The PRDM14-CtBP1/2-PRC2 complex regulates transcriptional repression during the transition from primed to naïve pluripotency.

The pluripotency-associated transcriptional network is regulated by a core circuitry of transcription factors. The PR domain-containing protein PRDM14 maintains pluripotency by activating and repressing transcription in a target gene-dependent manner. However, the mechanisms underlying dichotomic switching of PRDM14-mediated transcriptional control remain elusive. Here, we identified C-terminal binding protein 1 and 2 (CtBP1 and CtBP2; generically referred to as CtBP1/2) as components of the PRDM14-mediated repressive complex. CtBP1/2 binding to PRDM14 depends on CBFA2T2, a core component of the PRDM14 complex. The loss of Ctbp1/2 impaired the PRDM14-mediated transcriptional repression required for pluripotency maintenance and transition from primed to naïve pluripotency. Furthermore, CtBP1/2 interacted with the PRC2 complexes, and the loss of Ctbp1/2 impaired Polycomb repressive complex 2 (PRC2) and H3K27me3 enrichment at target genes after Prdm14 induction. These results provide evidence that the target gene-dependent transcriptional activity of PRDM14 is regulated by partner switching to ensure the transition from primed to naïve pluripotency.This article has an associated First Person interview with the first author of the paper.

Comparison of gingiva-derived and bone marrow mesenchymal stem cells for osteogenesis.

Presently, bone marrow is considered as a prime source of mesenchymal stem cells; however, there are some drawbacks and limitations. Compared with other mesenchymal stem cell (MSC) sources, gingiva-derived mesenchymal stem cells (GMSCs) are abundant and easy to obtain through minimally invasive cell isolation techniques. In this study, MSCs derived from gingiva and bone marrow were isolated and cultured from mice. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometry. Compared with bone marrow MSCs (BMSCs), the proliferation capacity was judged by CCK-8 proliferation assay. Osteogenic differentiation was assessed by ALP staining, ALP assay and Alizarin red staining. RT-qPCR was performed for ALP, OCN, OSX and Runx2. The results indicated that GMSCs showed higher proliferative capacity than BMSCs. GMSCs turned more positive for ALP and formed a more number of mineralized nodules than BMSCs after osteogenic induction. RT-qPCR revealed that the expression of ALP, OCN, OSX and Runx2 was significantly increased in the GMSCs compared with that in BMSCs. Moreover, it was found that the number of CD90-positive cells in GMSCs elevated more than that of BMSCs during osteogenic induction. Taking these results together, it was indicated that GMSCs might be a promising source in the future bone tissue engineering.

Indirect osteoblast differentiation by liposomal clodronate.

Bisphosphonates impair function of osteoclasts and prevent bone resorption, the mechanism of which has been studied extensively. However, the possible effects of bisphosphonates on chondroblast differentiation and calcium deposition by osteoblasts have only been demonstrated recently. Moreover, cells from monocytic lineage are capable of stimulating osteoblast proliferation. Hence, susceptibility of osteoblasts to various factors requires further investigation. A primary culture of bone marrow-derived stromal cells was treated with liposomal clodronate (0.1, 0.5, or 1.0 mg/ml) or conditioned medium from liposomal clodronate. Liposomal clodronate (0.25 mg) was injected into mouse femur for in vivo experiments. The effects of liposomal clodronate were examined by alkaline phosphatase staining and/or activity assay, and real-time RT-PCR was used for studying the effect on osteogenic gene expression. Administration of liposomal clodronate to bone marrow-derived mesenchymal stromal cell culture enhanced alkaline phosphatase activity and mRNA levels of Runx2 and Dlx5. In addition, conditioned medium from liposomal clodronate also stimulated osteogenic characteristics similar to those of observed in vitro, and the number of exosomes in the conditioned medium was highest when pre-treated with liposomal clodronate. Western blot analysis revealed the presence of RANK proteins in exosomes collected from conditioned medium of liposomal clodronate. Identical observations were obtained in vivo, as liposomal clodronate-injected mouse femur showed increased alkaline phosphatase activity and Runx2 and Dlx5 mRNA expressions, even though the numbers of monocytes and macrophages were reduced. In conclusion, osteoblast differentiation was promoted via soluble RANK-containing exosomes in response to clodronates.

Determination of Serum 25-Hydroxyvitamin D3 by LC/MS/MS and Its Monthly Variation in Sapporo Indoor Workers.

25-Hydroxyvitamin D3 (25(OH)D3) as the metabolite of vitamin D, is connected with various of diseases, and important to people with limited sunshine. Thus, the investigation of serum 25-hydroxyvitamin D and its variation in these people is necessary. In this study, a simple, precise, and accurate method for serum 25(OH)D3 determination by LC/MS/MS was developed. Serum samples were obtained monthly for one year from 11 male and 11 female indoor workers in Sapporo, Japan, and the overall 25(OH)D3 concentration was 12.9 ± 4.7 ng/mL. The 25(OH)D3 in females was significantly lower than that in males (14.0 ± 5.0 vs. 11.9 ± 4.3 ng/mL). The serum 25(OH)D3 concentration in males and females were both strongly correlated to UV-B radiation (r2 = 0.8477 and 0.7384, respectively), with a two-month's lag. Also the monthly change in 25(OH)D3 in males was more significant than that in females.

