京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23611号 / 医科博第134号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 小川 誠司, 教授 山中 伸弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cell dedifferentiation is a cell fate switching process in which a differentiated cell reverts to a status with competence for cell division and organ regeneration like an embryonic stem cell. Although the phenomenon of cell dedifferentiation has been known for over two and a half centuries in plants, little is known of the underlying mechanisms. Here, the proteome map of Arabidopsis cotyledons has been established and investigated the dynamic change of the cotyledon proteome in the time course of cell dedifferentiation. Among the 353 distinct genes, corresponding to 500 2-DE gel protein spots identified with high confidence, 12% have over twofold differential regulations within the first 48 h of induction of cell dedifferentiation. The distributions of these genes among different Gene Ontology categories and gene differential regulations within each of the categories have been examined. In addition, the cotyledon phosphoproteome has been investigated using Pro-Q Diamond Phosphoprotein in Gel Stain followed by mass spectrometry analyses. Among the 53 identified putative phosphoproteins, nine are differentially regulated during cell dedifferentiation. Arabidopsis cotyledon proteome at four different time points after the induction of cell dedifferentiation with MudPIT approach has been investigated and analyzed the protein quantity change using two labelree methods, the Spectral Count (SC) and SEQUEST Cross Correlation Coefficient (ÓXcorr) methods. Among the 662 MudPIT identified proteins, one hundred forty eight displayed differential regulation. The up-regulated proteins include transcription factors, calmodulins, translational regulators, and stress response proteins. The Spectral Count and the cross correlation coefficient quantification results are highly consistent in over 81% of the differentially regulated proteins. These studies have provided significant new insight into cell dedifferentiation process in Arabidopsis thaliana and also enhanced the Arabidopsis cotyledon proteome database established using gel based and non gel based methods. The results show that cell dedifferentiation involves extensive protein quantitative and qualitative changes in almost every cellular compartment and cellular process. Proteins like 14-3-3 proteins, Translational controlled tumor protein (TCTP) and its possible interaction protein-Translational elongation factor eEF1 alpha chain, GTP binding nuclear protein RAN2, GTP binding protein SAR1B and several other hypothetical and expressed proteins and nine other phosphoproteins showed significant differential expression during early dedifferentiation. Deciphering the molecular mechanisms regulating the cellular dedifferentiation certainly enhances the understandings and mechanisms of reprogramming all types of differentiated cells including animal cells.

A família Bromeliaceae apresenta importância ecológica e econômica, desta forma, o desenvolvimento de protocolos para a micropropagação de espécies dessa família, faz-se necessário, a fim de suprir sua demanda comercial e mesmo ecológica. A escolha do meio de cultura e do explante utilizado durante a micropropagação são fundamentais para um protocolo eficaz. Nesse contexto, o objetivo deste estudo foi avaliar as diferenças quantitativas e qualitativas no desenvolvimento de explantes de Neoregelia sp em meios de cultura e monitorar as vias morfogênicas dos propágulos obtidos em explantes foliares. Para tanto, brotos de microcepas e explantes foliares procedentes de um micro jardim clonal, foram transferidos para os meios de cultura de multiplicação MS, ½ MS e WPM, todos suplementados com 0,050 mg.L-1 ANA e 0,50 mg.L-1 de BAP, onde foram mantidos por 120 dias e submetidos a diversas análises morfofisiológicas. Paralelamente, explantes foliares foram mantidos em meio de cultura MS de multiplicação para o monitoramento das vias morfogênicas durante os processos regenerativos. Para os experimentos com brotos de microcepas verificou-se que o meio de cultura MS proporcionou a melhor taxa de multiplicação, maior crescimento dos brotos, obtendo os valores mais elevados de peso de matéria fresca e seca, além disso, apresentaram maior acúmulo de nitrogênio total e proteico. No entanto, os meios de cultura ½ MS e WPM promoveram uma taxa de multiplicação semelhante a do MS, mas