Etude du rôle des récepteurs Gabab dans l'anxiété, la dépression et l'addiction : approche pharmacologique et génétique.

Notre travail visait à caractériser le rôle des récepteurs GABAB dans l'anxiété, la dépression et l'addiction, prenant avantage du récent développement de souris GABAB knockout et d'un modulateur positif allostérique du récepteur GABAB (GS39783). Dans un premier temps, nous avons observé que l'ablation du gène codant pour le récepteur GABAB induisait un effet anxiogène. Inversement, la stimulation pharmacologique de ces récepteurs produisait une réponse anxiolytique. De plus, nous avons mis en évidence que l'inactivation génétique de ces récepteurs et leur blocage pharmacologique induisait un effet antidépresseur. Finalement, nous avons observé que le GS39783 s'opposait à la fois aux comportements et aux adaptations moléculaires associés à l'administration de psychostimulants. Ainsi, ces études nous permettent de conclure que les récepteurs GABAB pourraient représenter une cible dans le développement de nouvelles stratégies thérapeutique dans le traitement de ces trois pathologies.Taking advantage of the recent development of GABAB positive modulator GS39783 and mice lacking GABAB(1) or GABAB(2) receptor subunits, the studies in the present thesis are focus on addressing a broad hypothesis that GABAB receptors play a key role in the manifestation of psychiatric disorders, such as anxiety, depression and addiction. In a first time, we demonstrated that targetted deletion of either GABAB receptor subunit induced strong anxiety. Conversely, we also observed that GABAB activation, via administration of GS39783, induced anxiolytic-like effect in several paradigms. Secondly, we also demonstrated that genetic inactivation of either GABAB receptor subunit induced antidepressant-like effects. In confirmation of the genetic data, acute and chronic blockade of GABAB receptor decreased immobility in the forced swim test. Finnaly, we also demonstrated that pharmacological activation of GABAB receptor counteracted both molecular and behavioural adaptations elicited by a single administration of cocaine or nicotine. Altogether, our data support the role of GABAB receptor in anxiety, depression and addiction and that GABAB receptor might be considered as one of the most promising therapeuthic targets for treating these disorders

Behavioral Characterization of the Novel GABA B Receptor- Positive Modulator GS39783 (N,NЈ-Dicyclopentyl-2- methylsulfanyl-5-nitro-pyrimidine-4,6-diamine): Anxiolytic-Like Activity without Side Effects Associated with Baclofen or Benzodiazepines

ABSTRACT The role of GABA B receptors in various behavioral processes has been largely defined using the prototypical GABA B receptor agonist baclofen. However, baclofen induces sedation, hypothermia and muscle relaxation, which may interfere with its use in behavioral paradigms. Although there is much evidence for a role of the inhibitory neurotransmitter GABA in the pathophysiology of anxiety, the role of GABA B receptors in these disorders is largely unclear. We recently identified GS39783 (N,NЈ-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine) as a selective allosteric positive modulator at GABA B receptors. The aim of the present study was to broadly characterize the effects of GS39783 in well-validated rodent models for motor activity, cognition, and anxiety. The following tests were included: locomotor activity in rats and mice, rotarod and traction tests (including determinations of core temperature) in mice, passive avoidance in mice and rats, elevated plus maze in rats, elevated zero maze in mice and rats, stress-induced hyperthermia in mice, and pentobarbital-and ethanol-induced sleep in mice. Unlike baclofen and/or the benzodiazepine chlordiazepoxide, GS39783 had no effect in any of the tests for locomotion, cognition, temperature, or narcosis. Most interestingly, GS39783 had anxiolytic-like effects in all the tests used. Overall, the data obtained here suggest that positive modulation of GABA B receptors may serve as a novel therapeutic strategy for the development of anxiolytics, with a superior side effect profile to both baclofen and benzodiazepines

Epilepsy and intellectual disability linked protein Shrm4 interaction with GABA B Rs shapes inhibitory neurotransmission

