scholarly journals Notch Signaling Inhibition by LY411575 Attenuates Osteoblast Differentiation and Decreased Ectopic Bone Formation Capacity of Human Skeletal (Mesenchymal) Stem Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Nihal AlMuraikhi ◽  
Dalia Ali ◽  
Radhakrishnan Vishnubalaji ◽  
Muthurangan Manikandan ◽  
Muhammad Atteya ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Formation ◽  
Notch Signaling ◽  
Global Gene Expression ◽  
Osteoblastic Differentiation ◽  
Ectopic Bone Formation ◽  
Ectopic Bone ◽  
Global Gene Expression Profiling ◽  
Mineralized Matrix

Background. Chemical biology approaches using small molecule inhibitors targeting specific signaling pathways are useful tools to dissect the molecular mechanisms governing stem cell differentiation and for their possible use in therapeutic interventions. Methods. Stem cell signaling small molecule library functional screen was performed employing human bone marrow skeletal (mesenchymal) stem cells (hBMSCs). Alkaline phosphatase (ALP) activity and formation of mineralized matrix visualized by Alizarin red staining were employed as markers for osteoblastic differentiation. Global gene expression profiling was conducted using the Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. Pathway analyses were conducted using the Ingenuity Pathway Analysis (IPA) tool. In vivo ectopic bone formation was performed using hBMSC mixed with hydroxyapatite–tricalcium phosphate granules that were implanted subcutaneously in 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius red staining were performed to identify bone formation in vivo. Results. Among the tested molecules, LY411575, a potent γ-secretase and Notch signaling inhibitor, exhibited significant inhibitory effects on osteoblastic differentiation of hBMSCs manifested by reduced ALP activity, mineralized matrix formation, and decreased osteoblast-specific gene expression as well as in vivo ectopic bone formation. Global gene expression profiling of LY411575-treated cells revealed changes in multiple signaling pathways, including focal adhesion, insulin, TGFβ, IL6, and Notch signaling, and decreased the expression of genes associated with functional categories of tissue development. Among the affected signaling networks were TGFβ1, SPP1, and ERK regulatory networks. Conclusions. We identified γ-secretase inhibitor (LY411575) as a potent regulator of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with ectopic bone formation.

scholarly journals Hedgehog Signaling Inhibition by Smoothened Antagonist BMS-833923 Reduces Osteoblast Differentiation and Ectopic Bone Formation of Human Skeletal (Mesenchymal) Stem Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Nihal AlMuraikhi ◽  
Nuha Almasoud ◽  
Sarah Binhamdan ◽  
Ghaydaa Younis ◽  
Dalia Ali ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Global Gene Expression ◽  
Ectopic Bone Formation ◽  
Ectopic Bone ◽  
In Vitro Mineralization ◽  
Global Gene Expression Profiling

Background. Hedgehog (Hh) signaling is essential for osteoblast differentiation of mesenchymal progenitors during endochondral bone formation. However, the critical role of Hh signaling during adult bone remodeling remains to be elucidated. Methods. A Smoothened (SMO) antagonist/Hedgehog inhibitor, BMS-833923, identified during a functional screening of a stem cell signaling small molecule library, was investigated for its effects on the osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSC). Alkaline phosphatase (ALP) activity and Alizarin red staining were employed as markers for osteoblast differentiation and in vitro mineralization capacity, respectively. Global gene expression profiling was performed using the Agilent® microarray platform. Effects on in vivo ectopic bone formation were assessed by implanting hMSC mixed with hydroxyapatite-tricalcium phosphate granules subcutaneously in 8-week-old female nude mice, and the amount of bone formed was assessed using quantitative histology. Results. BMS-833923, a SMO antagonist/Hedgehog inhibitor, exhibited significant inhibitory effects on osteoblast differentiation of hMSCs reflected by decreased ALP activity, in vitro mineralization, and downregulation of osteoblast-related gene expression. Similarly, we observed decreased in vivo ectopic bone formation. Global gene expression profiling of BMS-833923-treated compared to vehicle-treated control cells, identified 348 upregulated and 540 downregulated genes with significant effects on multiple signaling pathways, including GPCR, endochondral ossification, RANK-RANKL, insulin, TNF alpha, IL6, and inflammatory response. Further bioinformatic analysis employing Ingenuity Pathway Analysis revealed significant enrichment in BMS-833923-treated cells for a number of functional categories and networks involved in connective and skeletal tissue development and disorders, e.g., NFκB and STAT signaling. Conclusions. We identified SMO/Hedgehog antagonist (BMS-833923) as a powerful inhibitor of osteoblastic differentiation of hMSC that may be useful as a therapeutic option for treating conditions associated with high heterotopic bone formation and mineralization.

