Stem cell treatments for spinal cord injury (SCI) are effective in pre-clinical animal model research but not yet for humans. Two promising stem cell repair strategies involve (1) endogenous neural stem/progenitor cells (NSPCs) and (2) induced pluripotent stem cells (iPSCs). Delineating species differences in spinal cord NSPC biology is essential to inform human SCI endogenous regeneration and repair. Understanding the phenotypic differences between iPSC-derived NSPCs and primary spinal cord NSPCs would also improve the clinical application of iPSC-derived NSPC therapy in human SCI. To directly compare the molecular and functional attributes of spinal cord NSPCs between humans and animal models of SCI, we designed an in vitro model that allows the characterization of primary human, pig, and rat NSPCs under identical conditions. We found an enrichment of transcription factors in NSPCs of either species that may underlie their differentiation and proliferation potentials. Specifically, human NSPCs are neurogenic, whereas pig and rat NSPCs are gliogenic. Also, the proliferation rate of human and pig NSPCs is less than rat NSPCs. Subsequently, we expanded our in vitro model to examine the responses of NSPCs to inflammation and regenerative factors. Surprisingly, inflammation had induced glial scarring mechanisms from pig and rat NSPCs but potentiated neurogenesis of human NSPCs. We also found species-specific responses to regenerative factors that depend on the type of factor used, concentration, and duration of treatment. To assess the extent that iPSC-derived NSPCs phenocopy primary spinal cord NSPCs, we created iPSC-derived NSPCs with a previously reported brain or spinal cord phenotype and directly compared them with isogenic primary NSPCs. We found that iPSC-derived NSPCs exhibit an earlier developmental stage and a greater proliferation rate. We also found that primary NSPCs possess a unique differentiation potential and regional polarity along the rostral-caudal and dorsoventral axes. In summary, we discovered that species differences in NSPC biology exist between human and animal primary spinal cord NSPCs and that iPSC-derived NSPCs do not recapitulate the transcriptional nor functional attributes of primary spinal cord NSPCs. This thesis highlights the translational gap between pre-clinical research and the clinical application of stem cell treatments that target endogenous NSPCs or transplant iPSC-derived NSPCs.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23777号 / 医博第4823号 / 新制||医||1057(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 寺田 智祐, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The cells from the inner cell mass (ICM) of an early embryo have the potential to differentiate into all the different cell types present in an adult organism. Cells from the ICM can be isolated and cultured in vitro, becoming embryonic stem cells (ESCs). ESCs have several properties that make them unique: they are unspecialized, can self-renew indefinitely in culture, and given the appropriate cues can differentiate into cells from all three germ layers (ecto-, meso-, and endoderm), including the germline, both in vivo and in vitro. Induced pluripotent stem cells (iPSCs) can be generated from adult, terminally differentiated somatic cells by transient exogenous expression of four transcription factors (Oct4, Sox2, Klf4, and cMyc; OSKM) present normally in ESCs. It has been shown that iPSCs are equivalent to ESCs in terms of morphology, gene expression, epigenetic signatures, in vitro proliferation capacity, and in vitro and in vivo differentiation potential. However, unlike ESCs, iPSCs can be obtained from a specific individual without the need for embryos. This makes them a promising source of pluripotent cells for regenerative medicine, tissue engineering, drug discovery, and disease modelling; additionally, in livestock species such as the bovine, they also have applications in genetic selection, production of transgenic animals for agricultural and biomedical purposes, and species conservancy. Nevertheless, ESC and iPSC lines that meet all pluripotency criteria have, to date, only been successfully produced in mice, rats, humans, and non-human primates. In the first part of this dissertation, we attempted reprogramming of three types of bovine somatic cells: fetal fibroblasts (bFFs), adult fibroblasts (bAFs), and bone marrow-derived mesenchymal stem cells (bMSCs), using six different culture conditions adapted from recent work in mice and humans. Using basic mouse reprogramming conditions, we did not succeed in inducing formation of ESC-like colonies in bovine somatic cells. The combination of 2i/LIF plus ALK5 inhibitor II and ascorbic acid, induced formation of colony-like structures with flat morphology, that occasionally produced trophoblast-like structures. These trophoblast-like vesicles did not appear when an inhibitor of