Shrm4, a protein expressed only in polarized tissues, is encoded by the KIAA1202 gene, whose mutations have been linked to epilepsy and intellectual disability. However, a physiological role for Shrm4 in the brain is yet to be established. Here, we report that Shrm4 is localized to synapses where it regulates dendritic spine morphology and interacts with the C terminus of GABA B receptors (GABA B Rs) to control their cell surface expression and intracellular trafficking via a dynein-dependent mechanism. Knockdown of Shrm4 in rat severely impairs GABA B R activity causing increased anxiety-like behaviour and susceptibility to seizures. Moreover, Shrm4 influences hippocampal excitability by modulating tonic inhibition in dentate gyrus granule cells, in a process involving crosstalk between GABA B Rs and extrasynaptic \uce-subunit-containing GABA A Rs. Our data highlights a role for Shrm4 in synaptogenesis and in maintaining GABA B R-mediated inhibition, perturbation of which may be responsible for the involvement of Shrm4 in cognitive disorders and epilepsy

Foxp2 Regulates Gene Networks Implicated in Neurite Outgrowth in the Developing Brain

Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP–chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections

Prevention of 5-hydroxytryptamine2C receptor RNA editing and alternate splicing in C57BL/6 mice activates the hypothalamic-pituitary-adrenal axis and alters mood

The 5-hydroxytryptamine2C (5-HT)2C receptor is widely implicated in the aetiology of affective and eating disorders as well as regulation of the hypothalamo-pituitary-adrenal axis. Signalling through this receptor is regulated by A-to-I RNA editing, affecting three amino acids in the protein sequence, with unedited transcripts encoding a receptor (INI) that, in vitro, is hyperactive compared with edited isoforms. Targeted alteration (knock-in) of the Htr2c gene to generate 'INI' mice with no alternate splicing, solely expressing the full-length unedited isoform, did not produce an overt metabolic phenotype or altered anxiety behaviour, but did display reduced depressive-like and fear-associated behaviours. INI mice exhibited a hyperactive hypothalamo-pituitary-adrenal axis, with increased nadir plasma corticosterone and corticotrophin-releasing hormone expression in the hypothalamus but responded normally to chronic stress and showed normal circadian activity and activity in a novel environment. The circadian patterns of 5-HT2C receptor mRNA and mbii52, a snoRNA known to regulate RNA editing and RNA splicing of 5-HT2C receptor pre-mRNA, were altered in INI mice compared with wild-type control mice. Moreover, levels of 5-HT1A receptor mRNA were increased in the hippocampus of INI mice. These gene expression changes may underpin the neuroendocrine and behavioural changes observed in INI mice. However, the phenotype of INI mice was not consistent with a globally hyperactive INI receptor encoded by the unedited transcript in the absence of alternate splicing. Hence, the in vivo outcome of RNA editing may be neuronal cell type specific

Fetal brain 11β-hydroxysteroid dehydrogenase type 2 selectively determines programming of adult depressive-like behaviors and cognitive function, but not anxiety behaviors in male mice

Stress or elevated glucocorticoids during sensitive windows of fetal development increase the risk of neuropsychiatric disorders in adult rodents and humans, a phenomenon known as glucocorticoid programming. 11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2), which catalyses rapid inactivation of glucocorticoids in the placenta, controls access of maternal glucocorticoids to the fetal compartment, placing it in a key position to modulate glucocorticoid programming of behavior. However, the importance of the high expression of 11β-HSD2 within the midgestational fetal brain is unknown. To examine this, a brain-specific knockout of 11β-HSD2 (HSD2BKO) was generated and compared to wild-type littermates. HSD2BKO have markedly diminished fetal brain 11β-HSD2, but intact fetal body and placental 11β-HSD2 and normal fetal and placental growth. Despite normal fetal plasma corticosterone, HSD2BKO exhibit elevated fetal brain corticosterone levels at midgestation. As adults, HSD2BKO show depressive-like behavior and have cognitive impairments. However, unlike complete feto-placental deficiency, HSD2BKO show no anxiety-like behavioral deficits. The clear mechanistic separation of the programmed components of depression and cognition from anxiety implies distinct mechanisms of pathogenesis, affording potential opportunities for stratified interventions

Evaluation of commercial soy sauce koji strains of Aspergillus oryzae for γ-aminobutyric acid (GABA) production

