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[Effect of bone morphogenetic protein-4 overexpression on the biological activity of mouse induced pluripotent stem cells].

ABSTRACT: OBJECTIVE:This study aimed to construct the expression of bone morphogenetic protein-4 (BMP4) lentiviral vector gene and explore its influence on the biological activity of mouse induced pluripotent stem (iPS) cells. METHODS:iPS cell lines stably overexpressing BMP4 were constructed by lentivirus transfection (BMP4-overexpressing group). Cells without transfection served as the blank group, and cells with only vector transfection served as the empty-vector group. Cell proliferation was detected by CCK8, and the expression levels of ameloblastin (AMBN), cytokeratin (CK) 14, dentin sialophospho-protein (DSPP), bone sialoprotein (BSP), and Runx2 mRNA were detected by quantitative polymerase chain reaction. Alkaline phosphatase (ALP) activity was used to detect the degree of cell differentiation. RESULTS:Compared with blank and empty-vector groups, proliferation activity and ALP activity of BMP4-overexpressing group obvious increased (P<0.05), BMP4, AMBN, CK14, DSPP, BSP, Runx2 mRNA expression also increased (P<0.05). CONCLUSIONS:BMP4 can significantly promote the odontogenic differentiation of iPS.

SUBMITTER: Qiu J

PROVIDER: S-EPMC7030344 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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[Effect of bone morphogenetic protein-4 overexpression on the biological activity of mouse induced pluripotent stem cells].

Qiu Jin J Zhang Hui-Yu HY Liu Li L Tan Ying-Hui YH

Hua xi kou qiang yi xue za zhi = Huaxi kouqiang yixue zazhi = West China journal of stomatology 20180401 2

<h4>Objective</h4>This study aimed to construct the expression of bone morphogenetic protein-4 (BMP4) lentiviral vector gene and explore its influence on the biological activity of mouse induced pluripotent stem (iPS) cells.<h4>Methods</h4>iPS cell lines stably overexpressing BMP4 were constructed by lentivirus transfection (BMP4-overexpressing group). Cells without transfection served as the blank group, and cells with only vector transfection served as the empty-vector group. Cell proliferatio ...[more]

PMID: 29779282

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Project description:Microspace culture is promising for self-organization of induced pluripotent stem cells (iPSCs). However, the optimal size of microspaces for osteogenic differentiation is unclear. We hypothesized that a specific microspace size could facilitate self-organizing iPSC differentiation to form bone-like tissue in vitro. The objectives of this study were to investigate such effects of microspace size and to evaluate bone regeneration upon transplantation of the resulting osteogenic constructs. Dissociated mouse gingival fibroblast-derived iPSCs were plated in ultra-low-attachment microspace culture wells containing hundreds of U-bottom-shaped microwell spots per well to form cell aggregates in growth medium. The microwells had different aperture diameters/depths (400/560??m (Elp400), 500/700??m (Elp500), and 900/700??m (Elp900)) (Kuraray; Elplasia). After 5 days of aggregation, cells were maintained in osteogenic induction medium for 35 days. Only cells in the Elp500 condition tightly aggregated and maintained high viability during osteogenic induction. After 10 days of induction, Elp500 cell constructs showed significantly higher gene expression of Runx2, Osterix, Collagen 1a1, Osteocalcin, Bone sialoprotein, and Osteopontin compared to constructs in Elp400 and Elp900. In methylene blue-counterstained von Kossa staining and Movat's pentachrome staining, only Elp500 constructs showed robust osteoid formation on day 35, with high expression of type I collagen (a major osteoid component) and osteocalcin proteins. Cell constructs were transplanted into rat calvarial bone defects, and micro-CT analysis after 3 weeks showed better bone repair with significantly higher bone mineral density in the Elp500 group compared to the Elp900 group. These results suggest that microspace size affects self-organized osteogenic differentiation of iPSCs. Elp500 microspace culture specifically induces mouse iPSCs into osteoid-rich bone-like tissue possessing high bone regeneration capacity.

Project description:BackgroundEstablishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current (If) flowing through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and attenuation of the inward rectifier K+ current (IK1) flowing through inward rectifier potassium (Kir) channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs) and induced cardiomyocytes that originally show poor IK1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs) function as a biological pacemaker in vitro.Methods and resultsThe rabbit Hcn4 gene was transfected into mESCs, and stable clones were selected. mESC-CMs were generated via embryoid bodies and purified under serum/glucose-free and lactate-supplemented conditions. Approximately 90% of the purified cells were troponin I-positive by immunostaining. In mESC-CMs, expression level of the Kcnj2 gene encoding Kir2.1, which is essential for generation of IK1 currents that are responsible for stabilizing the resting membrane potential, was lower than that in an adult mouse ventricle. HCN4-overexpressing mESC-CMs expressed about a 3-times higher level of the Hcn4 gene than did non-overexpressing mESC-CMs. Expression of the Cacna1h gene, which encodes T-type calcium channel and generates diastolic depolarization in the sinoatrial node, was also confirmed. Additionally, genes required for impulse conduction including Connexin40, Connexin43, and Connexin45 genes, which encode connexins forming gap junctions, and the Scn5a gene, which encodes sodium channels, are expressed in the cells. HCN4-overexpressing mESC-CMs showed significantly larger If currents and more rapid spontaneous beating than did non-overexpressing mESC-CMs. The beating rate of HCN4-overexpressing mESC-CMs responded to ivabradine, an If inhibitor, and to isoproterenol, a beta-adrenergic receptor agonist. Co-culture of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with aggregates composed of mESC-CMs resulted in synchronized contraction of the cells. The beating rate of hiPSC-CMs co-cultured with aggregates of HCN4-overexpressing mESC-CMs was significantly higher than that of non-treated hiPSC-CMs and that of hiPSC-CMs co-cultured with aggregates of non-overexpressing mESC-CMs.ConclusionsWe generated HCN4-overexpresssing mESC-CMs expressing genes required for impulse conduction, showing rapid spontaneous beating, responding to an If inhibitor and beta-adrenergic receptor agonist, and having pacing ability in an in vitro co-culture system with other excitable cells. The results indicated that these cells could be applied to a biological pacemaker.

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[Effect of bone morphogenetic protein-4 overexpression on the biological activity of mouse induced pluripotent stem cells].

Publications

[Effect of bone morphogenetic protein-4 overexpression on the biological activity of mouse induced pluripotent stem cells].

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