Rho-associated, coiled-coil containing protein kinase 1 (ROCK) was included in the medium. We screened for expression of exogenous OSKM vector with RT-PCR and found upregulation of OSKM vector 24h after Dox was added to the medium; however, expression was sharply decreased on day 2 after Dox induction, and was not detectable after day 3. In a separate experiment, we induced reprogramming of bFF and bAFs using medium supplemented with 50% of medium conditioned by co-culture with the bovine trophoblast CT1 line. These cells expressed both OCT4 and the OSKM vector 24h after Dox induction. However, similar to our previous observations, both markers decreased expression until no signal was detected after day 3. In summary, we were unable to produce fully reprogrammed bovine iPSCs using mouse and human protocols, and the exact cause of our lack of success is unclear. It is possible that a different method of transgene expression could play a role in reprogramming. However, these ideas would be driven by a rather empirical reasoning, extrapolating findings from other species, and not contributing in our understanding of the particular differences of pluripotecy in ungulates. Our inability to produce bovine iPSCs, combined with the only partial reprogramming observed by others, justifies the need for in depth study of bovine pluripotency mechanisms, before meaningful attempts to reprogram bovine somatic cells to plutipotency are made. Therefore, we focused on getting a better understanding of bovine nuclear reprogramming. This would allow us to rationally target the specific requirements of potential bovine pluripotent cells. Cell fusion is a process that involves fusion of the membrane of two or more cells to form a multinucleated cell. Fusion of a somatic cell to an ESC is known to induce expression of pluripotency markers in the somatic nucleus. In the second part of this dissertation, we hypothesized that fusion of bFFs to mouse ESCs (mESCs) would induce expression of pluripotency markers in the bFF nucleus. We first optimized a cell fusion protocol based on the use of polyethylene glycol (PEG), and obtained up to 11.02% of multinucleated cells in bFFs. Next, we established a method to specifically select for multinucleated cells originated from the fusion of mESCs with bFFs (heterokaryons), using indirect immunofluorescence. With this in place, flow cytometry was used to select 200 heterokaryons which were further analyzed using RNA-seq. We found changes in bovine gene expression patterns between bFFs and heterokaryons obtained 24h after fusion. Focusing on the bovine transcriptome, heterokaryons presented upregulation of early pluripotency markers OCT4 and KLF4, as well as hypoxia response genes, contrasted with downregulation of cell cycle inhibitors such as SST. The cytokine IL6, known to increase survival of early embryos in vitro, was upregulated in heterokaryons, although its role and mechanism of action is still unclear. This indicates that the heterokaryon cell fusion model recapitulates several of the events of early reprogramming, and can therefore be used for further study of pluripotency in the bovine. The cell fusion model presented here can be used as a tool to characterize early changes in bovine somatic nuclear reprogramming, and to study the effect of different reprogramming conditions on the bovine transcriptome. / Ph. D.

Spinal cord injuries result in permanent loss of motor function, leaving those affected with long term physical and financial burdens. Strategies for spinal cord injury repair must overcome unique challenges due to scar tissue that seals off the injury site, preventing regeneration. Tissue engineering can address these challenges with scaffolds that serve as cell- and drug-delivery tools, replacing damaged tissue while simultaneously addressing the inhibitory environment on a biochemical level. To advance this approach, the choice of cells, biomaterial matrix, and drug delivery system must be investigated and evaluated. This research seeks to evaluate (1) the behaviour of murine induced pluripotent stem cells in previously characterized 3D fibrin matrices; (2) the 3D fibrin matrix as a platform to support the differentiation of human induced pluripotent stem cells; and (3) the ability of an affinity-based drug delivery system to control the release of emerging spinal cord injury therapeutic, heat shock protein 70 from fibrin scaffolds. / Graduate / 0541 / amy.lynn.montgomery@gmail.com