In this study, four selected commercial strains of Aspergillus oryzae were collected from soy sauce koji. These A. oryzae strains designated as NSK, NSZ, NSJ and NST shared similar morphological characteristics with the reference strain (A. oryzae FRR 1675) which confirmed them as A. oryzae species. They were further evaluated for their ability to produce γ-aminobutyric acid (GABA) by cultivating the spore suspension in a broth medium containing 0.4 % (w/v) of glutamic acid as a substrate for GABA production. The results showed that these strains were capable of producing GABA; however, the concentrations differed significantly (P < 0.05) among themselves. Based on the A. oryzae strains, highest GABA concentration was obtained from NSK (194 mg/L) followed by NSZ (63 mg/L), NSJ (51.53 mg/L) and NST (31.66 mg/L). Therefore, A. oryzae NSK was characterized and the sequence was found to be similar to A. oryzae and A. flavus with 99 % similarity. The evolutionary distance (K nuc) between sequences of identical fungal species was calculated and a phylogenetic tree prepared from the K nuc data showed that the isolate belonged to the A. oryzae species. This finding may allow the development of GABA-rich ingredients using A. oryzae NSK as a starter culture for soy sauce production

Mechanically-induiced differentiation of stem cells in three dimensions by quantitative optical imaging study

Une cellule souche est une cellule qui a la capacité de s'auto-renouveler, de se multiplier à l'infini et de se différencier en des cellules spécialisées. Placée dans son microenvironnement, elle est soumise à une myriade de signaux chimiques et physiques qui régulent son destin cellulaire. De nombreuses études, conduites in vitro sur des cellules souches isolées ou en monocouche, ont mis en évidence le rôle crucial des forces extérieures ou des propriétés mécaniques des substrats dans le déclenchement et l’orientation de la différenciation cellulaire via des processus de mécanotransduction ou mécanosensation dont certains acteurs moléculaires ont été élucidés. La validation ou infirmation de ces mécanismes dans un environnement tridimensionnel (3D), qui récapitule plus fidèlement l’architecture des tissus biologiques, exige cependant le développement de nouvelles approches. En effet, il n’y a plus de substrat proprement dit, et comment appliquer ces forces pendant une durée suffisante pour influencer la différenciation.Notre stratégie est de générer des organoïdes encapsulés dans des coques d'hydrogel transparentes, perméables et élastiques. Ce système de culture cellulaire à 3D nous permet d’étudier par microscopie optique la croissance de l’organoïde en conditions de croissance libre ou confinée élastiquement. Pour permettre le suivi en temps réel de la différenciation, nous avons choisi comme système cellulaire d’étude des cellules souches adipeuses (ou pré-adipocytes) qui ont la particularité de subir une forte augmentation de volume (hypertrophie) due à l’apparition et l’accumulation de gouttelettes lipidiques au cours de leur différenciation en adipocytes. Ainsi, la croissance de l’organoïde est a priori déterminée par la combinaison de deux effets : l’augmentation du nombre de cellules souches adipeuses par prolifération et l’augmentation du volume cellulaire des pré-adipocytes en cours de différenciation. Par une méthode tout-optique associant suivi morphométrique par microscopie à contraste de phase et suivi structural par microscopie de fluorescence à sectionnement optique, nous avons montré, en l’absence de signal chimique adipogénique, que la prolifération