Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10452

REVIEW

Behavior of stem cells under outer-space microgravity and ground-based microgravity simulation Cui Zhang, Liang Li, Jianling Chen and Jinfu Wang* Institute of Cell and Development Biology, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, P. R. China

Abstract With rapid development of space engineering, research on life sciences in space is being conducted extensively, especially cellular and molecular studies on space medicine. Stem cells, undifferentiated cells that can differentiate into specialized cells, are considered a key resource for regenerative medicine. Research on stem cells under conditions of microgravity during a space flight or a ground-based simulation has generated several excellent findings. To help readers understand the effects of outer space and ground-based simulation conditions on stem cells, we reviewed recent studies on the effects of microgravity (as an obvious environmental factor in space) on morphology, proliferation, migration, and differentiation of stem cells. Keywords: development; differentiation; microgravity; stem cells; tissue engineering

Introduction A space flight can result in physiological and pathological changes within the human body, such as cardiovascular dysfunction, loss of bone density, muscle atrophy, degradation of immune function, endocrine disorders, and space movement disorder. These physiological and pathological changes in living beings take place at the cellular level. Many studies have demonstrated that microgravity is an influential factor in outer space. Microgravity strongly affects morphology, proliferation, differentiation, and signal transduction in cells. Therefore, research attention is currently focused on the influence of this environmental factor on isolated cells (in vitro). Stem cells, as multipotent cells, are capable of not only self-proliferation but also differentiation into a variety of terminal cell types, and are being used clinically. A lot of research into stem cells is also taking place in the field of space medicine. Here, we review the effects of spatial or simulated microgravity on various stem cells, as reported in recent years.

Simulated microgravity alters the adherence and differentiation ability of embryonic stem cells (ESCs) Embryonic stem cells (ESCs) are a type of stem cells isolated from early embryos (before the gastrula stage) or from a



primitive gonad. ESCs have the characteristics of infinite proliferation, self-renewal, and multidirectional differentiation in vitro. ESCs can be induced to differentiate into all kinds of cells in vivo or in vitro. Therefore, ESCs are used as a perfect cell model or seed cell source to study the development of cells and the tissue engineering in a microgravity environment. The first ESCs that were used to study various biological effects of microgravity (e.g., on cell proliferation, cell cycle distribution, cell differentiation, cell adhesion, apoptosis, genomic integrity, and on DNA damage repair) were mouse embryonic stem cells (mESCs). Wang et al. (2011) used a three-dimensional (3D)-clinostat, a multidirectional G force generator, to produce an environment with an average G of 10
3, thus simulating microgravity conditions by employing simultaneous rotation around two axes. They found that the total number of mESCs cultured under simulated microgravity significantly decreases in comparison with that of mESCs cultured under normal gravity. Nonetheless, the cell cycle showed no obvious differences between simulated microgravity and normal gravity. Therefore, it could be concluded that microgravity seems to negatively affect adherence of cells rather than their proliferation. A longer treatment (10 h), however, with simulated microgravity caused the adherence ability to return to the normal level quickly, indicating that the cells made quick adjustments to

Corresponding author: e-mail: [email protected]

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enhance their adherence capacity. Meanwhile, microgravity could not damage DNA but inhibited the repair of DNA damaged by radiation. In short, mESCs are sensitive to simulated microgravity at the early stage of treatment, and the major alterations in cellular events are a decrease of adhesion, increased apoptosis, and a delay in the DNA repair process (Wang et al., 2011). These results provided new information on the effects of microgravity on ESCs and should be helpful for ESC-based regenerative medicine. Differentiation of ESCs is also significantly affected by microgravity. Wang et al. (2012) examined generation of functional hepatocyte-like cells from mESCs by using a biodegradable polymer scaffold and a rotating bioreactor that allows for simulation of microgravity. The mESCs cultured in the rotating microgravity bioreactor could differentiate into hepatocyte-like cells with morphological characteristics of typical mature hepatocytes. The cells that differentiated on scaffolds exhibited morphological traits and biomarkers characteristic of liver cells, including albumin production, cytochrome P450 activity, and lowdensity lipoprotein uptake. When these cells were transplanted into mice with severe combined immunodeficiency, the cells could undergo further differentiation and maturation as hepatocyte-like cells. The multidirectional differentiation is one of the major characteristics of ESCs just as infinite proliferation and self-renewal are. The formation and development of embryoid bodies from ESCs is a classic biological research technique for multidirectional differentiation. Liu et al. studied formation and development of embryoid bodies from mESCs under microgravity and found that ESCs under simulated microgravity can quickly form a large number of embryoid bodies, accompanied by differentiation into endothelial cells, fibroblasts, hepatocytelike cells, blood vessel cells, heart muscle cells, and other types of tissue-forming cells (Liu et al., 2008). This finding is suggestive of stimulatory effects of microgravity on multidirectional differentiation of ESCs. In addition, research into the effects of microgravity on stem cells changed the most basic assumptions about these cells. For example, leukemia inhibitory factor (LIF) is indispensable for maintenance of ESC pluripotency. ESCs are typically maintained on a feeder layer of mouse embryonic fibroblasts (MEFs). It was demonstrated that MEFs inhibit ESC differentiation by producing an IL-6 family cytokine, LIF. On the other hand, Kawahara et al. used a 3D clinostat to simulate an environment, with an average of 10
3 G for cultured rat embryonic stem cells (rESCs) in this clinostat. They found that rESCs in this environment form many small spheres after 3 days of culture, even without the usual support of LIF or serum and the feeder layer, and these floating spheres grow during the culture period. After 7 days of culture, the number of cells in the simulated-microgravity environment was 8 times the 648

