NHLBI iPSC Core Facility

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

In FY25, we received more requests for iPSC gene-editing services than any year before. Totally we completed 77 gene-editing projects, more than the number of reprogramming projects for the first time since the establishment of iPSC Core in 2011. These projects include 11 gene knockout/deletion,20 gene-correction, 23 mutation or short-tag knock-in, 5 safe harbor knockin, and 18 gene knockin-in projects. Near the end of FY25, we again received many requests for reporter or tag knockin projects, indicating continuous interests from NIH labs in using these lines for basic research and drug screening projects. NCATS labs requested most of the gene-editing projects (32 projects), followed by NHLBI (15 projects) , NIDDK (7 projects), NINDS (6 projects), NIMH (3 projects), NEI (2 projects), and NICHD (1 project). We also made 11 reproter knockin lines as a collaboration with U. Penn. These gene-editing projects are completed by Core Director, one staff scientist, and one biologist. In FY25, we continued to provide high-quality, integration-free iPSC generation services. In total, we reprogrammed 68 samples that included 10 skin fibroblast and 58 PBMC samples. NIMH labs requested most of the iPSC generation projects (31 projects), followed by NHLBI (14 projects), NCATS (10 projects), NICHD (8 projects), NINDS (2 projects), NIAMS (2 projects), and NIAID (1 project). We had a new technician, who after training successfully completed >30 reprogramming projects with minimum supervision. We also successfully established T cell expansion and Sendai Virus reprogramming method for old PBMC samples that failed to expand erythroblast. The trend of fewer reprogramming projects than gene editing projects prompts us to shift our technologies towards more sophosicated gene editing, comprehensive iPSC characterization, and differentiation. In FY25, we continued our iPSC characterization services that include Scorecard assay, G-banded karyotyping, STR DNA fingerprinting, SeV qRT-PCR, mycoplasma test, and flow cytometry analysis of pluripotency markers. These services make a comprehensive package of standard iPSC characterization assays, which helped our users to publish their papers and share iPSC lines faster. With the increase of gene-editing projects, we noticed more clones with unwanted “on-target effects” that includes large deletion and copy-neutral loss of heterozygosity (cn-LOH). The large deletion results in hemizygous clone, which appears like homozygous clone due to the failure of PCR primer binding to the target sequence. This is detrimental in gene correction projects, and we found up to 20% corrected “homozygous” clones are actually hemizygous clone. The on-target effects can also create “homozygous” knockin clonen that contains a correct knockin allele and a deletion allele. To screen these large deletion clones, we added ddPCR gene copy number assay to eliminate such unwanted clones. The cn-LOH on-target effects usually don’t affect protein product and gene function, therefore we rely on the detection of nearby SNP to screen cn-LOH clones. In FY25, we explored hematopoietic differentition including megakaryocyte, platelet, and hematopoieitc stem cell (HSC) differentiation. We tested commerical kits from Stem Cell Technologies and home-made media based on published papaper. The commerical kits perform more robustly than home-made media for these hematopoietic differentiation, but cost more and couldn’t generate multi-lineage engraftment HSC. We also observed huge variation in differentiatio efficiency among different iPSC lines, and identified at least one male and one female control iPSC lines with high hematopoietic differentiation efficiency. In order to provide robust and cryopreservable products that the users can thaw and use easily, we plan to provide iPSC-derived megakaryocyte progenitors (iMKP) and megakaryocyte (iMK) service in FY26. We have promising results in iPSC-HSC differentiation which showed >90%CD34+CD43+CD45+CD90+ cells that express HSC-specific genes and are capable myeoloid and erythroid differentiation in CFC assay. In FY25, we served 10 NIH ICs that include NHLBI, NCATS, NEI, NIAID, NIAMS, NICHD, NIDDK, NIMH, NIDDS, and NIBIB. Among them, NCATS, NIAMS, NICHD, NIDDK, NIMH, and NINDS signed MOU with NHLBI to gain priority access to our services. The MOU benefits non-NHLBI PIs who now pay low NHLBI-rate service fees and are treated like NHLBI PIs. In addition, we collected membership fee for NHLBI OSD to reduce the cost of running iPSC Core. We were able to collect >$250,000 service fees to completely cover the operating cost of iPSC Core, including maintaining and upgrading equipment as well as purchasing all the reagents and supplies for all the exploratory and service projects. In return, we helped >50 NIH PIs and saved >$750,000 for them as our service fees are 4-5x lower than commerical service fees and we provide the most comprehensive iPSC services and the most sophiscated gene editing services that no commerical company provides. Among the service fees we collected, 74% were from gene-editing services; 18% from iPSC generation services; 8% from iPSC characterization services, reflecting continuous interests in gene-editing services from NIH PIs. The top 4 ICs who used our services are NHLBI, NCATS, NICHD, and NIMH. In FY25, we published 7 papers (https://www.ncbi.nlm.nih.gov/myncbi/jizhong.zou.1/bibliography/public/ ) that showed generation and characterization of new iPSC lines from patients with Algille syndrome or CLN3-related conditions, demonstrated robust differentiation of NK cells from engineered MSLN.CAR-IL-15 iPSC lines, revealed that maintenance of neuronal TDP-43 expression requires axonal lysosome transport, illuciated how CHIP protects lysomsomes from CLN4 mutant-induced membrane damage, and demonstrated human iPSC-derived microglia integrate into mouse retina and recapitulate features of endogenous microglia. In FY25, Addgene distributed 88 plasmid samples we deposited there ( https://www.addgene.org/Jizhong_Zou/ ). To date, Addgene has distributed 1187 samples from our lab’s plasmid collection.