cellulaire est considérablement réduite à 3D par rapport à 2D et que le processus de différenciation des cellules souches adipeuses est déclenché par simple agrégation dès la formation de l’organoïde. Alors que cette différenciation « spontanée » n’apparait à 2D qu’une fois la confluence atteinte et la prolifération complètement arrêtée, la formation d’un organoïde place intrinsèquement les cellules dans un contexte de confluence. En bref, par le développement d’une pipeline méthodologique d’analyse d’organoïdes en cours de différenciation, nous apportons, sur une étude de cas, une preuve nouvelle de l’influence cruciale de la dimensionalité sur le destin cellulaire. Enfin, ce travail se termine par une partie exploratoire indépendante autour de nouvelles méthodes d’encapsulation visant à concevoir des capsules d’hydrogel présentant une plus grande variété de propriétés physico-chimiques.A stem cell is a cell that has the ability to self-renew and differentiate into specialised cells. Placed in its microenvironment, a stem cell is exposed to a myriad of chemical and physical cues that regulate its cellular fate. Numerous studies, performed in vitro on individual cells or cell monolayers, have highlighted the crucial role of external forces or mechanical properties of substrates in triggering and guiding cell differentiation via mechanostransduction or mechanosensation processes, some of whose molecular players have been elucidated. The validation or disproof of these mechanisms in a three-dimensional (3D) environment, which more faithfully recapitulates the architecture of biological tissues, requires however the development of new approaches. Indeed, there is no longer a substrate as such, and the question arises as how to apply these forces for a sufficient duration to influence differentiation.Our strategy is to generate organoids encapsulated in transparent, permeable and elastic hydrogel shells. This 3D cell culture system allows to study organoid growth under free or elastically confined growth conditions by optical microscopy. To perform real-time monitoring of differentiation, we selected adipose stem cells (or pre-adipocytes), which have the peculiarity of drastically increasing in volume (hypertrophy) because of the appearance and accumulation of lipid droplets during their differentiation into adipocytes. Thus, the growth of the organoid is a priori determined by the combination of two effects: the increase in the number of adipose stem cells by proliferation and the increase in cell volume of the differentiating pre-adipocytes. Using an all-optical method combining morphometric monitoring by phase contrast microscopy and structural monitoring by fluorescence optical sectioning microscopy, we have shown, in the absence of a chemical adipogenic cue, that cell proliferation is considerably reduced in 3D compared to 2D and that the differentiation process of adipose stem cells is solely triggered by aggregation upon organoid formation. Whereas this "spontaneous" differentiation only occurs in 2D once confluence is reached and proliferation has stopped completely, organoid formation intrinsically places the cells in a confluent context. In conclusion, through the development of a methodological pipeline for the analysis of differentiating organoids, we provide new evidence for the crucial influence of dimensionality on cell fate in a case study. Finally, this thesis work ends with an independent exploratory part around new encapsulation methods aiming at designing hydrogel capsules with a wider variety of physicochemical properties

