Os tecidos da polpa dentária e vibrissa são dois microambientes celulares que compartilham a mesma origem embrionária. Ambos possuem o seu nicho especifico pós-natal que abrigam células tronco adultas (CTA). O objetivo do trabalho foi investigar a expressão diferencial dos marcadores de células pluripotentes, mesenquimais e neuroepiteliais nas populações de células tronco isoladas das vibrissas (CTV) e polpa de dente (CTPD) de camundongos C57BL-6. Resultados obtidos no presente trabalho, utilizando o método de imunofluorescência, revelaram que as CTA de ambos tecidos expressam um amplo painel de marcadores de pluripotência (Oct4, Nanog e Sox2), mesenquimais (CD73, CD90 e CD105), hematopoiético (CD34), crista neural (CKit), neuronal (Nestina) e epitelial (Integrina α6, LGR5 e LGR6) e indica possível potencial destas células em diversas linhagens celulares. Desta forma, células isoladas destes tecidos podem ser interessantes para serem aplicadas em diversos tratamentos na medicina regenerativa. Portanto, o estudo comparativo da expressão de um amplo painel de marcadores de células tronco em CTV e CTPD pode vir a aumentar o leque de possibilidades de sua utilização na terapia celular. Com isso, as células isoladas de polpa dentária foram submetidas à análise de expressão de marcadores já citados e a outros conhecidamente positivos e específicos para os folículos piloso como Citoqueratina 15 (CK15), LRig1 e Blimp1. Foram realizados ensaios de imunofluorescência, imunohistoquímica, citometria de fluxo e RT-PCR. Os dados obtidos nos permitiram concluir, que as CTA isoladas das ambas as fontes são bastante semelhantes em relação ao seu imunofenótipo, porém as características da sua diferenciação precisam ainda ser analisadas. / The tissues of the dental pulp and vibrissae are two cellular microenvironments that share the same embryonic origin. Both have their postnatal specific niche that keep adult stem cells (ASC). The objective of this study was to investigate the differential expression of pluripotent markers, mesenchymal and neuroepithelial in populations of stem cells isolated from whiskers (WSC) and dental pulp (DPSC) C57BL-6 mice. Results obtained in this study, using immunofluorescence, revealed that the ASC both tissues express a broad panel of pluripotency markers (Oct4, Nanog and Sox2), mesenchymal cells (CD73, CD90 and CD105), hematopoietic (CD34), crest neural (cKIT), neuronal (Nestin) and epithelial (Integrin α6, LGR5 and LGR6) and indicates possible potential of these cells in several cell lines. Thus, isolated cells of these tissues may be interesting for application in various treatments in regenerative medicine. Therefore, the comparative study of the expression of a broad panel of stem cell markers in WSC and DPSC could increase the range of possibilities for their use in cell therapy. Thus, the isolated cells were subjected to the dental pulp marker expression analysis cited above and others known to be positive and specific to hair follicles as Cytokeratin 15 (CK15), and LRig1 Blimp1. Immunofluorescence assays were performed, immunohistochemistry, flow cytometry and RT-PCR. The data allowed us to conclude that the ASC isolated from both sources are quite similar with respect to their immunophenotype, but the characteristics of differentiation remain to be analyzed.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24485号 / 医博第4927号 / 新制||医||1063(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 江藤 浩之, 教授 湊谷 謙司, 教授 大鶴 繁 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

