Compactin Enhances Osteogenesis in Murine Embryonic Stem Cells

doi:10.1006/bbrc.2001.4987

Biochemical and Biophysical Research Communications

Volume 284, Issue 2, 8 June 2001, Pages 478-484

https://doi.org/10.1006/bbrc.2001.4987 Get rights and content

Abstract

Embryonic stem (ES) cells have the capacity to differentiate into various cell types in vitro. In this study, we show that retinoic acid is important for the commitment of ES cells into osteoblasts. Culturing retinoic acid treated ES cells in the presence of the osteogenic supplements ascorbic acid and β-glycerophosphate resulted in the expression of several osteoblast marker genes, osteocalcin, alkaline phosphatase, and osteopontin. However, there was only a slight amount of mineralized matrix secretion. Addition of bone morphogenic protein-2 or compactin, a drug of the statin family of HMG-CoA reductase inhibitors, resulted in a greatly enhanced formation of bone nodules. Compactin did not modify the expression of osteogenic markers, but at the late stage of differentiation promoted an increase in BMP-2 expression. These results establish ES-cell derived osteogenesis as an effective model system to study the molecular mechanisms by which the statin compactin promotes osteoblastic differentiation and bone nodule formation.

References (35)

D. Jia et al.
Insulin-like growth factor-1 and -2 stimulate osteoprogenitor proliferation and differentiation and adipocyte formation in cell populations derived from adult rat bone
Bone
(2000)
J.A. Roth et al.
Melatonin promotes osteoblast differentiation and bone formation
J. Biol. Chem.
(1999)
T. Katagiri et al.
The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2
Biochem. Biophys. Res. Commun.
(1990)
N. Yamamoto et al.
Smad1 and Smad5 act downstream of intracellular signallings of BMP-2 that inhibits myogenic differentiation in C2C12 myoblasts
Biochem. Biophys. Res. Commun.
(1997)
M. Sugiyama et al.
Compactin and simvastatin, but not pravastatin, induce bone morphogenetic protein-2 in human osteosarcoma cells
Biochem. Biophys. Res. Commun.
(2000)
A.G. Smith
Mouse embryo stem cells: Their identification, propagation and manipulation
Semin. Cell Biol.
(1992)
J. Kramer et al.
Embryonic stem cell-derived chondrogenic differentiation in vitro: Activation by BMP-2 and BMP-4
Mech. Dev.
(2000)
G. Bain et al.
Embryonic stem cells express neuronal properties in vitro
Dev. Biol.
(1995)
P. Chomczynski et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction
Anal. Biochem.
(1987)
J. Aubert et al.
Leukemia inhibitory factor and its receptor promote adipocyte differentiation via the mitogen-activated protein kinase cascade
J. Biol. Chem.
(1999)

H. Wong et al.

Monitoring mRNA expression by polymerase chain reaction: The ‘primer-dropping’ method

Anal. Biochem.

(1994)

S. Pease et al.

Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor (LIF)

Dev. Biol.

(1990)

T. Maeda et al.

Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells

Biochem. Biophys. Res. Commun.

(2001)

D. Perez-Sala et al.

Inhibition of isoprenoid biosynthesis induces apoptosis in human promyelocytic HL-60 cells

Biochem. Biophys. Res. Commun.

(1994)

M. Alvarez de Sotomayor et al.

Simvastatin and Ca(2+) signaling in endothelial cells: Involvement of rho protein

Biochem. Biophys. Res. Commun.

(2001)

K. Tomiyama et al.

Both wortmannin and simvastatin inhibit the adipogenesis in 3T3-L1 cells during the late phase of differentiation

Jpn. J. Pharmacol.

(1999)

G. Karsenty

The genetic transformation of bone biology

Genes Dev.