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number of cells in the 1 G environment. These results suggest that simulated microgravity can be a straightforward and effective means of rESC culture, and this culture technique is expected to be just as effective for human ESC culture (Kawahara et al., 2009). Further studies are needed, however, to determine how and why ESCs remain undifferentiated in the simulated-microgravity environment. Mesenchymal stem cells (MSCs) under conditions of microgravity Mesenchymal stem cells (MSCs) are an important member of the stem cell family and can be found in most postnatal organs and tissues. MSCs can be induced to differentiate into adipocytes, osteocytes, cartilage cells, neurons, hepatocytes, endothelial cells, myocardial cells, and other terminal cell types via in vitro induction. Because of differentiation multipotency and ease of collection and culture, MSCs have been widely used in space life sciences. The effect of microgravity on the morphology and cytoskeleton of MSCs is easily visible. In a simulatedmicrogravity environment, the cytoskeleton and the adherence ability of MSCs change conspicuously. Simulated-microgravity experiments showed that human bone marrow-derived MSCs (hMSCs) that are cultured in a microgravity environment simulated by prolonged clinorotation appear more flattened and reach confluence at lower cell density in comparison with cells cultured under normal gravity. These results suggest that hMSCs may sense changes of gravity and respond to microgravity by altering their functional activities (Merzlikina et al., 2004). Gershovich and Buravkova studied the effects of long-term (20-day) simulated microgravity (clinostatic exposure) as well as osteogenic differentiation stimuli on cultured hMSCs isolated from human bone marrow. Those researchers found that the clinostatic exposure significantly increases the number of large flat cells and that the actin cytoskeleton of hMSCs cultured under the simulated microgravity is disrupted, vinculin is redistributed, and the expression of integrin a2 is elevated. Moreover, the number of cells expressing VCAM-1 (vascular cell adhesion molecule 1) increases, and the expression level of VCAM-1 in these cells changes. These results suggest that microgravity can cause a reversible change in microfilaments and in cell adhesion of MSCs, thereby altering biological characteristics of MSCs (Gershovich and Buravkova, 2007). There have been two reports of opposite results regarding the effect of microgravity on the proliferation potential of MSCs. Zhang et al. (2006) cultured dog MSCs (dMSCs) under microgravity simulated by a horizontal cyclotron bioreactor; they tested the morphology and proliferation of dMSCs by using scanning electron microscopy. Those Cell Biol Int 39 (2015) 647–656 © 2015 International Federation for Cell Biology

