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ZBTB16 as a Downstream Target Gene of Osterix Regulates Osteoblastogenesis of Human Multipotent Mesenchymal Stromal Cells.

ABSTRACT: Human multipotent mesenchymal stromal cells (hMSCs) possess the ability to differentiate into osteoblasts, and they can be utilized as a source for bone regenerative therapy. Osteoinductive pretreatment, which induces the osteoblastic differentiation of hMSCs in vitro, has been widely used for bone tissue engineering prior to cell transplantation. However, the molecular basis of osteoblastic differentiation induced by osteoinductive medium (OIM) is still unknown. Therefore, we used a next-generation sequencer to investigate the changes in gene expression during the osteoblastic differentiation of hMSCs. The hMSCs used in this study possessed both multipotency and self-renewal ability. Whole-transcriptome analysis revealed that the expression of zinc finger and BTB domain containing 16 (ZBTB16) was significantly increased during the osteoblastogenesis of hMSCs. ZBTB16 mRNA and protein expression was enhanced by culturing the hMSCs with OIM. Small interfering RNA (siRNA)-mediated gene silencing of ZBTB16 decreased the activity of alkaline phosphatase (ALP); the expression of osteogenic genes, such as osteocalcin (OCN) and bone sialoprotein (BSP), and the mineralized nodule formation induced by OIM. siRNA-mediated gene silencing of Osterix (Osx), which is known as an essential regulator of osteoblastic differentiation, markedly downregulated the expression of ZBTB16. In addition, chromatin immunoprecipitation (ChIP) assays showed that Osx associated with the ZBTB16 promoter region containing the GC-rich canonical Sp1 sequence, which is the specific Osx binding site. These findings suggest that ZBTB16 acts as a downstream transcriptional regulator of Osx and can be useful as a late marker of osteoblastic differentiation. J. Cell. Biochem. 117: 2423-2434, 2016. © 2016 Wiley Periodicals, Inc.

SUBMITTER: Onizuka S

PROVIDER: S-EPMC5094493 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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ZBTB16 as a Downstream Target Gene of Osterix Regulates Osteoblastogenesis of Human Multipotent Mesenchymal Stromal Cells.

Onizuka Satoru S Iwata Takanori T Park Sung-Joon SJ Nakai Kenta K Yamato Masayuki M Okano Teruo T Izumi Yuichi Y

Journal of cellular biochemistry 20160629 10

Human multipotent mesenchymal stromal cells (hMSCs) possess the ability to differentiate into osteoblasts, and they can be utilized as a source for bone regenerative therapy. Osteoinductive pretreatment, which induces the osteoblastic differentiation of hMSCs in vitro, has been widely used for bone tissue engineering prior to cell transplantation. However, the molecular basis of osteoblastic differentiation induced by osteoinductive medium (OIM) is still unknown. Therefore, we used a next-genera ...[more]

PMID: 27335174

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Project description:Multipotent mesenchymal stromal cells (MSC) have emerged as therapeutic tools for a wide range of pathological conditions. Yet, the still existing deficits regarding MSC phenotype characterization and the resulting heterogeneity of MSC used in different preclinical and clinical studies hamper the translational success. In search for novel MSC characterization approaches to complement the traditional trilineage differentiation and immunophenotyping assays reliably across species and culture conditions, this study explored the applicability of lipid phenotyping for MSC characterization and discrimination. Human peripheral blood mononuclear cells (PBMC), human fibroblasts, and human and equine adipose-derived MSC were used to compare different mesodermal cell types and MSC from different species. For MSC, cells cultured in different conditions, including medium supplementation with either fetal bovine serum or platelet lysate as well as culture on collagen-coated dishes, were additionally investigated. After cell harvest, lipids were extracted by chloroform/methanol according to Bligh and Dyer. The lipid profiles were analysed by an untargeted approach using liquid chromatography coupled to mass spectrometry (LC-MS) with a reversed phase column and an ion trap mass spectrometer. In all samples, phospholipids and sphingomyelins were found, while other lipids were not detected with the current approach. The phospholipids included different species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in all cell types, whereas phosphatidylglycerol (PG) species were only present in MSC. MSC from both species showed a higher phospholipid species diversity than PBMC and fibroblasts. Few differences were found between MSC from different culture conditions, except that human MSC cultured with platelet lysate exhibited a unique phenotype in that they exclusively featured PE O-40:4, PG 38:6 and PG 40:6. In search for specific and inclusive candidate MSC lipid markers, we identified PE O-36:3 and PG 40:7 as potentially suitable markers across culture conditions, at which PE O-36:3 might even be used across species. On that basis, phospholipid phenotyping is a highly promising approach for MSC characterization, which might condone some heterogeneity within the MSC while still achieving a clear discrimination even from fibroblasts. Particularly the presence or absence of PG might emerge as a decisive criterion for future MSC characterization.