com brotos menores e menos vigorosos, porém, mais homogêneos, com isso, na dependência do objetivo do cultivo in vitro, não deve ser desconsiderada a possibilidade de utilização dos meios de cultura ½ MS e WPM. Os explantes foliares não se desenvolveram bem no meio de cultura WPM, não havendo diferença entre os meios MS e ½ MS, visto que ambos apresentaram resultados satisfatórios. As análises histológicas e histoquímicas identificaram células parenquimáticas, que atuam como células-tronco, manifestando capacidade morfogênica para toti ou pluripotência, dando origem respectivamente a embriões somáticos e gemas adventícias, em resposta aos estímulos in vitro. / The Bromeliaceae family has an ecological and economic importance, therefore, the protocols development for micropropagation of species of this family becomes necessary in order to meet its business and even its ecological demand. The choice of culture medium and the explant used during micropropagation are essential for an effective protocol. Thus, the aim of this study was to evaluate the quantitative and qualitative differences in the explants development of Neoregelia sp in the culture media and monitor the morphogenetic pathways of obtained propagules from leaf explants. Consequently, shoots and leaf explants coming from microcloning garden were transferred to the MS, ½ MS and WPM multiplication culture media, all supplemented with 0.050 mg.L-1 NAA and 0.50 mg.L-1 BAP, where they were held for 120 days and submitted to morphological and physiological analysis. Therefore, leaf explants were kept on MS-medium multiplication for monitoring morphogenetic pathways during the regenerative processes. Furthermore, MS medium showed the best multiplication rate for the sprouts of the microstumps, increased growth of shoots, obtaining the highest values of fresh and dry matter weight, and also showed higher accumulation of total nitrogen and protein. However, the ½ MS and WPM culture media promoted a similar multiplication rate to the MS medium, with the development of the smaller and less vigorous shoots, but with greater homogeneity. This way, depending on the purpose of in vitro culture, their use in the micropropagation for this species should not be disregarded. The leaf explants are not well developed in WPM medium, and don\'t had significant difference between the MS and ½ MS culture medium, as both showed satisfactory results. The histological and histochemical analysis identified the presence of the parenchymatic cells, which act as stem cells, expressing morphogenic ability for toti or pluripotency, leading respectively to somatic embryogenesis or adventitious organogenesis in response to in vitro stimuli.

A família Bromeliaceae apresenta importância ecológica e econômica, desta forma, o desenvolvimento de protocolos para a micropropagação de espécies dessa família, faz-se necessário, a fim de suprir sua demanda comercial e mesmo ecológica. A escolha do meio de cultura e do explante utilizado durante a micropropagação são fundamentais para um protocolo eficaz. Nesse contexto, o objetivo deste estudo foi avaliar as diferenças quantitativas e qualitativas no desenvolvimento de explantes de Neoregelia sp em meios de cultura e monitorar as vias morfogênicas dos propágulos obtidos em explantes foliares. Para tanto, brotos de microcepas e explantes foliares procedentes de um micro jardim clonal, foram transferidos para os meios de cultura de multiplicação MS, ½ MS e WPM, todos suplementados com 0,050 mg.L-1 ANA e 0,50 mg.L-1 de BAP, onde foram mantidos por 120 dias e submetidos a diversas análises morfofisiológicas. Paralelamente, explantes foliares foram mantidos em meio de cultura MS de multiplicação para o monitoramento das vias morfogênicas durante os processos regenerativos. Para os experimentos com brotos de microcepas verificou-se que o meio de cultura MS proporcionou a melhor taxa de multiplicação, maior crescimento dos brotos, obtendo os valores mais elevados de peso de matéria fresca e seca, além disso, apresentaram maior acúmulo de nitrogênio total e proteico. No entanto, os meios de cultura ½ MS e WPM promoveram uma taxa de multiplicação semelhante a do MS, mas com brotos menores e menos vigorosos, porém, mais homogêneos, com isso, na dependência do objetivo do cultivo in vitro, não deve ser desconsiderada a possibilidade de utilização dos meios de cultura ½ MS e WPM. Os explantes foliares não se desenvolveram bem no meio de cultura WPM, não havendo diferença entre os