Endothelial Cells - Embryonic stem cells - Adult stem cells - Osteogenic differentiation - Adipogenic differentiation - Chondrogenic differentiation - Targeting bone loss
Endothelial CellsFor plastic surgery purposes, small vessels or pieces of tissues are often needed. However on Earth, endothelial cells grow in monolayers and they do not fully exhibit the vascular characteristics that are seen in vivo. Tissue engineering approaches such as culturing endothelial cells with other cells and using biodegradable matrices are more efficient for vascularization; however choosing the biocompatible and biodegradable material can be problematic3. A potential solution to this problem could be the tissues or vessels that are engineered under weightlessness at low shear stress since they are formed without requiring any artificial matrix or scaffolds3. The advantage of this would be not introducing any foreign materials to the human body.
In microgravity, endothelial cells grew into spheroids and formed 3D tubular structures after 72 hours4. Moreover, endothelial cells experienced apoptosis and gene expression alterations especially in terms of cytoskeletal rearrangements. The research performed by Grosse et al with endothelial cells in a parabolic flight indicated that tubulin expression was downregulated and tubulin structures were rearranged5. Furthermore, alteration of expression of 3605 genes occurred in endothelial cells after parabolic flight5. Likewise after 24h of clinorotation, tube formation and migration and endothelial nitric oxide synthase were significantly increased6.
Vascularization studies performed in microgravity showed that vascular endothelial growth factor (VEGF) supplement would be promising. Aleshcheva and colleagues cultured CD34-positive stem cells, which are involved in vascularization, in microgravity and supplemented them with vascular endothelial growth factor. As a result, the cells showed increased growth and differentiation into a vascular endothelial phenotype after 4 days as indicated by gene expression differences7. Another research showed that co-culture of endothelial cells with vascular smooth muscle cells and fibroblasts for 21 days was also a promising candidate for engineering a complete, functional vessel3.
Embryonic stem cellsMicrogravity also alters the differentiation and proliferation processes of embryonic stem cells and adult stem cells. Since these cells have the ability to differentiate into a number of lineage-specific terminally differentiated cells, altered stem cell function affects cell lineages throughout the body8. Previously, mouse embryonic stem cells were also cultured in microgravity to test their differentiation by a study performed by Shinde et al. It is found that after 3 days in simulated microgravity and 7 days in 1g, cells were differentiated into different somatic cell types. However, microgravity affected several genes that belong to MAP kinase and focal adhesion signal transduction pathways9. As a result, cardiomyogenesis was found to be one of the most prominent biological processes that is affected by simulated microgravity as illustrated by inhibition of the expression of cardiomyocyte specific genes after 3 days in microgravity and reduced heart beating activity of the embryoid bodies9.
Moreover, culturing stem cells in microgravity generated bigger cellular spheres and prolong proliferative capacity further compared to Earth controls as illustrated by the research performed by Kawahara et al. Based on their findings, in microgravity mouse embryonic stem cell cultures formed bigger spheres and held a longer process of undifferentiation like 7 days compared to 1g controls in feader-free and serum-free media without LIF 10. Moreover, the pluripotency of the culture was illustrated by the fact that when the cells were injected in mice, they formed teratomas10.
Adult stem cellsThere have been several researches in different fields in microgravity regarding adult stem cells for tissue engineering purposes. The study performed by Yuge et al showed that, when human mesenchymal stem cells cultured in microgravity were implanted in cartilage-defective mice, they formed hyaline cartilage after 7 days whereas the control 1g groups formed mostly non-cartilage tissue after implantation11. Thus, this might suggest that microgravity cultures have strong proliferative characteristics and can differentiate into destined tissue more efficiently. Another research group from China, studied the effects of microgravity on adipose-derived stem cell spheroids. According to the study, ADSCs spontaneously formed three-dimensional spheroids and the expression levels of E-cadherin and pluripotent markers were significantly upregulated in microgravity12. Moreover, when administered to the mice with carbon tetrachloride-induced acute liver failure, spheroid-derived ADSCs showed more effective potentials to rescue liver failure compared to control 1g culture12. Finally, a very recent research performed by Xu lab, seems to produce highly enriched cardiomyocytes through culturing the cardiac progenitors in microgravity13. The cardiomyocytes cultured in microgravity may potentially be more desirable for regenerative medicine compared to 1g control based on their higher viability and proliferation as well as survival at the early stage of differentiation. These could increase the cardiomyocyte survival and improve graft survival13.