Différenciation induite mécaniquement de cellules souches à trois dimensions : étude par imagerie optique quantitative.

A stem cell is a cell that has the ability to self-renew and differentiate into specialised cells. Placed in its microenvironment, a stem cell is exposed to a myriad of chemical and physical cues that regulate its cellular fate. Numerous studies, performed in vitro on individual cells or cell monolayers, have highlighted the crucial role of external forces or mechanical properties of substrates in triggering and guiding cell differentiation via mechanostransduction or mechanosensation processes, some of whose molecular players have been elucidated. The validation or disproof of these mechanisms in a three-dimensional (3D) environment, which more faithfully recapitulates the architecture of biological tissues, requires however the development of new approaches. Indeed, there is no longer a substrate as such, and the question arises as how to apply these forces for a sufficient duration to influence differentiation.Our strategy is to generate organoids encapsulated in transparent, permeable and elastic hydrogel shells. This 3D cell culture system allows to study organoid growth under free or elastically confined growth conditions by optical microscopy. To perform real-time monitoring of differentiation, we selected adipose stem cells (or pre-adipocytes), which have the peculiarity of drastically increasing in volume (hypertrophy) because of the appearance and accumulation of lipid droplets during their differentiation into adipocytes. Thus, the growth of the organoid is a priori determined by the combination of two effects: the increase in the number of adipose stem cells by proliferation and the increase in cell volume of the differentiating pre-adipocytes. Using an all-optical method combining morphometric monitoring by phase contrast microscopy and structural monitoring by fluorescence optical sectioning microscopy, we have shown, in the absence of a chemical adipogenic cue, that cell proliferation is considerably reduced in 3D compared to 2D and that the differentiation process of adipose stem cells is solely triggered by aggregation upon organoid formation. Whereas this "spontaneous" differentiation only occurs in 2D once confluence is reached and proliferation has stopped completely, organoid formation intrinsically places the cells in a confluent context. In conclusion, through the development of a methodological pipeline for the analysis of differentiating organoids, we provide new evidence for the crucial influence of dimensionality on cell fate in a case study. Finally, this thesis work ends with an independent exploratory part around new encapsulation methods aiming at designing hydrogel capsules with a wider variety of physicochemical properties.Une cellule souche est une cellule qui a la capacité de s'auto-renouveler, de se multiplier à l'infini et de se différencier en des cellules spécialisées. Placée dans son microenvironnement, elle est soumise à une myriade de signaux chimiques et physiques qui régulent son destin cellulaire. De nombreuses études, conduites in vitro sur des cellules souches isolées ou en monocouche, ont mis en évidence le rôle crucial des forces extérieures ou des propriétés mécaniques des substrats dans le déclenchement et l’orientation de la différenciation cellulaire via des processus de mécanotransduction ou mécanosensation dont certains acteurs moléculaires ont été élucidés. La validation ou infirmation de ces mécanismes dans un environnement tridimensionnel (3D), qui récapitule plus fidèlement l’architecture des tissus biologiques, exige cependant le développement de nouvelles approches. En effet, il n’y a plus de substrat proprement dit, et comment appliquer ces forces pendant une durée suffisante pour influencer la différenciation.Notre stratégie est de générer des organoïdes encapsulés dans des coques d'hydrogel transparentes, perméables et élastiques. Ce système de culture cellulaire à 3D nous permet d’étudier par microscopie optique la croissance de l’organoïde en conditions de croissance libre ou confinée élastiquement. Pour permettre le suivi en temps réel de la différenciation, nous avons choisi comme système cellulaire d’étude des cellules souches adipeuses (ou pré-adipocytes) qui ont la particularité de subir une forte augmentation de volume (hypertrophie) due à l’apparition et l’accumulation de gouttelettes lipidiques au cours de leur différenciation en adipocytes. Ainsi, la croissance de l’organoïde est a priori déterminée par la combinaison de deux effets : l’augmentation du nombre de cellules souches adipeuses par prolifération et l’augmentation du volume cellulaire des pré-adipocytes en cours de différenciation. Par une méthode tout-optique associant suivi morphométrique par microscopie à contraste de phase et suivi structural par microscopie de fluorescence à sectionnement optique, nous avons montré, en l’absence de signal chimique adipogénique, que la prolifération cellulaire est considérablement réduite à 3D par rapport à 2D et que le processus de différenciation des cellules souches adipeuses est déclenché par simple agrégation dès la formation de l’organoïde. Alors que cette différenciation « spontanée » n’apparait à 2D qu’une fois la confluence atteinte et la prolifération complètement arrêtée, la formation d’un organoïde place intrinsèquement les cellules dans un contexte de confluence. En bref, par le développement d’une pipeline méthodologique d’analyse d’organoïdes en cours de différenciation, nous apportons, sur une étude de cas, une preuve nouvelle de l’influence cruciale de la dimensionalité sur le destin cellulaire. Enfin, ce travail se termine par une partie exploratoire indépendante autour de nouvelles méthodes d’encapsulation visant à concevoir des capsules d’hydrogel présentant une plus grande variété de propriétés physico-chimiques

Mechanically-induiced differentiation of stem cells in three dimensions by quantitative optical imaging study