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researchers found that the growth rate and morphology of dMSCs cultured in the bioreactor are better compared to those of cells cultured under conditions of normal gravity (Zhang et al., 2006). A similar conclusion was obtained by Yuge et al. (2006), who cultured hMSCs in a 3D gyroscope and found that hMSCs cultured in the simulated-microgravity environment show marked proliferation (a 13-fold increase in the cell number in a week) compared to that observed in cells cultured under the normal 1 G environment (fourfold in a week; Yuge et al., 2006). On the other hand, Dai et al. (2007) obtained an opposite result: proliferation of hMSCs that are cultured in a gyroscope is inhibited and the cell cycle is blocked in the G0/G1 phase. This finding was confirmed by an experiment during the KUBIK Space Mission ISS12s with hMSCs cultured in the space environment; the decrease in the proliferation potential of hMSCs was attributed to the reduced expression of cell cycle genes (Monticone et al., 2010). One probable explanation for these discrepancies may be that MSCs were cultured under different growth conditions, and there were differences in the experimental methods. Nevertheless, these results may serve as a reference frame for the culture of MSCs under conditions of simulated or outer-space microgravity, and such findings hold great promise for regenerative medicine and developmental biology. Gravity is a factor that can strongly influence differentiation of MSCs. Hypergravity can induce MSCs to differentiate into force-sensitive cells such as osteoblasts and myocardial cells, whereas microgravity causes MSCs to differentiate into force-insensitive cells such as adipocytes (Huang et al., 2009). Nevertheless, the skeletal changes that are observed in astronauts in outer space are a serious damage to health and have been a focus of many studies. The osteogenic differentiation of MSCs under microgravity is at the center of attention with respect to musculoskeletal health of astronauts. Simulated-microgravity experiments showed that hMSCs in a rotating wall vessel (RWV) bioreactor cannot express alkaline phosphatase (ALP), collagen-I (COL-1), and osteonectin (OS), which are hallmark proteins of osteoblasts; expression of RUNT-related transcription factor 2 (RUNX2), which regulates osteogenic differentiation, is suppressed. In contrast, adipsin, leptin, glucose transporter 4, and peroxisome proliferator-activated receptor g (PPARg2), which is important for adipocyte differentiation, are strongly upregulated in response to simulated microgravity (Zayzafoon et al., 2004; Zheng et al., 2007). According to these results, we can conclude that simulated microgravity inhibits osteoblastic differentiation of hMSCs and induces development of phenotypes resembling the adipocyte lineage. In addition, one study showed that microgravity decreases phosphorylation of ERK and increases phosphorylation of p38, which is related to the regulation of activity of RUNX2 and PPARg2 (Zayzafoon Cell Biol Int 39 (2015) 647–656 © 2015 International Federation for Cell Biology

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et al., 2004). Meyers et al. (2004) found that under simulated microgravity, the inhibition of the integrin/MAPK signaling pathway has a significant effect on osteogenesis of hMSCs. In addition, their study showed that microgravity inhibits osteogenic differentiation and induces adipogenic differentiation of hMSCs by suppressing microfilament formation and RhoA activity in these cells (Meyers et al., 2005). In addition, telomerase activity is also an important factor that influences the differentiation potential of MSCs. Sun et al. (2008) reported that telomerase activity decreases significantly in MSCs under simulated microgravity; those authors hypothesized that the reduced bone formation during a space flight may partly be due to the altered potential for differentiation of MSCs, which is linked to telomerase activity. The latter plays a key role in the regulation of differentiation and of the lifespan of cells. According to the available knowledge about the signaling pathways involved in the effects of microgravity on the osteogenic and adipogenic differentiation of MSCs, these effects of microgravity can be reversed. Zheng et al. (2007) were able to regulate osteogenic and adipogenic differentiation of hMSCs under simulated microgravity by controlling the expression of RUNX2 and PPARg2 as well as phosphorylation of ERK and P38 MAPK via addition of BMP (a factor stimulating expression of RUNX2), FGF2 (ERK phosphorylation-stimulating factor), and SB203580 (P38/MAPK inhibitory factor). The results mean that the combination of these inhibitors and activators reverses the effects of simulated microgravity on osteogenic and adipogenic differentiation of hMSCs (increases the osteogenic potential and decreases the adipogenic potential). This study is indicative of an experimental model for the prevention and treatment of skeletal abnormalities in astronauts, with potential implications for similar medical conditions in the elderly. In addition, the above findings indicate that microgravity inhibits osteogenic differentiation and induces adipogenic differentiation through different signaling pathways. Figure 1 shows that simulated microgravity increases phosphorylation of p38 MAPK, which can ultimately increase activity of PPARg2 and the expression of its target genes: adipsin and leptin. The simulated microgravity, however, increases the expression of PPARg2 mRNA through an unknown signaling pathway that is different from the p38 MAPK pathway. BMP-2 performs an important function in activation of the SMAD pathway. Under simulated microgravity, BMP-2 increases expression of Runx2 mRNA but cannot reverse the effects of simulated microgravity on osteogenic differentiation of hMSCs. FGF2, an activator of the RAS pathway through activation of the receptor tyrosine kinase (RTK), which is the FGF2 receptor, increases phosphorylation of ERK but does not significantly increase the expression of osteocytic gene markers. Therefore, this observation suggests that the effects of 649

