NHLBI iPSC Core Facility

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

In FY22, we continued to see increasing requests for HDR-mediated gene-editing projects including gene-correction, mutation knock-in, short-tag knock-in, safe harbor knockin, and reporter knockin-in. These projects reflect that more NIH labs are interested in using gene-editing technologies to create sophisticated iPSC disease models. In FY22, we also expanded our gene-editing services to cell lines other than human iPSCs. These cell lines include immortalized human cell line, human lymphoma cell line, and dog epithelial cell line. Using our established high-efficiency gene-editing technologies, we were able to 17 human iPSC gene-editing projects and 7 non-iPSC gene-editing projects that included gene knockout, gene-correction, mutation knock-in, short-tag knock-in, safe harbor knockin, and reporter knockin-in. These iPSC gene-editing projects were done by Core Director and one full-time biologist for 7 NHLBI labs and 4 labs from NCATS, NEI, NHGRI, and NIDDK. The genetically modified iPSC lines are being used as cell line models to study human diseases. In FY22, we continued to provide high-quality, integration-free iPSC generation services. When our users prefer or only have lymphoblastoid cell lines (LCL) that are available in many large cell repositories, we developed episomal vector mediated integration-free iPSC generation service. Although LCL reprogramming efficiency is much lower (<10-4) than fibroblast or PBMC reprogramming efficiency (>10-3), we were able to achieve 100% successful rate in all the iPSC generation projects no matter what starting material was used. Due to continued COVID-19 impact on our users research and budget, we received and reprogrammed 64 samples that included 34 skin fibroblast, 17 PBMC, and 13 LCL samples. These reprogramming projects were for 3 NHLBI labs and 8 labs from NEI, NIMH, NHGRI, NCATS, NINDS, NIEHS, and CC. The iPSC reprogramming projects were mostly done by one half-time biologist with the help from other Core staff. Since the Core was established in 2011, we have generated >1000 iPSC lines. Working with Kapil Bhartis lab in NEI, we established a robust iPSC-RPE differentiation protocol. Following differentiation, immature iPSC-RPEs can be sorted on day 40 of differentiation to reach >90% purity and cryopreserved as an off-the-shelf service product, which is comparable to commercial iRPE product from Fujifilm/CDI but costs much less. The users culture these immature iPSC-RPEs for expansion, followed by a 35-day culture for full maturation and use in functional assays. In FY22, we have provided 150 million iRPE to a user in NIH CC. We also provided >300 million off-the-shelf iPSC-CM to 3 NHLBI labs and >100 million custom iPSC-CM to an NCI lab in FY22. In FY22, we expanded our iPSC characterization services to include karyotyping, STR DNA fingerprinting, SeV qRT-PCR, mycoplasma test, teratoma formation, and flow cytometry analysis of pluripotency markers. These services were responding to the demand from NIH users for many years. In FY22, we established MOU with NCATS, NIMH, NHGRI, and NEI to collect >$250,000 for NHLBI DIR to subsidize the personnel cost of iPSC Core. In addition, we collected >$200,000 service fees to completely cover the operating cost of iPSC Core. Among the service fees we collected, 40% were from gene-editing services; 40% from iPSC generation services; 10% were from iPSC differentiation services; 10% from iPSC characterization services. In FY22, we published 11 papers (https://www.ncbi.nlm.nih.gov/myncbi/jizhong.zou.1/bibliography/public/ ).