Alice Wong

Comparison of the osteogenic potential of equine mesenchymal stem cells from bone marrow, adipose tissue, umbilical cord blood, and umbilical cord tissue

OBJECTIVE To determine the optimal osteogenic source of equine mesenchymal stem cells (eMSCs) and optimize collection of and expansion conditions for those cells. ANIMALS 10 adult Quarter Horses and 8 newborn Thoroughbred foals. PROCEDURES eMSCs were isolated from bone marrow (BM), adipose tissue, and umbilical cord blood and tissue, and the osteogenic potential of each type was assessed. Effects of anatomic site, aspiration volume, and serum type on eMSC yield from BM were investigated. RESULTS BM-eMSCs had the highest overall expression of the osteogenic genes Cbfa1, Osx, and Omd and staining for ALP activity and calcium deposition. There was no significant difference in BM-eMSC yield from the tuber coxae or sternum, but yield was significantly greater from the first 60-mL aspirate than from subsequent aspirates. The BM-eMSC expansion rate was significantly higher when cells were cultured in fetal bovine serum instead of autologous serum (AS). CONCLUSIONS AND CLINICAL RELEVANCE eMSCs from BM possessed the highest in vitro osteogenic potential; eMSCs from adipose tissue also had robust osteogenic potential. The tuber coxae and the sternum were viable sources of BM-eMSCs in yearlings, and 60 mL of BM aspirate was sufficient for culture and expansion. Expanding BM-eMSCs in AS to avoid potential immunologic reactions decreased the total yield because BM-eMSCs grew significantly slower in AS than in fetal bovine serum. Additional studies are needed to determine optimal ex vivo eMSC culture and expansion conditions, including the timing and use of growth factor—supplemented AS.

Hypoxia Decreases Sclerostin Expression and Increases Wnt Signaling in Osteoblasts

Mutations in sclerostin function or expression cause sclerosing bone dysplasias, involving decreased antagonism of Wnt/Lrp5 signaling. Conversely, deletion of the VHL tumor suppressor in osteoblasts, which stabilize HIF‐α isoforms and thereby enables HIF‐α/β‐driven gene transcription, increases bone mineral content and cross‐sectional area compared to wild‐type controls. We examined the influence of cellular hypoxia (1% oxygen) upon sclerostin expression and canonical Wnt signaling. Osteoblasts and osteocytes cultured under hypoxia revealed decreased sclerostin transcript and protein, and increased expression and nuclear localization of activated β‐catenin. Similarly, both hypoxia and the hypoxia mimetic DFO increased β‐catenin gene reporter activity. Hypoxia and its mimetics increased expression of the BMP antagonists gremlin and noggin and decreased Smad‐1/5/8 phosphorylation. As a partial explanation for the mechanism of regulation of sclerostin by oxygen, MEF2 reporter assays revealed decreased activity. Modulation of VEGF signaling under normoxia or hypoxia revealed no influence upon Sost transcription. These data suggest that hypoxia inhibits sclerostin expression, through enhanced antagonism of BMP signaling independent of VEGF. J. Cell. Biochem. 110: 457–467, 2010.

Hypoxic regulation of mesenchymal stem cell migration: the role of RhoA and HIF-1α

A variety of pathologies such as skeletal fracture, neoplasia and inflammation compromise tissue perfusion and thereby decrease tissue oxygen tension. We and others have demonstrated that hypoxia is a potent stimulant for MSC (mesenchymal stem cell) recruitment and differentiation, yet to date little research has focused on the effects of oxygen tension on MSC migration. In the present study, we examined the effects of hypoxia and the potential role of the GTPase RhoA and HIF‐1α (hypoxia‐inducible factor 1α) on MSC migration. Our results demonstrate that hypoxia decreases MSC migration through an HIF‐1α and RhoA‐mediated pathway. The active GTP‐bound form of RhoA was reduced in 1% oxygen, whereas activation of RhoA under hypoxic conditions rescued migration. Furthermore, stabilization of HIF‐1α under normoxic conditions attenuated cell migration similar to that of hypoxia. These results suggest that hypoxia negatively affects MSC migration by regulating activation of GTPases. These results highlight the importance of oxygen in regulating the recruitment of progenitor cells to areas of ischaemic tissue damage.

Osteogenic proliferation and differentiation of canine bone marrow and adipose tissue derived mesenchymal stromal cells and the influence of hypoxia

The aim of this study was to compare the osteogenic and proliferative potential of canine mesenchymal stromal cells (cMSCs) derived from bone marrow (BM-cMSCs) and adipose tissue (AT-cMSCs). Proliferation potential was determined under varying oxygen tensions (1%, 5%, and 21% O(2)). Effects of reduced oxygen levels on the osteogenic differentiation of AT-cMSCs were also investigated. AT-cMSCs proliferated at a significantly faster rate than BM-cMSCs, although both cell types showed robust osteogenic differentiation. Culture in 5% and 1% O(2) impaired proliferation in cMSC from both sources and osteogenic differentiation in AT-cMSCs. Our data suggests that AT-cMSCs might be more suitable for use in a clinical situation, where large cell numbers are required for bone repair, due to their rapid proliferation combined with robust osteogenic potential. Our data also suggests that the inhibitory effects of hypoxia on both cell proliferation and differentiation should be considered when using MSCs in a potentially hypoxic environment such as a fracture site.

Long-term administration of AMD3100, an antagonist of SDF-1/CXCR4 signaling, alters fracture repair.

Fracture healing involves rapid stem and progenitor cell migration, homing, and differentiation. SDF‐1 (CXCL12) is considered a master regulator of CXCR4‐positive stem and progenitor cell trafficking to sites of ischemic (hypoxic) injury and regulates their subsequent differentiation into mature reparative cells. In this study, we investigated the role of SDF‐1/CXCR4 signaling in fracture healing where vascular disruption results in hypoxia and SDF‐1 expression. Mice were injected with AMD3100, a CXCR4 antagonist, or vehicle twice daily until euthanasia with the intent to impair stem cell homing to the fracture site and/or their differentiation. Fracture healing was evaluated using micro‐computed tomography, histology, quantitative PCR, and mechanical testing. AMD3100 administration resulted in a significantly reduced hyaline cartilage volume (day 14), callus volume (day 42) and mineralized bone volume (day 42) and reduced expression of genes associated with endochondral ossification including collagen Type 1 alpha 1, collagen Type 2 alpha 1, vascular endothelial growth factor, Annexin A5, nitric oxide synthase 2, and mechanistic target of rapamycin. Our data suggest that the SDF‐1/CXCR4 signaling plays a central role in bone healing possibly by regulating the recruitment and/or differentiation of stem and progenitor cells.

Hypoxia increases Annexin A2 expression in osteoblastic cells via VEGF and ERK

Vascular endothelial growth factor (VEGF)-stimulated angiogenesis is critical for endochondral ossification that occurs during bone development and bone repair. Under these circumstances, VEGF production appears to be driven by low oxygen tension, under the control of the hypoxia-inducible factor-α family of transcription factors (HIF-α). Annexin 2 (AnxA2) a calcium-dependent phospholipid binding protein has been implicated in VEGF-mediated retinal neovascularization and is upregulated by VEGF in choroid retinal endothelial cells. AnxA2 is also expressed in cells of the osteoblast lineage and chondrocytes and may play a role in matrix mineralization. In this paper, we examined the effects of hypoxia (1% O(2)) and VEGF on the expression of AnxA2 in osteoblastic MC3T3-E1 cells. Hypoxia, desferrioxamine (hypoxia mimetic), and recombinant VEGF all increased AnxA2 mRNA and protein levels in osteoblastic cells. The hypoxia-induced increase in AnxA2 was inhibited by a blocking antibody to VEGF-R1; however, VEGF(120), a VEGF-R1 agonist, demonstrated no influence upon Anxa2 expression. This suggests that VEGF induction of Annexin A2 is not mediated via VEGF-R1 agonism alone but by VEGF-R1 and Neuropilin-1 or Neuropilin-2 heterodimers. In addition, we demonstrated that VEGF-stimulated changes in AnxA2 expression via a pathway involving Src and MEK kinase. These data demonstrate that AnxA2 expression in osteoblasts is under the control of VEGF, which may have implications for both angiogenesis and bone mineralization under low oxygen conditions.

Mobilization of endogenous stem cell populations enhances fracture healing in a murine femoral fracture model

BACKGROUND AIMS Delivery of bone marrow-derived stem and progenitor cells to the site of injury is an effective strategy to enhance bone healing. An alternate approach is to mobilize endogenous, heterogeneous stem cells that will home to the site of injury. AMD3100 is an antagonist of the chemokine receptor 4 (CXCR4) that rapidly mobilizes stem cell populations into peripheral blood. Our hypothesis was that increasing circulating numbers of stem and progenitor cells using AMD3100 will improve bone fracture healing. METHODS A transverse femoral fracture was induced in C57BL/6 mice, after which they were subcutaneously injected for 3 d with AMD3100 or saline control. Mesenchymal stromal cells, hematopoietic stem and progenitor cells and endothelial progenitor cells in the peripheral blood and bone marrow were evaluated by means of flow cytometry, automated hematology analysis and cell culture 24 h after injection and/or fracture. Healing was assessed up to 84 d after fracture by histomorphometry and micro-computed tomography. RESULTS AMD3100 injection resulted in higher numbers of circulating mesenchymal stromal cells, hematopoietic stem cells and endothelial progenitor cells. Micro-computed tomography data demonstrated that the fracture callus was significantly larger compared with the saline controls at day 21 and significantly smaller (remodeled) at day 84. AMD3100-treated mice have a significantly higher bone mineral density than do saline-treated counterparts at day 84. CONCLUSIONS Our data demonstrate that early cell mobilization had significant positive effects on healing throughout the regenerative process. Rapid mobilization of endogenous stem cells could provide an effective alternative strategy to cell transplantation for enhancing tissue regeneration.

HIF‐1α regulates hypoxia‐induced EP1 expression in osteoblastic cells

Changes in regional oxygen tension that occur during skeletal development and fracture stimulate local bone cell activity to regulate bone formation, maintenance, and repair. The adaptive responses of bone cells to hypoxia are only beginning to be understood. The transcription factor hypoxia‐inducible factor‐1α (HIF‐1α) is activated under hypoxia and promotes expression of genes required for adaptation and cell survival, and also regulates both bone development and fracture repair. We have previously demonstrated that hypoxic osteoblasts increase PGE2 release and expression of the PGE2 receptor EP1. In the present studies, we investigated the impact of altered HIF‐1α activity and expression on EP1 expression in osteoblasts. HIF‐1α stabilization was induced in cells cultured in 21% oxygen by treatment with dimethyloxaloglycine (DMOG) or siRNA targeted against PHD2. To implicate HIF‐1α in hypoxia‐induced EP1 expression, osteoblastic cells were treated with siRNA targeted against HIF‐1α prior to exposure to hypoxia. EP1 expression was significantly increased in cells cultured in 21% oxygen with DMOG or PHD2 siRNA treatment compared to controls. Hypoxia responsive element (HRE) activation in hypoxia was attenuated in cells treated with HIF‐1α siRNA compared to controls, indicating HIF‐1α as the functional HIF‐α isoform in this system. Furthermore, hypoxic cells treated with HIF‐1α siRNA demonstrated reduced EP1 expression in hypoxia compared to controls. Inhibition of SAPK/JNK activity significantly reduced hypoxia‐induced EP1 expression but had no impact on HIF‐1α expression or activity. These data strongly implicate a role for HIF‐1α in hypoxia‐induced EP1 expression and may provide important insight into the mechanisms by which HIF‐1α regulates bone development and fracture repair. J. Cell. Biochem. 107: 233–239, 2009.

Regulation of Tenascin Expression in Bone

Tenascins regulate cell interaction with the surrounding pericellular matrix. Within bone, tenascins C and W influence osteoblast adhesion and differentiation, although little is known about the regulation of tenascin expression. In this study we examined the effect of osteogenic differentiation, bone morphogenetic protein (BMP) and Wnt growth factors, and mechanical loading on tenascin expression in osteogenic cells. Osteogenic differentiation increased tenascin C (TnC), and decreased tenascin W (TnW), expression. Both growth factors and mechanical loading increased both TnC and TnW expression, albeit via distinct signaling mechanisms. Both BMP‐2 and Wnt5a induction of tenascin expression were mediated by MAP kinases. These data establish a role for BMP, Wnts, and mechanical loading in the regulation of tenascin expression in osteoblasts. J. Cell. Biochem. 112: 3354–3363, 2011.

Impaired osteoblast differentiation in annexin A2- And -A5-deficient cells

Annexins are a class of calcium-binding proteins with diverse functions in the regulation of lipid rafts, inflammation, fibrinolysis, transcriptional programming and ion transport. Within bone, they are well-characterized as components of mineralizing matrix vesicles, although little else is known as to their function during osteogenesis. We employed shRNA to generate annexin A2 (AnxA2)- or annexin A5 (AnxA5)-knockdown pre-osteoblasts, and determined whether proliferation or osteogenic differentiation was altered in knockdown cells, compared to pSiren (Si) controls. We report that DNA content, a marker of proliferation, was significantly reduced in both AnxA2 and AnxA5 knockdown cells. Alkaline phosphatase expression and activity were also suppressed in AnxA2- or AnxA5-knockdown after 14 days of culture. The pattern of osteogenic gene expression was altered in knockdown cells, with Col1a1 expressed more rapidly in knock-down cells, compared to pSiren. In contrast, Runx2, Ibsp, and Bglap all revealed decreased expression after 14 days of culture. In both AnxA2- and AnxA5-knockdown, interleukin-induced STAT6 signaling was markedly attenuated compared to pSiren controls. These data suggest that AnxA2 and AnxA5 can influence bone formation via regulation of osteoprogenitor proliferation, differentiation, and responsiveness to cytokines in addition to their well-studied function in matrix vesicles.