Heterotypic Cell Culture from Mouse Bone Marrow under Simulated Microgravity: Lessons for Stromal Lineage Functions.

Muscle and skeleton structures are considered most susceptible to negative factors of spaceflights, namely microgravity. Three-dimensional clinorotation is a ground-based simulation of microgravity. It provides an opportunity to elucidate the effects of microgravity at the cellular level. The extracellular matrix (ECM) content, transcriptional profiles of genes encoding ECM and remodelling molecules, and secretory profiles were investigated in a heterotypic primary culture of bone marrow cells after 14 days of 3D clinorotation. Simulated microgravity negatively affected stromal lineage cells, responsible for bone tissue formation. This was evidenced by the reduced ECM volume and stromal cell numbers, including multipotent mesenchymal stromal cells (MSCs). ECM genes encoding proteins responsible for matrix stiffness and cell-ECM contacts were downregulated. In a heterotypic population of bone marrow cells, the upregulation of genes encoding ECM degrading molecules and the formation of a paracrine profile that can stimulate ECM degradation, may be mechanisms of osteodegenerative events that develop in real spaceflight.

Stromal Lineage Precursors from Rodent Femur and Tibia Bone Marrows after Hindlimb Unloading: Functional Ex Vivo Analysis.

Rodent hindlimb unloading (HU) model was developed to elucidate responses/mechanisms of adverse consequences of space weightlessness. Multipotent mesenchymal stromal cells (MMSCs) were isolated from rat femur and tibia bone marrows and examined ex vivo after 2 weeks of HU and subsequent 2 weeks of restoration of load (HU + RL). In both bones, decrease of fibroblast colony forming units (CFU-f) after HU with restoration after HU + RL detected. In CFU-f and MMSCs, levels of spontaneous/induced osteocommitment were similar. MMSCs from tibia initially had greater spontaneous mineralization of extracellular matrix but were less sensitive to osteoinduction. There was no recovery of initial levels of mineralization in MMSCs from both bones during HU + RL. After HU, most bone-related genes were downregulated in tibia or femur MMSCs. After HU + RL, the initial level of transcription was restored in femur, while downregulation persisted in tibia MMSCs. Therefore, HU provoked a decrease of osteogenic activity of BM stromal precursors at transcriptomic and functional levels. Despite unidirectionality of changes, the negative effects of HU were more pronounced in stromal precursors from distal limb-tibia. These observations appear to be on demand for elucidation of mechanisms of skeletal disorders in astronauts in prospect of long-term space missions.

Short-Term Reloading After Prolonged Unloading Ensures Restoration of Stromal but Not Hematopoietic Precursor Activity in Tibia Bone Marrow of C57Bl/6N Mice.

Bone and muscle tissues are mostly susceptible to different kinds of hypodynamia, including real and simulated microgravity (sµg). To evaluate the effect of sµg on bone marrow (BM), male C57Bl/6N mice were divided into three groups: vivarium control (VC), 30-day hindlimb suspension (HS), and subsequent 12-h short-term support reloading (RL). The effects on BM total mononucleated cells (MNCs) as well as stromal and hematopoietic progenitors from murine tibia were studied. The number of BM MNCs, immunophenotype, proliferation, colony-forming units (CFUs), differentiation and secretory activity of hematopoietic and stromal BM cells were determined. HS led to a twofold decrease in MNCs, alteration of surface molecule expression profiles, suppression of proliferative activity of BM cells, and change of soluble mediators' levels. The stromal compartment was characterized by a decrease of CFU of fibroblasts and suppression of spontaneous osteo-commitment after HS. Among the hematopoietic precursors, a decrease in the total number of CFUs was found mainly at the expense of suppression of CFU-GM and CFU-GEMM. After RL, restoration of the stromal precursor's functional activity to control levels and overabundance of paracrine mediator's production were detected, whereas the complete recovery of hematopoietic precursor's activity did not occur. These data demonstrate the fast functional reaction of the stromal compartment on restoration of loading support.

Stromal and Hematopoietic Progenitors from C57/BI/6N Murine Bone Marrow After 30-Day "BION-M1" Spaceflight.

Elucidation of the spaceflight (SF) effects on the adult stem and progenitor cells is an important goal in space biology and medicine. A unique opportunity for this was provided by project "BION-M1". The purpose of this study was to evaluate the effects of 30-day SF on biosatellite, 7-day recovery (SFR), and subsequent ground control (GC) experiment on the mononuclear cells (MNCs) from C57/BI/6N murine tibia bone marrow. Also, hematopoietic and stromal precursor functions were characterized ex vivo. There was no significant difference in the total MNC number between experimental groups. After SF, immunophenotyping revealed an increase of large-sized CD45+MNCs corresponded to committed hematopoietic progenitors. The total hematopoietic colony-forming unit (CFU) number decreased after SF and did not restore after 7 day of recovery due to predominant reduction of bi- and multipotent CFUs and primitive burst-forming units in favor of unipotent CFUs. Functional activity of stromal precursors in vitro was only slightly altered. SF cells displayed the enhanced expression of alkaline phosphatase. The data of the GC experiment demonstrated the preservation of the functional activity of progenitor cells from mice bone marrow. The activation of erythropoiesis in expense of burst-forming units of erythrocytes elevation was detected. After 7 days of recovery, the number of colony-forming units of fibroblast (CFUs-f) was similar to the vivarium control, while the proliferative activity of bone marrow stromal precursors decreased. The present study demonstrated that certain hematopoietic progenitors are susceptible to SF factors, while the stromal precursors displayed a certain degree of resistance. These data indicate mild and reversible alterations of bone marrow progenitors after SF.