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Figure 1 Putative signaling pathways involved in osteoblastic differentiation of human mesenchymal stem cells (hMSCs) under simulated microgravity (adapted from Zheng et al., 2007, with permission). BMP, bone morphogenetic protein; BMPR, BMP receptor; SMAD, Sma- and MAD-related protein; RUNX2, runt-related transcription factor 2; ECM, extracellular matrix; FAK, focal adhesion kinase; FGF2, fibroblast growth factor 2; FGFR, FGF receptor; RTK, receptor tyrosine kinase; RAS, rat sarcoma protein; MEK, MAPK/ERK kinase; ERK,extracellular signal-regulated kinase; PI3K, phosphoinositol 3-kinase; p38 MAPK, p38 mitogen-activated protein kinase; PPARg2, peroxisome proliferator-activated receptor g2; OSE2, osteoblastspecific element 2; ARE, adipocyte regulatory element.

simulated microgravity on hMSC differentiation may be due to the simultaneous decrease in ERK activity and Runx2 mRNA expression, as well as a combined increase in p38 MAPK activity and PPARg2 mRNA expression. The combination of SB203580, BMP-2, and FGF2 completely reverses the effects of simulated microgravity on osteogenesis of hMSCs and increases the expression levels of osteocytic gene markers. Further, studies are needed to determine (1) which signaling pathways are involved in the simulated microgravity-induced upregulation of PPARg2 mRNA and downregulation of Runx2 mRNA, (2) whether there is another yet unknown pathway that affects the phosphorylation of ERK besides the RAS pathway under simulated microgravity, and (3) which signaling pathway is responsible for the increased phosphorylation of p38 MAPK under simulated microgravity. Microgravity also has effects on other differentiation directions of MSCs, such as cartilage, nerve, and endothelial lineages. Microgravity promotes cartilage differentiation of hMSCs. Compared to cells cultured in a stationary state, the expression of collagen II and proteoglycan in hMSCs cultured in a dynamic 3D environment (simulated micro650

gravity) markedly increases, which points to the positive role of microgravity in the cartilage differentiation of hMSCs (Wang et al., 2007a). In addition, the function of the p38 MAPK pathway in the cartilage differentiation of human adipose-derived MSCs (ADSCs) was evaluated under simulated microgravity. The p38 MAPK pathway that is activated by TGF-b is further stimulated by microgravity. These findings suggest that p38 MAPK signaling acts as an essential mediator of the microgravity-induced chondrogenesis of ADSCs (Yu et al., 2011). Yuge et al. (2006) transplanted pellets of hMSCs from microgravity culture into cartilage-defective mice and found that these transplants form hyaline cartilage after 7 days, whereas the transplants from normal culture form only noncartilage tissue containing a small number of cells. These results show that hMSCs that are cultured in a simulated-microgravity environment retain their ability to differentiate into hyaline cartilage after transplantation. In recent years, the neural differentiation of MSCs under microgravity conditions has also been studied extensively. Chen et al. (2011) cultured rMSCs in the neural differentiation medium under microgravity simulated by a clinostat and found that Cell Biol Int 39 (2015) 647–656 © 2015 International Federation for Cell Biology