Given the rising demand for diverse cell types in regenerative and transfusion medicines, such as human induced pluripotent stem cell-derived neurons (iPSC-Ns), human T/chimeric antigen receptor (CAR) T cells, and human natural killer (NK) cells, the ability to cryopreserve cells has become increasingly important. In regenerative medicine, iPSC-Ns are powerful tools for treating and modelling neurodegenerative diseases. Moreover, transplants/transfusions of T/CAR T cells or NK cells offer promising treatment for numerous types of tumors, such as leukemia and multiple myeloma. Cryopreservation of cells at sub-zero temperatures (-80 to -196 °C) allows for the development of master cell banks that can be used for clinical applications. Conventional cryoprotective agents (CPAs), such as dimethylsulfoxide (DMSO) and glycerol, are utilized to protect cells from cryoinjuries associated with the freezing process. However, the use of high concentrations of DMSO (i.e., 10 to 20%) has been shown to be accompanied with toxic effects on patients receiving cell therapies if it is not removed or diluted prior to transfusion. Moreover, DMSO does not prevent the occurrence of the cryoinjury associated with ice recrystallization, which is one of the major causes of cell death/damage during cryopreservation. As a result, there is a surge of attention toward developing new non-toxic cryo-additives that inhibit ice recrystallization during cryopreservation to permit future advancement in regenerative and transfusion medicines. Moreover, the use of ice recrystallization inhibitors (IRIs) as novel CPAs has become a promising strategy to improve cell viability and function post-thaw. The Ben laboratory heavily invested in synthesizing several classes of carbohydrate-based small molecule IRIs (i.e., O-linked alkyl and aryl glycosides, and N-aryl-D-gluconamides), and studying the correlation between their IRI activity and molecular properties, such as polar surface area to molecular surface area (PSA/MSA) ratio. Moreover, compounds that belong to the O-linked aryl glycosides and N-aryl-D-gluconamides classes of IRIs have been shown to enhance the viability and functionality of red blood cells (RBCs), hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSCs) after thawing. Part of the research presented throughout this thesis focuses on structure-activity relationship (SAR) studies of alkyl pyranoses with modified alkyl chain lengths to explore any correlations between the IRI activity and the net polarity (i.e., PSA/MSA ratio) of the IRI candidates. O- and C-linked alkyl pyranose derivatives with different alkyl chain lengths were synthesized and their IRI activity was assessed using the modified splat cooling assay. While the IRI activity of the O- and C-linked alkyl glucosides did differ as the length of the alkyl chain increased, no correlation between the PSA/MSA ratios and their IRI activity was observed. In addition, this work allowed for investigation into the effect of the type of the glycosidic bond (i.e., C-O and C-C bonds) at the anomeric position, on the IRI activity of the different compounds. The O-linked alkyl glucosides appeared to be more IRI active than the C-linked compounds, suggesting the nature of the glycosidic bond is important for IRI activity. The second part of the research presented in this thesis focuses on examining the potential for IRIs to cryopreserve iPSC-Ns, T/CAR T cells, and NK cells. 2-fluorophenyl-D-gluconamides (2FA), which is one of the most active IRIs from the N-aryl-Dgluconamides, has shown promising results in maintaining a high number of viable and functional HSCs and iPSCs post-thaw, and therefore it was employed in the cryopreservation protocol of iPSC-Ns, human-derived T/CAR T cells, and human-derived NK cells. The efficacy of the cryopreservation protocol being constructed was evaluated by assessing the post-thaw viability and recovery rate, as well as the functionality of iPSCNs, T/CAR T cells, and NK cells post-thaw. These studies showed that protecting against ice recrystallization during cryopreservation with IRIs increases the number of viable and functional iPSC-Ns, and T/CAR T cells. It was also observed that employing IRI technology in the cryopreservation protocol of NK cells does not compromise their functionality compared to fresh, non-frozen NK cells. Overall, inhibition of ice recrystallization using IRIs appeared to enhance the cryopreservation outcomes of the different cell types, which will allow for the development of off-the-shelf cell therapy products and improvement of the delivery of efficacious cell products to clinics and hospitals.

