Elsevier

Journal of Biomechanics

Volume 48, Issue 10, 16 July 2015, Pages 1915-1921
Journal of Biomechanics

Embryonic stem cell-derived osteocytes are capable of responding to mechanical oscillatory hydrostatic pressure

https://doi.org/10.1016/j.jbiomech.2015.04.015Get rights and content

Abstract

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.

Introduction

Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of blastocysts that can be forced to undergo differentiation in vitro, via embryoid bodies, into cell types of all three germ layers. Osteoblasts that secrete a mineralized matrix have been shown to differentiate from murine and human ESCs in the presence of ascorbic acid and β-glycerophosphate (Buttery et al., 2001, Phillips et al., 2001, Sottile et al., 2003). Dexamethasone or 1,25α(OH)2 vitamin D3 (VD3) are additional triggers to induce osteogenesis (Buttery et al., 2001, zur Nieden et al., 2003). ESC-derived osteoblasts are a potentially promising cellular candidate for stem cell therapy of degenerative bone and joint disorders or skeletal injuries.

In the past few years, our understanding of osteogenesis from ESCs has made tremendous progress. Differentiation occurs in at least four distinct temporal phases: exit from pluripotency, primitive-streak formation (gastrulation) and/or neural crest induction, followed by mesenchymal commitment and calcium mineralization (Davis and zur Nieden, 2008). Following mineralization, osteoblasts mature into osteocytes in several distinct stages, first becoming embedded in the bone matrix by invading the osteoid tissue, then continuing to control mineralization while forming connections with the bone surface (Bonewald, 2011). All of these phases are characterized by the expression of a number of genes, as well as distinct morphological features.

In the past decade, the potential of ESCs to differentiate into osteoblasts has been proven by a number of groups (Buttery et al., 2001, Kramer et al., 2000, Trettner et al., 2014, zur Nieden et al., 2003) and research has shifted towards the purification of progenitors with osteogenic capacity or the identification of factors that can improve the osteogenic output (Dawson et al., 2013, Ochiai-Shino et al., 2014). For example, we and others have been using Affymetrix gene microarrays to identify pathways associated with specific differentiations to assist with development of improved culture conditions (Bourne et al., 2004, zur Nieden et al., 2007). Manipulation of identified signaling cascades has led to significant enhancement in osteogenic differentiation from 49% to over 90% (zur Nieden et al., 2007).

Efficiency, however, is not the only essential element in the successful development of cells for therapeutic use. Bone is a load-bearing tissue that has to perform under dynamic pressure changes during daily movement, and the success of the skeleton as a structure is ultimately a product of its mechanical properties. While ESC-derived osteogenic cells are now beginning to be applied to restore bone function in animal models (Bilousova et al., 2011, Kang et al., 2008, Li and Niyibizi, 2012) research is still lacking on whether osteogenic cultures mature into osteocytes and whether these cells are capable of adaptively responding to mechanical loading. In the end, only sources of cells that may effectively withstand the extremes of physical activity will be useful in therapeutic approaches.

Section snippets

Tissue culture

D3 murine ESCs were purchased from ATCC and routinely passaged every two days into high glucose DMEM, 15% fetal bovine serum (selected batch), 0.1 mM β-mercaptoethanol, 1% non-essential amino acids, 50 U/ml penicillin and 50 µg/ml streptomycin (all Invitrogen). Differentiation was induced in medium without LIF through embryoid body formation as described (Trettner et al., 2011, zur Nieden et al., 2003). EBs were dispersed into a single cell suspension on day 5 of the induction and transferred to

Results

To examine the capability of ESCs to differentiate into osteocytes, we first examined the levels of bone-specific mRNAs over time. Osteocytes are characterized by a decrease in the expression of the osteoblast-specific osteocalcin (Ocn) as well as the bone specific transcription factor Runx2 (Bonewald, 2011), while the osteocyte-specific markers Sclerostin (Sost) and Dmp1 are increased (Bertin et al., 2014, Compton and Lee, 2014, Feng et al., 2006, Poole et al., 2005). Additionally, osteocyte

Discussion

Osteoblast-like cells can be derived from ESCs by a 30-day differentiation process, whereupon cells spontaneously differentiate upon removal of LIF and subsequently respond to exogenous cues such as VD3 (zur Nieden et al., 2003). Osteogenic cultures show many features of bone cells: they express a panel of bone marker genes, show high alkaline phosphatase activity early on, and later deposit calcium into their matrix in order to mature and mineralize (Guo et al., 2010, zur Nieden et al., 2003,

Conflict of interest statement

The authors do not have any conflict of interest to declare.

Acknowledgments

The authors would like to acknowledge the financial support from Alberta Innovates Health Solutions and its predecessor, the Alberta Heritage Foundation for Medical Research, through a postdoctoral fellowship for NzN, a Youth Researcher Summer Fellowship for FDP and a Senior Scholarship for DER. NGS was supported by the Killam Trust. We are further deeply grateful to Kent Paulsen for servicing the compression chamber setup and providing excellent technical assistance. We would also like to

References (78)

  • T. Majima et al.

