Embryonic stem cell-derived osteocytes are capable of responding to mechanical oscillatory hydrostatic pressure
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)
- et al.
Testing the daily stress stimulus theory of bone adaptation with natural and experimentally controlled strain histories
J. Biomech.
(1997) - et al.
Endothelial nitric oxide synthase gene-deficient mice demonstrate marked retardation in postnatal bone formation, reduced bone volume, and defects in osteoblast maturation and activity
Am. J. Pathol.
(2001) - et al.
Nitric oxide production by bone cells is fluid shear stress rate dependent
Biochem. Biophys. Res. Commun.
(2004) - et al.
Enhancing the osteogenic efficacy of human bone marrow aspirate: concentrating osteoprogenitors using wave-assisted filtration
Cytotherapy
(2013) - et al.
Biochemical signal transduction of mechanical strain in osteoblast-like cells
Biomaterials
(1991) - et al.
Activation of β-catenin signaling in MLO-Y4 osteocytic cells versus 2T3 osteoblastic cells by fluid flow shear stress and PGE2: implications for the study of mechanosensation in bone
Bone
(2010) - et al.
Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways
Bone
(2003) - et al.
Mechanosensation and transduction in osteocytes
Bone
(2013) - et al.
Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4
Mech. Dev.
(2000) - et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method
Methods
(2001)