Human induced pluripotent stem cells (iPSCs) represent a scalable cell source for the generation of hematopoietic progenitor cells (iHPCs); however, a lack of efficient iHPC expansion in vitro currently limits translational applications. To address this translational bottleneck, we assessed a panel of stem cell agonist cocktails (SCACs), originally developed to enhance cord-blood derived HSPC (CB-HSPC) expansion, on iHPC expansion. Three SCACs and GAS6 (X2A, X2A+GAS6, SM6, or SMA) were supplemented during iHPC differentiation and subsequent expansion using the STEMdiff™ Hematopoietic Kit. This monolayer differentiation strategy yielded a population of CD34⁺CD43⁺ and CD45⁺CD34⁺ iHPC. SCAC supplementation during iHPC differentiation yielded up to 2.5-fold higher frequency of CD34⁺CD43⁺ hematopoietic progenitors and up to 2.9-fold higher frequency of CD45⁺CD34⁺CD45RA⁻CD90⁺ HSC-like cells compared to non-treated controls. Subsequent SCAC supplementation during 2 weeks of expansion culture also significantly increased iHPC expansion (X2A+GAS6: 3.8-fold, X2A: 3.5-fold, SM6: 2.8-fold, SMA: 2.0-fold). The expanded iHPCs retained high levels of CD34⁺CD43⁺ expression but we observed an increase in the expansion of HSC-like cell fraction. The collective expansion observed with the SCACs was 1.5- to 2.8-fold higher than UM171 treatment alone. Furthermore, all SCAC-supplemented iHPCs retained multilineage potency, producing erythroid and granulocyte-macrophage progenitors in CFU assays. However, prolonged expansion, beyond 7 days, reduced multilineage potential, indicating a limited expansion window. Although optimal timing and composition of SCAC supplementation remains to be refined, these results highlight that exploiting the additive and synergistic effects of multiple small molecules represents a promising approach for enhancing iHPC expansion yields and biomanufacturing.

Mutations in TREX1, encoding three prime repair exonuclease 1, cause Aicardi-Goutières syndrome (AGS) 1, an autoinflammatory disease characterized by neurodegeneration and constitutive activation of the antiviral cytokine type I interferon. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts from two AGS patients with biallelic TREX1 mutations. These cell lines offer a unique resource to investigate disease processes in a cell-type specific manner.

A tranferência nuclear de células somáticas (TNCS) está sendo utilizada para produzir cavalos de elite. No entanto, durante este procedimento pode ocorrer a perfuração da zona pelúcida, levando, ocasionalmente, à secção da massa celular interna, e conseqüente derivação de gêmeos monozigóticos. Além de serem relatadas alterações no processo de imprinting genômico, que conduzem ao desenvolvimento de doenças. Com a descoberta da possibilidade de reprogramar as células somáticas a um estado de pluripotência (iPSCs), estas células passaram a ser muito utilizadas em pesquisas de neurociência. Contudo, também ocorrem modificações epigenéticas durante esta reprogramação celular. Portanto, nossas hipóteses são que os gêmeos eqüinos gerados pela TNCS podem levar às irregularidades no desenvolvimento do sistema nervoso. O padrão de metilação do SNRPN nas estruturas dos fetos muares clonados, e as células iPSCs são diferentes dos padrões encontrados nos muares analisados. A expressão dos genes SNRPN, Necdin e UBE3A são maiores no cérebro, enquanto a expressão do H19 é maior nas membranas extra-embrionárias. Em nosso estudo, obtivemos duas gestações gemelares equinas derivadas da TNCS, que foram interrompidas com 40 e 60 dias de gestação, e comparados com gestações eqüinas únicas de idade similar. Diferenças no comprimento entre os embriões gêmeos foram observadas aos 40 (2.0 e 2.2 cm 10%) e aos 60 (6,5 e 8,5 cm 24%) dias de gestação. Somente o plexo coróide do quarto ventrículo apresentou-se mais desenvolvido nos fetos com maior comprimento. Ao analisarmos fetos muares clonados em diferentes idades gestacionais e compará-los com muares, nos períodos embrionário, fetal e adulto, não foi observada diferença no padrão de metilação do gene SNRPN. No entanto, na décima passagem das células iPSC o padrão de metilação alterou, em relação aos muares estudados e ao padrão observado nos fibroblastos. Ao analisarmos os fetos clonados nas diferentes idades gestacionais observou-se no cérebro menor expressão dos gene H19 e UBE3A, e maior expressão do gene SNRPN. Contudo, a expressão do gene Necdin variou entre as estruturas estudadas. Em conclusão, apesar dos gêmeos eqüinos provenientes de TNCS diferirem quanto ao tamanho, morfologicamente são iguais. Dentre as estruturas cerebrais o plexo coróide se apresentou mais