ABSTRACT
Stalled cell division in precursor bone cells and reduced osteoblast function are considered responsible for the microgravity-induced bone loss observed during spaceflight. However, underlying molecular mechanisms remain unraveled. Having overcome technological difficulties associated with flying cells in a space mission, we present the first report on the behavior of the potentially osteogenic murine bone marrow stromal cells (BMSC) in a 3D culture system, flown inside the KUBIK aboard space mission ISS 12S (Soyuz TMA-8 + Increment 13) from March 30 to April 8, 2006 (experiment "Stroma-2"). Flight 1g control cultures were performed in a centrifuge located within the payload. Ground controls were maintained on Earth in another KUBIK payload and in Petri dishes. Half of the cultures were stimulated with osteo-inductive medium. Differences in total RNA extracted suggested that cell proliferation was inhibited in flight samples. Affymetrix technology revealed that 1,599 genes changed expression after spaceflight exposure. A decreased expression of cell-cycle genes confirmed the inhibition of cell proliferation in space. Unexpectedly, most of the modulated expression was found in genes related to various processes of neural development, neuron morphogenesis, transmission of nerve impulse and synapse, raising the question on the lineage restriction in BMSC.
Subject(s)
Nervous System/growth & development , Space Flight , Transcriptional Activation , Weightlessness/adverse effects , Animals , Bone Marrow , Cell Cycle Proteins/genetics , Cell Proliferation , Cells, Cultured , Gene Expression Regulation , Mesenchymal Stem Cells , Mice , Neurons , RNA/analysis , Synapses/genetics , Synaptic Transmission/geneticsABSTRACT
There is increasing interest in developing new in vitro tissue models using typical tissue engineering approaches. This study was designed to (1) develop a novel three-dimensional (3D) in vitro model of bone by seeding murine primary osteoblasts and osteoclast precursors on a resorbable porous ceramic scaffold based on silicon-stabilized tricalcium phosphate (Skelite), and (2) investigate bone cell interactions in a 3D environment mimicking an in vivo condition and compare it to traditional two-dimensional (2D) cultures. Murine primary osteoblasts from C57Bl6/J mice and osteoclast precursors from C57Bl/6-Tg(ACTB-EGFP)1Osb/J mice were co-cultured on 3D Skelite scaffolds and on standard plastic culture dishes. The differentiation of these cells in both culture conditions was compared by histology (hematoxylin-eosin staining and polarized light analysis), immunohistochemistry (collagen type I), and gene expression analysis by real-time PCR for Runt-related transcription factor 2, osterix, osteocalcin, cathepsin K, and tartrate resistant acid phosphatase. To analyze and compare bone turnover in 3D and 2D co-cultures, we evaluated the modulation of RANKL and OPG mRNA expression. We observed an enhancement of osteoblast differentiation in the 3D mineralized environment that in turn promoted earlier osteoclast differentiation. In this paper, we also report that the increased osteoblast differentiation in the 3D model led to a deposition of extracellular matrix that faithfully reflected the morphology of bone tissue.
Subject(s)
Bone and Bones/cytology , Cell Differentiation , Models, Biological , Osteoblasts/cytology , Osteoclasts/cytology , Animals , Bone Matrix/cytology , Bone Matrix/metabolism , Bone and Bones/metabolism , Cells, Cultured , Coculture Techniques , Extracellular Matrix/metabolism , Gene Expression Regulation , Green Fluorescent Proteins/metabolism , Mice , Mice, Inbred C57BL , Osteoblasts/metabolism , Osteoclasts/metabolism , Osteogenesis , Osteoprotegerin/genetics , Osteoprotegerin/metabolism , RANK Ligand/genetics , RANK Ligand/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tissue ScaffoldsABSTRACT
Summary The purpose of this study was to evaluate telomere length in peripheral blood granulocytes and mononuclear cells collected from 22 women with polycythaemia vera (PV) and essential thrombocythaemia (ET). PV and ET are chronic myeloproliferative diseases whose heterogeneity of stem cell origin and clonal development has been established through analysis of X-chromosome inactivation patterns. The results from clonality assay and determination of telomere length show that only clonal granulocytes have shortened telomeres.
Subject(s)
Granulocytes/ultrastructure , Polycythemia Vera/immunology , Telomere/ultrastructure , Thrombocythemia, Essential/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Case-Control Studies , Clone Cells , Female , Hematopoietic Stem Cells/ultrastructure , Humans , Image Processing, Computer-Assisted , Leukocytes, Mononuclear/ultrastructure , Middle Aged , Polycythemia Vera/complications , Thrombocythemia, Essential/complicationsABSTRACT
Essential thrombocythemia (ET) and polycythemia vera (PV) are chronic myeloproliferative disorders that share the involvement of a multipotent progenitor cell and dominance of the transformed clone over normal hematopoiesis. On the other hand, the heterogeneity of these diseases with respect to clonal development from a common progenitor has been well established. To identify useful prognostic indicators, we analyzed telomerase activity (TA), a known marker of neoplastic proliferation, in granulocytes (PMNs) and mononuclear cells (MNCs) from 22 female patients with ET and PV. Clonality status was determined by investigation of X chromosome inactivation patterns (XCIPs). We found a statistically significant positive correlation between high TA and monoclonal pattern of XCIP. Therefore, our data suggest that the use of multiple tumor markers may contribute to a better understanding of the deregulated physiology of these disorders and provide useful prognostic factors.