Microarray analysis of human adipose-derived stem cells in three-dimensional collagen culture: osteogenesis inhibits bone morphogenic protein and Wnt signaling pathways, and cyclic tensile strain causes upregulation of proinflammatory cytokine regulators and angiogenic factors.
ABSTRACT: Human adipose-derived stem cells (hASC) have shown great potential for bone tissue engineering. However, the molecular mechanisms underlying this potential are not yet known, in particular the separate and combined effects of three-dimensional (3D) culture and mechanical loading on hASC osteogenesis. Mechanical stimuli play a pivotal role in bone formation, remodeling, and fracture repair. To further understand hASC osteogenic differentiation and response to mechanical stimuli, gene expression profiles of proliferating or osteogenically induced hASC in 3D collagen I culture in the presence and absence of 10% uniaxial cyclic tensile strain were examined using microarray analysis. About 847 genes and 95 canonical pathways were affected during osteogenesis of hASC in 3D culture. Pathway analysis indicated the potential roles of Wnt/?-catenin signaling, bone morphogenic protein (BMP) signaling, platelet-derived growth factor (PDGF) signaling, and insulin-like growth factor 1 (IGF-1) signaling in hASC during osteogenic differentiation. Application of 10% uniaxial cyclic tensile strain suggested synergistic effects of strain with osteogenic differentiation media on hASC osteogenesis as indicated by significantly increased calcium accretion of hASC. There was no significant further alteration in the four major pathways (Wnt/?-catenin, BMP, PDGF, and IGF-1). However, 184 transcripts were affected by 10% cyclic tensile strain. Function and network analysis of these transcripts suggested that 10% cyclic tensile strain may play a role during hASC osteogenic differentiation by upregulating two crucial factors in bone regeneration: (1) proinflammatory cytokine regulators interleukin 1 receptor antagonist and suppressor of cytokine signaling 3; (2) known angiogenic inductors fibroblast growth factor 2, matrix metalloproteinase 2, and vascular endothelial growth factor A. This is the first study to investigate the effects of both 3D culture and mechanical load on hASC osteogenic differentiation. A complete microarray analysis investigating both the separate effect of soluble osteogenic inductive factors and the combined effects of chemical and mechanical stimulation was performed on hASC undergoing osteogenic differentiation. We have identified specific genes and pathways associated with mechanical response and osteogenic potential of hASC, thus providing significant information toward improved understanding of our use of hASC for functional bone tissue engineering applications.
Project description:We have shown that the uniaxial cyclic tensile strain of magnitude 10% promotes and enhances osteogenesis of human mesenchymal stem cells (hMSC) and human adipose-derived stem cells (hASC) from normal, nonosteoporotic donors. In the present study, MSC from osteoporotic donors were analyzed for changes in mRNA expression in response to 10% uniaxial tensile strain to identify potential mechanisms underlying the use of this mechanical loading paradigm for prevention and treatment of osteoporosis. Human MSC isolated from three female, postmenopausal osteoporotic donors were analyzed for their responses to mechanical loading using microarray analysis of over 47,000 gene probes. Human MSC were seeded in three-dimensional collagen type I constructs to mimic the organic extracellular matrix of bone and 10% uniaxial cyclic tensile strain was applied to promote osteogenesis. Seventy-nine genes were shown to be regulated within hMSC from osteoporotic donors in response to 10% cyclic tensile strain. Upregulation of six genes were further confirmed with real-time RT-PCR: jun D proto-oncogene (JUND) and plasminogen activator, urokinase receptor (PLAUR), two genes identified as potential key molecules from network analysis; phosphoinositide-3-kinase, catalytic, delta polypeptide (PIK3CD) and wingless-type MMTV integration site family, member 5B (WNT5B), two genes with known importance in bone biology; and, PDZ and LIM domain 4 (PDLIM4) and vascular endothelial growth factor A (VEGFA), two genes that we have previously shown are significantly regulated in hASC in response to this mechanical stimulus. Function analysis indicated that 10% cyclic tensile strain induced expression of genes associated with cell movement, cell proliferation, and tissue development, including development in musculoskeletal and cardiovascular systems. Our results demonstrate that hMSC from aged, osteoporotic donors are capable of enhanced osteogenic differentiation in response to 10% cyclic tensile strain with significant increases in the expression of genes associated with enhanced cell proliferation, musculoskeletal development, and angiogenesis. Surprisingly, cyclic tensile strain of magnitude 10% not only enhanced osteogenesis in hMSC from osteoporotic donors, but also enhanced expression of angiogenic factors. Better understanding and methodologies to promote osteogenesis in hMSC from elderly, osteoporotic donors may greatly facilitate achieving long-term success in bone regeneration and functional bone tissue engineering for this ever-growing patient population.