desenvolvido nos fetos de maior comprimento. Os fetos muares clonados não apresentaram diferença no padrão de metilação do gene SNRPN. No entanto, as iPSCs apresentaram alteração no padrão de metilação deste gene na décima passagem. Embora os genes SNRPN, Necdin e UBE3A sejam expressos no cérebro, o SNRPN apresentou-se prevalente nessa estrutura / The nuclear transfer of somatic cells (SCNT) is being used to produce elite horses. However, during this procedure can occur drilling of the zona pellucida, leading occasionally to the section of the inner cell mass, and subsequent derivation of monozygotic twins. Besides being related changes in genomic imprinting process, leading to the development of diseases. With the discovery of the possibility to reprogram somatic cells to a pluripotent state (iPSCs), these cells have become widely used in neuroscience research. However, also occur epigenetic changes during this cellular reprogramming. Therefore, our hypothesis is that equine twins caused by equine ART could lead to developmental irregularities of the nervous system. The patterns of SNRPN methylation in the structures of cloned mule fetuses and in iPSCs are different from the patterns found in the analyzed mules. And the expression of SNRPN, Necdin and UBE3A genes are higher in the brain, while the higher expression of H19 gene occurs in the extraembryonic membranes. In our study we derived two equine twin SCNT pregnancies that were interrupted at 40 and 60 days of gestation and compared to singleton fetuses of similar age. Differences in lengths between twin embryos were observed at both 40 (2.0 and 2.2 cm 10%) and 60 (6.5 and 8.5 cm 24%) days of gestation. Only the choroid plexus in the fourth ventricle more developed in the twins with the greatest length. Analyzing mules cloned fetuses at different gestational ages, and compare them with mules at embryonic, fetal and adult period; there was no difference in the pattern of methylation in SNRPN gene. However, in the tenth passage of the iPSCs the methylation pattern was altered in relation to the studied mules and the pattern observed in fibroblasts. When the cloned fetuses at different gestational ages were analyzed, the brain presented lower expression of H19 and UBE3A genes, and higher expression of SNRPN gene. However, the expression of Necdin gene varied among the structures studied. In conclusion, despite the twin horses from SCNT differ in size, they are morphologically identical. Among the brain structures the choroid plexus performed more developed in the fetuses of greater length. Cloned mules fetuses showed no difference in the pattern of methylation SNRPN gene. However, iPSCs have changes in the pattern of methylation of this gene in the tenth passage. Although SNRPN, Necdin and Ube3A genes are expressed in the brain, SNRPN is prevalent in this structure

A tranferência nuclear de células somáticas (TNCS) está sendo utilizada para produzir cavalos de elite. No entanto, durante este procedimento pode ocorrer a perfuração da zona pelúcida, levando, ocasionalmente, à secção da massa celular interna, e conseqüente derivação de gêmeos monozigóticos. Além de serem relatadas alterações no processo de imprinting genômico, que conduzem ao desenvolvimento de doenças. Com a descoberta da possibilidade de reprogramar as células somáticas a um estado de pluripotência (iPSCs), estas células passaram a ser muito utilizadas em pesquisas de neurociência. Contudo, também ocorrem modificações epigenéticas durante esta reprogramação celular. Portanto, nossas hipóteses são que os gêmeos eqüinos gerados pela TNCS podem levar às irregularidades no desenvolvimento do sistema nervoso. O padrão de metilação do SNRPN nas estruturas dos fetos muares clonados, e as células iPSCs são diferentes dos padrões encontrados nos muares analisados. A expressão dos genes SNRPN, Necdin e UBE3A são maiores no cérebro, enquanto a expressão do H19 é maior nas membranas extra-embrionárias. Em nosso estudo, obtivemos duas gestações gemelares equinas derivadas da TNCS, que foram interrompidas com 40 e 60 dias de gestação, e comparados com gestações eqüinas únicas de idade similar. Diferenças no comprimento entre os embriões gêmeos foram observadas aos 40 (2.0 e 2.2 cm 10%) e aos 60 (6,5 e 8,5 cm 24%) dias de gestação. Somente o plexo coróide do quarto ventrículo apresentou-se mais desenvolvido nos fetos com maior comprimento. Ao analisarmos fetos muares clonados em diferentes idades gestacionais e compará-los com muares, nos períodos embrionário, fetal e adulto, não foi observada diferença no padrão de metilação do gene SNRPN. No entanto, na décima passagem das células iPSC o padrão de