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1.
Sci Rep ; 10(1): 12794, 2020 07 30.
Article in English | MEDLINE | ID: mdl-32732907

ABSTRACT

Articular cartilage damage does not heal spontaneously and causes joint dysfunction. The implantation of induced pluripotent stem cell (iPSC)-derived cartilage (iPS-Cart) is one candidate treatment to regenerate the damaged cartilage. However, concerns of tumorigenicity are associated with iPS-Cart, because the iPSC reprogramming process and long culture time for cartilage induction could increase the chance of malignancy. We evaluated the tumorigenic risks of iPS-Cart using HeLa cells as the reference. Spike tests revealed that contamination with 100 HeLa cells in 150 mg of iPS-Cart accelerated the cell growth rate. On the other hand, 150 mg of iPS-Cart without HeLa cells reached growth arrest and senescence after culture, suggesting less than 100 tumorigenic cells, assuming they behave like HeLa cells, contaminated iPS-Cart. The implantation of 10,000 or fewer HeLa cells into joint surface defects in the knee joint of nude rat did not cause tumor formation. These in vitro and in vivo studies collectively suggest that the implantation of 15 g or less iPS-Cart in the knee joint does not risk tumor formation if assuming that the tumorigenic cells in iPS-Cart are equivalent to HeLa cells and that nude rat knee joints are comparable to human knee joints in terms of tumorigenicity. However, considering the limited immunodeficiency of nude rats, the clinical amount of iPS-Cart for implantation needs to be determined cautiously.


Subject(s)
Carcinogenesis , Cartilage, Articular/pathology , Induced Pluripotent Stem Cells/pathology , Animals , B-Lymphocyte Subsets , Cartilage, Articular/cytology , Cell Proliferation , HeLa Cells , Humans , Induced Pluripotent Stem Cells/cytology , Knee Joint/cytology , Knee Joint/pathology , Rats, Nude , Risk
2.
Bioorg Med Chem ; 26(12): 3551-3558, 2018 07 23.
Article in English | MEDLINE | ID: mdl-29807700

ABSTRACT

Higher-order structures of nucleic acids have become widely noted for their biological consequences and the discovery of an alkylating small molecule for these structures has been of interest due to its therapeutic potential. We previously developed the vinyldiaminotriazine (VDAT)-acridine conjugate as a T-T mismatch alkylating agent. In this report, we focused on the finding of the alkylation to the G-quadruplex (G4) DNA with VDAT-acridine conjugates. The VDAT-acridine conjugates exhibited a considerable alkylation ability to G4 under mild conditions. Moreover, the investigation of properties with the alkylated G4 revealed that alkylation by this conjugate significantly increased the stability of the G4 structure. This study provides a starting point in the further development of selective G4 alkylating small molecules.


Subject(s)
Acridines/chemistry , Alkylating Agents/chemistry , G-Quadruplexes , Triazines/chemistry , Alkylation , Base Pair Mismatch , Base Sequence , Thymine/chemistry , Thymine/metabolism , Transition Temperature
3.
Nucleic Acids Res ; 46(3): 1059-1068, 2018 02 16.
Article in English | MEDLINE | ID: mdl-29309639

ABSTRACT

The alkylation of the specific higher-order nucleic acid structures is of great significance in order to control its function and gene expression. In this report, we have described the T-T mismatch selective alkylation with a vinyldiaminotriazine (VDAT)-acridine conjugate. The alkylation selectively proceeded at the N3 position of thymidine on the T-T mismatch. Interestingly, the alkylated thymidine induced base flipping of the complementary base in the duplex. In a model experiment for the alkylation of the CTG repeats DNA which causes myotonic dystrophy type 1 (DM1), the observed reaction rate for one alkylation increased in proportion to the number of T-T mismatches. In addition, we showed that primer extension reactions with DNA polymerase and transcription with RNA polymerase were stopped by the alkylation. The alkylation of the repeat DNA will efficiently work for the inhibition of replication and transcription reactions. These functions of the VDAT-acridine conjugate would be useful as a new biochemical tool for the study of CTG repeats and may provide a new strategy for the molecular therapy of DM1.