Project description:Non-motile primary cilia are dynamic cellular sensory structures and are expressed in adipose-derived stem cells (ASCs). We have previously shown that primary cilia are involved in chemically-induced osteogenic differentiation of human ASC (hASCs) in vitro. Further, we have reported that 10% cyclic tensile strain (1 Hz, 4 hours/day) enhances hASC osteogenesis. We hypothesize that primary cilia respond to cyclic tensile strain in a lineage dependent manner and that their mechanosensitivity may regulate the dynamics of signaling pathways localized to the cilium. We found that hASC morphology, cilia length and cilia conformation varied in response to culture in complete growth, osteogenic differentiation, or adipogenic differentiation medium, with the longest cilia expressed in adipogenically differentiating cells. Further, we show that cyclic tensile strain both enhances osteogenic differentiation of hASCs while it suppresses adipogenic differentiation as evidenced by upregulation of RUNX2 gene expression and downregulation of PPARG and IGF-1, respectively. This study demonstrates that hASC primary cilia exhibit mechanosensitivity to cyclic tensile strain and lineage-dependent expression, which may in part regulate signaling pathways localized to the primary cilium during the differentiation process. We highlight the importance of the primary cilium structure in mechanosensing and lineage specification and surmise that this structure may be a novel target in manipulating hASC for in tissue engineering applications.
Project description:INTRODUCTION:Mechanical forces play critical roles in the development and remodelling process of bone. As an alternative cell source for bone engineering, adipose-derived stem cells (ASCs) should be fully investigated for their responses to mechanical stress and the mechanisms responsible for osteogenic induction in response to mechanical signals. MATERIAL AND METHODS:We hypothesized that appropriate application of uniaxial cyclic tensile strain to ASCs could increase bone morphogenetic protein-2 (BMP-2) expression and improve osteogenesis of ASCs. To test our hypothesis, ASCs from the same flask of the same donor were subjected to tensile strain with different patterns in order to eliminate the difference of donor site and passage. After surface markers investigation, the osteo-induced ASCs were subjected to uniaxial cyclic tensile stretch with the following two loading patterns: long duration continuous pattern (6 h, 1 HZ, 2000 µ?) and short duration consecutive pattern (17 min every day for 10 consecutive days, 1 HZ, 2000 µ?). Then osteogenic related genes were analysed by real-time PCR. RESULTS:The ASCs were positive for the markers STRO-1, CD90 and CD44 and negative for CD34. Cyclic tensile strain of 6 continuous h' duration significantly increased gene expressions of BMP-2 and Runx2, and depressed OCN mRNA expression. In contrast, mechanical loading of 17 min every day did not significantly affect gene expression of BMP-2, Runx2, OCN or ALP. CONCLUSIONS:We indicate that ASCs may sense mechanical loading in a duration-dependent manner and cyclic tensile stretch may modulate the osteogenic differentiation of ASCs via the BMP-2 signalling pathway.
