In FY17, we continue to improve and add new technologies and services related to iPSC research: We generated >135 iPSC lines from >45 human fibroblast and >90 blood samples, surpassing FY16's record while maintaining nearly 100% success rate. This is also the first year that the number of blood-derived iPSC lines surpassed that of fibroblast-derived iPSC lines, proving that blood reprogramming technique we developed can meet the demands from NIH users. Now we can use as little as 50,000 PBMCs, equivalent to the cells from one drop of blood, to derive iPSCs. We have generated human iPSC lines from patients with more than 20 diseases, including but not limited to bone marrow failure syndromes, Parkinson disease, CADASIL syndrome, ACDC, Li-Fraumeni syndrome, immunodeficiency disease, Spinal and Bulbar Muscular Atrophy, Autism, Niemann-Pick disease, Wolman disease, and Smith-Lemli-Opitz syndrome, for NHLBI, NCI, NINDS, NEI, NICHD, NCATS, NHGRI, NIMH, NIAMS, NIDDK, NIA, and FDA laboratories. These lines are being used as human disease models and have led to publications by NIH investigators. We established robust CRISPR/Cas9 methods to accomplish various gene editing projects, including gene knockout (20-80% efficiency), precision gene correction and mutation knockin (0.5-5% efficiency), and AAVS1 safe harbor knockin (50-100% efficiency). In FY17, we have completed 21 gene-editing projects that include 17 gene knockout projects, 3 gene correction projects and 1 mutation knockin project, generating >50 gene-edited lines for 8 NIH DIR Laboratories. These genetically modified iPSC lines are being used as isogenic control lines to model human hematopoietic, neurological, or metabolic diseases. We have developed chemically defined media and a rapid 7-day protocol to differentiate human iPSC into >90% pure human cardiomyocytes (CM). In FY17, we provided iPSC-CM differentiation media kit and >700 million custom iPSC-CMs to 8 NIH laboratories, who utilized iPSC-CM to study mitochondrial functions, cancer drug-induced cardiomyopathies and cardiotoxicity, gene expression during cardiac lineage differentiation, and protein degradation and synthesis in iPSC-CM. We have studies electrophysiology properties of iPSC-CM using conventional patch clamp method and high-throughput optical imaging method. Our iPSC-CMs resemble immature embryonic or fetal cardiomyocytes, display high resting potential, slow upstroke velocity, and electrochemical coupling, beat spontaneously and mature into more ventricular cardiomyocytes under in vitro culture condition. We have also developed defined methods to derive smooth muscle cells (SMC) from human iPSCs at >90% purity. iPSC-SMCs express classic SMC-specific markers, can proliferate up to 10 passages as synthetic SMCs or future mature into contractile SMCs, both of which can be identified by subtype-specific markers. iPSC-SMCs can be used to study cardiovascular and aging diseases. We continue to provide validated control iPSC lines and various validated iPSC culture reagents to NIH investigators. We continue to use iLab system to document and manage Core services, and were able to recover >20% of our total budget . We continue to make significant contribution to Autologous iPSC-derived cardiomyocytes to treat heart disease project, which is supported by NHLBI Office of Scientific Director (OSD) and involves 7 NHLBI investigators and 2 extramural collaborators. The project is a preclinical study that uses rhesus monkey iPSC (RhiPSC)-derived cardiomyocytes labelled with reporter gene such as CD19 or NIS to rescue cardiac function in an autologous myocardial infarction (MI) model, in order to evaluate feasibility, safety, and efficacy of future human iPSC-CM cellular therapies for patients with cardiac failure. For this collaboration, the iPSC Core developed a SeV reprogramming protocol for generating integration-free RhiPSCs, a rhesus CRISPR/Cas9 gene editing method to allow stable expression of a non-immunogenic CD19 or NIS reporter gene at a safe harbor locus, and a new chemically defined method to differentiate RhiPSCs into cardiomyocytes with high efficiency (>85%). We have generated iPSC lines from 8 monkeys, engineered 6 reporter RhiPSC lines, and produced >500 million RhiPSC-CMs that are ready for cell injection. The RhiPSC-CM demonstrated cardiomyocyte-specific markers and gene expression profile, and normal electrophysiological and contractile functions. We have shared control and reporter human iPSC lines through fully executed MTAs. We have deposited our iPSC gene editing vectors in non-profit repository Addgene (www.addgene.org/Jizhong_Zou/ ), who has distributed our vectors for 266 times to 156 laboratories in 131 non-profit research institutes in 24 countries worldwide. We have taught 3 FAES course on iPSC generation, gene editing, and cardiac differentiation. We gave talks and presented posters at NHLBI DIR Research Festivals, NHLBI cardiovascular regenerative medicine symposia, and annual ASGCT and ISSCR meeting. We co-authored 9 papers in FY17.
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