meios MS e ½ MS, visto que ambos apresentaram resultados satisfatórios. As análises histológicas e histoquímicas identificaram células parenquimáticas, que atuam como células-tronco, manifestando capacidade morfogênica para toti ou pluripotência, dando origem respectivamente a embriões somáticos e gemas adventícias, em resposta aos estímulos in vitro. / The Bromeliaceae family has an ecological and economic importance, therefore, the protocols development for micropropagation of species of this family becomes necessary in order to meet its business and even its ecological demand. The choice of culture medium and the explant used during micropropagation are essential for an effective protocol. Thus, the aim of this study was to evaluate the quantitative and qualitative differences in the explants development of Neoregelia sp in the culture media and monitor the morphogenetic pathways of obtained propagules from leaf explants. Consequently, shoots and leaf explants coming from microcloning garden were transferred to the MS, ½ MS and WPM multiplication culture media, all supplemented with 0.050 mg.L-1 NAA and 0.50 mg.L-1 BAP, where they were held for 120 days and submitted to morphological and physiological analysis. Therefore, leaf explants were kept on MS-medium multiplication for monitoring morphogenetic pathways during the regenerative processes. Furthermore, MS medium showed the best multiplication rate for the sprouts of the microstumps, increased growth of shoots, obtaining the highest values of fresh and dry matter weight, and also showed higher accumulation of total nitrogen and protein. However, the ½ MS and WPM culture media promoted a similar multiplication rate to the MS medium, with the development of the smaller and less vigorous shoots, but with greater homogeneity. This way, depending on the purpose of in vitro culture, their use in the micropropagation for this species should not be disregarded. The leaf explants are not well developed in WPM medium, and don\'t had significant difference between the MS and ½ MS culture medium, as both showed satisfactory results. The histological and histochemical analysis identified the presence of the parenchymatic cells, which act as stem cells, expressing morphogenic ability for toti or pluripotency, leading respectively to somatic embryogenesis or adventitious organogenesis in response to in vitro stimuli.

The zygote and blastomeres of cleavage stage mouse embryos have the capacity to differentiate to the embryonic and both extra-embryonic lineages and are considered functionally totipotent. Until now, it has not been possible to establish stable cell lines that resemble these totipotent-like cells. In this work, I hypothesised that by modulating signalling pathways known to be important in early embryonic development it may be possible to capture in vitro a self-renewing state that possessed features of pre-implantation blastomeres. I succeeded in formulating a novel hypothesis driven cell culture medium which allowed the establishment of a stem cell state that possessed expanded differentiation potential to the embryonic and both extra-embryonic lineages in vitro and in vivo. These cells were isolated directly from in vitro culture of mouse pre-implantation embryos or single cell blastomeres, reprogrammed from somatic cells or converted from mouse ES cells. With these cells, I generated single cell chimeras which demonstrated extensive contribution to all lineages in the developing organism providing additional evidence that this chemical medium maintained a homogenous stem cell population. I demonstrated that the transcriptome of these cells was enriched with an early pre-implantation blastomere signature, distinct from other rare published totipotent-like cells. Finally, I demonstrated that the same chemical formulation permitted the establishment in vitro of a human stem cell state that possessed expanded differentiation potential to the embryonic and extra-embryonic lineage in vitro. My work has shown for the first time that through chemical modulation of pathways implicated to be involved in pre-implantation development, a novel homogenous stem cell state that possesses a pre-implantation transcriptional signature and expanded differentiation potential to both the embryonic and extra-embryonic lineage can be established and maintained in vitro in both mouse and human, suggesting a possible interspecies conservation of the signalling networks involved in early embryonic development.