scholarly journals Tankyrase inhibitor XAV-939 enhances osteoblastogenesis and mineralization of human skeletal (mesenchymal) stem cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Nuha Almasoud ◽  
Sarah Binhamdan ◽  
Ghaydaa Younis ◽  
Hanouf Alaskar ◽  
Amal Alotaibi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Expression Profiling ◽  
Osteoblast Differentiation ◽  
Global Gene Expression ◽  
Osteoblastic Differentiation ◽  
Global Gene Expression Profiling ◽  
Mineralized Matrix

Abstract Tankyrase is part of poly (ADP-ribose) polymerase superfamily required for numerous cellular and molecular processes. Tankyrase inhibition negatively regulates Wnt pathway. Thus, Tankyrase inhibitors have been extensively investigated for the treatment of clinical conditions associated with activated Wnt signaling such as cancer and fibrotic diseases. Moreover, Tankyrase inhibition has been recently reported to upregulate osteogenesis through the accumulation of SH3 domain-binding protein 2, an adaptor protein required for bone metabolism. In this study, we investigated the effect of Tankyrase inhibition in osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSCs). A Tankyrase inhibitor, XAV-939, identified during a functional library screening of small molecules. Alkaline phosphatase activity and Alizarin red staining were employed as markers for osteoblastic differentiation and in vitro mineralized matrix formation, respectively. Global gene expression profiling was performed using the Agilent microarray platform. XAV-939, a Tankyrase inhibitor, enhanced osteoblast differentiation of hBMSCs as evidenced by increased ALP activity, in vitro mineralized matrix formation, and upregulation of osteoblast-related gene expression. Global gene expression profiling of XAV-939-treated cells identified 847 upregulated and 614 downregulated mRNA transcripts, compared to vehicle-treated control cells. It also points towards possible changes in multiple signaling pathways, including TGFβ, insulin signaling, focal adhesion, estrogen metabolism, oxidative stress, RANK-RANKL (receptor activator of nuclear factor κB ligand) signaling, Vitamin D synthesis, IL6, and cytokines and inflammatory responses. Further bioinformatic analysis, employing Ingenuity Pathway Analysis identified significant enrichment in XAV-939-treated cells of functional categories and networks involved in TNF, NFκB, and STAT signaling. We identified a Tankyrase inhibitor (XAV-939) as a powerful enhancer of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with low bone formation.

Gene Expression Analysis of Ectopic Bone Formation Induced by Electroporatic Gene Transfer of BMP4

2006 ◽  
Vol 111 (2) ◽  
pp. 231-242 ◽  
Author(s):  
Satoshi Kotajima ◽  
Koshi N. Kishimoto ◽  
Munenori Watanuki ◽  
Masahito Hatori ◽  
Shoichi Kokubun
Keyword(s):  
Gene Expression ◽  
Gene Transfer ◽  
Bone Formation ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Ectopic Bone Formation ◽  
Ectopic Bone

Biologic properties of nano-hydroxyapatite: An in vivo study of calvarial defects, ectopic bone formation and bone implantation

2015 ◽  
Vol 25 (1) ◽  
pp. 25-38 ◽  
Author(s):  
Kang-Mi Pang ◽  
Jeong-Keun Lee ◽  
Young-Kwon Seo ◽  
Soung-Min Kim ◽  
Myung-Jin Kim ◽  
...  
Keyword(s):  
Bone Formation ◽  
Ectopic Bone Formation ◽  
In Vivo Study ◽  
Ectopic Bone ◽  
Calvarial Defects

scholarly journals 3579 The role of CypD/mPTP during osteogenic differentiation - a potential target to prevent bone loss