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microtubule-associated protein 2 (MAP-2), tyrosine hydroxylase (TH), and choline acetyltransferase (CHAT) in cells under simulated microgravity are expressed at higher levels compared to those observed at normal gravity conditions. They also found that secretion of neurotrophins like nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF) is increased (Chen et al., 2011). The above results point to a stimulatory effect of microgravity on the neural differentiation potential of MSCs. In space flight experiments, the expression of most genes specific to some processes, such as neural development, neuron morphogenesis, transmission of nerve impulses, and synapse structure, undergoes changes (Monticone et al., 2010). Moreover, simulated microgravity promotes differentiation toward a nucleus pulposus-like phenotype (Luo et al., 2011); TGF-b1 can enhance this differentiation potential (Han and Jiang, 2011). In addition, under special induction conditions, microgravity can cause MSCs to differentiate into endothelial cells (Zhang et al., 2013) and myocardial cells (Huang et al., 2005). In recent years, developments in space biomedical research and space life sciences were actively utilized for experiments on Earth. The research into microgravity effects on tissue engineering and into regenerative medicine involving MSCs as seed cells represents some of the fields of spatial biological engineering. For instance, the cartilage differentiation potential of MSCs that are cultured under microgravity is noticeably enhanced in comparison with that observed under normal gravity. The 3D dynamic microgravity conditions can improve the differentiation potential of MSCs, which is expected to facilitate treatment of articular cartilage injury (Wang et al., 2007b). Hwang et al. (2009) developed an efficient and integrated 3D bioprocess based on the encapsulation of undifferentiated mESCs within alginate hydrogels and culturing of ESC-derived MSCs in a rotary cell culture microgravity bioreactor for application to bone tissue engineering in the context of macroscopic bone formation. 3D mineralized constructs were generated by means of this 3D bioprocess and displayed morphological, phenotypic, and molecular attributes of the osteogenic lineage, as well as mechanical strength and mineralized calcium/phosphate deposition. This bioprocess can serve as an efficient, automatable, scalable, and fully functional culture system for application to bone tissue engineering in the context of macroscopic bone formation. Microgravity inhibits proliferation and migration of hematopoietic stem cells (HSCs) and alters their differentiation directions Hematopoietic stem cells (HSCs) are pluripotent and selfrenewing cells with the membrane-bound surface antigen CD34 in bone marrow and have the potential to differentiate Cell Biol Int 39 (2015) 647–656 © 2015 International Federation for Cell Biology

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into various blood cell precursors and then to generate a variety of blood cells, including red blood cells, white blood cells, and platelets. Human space flight missions have resulted in some hematologic anomalies. Therefore, the effects of microgravity on the hematologic function have drawn the attention of researchers to the health of astronauts. Earlier studies showed that microgravity changes the migration capacity of bone marrow CD34þ cells by altering the cytoskeleton and cell cycle (Plett et al., 2004). Culture of bone marrow CD34þ cells under simulated microgravity results in a significant reduction of migration mediated by stromal cell-derived factor 1 (SDF-1a), which is linked to the decreased expression of F-actin and alteration of cell cycle kinetics, for example, prolongation of the S phase and a reduction of cyclin A expression. The above results demonstrate that microgravity significantly inhibits the migration ability and cell cycle of bone marrow CD34þ cells; this effect is regarded as the possible reason for the hematological abnormalities observed in humans during a space flight. CD34þ cells that are cultured under simulated microgravity exit the G0/G1 phase of the cell cycle at a slower rate than do cells cultured under normal gravity. This is a possible reason why the cells cultured under normal gravity increase in number threefold by day 4–6, whereas cells cultured under microgravity do not proliferate much (Plett et al., 2004). While studying human erythroleukemia cells (K562) cultured in RCCS (rotary cell culture system), Long et al. found that microgravity inhibits proliferation of K562 cells and arrests their cell cycle in the G0/G1 phase (Long et al., 2011). One possible reason is that the phosphorylation level of ERK1/2 decreases under microgravity. After a 14-day flight onboard the Cosmos2044 Biological Experimental Satellite, the numbers of rat bone marrow cells, granulocytes, macrophages, and hematopoietic progenitor cells were significantly lower in comparison with cells cultured on the ground (regular conditions) (Vacek et al., 1991). During the Space Shuttle Missions STS-63 (Discovery) and STS-69 (Endeavor) after an 11- to 13-day period, the total number of cells cultured during the flight increased only 10.1- to 17.6-fold: a 57–84% decrease compared to the cells cultured on the ground (41.0to 65.5-fold proliferation). The number of myeloid progenitor cells expanded 2.6- to 17.5-fold on the ground compared to 0.9- to 7.0-fold during the flight, and the number of erythroid progenitor cells expanded 2.0- to 4.1fold on the ground compared to the actual decline of the number of cells observed under microgravity (>83% reduction; Davis et al., 1996). In addition, the number of HSCs, monocyte-macrophage precursors, and red blood cells decreased (Domaratakaya et al., 2002). It was shown that proliferation of bone marrow CD34þ cells is restrained after culture for 4–6 days in RWVB (rotating wall vessel bioreactor; Plett et al., 2001). In summary, microgravity can 651

