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The ability to reprogram adult somatic cells into induced pluripotent stem (iPS) cells could provide a valuable implement for in vitro disease modeling and drug discovery. More importantly, they may potentially serve as an unlimited source of cells for regenerative medicine. However, most of the iPS cells have been generated by retroviral vectors, and therefore they carry the risk of viral integration into the host genome. This problem prevents their use for clinical applications and regenerative medicine. mRNA-mediated delivery of reprogramming factors is an alternative approach for cellular reprogramming. mRNA-based reprogramming offers the advantage of being completely free of genomic integration and is therefore highly suitable for clinical translation. However, there are some limitations which must be overcome so that mRNA can be widely used for successful cellular reprogramming. In the current thesis, the attempt was to generate stable mRNA-iPS cells through overcoming those limitations. Several human donor cells were transfected with mRNA encoding reprogramming factors and the generation of two stable mRNA-iPS cell lines was shown. The resultant mRNA-iPS colonies were assessed for pluripotency markers. Their pluripotency features were evaluated by the viral-iPS cells produced by conventional retroviral vectors. It was noticed that the generation of mRNA-iPS cells was largely affected by the parental cells from which they were derived. However, characterization and evaluation of the generated mRNA-iPS cells proved their pluripotency states comparable to the viral-iPS cells. On the other hand, the aging hallmarks of the iPS cells were assessed in the second part of this thesis. The potential aging signatures of the iPS cells should be conducted before their use in clinical applications. Currently, there are controversial data regarding the ability of reprogramming to fully rejuvenate an aged somatic cell and reverse agerelated changes such as shortened telomeres, dysfunctional mitochondria and DNA damage. Moreover, mixed findings have been published regarding whether the iPS cells are fully rejuvenated or they might retain some of the aging hallmarks from the cells which they were derived. This thesis studied these controversies through the investigation of three hallmarks of aging including telomere length, mitochondrial alteration and DNA damage. Telomere elongation was indicated in the iPS cells. Furthermore, mitochondrial morphology and function were improved into more immature features in iPS cell lines than their corresponding fibroblasts. Moreover, the iPS cell lines were shown to have less amount of DNA damage compared to their parental fibroblasts. In summary, it can be concluded that generation of mRNA-iPS cells is largely affected by the primary donor cells from which they are derived. Furthermore, it seems that reprogramming enables reversion of aging signatures to a more youthful state.

Background and objectives: In 2006 Shinya Yamanaka successfully reprogrammed mouse fibroblasts back to an embryonic stem cell-like state (called induced pluripotent cells, iPS cells) using retrovirus to introduce four genes that encode critical transcription factor proteins (Oct4, Sox2, KLF4, and c-Myc). This ability to reprogram has promising future applications in clinical and biomedical research for study of diseases, development of candidate drugs and to support therapeutic treatments in regenerative medicine. However, the clinical applications have to meet GMP requirements without the risk of insertional mutagenesis associated with retrovirus. Chromatin modifying agents are widely used in many protocols to generate iPS cells and culture of blood CD34+ cells with chromatin-modifying agents can lead to an increase in marrow repopulating cells and in the case of valproic acid increased erythroid cell colony formation. We undertook research to help understand what effects these reagents have on mobilised peripheral blood (mPB) CD34+ cells and optimised the expansion medium protocol to facilitate reprogramming work. This project aims to utilize peripheral blood mononuclear cells (MNC), one of the most easily accessible tissues to generate iPS cells using an efficient non-viral, feeder cell free methodology, with the ultimate goal of moving this methodology towards clinical use. Materials and Methods: G-CSF mobilised peripheral blood, buffy coat, cord blood and fetal liver were obtained from patients and donors under informed consent and ethics committee approval. Haematopoietic stem/progenitor cells CD34+ or CD133+) isolated by magnetic separation were flow cytometry sorted into CD34+/CD133+, CD34+/CD133-, and CD34-/CD133+ sub-populations and their lineage potential were assessed in colony forming unit assays. The effect of epigenetic modifiers valproic acid and 5-aza-2-deoxycytidine used singly or in combination with each other and with IL3 on phenotype and lineage potential of cultured CD34+ cells from mobilised peripheral blood were assessed by flow cytometry and colony-forming unit assays. Prior to reprogramming mononuclear cells from peripheral blood or CD34+ cells from blood were expanded in culture medium supplemented with stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (Flt3L) and