Ultrastructural analysis of organization of roots obtained from cell cultures at clinostating and under microgravity.

Data are presented of a comparative analysis on rhizogenesis in the Arabidopsis thaliana tissue culture growing in a solid nutrient medium under stationary conditions, clinostatic conditions and microgravity. Tissue samples weighing 100 mg. were set in the Petri dishes and placed in a horizontal slow clinostat /2 revs/min/. After 14 days of growth they were analyzed. On clinostating the number of roots formed from the callus cells was approximately one half the control. The formed root cap manifested no essential differences, in comparison with the stationary control, in the number of layers and cell sizes in its layers. In callusogenic roots, formed from clinostated cells, differentiation including root cap cells, proceeds without noticeable deviations from the norm. At the same time, gravireceptor cells do not function under these conditions. This is clearly displayed at a structural level in the location of amyloplasts-statoliths throughout the cytoplasm. The callus cell cultures experienced microgravity for 8 days. The number of formed roots under the influence of this factor was 36% relative to the stationary control. Root cap formation was abnormal. Gravireceptor cells did not formed under microgravity.     

Development of plant protoplasts during the IML-1 mission.

During the 8 day IML-1 mission, regeneration of cell walls and cell divisions in rapeseed protoplasts were studied using the Biorack microscope onboard the Space Shuttle "Discovery". Samples from microgravity and 1g protoplast cultures were loaded on microscope slides. Visual microscopic observations were reported by the payload specialist Roberta Bondar, by down-link video transmission and by use of a microscope camera. Protoplasts grown under microgravity conditions do regenerate cell walls but to a lesser extent than under 1g. Cell divisions are delayed under microgravity. Few cell aggregates with maximum 4-6 cells per aggregate are formed under microgravity conditions, indicating that microgravity may have a profound influence on plant cell differentiation.     

Simulated microgravity inhibits cell wall regeneration of Penicillium decumbens protoplasts

This work compares cell wall regeneration from protoplasts of the fungus Penicillium decumbens under rotary culture (simulated microgravity) and stationary cultures. Using an optimized lytic enzyme mixture, protoplasts were successfully released with a yield of 5.3 × 10⁵ cells/mL. Under simulated microgravity conditions, the protoplast regeneration efficiency was 33.8%, lower than 44.9% under stationary conditions. Laser scanning confocal microscopy gave direct evidence for reduced formation of polysaccharides under simulated conditions. Scanning electron microscopy showed the delayed process of cell wall regeneration by simulated microgravity. The delayed regeneration of P. decumbens cell wall under simulated microgravity was likely caused by the inhibition of polysaccharide synthesis. This research contributes to the understanding of how gravitational loads affect morphological and physiological processes of fungi.     

Simulated microgravity does not alter epithelial cell adhesion to matrix and other molecules.

Microgravity has advantages for the cultivation of tissues with high fidelity; however, tissue formation requires cellular recognition and adhesion. We tested the hypothesis that simulated microgravity does not affect cell adhesion. Human colorectal carcinoma cells were cultured in the NASA Rotating Wall Vessel (RWV) under low shear stress with randomization of the gravity vector that simulates microgravity. After 6-7 days, cells were assayed for binding to various substrates and compared to cells grown in standard tissue culture flasks and static suspension cultures. The RWV cultures bound as well to basement membrane proteins and to CEA, an intercellular adhesion molecule, as control cultures did. Thus, microgravity does not alter epithelial cell adhesion and may be useful for tissue engineering.     

Inhibitory effect of simulated microgravity on differentiating preosteoblasts

The bone loss induced by microgravity is partly due to the decrease of mature osteoblasts. In the present study, we employed the random positioning machine (RPM) to simulate microgravity and investigated the acute effects of simulated microgravity on the differentiation of 2T3 preosteoblasts. Following 7 days’ culture under normal (1 g) condition, cells were exposed to simulated microgravity for 24 h. The results showed that 24 h treatment of simulated microgravity significantly decreased alkaline phosphatase (ALP) activity without changing the cell morphology. In addition, the mRNA expressions of osteogenic genes, including runt-related gene 2 (Runx2), osterix, osteocalcin (OC), type I collagen (Col I) and bone morphogenetic protein (BMP), were dramatically downregulated. Moreover, western blot analysis of total extracellular signal-regulated kinase (Erk) and phosphorylated Erk (p-Erk) indicated that p-Erk level, which represents the Erk activation status, was increased. Taken together, our results suggested that acute exposure to simulated microgravity inhibited osteoblast differentiation through modulating the expression of osteogenic genes and the Erk activity. These findings provide new insight for bone loss due to microgravity and unloading.     