Recently, it has been argued that the effect that microgravity exerts on stem cells can be age-dependent. The research performed by Fuentes et al using cardiovascular progenitors indicated that, progenitors from neonatal and adult heart demonstrated different responses in cell differentiation in microgravity14. Adult cardiovascular progenitors showed an increased expression of MLC2v and Troponin T, indicating that endothelial cell differentiation is favored under microgravity14. On the other hand, neonatal progenitors did not form tubes effectively, showed increased stemness and cell survival under microgravity13. Moreover, high levels of telomerase reverse transcriptase and DNA repair proteins were found in neonatal progenitors14. Based on the results, age-dependent differences can be advantageous and can provide greater regenerative capacity for tissue engineering purposes.
Osteogenic differentiationIn microgravity, osteoblast differentiation and the overexpression of some osteogenic transcripts are induced only when the osteogenic cocktail is added to the culture15. For example; a research indicated that osteoblast differentiation from dental stem cells is enhanced in 3D rotary cell culture systems mimicking microgravity. This is shown by increased expression of osteogenic genes such as Chfa1, collagen I, osterix and BSPII16. Another research performed by Hwang et al showed that encapsulation of undifferentiated mESCs within alginate hydrogels and culture in a rotary cell culture simulating microgravity is efficient to differentiate them into osteogenic lineage as the cells were tested for morphological, phenotypical and molecular properties17. Likewise, Koç et al studied in vitro osteogenic differentiation of rat mesenchymal stem cells in a microgravity bioreactor and the findings seem to illustrate an efficient differentiation as supported by mineralization, morphology and gene expression testing18.
A recent research showed that endothelial cell-osteoblast crosstalk is also important for osteogenic differentiation in simulated microgravity. According to the study, conditioned media from endothelial cells, which are subjected to microgravity, enhanced the expression of mechano-responsive molecules such as interleukin-1b, lipocalin 2 and nitric oxide synthase 2 in osteoblasts19. As a result, osteogenesis becomes insufficient because of the fact that the proliferation of osteoblasts increases and their differentiation is impaired19. This study indicates in addition to mechanical forces, endothelial cells also contribute to remodeling of the bone.
Adipogenic differentiationEven though efficient osteoblast differentiation occurs in microgravity cultures, previous research indicated that the preferred lineage of human mesenchymal stem cells is adipocytes. This is clearer, when the osteogenic cocktail support is not available such as the case of astronauts in microgravity. A study by Zayzafoon et al, indicated that hMSC differentiation into osteoblasts was suppressed as indicated by no expression of ALP, collagen I and osteonectin and instead adipocyte differentiation was promoted20. Likewise, a study performed by Gershovich et al, identified that exposure of human mesenchymal cell cultures to simulated microgravity for 20 days resulted in significant changes in the expression of 144 genes which take part in inflammatory responses, intercellular interactions, matrix and adhesion, metabolic processes and signaling and regulation21. Expression of genes that have a key role in osteogenic differentiation such as COL15A1, CXCL12, DPT and WISP2 were also inhibited21.
Mesenchymal stem cell differentiation in microgravity was also studied in spaceflights. A very recent research investigating the effects of a 14-day spaceflight on bone in rats, showed that compared with ground controls, rats flown in space had 32% lower cancellous bone area and 30% higher marrow adipose tissue (MAT), which was due to an increase in adipocyte number and size22. Mineral apposition rate and osteoblast turnover were unchanged during flight. Cancellous bone loss in rat lumbar vertebrae during spaceflight was determined by increased bone resorption and MAT but there were no change in bone formation capacity22. The results indicate that, during spaceflight mesenchymal stem cells are diverted to adipocytes with the loss of osteoblasts.
Chondrogenic differentiationSimulated microgravity reduces chondrogenic potential of hMSCs via decreased expressions of COLXA1 and COL2A123. However application of low frequency electromagnetic fields seem to increase the COL2A1 expression even though the results were not significantly superior than control groups. Another research performed by Cerwinka et al, examined the differentiation of human mesenchymal stem cells in gelatin scaffolds. Spheroids cultured in adipogenic and osteogenic differentiation media showed differential potential detected by calcification and gene expression pattern. However, chondrogenic differentiation was not successful24.