Project description:Development and maintenance of a vascular network are critical for bone growth and homeostasis; strategies that promote vascular function are critical for clinical success of tissue-engineered bone constructs. Co-culture of endothelial cells (ECs) with mesenchymal stem cells (MSCs) and exposure to 10% cyclic tensile strain have both been shown to regulate osteogenesis in isolation, but potential synergistic effects have yet to be explored. The objective of this study was to expose an MSC-EC co-culture to 10% cyclic tensile strain to examine the role of this mechanical stimulus on MSC-EC behavior. We hypothesized that paracrine signaling from ECs would stimulate osteogenesis of MSCs, and exposure to 10% cyclic tensile strain would enhance this anabolic signal. Human umbilical vein ECs and human bone marrow-derived MSCs were either monocultured or co-cultured at a 1:1 ratio in a mixed osteo/angiogenic medium, exposed to 10% cyclic tensile strain at 1?Hz for 4?h/day for 2 weeks, and biochemically and histologically analyzed for endothelial and osteogenic markers. While neither 10% cyclic tensile strain nor co-culture alone had a significant effect on osteogenesis, the concurrent application of strain to an MSC-EC co-culture resulted in a significant increase in calcium accretion and mineral deposition, suggesting that co-culture and strain synergistically enhance osteogenesis. Neither co-culture, 10% cyclic tensile strain, nor a combination of these stimuli affected endothelial markers, indicating that the endothelial phenotype remained stable, but unresponsive to the stimuli evaluated in this study. This study is the first to investigate the role of cyclic tensile strain on the complex interplay between ECs and MSCs in co-culture. The results of this study provide key insights into the synergistic effects of 10% cyclic tensile strain and co-culture on osteogenesis. Understanding mechanobiological factors affecting MSC-EC crosstalk will help enhance strategies for creating vascularized tissues in tissue engineering and regenerative medicine.
Project description:Human adipose-derived stem cells (hASCs) are multipotent progenitor cells with self-renewal capabilities and multilineage differentiation potential, including osteogenesis. Although protein deubiquitinases have been linked to stem cell fate determination, whether protein deubiquitination contributes to lineage commitment during osteogenic differentiation of hASCs remains to be investigated. The objective of this study was to evaluate the effects of the ubiquitin specific protease 7 (USP7) on osteogenic differentiation of hASCs.An osteocalcin promoter driven luciferase reporter system was established to initially discover the potential association between USP7 and hASC osteogenesis. To further characterize the function of USP7 in osteogenic differentiation of hASCs, a combination of in vitro and in vivo experiments were carried out through genetic depletion or overexpression of USP7 using a lentiviral strategy. Moreover, HBX 41,108, a cyanoindenopyrazine-derived deubiquitinase inhibitor of USP7, was utilized at different doses to further examine whether USP7 regulated osteogenic differentiation of hASCs through its enzymatic activity.We demonstrated that USP7 depletion was associated with remarkable downregulation of the reporter gene activity. Genetic depletion of USP7 by lentiviral RNAi markedly suppressed hASC osteogenesis both in vitro and in vivo, while overexpression of USP7 enhanced the osteogenic differentiation of hASCs. Notably, chemical blockade via the small molecular inhibitor HBX 41,108 could efficiently mimic the effects of USP7 genetic depletion in a dose-dependent manner.Taken together, our study revealed that protein deubiquitinase USP7 is an essential player in osteogenic differentiation of hASCs through its catalytic activity, and supported the pursuit of USP7 as a potential target for modulation of hASC-based stem cell therapy and bone tissue engineering.