Effects of altered gravity on plant cell processes: results of recent space and clinostatic experiments.

Space and clinostatic experiments revealed that plant cell structure and metabolism rearrangements depend on taxonomical position and physiological state of objects, growth phase and real or simulated microgravity influence duration. It was shown that clinostat conditions reproduce only a part of microgravity biological effects. It is established that various responses occur in microgravity: 1) rearrangements of cytoplasmic organelles ultrastructure and calcium balance; 2) physical-chemical properties of the plasmalemma are changed; 3) enzymes activity is often enhanced. These events provoke the acceleration of growth and differentiation of cells and their aging as a result; at the same time some responses can be considered as cell adaptation to microgravity.     

Upregulation of erythropoietin receptor in UT-7/EPO cells inhibits simulated microgravity-induced cell apoptosis

Hematopoietic progenitor cell proliferation can be altered in either spaceflight or under simulated microgravity experiments on the ground, however, the underlying mechanism remains unknown. Our previous study showed that exposure of the human erythropoietin (EPO)-dependent leukemia cell line UT-7/EPO to conditions of simulated microgravity significantly inhibited the cellular proliferation rate and induced cell apoptosis. We postulated that the downregulation of the erythropoietin receptor (EPOR) expression in UT-7/EPO cells under simulated microgravity may be a possible reason for microgravity triggered apoptosis. In this paper, a human EPOR gene was transferred into UT-7/EPO cells and the resulting expression of EPOR on the surface of UT-7/EPO cells increased approximately 61% (p < 0.05) as selected by the antibiotic G418. It was also shown through cytometry assays and morphological observations that microgravity-induced apoptosis markedly decreased in these UT-7/EPO–EPOR cells. Thus, we concluded that upregulation of EPOR in UT-7/EPO cells could inhibit the simulated microgravity-induced cell apoptosis in this EPO dependent cell line.     

Cortical Microtubule Reorientation and Its Relation to Cell Surface Texture of Epidermal Cells of Arabidopsis Thaliana Hypocotyls under Simulated Microgravity Conditions

Gravitropic curvature growth of Arabidopsis hypocotyls mainly occurred in the rapid growing Elongation Zone (EZI), not in the slow-growing Elongation Zone (EZⅡ). By examining reorientation of Microtubules (MT) and phenotype of the cell wall in the EZI and the EZⅡ of Arabidopsis hypocotyls under normal gravitational condition, it is found that MTs in the rapid growing epidermal cells were mainly in the transverse direction, while those in the non-growing epidermal cells were in the longitudinal directions. However, this difference in cortical MT arrays between the EZI and EZⅡ cells disappeared when the seedlings were exposed to the simulated microgravity condition on a horizontal clinostat. Field emission scanning electron microscopy revealed that the surface texture of epidermal cells, like the direction of the MT, in the EZI and the EZⅡ also became similar when exposed to the simulated microgravity condition. This result indicated that simulate microgravity could modify the potential differentiation between the EZI and the EZⅡ by affecting the orientation of cortical MT in the epidermal cells.      

Thin film bioreactors in space.

Studies from the Skylab, SL-3 and D-1 missions have demonstrated that biological organisms grown in microgravity have changes in basic cellular functions such as DNA, mRNA and protein synthesis, cytoskeleton synthesis, glucose utilization and cellular differentiation. Since microgravity could affect prokaryotic and eukaryotic cells at a subcellular and molecular level, space offers us an opportunity to learn more about basic biological systems with one important variable removed. The thin film bioreactor will facilitate the handling of fluids in microgravity, under constant temperature and will allow multiple samples of cells to be grown with variable conditions. Studies on cell cultures grown in microgravity would enable us to identify and quantify changes in basic biological function in microgravity which are needed to develop new applications of orbital research and future biotechnology.     

