RNA-seq analysis in simulated microgravity unveils down-regulation of the beta-rhizobial siderophore phymabactin.

Exploiting the symbiotic interaction between crops and nitrogen-fixing bacteria is a simple and ecological method to promote plant growth in prospective extraterrestrial human outposts. In this study, we performed an RNA-seq analysis to investigate the adaptation of the legume symbiont Paraburkholderia phymatum STM815T to simulated microgravity (s0-g) at the transcriptome level. The results revealed a drastic effect on gene expression, with roughly 23% of P. phymatum genes being differentially regulated in s0-g. Among those, 951 genes were upregulated and 858 downregulated in the cells grown in s0-g compared to terrestrial gravity (1 g). Several genes involved in posttranslational modification, protein turnover or chaperones encoding were upregulated in s0-g, while those involved in translation, ribosomal structure and biosynthesis, motility or inorganic ions transport were downregulated. Specifically, the whole phm gene cluster, previously bioinformatically predicted to be involved in the production of a hypothetical malleobactin-like siderophore, phymabactin, was 20-fold downregulated in microgravity. By constructing a mutant strain (ΔphmJK) we confirmed that the phm gene cluster codes for the only siderophore secreted by P. phymatum as assessed by the complete lack of iron chelating activity of the P. phymatum ΔphmJK mutant on chrome azurol S (CAS) agar plates. These results not only provide a deeper understanding of the physiology of symbiotic organisms exposed to space-like conditions, but also increase our knowledge of iron acquisition mechanisms in rhizobia.

Effects of microgravity on neural crest stem cells.

Exposure to microgravity (µg) results in a range of systemic changes in the organism, but may also have beneficial cellular effects. In a previous study we detected increased proliferation capacity and upregulation of genes related to proliferation and survival in boundary cap neural crest stem cells (BC) after MASER14 sounding rocket flight compared to ground-based controls. However, whether these changes were due to µg or hypergravity was not clarified. In the current MASER15 experiment BCs were exposed simultaneously to µg and 1 g conditions provided by an onboard centrifuge. BCs exposed to µg displayed a markedly increased proliferation capacity compared to 1 g on board controls, and genetic analysis of BCs harvested 5 h after flight revealed an upregulation, specifically in µg-exposed BCs, of Zfp462 transcription factor, a key regulator of cell pluripotency and neuronal fate. This was associated with alterations in exosome microRNA content between µg and 1 g exposed MASER15 specimens. Since the specimens from MASER14 were obtained for analysis with 1 week's delay, we examined whether gene expression and exosome content were different compared to the current MASER15 experiments, in which specimens were harvested 5 h after flight. The overall pattern of gene expression was different and Zfp462 expression was down-regulated in MASER14 BC µg compared to directly harvested specimens (MASER15). MicroRNA exosome content was markedly altered in medium harvested with delay compared to directly collected samples. In conclusion, our analysis indicates that even short exposure to µg alters gene expression, leading to increased BC capacity for proliferation and survival, lasting for a long time after µg exposure. With delayed harvest of specimens, a situation which may occur due to special post-flight circumstances, the exosome microRNA content is modified compared to fast specimen harvest, and the direct effects from µg exposure may be partially attenuated, whereas other effects can last for a long time after return to ground conditions.

Omics Studies of Tumor Cells under Microgravity Conditions.