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inhibit proliferation and differentiation of hematopoietic precursors, especially precursors of red blood cells. Microgravity also alters the differentiation mode of bone marrow CD34þ cells. Bone marrow CD34þ cells differentiate into myeloid cells instead of erythrocytes after culture for 13–14 days under microgravity (Plett et al., 2001). The results of hematological experiments during a space flight showed significant downregulation of hematocrit, red blood cells, and hemoglobin in rats, slight neutrophilia and lymphopenia, and no significant changes in the differentiation potential of bone marrow and spleen cells or in the level of erythropoietin (Lange et al., 1987). Culture of bone marrow progenitor cells that are harvested from rats undergoing a space flight revealed that not only the total number of leukocytes but also the numbers of lymphocytes and monocytes decrease in comparison with those observed in rats on the ground. Nonetheless, neutrophil numbers are increased. Compared to rats on the ground, rats on the space flight have lower numbers of CD4, CD8, CD2, CD3, and B cells in peripheral blood, but no differences in spleen lymphocytes (Chiki et al., 1996). In addition, microgravity inhibits differentiation of K562 cells that is induced by hemin, especially into erythrocytes; this effect may be one of the reasons for the development of anemia under microgravity. Some researchers also hypothesized that inhibition of K562 differentiation by microgravity has nothing to do with GATA because microgravity does not inhibit the expression of GATA-1(Long et al., 2011). According to one study, microgravity also affects proliferation and endothelial differentiation of human umbilical blood stem cells (Chiu et al., 2005). CD34þ mononuclear cells were isolated from human umbilical cord blood and cultured under simulated microgravity for 14 days (Chiu et al., 2005). The cells cultured under microgravity exhibited a significant increase in proliferation and formed 3D tissue-like aggregates after incubation with vascular endothelial growth factor (VEGF). On day 4, CD34þ cells developed into vascular tubular assemblies and expressed endothelial phenotypic markers. These data suggest that human umbilical cord blood CD34þ progenitors are capable of transdifferentiation into the vascular endothelial cell phenotype and can assemble into 3D tissue structures. Culture of CD34þ cells under simulated microgravity may benefit research in the fields of stem cell biology and somatic cell therapy. Microgravity promotes proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) PDLSCs are the most reliable seed cells for periodontal tissue regeneration and have been used for cell therapy and gene therapy of periodontal defects. PDLSCs play an important role in the treatment of periodontal diseases, repair of 652

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cartilage tissue defects around the implant, and tissue repair and reconstruction during orthodontic tooth movement. Accordingly, simulated microgravity has also been studied with respect to its effects on the biological characteristics of PDLSCs. These effects involve promotion of proliferation and viability of PDLSCs. Li et al. (2009) evaluated the biological effects of 3D dynamic simulated microgravity induced by a rotary cell culture system (RCCS) on human PDLSCs (hPDLSCs) isolated from surgically extracted human teeth (these cells were enriched by collecting multiple colonies). The results showed that simulated microgravity affected the biology of hPDLSCs judging by the promotion of proliferation and viability, alterations of morphology, and disorganization of the microfilament system. These changes in microfilaments were time dependent and reduced the migration capacity of the cells (Dong et al., 2012). Ma et al. found that PDLSCs under microgravity show hemispherical morphology, and some of them spread into the irregular flat or long shuttle shape. In addition, the growth rate of these cells noticeably increases (Ma et al., 2011). Furthermore, simulated microgravity-treated hPDLSCs exhibit increased matrix mineralization and enhanced expression of mineralization-associated genes (Li et al., 2009). These studies may provide insights into variations of cellular responses in a 3D environment and can help to achieve desirable periodontal regeneration by means of PDLSCs-based tissue engineering approaches. A large number of PDLSCs can be obtained by culturing cells under microgravity; this abundance is important for periodontal tissue engineering. In addition to the enhanced expression of mineralizationspecific genes in hPDLSCs that are incubated in the osteogenic medium under simulated microgravity (Li et al., 2009), another study showed that the expression of SMAD2, -3, and -4 significantly increases in a timedependent manner under microgravity. The results also show that the level of p-SMADs begins increasing at 30 min and reaches its peak at 2 h (91.32%). Nevertheless, the expression of COL2, ALP, OCN, and p-Smad decreases after addition of SIS3 (a specific inhibitor of SMAD3 phosphorylation; Li et al., 2012a). These findings are indicative of the stimulatory role of SMAD signaling in osteogenic differentiation of PDLSCs under microgravity. Similar conclusions were reached by Li et al. (2012b). They also found that TGF-b1 promotes osteogenic/cementum differentiation of PDLSCs via the SMAD signaling pathway. Li et al. (2009) found that IGF-1 can promote the proliferation and osteogenic differentiation of PDLSCs under microgravity and deduced that IGF-1 should be involved in the formation of osteoclasts by regulating the expression of OPG/RANKL in the stem cells. The studies on the differentiation mechanism of PDLSCs may be helpful for the periodontal tissue engineering under microgravity. Cell Biol Int 39 (2015) 647–656 © 2015 International Federation for Cell Biology