Structural and functional organisation of regenerated plant protoplasts exposed to microgravity on Biokosmos 9.

Preparatory experiments for the IML-1 mission using plant protoplasts, were flown on a 14-day flight on Biokosmos 9 in September 1989. Thirty-six hours before launch of the biosatellite, protoplasts were isolated from hypocotyl cells of rapeseed (Brassica napus) and suspension cultures of carrot (Daucus carota). Ultrastructural and fluorescence analysis of cell aggregates from these protoplasts, cultured under microgravity conditions, have been performed. In the flight samples as well as in the ground controls, a portion of the total number of protoplasts regenerated cell walls. The processes of cell differentiation and proliferation under micro-g did not differ significantly from those under normal gravity conditions. However, in micro-g differences were observed in the ultrastructure of some organelles such as plastids and mitochondria. There was also an increase in the frequency of the occurrence of folds formed by the plasmalemma together with an increase in the degree of complexity of these folds. In cell cultures developed under micro-g conditions, the calcium content tends to decrease, compared to the ground control. Different aspects of using isolated protoplasts for clarifying the mechanisms of biological effects of microgravity are discussed.     

Simulated microgravity inhibits the proliferation of K562 erythroleukemia cells but does not result in apoptosis

Astronauts and experimental animals in space develop the anemia of space flight, but the underlying mechanisms are still unclear. In this study, the impact of simulated microgravity on proliferation, cell death, cell cycle progress and cytoskeleton of erythroid progenitor-like K562 leukemia cells was observed. K562 cells were cultured in NASA Rotary Cell Culture System (RCCS) that was used to simulate microgravity (at 15 rpm). After culture for 24 h, 48 h, 72 h, and 96 h, the cell densities cultured in RCCS were only 55.5%, 54.3%, 67.2% and 66.4% of the flask-cultured control cells, respectively. The percentages of trypan blue-stained dead cells and the percentages of apoptotic cells demonstrated no difference between RCCS-cultured cells and flask-cultured cells at every time points (from 12 h to 96 h). Compared with flask-cultured cells, RCCS culture induced an accumulation of cell number at S phase concomitant with a decrease at G0/G1 and G2/M phases at 12 h. But 12 h later (from 24 h to 60 h), the distribution of cell cycle phases in RCCS-cultured cells became no difference compared to flask-cultured cells. Consistent with the changes of cell cycle distribution, the levels of intercellular cyclins in RCCS-cultured cells changed at 12 h, including a decrease in cyclin A, and the increasing in cyclin B, D1 and E, and then (from 24 h to 36 h) began to restore to control levels. After RCCS culture for 12–36 h, the microfilaments showed uneven and clustered distribution, and the microtubules were highly disorganized. These results indicated that RCCS-simulated microgravity could induce a transient inhibition of proliferation, but not result in apoptosis, which could involve in the development of space flight anemia. K562 cells could be a useful model to research the effects of microgravity on differentiation and proliferation of hematopoietic cells.     

Chondrogenesis in aggregates of embryonic limb cells grown in a rotating wall vessel.

Previous studies in this lab have shown that chondrogenesis is affected in growth plates of rats exposed to microgravity, and in micromass cultures of embryonic limb mesenchyme differentiating in space. In order to provide a three dimensional aspect not seen in the micromass system, and a tissue homogeneity not possible with explants of limb or limb elements, and to alleviate certain difficulties regarding crew time and stowage, we began culturing embryonic limb cells in Rotating Wall Vessels (RWV). First, these cells were attached to beads, and grown for up to 65 days in a type of RWV known as STLV at the Johnson Space Center. During this time, the cells and beads aggregated and the aggregates continued to increase in size, and differentiated into Alcian blue staining chondrocytes. Because our intent was to use these aggregates for implanting into bony defects in addition to their use in studies of chondrogenic regulation at 1g and microgravity, aggregates of these cells without beads were grown in the commercially available version of the STLV, and their ability to ossify when subcutaneously implanted assessed.     

Effects of spaceflight (STS-87) on tropisms and plastid positioning in protonemata of the moss Ceratodon purpureus.