Osteogenic Potential of Adipose-Derived Macrospheroids Cocultured with CD11b+ Monocytes.

PURPOSE: Among potential cell-based therapies, adipose-derived stem cells (ASCs) have been proposed as a promising source of stem cells for tissue regeneration. Although many recent clinical trials have investigated the use of adipose tissue or ASCs in transplantation, analysis of the microstructures of outgrowing macrosized spheroids (macrospheroids) or three-dimensional coculture of ASC spheroids and monocyte/macrophage lineages has not been performed. The aim of this study was to analyze the microstructures of murine-derived ASC macrospheroids and the growth and osteogenic potential of these macrospheroids in a three-dimensional environment and after calcification induction by coculture with monocytes. MATERIALS AND METHODS: The histologic structures of murine-derived ASC macrospheroids and the expression of marker genes for multipotency within these macrospheroids were analyzed by hematoxylin and eosin staining and in situ hybridization. ASC macrospheroid microstructures were observed by transmission electron microscopy, and cell proliferation in the spheroids was analyzed. Additionally, the growth and osteogenic potential of these macrospheroids were assessed in two-dimensional and three-dimensional environments and after calcification induction by coculture with monocytes. RESULTS: The expression of Oct3/4, Nanog, and Sox2 was detected even in the deep zone of spheroids, although higher expression was observed at the surface. Cell proliferation was detected within the spheroid centers. Observation of spheroid microstructure revealed extracellular matrix production within the spheroid architecture. Transplantation of a spheroid on the hydroxyapatite disc resulted in three-dimensional cell growth, filling the disc. Coculture of the spheroids with monocytes led to the formation of many osteoclast-like, multinucleated cells, and calcification was observed after 3 weeks of coculture. CONCLUSION: ASC spheroids exhibited high capacity for dynamic three-dimensional growth and osteogenic differentiation. Furthermore, ASC spheroids promoted monocyte differentiation into osteoclast-like cells, which may enhance the osteogenic potential of ASC spheroids.

Osteogenic Potential of Mouse Periosteum-Derived Cells Sorted for CD90 In Vitro and In Vivo.

The treatment of bone defects still presents complex problems, although various techniques have been developed. The periosteum is considered a good source of osteogenic precursor cells for new bone formation. It can be collected easily in the clinical setting and is less invasive to the donor site. However, the murine skull periosteum has a poor cellular component, and growth is very slow, making it important to identify a culture method for efficient growth. In the present study, we used three-dimensional cell migration with atelocollagen and gelatin media and found that both were effective for promoting the proliferation of periosteum-derived cells. Moreover, atelocollagen medium is expected to provide an added benefit as a scaffold structure in the ambient temperature of the human body. The selection of a proper surface marker for osteogenesis is imperative for bone regeneration. CD90 is a mesenchymal stem cell marker. Periosteum-derived cells sorted with CD90 showed higher proliferative capacity and osteogenic potential than that of unsorted periosteum-derived cells in vivo and in vitro. Thus, periosteum-derived cells sorted with CD90 are expected to be a good source for bone regeneration. Significance: Periosteum-derived cells showed higher proliferative capacity and osteogenic potential. Periosteum can be collected easily in the clinical setting and is less invasive to the donor site. Thus, periosteum-derived cells can be expected to be a good source for bone regeneration.

Osteogenic Potential of Mouse Adipose-Derived Stem Cells Sorted for CD90 and CD105 In Vitro.

Adipose tissue-derived stromal cells, termed ASCs, play an important role in regenerative applications. They resemble mesenchymal stem cells owing to their inexhaustibility, general differentiation potential, and plasticity and display a series of cell-specific and cluster-of-differentiation (CD) marker profiles similar to those of other somatic stem cells. Variations in phenotypes or differentiation are intimately associated with CD markers. The purpose of our study was to exhibit distinct populations of ASCs with differing characteristics for osteogenic differentiation. The primary cell batch of murine-derived ASCs was extracted from subcutaneous adipose tissue and the cells were sorted for the expression of the surface protein molecules CD90 and CD105 using flow cytometry. Each cell population sorted for CD90 and CD105 was analyzed for osteogenic potency after cell culture. The results suggested that ASCs exhibit distinct populations with differing characteristics for osteogenic differentiation: unsorted ASCs stimulated comparable mineralized nodule formation as bone marrow stromal cells (BMSCs) in osteogenic medium and viral transfection for BMP2 accelerated the formation of mineralized nodules in CD90 and/or CD105 positive ASCs with observation of decrease in CD105 expression after 14 days. Future studies assessing different immunophenotypes of ASCs should be undertaken to develop cell-based tissue engineering.