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Microgravity is beneficial for the survival and stemness maintenance of liver stem cells The definition of liver stem cells was proposed by Peteoen in 1999 (as hepatogenic stem cells). These cells mainly exist in the liver owing to the 2-directional differentiation potential into either hepatocytes or bile duct epithelial cells. Liver tissue engineering using liver stem cells as seed cells has been explored in various environments. Yao et al. (2011) cultured human liver stem cells (isolated from the human fetal liver) under microgravity simulated by a 3D rotary cell culture system and found that the liver stem cells under microgravity efficiently adhere to the collagencoated microcarriers and form complex multilayered 3D aggregates. A massive extracellular matrix and microvillus were observed on the surface of these 3D aggregates. Glucose consumption from the culture medium increased on day 9 and reached its peak on day 17, and the LDH activity remained low. RNA isolated from the cells cultured under the simulated-microgravity environment demonstrated higher levels of EpCAM, ALB, CK19, HNF6, and CYP3A7 transcripts, but not of AFP or CYP3A4 transcripts, in comparison with that observed in cells cultured in the normal-gravity environment. These results indicate that liver stem cells proliferate more efficiently with a more stable phenotype in the simulated microgravity 3D culture system than under control conditions. Therefore, it was concluded that microgravity that is simulated by a 3D culture system provides superior conditions for liver stem cell survival and proliferation and should be useful for production of cells that could be used in bioartificial liver support systems or tissueengineered liver grafts. Besides, the culture of WB-F344 liver stem cells under the simulated microgravity also showed that proliferation of WB-F344 cells increases in comparison with the stationary state (Qu et al., 2007). However, no differences in cell proliferation were observed between PICM-19 pig liver stem cells cultured during a space flight (STS-126 Mission) and the cells on the ground (Talbot et al., 2010). The PICM pig liver stem cell line was cultured in space for nearly 16 days on the STS-126 mission to assess the effects of a space flight on the ability of liver parenchymal cells to differentiate into either monolayers of fetal hepatocytes or 3D bile ductules (cholangiocytes). In the comparison between flight and ground-control cultures 17 h after the shuttle returned to earth, no differences were found between two types of culture with the exception of some differentially expressed genes. The cells in the space flight grew to ~75% confluence and showed no signs of apoptosis or necrosis (Talbot et al., 2010) and either differentiated into monolayer patches of hepatocytes (with biliary canaliculi visible between cells) or into 3D bile ductules with a well-defined lumen. Ultrastructural features of the flight and groundcontrol cells were similar: PICM-19 cells displayed numerCell Biol Int 39 (2015) 647–656 © 2015 International Federation for Cell Biology

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ous mitochondria, a Golgi apparatus, smooth and rough endoplasmic reticulum, vesicular bodies, and occasional lipid vacuoles. Cell-to-cell contacts were typical in the flight and ground-control samples; biliary canaliculi were well formed between the cells, and these cells were sandwiched between the STO feeder cells. PICM-19 cells in both environments displayed inducible P450 activities and produced urea in a glutamine-free medium and in response to ammonia. This experiment demonstrated that the PICM19 cell line is a good in vitro model for studies of liver function under microgravity and that the difference between flight and ground-control cultures is minor. Simulated microgravity can maintain the characteristics of liver stem cells. In the study on the effects of microgravity on the expression of WB-F334 liver stem cell-specific molecules, the expression level of a-fetal protein (AFP) gene (specific for liver stem cells) and the number of AFP-positive cells under microgravity were significantly higher compared to those observed in static culture; however, the expression level of albumin (ACB) mRNA and the number of ACBpositive cells was lower compared to those observed in static culture (Qu et al., 2007). This finding indicates that simulated microgravity may provide an appropriate environment to sustain stemness of liver stem cells and is beneficial for large-scale culture of such cells. Effects of microgravity on other stem cells The structure and function of the human nervous system are altered in space. To directly assess the influence of simulated microgravity conditions that may be beneficial for cultivation and proliferation of human neural stem cells (hNSCs), Chiang et al. (2012) used the rotary cell culture system (RCCS) developed by NASA to create a unique microgravity environment that is characterized by low shear force and high mass transfer and enables 3D cell culture of various cell types. The results showed that this simulated microgravity environment influences the proliferation of human neural stem cells; this effect is mediated by the function of mitochondria. Microgravity also induces the expression of b-adrenoreceptor, upregulates formation of cAMP, and activates both PKA and CREB (cAMP response elementbinding protein) pathways. The expression of intracellular mitochondrial genes that are regulated by CREB, including PGC1a (PPAR coactivator 1a), nuclear respiratory factors 1 and 2 (NRF1 and NRF2), and mitochondrial transcription factor A (TFAM), is significantly increased 72 h after the onset of microgravity. Thus, the ATP level and amount of mitochondrial mass were also increased. Therefore, it appears that microgravity promotes proliferation of neural stem cells by enhancing the function of mitochondria (Chiang et al., 2012). In addition, simulated microgravity may support the growth of neural stem cells, which hold 653