Project description:Mechanical stimulation and histone deacetylases (HDACs) have essential roles in regulating the osteogenic differentiation of bone marrow stromal cells (BMSCs) and bone formation. However, little is known regarding what regulates HDAC expression and therefore the osteogenic differentiation of BMSCs during osteogenesis. In this study, we investigated whether mechanical loading regulates HDAC expression directly and examined the role of HDACs in mechanical loading-triggered osteogenic differentiation and bone formation. We first studied the microarrays of samples from patients with osteoporosis and found that the NOTCH pathway and skeletal development gene sets were downregulated in the BMSCs of patients with osteoporosis. Then we demonstrated that mechanical stimuli can regulate osteogenesis and bone formation both in vivo and in vitro. NOTCH signaling was upregulated during cyclic mechanical stretch (CMS)-induced osteogenic differentiation, whereas HDAC1 protein expression was downregulated. The perturbation of HDAC1 expression also had a significant effect on matrix mineralization and JAG1-mediated Notch signaling, suggesting that HDAC1 acts as an endogenous attenuator of Notch signaling in the mechanotransduction of BMSCs. Chromatin immunoprecipitation (ChIP) assay results suggest that HDAC1 modulates the CMS-induced histone H3 acetylation level at the JAG1 promoter. More importantly, we found an inhibitory role of Hdac1 in regulating bone formation in response to hindlimb unloading in mice, and pretreatment with an HDAC1 inhibitor partly rescued the osteoporosis caused by mechanical unloading. Our results demonstrate, for the first time, that mechanical stimulation orchestrates genes expression involved in the osteogenic differentiation of BMSCs via the direct regulation of HDAC1, and the therapeutic inhibition of HDAC1 may be an efficient strategy for enhancing bone formation under mechanical stimulation.
Project description:Adipose tissue is an attractive stem cell source for soft and bone tissue engineering applications and stem cell therapies. The adipose-derived stromal/stem cells (ASCs) have a multilineage differentiation capacity that is regulated through extracellular signals. The cellular events related to cell adhesion and cytoskeleton have been suggested as central regulators of differentiation fate decision. However, the detailed knowledge of these molecular mechanisms in human ASCs remains limited. This study examined the significance of focal adhesion kinase (FAK), Rho-Rho-associated protein kinase (Rho-ROCK), and their downstream target extracellular signal-regulated kinase 1/2 (ERK1/2) on hASCs differentiation towards osteoblasts and adipocytes. Analyses of osteogenic markers RUNX2A, alkaline phosphatase, and matrix mineralization revealed an essential role of active FAK, ROCK, and ERK1/2 signaling for the osteogenesis of hASCs. Inhibition of these kinases with specific small molecule inhibitors diminished osteogenesis, while inhibition of FAK and ROCK activity led to elevation of adipogenic marker genes AP2 and LEP and lipid accumulation implicating adipogenesis. This denotes to a switch-like function of FAK and ROCK signaling in the osteogenic and adipogenic fates of hASCs. On the contrary, inhibition of ERK1/2 kinase activity deceased adipogenic differentiation, indicating that activation of ERK signaling is required for both adipogenic and osteogenic potential. Our findings highlight the reciprocal role of cell adhesion mechanisms and actin dynamics in regulation of hASC lineage commitment. This study enhances the knowledge of molecular mechanisms dictating hASC differentiation and thus opens possibilities for more efficient control of hASC differentiation.
Project description:Human adipose stem cells (hASCs) are an attractive cell source for bone tissue engineering applications. However, a critical issue to be addressed before widespread hASC clinical translation is the dramatic variability in proliferative capacity and osteogenic potential among hASCs isolated from different donors. The goal of this study was to test our hypothesis that electrical cell-substrate impedance spectroscopy (ECIS) could track complex bioimpedance patterns of hASCs throughout proliferation and osteogenic differentiation to better understand and predict variability among hASC populations. Superlots composed of hASCs from young (aged 24-36 years), middle-aged (aged 48-55 years), and elderly (aged 60-81 years) donors were seeded on gold electrode arrays. Complex impedance measurements were taken throughout proliferation and osteogenic differentiation. During osteogenic differentiation, four impedance phases were identified: increase, primary stabilization, drop phase, and secondary stabilization. Matrix deposition was first observed 48-96 hours after the impedance maximum, indicating, for the first time, that ECIS can identify morphological changes that correspond to late-stage osteogenic differentiation. The impedance maximum was observed at day 10.0 in young, day 6.1 in middle-aged, and day 1.3 in elderly hASCs, suggesting that hASCs from younger donors require a longer time to differentiate than do hASCs from older donors, but young hASCs proliferated more and accreted more calcium long-term. This is the first study to use ECIS to predict osteogenic potential of multiple hASC populations and to show that donor age may temporally control onset of osteogenesis. These findings could be critical for development of patient-specific bone tissue engineering and regenerative medicine therapies. Stem Cells Translational Medicine 2017;6:502-511.