There have been some studies in microgravity simulation indicating successful chondrocyte proliferation. For example; a study showed when human chondrocytes that are derived from hip joint cartilage or distal femur were cultured in simulated microgravity, chondrocytes grew in 3D in this scaffold-free environment7. Moreover, another research performed by Yuge et al, showed that human mesenchymal stem cells, which are grown in simulated microgravity environment, showed higher proliferation and increased number of undifferentiated cell population11. Additionally, hMSCs grown in microgravity when implanted in cartilage-defective mice, formed hyaline cartilage after 7 days whereas the cells grown in 1g control formed only noncartilage tissue containing a small number of cells11.
Additionally, in literature there are two patents regarding cartilage tissue engineering: one involving three-dimensional cartilage tissue engineering using bone marrow cells in simulated microgravity environment (US20070116676) and the other is a method for cartilage tissue regeneration via simulated microgravity culture using scaffolds (US21010221835)1. Therefore, the cartilage tissue cultures grown in microgravity seems to be interesting and potentially very beneficial for future regenerative medicine applications especially for patients suffering from osteochondrosis or injuries with cartilage damage.
Targeting bone lossTo be able to understand bone loss mechanisms that occur in microgravity and develop countermeasures for it, researchers have been performing experiments with osteoclast and osteoblast cells using different model organisms. For example; a research team led by Shuxun Hou, discovered that AQP9 gene could be a novel target for bone loss induced by microgravity based on hind-limb suspension mice model. In this study, under microgravity, AQP9 knockout mice attenuated bone loss and inhibited osteoclastogenesis25. Moreover, femur AQP9 mRNA levels were negatively related to femur bone mineral density25.
Another recent research performed by Chen et al, investigated whether the abnormal regulation of actin cytoskeleton induced by microgravity causes the inhibited osteogenesis of bone marrow mesenchymal stem cells (BMSCs). The researchers found out that the osteogenic process of BMSCs requires a polymerized actin cytoskeleton to facilitate TAZ nuclear translocation26. Thus, stabilizing the actin cytoskeleton induced by Jasplakinolide significantly rescued TAZ nuclear translocation and recovered osteogenic differentiation of BMSCs26.
Likewise, the research performed by Sambandam et al, found that microgravity significantly enhanced the expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) in mice preosteoclast cells27. This factor has been shown to induce osteoclastogenesis together with other molecules such as Pim-1. Since microgravity has an effect on only TRAIL, the researchers concluded that microgravity induced TRAIL expression in preosteoclast cells promotes osteoclastogenesis and therefore could be a potential target for bone loss in space27. Furthermore, the research performed at the international space station with medaka fish, showed that osteoclasts were more affected by microgravity than osteoblasts based on the morphological alterations in osteoclast mitochondria28. As a result, glucocorticoid receptor pathway was induced and the expression levels of FKBP5 and DDIT4 genes were increased, which results in up-regulation of bone resorption28.
Additionally, several micro RNAs have been discovered in simulated microgravity as potential therapeutic targets for bone loss. For example: miR-33-5p is a recently found miRNA, which is sensitive to microgravity and can promote osteoblast differentiation by targeting Hmga229. Another miRNA is miRNA-132-3p, which is discovered to participate in the bone loss mechanisms that are induced by microgravity by inhibiting osteoblast differentiation30. Moreover, it is discovered that a novel miR-103 may have a role in regulating L-type voltage-sensitive calcium channels (LTCCs) in osteoblasts under microgravity31. Since LTCCs have an important role in osteoblast function, miR-103 might be a potential target for bone loss in microgravity31. Further research involving miRNAs can lead to development of certain targets for counteracting decreases in bone formation.
Finally, human bone marrow mesenchymal stem cells will fly to space for the first time through SCD-Stem Cell Differentiation experiment supported by the European Space Agency. This experiment help us to further understand the molecular mechanisms governing BMSC growth and differentiation and to develop new countermeasures against astronaut bone loss will be discovered32.
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