DNA Methylation Is Dispensable for the Growth and Survival of the Extraembryonic Lineages

SummaryDNA methylation regulates development and many epigenetic processes in mammals [1], and it is required for somatic cell growth and survival [2, 3]. In contrast, embryonic stem (ES) cells can self-renew without DNA methylation [4–6]. It remains unclear whether any lineage-committed cells can survive without DNA-methylation machineries. Unlike in somatic cells, DNA methylation is dispensable for imprinting and X-inactivation in the extraembryonic lineages [7–12]. In ES cells, DNA methylation prevents differentiation into the trophectodermal fate [13]. Here, we created triple-knockout (TKO) mouse embryos deficient for the active DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b (TKO) by nuclear transfer (NT), and we examined their development. In chimeric TKO-NT and WT embryos, few TKO cells were found in the embryo proper, but they contributed to extraembryonic tissues. TKO ES cells showed increasing cell death during their differentiation into epiblast lineages, but not during differentiation into extraembryonic lineages. Furthermore, we successfully established trophoblastic stem cells (ntTS cells) from TKO-NT blastocysts. These TKO ntTS cells could self-renew, and they retained the fundamental gene expression patterns of stem cells. Our findings indicated that extraembryonic-lineage cells can survive and proliferate in the absence of DNA methyltransferases and that a cell's response to the stress of epigenomic damage is cell type dependent

Immune responses to repetitive adenovirus-mediated gene transfer and restoration of gene expression by cyclophosphamide or etoposide

Abstract Background. One major concern about adenoviral vectors for repetitive gene delivery is the induction of an immune response to the vector, thus impeding effective gene transduction. Methods. To assess the immune response to the adenoviral vector, repetitive gene dosing was performed into rhesus monkey cervix and C3H mouse skin using the adenoviral vector carrying the lacZ gene. Three repetitive intracervical injections of adenovirus-lacZ were done in the rhesus monkey at the intervals of 4 weeks. Gene expression on the second and third injection was completely suppressed. Results. Anti-adenovirus IgG levels and neutralizing antibody titers in the rhesus monkey significantly increased after the first injection of adenovirus. In the C3H mouse, neutralizing antibody titers significantly increased after the first injection of adenovirus-lacZ at more than 10 8 plaque-forming unit (PFU). The repetitive expression of lacZ gene in the mouse skin markedly decreased when the second injection is done more than 2 weeks after the first injection. Chronic low-dose treatment with cyclophosphamide or etoposide markedly suppressed neutralizing antibody titers in the mouse serum and restored the gene expression in the mouse skin on the second and third injection. Conclusions. It is suggested that repetitive gene expression by adenovirus-mediated transfer may be reduced by circulating neutralizing antibodies and could be restored by chronic low-dose treatment with cyclophosphamide or etoposide.

IGF-1 Gene Transfer to Human Synovial MSCs Promotes Their Chondrogenic Differentiation Potential without Induction of the Hypertrophic Phenotype

Mesenchymal stem cell- (MSC-) based therapy is a promising treatment for cartilage. However, repair tissue in general fails to regenerate an original hyaline-like tissue. In this study, we focused on increasing the expression levels for insulin-like growth factor-1 (IGF-1) to improve repair tissue quality. The IGF-1 gene was introduced into human synovial MSCs with a lentiviral vector and examined the levels of gene expression and morphological status of MSCs under chondrogenic differentiation condition using pellet cultures. The size of the pellets derived from IGF-1-MSCs were significantly larger than those of the control group. The abundance of glycosaminoglycan (GAG) was also significantly higher in the IGF-1-MSC group. The histology of the IGF-1-induced pellets demonstrated similarities to hyaline cartilage without exhibiting features of a hypertrophic chondrocyte phenotype. Expression levels for the Col2A1 gene and protein were significantly higher in the IGF-1 pellets than in the control pellets, but expression levels for Col10, MMP-13, ALP, and Osterix were not higher. Thus, IGF-1 gene transfer to human synovial MSCs led to an improved chondrogenic differentiation capacity without the detectable induction of a hypertrophic or osteogenic phenotype

DS_10.1177_0363546518781825 – Supplemental material for First-in-Human Pilot Study of Implantation of a Scaffold-Free Tissue-Engineered Construct Generated From Autologous Synovial Mesenchymal Stem Cells for Repair of Knee Chondral Lesions

<p>Supplemental material, DS_10.1177_0363546518781825 for First-in-Human Pilot Study of Implantation of a Scaffold-Free Tissue-Engineered Construct Generated From Autologous Synovial Mesenchymal Stem Cells for Repair of Knee Chondral Lesions by Kazunori Shimomura, Yukihiko Yasui, Kota Koizumi, Ryota Chijimatsu, David A. Hart, Yasukazu Yonetani, Wataru Ando, Takashi Nishii, Takashi Kanamoto, Shuji Horibe, Hideki Yoshikawa and Norimasa Nakamura in The American Journal of Sports Medicine</p