Identification of mesenchymal stem cell (MSC)‐transcription factors by microarray and knockdown analyses, and signature molecule‐marked MSC in bone marrow by immunohistochemistry

Kubo, Hiroshi; Shimizu, Masakazu; Taya, Yuji; Kawamoto, Takeshi; Michida, Masahiko; Kaneko, Emi; Igarashi, Akira; Nishimura, Masahiro; Segoshi, Kazumi; Shimazu, Yoshihito; Tsuji, Kiichiro; Aoba, Takaaki; Kato, Yukio

doi:10.1111/j.1365-2443.2009.01281.x

Cited by 106 publications

(92 citation statements)

References 64 publications

(53 reference statements)

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“…We identified 99 genes that were reported to be bound by EZH2 in MSC, but not following osteogenic differentiated in vitro [26], and were found to have a loss of the repressive modification H3K27me3 during osteogenic differentiation of MSCs in the independent ChIP-Seq data set GSE35576 [28]. Assessment of differential gene expression in the independent microarray data set GSE9451 [27] identified that, of these 99 genes, 6 genes (ZBTB16, HOPX, ROR2, MYADM, FHL1, MX1) were significantly upregulated (>2-fold change; P £ 0.05, LIMMA), while no genes were significantly downregulated, under osteogenic differentiation, when compared with undifferentiated MSC (Fig. 1).…”

Section: Identification Of Novel Ezh2 Targets During Msc Osteogenic Dmentioning

confidence: 99%

“…These data sets compared primary cultured human bone marrow-derived MSC versus in vitro differentiated osteoblasts (OB) differentiated with dexamethasone 100 nM (Dex) and 10 mM b-glycerophosphate and with the addition of 50 mg/mL ascorbic acid for 28 days (GSE9451 [27]; [26]) or 50 nM ascorbic acid for 16 days (GSE27900 [28]). GSE9451 data set contained three independent MSC lines.…”

Section: Bioinformatic Analysismentioning

confidence: 99%

“…To assess the differential expression of the genes identified in the ChIP data sets, two data sets were used, both conducted on Affymetrix GeneChip Human Genome U133 plus 2.0 arrays. Microarray (Affymetrix U133A) GEO data set GSE9451 [27] was normalized using RMA (RMA Express program http://rmaexpress.bmbolstad.com/).…”

Section: Bioinformatic Analysismentioning

confidence: 99%

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Identification of Novel EZH2 Targets Regulating Osteogenic Differentiation in Mesenchymal Stem Cells

Hemming

Cakouros

Vandyke

et al. 2016

Stem Cells and Development

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Histone three lysine 27 (H3K27) methyltransferase enhancer of zeste homolog 2 (EZH2) is a critical epigenetic modifier, which regulates gene transcription through the trimethylation of the H3K27 residue leading to chromatin compaction and gene repression. EZH2 has previously been identified to regulate human bone marrow-derived mesenchymal stem cells (MSC) lineage specification. MSC lineage specification is regulated by the presence of EZH2 and its H3K27me3 modification or the removal of the H3K27 modification by lysine demethylases 6A and 6B (KDM6A and KDM6B). This study used a bioinformatics approach to identify novel genes regulated by EZH2 during MSC osteogenic differentiation. In this study, we identified the EZH2 targets, ZBTB16, MX1, and FHL1, which were expressed at low levels in MSC. EZH2 and H3K27me3 were found to be present along the transcription start site of their respective promoters. During osteogenesis, these genes become actively expressed coinciding with the disappearance of EZH2 and H3K27me3 on the transcription start site of these genes and the enrichment of the active H3K4me3 modification. Overexpression of EZH2 downregulated the transcript levels of ZBTB16, MX1, and FHL1 during osteogenesis. Small interfering RNA targeting of MX1 and FHL1 was associated with a downregulation of the key osteogenic transcription factor, RUNX2, and its downstream targets osteopontin and osteocalcin. These findings highlight that EZH2 not only acts through the direct regulation of signaling modules and lineage-specific transcription factors but also targets many novel genes important for mediating MSC osteogenic differentiation.