2019 ◽  
Vol 3 (s1) ◽  
pp. 24-24
Author(s):  
Rubens Sautchuk ◽  
Brianna H. Shares ◽  
Roman A. Eliseev
Keyword(s):  
Bone Loss ◽  
Bone Formation ◽  
Mitochondrial Function ◽  
Ectopic Bone Formation ◽  
Regulatory Mechanisms ◽  
Mrna Levels ◽  
Prevent Bone Loss ◽  
Ectopic Bone

OBJECTIVES/SPECIFIC AIMS: The study aims to further investigate how cyclophilin D (CypD), the key mPTP opening regulator, affects BMSCs fate and to determine potential regulatory mechanisms involved in CypD regulation during osteogenesis. METHODS/STUDY POPULATION: We evaluated CypD mRNA expression in mouse BMSCs and in osteogenic-like (OL) cells during the course of OB differentiation. CypD protein level was also probed. Moreover, BMSCs had their mPTP activity recorded during osteoinduction. We further analyzed the effect of CypD genetic deletion on osteogenesis in vitro and in vivo. For our in vivo model, we performed the ectopic bone formation assay to asses differences in ossicle formation when CypD KO BMSCs were transplanted compared to wild type littermate BMSCs. In our in vitro model, we transfected OL cells with either CypD gain of function or CypD loss of function vector and measured their osteogenic differentiation potential. Additionally, we treated BMSCs with CypD inhibitor and compare to non-treated BMSCs for mineralization level. To determine potential regulatory mechanisms involved in CypD regulation, we analyzed the CypD gene (Ppif) promoter for potential transcription factor (TF) binding sites and found multiple Smad-binding elements within this promoter. Smads (Smad1, 5, 8) are TFs downstream from Bone Morphogenic Protein (BMP) signaling pathway that transmit cell differentiation signaling, and exert either activating or inhibitory effects on a variety of genes. We also transfect OL cells with Smad1 vector and analyzed for CypD mRNA levels. RESULTS/ANTICIPATED RESULTS: - Our data showed that CypD mRNA levels decreased in both primary cells and OL cells at day 7 and day 14 in osteogenic media. - Osteogenic induction also decreased mPTP activity. - In vivo ectopic bone formation assay showed increased ossicle fo DISCUSSION/SIGNIFICANCE OF IMPACT: Our data suggest that downregulation of CypD increases OB differentiation due to improved OxPhos activity led by mPTP closure. Our results corroborate reports of CypD downregulation and mPTP closure during neuronal differentiation in developing rat brains as well as in cardiomyocyte differentiation in developing mouse hearts. Our studies also suggest a yet unknown mechanism linking differentiation signaling with mitochondrial function – BMP/Smad mediated downregulation of CypD transcription. As initially mentioned, in a previous study, our lab showed that CypD KO mice present higher mitochondrial function and osteogenicity in aged BMSCs and less osteoporosis burden. Taken together, these results suggest that CypD can be a potential target to prevent bone loss in aging.

scholarly journals In vivo bioluminescence imaging study to monitor ectopic bone formation by luciferase gene marked mesenchymal stem cells

2008 ◽  
Vol 26 (7) ◽  
pp. 901-909 ◽  
Author(s):  
Cristina Olivo ◽  
Jacqueline Alblas ◽  
Vivienne Verweij ◽  
Anton-Jan Van Zonneveld ◽  
Wouter J. A. Dhert ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Bone Formation ◽  
Bioluminescence Imaging ◽  
Imaging Study ◽  
Ectopic Bone Formation ◽  
Luciferase Gene ◽  
Ectopic Bone ◽  
In Vivo Bioluminescence Imaging

Alginate Bead-Encapsulated PEDF Induces Ectopic Bone Formation In Vivo in the Absence of Co-Administered Mesenchymal Stem Cells

2018 ◽  
Vol 19 (5) ◽  
pp. 467-478 ◽  
Author(s):  
Mina Elahy ◽  
Michael R. Doschak ◽  
Jeffery D. Hughes ◽  
Swati Baindur-Hudson ◽  
Crispin R. Dass
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Bone Formation ◽  
Alginate Bead ◽  
Ectopic Bone Formation ◽  
Ectopic Bone

scholarly journals Disrupted PGR-B and ESR1 signaling underlies preconceptional defective decidualization linked to severe preeclampsia