Une cellule souche est une cellule qui a la capacité de s'auto-renouveler, de se multiplier à l'infini et de se différencier en des cellules spécialisées. Placée dans son microenvironnement, elle est soumise à une myriade de signaux chimiques et physiques qui régulent son destin cellulaire. De nombreuses études, conduites in vitro sur des cellules souches isolées ou en monocouche, ont mis en évidence le rôle crucial des forces extérieures ou des propriétés mécaniques des substrats dans le déclenchement et l’orientation de la différenciation cellulaire via des processus de mécanotransduction ou mécanosensation dont certains acteurs moléculaires ont été élucidés. La validation ou infirmation de ces mécanismes dans un environnement tridimensionnel (3D), qui récapitule plus fidèlement l’architecture des tissus biologiques, exige cependant le développement de nouvelles approches. En effet, il n’y a plus de substrat proprement dit, et comment appliquer ces forces pendant une durée suffisante pour influencer la différenciation.Notre stratégie est de générer des organoïdes encapsulés dans des coques d'hydrogel transparentes, perméables et élastiques. Ce système de culture cellulaire à 3D nous permet d’étudier par microscopie optique la croissance de l’organoïde en conditions de croissance libre ou confinée élastiquement. Pour permettre le suivi en temps réel de la différenciation, nous avons choisi comme système cellulaire d’étude des cellules souches adipeuses (ou pré-adipocytes) qui ont la particularité de subir une forte augmentation de volume (hypertrophie) due à l’apparition et l’accumulation de gouttelettes lipidiques au cours de leur différenciation en adipocytes. Ainsi, la croissance de l’organoïde est a priori déterminée par la combinaison de deux effets : l’augmentation du nombre de cellules souches adipeuses par prolifération et l’augmentation du volume cellulaire des pré-adipocytes en cours de différenciation. Par une méthode tout-optique associant suivi morphométrique par microscopie à contraste de phase et suivi structural par microscopie de fluorescence à sectionnement optique, nous avons montré, en l’absence de signal chimique adipogénique, que la prolifération cellulaire est considérablement réduite à 3D par rapport à 2D et que le processus de différenciation des cellules souches adipeuses est déclenché par simple agrégation dès la formation de l’organoïde. Alors que cette différenciation « spontanée » n’apparait à 2D qu’une fois la confluence atteinte et la prolifération complètement arrêtée, la formation d’un organoïde place intrinsèquement les cellules dans un contexte de confluence. En bref, par le développement d’une pipeline méthodologique d’analyse d’organoïdes en cours de différenciation, nous apportons, sur une étude de cas, une preuve nouvelle de l’influence cruciale de la dimensionalité sur le destin cellulaire. Enfin, ce travail se termine par une partie exploratoire indépendante autour de nouvelles méthodes d’encapsulation visant à concevoir des capsules d’hydrogel présentant une plus grande variété de propriétés physico-chimiques.A stem cell is a cell that has the ability to self-renew and differentiate into specialised cells. Placed in its microenvironment, a stem cell is exposed to a myriad of chemical and physical cues that regulate its cellular fate. Numerous studies, performed in vitro on individual cells or cell monolayers, have highlighted the crucial role of external forces or mechanical properties of substrates in triggering and guiding cell differentiation via mechanostransduction or mechanosensation processes, some of whose molecular players have been elucidated. The validation or disproof of these mechanisms in a three-dimensional (3D) environment, which more faithfully recapitulates the architecture of biological tissues, requires however the development of new approaches. Indeed, there is no longer a substrate as such, and the question arises as how to apply these forces for a sufficient duration to influence differentiation.Our strategy is to generate organoids encapsulated in transparent, permeable and elastic hydrogel shells. This 3D cell culture system allows to study organoid growth under free or elastically confined growth conditions by optical microscopy. To perform real-time monitoring of differentiation, we selected adipose stem cells (or pre-adipocytes), which have the peculiarity of drastically increasing in volume (hypertrophy) because of the appearance and accumulation of lipid droplets during their differentiation into adipocytes. Thus, the growth of the organoid is a priori determined by the combination of two effects: the increase in the number of adipose stem cells by proliferation and the increase in cell volume of the differentiating pre-adipocytes. Using an all-optical method combining morphometric monitoring by phase contrast microscopy and structural monitoring by fluorescence optical sectioning microscopy, we have shown, in the absence of a chemical adipogenic cue, that cell proliferation is considerably reduced in 3D compared to 2D and that the differentiation process of adipose stem cells is solely triggered by aggregation upon organoid formation. Whereas this "spontaneous" differentiation only occurs in 2D once confluence is reached and proliferation has stopped completely, organoid formation intrinsically places the cells in a confluent context. In conclusion, through the development of a methodological pipeline for the analysis of differentiating organoids, we provide new evidence for the crucial influence of dimensionality on cell fate in a case study. Finally, this thesis work ends with an independent exploratory part around new encapsulation methods aiming at designing hydrogel capsules with a wider variety of physicochemical properties