De l’organisme unicellulaire aux organismes multicellulaires complexes, la spécification cellulaires est un aspect fondamental de la biologie de l'adaptation et du développement. Les cellules souches pluripotentes (CSP) telles que les embryonnaires (CSE) fournissent un modèle approprié pour étudier les mécanismes de régulation et la spécification du sort des cellules chez les mammifères. Les ESC de souris sont connus pour être de nature hétérogènes et sont rapportées comme étant composées de multiples états de cellules souches ressemblant à des stades distincts du développement embryonnaire précoce, tels que totipotentes, pluripotentes, préparées et endoderme primitif. Malgré des études approfondies sur les CSE, les mécanismes moléculaires régulant leur hétérogénéité et l'état totipotent, en particulier, ne sont pas bien compris. Le travail présenté dans cette thèse utilise les CSE de souris comme modèle intéressant pour déterminer les mécanismes de signalisation et de régulation génique qui conduisent à l'hétérogénéité cellulaire et l'état cellulaire totipotent des CSE. Dans une première étude, nous avons utilisé la cytométrie en flux de masse pour analyser simultanément de multiples protéines régulatrices des cellules souches, en mettant l'accent sur les facteurs de transcription clés, les protéines de signalisation et les modificateurs de la chromatine qui régissent les CSE de souris. Les données de cytométrie en flux de masse ont révélé des variations dans les niveaux protéiques cellulaires individuels des régulateurs des cellules souches et ont souligné la vaste coactivation des voies de signalisation cellulaire dans des conditions de culture définies des CSE. De plus, l'application de la cytométrie en flux de masse a facilité l'identification d'états cellulaires distincts et de leurs caractéristiques moléculaires au sein des CSE, offrant des aperçus de leurs variations selon différentes conditions de culture, validant ainsi la présence d'hétérogénéité cellulaire dans les CSE de souris. Dans une deuxième étude, nous avons identifié la signalisation du facteur de croissance des os (BMP) comme inducteur de l'état totipotent. Nous avons également constaté que le rôle du BMP dans la totipotence est réprimé par la coactivation des voies FGF, NODAL et WNT. En inhibant ces voies coactivées, nous démontrons l'amélioration de l'induction de cellules totipotentes et la suppression des états préparés et d'endoderme primitif. Nous avons validé les changements d'état cellulaire au niveau cellulaire unique grâce à un séquençage d'ARNm à cellule unique. De plus, nous avons également démontré que les cellules totipotentes reprogrammées in vitro imitent les cellules totipotentes de l'embryon préimplantatoire avec la capacité de générer des blastocystes in vitro (Blastoïdes) et de s'intégrer dans les lignées embryonnaires et extra-embryonnaires chez la souris. Ensemble, ces résultats ont révélé les mécanismes de signalisation du BMP pour réguler à la fois l'état totipotent et l'hétérogénéité des CSE. Pour la troisième étude, nous avons utilisé les observations clés de nos données de cytométrie en flux de masse (première étude) pour évaluer le rôle des protéines régulatrices clés pour promouvoir l'état cellulaire totipotent. Ici, nous démontrons que NACC1, un régulateur transcriptionnel des CSE, agit également comme un régulateur important des cellules totipotentes. Après avoir identifié NACC1 comme un régulateur potentiel à partir de données de protéines cellulaires à cellule unique et de transcriptome en vrac, nous avons validé sa fonction en utilisant une suppression médiée par CRISPR en combinaison avec des conditions de reprogrammation cellulaire pluripotente à totipotente. Ensuite, nous avons intégré une combinaison d'approches génomiques pour étudier les changements au niveau du système dépendants de NACC1 dans le transcriptome, l'accessibilité à la chromatine et la liaison à l'ADN génomique. Ensemble, ces données ont révélé que NACC1 induit à la fois les programmes d'expression génique codant et de gènes de rétrotransposons pour promouvoir l'état cellulaire totipotent. Enfin, nous avons montré que NACC1 régule les éléments rétrotransposables MERVL-int et MT2_Mm pour moduler l'expression des gènes codants de l'état totipotent. En conclusion, cette thèse révèle la nature hétérogène des CSE de souris au niveau protéique à cellule unique, élucide le rôle significatif et les mécanismes de la voie de signalisation BMP pour réguler l'état totipotent et l'hétérogénéité des CSE, et dévoile les mécanismes de régulation génique dépendants de NACC1 pour promouvoir l'état totipotent. Ces résultats ouvrent la voie à des études ultérieures visant