Cancer is defined as a group of diseases characterized by abnormal cell growth, expansion, and progression with metastasis. Various signaling pathways are involved in its development. Malignant tumors exhibit a high morbidity and mortality. Cancer research increased our knowledge about some of the underlying mechanisms, but to this day, our understanding of this disease is unclear. High throughput omics technology and bioinformatics were successful in detecting some of the unknown cancer mechanisms. However, novel groundbreaking research and ideas are necessary. A stay in orbit causes biochemical and molecular biological changes in human cancer cells which are first, and above all, due to microgravity (µg). The µg-environment provides conditions that are not reachable on Earth, which allow researchers to focus on signaling pathways controlling cell growth and metastasis. Cancer research in space already demonstrated how cancer cell-exposure to µg influenced several biological processes being involved in cancer. This novel approach has the potential to fight cancer and to develop future cancer strategies. Space research has been shown to impact biological processes in cancer cells like proliferation, apoptosis, cell survival, adhesion, migration, the cytoskeleton, the extracellular matrix, focal adhesion, and growth factors, among others. This concise review focuses on publications related to genetic, transcriptional, epigenetic, proteomic, and metabolomic studies on tumor cells exposed to real space conditions or to simulated µg using simulation devices. We discuss all omics studies investigating different tumor cell types from the brain and hematological system, sarcomas, as well as thyroid, prostate, breast, gynecologic, gastrointestinal, and lung cancers, in order to gain new and innovative ideas for understanding the basic biology of cancer.

The androgen system across the menstrual cycle: Hormonal, (epi-)genetic and psychometric alterations.

The menstrual cycle is characterized by various hormonal alterations and associations with mental and physical conditions have been postulated. Among endocrine factors, the androgen system has been a target of major interest in males and to a lesser extent in females and may influence emotion, cognition, behavior and somatic factors. Only few studies investigated alterations of these parameters throughout the menstrual cycle and there is a lack of studies exploring a link towards epigenetic and genetic regulation. This multisite longitudinal study examines behavioral parameters including affectivity, stress perception and various diary parameters of mental and physical well-being in conjunction with testosterone and LH plasma levels in 87 menstruating women. Additionally, Cysteine-Adenenine-Guanin (CAG) repeat length and methylation of the androgen receptor gene collected at four time points across two cycles comprising the menstrual, pre-ovulatory, mid-luteal and premenstrual phase were assesed. There was a significant increase of LH and testosterone plasma levels during the pre-ovulatory phase as well as a decrease of methylation of the androgen receptor at mid-luteal phase. Subjective ratings of physical condition and sexual interest peaked during the pre-ovulatory phase and the former correlated negatively with the androgen receptor gene methylation level. This longitudinal study shows alterations of the androgen system including epigenetic measurements throughout the menstrual cycle. While a link between peripheral testosterone and sexual activity and between increased physical condition and an upregulation of testosterone receptor protein expression can be assumed, the majority of parameters remained unchanged. These initial findings need validation by subsequent studies.

The Fight against Cancer by Microgravity: The Multicellular Spheroid as a Metastasis Model.

Cancer is a disease exhibiting uncontrollable cell growth and spreading to other parts of the organism. It is a heavy, worldwide burden for mankind with high morbidity and mortality. Therefore, groundbreaking research and innovations are necessary. Research in space under microgravity (µg) conditions is a novel approach with the potential to fight cancer and develop future cancer therapies. Space travel is accompanied by adverse effects on our health, and there is a need to counteract these health problems. On the cellular level, studies have shown that real (r-) and simulated (s-) µg impact survival, apoptosis, proliferation, migration, and adhesion as well as the cytoskeleton, the extracellular matrix, focal adhesion, and growth factors in cancer cells. Moreover, the µg-environment induces in vitro 3D tumor models (multicellular spheroids and organoids) with a high potential for preclinical drug targeting, cancer drug development, and studying the processes of cancer progression and metastasis on a molecular level. This review focuses on the effects of r- and s-µg on different types of cells deriving from thyroid, breast, lung, skin, and prostate cancer, as well as tumors of the gastrointestinal tract. In addition, we summarize the current knowledge of the impact of µg on cancerous stem cells. The information demonstrates that µg has become an important new technology for increasing current knowledge of cancer biology.

Retrograde Analysis of Calcium Signaling by CaMPARI2 Shows Cytosolic Calcium in Chondrocytes Is Unaffected by Parabolic Flights.

Calcium (Ca2+) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca2+ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca2+ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca2+ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight.

The Cellular Composition of Bovine Coccygeal Intervertebral Discs: A Comprehensive Single-Cell RNAseq Analysis.

