Generation of reporter hESCs by targeting <i>EGFP</i> at the CD144 locus to facilitate the endothelial differentiation

Hu, Zhiqing; Wu, Yong; Zhou, Miaojin; Xiao-lin, Wang; Pang, Jialun; Li, Zhuo; Feng, Mai; Wang, Yanchi; Hu, Qian; Zhao, Junya; Liu, Xionghao; Wu, Lingqian

doi:10.1111/dgd.12433

Cited by 2 publications

(3 citation statements)

References 51 publications

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“…The 50-bp targeted deletion was expected to reframe the 4-bp frameshift deletion in the F8 B domain and restore the function of the FVIII protein. Because human ECs can secrete FVIII, the C-iPSCs (2-6-iPSCs and 2-46-iPSCs), HA-iPSCs, and N-iPSCs were differentiated into C-iECs, HA-iECs, and N-iECs (Figure 3A) via the optimized OP9 coculture method, to examine the recovery of FVIII secretion 34 . A deleted F8 transcript was detected, indicating that there was no NMD of the 54-bp deletion mRNA in the C-iECs (2-6-iECs and 2-46-iECs) (Figure 3B).…”

Section: Resultsmentioning

confidence: 99%

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ssODN-Mediated In-Frame Deletion with CRISPR/Cas9 Restores FVIII Function in Hemophilia A-Patient-Derived iPSCs and ECs

Zhou

et al. 2019

Molecular Therapy - Nucleic Acids

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Given that the cDNA of F8 is too large to be packaged into adeno-associated virus (AAV) capsids, gene transfer of some versions of B-domain-deleted F8 (BDD- F8 ) for hemophilia A (HA) treatment has been attempted with promising results. Here, we describe an efficient gene correction via single-stranded-oligodeoxynucleotide (ssODN)-mediated in-frame deletion within the B domain of F8 with CRISPR/Cas9 in HA-patient-derived induced pluripotent stem cells (HA-iPSCs). The expression and activity of FVIII was restored in corrected HA-iPSC-derived induced endothelial progenitor cells (C-iEPCs) in vitro and in vivo . The bleeding phenotype was rescued in HA mice after C-iEPC infusion. Our results demonstrate an efficient approach for in situ gene correction via introduction of a tiny deletion using ssODN and CRISPR/Cas9 to reframe the F8 transcript and restore FVIII function in HA-iPSC-derived EPCs with potential clinical impact in HA gene therapy. For the first time, we demonstrated in vitro and in vivo the FVIII function that is encoded by the endogenous F8 gene with a partially deleted B domain. This work also suggests an applicable strategy for genetic correction of other gene frameshift mutations.

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Section: Resultsmentioning

confidence: 99%

“…The differentiation of hiPSCs to ECs was carried out as previously described 34 . Briefly, hiPSCs were seeded on Matrigel with OP9-diff-conditioned medium (OP9-diff-CM).…”

Section: Methodsmentioning

confidence: 99%

ssODN-Mediated In-Frame Deletion with CRISPR/Cas9 Restores FVIII Function in Hemophilia A-Patient-Derived iPSCs and ECs

Zhou

et al. 2019

Molecular Therapy - Nucleic Acids

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“…Drug testing using heterogeneous cell populations may lead to an inaccurate conclusion, especially if it is based on ensemble measurements (Altschuler and Wu, 2010). To tackle this issue, a variety of strategies can be employed, such as optimization of the differentiation protocol (Wang et al, 2020), purification using genetically engineered reporter cell lines (Hu et al, 2018), or magnetic-activated cell sorting (MACS) based on the desired surface marker (Li et al, 2017).…”

Section: Moving Forward-what Is Next?mentioning

confidence: 99%

Human Induced Pluripotent Stem Cells as a Screening Platform for Drug-Induced Vascular Toxicity

Cunningham

Zhang

et al. 2021

Front. Pharmacol.

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Evaluation of potential vascular injury is an essential part of the safety study during pharmaceutical development. Vascular liability issues are important causes of drug termination during preclinical investigations. Currently, preclinical assessment of vascular toxicity primarily relies on the use of animal models. However, accumulating evidence indicates a significant discrepancy between animal toxicity and human toxicity, casting doubt on the clinical relevance of animal models for such safety studies. While the causes of this discrepancy are expected to be multifactorial, species differences are likely a key factor. Consequently, a human-based model is a desirable solution to this problem, which has been made possible by the advent of human induced pluripotent stem cells (iPSCs). In particular, recent advances in the field now allow the efficient generation of a variety of vascular cells (e.g., endothelial cells, smooth muscle cells, and pericytes) from iPSCs. Using these cells, different vascular models have been established, ranging from simple 2D cultures to highly sophisticated vascular organoids and microfluidic devices. Toxicity testing using these models can recapitulate key aspects of vascular pathology on molecular (e.g., secretion of proinflammatory cytokines), cellular (e.g., cell apoptosis), and in some cases, tissue (e.g., endothelium barrier dysfunction) levels. These encouraging data provide the rationale for continuing efforts in the exploration, optimization, and validation of the iPSC technology in vascular toxicology.

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Generation of reporter hESCs by targeting EGFP at the CD144 locus to facilitate the endothelial differentiation

Cited by 2 publications

References 51 publications

ssODN-Mediated In-Frame Deletion with CRISPR/Cas9 Restores FVIII Function in Hemophilia A-Patient-Derived iPSCs and ECs

ssODN-Mediated In-Frame Deletion with CRISPR/Cas9 Restores FVIII Function in Hemophilia A-Patient-Derived iPSCs and ECs

Human Induced Pluripotent Stem Cells as a Screening Platform for Drug-Induced Vascular Toxicity

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