à comprendre la spécification de l'état des cellules souches et leur transition via la modulation des voies de signalisation / facteurs de transcription. De plus, ces mécanismes peuvent réguler l'état cellulaire totipotent chez l'homme, éclairant l'hétérogénéité cellulaire dans les CSE humaines et dans des contextes pathologiques, tels que le cancer. / From unicellular entities to intricate multicellular organisms, the omnipresent process of cell fate specification is a fundamental aspect of adaptation and developmental biology. Pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) provide a suitable model to study the regulatory mechanisms and cell fate specification in mammals. Intriguingly, mouse ESCs are known to be heterogenous in nature and are reported to consist of multiple stem cell states resembling distinct stages of early embryogenesis, such as totipotent, pluripotent, primed, and primitive endoderm. Despite extensive study of ESCs, the molecular mechanisms regulating their heterogeneity and the totipotent state in particular are not well understood. The work presented in this thesis utilizes mouse ESCs as an attractive model to delineate the signaling and gene regulatory mechanisms driving the cellular heterogeneity and the totipotent cell state of ESCs. In the first study, we utilized mass cytometry (cytometry by time of flight) to concurrently analyse multiple stem cell regulatory proteins, focusing on key transcription factors, signaling proteins, and chromatin modifiers that govern mouse ESCs. Mass cytometry data revealed variations in the single-cell protein levels of stem cell regulators and highlighted the extensive cross-activation of cell signaling pathways across defined culture conditions of ESCs. Furthermore, the application of mass cytometry facilitated the identification of distinct cell states and their molecular features within ESCs, offering insights into their variations across different culture conditions, thereby validating the presence of cellular heterogeneity in mouse ESCs. In the second study, we identified bone morphogenetic protein (BMP) signaling as an inducer of the totipotent state. We also found that, BMP’s role for totipotency is repressed by the cross-activation of FGF, NODAL, and WNT pathways. Through rational inhibition of these cross-activated pathways, we demonstrate the enhancement in the induction of totipotent cells and suppression of primed and primitive endoderm states. We validated the cell state changes at the single-cell level through single-cell mRNA sequencing. Furthermore, we also demonstrate that the in-vitro reprogrammed totipotent cells mimic the totipotent cells of preimplantation embryo with the potency to generate in-vitro blastocyst (Blastoids) and to integrate into both embryonic and extra-embryonic lineages in the mice. Together these results revealed BMP signaling mechanisms to regulate both the totipotent state and the heterogeneity of ESCs. For our third study, we utilized the key observations from our mass cytometry data (first study) to evaluate the role of key regulatory proteins to promote the totipotent cell state. Here, we demonstrate that NACC1, a transcriptional regulator of ESCs, also acts as an important regulator of totipotent cells. Following identification of NACC1 as a potential regulator from both single-cell protein and bulk transcriptome data, we validated its function using CRISPR-mediated knock-out in combination with pluripotent-to-totipotent cell reprogramming conditions. Next, we integrated a combination of genomic approaches to study the NACC1 dependent system’s level changes in the transcriptome, chromatin accessibility and genomic DNA binding. Together, these data revealed that NACC1 induces both the coding gene and retrotransposon gene expression programs to promote the totipotent cell state. Finally, we showed that NACC1 regulates MERVL-int and MT2_Mm retrotransposable elements to modulate the expression of coding genes of the totipotent state. In conclusion, this thesis reveals the heterogeneous nature of mouse ESCs at the single-cell protein level, elucidates the significant role and mechanisms of BMP signaling pathway to regulate the totipotent state and ESC heterogeneity, and unveils NACC1 dependent gene regulatory mechanisms to promote the totipotent state. These findings open the door for subsequent studies aimed at understanding stem cell state specification and their transition occurring via modulation of signaling pathways / transcription factors. Moreover, these mechanisms may regulate the totipotent cell state in humans, shedding light on the cellular heterogeneity in human ESCs and in disease contexts, such as cancer.