In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model

Luce, Eléanor; Steichen, Clara; Allouche, Michèle; Messina, Antonietta; Heslan, Jean‐Marie; Lambert, Thierry; Weber, Anne; Nguyen, Tuan Huy; Olivier, Christophe; Dubart‐Kupperschmitt, Anne

doi:10.1002/hep.32211

Cited by 11 publications

(16 citation statements)

References 36 publications

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“…In the hepatocyte-like cell differentiation system, efficiency was not evident although the current study differentiated mESCs into hepatocyte-like cells, and the expression level of liver markers was lower than that previously reported [ 31 , 32 ]. On the one hand, the presence of undifferentiated cells in the sample was considered without segregating them from differentiated cells.…”

Section: Discussioncontrasting

confidence: 85%

Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology

Sun

Zhao

et al. 2022

Stem Cell Res Ther

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Background Hemophilia B is a rare inherited genetic bleeding disorder caused by a deficiency or lack of coagulation factor IX, the gene for which (F9) is located on the X chromosome. Hemophilia B is currently incurable and the standard treatment is coagulation factor replacement therapy. Although gene therapy has the potential to cure hemophilia, significant barriers are still needed to be overcome, e.g., off-target effects and immunoreactivity, so new approaches must be explored. Nonsense mutations account for 8% of all the hemophilia B mutation types and can result in the development of coagulation factor inhibitors. In this study, CRISPR/Cas9 technology was used to construct a mouse embryonic stem cell model with a hemophilia B nonsense mutation (F9 c.223C > T) in humans to investigate the pathogenesis and treatment of nonsense mutations in hemophilia B. Methods First, a donor plasmid with a mutation (F9 c.223 C > T) and sgRNAs were constructed. Second, both the donor plasmid and the px330-sgRNA were electroporated into mouse embryonic stem cell, and the mutant cells were then screened using puromycin and red fluorescence. Third, the mutant cell lines were tested for pluripotency and the ability to differentiate into three layers. Finally, the effect of mutation on gene function was studied in the differentiation system. Results The mutant vector and effective sgRNA were constructed, and the mutant cell line was screened. This mutant cell line exhibited pluripotency and the ability to differentiate into three layers. This point mutation affects F9 expression at both the RNA and protein levels in the differentiation system. Conclusions The mutant cell line obtained in the current study had a single-base mutation rather than a base deletion or insertion in the exon, which is more similar to clinical cases. In addition, the mutant has the characteristics of mouse embryonic stem cells, and this point mutation affects F9 gene transcription and translation, which can be used as a disease model for studying the pathogenesis and treatment of hemophilia at the stem cell level.

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Section: Discussioncontrasting

confidence: 85%

Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology

Sun

Zhao

et al. 2022

Stem Cell Res Ther

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“…Indeed, we developed an in vitro model of hemophilia B hepatocytes using iPSCs generated from fibroblasts of a severe HB patient. Through the 3D culture and by optimizing the maturation step of the differentiation protocol, we produced iHeps secreting the active form of the coagulation factor IX, specific ability of adult hepatocytes [34].…”

Section: Discussionmentioning

confidence: 99%

Evidence of Adult Features and Functions of Hepatocytes Differentiated from Human Induced Pluripotent Stem Cells and Self-Organized as Organoids

Messina

Luce

Benzoubir

et al. 2022

Cells

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Background: Human-induced pluripotent stem cell-derived hepatocytes (iHeps) have been shown to have considerable potential in liver diseases, toxicity, and pharmacological studies. However, there is a growing need to obtain iHeps that are truly similar to primary adult hepatocytes in terms of morphological features and functions. We generated such human iHeps, self-assembled as organoids (iHep-Orgs). Methods: iPSC-derived hepatoblasts were self-assembled into spheroids and differentiated into mature hepatocytes modulating final step of differentiation. Results: In about four weeks of culture, the albumin secretion levels and the complete disappearance of α-fetoprotein from iHep-Orgs suggested the acquisition of a greater degree of maturation than those previously reported. The expression of apical transporters and bile acid secretion evidenced the acquisition of complex hepatocyte polarity as well as the development of a functional and well-defined bile canalicular network confirmed by computational analysis. Activities recorded for CYP450, UGT1A1, and alcohol dehydrogenase, response to hormonal stimulation, and glucose metabolism were also remarkable. Finally, iHep-Orgs displayed a considerable ability to detoxify pathological concentrations of lactate and ammonia. Conclusions: With features similar to those of primary adult hepatocytes, the iHep-Orgs thus produced could be considered as a valuable tool for the development and optimization of preclinical and clinical applications.

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“…The differentiation of iPSCs into hepatocytes was carried out by supplier (iRegene Therapeutics Ltd.) as described previously ( 48 , 49 ). qRT-PCR was used to detect the expression of OCT4 , GATA4 , and FOXA2 on day 4; PROX1 and AFP on day 8; and ALB and HNF4 α on day 14 of induction.…”

Section: Methodsmentioning

confidence: 99%

Gene editing of human iPSCs rescues thrombophilia in hereditary antithrombin deficiency in mice

et al. 2022

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Hereditary antithrombin deficiency is caused by SERPINC1 gene mutations and predisposes to recurrent venous thromboembolism that can be life-threatening. Therefore, lifelong anticoagulation is required, which has side effects and may not be effective. In this study, peripheral blood mononuclear cells from a patient with severe antithrombin deficiency were reprogrammed into induced pluripotent stem cells (iPSCs). The mutation was corrected using CRISPR-Cas9 and Cre/LoxP genome editing. iPSCs were differentiated into hepatocytes, which were injected into the spleen of antithrombin knockout mice to restore the activity of antithrombin and reduce the thrombophilic state. Human iPSC-differentiated hepatocytes colonized mice and secreted antithrombin stably, normalizing antithrombin in plasma (activity: from 46.8 ± 5.7% to 88.6 ± 7.6%, P < 0.0001; antigen: from 146.9 ± 19.5 nanograms per milliliter to 390.7 ± 16.1 nanograms per milliliter, P < 0.0001). In venous thrombosis model, the rate of thrombosis in mice treated with edited hepatocytes, parental hepatocytes, and wild-type mice were 60, 90, and 70%, respectively. The thrombus weight was much lighter in mice treated with edited hepatocytes compared with parental hepatocytes (7.25 ± 2.00 milligrams versus 15.32 ± 2.87 milligrams, P = 0.0025) and showed no notable difference compared with that in wild-type mice (10.41 ± 2.91 milligrams). The activity and concentration of antithrombin remained high for 3 weeks after injection. The liver and kidney function markers showed no obvious abnormality during the observation period. This study provides a proof of principle for correction of mutations in patient-derived iPSCs and potential therapeutic applications for hereditary thrombophilia.

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In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model

Cited by 11 publications

References 36 publications

Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology

Generation of an mESC model with a human hemophilia B nonsense mutation via CRISPR/Cas9 technology

Evidence of Adult Features and Functions of Hepatocytes Differentiated from Human Induced Pluripotent Stem Cells and Self-Organized as Organoids

Gene editing of human iPSCs rescues thrombophilia in hereditary antithrombin deficiency in mice

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