(1999)

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Osteoblasts can be derived from embryonic stem cells (ESCs) by a 30 day differentiation process, whereupon cells spontaneously differentiate upon removal of LIF and respond to exogenously added 1,25α(OH)₂ vitamin D₃ with enhanced matrix mineralization. However, bone is a load-bearing tissue that has to perform under dynamic pressure changes during daily movement, a capacity that is executed by osteocytes. At present, it is unclear whether ESC-derived osteogenic cultures contain osteocytes and whether these are capable of responding to a relevant cyclic hydrostatic compression stimulus.
Here, we show that ESC-osteoblastogenesis is followed by the generation of osteocytes and then mechanically load ESC-derived osteogenic cultures in a compression chamber using a cyclic loading protocol. Following mechanical loading of the cells, iNOS mRNA was upregulated 31-fold, which was consistent with a role for iNOS as an immediate early mechanoresponsive gene. Further analysis of matrix and bone-specific genes suggested a cellular response in favor of matrix remodeling. Immediate iNOS upregulation also correlated with a concomitant increase in Ctnnb1 and Tcf7l2 mRNAs along with increased nuclear TCF transcriptional activity, while the mRNA for the repressive Tcf7l1 was downregulated, providing a possible mechanistic explanation for the noted matrix remodeling. We conclude that ESC-derived osteocytes are capable of responding to relevant mechanical cues, at least such that mimic oscillatory compression stress, which not only provides new basic understanding, but also information that likely will be important for their use in cell-based regenerative therapies.
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Oxysterols, oxidized derivatives of cholesterol, are biologically active molecules. Specific oxysterols have potent osteogenic properties that act on osteoprogenitor cells. However, the molecular mechanisms underlying these osteoinductive effects on embryonic stem cells (ESCs) are unknown. This study investigated the effect of an oxysterol combination of 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol (SS) on osteogenic differentiation of ESCs and the alterations to mitochondrial activity during differentiation. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, matrix mineralization, mRNA expression of osteogenic factors, runt-related transcription factor 2, osterix, and osteocalcin, and protein levels of collagen type IA (COLIA) and osteopontin (OPN). Treatment of cells with SS increased osteoinductive activity compared to the control group. Intracellular reactive oxygen species production, intracellular ATP content, mitochondrial membrane potential, mitochondrial mass, mitochondrial DNA copy number, and mRNA expression of peroxisome proliferator-activated receptor-γ coactivators 1α and β, transcription factors involved in mitochondrial biogenesis, were significantly increased during osteogenesis, indicating upregulation of mitochondrial activity. Oxysterol combinations also increased protein levels of mitochondrial respiratory complexes I–V. We also found that SS treatment increased hedgehog signaling target genes, Smo and Gli1 expression. Inhibition of Hh signaling by cyclopamine suppressed mitochondrial biogenesis and ESC osteogenesis. Subsequently, oxysterol-induced Wnt/β-catenin pathways were inhibited by repression of Hh signaling and mitochondrial biogenesis. Transfection of β-catenin specific siRNA decreased the protein levels of COLIA and OPN, as well as ALP activity. Collectively, these data suggest that lipid-based oxysterols enhance differentiation of ESCs toward the osteogenic lineage by regulating mitochondrial activity, canonical Hh/Gli, and Wnt/β-catenin signaling.
Small molecules and their controlled release that induce the osteogenic/chondrogenic commitment of stem cells
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Citation Excerpt :
Statins, also known as 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase suppressants, are widely used as cholesterol-lowering drugs because they inhibit HMG-CoA conversion to mevalonate (Horiuchi and Maeda, 2006). However, statins can also induce the osteogenic differentiation of stem cells in vivo and ex vivo, according to a number of reports (Garrett and Mundy, 2002; Kim et al., 2009; Maeda et al., 2001; Mundy et al., 1999; Mundy, 2001; Pagkalos et al., 2010; Phillips et al., 2001; Sonobe et al., 2005). For instance, mevinolin, which is a member of the drug class of statins, promoted the expression of OCN, CD44, CD47 and CD51 and increased the ALP activity of D1 cells that were cloned from mouse multipotent bone MSCs (Kim et al., 2009).
Stem cell-based tissue engineering plays a significant role in skeletal system repair and regenerative therapies. However, stem cells must be differentiated into specific mature cells prior to implantation (direct implantation may lead to tumour formation). Natural or chemically synthesised small molecules provide an efficient, accurate, reversible, and cost-effective way to differentiate stem cells compared with bioactive growth factors and gene-related methods. Thus, investigating the influences of small molecules on the differentiation of stem cells is of great significance. Here, we review a series of small molecules that can induce or/and promote the osteogenic/chondrogenic commitment of stem cells. The controlled release of these small molecules from various vehicles for stem cell-based therapies and tissue engineering applications is also discussed. The extensive studies in this field represent significant contributions to stem cell-based tissue engineering research and regenerative medicine.