Project description:Translation of multipotent mesenchymal stromal cell (MSC)-based therapies is advancing in human and veterinary medicine. One critical issue is the in vitro culture of MSC before clinical use. Using fetal bovine serum (FBS) as supplement to the basal medium is still the gold standard for cultivation of many cell types including equine MSC. Alternatives are being explored, with substantial success using platelet lysate-supplemented media for human MSC. However, progress lags behind in the veterinary field. The aim of this study was to establish a scalable protocol for equine platelet lysate (ePL) production and to test the ePL in equine MSC culture. Whole blood was harvested into blood collection bags from 20 healthy horses. After checking sample materials for pathogen contamination, samples from 19 animals were included. Platelet concentrates were prepared using a buffy coat method. Platelets, platelet-derived growth factor BB, and transforming growth factor β1 concentrations were increased in the concentrates compared with whole blood or serum (p < 0.05), while white blood cells were reduced (p < 0.05). The concentrates were lysed using freeze/thaw cycles, which eliminated the cells while growth factor concentrations were maintained. Donor age negatively correlated with platelet and growth factor concentrations after processing (p < 0.05). Finally, all lysates were pooled and the ePL was evaluated as culture medium supplement in comparison with FBS, using adipose-derived MSC from four unrelated donor horses. MSC proliferated well in 10% FBS as well as in 10% ePL. However, using 5 or 2.5% ePL entailed highly inconsistent proliferation or loss of proliferation, with significant differences in generation times and confluencies (p < 0.05). MSC expressed the surface antigens CD90, CD44, and CD29, but CD73 and CD105 detection was low in all culture media. Adipogenic and osteogenic differentiation led to similar results in MSC from different culture media. The buffy coat method is useful to produce equine platelet concentrate with increased platelet and reduced white blood cell content in large scales. The ePL obtained supports MSC expansion similar as FBS when used at the same concentration (10%). Further investigations into equine MSC functionality in culture with ePL should follow.

Project description:BACKGROUND: A reciprocal relationship between bone and fat development in osteoporosis is clinically well established. Some of the key molecular regulators involved in this tissue replacement process have been identified. The detailed mechanisms governing the differentiation of mesenchymal stem cells (MSC) - the key cells involved - are however only now beginning to emerge. In an attempt to address the regulation of the adipocyte/osteoblast balance at the level of gene transcription in a comprehensive and unbiased manner, we performed a large-scale gene expression profiling study using a unique cellular model, human multipotent adipose tissue-derived stem cells (hMADS). RESULTS: The analysis of 1606 genes that were found to be differentially expressed between adipogenesis and osteoblastogenesis revealed gene repression to be most prevalent prior to commitment in both lineages. Computational analyses suggested that this gene repression is mediated by miRNAs. The transcriptional activation of lineage-specific molecular processes in both cases occurred predominantly after commitment. Analyses of the gene expression data and promoter sequences produced a set of 65 genes that are candidates for genes involved in the process of adipocyte/osteoblast commitment. Four of these genes were studied in more detail: LXRalpha and phospholipid transfer protein (PLTP) for adipogenesis, the nuclear receptor COUP-TF1 and one uncharacterized gene, TMEM135 for osteoblastogenesis. PLTP was secreted during both early and late time points of hMADS adipocyte differentiation. LXRalpha, COUP-TF1, and the transmembrane protein TMEM135 were studied in primary cultures of differentiating bone marrow stromal cells from healthy donors and were found to be transcriptionally activated in the corresponding lineages. CONCLUSION: Our results reveal gene repression as a predominant early mechanism before final cell commitment. We were moreover able to identify 65 genes as candidates for genes controlling the adipocyte/osteoblast balance and to further evaluate four of these. Additional studies will explore the precise role of these candidate genes in regulating the adipogenesis/osteoblastogenesis switch.

Dataset Information

ZBTB16 as a Downstream Target Gene of Osterix Regulates Osteoblastogenesis of Human Multipotent Mesenchymal Stromal Cells.

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ZBTB16 as a Downstream Target Gene of Osterix Regulates Osteoblastogenesis of Human Multipotent Mesenchymal Stromal Cells.

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