Examination of the "Osteopontin Hypothesis" (OSTEOPONTIN) - 09.17.14
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To clarify whether osteopontin is an important factor regulating microgravity induced bone loss, we evaluate the osteoblastic differentiation of bone marrow stromal cells obtained form osteopontin deficient mice and wild type control mice, by culturing with minerarizing condition in the space.
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Japan Aerospace Exploration Agency (JAXA), Tsukuba, , Japan
Sponsoring Space Agency
Japan Aerospace Exploration Agency (JAXA)
ISS Expedition Duration
March 2015 - October 2015
Previous ISS Missions
- The mechanisms of microgravity induced osteoporosis are not well understood. Osteopontin (OPN), one of the abundant proteins in bone matrix, is believed to be involved in this process. Thus, we aimed to examine the influence of microgravity on the mineralization of cultured bone marrow cells derived from osteopontin-deficient mice and control mice, in the ISS.
- Bone marrow stromal cells shall be prepared, and frozen on the ground. Frozen cells shall be brought up to the ISS, and shall be cultured under the microgravity of the space for 3 weeks. Cultured medium shall be collected at day10 when the medium shall be changed. After 21 days of culture, the cells added with "RNA later" reagent shall be preserved in the freezer. Chemically fixed cells shall be kept under the 4ºC condition. After recovery on the ground, these samples shall be analyzed for mineral, protein, RNA expressing and image analysis for Alizarin Red staining for the mineralization nodules.
- Our study would be considered to be beneficial for the development of novel drugs which would be expected to be valuable for many people facing to immobility induced osteoporosis.
The mechanisms causing the unloading-induced osteoporosis by microgravity environment is not well understood, and thus no sufficient treatment is available at this point. Our study testing the hypothesis that Osteopontin is an important regulator of this process in the bone forming cells may provide a novel findings contributing to development of effective therapeutics for unloading-induced osteoporosis.
Unloading-induced osteoporosis by microgravity environment mimics to the immobilization-induced osteoporosis, which occurs commonly in elederlies suffering from various chronic diseases including heart diseases, strokes and neurodegenerative diseases. Pathophysiological understandings and development of novel therapeutics for these pathological conditions are thought to be mutually related, and thus beneficial each other.
This experiment requires launching 24-lines (included 8-line backups) of frozen bone marrow stromal cells. For culturing these cells, it requires CBEF and five sets of the PFK-III. For sample storage after the experiment, it requires freezer space for used culture medium from day 10 of the culture, and the cells soaked with the RNAlater reagent after culture completion at day 21. It also requires storage space in the refrigerator for the paraformaldehyde fixed cells after completion of the culture at day 21. Downlink of the microscopic images at day 21 is required.
In this experiment, 16-lines of frozen bone marrow stromal cells from Osteopontin –deficient mice or control mice will be launched. Procedures should be added by a crew member, at three time points, as follows.
(1) The frozen syringes should be picked up from the storage, to plate the cells on the Meas Exp Culture Chamber, by adding warmed culture medium. Each cell-suspension should be plated on two chambers, and the cells in total of 32 chambers should be incubated in the Meas Exp Culture Cage at 37C, using CBEF. Each half of the duplicated chambers should be cultured in the microgravity condition, and the other half should be cultured under the artificial 1-gravity environment.
(2) At 10 day of the culture; culture medium will be exchanged.
(3) At 21 day of the culture; Microscopic images should be taken for several chambers to downlink the data to the ground. 12 chambers should be frozen in the freezer, after exchanging the culture medium to the RNAlater for preserving RNA. Rest 20 chambers should be stored in the refrigerator, after exchanging the medium to 0.99% paraformaldehyde. These specimens should be used for alizarin red staining later. The used medium should be collected to be stored in the freezer. These samples should be applied for calcium and phosphorous measurement, later.