Apical cells of moss protonemata represent a single-celled system that perceives and reacts to light (positive and negative phototropism) and to gravity (negative gravitropism). Phototropism completely overrides gravitropism when apical cells are laterally irradiated with relatively high red light intensities, but below a defined light intensity threshold gravitropism competes with the phototropic reaction. A 16 day-long exposure to microgravity conditions demonstrated that gravitropism is allowed when protonemata are laterally illuminated with light intensities below 140 nmol m-2s-1. Protonemata that were grown in darkness in microgravity expressed an endogenous tendency to grow in arcs so that the overall culture morphology resembled a clockwise spiral. However this phenomenon only was observed in cultures that had reached a critical age and/or size. Organelle positioning in dark-grown apical cells was significantly altered in microgravity. Gravisensing most likely involves the sedimentation of starch-filled amyloplasts in a well-defined area of the tip cell. Amyloplasts that at 1-g are sedimented were clustered at the apical part of the sedimentation zone in microgravity. Clustering observed in microgravity or during clino-rotation significantly differs from sedimentation-induced plastid aggregations after inversion of tip cells at 1-g.     

Microgravity effects on neural retina regeneration in the newt.

Data on forelimb and eye lens regeneration in urodeles under spaceflight conditions (SFC) have been obtained in our previous studies. Today, evidence is available that SFC stimulate regeneration in experimental animals rather than inhibit it. The results of control on-ground experiments with simulated microgravity suggest that the stimulatory effect of SFC is due largely to weightlessness. An original experimental model is proposed, which is convenient for comprehensively analyzing neural regeneration under SFC. The initial results described here concern regeneration of neural retina in Pleurodeles waltl newts exposed to microgravity simulated in radial clinostat. After clinorotation for seven days (until postoperation day 16), a positive effect of altered gravity on structural restoration of detached neural retina was confirmed by a number of criteria. Specifically, an increased number of Mullerian glial cells, an increased relative volume of the plexiform layers, reduced cell death, advanced redifferentiation of retinal pigment epithelium, and extended areas of neural retina reattachment were detected in experimental newts. Moreover, cell proliferation in the inner nuclear layer of neural retina increased as compared with control. Thus, low gravity appears to intensify natural cytological and molecular mechanisms of neural retina regeneration in lower vertebrates.     

Structure of cress root statocytes in microgravity (Bion-10 mission).

Experiments on primary roots of Lepidium sativum L. have been performed on board the Bion-10 satellite. The experimental set-up was extremely miniaturized and completely automatic. The results demonstrate the effectiveness of the instrumentation. The spatial orientation, growth, root cap differentiation and statocyte structure of roots grown under microgravity (MG) have been compared with control roots grown on the ground (GC) and in a 1G-reference centrifuge in space (RC). Root length and cap shape did not differ between MG and control samples. Under MG, the mean distance of the statoliths from the distal cell wall of the statocytes increased significantly, the mean distance of the mitochondria decreased and the nucleus did not change its position in comparison to both controls. The number and the shape of the amyloplasts (statoliths) were not influenced by the space flight factors, but their size as well as their relative area in the cell decreased. The number of starch grains per statolith as well as their size and shape changed under MG. In MG and RC samples the number of lipid bodies in the statocytes was higher and the relative area larger than in GC samples. The relative area occupied by vacuoles was greater in RC statocytes than in GC and MG statocytes. These results partly confirm and, in addition, extend the data from earlier experiments in space.     

Ontogeny of plants under various gravity condition.

The results of experiments performed under conditions of microgravity (MG) or under its simulation on the horizontal clinostat (HC) with the callus, seedlings of various species and embryogenic structures have revealed a definite role of gravity as an ecological factor in the processes of cytomorphogenesis, growth, and development. The transformation of differentiated somatic cells of arabidopsis seed into undifferentiated callus was not inhibited under MG, though modifications of the whole callus morphology and of mean cell and nucleus size were observed. The morphogenesis of polar structures such as root-hair bearing cells of Lactuca primary root has been shown to be modified in the course of differentiation under mass acceleration diminished below 0.1 g. Seed germination and seedling morphogenesis under MG follow their normal course, but a significant stimulation of shoot growth with no effect on primary root growth has been determined. A successful in vitro regeneration of Nicotiana tabacum plantlets from leaf cells and subsequent formation of shoots and roots on a continuously rotating HC as well as the formation of viable seeds during seed-to-seed growth of Arabidopsis plants under MG have indicated that gravity plays but a limited role in the processes of embryogenesis and organogenesis.     

