Spaceflight-induced contractile and mitochondrial dysfunction in an automated heart-on-a-chip platform.

With current plans for manned missions to Mars and beyond, the need to better understand, prevent, and counteract the harmful effects of long-duration spaceflight on the body is becoming increasingly important. In this study, an automated heart-on-a-chip platform was flown to the International Space Station on a 1-mo mission during which contractile cardiac function was monitored in real-time. Upon return to Earth, engineered human heart tissues (EHTs) were further analyzed with ultrastructural imaging and RNA sequencing to investigate the impact of prolonged microgravity on cardiomyocyte function and health. Spaceflight EHTs exhibited significantly reduced twitch forces, increased incidences of arrhythmias, and increased signs of sarcomere disruption and mitochondrial damage. Transcriptomic analyses showed an up-regulation of genes and pathways associated with metabolic disorders, heart failure, oxidative stress, and inflammation, while genes related to contractility and calcium signaling showed significant down-regulation. Finally, in silico modeling revealed a potential link between oxidative stress and mitochondrial dysfunction that corresponded with RNA sequencing results. This represents an in vitro model to faithfully reproduce the adverse effects of spaceflight on three-dimensional (3D)-engineered heart tissue.

Spiders in space-orb-web-related behaviour in zero gravity.

Gravity is very important for many organisms, including web-building spiders. Probably the best approach to study the relevance of gravity on organisms is to bring them to the International Space Station. Here, we describe the results of such an experiment where two juvenile Trichonephila clavipes (L.) (Araneae, Nephilidae) spiders were observed over a 2-month period in zero gravity and two control spiders under otherwise identical conditions on Earth. During that time, the spiders and their webs were photographed every 5 min. Under natural conditions, Trichonephila spiders build asymmetric webs with the hub near the upper edge of the web, and they always orient themselves downwards when sitting on the hub whilst waiting for prey. As these asymmetries are considered to be linked to gravity, we expected the spiders experiencing no gravity to build symmetric webs and to show a random orientation when sitting on the hub. We found that most, but not all, webs built in zero gravity were indeed quite symmetric. Closer analysis revealed that webs built when the lights were on were more asymmetric (with the hub near the lights) than webs built when the lights were off. In addition, spiders showed a random orientation when the lights were off but faced away from the lights when they were on. We conclude that in the absence of gravity, the direction of light can serve as an orientation guide for spiders during web building and when waiting for prey on the hub.

International Space Station conditions alter genomics, proteomics, and metabolomics in Aspergillus nidulans.

The first global genomic, proteomic, and secondary metabolomic characterization of the filamentous fungus Aspergillus nidulans following growth onboard the International Space Station (ISS) is reported. The investigation included the A. nidulans wild-type and three mutant strains, two of which were genetically engineered to enhance secondary metabolite production. Whole genome sequencing revealed that ISS conditions altered the A. nidulans genome in specific regions. In strain CW12001, which features overexpression of the secondary metabolite global regulator laeA, ISS conditions induced the loss of the laeA stop codon. Differential expression of proteins involved in stress response, carbohydrate metabolic processes, and secondary metabolite biosynthesis was also observed. ISS conditions significantly decreased prenyl xanthone production in the wild-type strain and increased asperthecin production in LO1362 and CW12001, which are deficient in a major DNA repair mechanism. These data provide valuable insights into the adaptation mechanism of A. nidulans to spacecraft environments.

Development of a kidney microphysiological system hardware platform for microgravity studies.

Determining the physiological effects of microgravity on the human kidney is limited to relatively insensitive tests of biofluids (blood and urine) that do not return abnormal results until more than 50% of kidney function is lost. We have developed an "organ on chip" microphysiological model of the human kidney proximal tubule (PT-MPS) that can recapitulate many kidney functions and disease states and could play a critical role in determining mechanisms of early kidney dysfunction in microgravity. However, the ground-based PT-MPS system is incompatible with spaceflight as it requires a large pneumatic system coupled to a cell incubator for perfusion and intensive hand-on manipulation. Herein, we report the hardware engineering and performance of the Kidney Chip Perfusion Platform (KCPP), a small, advanced, semi-autonomous hardware platform to support kidney microphysiological model experiments in microgravity. The KCPP is composed of five components, the kidney MPS, the MPS housing and valve block, media cassettes, fixative cassettes, and the programable precision syringe pump. The system has been deployed twice to the ISSNL (aboard CRS-17 and CRS-22). From each set of ISSNL experiments and ground-based controls, we were able to recover PT-MPS effluent for biomarker analysis and RNA suitable for transcriptomics analysis demonstrating the usability and functionality of the KCPP.

Modeling cellular responses to serum and vitamin D in microgravity using a human kidney microphysiological system.