metilação alterou, em relação aos muares estudados e ao padrão observado nos fibroblastos. Ao analisarmos os fetos clonados nas diferentes idades gestacionais observou-se no cérebro menor expressão dos gene H19 e UBE3A, e maior expressão do gene SNRPN. Contudo, a expressão do gene Necdin variou entre as estruturas estudadas. Em conclusão, apesar dos gêmeos eqüinos provenientes de TNCS diferirem quanto ao tamanho, morfologicamente são iguais. Dentre as estruturas cerebrais o plexo coróide se apresentou mais desenvolvido nos fetos de maior comprimento. Os fetos muares clonados não apresentaram diferença no padrão de metilação do gene SNRPN. No entanto, as iPSCs apresentaram alteração no padrão de metilação deste gene na décima passagem. Embora os genes SNRPN, Necdin e UBE3A sejam expressos no cérebro, o SNRPN apresentou-se prevalente nessa estrutura / The nuclear transfer of somatic cells (SCNT) is being used to produce elite horses. However, during this procedure can occur drilling of the zona pellucida, leading occasionally to the section of the inner cell mass, and subsequent derivation of monozygotic twins. Besides being related changes in genomic imprinting process, leading to the development of diseases. With the discovery of the possibility to reprogram somatic cells to a pluripotent state (iPSCs), these cells have become widely used in neuroscience research. However, also occur epigenetic changes during this cellular reprogramming. Therefore, our hypothesis is that equine twins caused by equine ART could lead to developmental irregularities of the nervous system. The patterns of SNRPN methylation in the structures of cloned mule fetuses and in iPSCs are different from the patterns found in the analyzed mules. And the expression of SNRPN, Necdin and UBE3A genes are higher in the brain, while the higher expression of H19 gene occurs in the extraembryonic membranes. In our study we derived two equine twin SCNT pregnancies that were interrupted at 40 and 60 days of gestation and compared to singleton fetuses of similar age. Differences in lengths between twin embryos were observed at both 40 (2.0 and 2.2 cm 10%) and 60 (6.5 and 8.5 cm 24%) days of gestation. Only the choroid plexus in the fourth ventricle more developed in the twins with the greatest length. Analyzing mules cloned fetuses at different gestational ages, and compare them with mules at embryonic, fetal and adult period; there was no difference in the pattern of methylation in SNRPN gene. However, in the tenth passage of the iPSCs the methylation pattern was altered in relation to the studied mules and the pattern observed in fibroblasts. When the cloned fetuses at different gestational ages were analyzed, the brain presented lower expression of H19 and UBE3A genes, and higher expression of SNRPN gene. However, the expression of Necdin gene varied among the structures studied. In conclusion, despite the twin horses from SCNT differ in size, they are morphologically identical. Among the brain structures the choroid plexus performed more developed in the fetuses of greater length. Cloned mules fetuses showed no difference in the pattern of methylation SNRPN gene. However, iPSCs have changes in the pattern of methylation of this gene in the tenth passage. Although SNRPN, Necdin and Ube3A genes are expressed in the brain, SNRPN is prevalent in this structure

ROS are frequently associated with deleterious effects caused by oxidative stress. Despite the harmful effects of non-specific oxidation, ROS also function as signal transduction molecules that regulate various biological processes, including stem cell proliferation and differentiation. Here we show that mitochondrial ROS level determines cell fate during differentiation of the pluripotent stem cell line P19. As stem cells in general, P19 cells are characterized by a low respiration activity, accompanied by a low level of ROS formation. Nevertheless, we found that P19 cells contain fully assembled mitochondrial electron transport chain supercomplexes (respirasomes), suggesting that low respiration activity may serve as a protective mechanism against ROS. Upon elevated mitochondrial ROS formation, the proliferative potential of P19 cells is decreased due to longer S phase of the cell cycle. Our data show that besides being harmful, mitochondrial ROS production regulates the differentiation potential of P19 cells: elevated mitochondrial ROS level favours trophoblast differentiation, whereas preventing neuron differentiation. Therefore, our results suggest that mitochondrial ROS level serves as an important factor that directs differentiation towards certain cell types while preventing others.

