Generation of a human induced pluripotent stem cell line, YCMi002-A, from a Factor VII deficiency patient carrying F7 mutations

Factor VII (FVII) deficiency is the most common among the rare bleeding disorders, which is caused by mutations in coagulation factor VII. Clinical features caused by FVII deficiency vary from mild or asymptomatic to fatal cerebral hemorrhage. We generated an induced pluripotent stem cell (iPSC) line, YCMi002-A, from FVII deficiency patient-derived fibroblasts. YCMi002-A cells are characterized by novel compound heterozygous mutations. The c.345C > A; p.C115X is well known and the second one, c.1276C > T; p.Q426X, remains novel. YCMi002-A cells may help researchers to understand correlation between these mutations and the symptoms of FVII deficiency.ope

Reversion of FMR1 Methylation and Silencing by Editing the Triplet Repeats in Fragile X iPSC-Derived Neurons

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from a CGG repeat expansion in the fragile X mental retardation 1 (FMR1) gene. Here, we report a strategy for CGG repeat correction using CRISPR/Cas9 for targeted deletion in both embryonic stem cells and induced pluripotent stem cells derived from FXS patients. Following gene correction in FXS induced pluripotent stem cells, FMR1 expression was restored and sustained in neural precursor cells and mature neurons. Strikingly, after removal of the CGG repeats, the upstream CpG island of the FMR1 promoter showed extensive demethylation, an open chromatin state, and transcription initiation. These results suggest a silencing maintenance mechanism for the FMR1 promoter that is dependent on the existence of the CGG repeat expansion. Our strategy for deletion of trinucleotide repeats provides further insights into the molecular mechanisms of FXS and future therapies of trinucleotide repeat disorders.ope

Restoration of FVIII expression by targeted gene insertion in the FVIII locus in hemophilia A patient-derived iPSCs

Target-specific genome editing, using engineered nucleases zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is considered a promising approach to correct disease-causing mutations in various human diseases. In particular, hemophilia A can be considered an ideal target for gene modification via engineered nucleases because it is a monogenic disease caused by a mutation in coagulation factor VIII (FVIII), and a mild restoration of FVIII levels in plasma can prevent disease symptoms in patients with severe hemophilia A. In this study, we describe a universal genome correction strategy to restore FVIII expression in induced pluripotent stem cells (iPSCs) derived from a patient with hemophilia A by the human elongation factor 1 alpha (EF1α)-mediated normal FVIII gene expression in the FVIII locus of the patient. We used the CRISPR/Cas9-mediated homology-directed repair (HDR) system to insert the B-domain deleted from the FVIII gene with the human EF1α promoter. After gene targeting, the FVIIIgene was correctly inserted into iPSC lines at a high frequency (81.81%), and these cell lines retained pluripotency after knock-in and neomycin resistance cassette removal. More importantly, we confirmed that endothelial cells from the gene-corrected iPSCs could generate functionally active FVIII protein from the inserted FVIII gene. This is the first demonstration that the FVIII locus is a suitable site for integration of the normal FVIII gene and can restore FVIII expression by the EF1α promoter in endothelial cells differentiated from the hemophilia A patient-derived gene-corrected iPSCs.ope

Targeted inversion and reversion of the blood coagulation factor 8 gene in human iPS cells using TALENs

Hemophilia A, one of the most common genetic bleeding disorders, is caused by various mutations in the blood coagulation factor VIII (F8) gene. Among the genotypes that result in hemophilia A, two different types of chromosomal inversions that involve a portion of the F8 gene are most frequent, accounting for almost half of all severe hemophilia A cases. In this study, we used a transcription activator-like effector nuclease (TALEN) pair to invert a 140-kbp chromosomal segment that spans the portion of the F8 gene in human induced pluripotent stem cells (iPSCs) to create a hemophilia A model cell line. In addition, we reverted the inverted segment back to its normal orientation in the hemophilia model iPSCs using the same TALEN pair. Importantly, we detected the F8 mRNA in cells derived from the reverted iPSCs lines, but not in those derived from the clones with the inverted segment. Thus, we showed that TALENs can be used both for creating disease models associated with chromosomal rearrangements in iPSCs and for correcting genetic defects caused by chromosomal inversions. This strategy provides an iPSC-based novel therapeutic option for the treatment of hemophilia A and other genetic diseases caused by chromosomal inversions.ope