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promise for cell replacement therapy in neurological disorders. The skin is susceptible to various injuries and diseases. The major problem in skin tissue engineering is how to develop a functional 3D substitute for damaged skin. There are studies of 3D dynamic simulated-microgravity culture systems as a “stimulatory” environment for the proliferation and differentiation of stem cells. For example, Lei et al. (2011) utilized a NASA-approved rotary bioreactor to evaluate the proliferation and differentiation of human epidermal stem cells (hEpSCs). These investigators enriched epidermal stem cell colonies isolated from children’s foreskin. Cytodex-3 microcarriers and hEpSCs were cocultured in the rotary bioreactor for 15 days. The results showed that hEpSCs that are cultured in a rotary bioreactor exhibited enhanced proliferation and viability compared to cells cultured under static conditions. Additionally, immunostaining demonstrated a higher percentage of Ki67-positive cells in the rotary bioreactor compared to the static culture. Cells in the rotary bioreactor also display low expression of involucrin on day 10. Histological analysis revealed that the cells cultured in rotary bioreactor aggregate on the microcarriers and form multilayer 3D epidermis structures (Lei et al., 2011). These findings mean that simulated microgravity can support proliferation of hEpSCs and represents a new strategy for the formation of a multilayer epidermis structure.

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experimental methods and new techniques for cell culture. The program of the SJ-10 satellite was organized and started by the Chinese National Space Administration (CNSA) in 2011, with 10 experiments planned in space life sciences (Hu et al., 2014). The satellite will be launched at the end of 2015. During this mission, 3D culture of neural stem cells and hematopoietic stem cells will be performed in space, and the capacity for and the molecular mechanisms of osteogenic differentiation will be assessed in human bone marrowderived MSCs. This project should advance stem cell-based tissue engineering as well as differentiation and developmental biology; thus, molecular mechanisms underlying the changes in stem cells under real weightlessness may be finally deciphered. Acknowledgments and funding The authors thank Dr. Hongbin Zhang in Harvard University, who critically read this manuscript, and acknowledge support from National Basic Research Program of China (2012CB967902, 2014CB541705), National Development Program of Important Scientific Instrument (2013YQ030595), Strategically Guiding Scientific Special Project from Chinese Academy of Sciences (XDA0402020223), Opening Foundation of the State Key Laboratory of Space Medicine Fundamentals and Application (SMFA12K02), and TZ-1 Application Program (KYTZ010901-FB-003).

Conclusion In this article, we summarized the recently uncovered effects of microgravity on stem cells in outer space and in groundbased simulation environments. Although these studies involve many kinds of stem cells, most of these projects are limited to the assessment of phenotypic changes in morphology, proliferation, and the differentiation potential. Many specific mechanisms underlying the effects of a space environment on stem cells are still unclear. Furthermore, most studies on the effects of microgravity on stem cell biology involve microgravity simulation (on Earth), and the adequacy and authenticity of this simulation are always questioned. The differences between the ground-based simulations and the outer-space environment make it difficult to draw correct and convincing conclusions; these differences even lead to conflicting conclusions. Long-term microgravity experiments that can only be performed at outer-space facilities, such as a space station, space shuttle, or a satellite, are essential for studies of microgravity effects on life sciences. Therefore, in order to make such research less problematic and more accurate, both the ground-based simulation and flight experiments must involve adequate hardware and need new “smart” instruments or equipment for space experiments. This field also requires sensible 654

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