Intervertebral disc (IVD) degeneration and its medical consequences is still one of the leading causes of morbidity worldwide. To support potential regenerative treatments for degenerated IVDs, we sought to deconvolute the cell composition of the nucleus pulposus (NP) and the annulus fibrosus (AF) of bovine intervertebral discs. Bovine calf tails have been extensively used in intervertebral disc research as a readily available source of NP and AF material from healthy and young IVDs. We used single-cell RNA sequencing (scRNAseq) coupled to bulk RNA sequencing (RNAseq) to unravel the cell populations in these two structures and analyze developmental changes across the rostrocaudal axis. By integrating the scRNAseq data with the bulk RNAseq data to stabilize the clustering results of our study, we identified 27 NP structure/tissue specific genes and 24 AF structure/tissue specific genes. From our scRNAseq results, we could deconvolute the heterogeneous cell populations in both the NP and the AF. In the NP, we detected a notochordal-like cell cluster and a progenitor stem cell cluster. In the AF, we detected a stem cell-like cluster, a cluster with a predominantly fibroblast-like phenotype and a potential endothelial progenitor cluster. Taken together, our results illustrate the cell phenotypic complexity of the AF and NP in the young bovine IVDs.

Molecular genetic analysis of neural stem cells after space flight and simulated microgravity on earth.

Understanding how stem cells adapt to space flight conditions is fundamental for human space missions and extraterrestrial settlement. We analyzed gene expression in boundary cap neural crest stem cells (BCs), which are attractive for regenerative medicine by their ability to promote proliferation and survival of cocultured and co-implanted cells. BCs were launched to space (space exposed cells) (SEC), onboard sounding rocket MASER 14 as free-floating neurospheres or in a bioprinted scaffold. For comparison, BCs were placed in a random positioning machine (RPM) to simulate microgravity on earth (RPM cells) or were cultured under control conditions in the laboratory. Using next-generation RNA sequencing and data post-processing, we discovered that SEC upregulated genes related to proliferation and survival, whereas RPM cells upregulated genes associated with differentiation and inflammation. Thus, (i) space flight provides unique conditions with distinctly different effects on the properties of BC compared to earth controls, and (ii) the space flight exposure induces postflight properties that reinforce the utility of BC for regenerative medicine and tissue engineering.

Mathematical Modeling and Simulation Provides Evidence for New Strategies of Ovarian Stimulation.

New approaches to ovarian stimulation protocols, such as luteal start, random start or double stimulation, allow for flexibility in ovarian stimulation at different phases of the menstrual cycle. It has been proposed that the success of these methods is based on the continuous growth of multiple cohorts ("waves") of follicles throughout the menstrual cycle which leads to the availability of ovarian follicles for ovarian controlled stimulation at several time points. Though several preliminary studies have been published, their scientific evidence has not been considered as being strong enough to integrate these results into routine clinical practice. This work aims at adding further scientific evidence about the efficiency of variable-start protocols and underpinning the theory of follicular waves by using mathematical modeling and numerical simulations. For this purpose, we have modified and coupled two previously published models, one describing the time course of hormones and one describing competitive follicular growth in a normal menstrual cycle. The coupled model is used to test ovarian stimulation protocols in silico. Simulation results show the occurrence of follicles in a wave-like manner during a normal menstrual cycle and qualitatively predict the outcome of ovarian stimulation initiated at different time points of the menstrual cycle.

Cognitive function in association with high estradiol levels resulting from fertility treatment.

The putative association between hormones and cognitive performance is controversial. While there is evidence that estradiol plays a neuroprotective role, hormone treatment has not been shown to improve cognitive performance. Current research is flawed by the evaluation of combined hormonal effects throughout the menstrual cycle or in the menopausal transition. The stimulation phase of a fertility treatment offers a unique model to study the effect of estradiol on cognitive function. This quasi-experimental observational study is based on data from 44 women receiving IVF in Zurich, Switzerland. We assessed visuospatial working memory, attention, cognitive bias, and hormone levels at the beginning and at the end of the stimulation phase of ovarian superstimulation as part of a fertility treatment. In addition to inter-individual differences, we examined intra-individual change over time (within-subject effects). The substantial increases in estradiol levels resulting from fertility treatment did not relate to any considerable change in cognitive functioning. As the tests applied represent a broad variety of cognitive functions on different levels of complexity and with various brain regions involved, we can conclude that estradiol does not show a significant short-term effect on cognitive function.