Les mutations somatiques sur le variant d’histone H3.3 et les régulateurs épigénétiques associés à H3.3 ont été identifiés dans 30 % des glioblastomes pédiatriques. Ces mutations sont caractérisées par des substitutions d'acides aminés à des positions spécifiques dans la région N-terminale de l'histone H3.3 telles que la glycine 34 en valine/arginine (G34V/R), l'alanine 29 en proline (A29P), ou une haplo-insuffisance de la protéine Zinc Finger MYND-Type Containing 11 (ZMYND11). ZMYND11 est un co-répresseur de la transcription qui se lie spécifiquement à H3.3K36me3 pour moduler l'activité de l'ARN polymérase II. De plus, il est intéressant de mentionner que l’interaction entre ZMYND11 et H3.3K36me3 est altérée lorsque le résidu G34 est muté en G34V. Récemment, les mutations germinales H3.3G34V, H3.3A29P et ZMYND11 ont été identifiées chez des patients présentant une déficience neurologique. Nous émettons l'hypothèse que les mutants H3.3G34V et H3.3A29P empêchent ZMYND11 de se lier à H3.3K36me3 et pourrait converger mécaniquement avec la perte de fonction de ZMYND11, ce qui perturberait la neurogenèse. À l'aide de la technologie CRISPR Cas9, nous avons généré des modèles mutants isogéniques à partir de cellules souches pluripotentes (iPSC) pour H3F3B-A29P, H3F3B-G34V et ZMYND11-knock-out (KO). Par la suite, nous avons stimulé la différenciation de ces modèles vers des lignées neuronales afin d’identifier si ces mutations affectent la neurogenèse. Enfin, en utilisant des méthodes de séquençage à haut-débit nous avons analysé le profil épigénomique et transcriptomique pour déterminer comment l’interaction entre ZMYND11 et H3K36me3 est perturbée et à quels degrés ces mutations impactent sur les modifications post-traductionnelles des histones. Ce projet permettra de mieux comprendre les fonctions de ZMYND11 sur le remodelage de la chromatine et sa fonction biologique au cours du développement cérébral. / Somatic mutations on the histone 3 variant H3.3 and H3.3-associated chromatin modifiers have been identified in 30% of pediatric high-grade gliomas (pHGG). The mutations are characterized by amino acid substitutions at specific positions within the histone H3.3 tail such as glycine 34 to valine/arginine (G34V/R), alanine 29 to proline (A29P), or haploinsufficiency of the chromatin reader Zinc Finger MYND-Type Containing 11 (ZMYND11). ZMYND11 is a transcriptional co-repressor that specifically reads H3.3K36me3 to modulate RNA polymerase II activity. Notably, binding of ZMYND11 to H3.3K36me3 is altered when G34 residue is mutated to G34V. Recently, germline mutations of H3.3G34V, H3.3A29P, and ZMYND11 have been identified in patients with Intellectual disability. We hypothesize that H3.3 G34V and H3.3A29P mutants impede the binding of ZMYND11 to H3.3K36me3 and may mechanistically converge with ZMYND11 loss-of-function mutation to perturb neurogenesis. Using CRISPR Cas9-mediated gene editing, we will generate isogenic human induced pluripotent stem cell (iPSC) models for H3F3B-A29P, H3F3B-G34V and ZMYND11-KO, and perform in vitro neural differentiation to identify whether specific neural lineages are affected. Next, using epigenomic and transcriptomic profiling we will study whether binding between ZMYND11 and H3K36me3 is disrupted, and the downstream impact on Post-Translational Modifications of histones (PTMs) and transcription. This project will lead to a better understanding of the crucial role of the chromatin reader ZMYND11 on chromatin remodeling and the biological function during neural development.

Mutations in SAMHD1, encoding SAM and HD domain-containing protein 1, cause Aicardi-Goutières syndrome (AGS) 5, an infancy-onset autoinflammatory disease characterized by neurodegeneration and chronic activation of type I interferon. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts and peripheral blood mononuclear cells from three AGS patients with biallelic SAMHD1 mutations. These cell lines provide a valuable source to study disease mechanisms and to assess therapeutic molecules.