Les porphyries érythropoïétiques (PE) : Porphyrie Erythropoïétique Congénitale -PEC- et Protoporphyrie Erythropoïétique -PPE- sont caractérisées par le déficit d’une des enzymes de la voie de biosynthèse de l’hème. Le traitement curatif des formes sévères de PE est la transplantation de moelle osseuse allogénique (TMOA). La PPE est parfois compliquée d’une insuffisance hépatique majeure nécessitant une greffe hépatique. Dans un modèle murin de PPE (Fechm1Pas/Fechm1Pas), nous avons démontré l’apparition progressive de lésions hépatiques dès la 2ème semaine de vie. Une TMO précoce (nouveau-né) a permis de prévenir l’apparition de ces lésions hépatiques et de corriger la photosensibilité cutanée démontrant l’efficacité de cette approche thérapeutique pour les formes sévères de PPE. La thérapie génique par greffe de cellules souches hématopoïétiques autologues corrigées représente une alternative à la TMOA en l’absence de donneur HLA-compatible. Nous avons développé des cellules souches pluripotentes induites (iPS) à partir de cellules épidermiques issues de modèles murins de PE et d’un patient PEC. La correction génique a été obtenue par transfert du gène lentiviral (ferrochélatase ou uroporphyrinogène III synthase (UROS). La pluripotence des cellules iPS a été caractérisée in vitro par la formation de corps embryoïdes et in vivo par la formation de tératomes. In vitro, la correction métabolique a été obtenue après différenciation des cellules iPS humaines en progéniteurs hématopoïétiques. Enfin dans une dernière partie, nous nous sommes intéressés à une approche pharmacologique de la PEC. Nous avons montré que les mutations C73R et P248Q entraînaient une instabilité et une dégradation accélérée de l’UROS par la voie du protéasome. Le traitement de souris UrosP248Q par un inhibiteur du protéasome (Velcade®) a permis la correction de la photosensibilité cutanée. Ces travaux ouvrent de nouvelles perspectives pour le traitement des porphyries érythropoïétiques. / Erythropoietic porphyrias (EP) : Congenital Erythropoietic Porphyria -CEP- and Erythropoietic Protoporphyria -EPP-) are characterized by a deficit of one enzyme implicated in heme biosynthetic pathway. The curative therapy for severe cases of EP is an HLA-compatible Bone Marrow Transplantation (BMT). EPP is sometimes complicated by a major hepatic failure requiring hepatic graft. In a murine model of EPP (Fechm1Pas/Fechm1Pas), we have demonstrated that hepatic lesions progressively appear 2 weeks after birth. Early BMT (in neonates) has made it possible to prevent hepatic lesions and correct skin photosensitivity, demonstrating the efficiency of this therapeutic approach in severe cases of EPP. The gene therapy by graft of corrected autologous hematopoietic stem cells represents an alternative to BMT when HLA-compatible donors are lacking. We have developed induced pluripotent stem cells (iPSC) from epidermic cells of murine models of EP and of one PEC patient. The gene correction was obtained by lentiviral gene transfer (ferrochelatase and uroporphyrinogen III synthase -UROS). The pluripotency of iPSC was characterized in vitro by the formation of embryoid bodies and in vivo by the formation of teratomas. In vitro, the metabolic correction was obtained after differentiation of human IPSC into hematopoietic progenitors. In the last part of this thesis, we have focused on a pharmacological approach of CEP. We have shown that C73R and P248Q mutations lead to instability and accelerated degradation of the UROS protein via the proteasome. Treating UrosP248Q mice with a proteasome inhibitor (Velcade®) has allowed the correction of skin photosensitivity. These works offer new prospects for the treatment of erythropoietic porphyrias.

A síndrome de Prader Willi (PWS) é uma doença de neurodesenvolvimento; a principal hipótese de causa de PWS é a ausência da expressão de SNORD116. O SNORD116 fica na região 15q11-q13 que apresenta vários genes com imprinting genômico e é conhecida por ser controlada pela região de controle de imprinting PWS (PWS-IC) que se localiza sobreposta à região promotora e ao exon 1 do gene SNRPN. Em camundongos, uma proteína zinc finger (Zfp57) foi descrita como importante para o estabelecimento e manutenção do imprinting no Snrpn. Através de análise do ENCODE do Genome Browser, verificamos que outra proteína zinc finger (ZNF274) se liga ao SNORD116. ZNF274 é conhecida por formar um complexo com TRIM28 e SETDB1 que inibe a expressão através da trimetilação da lisina 9 na histona 3 (H3K9me3). No atual estudo mostramos que ZNF274 se liga ao SNORD116 preferencialmente ao alelo materno nas células-tronco pluripotente induzidas (iPSCs). Adicionalmente, as proteínas TRIM28 e SETDB1, que formam um complexo com a ZNF274, estão presentes na região do SNORD116, e a modificação H3K9me3 ocorre preferencialmente no alelo materno nas iPSCs. Na análise funcional, mostramos que o knockdown de SETDB1 isoladamente ou combinado com o knockdown de ZNF274 causa aumento na expressão de SNRPN e SNORD116 nas iPSCs. Além disso, ocorre redução do H3K9me3 e aumento da modificação relacionada à ativação da transcrição, H3K4me2 (dimetilação da lisina 4 na histona 3), na PWS-IC. Os knockdowns também afetam a metilação de DNA, ocasionando o aumento de 5-hidroximetliação de citosinas na PWS-IC. Em outros tipos celulares estudados, neurônios