Scalable Microgravity Simulator Used for Long-Term Musculoskeletal Cells and Tissue Engineering.

We introduce a new benchtop microgravity simulator (MGS) that is scalable and easy to use. Its working principle is similar to that of random positioning machines (RPM), commonly used in research laboratories and regarded as one of the gold standards for simulating microgravity. The improvement of the MGS concerns mainly the algorithms controlling the movements of the samples and the design that, for the first time, guarantees equal treatment of all the culture flasks undergoing simulated microgravity. Qualification and validation tests of the new device were conducted with human bone marrow stem cells (bMSC) and mouse skeletal muscle myoblasts (C2C12). bMSC were cultured for 4 days on the MGS and the RPM in parallel. In the presence of osteogenic medium, an overexpression of osteogenic markers was detected in the samples from both devices. Similarly, C2C12 cells were maintained for 4 days on the MGS and the rotating wall vessel (RWV) device, another widely used microgravity simulator. Significant downregulation of myogenesis markers was observed in gravitationally unloaded cells. Therefore, similar results can be obtained regardless of the used simulated microgravity devices, namely MGS, RPM, or RWV. The newly developed MGS device thus offers easy and reliable long-term cell culture possibilities under simulated microgravity conditions. Currently, upgrades are in progress to allow real-time monitoring of the culture media and liquids exchange while running. This is of particular interest for long-term cultivation, needed for tissue engineering applications. Tissue grown under real or simulated microgravity has specific features, such as growth in three-dimensions (3D). Growth in weightlessness conditions fosters mechanical, structural, and chemical interactions between cells and the extracellular matrix in any direction.

The effects of microgravity on differentiation and cell growth in stem cells and cancer stem cells.

A spaceflight has enormous influence on the health of space voyagers due to the combined effects of microgravity and cosmic radiation. Known effects of microgravity (µg) on cells are changes in differentiation and growth. Considering the commercialization of spaceflight, future space exploration, and long-term manned flights, research focusing on differentiation and growth of stem cells and cancer cells exposed to real (r-) and simulated (s-) µg is of high interest for regenerative medicine and cancer research. In this review, we focus on platforms to study r- and s-µg as well as the impact of µg on cancer stem cells in the field of gastrointestinal cancer, lung cancer, and osteosarcoma. Moreover, we review the current knowledge of different types of stem cells exposed to µg conditions with regard to differentiation and engineering of cartilage, bone, vasculature, heart, skin, and liver constructs.

Ambient and supplemental magnetic fields promote myogenesis via a TRPC1-mitochondrial axis: evidence of a magnetic mitohormetic mechanism.

