was focused on the knowledge of the molecular effectors involved in the control of the balance between proliferation and quiescence in KSC. This scientific question was investigated in an in vitro model of KSC which were clonally derived and characterized as holoclones. Holoclones are controlled by mitogenic growth factors and also by antiproliferative signals. One of these regulators is the growth factor TGF β1 which plays an important role in the control of quiescence and cell proliferation within several adult stem cell systems. In the context of growth inhibition by TGF β1, I have studied the role of a downstream gene of the TGF β pathway, the transcription factor Klf4, whose role in adult stem cell biology remains unclear. In fact, Klf4 is mostly described for its involvement in the reprogramming process of somatic cells into iPS cells. The maintenance of holoclone sensitivity to cell growth inhibitors is a critical parameter of KSC normal physiology. Holoclones possess an extensive growth capacity, which is characteristic of KSC. Despite this high proliferation rate, holoclones are still responsive to the antiproliferative effect of TGF β1. These results allowed us to validate the use of holoclone as a relevant model of non-transformed KSC suitable for the characterization of the role of candidate stemness genes in KSC biology, such as Klf4. The holoclone model was exploited to perform a functional genomic approach to investigate the role of Klf4 in KSC. We have developed a shRNA-based gene knock-down method using lentiviral vectors to assess the impact of Klf4 down-modulation on holoclone functional properties. Our results show that Klf4 down modulation controls the expansion of the clonogenic compartment present within holoclone progeny. This gain-of-function, which is maintained at the long term level, leads to an increase in holoclone 3D epidermis reconstruction capacity. A major point of this project was to elucidate the molecular networks controlled by Klf4 in holoclones. Microarray data show that Klf4 regulates the expression of several genes related to pathways involved in the control of stem cell fate. In particular, we identified many transcripts related to TGF β/BMP and Wnt signallings. Interestingly, the majority of the modulated transcripts are involved in the regulation of cell cycle and in keratinocyte differentiation process. All together these results suggest a critical role Klf4 as a stemness censor of the most immature compartment activity. Klf4 is likely to be involved in cell cycle regulation of KSC compartment and in the control of KSC self-renewal process.

The relationship between cellular proliferation and differentiation is a major topic in cell biology. What we know comes from models of somatic cell differentiation, where it is widely viewed that cycling and differentiation are coupled, antagonistic phenomena linked at the G1 phase. The extension of this view to stem cells, however, is unclear. One potential possibility is that stem cells also tightly link their G1 phase with their differentiation, indicating a similarity between the differentiation of stem cells and the differentiation of more mature somatic cells. On the other hand, stem cells may utilize different mechanisms or adaptations that confer on them some aspect of uniqueness or "stemness." In this case, stem cells will not exhibit the same coupling with the cell cycle as in many somatic cell models. In this thesis, we examined mouse embryonic stem cells (mESCs), a stem cell that is pluripotent and rapidly cycling with a highly condensed G1 phase. Direct extension of the somatic view posits that elongation of their G1 phase to somatic lengths by cyclin-dependent kinase (CDK) activity inhibition should induce or increase differentiation of these stem cells. Evidence supporting this claim has been contradictory. We show that elongation of the cell cycle and elongation of G1 to somatic lengths is fully compatible with the pluripotent state of mESCs. Multiple methods that lengthen the cell cycle and that target CDK activity or that trigger putative downstream mechanisms (i.e. Rb and E2F activity) all fail to induce differentiation on their own or even to facilitate differentiation. These results indicates that the model of linkage between the G1 phase and differentiation in mESCs is incorrect and leads us to propose that "stemness" may have a physiological basis in the decoupling of cell cycling and differentiation. In summary, we provide evidence that there is a resistance of mESCs to differentiation induced by lengthening G1 and/or the cell cycle. This could allow for separate control of these events and provide new opportunities for investigation and application.

No description available.