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Section: Identification Of Novel Ezh2 Targets During Msc Osteogenic Dmentioning

confidence: 99%

Section: Bioinformatic Analysismentioning

confidence: 99%

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Identification of Novel EZH2 Targets Regulating Osteogenic Differentiation in Mesenchymal Stem Cells

Hemming

Cakouros

Vandyke

et al. 2016

Stem Cells and Development

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“…ETV1 (ER81), which is regulated by miR193b, has also been reported as a transcription factor unique to mesenchymal stem cells (MSCs). 24 In the study by Kubo et al, 24 ETV1 expression was upregulated by 5-10 times in MSC compared with fibroblasts, chondrocytes, synovial cells, and adipose cells. Kubo et al also reported that cell differentiation and juvenile properties are affected when ETV1 is knocked down using short interfering RNA; for example, the self-renewal capacity of MSCs was repressed.…”

mentioning

confidence: 99%

MicroRNA‐199a‐3p, microRNA‐193b, and microRNA‐320c are correlated to aging and regulate human cartilage metabolism

Ukai

Sato

Akutsu

et al. 2012

Journal Orthopaedic Research

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MicroRNAs (miRNAs) are small RNAs of $22 base pairs that regulate gene expression. We harvested cartilage tissue from patients with polydactylism, anterior cruciate ligament injury, and osteoarthritis undergoing total knee arthroplasty and used microarrays to identify miRNAs whose expression is upregulated or downregulated with age. The results were assessed by real-time PCR and MTT assay in a mimic group, in which synthetic double-stranded RNA from the isolated miRNA was transfected to upregulate expression, and in an inhibitor group, in which the miRNA was bound specifically to downregulate expression. The expression of two miRNAs (miR-199a-3p and miR-193b) was upregulated with age and that of one miRNA (miR-320c) was downregulated with age. A real-time PCR assay showed that type 2 collagen, aggrecan, and SOX9 expression were downregulated in the miR-199a-3p mimic group but was upregulated in the inhibitor group. Similar results were observed for miR-193b. By contrast, ADAMTS5 expression was downregulated in the miR-320c mimic group and upregulated in the inhibitor group. Cell proliferative activity was upregulated significantly in the miR-193b inhibitor group compared with the control group. We believe that miR-199a-3p and miR-193b are involved in the senescence of chondrocytes, and miR-320c is involved in the juvenile properties of chondrocytes. ß

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“…The total RNA was isolated, 24 h after the cultures reached confluency, using TRIzol (Life Technologies) and an RNeasy Mini kit (Qiagen, Chatsworth, CA, USA). In addition, total RNA was isolated from OBs, ADs and CHs, which were derived from BM-MSC (BM-MSCs-1, -2 and -3) cultures exposed to appropriate differentiation-inducing media for 28 days (13). DNA microarray analyses were carried out by a Kurabo GeneChip Custom Analysis Service with the Human Genome U133 Plus 2.0 chips (Affymetrix Inc., Santa Clara, CA, USA).…”

Section: Preparation Of Fbs and Mscsmentioning

confidence: 99%

Characteristic expression of MSX1, MSX2, TBX2 and ENTPD1 in dental pulp cells

et al. 2015

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Abstract. Dental pulp cells (DPCs) are a promising source of transplantable cells in regenerative medicine. However, DPCs have not been fully characterized at the molecular level. The aim of the present study was to distinguish DPCs from various source-derived mesenchymal stem cells (MSCs), fibroblasts (FBs) and other cells by the expression of several DPC-characteristic genes. DPCs were isolated from human pulp tissues by the explant method or the enzyme digestion method, and maintained with media containing 10% serum or 7.5% platelet-rich plasma. RNA was isolated from the cells and from dental pulp tissue specimens. The mRNA levels were determined by DNA microarray and quantitative polymerase chain reaction analyses. The msh homeobox 1, msh homeobox 2, T-box 2 and ectonucleoside triphosphate diphosphohydrolase 1 mRNA levels in DPCs were higher than that of the levels identified in the following cell types: MSCs derived from bone marrow, synovium and adipose tissue; and in cells such as FBs, osteoblasts, adipocytes and chondrocytes.The enhanced expression in DPCs was consistently observed irrespective of donor age, tooth type and culture medium. In addition, these genes were expressed at high levels in dental pulp tissue in vivo. In conclusion, this gene set may be useful in the identification and characterization of DPCs in basic studies and pulp cell-based regeneration therapy.

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Identification of mesenchymal stem cell (MSC)‐transcription factors by microarray and knockdown analyses, and signature molecule‐marked MSC in bone marrow by immunohistochemistry

Cited by 106 publications

References 64 publications

Identification of Novel EZH2 Targets Regulating Osteogenic Differentiation in Mesenchymal Stem Cells

Identification of Novel EZH2 Targets Regulating Osteogenic Differentiation in Mesenchymal Stem Cells

MicroRNA‐199a‐3p, microRNA‐193b, and microRNA‐320c are correlated to aging and regulate human cartilage metabolism

Characteristic expression of MSX1, MSX2, TBX2 and ENTPD1 in dental pulp cells

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