The microgravity environment aboard the International Space Station (ISS) provides a unique stressor that can help understand underlying cellular and molecular drivers of pathological changes observed in astronauts with the ultimate goals of developing strategies to enable long- term spaceflight and better treatment of diseases on Earth. We used this unique environment to evaluate the effects of microgravity on kidney proximal tubule epithelial cell (PTEC) response to serum exposure and vitamin D biotransformation capacity. To test if microgravity alters the pathologic response of the proximal tubule to serum exposure, we treated PTECs cultured in a microphysiological system (PT-MPS) with human serum and measured biomarkers of toxicity and inflammation (KIM-1 and IL-6) and conducted global transcriptomics via RNAseq on cells undergoing flight (microgravity) and respective controls (ground). Given the profound bone loss observed in microgravity and PTECs produce the active form of vitamin D, we treated 3D cultured PTECs with 25(OH)D3 (vitamin D) and monitored vitamin D metabolite formation, conducted global transcriptomics via RNAseq, and evaluated transcript expression of CYP27B1, CYP24A1, or CYP3A5 in PTECs undergoing flight (microgravity) and respective ground controls. We demonstrated that microgravity neither altered PTEC metabolism of vitamin D nor did it induce a unique response of PTECs to human serum, suggesting that these fundamental biochemical pathways in the kidney proximal tubule are not significantly altered by short-term exposure to microgravity. Given the prospect of extended spaceflight, more study is needed to determine if these responses are consistent with extended (>6 months) exposure to microgravity.

Surface tension enables induced pluripotent stem cell culture in commercially available hardware during spaceflight.

Low Earth Orbit (LEO) has emerged as a unique environment for evaluating altered stem cell properties in microgravity. LEO has become increasingly accessible for research and development due to progress in private spaceflight. Axiom Mission 2 (Ax-2) was launched as the second all-private astronaut mission to the International Space Station (ISS). Frozen human induced pluripotent stem cells (hiPSCs) expressing green fluorescent protein (GFP) under the SOX2 promoter, as well as fibroblasts differentiated from SOX2-GFP hiPSCs, were sent to the ISS. Astronauts then thawed and seeded both cell types into commercially available 96-well plates, which provided surface tension that reduced fluid movement out of individual wells and showed that hiPSCs or hiPSC-derived fibroblasts could survive either in suspension or attached to a Matrigel substrate. Furthermore, both cell types could be transfected with red fluorescent protein (RFP)-expressing plasmid. We demonstrate that hiPSCs and hiPSC-fibroblasts can be thawed in microgravity in off-the-shelf, commercially-available cell culture hardware, can associate into 3D spheroids or grow adherently in Matrigel, and can be transfected with DNA. This lays the groundwork for future biomanufacturing experiments in space.

CRISPR-based genetic diagnostics in microgravity.

Monitoring astronauts' health during space missions poses many challenges, including rapid assessment of crew health conditions. Sensitive genetic diagnostics are crucial for examining crew members and the spacecraft environment. CRISPR-Cas12a, coupled with isothermal amplification, has proven to be a promising biosensing system for rapid, on-site detection of genomic targets. However, the efficiency and sensitivity of CRISPR-based diagnostics have never been tested in microgravity. We tested the use of recombinase polymerase amplification (RPA) coupled with the collateral cleavage activity of Cas12a for genetic diagnostics onboard the International Space Station. We explored the detection sensitivity of amplified and unamplified target DNA. By coupling RPA with Cas12a, we identified targets in attomolar concentrations. We further assessed the reactions' stability following long-term storage. Our results demonstrate that CRISPR-based detection is a powerful tool for on-site genetic diagnostics in microgravity, and can be further utilized for long-term space endeavors to improve astronauts' health and well-being.

Development of a Kidney Microphysiological System Hardware Platform for Microgravity Studies.

Study of the physiological effects of microgravity on humans is limited to non-invasive testing of astronauts. Microphysiological models of human organs recapitulate many functions and disease states. Here we describe the development of an advanced, semi-autonomous hardware platform to support kidney microphysiological model experiments in microgravity.

Impact of microgravity on a three-dimensional microphysiologic culture of the human kidney proximal tubule epithelium: cell response to serum and vitamin D.

