In FY19, we continued to improve CRISPR/Cas gene-editing methods suitable for human iPSCs. Using single-cell sorting with chemcial supplement, we can obtain gene-edited clones in 1-2 months with high frequency of true single-cell clones. Besides achieving gene knockout at 20-80% efficiency, we can now accomplish precision gene correction or mutation knockin at 5-30% efficiency which is 5-10 fold increase from FY18. In FY19 we finished 13 gene knockout projects, 5 gene correction or mutation knockin projects, 2 gene-deletion projects, and 3 AAVS1 safe harbor knockin projects within this fiscal year. The total 23 projects made FY19 the most productive gene-editing year since we introduced iPSC gene editing service in FY15. These genetically modified iPSC lines are being used as isogenic control lines to model many human diseases. In FY19, we continued to provide high-quality, integration-free iPSC generation services. We reprogrammed 77 blood samples and 81 skin fibroblast samples, in addition to a dozen of myoblast, amniocyte, and LCL samples. With >170 iPSC lines, FY19 has the second highest number of iPSCl lines we generated in any fiscal year since the Core was established in 2011. Therefore, we have generated >800 iPSC lines in the past 8 years. While iPSC generation still account for the majority of our service projects and cost recovery (>60%), we saw 5% of reduction in iPSC generation but 7% increase in iPSC gene-editing in FY19 compared to those in FY18. In FY19, more sophisticated and elegant gene-editing projects such as gene-correction, mutation knockin, and gene deletion were requested more than simple gene KO projects for the first time. These trends reflect that NIH users are becoming experts in using iPSC technology to help their research. They realize that simply generating many iPSC lines and/or creating KO clones are not enough to accurately establish disease-specific research models for personalized medicine. For iPSC-cardiomyocyte services, we plan the experiment with the user in advance to plate the cells in any plate format and density that the user would like to have. We educate the user that iPSC-cardiomyocytes go through maturation during in vitro differentiation and gradually obtain fetal to adult cardiomyocyte characteristics. Then we can provide cells at various timepoints during the differentiation to fit specific study goals. Working with NHLBI investigators, we performed hematopoietic differentiation using iPSC derived from patients with GATA2 deficiency and examined their ability to commit to mesoderm, hemogenic endothelial precursors (HEPs), hematopoietic stem progenitor cells, and natural killer (NK) cells. Patient-derived iPSC, either derived from fibroblasts/marrow stromal cells or peripheral blood mononuclear cells, did not show significant defects in committing to mesoderm, HEP, hematopoietic stem progenitor, or NK cells. However, HEP derived from GATA2-mutant iPSC showed impaired maturation toward hematopoietic lineages. Hematopoietic differentiation was nearly abolished from homozygous GATA2 knockout (KO) iPSC lines and markedly reduced in heterozygous KO lines compared with isogenic controls. On the other hand, correction of the mutated GATA2 allele in patient-specific iPSC did not alter hematopoietic development consistently in our model. GATA2 deficiency usually manifests within the first decade of life. Newborn and infant hematopoiesis appears to be grossly intact; therefore, our iPSC model indeed may resemble the disease phenotype, suggesting that other genetic, epigenetic, or environmental factors may contribute to bone marrow failure in these patients following birth. We also used OGFOD1 knockout iPSC-cardiomyocytes to show that loss of OGFOD1 and the resultant alterations in protein translation modulates the cardiac proteome, shifting it towards higher protein amounts of sarcomeric proteins. Furthermore, we found a decrease of OGFOD1 during cardiomyocyte differentiation. These results suggest that loss of OGFOD1 modulates protein translation and splicing, thereby leading to alterations in the cardiac proteome and highlight the role of altered translation and splicing in regulating the proteome.. In FY19, we provided services to 38 NIH labs, 10 of which are from NHLBI. While only 9% cost recovery came from NHLBI labs, 63% of all the service projects were provided to NHLBI PIs. Thus, 6 of top 10 users were from NHLBI. We presented posters at NHLBI DIR Research Festivals and NIH Postbac Poster Day. We published 12 papers in FY19. To contribute to broad research community, we have deposited our iPSC gene editing vectors in non-profit repository Addgene ( ), who has distributed our top 10 vectors for 449 times to 289 laboratories in 221 non-profit research institutes worldwide.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Scientific Cores Intramural Research (ZIC)
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Lin, Yongshun; Liu, Huimin; Klein, Michael et al. (2018) Efficient differentiation of cardiomyocytes and generation of calcium-sensor reporter lines from nonhuman primate iPSCs. Sci Rep 8:5907
Sima, Ni; Li, Rong; Huang, Wei et al. (2018) Neural stem cells for disease modeling and evaluation of therapeutics for infantile (CLN1/PPT1) and late infantile (CLN2/TPP1) neuronal ceroid lipofuscinoses. Orphanet J Rare Dis 13:54
Li, Pingjuan; Marino, Michael P; Zou, Jizhong et al. (2018) Efficiency and Specificity of Targeted Integration Mediated by the Adeno-Associated Virus Serotype 2 Rep 78 Protein. Hum Gene Ther Methods 29:135-145
Lin, Yongshun; Linask, Kaari L; Mallon, Barbara et al. (2017) Heparin Promotes Cardiac Differentiation of Human Pluripotent Stem Cells in Chemically Defined Albumin-Free Medium, Enabling Consistent Manufacture of Cardiomyocytes. Stem Cells Transl Med 6:527-538
Yada, Ravi Chandra; Ostrominski, John W; Tunc, Ilker et al. (2017) CRISPR/Cas9-Based Safe-Harbor Gene Editing in Rhesus iPSCs. Curr Protoc Stem Cell Biol 43:5A.11.1-5A.11.14
Hong, So Gun; Yada, Ravi Chandra; Choi, Kyujoo et al. (2017) Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers. Mol Ther 25:44-53
Aguisanda, Francis; Yeh, Charles D; Chen, Catherine Z et al. (2017) Neural stem cells for disease modeling of Wolman disease and evaluation of therapeutics. Orphanet J Rare Dis 12:120
Chen, Guokai; Rao, Mahendra (2017) Derivation of Human-Induced Pluripotent Stem Cells in Chemically Defined Medium. Methods Mol Biol 1590:131-137
Han, Kim; Hassanzadeh, Shahin; Singh, Komudi et al. (2017) Parkin regulation of CHOP modulates susceptibility to cardiac endoplasmic reticulum stress. Sci Rep 7:2093
Sweeney, Colin L; Zou, Jizhong; Choi, Uimook et al. (2017) Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional Correction. Mol Ther 25:321-330

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