The ovarian surface epithelium (OSE) is a monolayer of cells surrounding the ovary that is ruptured during ovulation. After ovulation the wound is repaired, however this process, and the mechanisms to maintain OSE homeostasis after the wound is repaired are poorly understood. We have shown the mouse OSE (mOSE) contains a stem cell population that is expanded by Transforming Growth Factor Beta 1 (TGFB1), a factor present in follicular fluid. These data suggest that components in the follicular fluid such as TGFB1 may promote wound repair and OSE homeostasis through maintenance of the OSE stem cell population. Additionally, TGFB1 may promote wound repair through induction of an epithelial-to-mesenchymal transition (EMT) and activation of pro-survival pathways, as seen in other tissues. To elucidate the mechanism for TGFB1-mediated ovulatory wound repair, mOSE cells were treated with TGFB1, which induced an EMT seen with increased Snai1 expression and cell migration. Snai1 overexpression also increased cell migration and sphere formation (a stem cell characteristic). RNA sequencing results suggest this is at least in part through elevated collagen deposition in SNAI1 overexpressing cells. A TGFB signalling targets array identified Cox2 induction following TGFB1 treatment. Constitutive Cox2 expression did not promote an EMT, but enhanced sphere formation and cell survival. Finally, TGFB1 treatment decreased Brca1 expression, which when deleted from mOSE cells also increased sphere formation. RNA sequencing results suggest that Brca1 deletion promotes stemness through activation of the stem cell genes Ly6a and Lgr5. RNA sequencing was also used to compare mOSE cells cultured as monolayers and as spheroids, with and without TGFB1. These results validate our findings that TGFB1 promotes an EMT partially through Snail induction and the upregulation of Cox2. mOSE cells cultured as spheroids acquire a mesenchymal transcriptional profile that is further enhanced with TGFB1 treatment. These data suggest that TGFB1 may promote ovulatory wound repair and maintain OSE homeostasis through the induction of an EMT, maintenance of the stem cell population and activation of a pro-survival pathway. Interestingly, mOSE spheroids also decrease Brca1 expression and upregulate cancer associated genes such as Pax8 and Greb1. The induction of survival pathways, while simultaneously increasing stemness and repressing Brca1 could render cells more susceptible to transformation. This work provides novel insights as to why ovulation is the primary non-hereditary risk factor for ovarian cancer.

As células-tronco provenientes da polpa de dentes decíduos esfoliados (SHED) são uma fonte promissora de células-tronco para a terapia regenerativa. Já foi demonstrado que elas podem se diferenciar em células endoteliais, mas os mecanismos envolvidos nesse processo ainda são desconhecidos. Dessa forma, o presente estudo teve como objetivo elucidar os mecanismos moleculares relacionados à diferenciação endotelial dessas células. Para isso, um meio de cultura para células endoteliais (EGM-2MV), suplementado por 50ng/mL rhVEGF, foi utilizado com sucesso como estímulo para as SHED se diferenciarem em células endoteliais em cultura de monocamada. Uma vez que, elas expressaram os marcadores endoteliais VEGFR-2 e CD-31. Além disso, quando as SHED foram expostas ao meio de diferenciação, a fosforilação de STAT-3 (um marcador de células-tronco) foi inibida de acordo com a concentração utilizada, enquanto a fosforilação de ERK e AKT foi estimulada. Quando um inibidor de ERK foi adicionado ao meio, a fosforilação de STAT-3 aumentou. Por outro lado, quando um inibidor de STAT-3 foi adicionado ao meio de cultura, a fosforilação de ERK aumentou. A inibição de ERK também impediu a diferenciação endotelial. Para confirmar esse resultado, shRNA MEK-1 SHED também falharam em expressar VEGFR-2 quando estimuladas. Além disso, scaffolds de fatias dentais foram semeadas com shRNA VEGFR-1 SHED ou shRNA controle e implantadas no subcutâneo de camundongos imunodeficientes. Após recuperados, um tecido muito semelhante à polpa dental foi formado no interior das fatias. No entanto, havia menos células CD-31 positivas nos vasos sanguíneos dos implantes semeados com VEGFR-1 shRNA SHED, sugerindo um papel do VEGFR-1 na formação de vasos sanguíneos. Em conclusão, o VEGFR-1 e a via de sinalização de ERK estão envolvidos no processo de diferenciação das SHED em células endoteliais. / Stem Cells from Exfoliated Deciduous Teeth (SHED) are a promising source of stem cells for regenerative therapy. It was already shown they can differentiate into endothelial cells, but the mechanisms involved in this process remain unclear. Therefore, the following study aimed to elucidate the molecular mechanisms related to endothelial differentiation of SHED. For this purpose, a culture media for endothelial cells (EGM-2MV), supplemented with 50ng/ml rhVEGF, was successfully used as stimuli for SHED to undergo endothelial differentiation in monolayer culture, as they expressed the endothelial markers VEGFR-2 and CD- 31. Moreover, when SHED were exposed to the differentiation medium, STAT-3 phosphorilation (a stemness marker) was inhibited while the ERK and AKT phosphorilation was stimulated. When an ERK inhibitor was added to the medium, the STAT-3 phosphorilation increased in a dependent concentration manner. On the other hand, when a STAT-3 inhibitor was added the ERK phosphorilation increased. The ERK inhibition also arrested endothelial differentiation. To confirm this results, shRNA MEK-1 SHED also failed to express VEGFR-2 when stimulated. Additionally, tooth slice scaffolds were seeded with shRNA VEGFR-1 SHED or shRNA control SHED and implanted, subcutaneously, into immunodeficient mice. After retrieved, a dental pulp-like tissue had been formed inside the tooth slice. However, there were fewer CD-31 positive blood vessels in the shRNA VEGFR-1 implants suggesting a role of VEGFR-1 in the formation of blood vessels by SHED. In conclusion, the VEGFR-1 and ERK pathway are involved in the process of differentiation of SHED into endothelial cells.