2021 ◽  
Author(s):  
Tamara Garrido-Gomez ◽  
Nerea Castillo-Marco ◽  
Monica Clemente-Ciscar ◽  
Teresa Cordero ◽  
Irene Munoz-Blat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dynamic Network ◽  
Global Gene Expression ◽  
Severe Preeclampsia ◽  
Maternal Adaptation ◽  
Uterine Mucosa ◽  
Transcriptomic Signature ◽  
Global Gene Expression Profiling ◽  
Progesterone Receptor B

Decidualization of the uterine mucosa drives the maternal adaptation to invasion by the placenta. Appropriate depth of placental invasion is needed to support a healthy pregnancy; shallow invasion is associated with the development of severe preeclampsia (sPE). Maternal contribution to sPE through failed decidualization is an important determinant of placental phenotype. However, the molecular mechanism underlaying the in vivo defect linking decidualization to sPE is unknown. Here, we discover the footprint encoding this decidualization defect comprising of 166 genes using global gene expression profiling in decidua from women who developed sPE in a previous pregnancy. This signature allowed us to effectively segregate samples into sPE and control groups. Estrogen receptor 1 (ESR1) and progesterone receptor B (PGR-B) were found highly interconnected with the dynamic network of defective decidualization fingerprint. ESR1 and PGR-B gene expression and protein abundance were remarkably disrupted in sPE. Thus, the transcriptomic signature of impaired decidualization implicates dysregulated hormonal signaling in the decidual endometria in women who developed sPE. These findings reveal a potential footprint that may be leverage for a preconception or early prenatal screening of sPE risk, thus improving prevention and early treatments.

scholarly journals Disrupted PGR-B and ESR1 signaling underlies defective decidualization linked to severe preeclampsia

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Tamara Garrido-Gomez ◽  
Nerea Castillo-Marco ◽  
Mónica Clemente-Ciscar ◽  
Teresa Cordero ◽  
Irene Muñoz-Blat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dynamic Network ◽  
Interaction Network ◽  
Global Gene Expression ◽  
Severe Preeclampsia ◽  
Protein Abundance ◽  
Previous Pregnancy ◽  
Global Gene Expression Profiling ◽  
Phd Program

Background:Decidualization of the uterine mucosa drives the maternal adaptation to invasion by the placenta. Appropriate depth of placental invasion is needed to support a healthy pregnancy; shallow invasion is associated with the development of severe preeclampsia (sPE). Maternal contribution to sPE through failed decidualization is an important determinant of placental phenotype. However, the molecular mechanism underlying the in vivo defect linking decidualization to sPE is unknown.Methods:Global RNA sequencing was applied to obtain the transcriptomic profile of endometrial biopsies collected from nonpregnant women who suffer sPE in a previous pregnancy and women who did not develop this condition. Samples were randomized in two cohorts, the training and the test set, to identify the fingerprinting encoding defective decidualization in sPE and its subsequent validation. Gene Ontology enrichment and an interaction network were performed to deepen in pathways impaired by genetic dysregulation in sPE. Finally, the main modulators of decidualization, estrogen receptor 1 (ESR1) and progesterone receptor B (PGR-B), were assessed at the level of gene expression and protein abundance.Results:Here, we discover the footprint encoding this decidualization defect comprising 120 genes—using global gene expression profiling in decidua from women who developed sPE in a previous pregnancy. This signature allowed us to effectively segregate samples into sPE and control groups. ESR1 and PGR were highly interconnected with the dynamic network of the defective decidualization fingerprint. ESR1 and PGR-B gene expression and protein abundance were remarkably disrupted in sPE.Conclusions:Thus, the transcriptomic signature of impaired decidualization implicates dysregulated hormonal signaling in the decidual endometria in women who developed sPE. These findings reveal a potential footprint that could be leveraged for a preconception or early prenatal screening of sPE risk, thus improving prevention and early treatments.Funding:This work has been supported by the grant PI19/01659 (MCIU/AEI/FEDER, UE) from the Spanish Carlos III Institute awarded to TGG. NCM was supported by the PhD program FDGENT/2019/008 from the Spanish Generalitat Valenciana. IMB was supported by the PhD program PRE2019-090770 and funding was provided by the grant RTI2018-094946-B-100 (MCIU/AEI/FEDER, UE) from the Spanish Ministry of Science and Innovation with CS as principal investigator. This research was funded partially by Igenomix S.L.

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