We show that both supplemental and ambient magnetic fields modulate myogenesis. A lone 10 min exposure of myoblasts to 1.5 mT amplitude supplemental pulsed magnetic fields (PEMFs) accentuated in vitro myogenesis by stimulating transient receptor potential (TRP)-C1-mediated calcium entry and downstream nuclear factor of activated T cells (NFAT)-transcriptional and P300/CBP-associated factor (PCAF)-epigenetic cascades, whereas depriving myoblasts of ambient magnetic fields slowed myogenesis, reduced TRPC1 expression, and silenced NFAT-transcriptional and PCAF-epigenetic cascades. The expression levels of peroxisome proliferator-activated receptor γ coactivator 1α, the master regulator of mitochondriogenesis, was also enhanced by brief PEMF exposure. Accordingly, mitochondriogenesis and respiratory capacity were both enhanced with PEMF exposure, paralleling TRPC1 expression and pharmacological sensitivity. Clustered regularly interspaced short palindromic repeats-Cas9 knockdown of TRPC1 precluded proliferative and mitochondrial responses to supplemental PEMFs, whereas small interfering RNA gene silencing of TRPM7 did not, coinciding with data that magnetoreception did not coincide with the expression or function of other TRP channels. The aminoglycoside antibiotics antagonized and down-regulated TRPC1 expression and, when applied concomitantly with PEMF exposure, attenuated PEMF-stimulated calcium entry, mitochondrial respiration, proliferation, differentiation, and epigenetic directive in myoblasts, elucidating why the developmental potential of magnetic fields may have previously escaped detection. Mitochondrial-based survival adaptations were also activated upon PEMF stimulation. Magnetism thus deploys an authentic myogenic directive that relies on an interplay between mitochondria and TRPC1 to reach fruition.-Yap, J. L. Y., Tai, Y. K., Fröhlich, J., Fong, C. H. H., Yin, J. N., Foo, Z. L., Ramanan, S., Beyer, C., Toh, S. J., Casarosa, M., Bharathy, N., Kala, M. P., Egli, M., Taneja, R., Lee, C. N., Franco-Obregón, A. Ambient and supplemental magnetic fields promote myogenesis via a TRPC1-mitochondrial axis: evidence of a magnetic mitohormetic mechanism.

Associations Between Natural Physiological and Supraphysiological Estradiol Levels and Stress Perception.

Stress is a risk factor for impaired general, mental, and reproductive health. The role of physiological and supraphysiological estradiol concentrations in stress perception and stress processing is less well understood. We, therefore, conducted a prospective observational study to investigate the association between estradiol, stress perception, and stress-related cognitive performance within serial measurements either during the natural menstrual cycle or during fertility treatment, where estradiol levels are strongly above the physiological level of a natural cycle, and consequently, represent a good model to study dose-dependent effects of estradiol. Data from 44 women receiving in vitro fertilization (IVF) at the Department of Reproductive Endocrinology in Zurich, Switzerland was compared to data from 88 women with measurements during their natural menstrual cycle. The German version of the Perceived Stress Questionnaire (PSQ) and the Cognitive Bias Test (CBT), in which cognitive performance is tested under time stress were used to evaluate subjective and functional aspects of stress. Estradiol levels were investigated at four different time points during the menstrual cycle and at two different time points during a fertility treatment. Cycle phases were associated with PSQ worry and cognitive bias in normally cycling women, but different phases of fertility treatment were not associated with subjectively perceived stress and stress-related cognitive bias. PSQ lack of joy and PSQ demands related to CBT in women receiving fertility treatment but not in women with a normal menstrual cycle. Only strong changes of the estradiol level during fertility treatment were weakly associated with CBT, but not with subjectively experienced stress. Our research emphasizes the multidimensional character of stress and the necessity to adjust stress research to the complex nature of stress perception and processing. Infertility is associated with an increased psychological burden in patients. However, not all phases of the process to overcome infertility do significantly increase patient stress levels. Also, research on the psychological burden of infertility should consider that stress may vary during the different phases of fertility treatment. Clinical trial registration: ClinicalTrials.gov # NCT02098668.

Growth of Endothelial Cells in Space and in Simulated Microgravity - a Comparison on the Secretory Level.

