[Contribution to Science]
1. DNA methylation of ESC specific genes in ESCs and somatic cells:

Embryonic stem cells (ESCs) possess capacities to differentiate into all three germ layers and maintain long-term self-renewal. However, the molecular basis that support ESC specific feature was unclear. We compared published data of expressed sequence tags between ESCs and other cell types and identified ESC-associated transcripts (ECATs). In this project, we aimed to understand how ECATs differentially expressed among ESCs and somatic cells, and whether DNA methylation correlated with the expression features. Most of somatic cells lacked the expression of ECATs and their promoter regions were hypermethylated. Only male germ cells exhibited low levels of CpG methylation. This result provided the feature of male germ cells that brings forth the next pluripotent state after the fertilization, and it has been referenced in several articles related to stem cell biology and reproductive biology. My mission in this project was mainly bisulfite sequencing to profile CpG methylation in both of ESCs and male germ

a. Imamura M., Miura K., Iwabuchi K., Ichisaka T., Nakagawa M., Lee J., Kanatsu-Shinohara M., Shinohara T. & Yamanaka S. Transcriptional repression and DNA hypermethylation of a small set of ES cell marker genes in male germline stem cells. BMC Developmental Biology, 2006; 6: 34. Epub 2006 July 25. PMCID: PMC1564388

2. Functional analysis of ESC-associated transcripts in mouse iPSCs, ESCs and development:

Next, I started to analyze molecular mechanisms of individual ECATs, and focused on ECAT11/L1td1. ECAT11/L1td1 is a truncated repetitive elements originating in LINE-1 retrotransposon. These elements were predicted to possess transposase activity and/or RNA-binding activity from its putative motifs. The expression of retrotransposable elements and their truncated residue are often observed in pluripotent cells, however, their biological significance in stem cells were totally unknown. This study revealed that ECAT11/L1td1 lacks protein function in mouse and is dispensable for maintaining pluripotency and development but expresses in certain region of mouse embryo and starts to express in the early phase of induced-pluripotent stem cell (iPSC) generation. These results suggested that ECAT11/L1td1 could represent the early/primary phase of acquiring pluripotency. Also, this result showed that a potential of
ECAT11/L1td1 as early reprogramming marker of iPS generation. I performed most of the experiments and data analysis for this project.

a. Iwabuchi KA, Yamakawa T, Sato Y, et al. “ECAT11/L1td1 is enriched in ESCs and rapidly activated during iPSC generation, but it is dispensable for the maintenance and induction of pluripotency.” PLoS One, 2011; 6(5), e20461. Epub 2011 June 4. PMCID: PMC3102727
b. Patent International Application Number: PCT/JP2010/059490, Inventors: Shinya Yamanaka and Kumiko Iwabuchi, Title: “NEW MARKER FOR DETECTION OF INDUCED PLURIPOTENT STEM CELL” Publication, Date: December 2, 2010

3. Transcriptional analysis of reprogramming cells at single-cell level:

In the early phase of the technology development of iPSC, the reprogramming efficiency was extremely low (~0.01%) and its processes were poorly understood. The reprogramming population contained multiple types of cells and those populations obscured the authentic cells in the process of reprogramming. In the first half of this project, to enable to classify the reprogramming population into different stages, I designed a dual-marker reprogramming dissection system using the ECAT11-EGFP mouse line that was generated in the study described above and SSEA-1 as a stem cell marker. I also analyzed expression features of individual cells by Single-cell qPCR to see the expression dynamics clearly. I found that the cell populations on the track to iPSCs exhibit stepwise expression of pluripotentrelated genes including ECATs. In addition, I found Dppa4 as a marker of the matured iPSC state. I performed all of the experiments and data analysis for this project. In the second half of this project, we tried other cell-surface markers, CD44 and Icam1, and tested expression features in the same way as described above with collaborators. This study revealed that reprogramming cells pass through the stages that cells abundantly express epidermal genes. My role in this project was analyzing single-cell qPCR works and part of FACS analysis.

a. Iwabuchi KA, Ichisaka T and Yamanaka S, “The dissection of reprogramming process in murine induced pluripotent stem cells via expression patterns of SSEA-1 and ECAT11” Poster session, ISSCR 8th meeting, San Francisco, USA, June, 2010
b. O'Malley J, Skylaki S, Iwabuchi KA, Chantzoura E, Ruetz T, Johnsson A, Tomlinson SR, Linnarsson S, Kaji K. “High-resolution analysis with novel cell-surface markers identifies routes to iPS cells.” Nature, 2013; 499(7456), 88-91. Epub 2013 June 4. PMCID: PMC3743022

4. Reprogramming by somatic cell nuclear transfer (SCNT) and epigenetic road blocks:
Animal oocytes have the capability to reprogram somatic cells, similar to the combination of
transcription factors that are used to generate iPSCs. However, SCNT also has limited efficiency of reprogramming, and most of SCNT embryos fail to develop up to the 2-cell stage. We compared the transcriptome of normal early embryos and SCNT embryos, and identified differentially expressed genes. Among them, we identified a histone H3K9 demethylase, Kdm4a, which improves this resistance. We also found that genome that were resistant to reprogramming were enriched for H3K9me3 in donor somatic cells, suggesting this epigenetic barrier is a major obstacle for reprogramming by SCNT. In this project, I participated in epigenetic analyses (immunostaining and ChIP) of donor somatic cells.

a. Matoba S., Liu Y., Lu F., Iwabuchi K. A., Shen L., Inoue A. & Zhang Y. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation. Cell, 2014: 159(4), 884-895, Epub 2014 Nov 25.. PMCID: PMC4243038

5. Effects of exogenous glucosamine intake via Glut2 in embryonic stem cells:
The hexose transporter, Glut2, is a glucosamine (GlcN) transporter that increases substrate for the hexosamine biosynthetic pathway (HBSP). Also, Glut2 is known as one of critical regulators of mouse embryonic development and survival, but its role had been remained unknown. We utilized an in vitro mouse ESC model cultured in low-glucose (LG-ESC) that mimics physiological Glut2 expression to scrutinize the effect of GlcN intake to cellular function via Glut2. LG-ESCs cultured in the presence of GlcN showed increased number of AP+ colonies. Glut2 knocked-down LG-ESC lines compromised GlcN intake showed no altered number of colonies suggesting stimulated proliferation by GlcN is Glut2 dependent. We also found that increased O-GlcNAcylation caused by transported GlcN is not responsible for proliferation of LG-ESC but exogenous GlcN would stimulate metabolic pathways by shifting flux of fructose 6-PO4 and glutamine from HBSP toward glycolysis and increase biomass for proliferation. These results suggest that fine tuning of anabolic metabolic pathway would be important for development.

a. Jung JH, Iwabuchi K, Yang Z, Loeken MR. “Embryonic Stem Cell Proliferation Stimulated By Altered Anabolic Metabolism From Glucose Transporter 2-Transported Glucosamine” Scientific Reports, 2016; 17(6)28452, Epub 2016 Jun 17. PMCID: PMC4911601

6. Functional Enhancement of iPSC-derived T cells by editing inhibitory immune signaling using inducible CRISPR/Cas9 knock-out system:

Among therapeutic approaches available for cancer treatment, Adoptive Cell Therapy (ACT) with tumor antigen-specific CD8+ T cells is a promising approach. To gain a practical amount of T cells, we have aimed to regenerate rejuvenated T-cells from tumor infiltrated lymphocytes (TIL) using iPS technology that would provide an unlimited source of autologous T cells. These rejuvenated T cells exhibit T-cell receptor gene rearrangement patterns identical to the original T cell clones, and antigen-specific killing effector functions in vitro. Despite encouraging results in previous studies, poor survival of infused T cells and the existence of immune suppressive pathways appear to restrict the full potential of ACT. There are several inhibitory mechanisms of in vivo T cell function have been discovered and those expression features are different depending types of cancers. To maximize the treatment utilizing rejuvenated T cells, I have been aim to understand those inhibitory molecular mechanisms comprehensively. Also, the potential of iPSCs-delived T cells can be further enhanced and customized by genome engineering against inhibiting molecular mechanisms. I focused on establishing iPSCs from TILs and modifying inhibitory genes such as Pdcd-1, Tim-3 and Ctla4 using CRISPR-Cas9 system, and generating T cells from them.

a. Saito H, Iwabuchi K, Fusaki N, Ito F. “Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes.” Journal of Visualized Experiments. 2016

Expertized in:
• Mammalian cell culture and biology
• Flow cytometry and cell sorting (Becton Dickinson LSRII / FACSAria series)
• Single-cell based multiplex gene expression analysis (Fluidigm BioMark, Roche Light Cycler 480)
• Somatic nuclear reprogramming and induction of pluripotency (Mouse/Human cell lines)
• Expression vector construction (Bacterial/Mammal)
• Production of lentivirus/retrovirus for experimental infection (for cultured cells)
• Primary cell isolation and expansion (Skin fibroblasts/hepatocytes/lymphocytes/germline stem cells)
• Immunocytostaining
• Gene modification in various mammalian cells (CRISPR/cas9 based, BAC vector based, PiggyBac based)
• Mouse rearing, embryonic dissection
• DNA methylation analysis (Bisulfite sequencing, meDIP)
• General molecular biological methods : southernblotting, westernblotting, immunoprecipitation
(ChIP, RNA-IP), PCR and qPCR, plasmid modification (including BAC modification)
Experienced in:
• Library preparation for Single-cell based multiplex RNA-sequencing, CIRCLE-seq, SITE-seq
• Tissue fixation (formalin-based: teratoma, brain and testis) and staining (hematoxylin-eosin
staining and immunohistostaining)
• Two-dimensional proteome analysis
• Protein expression and purification (Mammalian cell based, Bacteria based)