Project description:Hydrogels represent an attractive material platform for realization of three-dimensional (3D) tissue-engineered constructs, as they have tunable mechanical properties, are compatible with different types of cells, and resemble elements found in natural extracellular matrices. So far, numerous hydrogel-cartilage/bone tissue engineering (TE)-related studies were performed by utilizing a single cell encapsulation approach. Although multicellular spheroid cultures exhibit advantageous properties for cartilage or bone TE, the chondrogenic or osteogenic differentiation potential of stem cell microspheroids within hydrogels has not been investigated much. This study explores, for the first time, how stiffness of gelatin-based hydrogels (having a storage modulus of 538, 3584, or 7263 Pa) affects proliferation and differentiation of microspheroids formed from telomerase-immortalized human adipose-derived stem cells (hASC/hTERT). Confocal microscopy indicates that all tested hydrogels supported cell viability during their 3- to 5-week culture period in the control, chondrogenic, or osteogenic medium. Although in the softer hydrogels cells from neighboring microspheroids started outgrowing and interconnecting within a few days, their protrusion was slower or limited in stiffer hydrogels or those cultured in chondrogenic medium, respectively. High expressions of chondrogenic markers (SOX9, ACAN, COL2A1), detected in all tested hydrogels, proved that the chondrogenic differentiation of hASC/hTERT microspheroids was very successful, especially in the two softer hydrogels, where superior cartilage-specific properties were confirmed by Alcian blue staining. These chondrogenically induced samples also expressed COL10A1, a marker of chondrocyte hypertrophy. Interestingly, the hydrogel itself (with no differentiation medium) showed a slight chondrogenic induction. Regardless of the hydrogel stiffness, in the samples stimulated with osteogenic medium, the expression of selected markers RUNX2, BGLAP, ALPL, and COL1A1 was not conclusive. Nevertheless, the von Kossa staining confirmed the presence of calcium deposits in osteogenically stimulated samples in the two softer hydrogels, suggesting that these also favor osteogenesis. This observation was also confirmed by Alizarin red quantification assay, with which higher amounts of calcium were detected in the osteogenically induced hydrogels than in their controls. The presented data indicate that the encapsulation of adipose-derived stem cell microspheroids in gelatin-based hydrogels show promising potential for future applications in cartilage or bone TE. Impact Statement Osteochondral defects represent one of the leading causes of disability in the world. Although numerous tissue engineering (TE) approaches have shown success in cartilage and bone tissue regeneration, achieving native-like characteristics of these tissues remains challenging. This study demonstrates that in the presence of a corresponding differentiation medium, gelatin-based hydrogels support moderate osteogenic and excellent chondrogenic differentiation of photo-encapsulated human adipose-derived stem cell microspheroids, the extent of which depends on hydrogel stiffness. Because photosensitive hydrogels are a convenient material platform for creating stiffness gradients in three dimensions, the presented microspheroid-hydrogel encapsulation strategy holds promise for future strategies of cartilage or bone TE.
Project description:Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into any mesenchymal tissue, including bone, cartilage, muscle, and fat. MSC differentiation can be influenced by a variety of stimuli, including environmental and mechanical stimulation, scaffold physical properties, or applied loads. Numerous studies have evaluated the effects of vibration or cyclic tensile strain on MSCs towards developing a mechanically based method of differentiation, but there is no consensus between studies and each investigation uses different culture conditions, which also influence MSC fate. Here we present an overview of the response of MSCs to vibration and cyclic tension, focusing on the effect of various culture conditions and strain or vibration parameters. Our review reveals that scaffold type (e.g., natural versus synthetic; 2D versus 3D) can influence cell response to vibration and strain to the same degree as loading parameters. Hence, in the efforts to use mechanical loading as a reliable method to differentiate cells, scaffold selection is as important as method of loading.