The microgravity environment aboard the International Space Station (ISS) provides a unique stressor that can help understand underlying cellular and molecular drivers of pathological changes observed in astronauts with the ultimate goals of developing strategies to enable long-term spaceflight and better treatment of diseases on Earth. We used this unique environment to evaluate the effects of microgravity on kidney proximal tubule epithelial cell (PTEC) response to serum exposure and vitamin D biotransformation capacity. To test if microgravity alters the pathologic response of the proximal tubule to serum exposure, we treated PTECs cultured in a microphysiological system (PT-MPS) with human serum and measured biomarkers of toxicity and inflammation (KIM-1 and IL-6) and conducted global transcriptomics via RNAseq on cells undergoing flight (microgravity) and respective controls (ground). We also treated 3D cultured PTECs with 25(OH)D3 (vitamin D) and monitored vitamin D metabolite formation, conducted global transcriptomics via RNAseq, and evaluated transcript expression of CYP27B1, CYP24A1, or CYP3A5 in PTECs undergoing flight (microgravity) and respective ground controls. We demonstrated that microgravity neither altered PTEC metabolism of vitamin D nor did it induce a unique response of PTECs to human serum, suggesting that these fundamental biochemical pathways in the kidney proximal tubule are not significantly altered by short-term exposure to microgravity. Given the prospect of extended spaceflight, more study is needed to determine if these responses are consistent with extended (> 6 month) exposure to microgravity.

Feasibility, potency, and safety of growing human mesenchymal stem cells in space for clinical application.

Growing stem cells on Earth is very challenging and limited to a few population doublings. The standard two-dimensional (2D) culture environment is an unnatural condition for cell growth. Therefore, culturing stem cells aboard the International Space Station (ISS) under a microgravity environment may provide a more natural three-dimensional environment for stem cell expansion and organ development. In this study, human-derived mesenchymal stem cells (MSCs) grown in space were evaluated to determine their potential use for future clinical applications on Earth and during long-term spaceflight. MSCs were flown in Plate Habitats for transportation to the ISS. The MSCs were imaged every 24-48 h and harvested at 7 and 14 days. Conditioned media samples were frozen at -80 °C and cells were either cryopreserved in 5% dimethyl sulfoxide, RNAprotect, or paraformaldehyde. After return to Earth, MSCs were characterized to establish their identity and cell cycle status. In addition, cell proliferation, differentiation, cytokines, and growth factors' secretion were assessed. To evaluate the risk of malignant transformation, the space-grown MSCs were subjected to chromosomal, DNA damage, and tumorigenicity assays. We found that microgravity had significant impact on the MSC capacity to secrete cytokines and growth factors. They appeared to be more potent in terms of immunosuppressive capacity compared to their identical ground control. Chromosomal, DNA damage, and tumorigenicity assays showed no evidence of malignant transformation. Therefore, it is feasible and potentially safe to grow MSCs aboard the ISS for potential future clinical applications.

Effects of Spaceflight on Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Structure and Function.

With extended stays aboard the International Space Station (ISS) becoming commonplace, there is a need to better understand the effects of microgravity on cardiac function. We utilized human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study the effects of microgravity on cell-level cardiac function and gene expression. The hiPSC-CMs were cultured aboard the ISS for 5.5 weeks and their gene expression, structure, and functions were compared with ground control hiPSC-CMs. Exposure to microgravity on the ISS caused alterations in hiPSC-CM calcium handling. RNA-sequencing analysis demonstrated that 2,635 genes were differentially expressed among flight, post-flight, and ground control samples, including genes involved in mitochondrial metabolism. This study represents the first use of hiPSC technology to model the effects of spaceflight on human cardiomyocyte structure and function.

Effects of intensive voice treatment (the Lee Silverman Voice Treatment [LSVT]) on ataxic dysarthria: a case study.

This study examined the effects of intensive voice treatment (the Lee Silverman Voice Treatment [LSVT]) on ataxic dysarthria in a woman with cerebellar dysfunction secondary to thiamine deficiency. Perceptual and acoustic measures were made on speech samples recorded just before the LSVT program was administered, immediately after it was administered, and at 9 months follow-up. Results indicate short- and long-term improvement in phonatory and articulatory functions, speech intelligibility, and overall communication and job-related activity following LSVT. This study's findings provide initial support for the application of LSVT to the treatment of speech disorders accompanying ataxic dysarthria. Potential neural mechanisms that may underlie the effects of loud phonation and LSVT are addressed.

Speech loudness and quality 12 months after intensive voice treatment (LSVT) for Parkinson's disease: a comparison with an alternative speech treatment.

Thirty-five individuals with idiopathic Parkinson's disease were enrolled in speech treatment. Twenty-two were enrolled in a high-effort phonatory-respiratory treatment program (Lee Silverman Voice Treatment, LSVT) and 13 were enrolled in a high-effort respiratory treatment program (RET). Perceptual judgments of speech loudness and quality were made independently by 6 listeners on recordings of the 'Rainbow Passage'. These recordings had been obtained just before treatment (pre) and at 12 months' follow-up (FU12). The speech samples in the LSVT group, but not in the RET group, were significantly more likely to be perceived 'louder' and 'better quality' at FU12 than at pre (p < 0.0001). These findings, along with others, suggest that the long-term effects of the LSVT are perceptible, clinically significant and treatment-specific.