In 2006, Kazutoshi Takahashi and Shinya Yamanaka demonstrated the ability of four transcription factors; Oct4, Sox2, Klf4 and c-Myc to 'reprogram' differentiated somatic cells to a pluripotent state. This technology holds huge potential in the field of regenerative medicine, but reprogramming also a model system by which to the common regulators of all forced cell identity changes, for example, transdifferentiation. Despite this, the mechanism underlying reprogramming remains poorly understood and the efficiency of induced pluripotent stem cell (iPSC) generation, inefficient. One powerful method for elucidating the gene components influencing a biological process, such as reprogramming, is screening for a phenotype of interest using genome-wide mutant libraries. Historically, large-scale knockout screens have been challenging to perform in diploid mammalian genomes, while other screening technologies such as RNAi can be disadvantaged by variable knockdown of target transcripts and off-target effects. Components of clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) prokaryote adaptive immunity systems have recently been adapted to edit genomic sequences at high efficiency in mammalian systems. Furthermore, the application of CRISPR-Cas components to perform proofof- principle genome-wide KO screens has been successfully demonstrated. I have utilised the CRISPR-Cas9 system to perform genome-wide loss-of-function screening in the context of murine iPSC reprogramming, identifying 18 novel inhibitors of reprogramming, in addition to four known inhibitors, Trp53, Cdkn1a, Jun, Dot1l and Gtf2i. Understanding how these novel reprogramming roadblocks function to inhibit the reprogramming process will provide insight into the molecular mechanisms underpinning forced cell identity changes.

Pluripotent stem cells (PSCs) are unique in their capability to self-renew and differentiate into cell types of all three embryonic germ layers. Since their discovery, PSCs have become an indispensable tool for modeling development, disease onset/progression, and drug discovery. The pluripotent state is known to be regulated by a core network of transcription factors including Oct4, Sox2, and Nanog. However, the roles of other contributing transcription factors remain understudied. Our research focused on defining the roles and molecular mechanisms of Rex1, a zinc finger transcription factor whose expression is strongly correlated with the pluripotent state. Attempts by our lab to elucidate the role of Rex1 in embryonic stem cells (ESCs) revealed the presence of two smaller protein products that result from the initiation of translation at downstream start codons within the REX1 open reading frame. We hypothesized that the full-length Rex1 protein and its shorter alternative translation isoforms were acting to regulate the expression of lineage-determining genes in PSCs. To evaluate this hypothesis, we generated mouse embryonic stem cell (mESC) lines expressing FLAG-tagged versions of the full-length Rex1 protein, and its isoforms, from the endogenous locus. Through the use of these lines, we demonstrated the formation of multiple Rex1 isoforms by alternative translation, a novel observation that has yet to be reported. Furthermore, our results indicate that Rex1 is a negative regulator of differentiation-related genes and endogenous retroviral elements, suggesting Rex1 is acting to maintain the tightly regulated transcriptional network of pluripotency, while also maintaining genomic integrity through the repression of repetitive elements. / Thesis / Master of Science (MSc)

Cell based therapy using stem cell derived cardiomyocytes, has emerged as a potential therapeutic approach for cardiac diseases such as myocardial infarction and heart failure. Induced pluripotent stem cells (iPS cells) could be an ideal source of cardiomyocytes (iPS-CM). Challenges facing cell therapy include the high number of viable cells needed to survive in pathological conditions. The Hippo signalling pathway has been described as a key pathway involved in regulating cardiomyocyte proliferation and survival in both embryonic and adult hearts. We hypothesise that modification of the Hippo pathway will enhance the efficiency of iPS-CM generation and will increase iPS-CM survival and viability in pathological conditions. Skin fibroblasts were reprogrammed to iPS cells and then differentiated to cardiomyocytes. The Hippo signalling pathway was modified by genetic ablation of MST1, a major upstream regulator of the Hippo pathway, or by overexpressing YAP, the main downstream effector of the pathway. Cell proliferation was analysed using an EdU incorporation assay and staining for cytokinesis markers Ki67 and phospho-histone H3. Cell death and viability were analysed by measuring caspase 3/7 and MTT activity and by trypan blue staining in both normal and hypoxic conditions (CoCl2 treatment). Analysis of cell proliferation shows that genetic ablation of Mst1 leads to significantly increased proliferation (+12±1.5% P < 0.001), survival and viability (+20±4.3% P < 0.001) of iPS cells in both normal and hypoxic (CoCl2 treatment) conditions compared to controls. In addition, overexpression of YAP, which is normally inhibited by upstream Hippo pathway components, and overexpression of mutated constitutively active form of YAP (S127A) increases cell proliferation in iPS-CM compared to control iPS-CM as shown with EdU assay (46±2.60% P < 0.01) and Ki67 staining (4.9±0.9% P < 0.001). Overexpression of YAP leads to up regulation of genes associated with inhibition of apoptosis and promotion of cell proliferation. Preliminary studies show mouse iPS-CM are retained in the myocardium following intra-cardiac injection and do not cause any adverse effects confirmed with histological, echocardiography and electrocardiogram analysis. In conclusion targeting the Hippo pathway in iPS cells and iPS-CM significantly increases proliferation and survival in both normal and hypoxic conditions. Therefore, modulation of the Hippo pathway could become a new strategy to enhance the therapeutic potential of iPS-CM.