Optimized osteogenic differentiation protocol from R1 mouse embryonic stem cells in vitro
2015, Differentiation
Embryonic stem cells (ESCs) are a unique model that allows the study of molecular pathways underlying commitment and differentiation. One such lineage is osteoblasts, which are responsible for forming bone tissue in the body. There are many osteogenic differentiation protocols in the literature utilizing different soluble factors. The goal of the present study was to increase the efficacy of our osteogenic differentiation protocol from R1 cells. We have studied the effects of the addition of the following factors: dexamethasone, retinoic acid, and peroxisome-proliferator-activated receptor-gamma inhibitor, which have been reported to enhance osteogenesis. We found that among the 6 different protocols that were tested, the addition of retinoic acid with later addition of dexamethasone gives the most enrichment of osteogenic lineage cells. Thus, our findings provide valuable guidelines for culture condition to differentiate mouse R1 ESCs to osteoblastic cells in vitro.
Exogenous nitric oxide enhances calcification in embryonic stem cell-derived osteogenic cultures
2014, Differentiation
Citation Excerpt :
Each of these phases is characterized by the expression of a number of genes and distinct morphological features, and the end result is the maturation of osteoblasts and the calcification of the extracellular matrix (Davis and zur Nieden, 2008). Now that a number of groups have demonstrated differentiation of ESCs and induced pluripotent stem cells into skeletal cell types (Buttery et al., 2001; Phillips et al., 2001; zur Nieden et al., 2003; Sottile et al., 2003; Li and Niyibizi, 2012; Egusa et al., 2014; Kanke et al., 2014; Trettner et al., 2014), research has shifted towards the identification of factors that can improve the output of these cell types, which remains insufficient in basic osteogenic medium. For example, we have recently demonstrated that the yield of chondrocytes within differentiated cultures can be increased to 57% simply through the continuous supplementation of culture with bone morphogenetic protein-2 (BMP-2) (zur Nieden et al., 2005).
While the involvement of nitric oxide in bone formation, homeostasis and healing has been extensively characterized, its role in directing pluripotent stem cells to the osteogenic lineage has not been described. Yet, the identification of chemical inducers that improve differentiation output to a particular lineage is highly valuable to the development of such cells for the cell-based treatment of osteo-degenerative diseases. This study aimed at investigating the instructive role of nitric oxide (NO) and its synthesizing enzymes on embryonic stem cell (ESC) osteogenic differentiation. Our findings showed that NO levels may support osteogenesis, but that the effect of nitric oxide on osteoblast differentiation may be specific to a particular time phase during the development of osteoblasts in vitro. Endogenously, nitric oxide was specifically secreted by osteogenic cultures during the calcification period. Simultaneously, messenger RNAs for both the endothelial and inducible nitric oxide synthase isoforms (eNOS and iNOS) were upregulated during this late phase development. However, the specific eNOS inhibitor L-N⁵-(1-Iminoethyl)ornithine dihydrochloride attenuated calcification more so than the specific iNOS inhibitor diphenyleneiodonium. Exogenous stage-specific supplementation of culture medium with the NO donor S-nitroso-N-acetyl-penicillamine increased the percentage of cells differentiating into osteoblasts and enhanced calcification. Our results point to a primary role for eNOS as a pro-osteogenic trigger in ESC differentiation and expand on the variety of supplements that may be used to direct ESC fate to the osteogenic lineage, which will be important in the development of cell-based therapies for osteo-degenerative diseases.
Stepwise differentiation of pluripotent stem cells into osteoblasts using four small molecules under serum-free and feeder-free conditions
2014, Stem Cell Reports
Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs) and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively) into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4′,3′:4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

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Regular ArticleCompactin Enhances Osteogenesis in Murine Embryonic Stem Cells

Abstract

Bone

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Semin. Cell Biol.

Mech. Dev.

Dev. Biol.

Anal. Biochem.

J. Biol. Chem.

Anal. Biochem.

Dev. Biol.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Jpn. J. Pharmacol.

The genetic transformation of bone biology

Genes Dev.

Regular Article
Compactin Enhances Osteogenesis in Murine Embryonic Stem Cells