ABCD2 Is a Direct Target of β-Catenin and TCF-4: Implications for X-Linked Adrenoleukodystrophy Therapy

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene that encodes the peroxisomal ATP-binding cassette (ABC) transporter subfamily D member 1 protein (ABCD1), which is referred to as the adrenoleukodystrophy protein (ALDP). Induction of the ABCD2 gene, the closest homolog of ABCD1, has been mentioned as a possible therapeutic option for the defective ABCD1 protein in X-ALD. However, little is known about the transcriptional regulation of ABCD2 gene expression. Here, through in silico analysis, we found two putative TCF-4 binding elements between nucleotide positions −360 and −260 of the promoter region of the ABCD2 gene. The transcriptional activity of the ABCD2 promoter was strongly increased by ectopic expression of β-catenin and TCF-4. In addition, mutation of either or both TCF-4 binding elements by site-directed mutagenesis decreased promoter activity. This was further validated by the finding that β-catenin and the promoter of the ABCD2 gene were pulled down with a β-catenin antibody in a chromatin immunoprecipitation assay. Moreover, real-time PCR analysis revealed that β-catenin and TCF-4 increased mRNA levels of ABCD2 in both a hepatocellular carcinoma cell line and primary fibroblasts from an X-ALD patient. Interestingly, we found that the levels of very long chain fatty acids were decreased by ectopic expression of ABCD2-GFP as well as β-catenin and TCF-4. Taken together, our results demonstrate for the first time the direct regulation of ABCD2 by β-catenin and TCF-4.ope

OECD 주요국간 정부회계제도의 비교 연구

학위논문(석사)--서울대학교 대학원 :경영학과 경영학전공,2000.Maste

Transplantation of PSA-NCAM-Positive Neural Precursors from Human Embryonic Stem Cells Promotes Functional Recovery in an Animal Model of Spinal Cord Injury

Background: Spinal cord injury (SCI) results in permanent impairment of motor and sensory functions at and below the lesion site. There is no therapeutic option to the functional recovery of SCI involving diverse injury responses of different cell types in the lesion that limit endogenous nerve regeneration. In this regard, cell replacement therapy utilizing stem cells or their derivatives has become a highly promising approach to promote locomotor recovery. For this reason, the demand for a safe and efficient multipotent cell source that can differentiate into various neural cells is increasing. In this study, we evaluated the efficacy and safety of human polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive neural precursor cells (hNPCsPSA-NCAM+) as a treatment for SCI. Methods: One hundred thousand hNPCsPSA-NCAM+ isolated from human embryonic stem cell-derived NPCs were transplanted into the lesion site by microinjection 7 days after contusive SCI at the thoracic level. We examined the histological characteristics of the graft and behavioral improvement in the SCI rats 10 weeks after transplantation. Results: Locomotor activity improvement was estimated by the Basso-Beattie-Bresnahan locomotor rating scale. Behavioral tests revealed that the transplantation of the hNPCsPSA-NCAM+ into the injured spinal cords of rats significantly improved locomotor function. Histological examination showed that hNPCsPSA-NCAM+ had differentiated into neural cells and successfully integrated into the host tissue with no evidence of tumor formation. We investigated cytokine expressions, which led to the early therapeutic effect of hNPCsPSA-NCAM+, and found that some undifferentiated NPCs still expressed midkine, a well-known neurotrophic factor involved in neural development and inflammatory responses, 10 weeks after transplantation. Conclusion: Our results demonstrate that hNPCsPSA-NCAM+ serve as a safe and efficient cell source which has the potential to improve impaired motor function following SCI.restrictio