derivados de iPSCs e SHEDs, ZNF274 e a modificação H3K9me3 ocorrem em ambos os alelos dentro do SNORD116. É possível que, nas iPSCs, este complexo proteja a região imprintada da desmetilação do DNA de proteína(s) que atue(m) nessa região somente em células pluripotentes. Nossos achados possibilitam melhor compreensão dos mecanismos envolvidos no imprinting da região 15q11-q13, principalmente do SNORD116, e, consequentemente, disponibiliza novas ferramentas para o desenvolvimento de futuras terapias para PWS. / Prader-Willi syndrome (PWS) is a neurodevelopmental disorder. Loss of paternal copies of the cluster of SNORD116 C/D box snoRNAs and their host transcript, 116HG, on human chromosome 15q11-q13 imprinted region is considered to be the major responsible for PWS. PWS-imprinting center (PWS-IC) regulates 15q11-q13 imprinting. PWS-IC is located upstream and in the exon 1 of SNURF-SNRPN gene. In mice, Zfp57 plays an important role in establishment and maintenance of Snrpn imprinting. In human, ENCODE database indicates that ZNF274 binds to SNORD116. Moreover, ZNF274 are C2H2/KRAB zinc finger proteins as Zfp57. We have investigated the mechanism of repression of the maternal SNORD116. Here, we report that the ZNF274, in association with the histone H3 lysine 9 (H3K9) methyltransferase SETDB1, is part of a complex that binds to the silent maternal but not to the active paternal alleles in induced pluripotent stem cells (iPSCs). Knockdown of SETDB1 in PWS-specific iPSCs causes a decrease in the accumulation of H3K9 trimethylation (H3K9me3) at SNORD116. We also show that upon knockdown of SETDB1 in PWS-specific iPSCs, expression of maternally silenced 116HG RNA is partially restored. SETDB1 knockdown in PWS iPSCs also disrupts DNA methylation at the PWS-IC where a decrease in 5-methylcytosine is observed in association with a concomitant increase in 5-hydroxymethylcytosine. In iPSCs-derived neurons and stem cells from human exfoliated teeth (SHEDs) ZNF274/SETDB1 complex binding and H3K9me3 modification occur in both alleles. These observations suggest that the ZNF274/SETDB1 complex bound to the SNORD116 cluster may protect the PWS-IC from DNA demethylation during early development, as indicated by iPSCs. Our findings reveal novel epigenetic mechanisms that function to repress the maternal 15q11-q13 region. The better understanding of epigenetic mechanisms provides new tools for future therapy research.

Os transtornos do espectro autista (TEA) são um grupo de doenças neuropsiquiátricas caracterizadas por um prejuízo na capacidade de comunicação e de interação social e por padrões comportamentais estereotipados. Os TEA são geneticamente heterogêneos o que dificulta a identificação das alterações genéticas que estão contribuindo para estes transtornos. No presente estudo, selecionamos como uma primeira abordagem o estudo de translocações cromossômicas, buscando encontrar genes candidatos para posteriores estudos funcionais. No primeiro caso, uma translocação de novo balanceada envolvendo os cromossomos 2q11 e Xq24, não identificamos nenhum candidato funcional rompido pelos pontos de quebra. Detectamos ainda a presença de uma isodissomia materna do cromossomo 5 nesta paciente. Este resultado sugere que, possivelmente, tanto a translocação cromossômica quanto a isodissomia devem estar contribuindo para a etiologia do TEA nesta paciente, caracterizando este como um caso de efeito poligênico. Já o estudo da translocação de novo balanceada (3,11)(p21,q22) revelou que o gene TRPC6, um canal de cálcio envolvido no desenvolvimento de dendritos e sinapses excitatórias, encontrava-se rompido no cromossomo 11 deste paciente. As análises dos neurônios e células progenitoras neurais deste paciente obtidas através da técnica de reprogramação celular e o estudo global de expressão gênica sugerem fortemente que o rompimento do gene TRPC6 é o fator etiológico do TEA neste caso. Por fim, nós também realizamos um estudo de expressão gênica global de pacientes autistas idiopáticos e verificamos que os genes diferencialmente expressos nestes pacientes estão principalmente envolvidos na regulação da dinâmica do citoesqueleto, indicando que este pode ser o processo biológico comumente afetado nos pacientes autistas. Nosso trabalho mostra que os estudos citogenéticos são importantes para a identificação de genes candidatos para os TEA e reforça a hipótese de que estes transtornos são causados por diferentes variantes genéticas mas que levam ao comprometimento de um processo biológico comum. Acreditamos que o modelo de reprogramação celular contribuirá para o entendimento da implicação de tais processos na etiologia dos TEA. / Autism spectrum disorders (ASD) are a group of neurodevelopmental diseases characterized by impairments in social and communicative skills and repetitive behaviors. The investigation of ASD causes is hampered by the genetic heterogeneity of these neurodevelopmental