Subject(s)
Acridines/chemistry , Base Pair Mismatch , DNA Replication , DNA/chemistry , Thymidine/chemistry , Triazines/chemistry , Vinyl Compounds/chemistry , Alkylation , Base Pairing , Base Sequence , DNA/metabolism , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Humans , Molecular Targeted Therapy , Myotonic Dystrophy/genetics , Myotonic Dystrophy/pathology , Myotonic Dystrophy/therapy , Thymidine/metabolism , Transcription, Genetic , Trinucleotide Repeats
4.
Biochem Biophys Res Commun ; 495(1): 1468-1475, 2018 01 01.
Article in English | MEDLINE | ID: mdl-29196261

ABSTRACT

The implantation of autologous chondrocytes is a therapeutic treatment for articular cartilage damage. However, the benefits are limited due to the expansion of chondrocytes in monolayer culture, which causes loss of chondrocytic characters. Therefore, culture conditions that enhance chondrocytic characters are needed. We screened 5822 compounds and found that A-674563 enhanced the transcription of several chondrocyte marker genes, including Col2a1, Acan and Col11a2, in mouse primary chondrocytes. Experiments using cycloheximide, MG132 and bafilomycin A1 have revealed that Sox9 is degraded through the ubiquitin-proteasome pathway and that A-674563 inhibits this degradation, resulting in larger amount of Sox9 protein. RNA sequencing transcriptome analysis showed that A-674563 increases the expression of the gene that encodes ubiquitin-specific peptidase 29, which is known to induce the deubiquitination of proteins. Although the precise mechanism remains to be determined, our findings indicated that A-674563 could contribute to culture conditions that expand chondrocytes without losing chondrocytic characters.


Subject(s)
Batch Cell Culture Techniques/methods , Cell Proliferation/physiology , Chondrocytes/drug effects , Chondrocytes/metabolism , Indazoles/administration & dosage , Pyridines/administration & dosage , SOX9 Transcription Factor/metabolism , Animals , Biomarkers/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Chondrocytes/cytology , Gene Expression Regulation/drug effects , Mice
6.
Nat Commun ; 7: 10959, 2016 Mar 24.
Article in English | MEDLINE | ID: mdl-27009967

ABSTRACT

Osteoarthritis is a common debilitating joint disorder. Risk factors for osteoarthritis include age, which is associated with thinning of articular cartilage. Here we generate chondrocyte-specific salt-inducible kinase 3 (Sik3) conditional knockout mice that are resistant to osteoarthritis with thickened articular cartilage owing to a larger chondrocyte population. We also identify an edible Pteridium aquilinum compound, pterosin B, as a Sik3 pathway inhibitor. We show that either Sik3 deletion or intraarticular injection of mice with pterosin B inhibits chondrocyte hypertrophy and protects cartilage from osteoarthritis. Collectively, our results suggest Sik3 regulates the homeostasis of articular cartilage and is a target for the treatment of osteoarthritis, with pterosin B as a candidate therapeutic.


Subject(s)
Antineoplastic Agents/pharmacology , Cartilage, Articular/metabolism , Chondrocytes/metabolism , Indans/pharmacology , Osteoarthritis, Knee/metabolism , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Aged , Aged, 80 and over , Animals , Blotting, Western , Cartilage, Articular/drug effects , Cartilage, Articular/pathology , Cells, Cultured , Chondrocytes/drug effects , Chondrocytes/pathology , Female , Humans , Hypertrophy , Immunoblotting , Male , Mice , Mice, Knockout , Middle Aged , Organ Size , Osteoarthritis, Knee/pathology , Phosphorylation , Protein Serine-Threonine Kinases/drug effects , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
7.
Stem Cell Reports ; 4(3): 404-18, 2015 Mar 10.
Article in English | MEDLINE | ID: mdl-25733017

ABSTRACT

Defects in articular cartilage ultimately result in loss of joint function. Repairing cartilage defects requires cell sources. We developed an approach to generate scaffoldless hyaline cartilage from human induced pluripotent stem cells (hiPSCs). We initially generated an hiPSC line that specifically expressed GFP in cartilage when teratoma was formed. We optimized the culture conditions and found BMP2, transforming growth factor ß1 (TGF-ß1), and GDF5 critical for GFP expression and thus chondrogenic differentiation of the hiPSCs. The subsequent use of scaffoldless suspension culture contributed to purification, producing homogenous cartilaginous particles. Subcutaneous transplantation of the hiPSC-derived particles generated hyaline cartilage that expressed type II collagen, but not type I collagen, in immunodeficiency mice. Transplantation of the particles into joint surface defects in immunodeficiency rats and immunosuppressed mini-pigs indicated that neocartilage survived and had potential for integration into native cartilage. The immunodeficiency mice and rats suffered from neither tumors nor ectopic tissue formation. The hiPSC-derived cartilaginous particles constitute a viable cell source for regenerating cartilage defects.


Subject(s)
Cell Differentiation , Hyaline Cartilage/cytology , Hyaline Cartilage/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Animals , Biomarkers , Cell Culture Techniques , Cell Line , Cell Transformation, Neoplastic , Chondrocytes/cytology , Chondrogenesis , Gene Expression , Gene Knock-In Techniques , Genes, Reporter , Humans , Mice , Mice, SCID , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Stem Cell Transplantation , Swine , Swine, Miniature , Transgenes
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