As células-tronco provenientes da polpa de dentes decíduos esfoliados (SHED) são uma fonte promissora de células-tronco para a terapia regenerativa. Já foi demonstrado que elas podem se diferenciar em células endoteliais, mas os mecanismos envolvidos nesse processo ainda são desconhecidos. Dessa forma, o presente estudo teve como objetivo elucidar os mecanismos moleculares relacionados à diferenciação endotelial dessas células. Para isso, um meio de cultura para células endoteliais (EGM-2MV), suplementado por 50ng/mL rhVEGF, foi utilizado com sucesso como estímulo para as SHED se diferenciarem em células endoteliais em cultura de monocamada. Uma vez que, elas expressaram os marcadores endoteliais VEGFR-2 e CD-31. Além disso, quando as SHED foram expostas ao meio de diferenciação, a fosforilação de STAT-3 (um marcador de células-tronco) foi inibida de acordo com a concentração utilizada, enquanto a fosforilação de ERK e AKT foi estimulada. Quando um inibidor de ERK foi adicionado ao meio, a fosforilação de STAT-3 aumentou. Por outro lado, quando um inibidor de STAT-3 foi adicionado ao meio de cultura, a fosforilação de ERK aumentou. A inibição de ERK também impediu a diferenciação endotelial. Para confirmar esse resultado, shRNA MEK-1 SHED também falharam em expressar VEGFR-2 quando estimuladas. Além disso, scaffolds de fatias dentais foram semeadas com shRNA VEGFR-1 SHED ou shRNA controle e implantadas no subcutâneo de camundongos imunodeficientes. Após recuperados, um tecido muito semelhante à polpa dental foi formado no interior das fatias. No entanto, havia menos células CD-31 positivas nos vasos sanguíneos dos implantes semeados com VEGFR-1 shRNA SHED, sugerindo um papel do VEGFR-1 na formação de vasos sanguíneos. Em conclusão, o VEGFR-1 e a via de sinalização de ERK estão envolvidos no processo de diferenciação das SHED em células endoteliais. / Stem Cells from Exfoliated Deciduous Teeth (SHED) are a promising source of stem cells for regenerative therapy. It was already shown they can differentiate into endothelial cells, but the mechanisms involved in this process remain unclear. Therefore, the following study aimed to elucidate the molecular mechanisms related to endothelial differentiation of SHED. For this purpose, a culture media for endothelial cells (EGM-2MV), supplemented with 50ng/ml rhVEGF, was successfully used as stimuli for SHED to undergo endothelial differentiation in monolayer culture, as they expressed the endothelial markers VEGFR-2 and CD- 31. Moreover, when SHED were exposed to the differentiation medium, STAT-3 phosphorilation (a stemness marker) was inhibited while the ERK and AKT phosphorilation was stimulated. When an ERK inhibitor was added to the medium, the STAT-3 phosphorilation increased in a dependent concentration manner. On the other hand, when a STAT-3 inhibitor was added the ERK phosphorilation increased. The ERK inhibition also arrested endothelial differentiation. To confirm this results, shRNA MEK-1 SHED also failed to express VEGFR-2 when stimulated. Additionally, tooth slice scaffolds were seeded with shRNA VEGFR-1 SHED or shRNA control SHED and implanted, subcutaneously, into immunodeficient mice. After retrieved, a dental pulp-like tissue had been formed inside the tooth slice. However, there were fewer CD-31 positive blood vessels in the shRNA VEGFR-1 implants suggesting a role of VEGFR-1 in the formation of blood vessels by SHED. In conclusion, the VEGFR-1 and ERK pathway are involved in the process of differentiation of SHED into endothelial cells.