diseases. In the present study, we mapped the breakpoints associated to chromosomal translocations found in two autistic patients as a first screening approach, trying to identify single candidate genes that could be further investigated by functional analysis. In the first case, a de novo balanced translocation involving the chromosomes 2q11 and Xq24, we did not find any functionally known relevant gene disrupted by the breakpoints but, surprisingly, SNP-array data showed that the patient also presents a maternally inherited isodisomy on chromosome 5. In this case, is possible that ASD is caused by the combination of the molecular results caused by the translocation and the UPD on chromosome 5, which would characterize this case as an example of polygenic effects on ASD etiology. On the other hand, the study of a second case, a boy with a de novo balanced translocation (3;11)(p21;q22), revealed that TRPC6, a calcium channel involved in dendritic spine and excitatory synapse formation, was disrupted by the translocation on chromosome 11. Making use of cellular reprogramming to generate neurons and neuronal progenitor cells from this patient and expression analysis, we demonstrated that TRPC6 disruption can respond for the phenotype seen in this patient. Finally, we also performed a genome-wide expression analysis to investigate idiopathic autistic patients and we verified that ASD DEGs are mainly implicated in cytoskeleton dynamics, suggesting that the regulation of this cellular structure can be one of the common mechanisms of ASD etiology. Our work shows that cytogenetic studies are important for the identification of ASD candidate genes and reinforces the hypothesis that these disorders are caused by different genetic variants that are implicated in a common biological process. We believe that cellular reprogramming will contribute for the understanding of the implication of such biological processes in the etiology of ASD.

Heart failure is one of the leading causes of death in the United States. Every year in the United States, more than 800,000 people are diagnosed with heart failure and more than 375,000 people die from heart disease. Current therapies such as heart transplants and bioartificial hearts are helpful, but not optimal. Decellularization of porcine whole hearts followed by recellularization with patient-specific human cells may provide the ultimate solution for patients with heart failure. Great progress has been made in the development of efficient processes for decellularization, and the design of automated bioreactors. In this study, the decellularization of porcine hearts was accomplished in 24 h with only 6 h of sodium dodecyl sulfate (SDS) exposure and 98% DNA removal. Automatically controlling the pressure during decellularization reduced the detergent exposure time while still completely removing immunogenic cell debris. Stimulation of macrophages was greatly reduced when comparing native tissue samples to the processed ECM. Complete cell removal was confirmed by analysis of DNA content. General collagen and elastin preservation was demonstrated by SEM and histology. The compression elastic modulus of the ECM after decellularization was lower than native at low strains but there was no significant difference at high strains. Polyurethane casts of the vasculature of native and decellularized hearts demonstrated that the microvasculature network was preserved after decellularization. A static blood thrombosis assay using bovine blood was also developed. A perfusion bioreactor was designed and right ventricle of the decellularized hearts were recellularized with human endothelial cells and cardiac fibroblasts. An effective, reliable, and relatively inexpensive assay based on human blood hemolysis was developed for determining the remaining cytotoxicity of the cECM and the results were consistent with a standard live/dead assay using MS1 endothelial cells incubated with the cECM. Samples from the left ventricle of the hearts were prepared with 300 µm thickness, mounted on 10 mm round glass coverslips. Human induced pluripotent stem cells were differentiated into cardiomyocytes (CMs) and 4 days after differentiation, cardiac progenitors were seeded onto the decellularized cardiac slices. After 10 days, the tissues started to beat spontaneously. Immunofluorescence images showed confluent coverage of CMs on the decellularized slices and the effect of the scaffold was evident in the arrangement of the CMs in the direction of fibers. This study demonstrated the biocompatibility of decellularized porcine hearts with human CMs and the potential of these scaffolds for cardiac tissue engineering. Further studies can be directed toward 3D perfusion recellularization of the hearts and improving repopulation of the scaffolds with various cell types as well as adding mechanical and electrical stimulations to obtain more mature CMs.