Master of Science / Department of Grain Science and Industry / X. Susan Sun / Human induced pluripotent stem cells (hiPSCs) are generated from human somatic cells using defined transcription factors. These cells possess characteristics very similar to that of human embryonic stem cells including the ability to differentiate into cell types of all three germ layers. HiPSCs show great potential in clinical researches like drug screening and regenerative medicine, that all require large amount of cells cultured under well-defined conditions. The most common culture methods used for hiPSCs are 2D culture methods using Matrigel or vitronectin coated culture plates or flasks. 2D culture methods require large surface area to produce the same amount of cells compared to 3D methods. In addition, cells cultured in 2D culture environment are far from that in vivo. In this study, we developed a robust 3D culture condition based on hiPSC-qualified PGmatrix (PGmatrix-hiPSC) hydrogel. This 3D culture system provide hiPSCs with well-defined, more in vivo-like environment that encapsulate cells in liquid rich hydrogel with appropriate oxygen supply that resembles the hypoxia condition in vivo. Two hiPSC lines grown continuously in PGmatrix-hiPSC showed higher total population expansion and higher viability, with more consistency compared to the same cell lines grown in 2D on Matrigel or Vitronectin-XF. After grown in 3D PGmatrix-hiPSC for over 25 passages, major pluripotency markers, such as Oct4, Sox2, Nanog, and SSEA4 are expressed in most hiPSCs examined by flow cytometry. RT-qPCR also confirmed adequate expression levels of major pluripotency related genes. In addition, karyotype analysis of hiPSC after 37 passages in 3D PGmatrix-hiPSC was found normal. The same hiPSC lines cultured continuously in parallel in 2D and 3D showed differences in gene expression and surface marker TRA-1-81 expression. These results indicated the 3D PGmatrix-hiPSC system is likely superior in maintaining hiPSC growth as well as pluripotency. The findings also suggest that it is very important to study cells in 3D culture environment to better understand the mechanism of pluripotency maintenance.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22121号 / 医科博第106号 / 新制||医科||7(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 濵﨑 洋子, 教授 河本 宏, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Shimamoto R, Amano N, Ichisaka T, Watanabe A, Yamanaka S, et al. (2014) Generation and Characterization of Induced Pluripotent Stem Cells from Aid-Deficient Mice. PLoS ONE 9(4): e94735. doi:10.1371/journal.pone.0094735 / 京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18515号 / 医科博第56号 / 新制||医科||4(附属図書館) / 31401 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 斎藤 通紀, 教授 平家 俊男, 教授 山田 泰広 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Systemic lupus erythematosus (SLE) develops when genetically susceptible individuals lose tolerance to autoantigens, likely as a result of an environmental insult. The list of identified genetic susceptibilities is expansive, however variants in the interferon regulatory factor 5 (IRF5) gene have consistently and convincingly been shown to be associated with an increased risk of developing SLE across all ethnic and racial groups examined. These genetic variants are hypothesized to produce a gain-of-function phenotype due to increased IRF5 mRNA and increased stability of the IRF5 protein; however, definitive functional studies examining these polymorphisms in primary human cells are not possible given the genetic variation from patient to patient. IRF5 is a transcription factor that is constitutively expressed in a number of immune cells including B cells and dendritic cells. IRF5 has cell type specific roles; in dendritic cells, it primarily controls a proinflammatory program which directs T cell polarization. Dysfunctional conventional dendritic cells (cDCs) have been implicated in the onset and development of SLE due to their high capacity to activate and interact with autoreactive lymphoid cells via a number of different pathways; the exact type of dysfunction and mechanisms underlying it are still debated. Study of primary cDCs either from SLE patients or healthy controls is complicated by the low frequency of cDCs in peripheral blood (<0.1%). To better evaluate the role IRF5 plays in cDC dysfunction in SLE, I developed a method for generating cDCs from induced pluripotent stem cells (iPSCs). The cDCs derived from this protocol are similar in many respects to primary human cDCs based on their gene expression profiles, cytokine production, and ability to act as antigen presenting cells to activate T cells. I also generated a library of iPSCs with and without the IRF5 risk haplotype to enable future studies to delineate the role of IRF5 polymorphisms in human cDCs. To facilitate these future studies, I also made an IRF5 deficient iPSC line which will be essential in discerning the role of IRF5 in cDC function. More broadly, we describe herein a platform to study gene function in an isogenic model of human conventional dendritic cells.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20562号 / 医博第4247号 / 新制||医||1022(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 山下 潤, 教授 江藤 浩之, 教授 開 祐司 / 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24502号 / 医博第4944号 / 新制||医||1064(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 長船 健二, 教授 朝長 啓造, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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