BACKGROUND/AIMS: Endothelial cells exposed to the Random Positioning Machine (RPM) reveal three different phenotypes. They grow as a two-dimensional monolayer and form three-dimensional (3D) structures such as spheroids and tubular constructs. As part of the ESA-SPHEROIDS project we want to understand how endothelial cells (ECs) react and adapt to long-term microgravity. METHODS: During a spaceflight to the International Space Station (ISS) and a subsequent stay onboard, human ECs (EA.hy926 cell line) were cultured for 12 days in real microgravity inside an automatic flight hardware, specially designed for use in space. ECs were cultivated in the absence or presence of vascular endothelial growth factor, which had demonstrated a cell-protective effect on ECs exposed to an RPM simulating microgravity. After cell fixation in space and return of the samples, we examined cell morphology and analyzed supernatants by Multianalyte Profiling technology. RESULTS: The fixed samples comprised 3D multicellular spheroids and tube-like structures in addition to monolayer cells, which are exclusively observed during growth under Earth gravity (1g). Within the 3D aggregates we detected enhanced collagen and laminin. The supernatant analysis unveiled alterations in secretion of several growth factors, cytokines, and extracellular matrix components as compared to cells cultivated at 1g or on the RPM. This confirmed an influence of gravity on interacting key proteins and genes and demonstrated a flight hardware impact on the endothelial secretome. CONCLUSION: Since formation of tube-like aggregates was observed only on the RPM and during spaceflight, we conclude that microgravity may be the major cause for ECs' 3D aggregation.

A Simple Method to Determine Cytotoxicity of Water-Soluble Organic Compounds and Solid Particles from Biomass Combustion in Lung Cells in Vitro.

Adverse health effects of condensable organic compounds (COC) and potential secondary organic aerosols from wood combustion emissions are difficult to determine. Hence, available information is usually limited to a small number of specific applications. Therefore, we introduced a simple, fast, and economic method where water-soluble COC (WSCOC) and WSCOC together with water-soluble primary solid particles (WSpSP) from wood combustion were sampled and subsequently exposed to cultured human lung cells. Comparing the cell viability of H187 human epithelial lung cells from five combustion devices, operated at different combustion conditions, no, or only a minor, cytotoxicity of WSCOC is found for stationary conditions in a grate boiler, a log wood boiler, and a pellet boiler. All combustion conditions in a log wood stove and unfavorable conditions in the other devices induce, however, significant cytotoxicity (median lethal concentration LC50 5-17 mg/L). Furthermore, a significant correlation between CO and cytotoxicity was found ( R2 ∼ 0.8) suggesting that the simply measurable gas phase compound CO can be used as a first indicator for the potential harmfulness of wood combustion emissions. Samples containing WSCOC plus WSpSP show no additional cytotoxicity compared to samples with COC only, indicating that WSCOC exhibit much higher cytotoxicity than WSpSP.

Simulated microgravity and the antagonistic influence of strigolactone on plant nutrient uptake in low nutrient conditions.

Human-assisted space exploration will require efficient methods of food production. Large-scale farming in presence of an Earth-like atmosphere in space faces two main challenges: plant yield in microgravity and plant nutrition in extraterrestrial soils, which are likely low in nutrients compared to terrestrial farm lands. We propose a plant-fungal symbiosis (i.e. mycorrhiza) as an efficient tool to increase plant biomass production in extraterrestrial environments. We tested the mycorrhization of Solanaceae on the model plant Petunia hybrida using the arbuscular mycorrhizal fungus Rhizophagus irregularis under simulated microgravity (s0-g) conditions obtained through a 3-D random positioning machine. Our results show that s0-g negatively affects mycorrhization and plant phosphate uptake by inhibiting hyphal elongation and secondary branching. However, in low nutrient conditions, the mycorrhiza can still support plant biomass production in s0-g when colonized plants have increased SL root exudation. Alternatively, s0-g in high nutrient conditions boosts tissue-specific cell division and cell expansion and overall plant size in Petunia, which has been reported for other plants species. Finally, we show that the SL mimic molecule rac-GR24 can still induce hyphal branching in vitro under simulated microgravity. Based on these results, we propose that in nutrient limited conditions strigolactone root exudation can challenge the negative microgravity effects on mycorrhization and therefore might play an important role in increasing the efficiency of future space farming.

TRPC6 in simulated microgravity of intervertebral disc cells.