Interleukin- 3 (IL-3) for several days. Actively proliferating cells were reprogrammed by electroporation using episomal vectors with an oriP/EBNA-1 backbone to deliver five reprogramming genes, Oct4, Sox2, Lin28, L-Myc, and Klf4. Electroporated cells were seeded onto matrigel coated plates immediately after transfection or were reseeded after three days’ culture. Subsequently, cells were cultured in specific medium on different days. When iPS colonies appeared, they were picked and cultured as for ES cells. Once established, iPS cell lines were immunophenotyped using flow cytometry and immunofluorescence and their potential to differentiate into the three germ layers was assessed in vitro. Results and Conclusion: The largest subpopulation of CD34+ cells was CD34+/CD133+ population which was essentially committed to myeloid colony production, while much smaller CD34+/CD133- subpopulation had a greater capacity to generate erythroid colonies. Optimised cytokine cocktail for expansion of CD34+ cells included IL-3, important in improving expansion and maintaining functionality of CD34+ cells. The optimised cytokine cocktail comprised 100 ng/ml SCF, 10 ng/ml Flt3L, and 20 ng/ml IL-3, which maintained CD34+ cells and MNC in an active proliferating state. In addition, valproic acid and IL3 were found to act synergistically, to increase the numbers of CD34+/CD36+ positive cells. However, we found that an apparent increase in red cell colony formation actually resulted from a decrease in white cell colonies, so no overall increase in red cell colonies was seen when equivalent numbers of CD34+ cells were plated. Proliferating MNC maintained in optimised cytokine cocktail were amenable to electroporation for the effective delivery of episomal transcription factors (Oct4, Sox2, Klf4, L-Myc, and Lin28) within a backbone of oriP/EBNA-1. We successfully developed an efficient and simple method for reprogramming MNC from fresh or frozen samples to generate induced pluripotent cells using episomal vectors in a feeder-free system without any requirement for small molecules and the highest reprogramming efficiency is 0.033% (65 colonies from 2 ◊ 105 seeding MNC). The cytokine cocktail and reprogramming methods work better in CD34+ cells from cord blood or fetal liver, and we obtained 148 iPS colonies from 105 seeding cells (0.148%) at most. In addition, fibroblasts from adult and fetal liver can be successfully reprogrammed using the same reprogramming method. The use of episomal vectors with an oriP/EBNA-1 backbone to deliver reprogramming genes, and efficient electroporation were the most important factors in efficiency of the reprogramming process. In addition, it is pivotal to initiate transfection when cells are actively proliferating. The iPS cell lines we generated maintained the successful expression of ES markers including Oct4, Nanog, SSEA3. SSEA4, TRA-1-60 and TRA-1-81, and had the capacity to successfully differentiate into cell types of ectoderm, mesoderm and endoderm layers in vitro.

Oshima N, Yamada Y, Nagayama S, Kawada K, Hasegawa S, et al. (2014) Induction of Cancer Stem Cell Properties in Colon Cancer Cells by Defined Factors. PLoS ONE 9(7): e101735. doi:10.1371/journal.pone.0101735 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18547号 / 医博第3940号 / 新制||医||1006(附属図書館) / 31447 / 京都大学大学院医学研究科医学専攻 / (主査)教授 千葉 勉, 教授 野田 亮, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23417号 / 医博第4762号 / 新制||医||1052(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 松田 秀一特定拠点, 教授 妻木 範行, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les syndromes progéroïdes regroupent un ensemble de pathologies caractérisées par un vieillissement précoce et accéléré. Le syndrome le plus connu et étudié est la progéria de Hutchinson-Gilford dont l'incidence est de 1 cas sur 8 millions ce qui en fait une maladie très rare. Nous avons étudié trois symptômes progéroïdes dont le syndrome HGPS, un syndrome HGPS-like ainsi qu'un syndrome APS. Ces pathologies ont de nombreux symptômes en commun dont une ostéolyse, une lipodystrophie, ainsi qu'une atteinte cardiovasculaire. Ces trois syndromes sont provoqués par différentes mutations du gène LMNA qui code pour les Lamines A et C. Nous avons utilisé le modèle des iPSCs afin d'étudier in vitro la physiopathologie de ces trois syndromes en les comparant à des cellules contrôles. Les cellules dérivées de la voie mésenchymateuse étant majoritairement altérées dans ces pathologies, nous avons créé des modèles in vitro d'étude de la différentiation en MSCs. De plus, ces patients présentant des altérations arterio-veineuses, nous avons analysé la différenciation en VSMCs. Le phénotype des ces cellules a été analysé et les profils transcriptomiques comparés pour les différentes lignées. Des gènes communs, impliqués dans le stress oxydatif et dans des systèmes de réparation géniques ont été retrouvés comme étant altérés. De plus, nous avons mis en évidence des altérations de voies de signalisation indispensables à la survie et à la prolifération cellulaire en comparant les cellules progéroïdes aux contrôles. Certaines de ces voies biologiques ouvrent de nouvelles perspectives dans la compréhension des symptômes observés chez ces patients. / Progeroid syndromes are a group of pathologies