    In-vitro cyclic tensile loading of an immobilized and mobilized ligament autograft selectively inhibits mRNA levels for collagenase (MMP-1)

    J. Orthop. Sci.

    (2000)
  • K.A. Myers et al.

    A novel apparatus applying long term intermittent cyclic hydrostatic pressure to in vitro cell cultures

    J. Biosci. Bioeng.

    (2007)
  • H. Nguyen et al.

    Tcf3 governs stem cell features and represses cell fate determination in skin

    Cell

    (2006)
  • B.W. Phillips et al.

    Compactin enhances osteogenesis in murine embryonic stem cells

    Biochem. Biophys. Res. Commun.

    (2001)
  • A.G. Robling et al.

    J. Biol. Chem.

    (2008)
  • A. Santos et al.

    Early activation of the beta-catenin pathway in osteocytes is mediated by nitric oxide, phosphatidyl inositol-3 kinase/Akt, and focal adhesion kinase

    Biochem. Biophys. Res. Commun.

    (2010)
  • S. Tatsumi et al.

    Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction

    Cell Metab.

    (2007)
  • X. Tu et al.

    Sost downregulation and local Wnt signaling are required for the osteogenic response to mechanical loading

    Bone

    (2012)
  • N.I. zur Nieden et al.

    In vitro differentiation of embryonic stem cells into mineralized osteoblasts

    Differentiation

    (2003)
  • N.I. zur Nieden et al.

    Comparing three novel endpoints for developmental osteotoxicity in the embryonic stem cell test

    Toxicol. Appl. Pharmacol.

    (2010)
  • D.M. Adyshev et al.

    Mechanical stress induces pre-B-cell colony-enhancing factor/NAMPT expression via epigenetic regulation by miR-374a and miR-568 in human lung endothelium

    Am. J. Respir. Cell Mol. Biol.

    (2014)
  • K.E. Armour et al.

    Defective bone formation and anabolic response to exogenous estrogen in mice with targeted disruption of endothelial nitric oxide synthase

    Endocrinology

    (2001)
  • K.J. Armour et al.

    Activation of the inducible nitric oxide synthase pathway contributes to inflammation-induced osteoporosis by suppressing bone formation and causing osteoblast apoptosis

    Arthritis Rheumatol.

    (2001)
  • A.D. Bakker et al.

    Endothelial nitric oxide synthase is not essential for nitric oxide production by osteoblasts subjected to fluid shear stress in vitro

    Calcif. Tissue Int.

    (2013)
  • A. Bertin et al.

    Cellular and molecular characterization of a novel primary osteoblast culture from the vertebrate model organism Xenopus tropicalis

    Histochem. Cell Biol.

    (2014)
  • G. Bilousova et al.

    Osteoblasts derived from induced pluripotent stem cells form calcified structures in scaffolds both in vitro and in vivo

    Stem Cells

    (2011)
  • L.F. Bonewald et al.

    Osteocytes, mechanosensing and Wnt signaling

    Bone

    (2009)
  • L.F. Bonewald

    The amazing osteocyte

    J. Bone Miner. Res.

    (2011)
  • S. Bourne et al.

    Osteogenic differentiation of mouse embryonic stem cells: differential gene expression analysis by cDNA microarray and purification of osteoblasts by cadherin-11 magnetically activated cell sorting

    Tissue Eng.

    (2004)
  • L.D. Buttery et al.

    Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells

    Tissue Eng.

    (2001)
  • B.P. Chen et al.

    DNA microarray analysis of gene expression in endothelial cells in response to 24 h shear stress

    Physiol. Genomics

    (2001)
  • J.C. Chen et al.

    Epigenetic changes during mechanically induced osteogenic lineage commitment

    J. Biomech. Eng.

    (2015)
  • J.T. Compton et al.

    A review of osteocyte function and the emerging importance of sclerostin

    J. Bone Joint Surg. Am. Ed.

    (2014)
  • S. Cuzzocrea et al.

    Inducible nitric oxide synthase mediates bone loss in ovariectomized mice

    Endocrinology

    (2003)
  • L.A. Davis et al.

    Mesodermal fate decisions of a stem cell: the Wnt switch

    Cell. Mol. Life Sci.

    (2008)
  • S. Damaraju et al.

    The effect of mechanical stimulation on mineralization in differentiating osteoblasts in collagen-I scaffolds

    Tissue Eng. Part A

    (2014)
  • V. Das-Gupta et al.

    Expression of endothelial nitric oxide synthase protein is not necessary for mechanical strain-induced nitric oxide production by cultured osteoblasts

    Osteoporos. Int.

    (2012)
  • H. Ding et al.

    NO/beta-catenin crosstalk modulates primitive streak formation prior to embryonic stem cell osteogenic differentiation

    J. Cell Sci.

    (2012)
  • Y.H. Du et al.

    Endothelium-specific GTP cyclohydrolase I overexpression attenuates blood pressure progression in salt-sensitive low-renin hypertension

    Circulation

    (2008)
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    Authors contributed equally as first authors.

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    Authors contributed equally as senior authors.

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