PURPOSE: Prolonged bed rest and microgravity in space cause intervertebral disc (IVD) degeneration. However, the underlying molecular mechanisms are not completely understood. Transient receptor potential canonical (TRPC) channels are implicated in mechanosensing of several tissues, but are poorly explored in IVDs. METHODS: Primary human IVD cells from surgical biopsies composed of both annulus fibrosus and nucleus pulposus (passage 1-2) were exposed to simulated microgravity and to the TRPC channel inhibitor SKF-96365 (SKF) for up to 5 days. Proliferative capacity, cell cycle distribution, senescence and TRPC channel expression were analyzed. RESULTS: Both simulated microgravity and TRPC channel antagonism reduced the proliferative capacity of IVD cells and induced senescence. While significant changes in cell cycle distributions (reduction in G1 and accumulation in G2/M) were observed upon SKF treatment, the effect was small upon 3 days of simulated microgravity. Finally, downregulation of TRPC6 was shown under simulated microgravity. CONCLUSIONS: Simulated microgravity and TRPC channel inhibition both led to reduced proliferation and increased senescence. Furthermore, simulated microgravity reduced TRPC6 expression. IVD cell senescence and mechanotransduction may hence potentially be regulated by TRPC6 expression. This study thus reveals promising targets for future studies. These slides can be retrieved under Electronic Supplementary Material.

Influence of Mechanical Unloading on Articular Chondrocyte Dedifferentiation.

Due to the limited self-repair capacity of articular cartilage, the surgical restoration of defective cartilage remains a major clinical challenge. The cell-based approach, which is known as autologous chondrocyte transplantation (ACT), has limited success, presumably because the chondrocytes acquire a fibroblast-like phenotype in monolayer culture. This unwanted dedifferentiation process is typically addressed by using three-dimensional scaffolds, pellet culture, and/or the application of exogenous factors. Alternative mechanical unloading approaches are suggested to be beneficial in preserving the chondrocyte phenotype. In this study, we examined if the random positioning machine (RPM) could be used to expand chondrocytes in vitro such that they maintain their phenotype. Bovine chondrocytes were exposed to (a) eight days in static monolayer culture; (b) two days in static monolayer culture, followed by six days of RPM exposure; and, (c) eight days of RPM exposure. Furthermore, the experiment was also conducted with the application of 20 mM gadolinium, which is a nonspecific ion-channel blocker. The results revealed that the chondrocyte phenotype is preserved when chondrocytes go into suspension and aggregate to cell clusters. Exposure to RPM rotation alone does not preserve the chondrocyte phenotype. Interestingly, the gene expression (mRNA) of the mechanosensitive ion channel TRPV4 decreased with progressing dedifferentiation. In contrast, the gene expression (mRNA) of the mechanosensitive ion channel TRPC1 was reduced around fivefold to 10-fold in all of the conditions. The application of gadolinium had only a minor influence on the results. This and previous studies suggest that the chondrocyte phenotype is preserved if cells maintain a round morphology and that the ion channel TRPV4 could play a key role in the dedifferentiation process.

Electrophysiological experiments in microgravity: lessons learned and future challenges.

Advances in electrophysiological experiments have led to the discovery of mechanosensitive ion channels (MSCs) and the identification of the physiological function of specific MSCs. They are believed to play important roles in mechanosensitive pathways by allowing for cells to sense their mechanical environment. However, the physiological function of many MSCs has not been conclusively identified. Therefore, experiments have been developed that expose cells to various mechanical loads, such as shear flow, membrane indentation, osmotic challenges and hydrostatic pressure. In line with these experiments, mechanical unloading, as experienced in microgravity, represents an interesting alternative condition, since exposure to microgravity leads to a series of physiological adaption processes. As outlined in this review, electrophysiological experiments performed in microgravity have shown an influence of gravity on biological functions depending on ion channels at all hierarchical levels, from the cellular level to organs. In this context, calcium signaling represents an interesting cellular pathway, as it involves the direct action of calcium-permeable ion channels, and specific gravitatic cells have linked graviperception to this pathway. Multiple key proteins in the graviperception pathways have been identified. However, measurements on vertebrae cells have revealed controversial results. In conclusion, electrophysiological experiments in microgravity have shown that ion-channel-dependent physiological processes are altered in mechanically unloaded conditions. Future experiments may provide a better understanding of the underlying mechanisms.