模拟失重对培养心肌细胞形态和结构的影响

本实验是利用回转器模拟失重对离体培养大鼠乳鼠的心肌细胞形态和结构的影响．在光学显微镜和荧光显微镜下观察发现,细胞的形态由细长梭形变成椭圆形甚至为圆形,并且通过荧光标记后的细胞骨架的排列由纵形变成辐射状．同时在对细胞进行测量发现,细胞体积缩小近40％,细胞长短径比例减少近70％．上述结果提示模拟失重对培养心肌细胞的形态和结构有显著影响．     

Simulated microgravity induce apoptosis and down-regulation of erythropoietin receptor of UT-7/EPO cells

Hematopoietic progenitor cell proliferation can be alternated on either spaceflight or under simulated microgravity experiments on the ground; however, the underlying mechanism remains largely unknown. In the present study, we have demonstrated that exposure of human erythropoietin (EPO)-dependent leukemia cell line UT-7/EPO cells to conditions of simulated microgravity with a rotary culture instrument significantly inhibited the cellular proliferation rate. Adding higher concentrations of EPO to the culture medium failed to improve the inhibitory status. Cell apoptosis was detected by fluorescence staining of cell nuclei and a flow cytometry assay using Annexin V/PI double staining. This microgravity-induced apoptosis in UT-7/EPO cells could be blocked by a pancaspase inhibitor Z-VAD-FMK. Immunoblotting demonstrated that rotary culture resulted in a reduction of the expression of Bcl-xL, an anti-apoptotic protein, and the cleavage of caspase-3. Furthermore, rotary culture reduced surface localization and protein content, as well as the mRNA expression of erythropoietin receptor (EPOR) of UT-7/EPO. Take together, the findings indicated that simulated microgravity may induce mitochondrial related apoptosis of UT-7/EPO cell through depressing the EPO–EPOR pathway.     

大鼠后肢去负荷体位调节装置设计与实验研究

通过对大鼠尾吊模型进行改进，研制出一种新型可调节体位的大鼠后肢去负荷悬吊装置，研究模拟微重力效应下体液分布变化对大鼠骨代谢的影响.将36只SD大鼠均分为对照组（CON）、头低位后肢去负荷组（HDT）、水平位后肢去负荷组（HH）和头高位后肢去负荷组（HUT）4组，实验21天后，利用DXA检测大鼠的骨密度（BMD）.模拟微重力效应下的三组大鼠后肢均发生严重骨丢失，其中HH和HUT组后肢BMD显著大于HDT组.实验结果表明，体液分布变化可能在模拟微重力效应导致的骨丢失中起到重要作用，新型大鼠后肢去负荷悬吊装置能够调节大鼠体位（体液）进行模拟微重力效应研究.      

Exposure to altered gravity affects all stages of endochondral cartilage differentiation.

Chondrogenesis has a number of well-defined steps: (1) condensation, which involves cell aggregation, adhesion and communication; (2) activation of cartilage genes, which is accompanied by rounding up of the cells and intracellular differentiation; and (3) production and secretion of cartilage specific matrix molecules. Our studies show that each of these steps is affected by exposure to gravitational changes. Clinorotation and centrifugation affected initial aggregation and condensation. In the CELLS experiment, where cells were exposed to microgravity after some condensation occurred preflight, intracellular differentiation and matrix production were delayed relative to controls. Once cartilage has developed, in rats, further differentiation (hypertrophy, matrix production) was also affected by spaceflight and hind limb suspension. For the process of chondrogenesis to proceed as we know it, loading and other factors present at 1g are required at each step of the process. This requirement means that not only will skeletal development and bone healing, processes involving chondrogenesis, be altered by long term exposure to microgravity, but that continuous intervention will be necessary to correct any defects produced by altered gravity environments.