characterized by accelerated and early aging. One of the most studied of these diseases is HGPS, with an estimated incidence of 1 in 8 millions birth making it an extremely rare disease. We focused our attention on three different progeroid syndromes including classic HGPS, a HGPS-like and an atypical progeroid syndrome. These pathologies share many symptoms, including osteolysis, lipodystrophy, and cardiovascular alterations. These 3 syndromes are caused by 3 different mutations in the LMNA gene that encodes A- and C-type lamins, inducing production of a truncated Lamin A in HGPS and HGPS-like and production of a mutated Lamin with a p.T528M substitution in APS. We produced hiPSCs to create a model of these different diseases and investigate in vitro the physiopathology of these syndromes by comparing them to control cells. Cells derived from mesenchymal stem cells being the most impaired type of tissue, we established in vitro models in order to study the differentiation of hiPSCs into MSCs. In addition given the massive cardiovascular defects in these patients, we also investigated differentiation toward the VSMCs. Cell phenotypes were carefully characterized and we compared the transcripttomic profile of the different cell types. We identified dysregulation in genes involved in oxidative stress response and in DNA repair in progeroid cells. In addition, pathways essential for cell survival and proliferation are also modified when comparing progeroid and controls cells. Altogether, these results might explain some of the symptoms observed in progeroid patients but also reveal pathways involved in ageing.

The ability to reprogram adult somatic cells into induced pluripotent stem (iPS) cells could provide a valuable implement for in vitro disease modeling and drug discovery. More importantly, they may potentially serve as an unlimited source of cells for regenerative medicine. However, most of the iPS cells have been generated by retroviral vectors, and therefore they carry the risk of viral integration into the host genome. This problem prevents their use for clinical applications and regenerative medicine. mRNA-mediated delivery of reprogramming factors is an alternative approach for cellular reprogramming. mRNA-based reprogramming offers the advantage of being completely free of genomic integration and is therefore highly suitable for clinical translation. However, there are some limitations which must be overcome so that mRNA can be widely used for successful cellular reprogramming. In the current thesis, the attempt was to generate stable mRNA-iPS cells through overcoming those limitations. Several human donor cells were transfected with mRNA encoding reprogramming factors and the generation of two stable mRNA-iPS cell lines was shown. The resultant mRNA-iPS colonies were assessed for pluripotency markers. Their pluripotency features were evaluated by the viral-iPS cells produced by conventional retroviral vectors. It was noticed that the generation of mRNA-iPS cells was largely affected by the parental cells from which they were derived. However, characterization and evaluation of the generated mRNA-iPS cells proved their pluripotency states comparable to the viral-iPS cells. On the other hand, the aging hallmarks of the iPS cells were assessed in the second part of this thesis. The potential aging signatures of the iPS cells should be conducted before their use in clinical applications. Currently, there are controversial data regarding the ability of reprogramming to fully rejuvenate an aged somatic cell and reverse agerelated changes such as shortened telomeres, dysfunctional mitochondria and DNA damage. Moreover, mixed findings have been published regarding whether the iPS cells are fully rejuvenated or they might retain some of the aging hallmarks from the cells which they were derived. This thesis studied these controversies through the investigation of three hallmarks of aging including telomere length, mitochondrial alteration and DNA damage. Telomere elongation was indicated in the iPS cells. Furthermore, mitochondrial morphology and function were improved into more immature features in iPS cell lines than their corresponding fibroblasts. Moreover, the iPS cell lines were shown to have less amount of DNA damage compared to their parental fibroblasts. In summary, it can be concluded that generation of mRNA-iPS cells is largely affected by the primary donor cells from which they are derived. Furthermore, it seems that reprogramming enables reversion of aging signatures to a more youthful state.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18870号 / 医博第3981号 / 新制||医||1008(附属図書館) / 31821 / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 瀬原 淳子, 教授 中畑 龍俊 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Final publication is available at http://www.cell.com/cell-stem-cell/abstract/S1934-5909(13)00012-X / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19622号 / 医博第4129号 / 新制||医||1015(附属図書館) / 32658 / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 伊佐 正, 教授 YOUSSEFIAN Shohab / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24129号 / 医博第4869号 / 新制||医||1059(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 平井 豊博, 教授 中島 貴子, 教授 森本 尚樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM