Center for iPS Cell Research and Application (CiRA) Kyoto University Department of
Life Science Frontiers Hotta Lab
Reliable and reproducible isolation of high-quality iPS cells is essential for future clinical applications of stem cell therapy. When patient has a genetic mutation, gene therapy by genetic correction would be desirable. My research group will challenge these issues by using several gene delivery vectors and genome engineering technologies.
In particular, we will focus on the long-term and stable expression of transgenes in human iPS cells with viral or non-viral delivery methods, and TALEN/CRISPR technologies to correct genetic mutations. These technologies will be useful not only for iPS cell research but for future gene therapy applications.
Gene Delivery by Non-viral Vectors
Gene correction of congenital disorder will open the door for understanding diseases, learning developmental and differentiation pathways, and also for iPS cell-based gene therapy approach.
"Genome Surgery" by Engineered Nucleases
Duchenne Muscular Dystrophy (DMD) is a severe muscle degeneration disease caused by the loss-of-function mutations in Dystrophin gene on X chromosome. Exon skipping to modulate mRNA splicing patterns using antisense oligonucleotide is a promising approach currently tested in clinical trials, however, the effect of antisense oligos is transient.
Recently, targeted genome editing using engineered nucleases, such as TALENs and CRISPR-Cas9 (right panel: Cas9, sgRNA, Target DNA strand, and non-target DNA strand), have been demonstrated to be effective and efficient to modify the target region of the genome in many organism.
Genome-wide shRNA library
iPS cells can be generated from adult somatic cells by introducing a cocktail of transcriptional factors, and have an enormous potential for future stem cell therapy. However, induction efficiency of iPS cells is still low (~0.02%), and heterogeneous nature of reprogramming makes difficult to control the quality of the iPS cell lines and their differentiation potentials.
When I was in the Ellis lab at Toronto, I have developed a novel selection system for human iPS cells by using a lentiviral vector that specifically express GFP (Green fluorescence protein) gene in pluripotent stem cells. By utilizing Early Transposon (ETn) promoter combined with Oct-4 (= Pou5f1) core enhancer elements, resultant EOS lentiviral vector can express GFP specifically in pluripotent stem cells, but extinguished after differentiation (Hotta et al., Nature Methods, 2009).
When the EOS vector was introduced into human fibroblasts, GFP was not activated as expected. However, after induction of iPS cell reprogramming by a forced expression of the Yamanaka factors (OCT-4, SOX2, KLF4, C-MYC), GFP expression was activated on emerged iPS cell colonies. By selecting for puromycin resistance gene which is under the control of the EOS cassette, only iPS cells can be grown, and enriched the percentage of hESC marker (i.e. TRA-1-81) positive iPSC population up to 70%. We also succeeded to isolate iPS cell lines from Rett Syndrome patient who has neurodevelopmental disorder due to a missence mutation in MeCP2 gene. The EOS vector is not only useful for selecting iPS cells, but also has a potential to utilize for optimizing novel induction methods and for screening small molecules to enhance reprogramming.