As células-tronco provenientes da polpa de dentes decíduos esfoliados (SHED) são uma fonte promissora de células-tronco para a terapia regenerativa. Já foi demonstrado que elas podem se diferenciar em células endoteliais, mas os mecanismos envolvidos nesse processo ainda são desconhecidos. Dessa forma, o presente estudo teve como objetivo elucidar os mecanismos moleculares relacionados à diferenciação endotelial dessas células. Para isso, um meio de cultura para células endoteliais (EGM-2MV), suplementado por 50ng/mL rhVEGF, foi utilizado com sucesso como estímulo para as SHED se diferenciarem em células endoteliais em cultura de monocamada. Uma vez que, elas expressaram os marcadores endoteliais VEGFR-2 e CD-31. Além disso, quando as SHED foram expostas ao meio de diferenciação, a fosforilação de STAT-3 (um marcador de células-tronco) foi inibida de acordo com a concentração utilizada, enquanto a fosforilação de ERK e AKT foi estimulada. Quando um inibidor de ERK foi adicionado ao meio, a fosforilação de STAT-3 aumentou. Por outro lado, quando um inibidor de STAT-3 foi adicionado ao meio de cultura, a fosforilação de ERK aumentou. A inibição de ERK também impediu a diferenciação endotelial. Para confirmar esse resultado, shRNA MEK-1 SHED também falharam em expressar VEGFR-2 quando estimuladas. Além disso, scaffolds de fatias dentais foram semeadas com shRNA VEGFR-1 SHED ou shRNA controle e implantadas no subcutâneo de camundongos imunodeficientes. Após recuperados, um tecido muito semelhante à polpa dental foi formado no interior das fatias. No entanto, havia menos células CD-31 positivas nos vasos sanguíneos dos implantes semeados com VEGFR-1 shRNA SHED, sugerindo um papel do VEGFR-1 na formação de vasos sanguíneos. Em conclusão, o VEGFR-1 e a via de sinalização de ERK estão envolvidos no processo de diferenciação das SHED em células endoteliais. / Stem Cells from Exfoliated Deciduous Teeth (SHED) are a promising source of stem cells for regenerative therapy. It was already shown they can differentiate into endothelial cells, but the mechanisms involved in this process remain unclear. Therefore, the following study aimed to elucidate the molecular mechanisms related to endothelial differentiation of SHED. For this purpose, a culture media for endothelial cells (EGM-2MV), supplemented with 50ng/ml rhVEGF, was successfully used as stimuli for SHED to undergo endothelial differentiation in monolayer culture, as they expressed the endothelial markers VEGFR-2 and CD- 31. Moreover, when SHED were exposed to the differentiation medium, STAT-3 phosphorilation (a stemness marker) was inhibited while the ERK and AKT phosphorilation was stimulated. When an ERK inhibitor was added to the medium, the STAT-3 phosphorilation increased in a dependent concentration manner. On the other hand, when a STAT-3 inhibitor was added the ERK phosphorilation increased. The ERK inhibition also arrested endothelial differentiation. To confirm this results, shRNA MEK-1 SHED also failed to express VEGFR-2 when stimulated. Additionally, tooth slice scaffolds were seeded with shRNA VEGFR-1 SHED or shRNA control SHED and implanted, subcutaneously, into immunodeficient mice. After retrieved, a dental pulp-like tissue had been formed inside the tooth slice. However, there were fewer CD-31 positive blood vessels in the shRNA VEGFR-1 implants suggesting a role of VEGFR-1 in the formation of blood vessels by SHED. In conclusion, the VEGFR-1 and ERK pathway are involved in the process of differentiation of SHED into endothelial cells.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22833号 / 医博第4672号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 濵﨑 洋子, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM