Ontology analysis of global gene expression differences of human bone marrow stromal cells cultured on 3D scaffolds or 2D films

Differences in gene expression of human bone marrow stromal cells (hBMSCs) during culture in three-dimensional (3D) nanofiber scaffolds or on two-dimensional (2D) films were investigated via pathway analysis of microarray mRNA expression profiles. Previous work has shown that hBMSC culture in nanofiber scaffolds can induce osteogenic differentiation in the absence of osteogenic supplements (OS). Analysis using ontology databases revealed that nanofibers and OS regulated similar pathways and that both were enriched for TGF-beta and cell-adhesion/ECM-receptor pathways. The most notable difference between the two was that nanofibers had stronger enrichment for cell-adhesion/ECM-receptor pathways. Comparison of nanofibers scaffolds with flat films yielded stronger differences in gene expression than comparison of nanofibers made from different polymers, suggesting that substrate structure had stronger effects on cell function than substrate polymer composition. These results demonstrate that physical (nanofibers) and biochemical (OS) signals regulate similar ontological pathways, suggesting that these cues use similar molecular mechanisms to control hBMSC differentiation. Published by Elsevier Ltd.

Substrate Strain and Proliferation & Differentiation of Bone Marrow Mesenchymal Stem Cells

New methods of surface modification of transparent silicone substrate were developed, and a new set of cell culture devices that provide homogeneous substrate strain was designed. Using the new device, effects of cyclic substrate strain on bone marrow mesenchymal stem cells(MSCs) were studied. It was found that cyclic strain influenced proliferation and differentiation of bone marrow MSCs in different ways.

Vascular endothelial growth factor regulates melanoma cell adhesion and growth in the bone marrow microenvironment via tumor cyclooxygenase-2

Background: Human melanoma frequently colonizes bone marrow (BM) since its earliest stage of systemic dissemination, prior to clinical metastasis occurrence. However, how melanoma cell adhesion and proliferation mechanisms are regulated within bone marrow stromal cell (BMSC) microenvironment remain unclear. Consistent with the prometastatic role of inflammatory and angiogenic factors, several studies have reported elevated levels of cyclooxygenase-2 (COX-2) in melanoma although its pathogenic role in bone marrow melanoma metastasis is unknown. Methods: Herein we analyzed the effect of cyclooxygenase-2 (COX-2) inhibitor celecoxib in a model of generalized BM dissemination of left cardiac ventricle-injected B16 melanoma (B16M) cells into healthy and bacterial endotoxin lipopolysaccharide (LPS)-pretreated mice to induce inflammation. In addition, B16M and human A375 melanoma (A375M) cells were exposed to conditioned media from basal and LPS-treated primary cultured murine and human BMSCs, and the contribution of COX-2 to the adhesion and proliferation of melanoma cells was also studied. Results: Mice given one single intravenous injection of LPS 6 hour prior to cancer cells significantly increased B16M metastasis in BM compared to untreated mice; however, administration of oral celecoxib reduced BM metastasis incidence and volume in healthy mice, and almost completely abrogated LPS-dependent melanoma metastases. In vitro, untreated and LPS-treated murine and human BMSC-conditioned medium (CM) increased VCAM-1-dependent BMSC adherence and proliferation of B16M and A375M cells, respectively, as compared to basal medium-treated melanoma cells. Addition of celecoxib to both B16M and A375M cells abolished adhesion and proliferation increments induced by BMSC-CM. TNF alpha and VEGF secretion increased in the supernatant of LPS-treated BMSCs; however, anti-VEGF neutralizing antibodies added to B16M and A375M cells prior to LPS-treated BMSC-CM resulted in a complete abrogation of both adhesion-and proliferation-stimulating effect of BMSC on melanoma cells. Conversely, recombinant VEGF increased adherence to BMSC and proliferation of both B16M and A375M cells, compared to basal medium-treated cells, while addition of celecoxib neutralized VEGF effects on melanoma. Recombinant TNFa induced B16M production of VEGF via COX-2-dependent mechanism. Moreover, exogenous PGE2 also increased B16M cell adhesion to immobilized recombinant VCAM-1. Conclusions: We demonstrate the contribution of VEGF-induced tumor COX-2 to the regulation of adhesion-and proliferation-stimulating effects of TNFa, from endotoxin-activated bone marrow stromal cells, on VLA-4-expressing

Intermolecular Multiple Quantum Coherences Enable Accurate Thermal Imaging of Red Bone Marrow During Thermal Therapy of Bone Metastases

Prostate and breast cancers are two of the most common types of cancer in the United States, and those cancers metastasize to bone in more than two thirds of patients. Recent evidence suggests that thermal therapy is effective at treating metastatic bone cancer. For example, thermal therapy enables targeted drug delivery to bone, ablation of cancer cells in bone marrow, and palliation of bone pain. Thermal therapy of bone metastases would be greatly improved if it were possible to image the temperature of the tissue surrounding the disease, which is usually red bone marrow (RBM). Unfortunately, current thermal imaging techniques are inaccurate in RBM.

This dissertation shows that many of the difficulties with thermal imaging of RBM can be overcome using a magnetic resonance phenomenon called an intermolecular multiple quantum coherence (iMQC). Herein, iMQCs are detected with a magnetic resonance imaging (MRI) pulse sequence called multi-spin-echo HOMOGENIZED with off resonance transfer (MSE-HOT). Compared to traditional methods, MSE-HOT provided ten-fold more accurate images of temperature change. Furthermore, MSE-HOT was translated to a human MRI scanner, which enabled imaging of RBM temperature during heating with a clinical focused ultrasound applicator. In summary, this dissertation develops a MRI technique that enables thermal imaging of RBM during thermal therapy of bone metastases.

CD45+ Cells Present Within Mesenchymal Stem Cell Populations Affect Network Formation of Blood-Derived Endothelial Outgrowth Cells.

Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) represent promising cell sources for angiogenic therapies. There are, however, conflicting reports regarding the ability of MSCs to support network formation of endothelial cells. The goal of this study was to assess the ability of human bone marrow-derived MSCs to support network formation of endothelial outgrowth cells (EOCs) derived from umbilical cord blood EPCs. We hypothesized that upon in vitro coculture, MSCs and EOCs promote a microenvironment conducive for EOC network formation without the addition of angiogenic growth supplements. EOC networks formed by coculture with MSCs underwent regression and cell loss by day 10 with a near 4-fold and 2-fold reduction in branch points and mean segment length, respectively, in comparison with networks formed by coculture vascular smooth muscle cell (SMC) cocultures. EOC network regression in MSC cocultures was not caused by lack of vascular endothelial growth factor (VEGF)-A or changes in TGF-β1 or Ang-2 supernatant concentrations in comparison with SMC cocultures. Removal of CD45+ cells from MSCs improved EOC network formation through a 2-fold increase in total segment length and number of branch points in comparison to unsorted MSCs by day 6. These improvements, however, were not sustained by day 10. CD45 expression in MSC cocultures correlated with EOC network regression with a 5-fold increase between day 6 and day 10 of culture. The addition of supplemental growth factors VEGF, fibroblastic growth factor-2, EGF, hydrocortisone, insulin growth factor-1, ascorbic acid, and heparin to MSC cocultures promoted stable EOC network formation over 2 weeks in vitro, without affecting CD45 expression, as evidenced by a lack of significant differences in total segment length (p=0.96). These findings demonstrate the ability of MSCs to support EOC network formation correlates with removal of CD45+ cells and improves upon the addition of soluble growth factors.

Ovarian function and spontaneous pregnancy after combined heterotopic and orthotopic cryopreserved ovarian tissue transplantation in a patient previously treated with bone marrow transplantation: case report

Cryopreservation of ovarian tissue has been proposed for storing gametes of young patients at high risk of premature ovarian failure. Autotransplantation has recently provided some promising results and is still the unique option to restore ovarian function from cryopreserved ovarian tissue in humans. In this article, we analyse data from the combined orthotopic and heterotopic transplantation of cryopreserved ovarian tissue that restored the ovarian function and fertility. Orthotopic transplantation of cryopreserved ovarian tissue at ovarian and peritoneal sites, together with a heterotopic transplantation at the abdominal subcutaneous site, was performed to restore the ovarian function of a 29-year-old woman previously treated with bone marrow transplantation (BMT) for Hodgkin's disease. Ovarian reserve markers progressively suppress within values 5 months after the transplantation (basal FSH 5 mUI/ml and inhibin B 119 ng/ml). Follicular development was observed at all transplantation sites but was predominant at the ovarian site. Six natural cycles were fully documented and analysed. The patient became spontaneously pregnant following the sixth cycle, but unfortunately she later miscarried. Combined orthotopic and heterotopic transplantations succeeded in the restoration of normal spontaneous cycles. Furthermore, this spontaneous pregnancy confirmed the efficiency of this procedure for restoring human fertility.

FES KINASE SIGNALING PROMOTES MAST CELL RECRUITMENT TO TUMOURS

FES protein-tyrosine kinase (PTK) activation downstream of the KIT receptor in mast cells (MC) promotes cell polarization and migration towards the KIT ligand Stem cell factor (SCF). A variety of tumours secrete SCF to promote MC recruitment and release of mediators that enhance tumour vascularization and growth. This study investigates whether FES promotes MC migration via regulation of microtubules (MTs), and if FES is required for MC recruitment to the tumour microenvironment. MT binding assays showed that FES has at least two MT binding sites, which likely contribute to the partial co-localization of FES with MTs in polarized bone marrow-derived mast cells (BMMCs). Live cell imaging revealed a significant defect in chemotaxis of FES-deficient BMMCs towards SCF embedded within an agarose drop, which correlated with less MT organization compared to control cells. To extend these results to a tumour model, mouse mammary carcinoma AC2M2 cells were engrafted under the skin and into the mammary fat pads of immune compromised control (nu/nu) or FES-deficient (nu/nu:fes-/-) mice. A drastic reduction in tumour-associated MCs was observed in FES-deficient mice compared to control in both mammary and skin tissue sections. This correlated with a trend towards reduced tumour volumes in FES-deficient mice. These results implicate FES signaling downstream of KIT, in promoting MT reorganization during cell polarization and for chemotaxis of MCs towards tumour-derived SCF. Thus, FES is a potential therapeutic target to limit recruitment of stromal mast cells or macrophages to solid tumours that enhance tumour progression.

Osteoclastogenesis induced by sRANKL and M-CSF from the non-adherent cell population of human bone marrow is inhibited by rhBMP-2 alone or together with rhVEGF

During bone development and repair, angiogenesis, osteogenesis and bone remodelling are closely associated processes that share some common mediators. In the present study non-adherent human bone marrow mononuclear cells under the induction of sRANKL and M-CSF, differentiated into osteoclasts with TRAP positive staining, VNR expression, and Ca-P resorptive activity. The effects of various combinations of rhBMP-2 (0, 3, 30, 300 ng/ml) and rhVEGF (0, 25 ng/ml) on osteoclastogenesis potentials were examined in this experimental system. The percentages of TRAP-positive multiple nucleated cells represent osteoclast differentiation potential and the percentages of resorptive areas in the Ca-P coated plates resemble osteoclast resorption capability. The presence of rhBMP-2 at 30 and 300 ng/ml showed inhibitory effects on osteoclast differentiation and their resorptive capability in the human osteoclast culture system. rhVEGF (25 ng/ml) enhanced the resorptive function of osteoclast whenever it was used alone or combined with 3 ng/ml rhBMP-2. However, rhVEGF induced resorptive function was inhibited by 30 ng/ml and 300 ng/ml rhBMP-2 at a dose-dependent manner. Statistical analysis demonstrated that an interactive effect exists between rhBMP-2 and rhVEGF on human osteoclastogenesis. These findings suggested that an interactive regulation may exist between BMPs and VEGF signaling pathways during osteoclastogenesis, exact mechanisms are yet to be elucidated.

Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells

Using a validated tetracycline (tet)-regulated MCF7-founder (MCF7F) expression system to modulate expression of CD44 standard form (CD44s), we report the functional importance of CD44s and that of a novel transcriptional target of hyaluronan (HA)/CD44s signaling, EMS1/cortactin, in underpinning breast cancer metastasis. In functional experiments, tet-regulated induction of CD44s potentiated the migration and invasion of MCF7F cells through HA-supplemented Matrigel. EMS1/cortactin was identified by expression profiling as a novel transcriptional target of HA/CD44 signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast cancer cell lines. The mechanistic basis underpinning CD44-promoted transcription of EMS1/cortactin was shown to be dependent upon a NFB mechanism, since pharmacological inhibition of IKinase-2 or suppression of p65 Rel A expression attenuated CD44-induced increases in cortactin mRNA transcript levels. Overexpression of a c-myc tagged murine cortactin construct in the weakly invasive, CD44-deficient MCF7F and T47D cells potentiated their invasion. Furthermore, the functional importance of cortactin to CD44s-promoted metastasis was demonstrated by selective suppression of cortactin in CD44-expressing MCF7F-B5 and MDA-MB-231 breast cancer cells using RNAi, which was shown to result in attenuated CD44-promoted invasion and CD44-promoted adhesion to bone marrow endothelial cells (BMECs).