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

Abstract 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.

27 February 2020 gave 9010 matches for the term "weightlessness," 11 338 matches for "microgravity," 549 matches for "microgravity and differentiation," and 273 matches for "microgravity and stem cells." An important aspect is the differentiation and redifferentiation potential of benign and malignant cells grown under simulated and real μg conditions. Endothelial progenitor cells exposed to μg revealed an improved angiogenic potential. 11 Human-induced pluripotent stem cellderived cardiomyocytes (hiPSC-CMs) cultured aboard the ISS exhibited alterations in calcium handling and showed 2635 differentially expressed genes among flight, postflight, and ground control samples. 12 In particular, genes involved in mitochondrial metabolism were differentially regulated. 12 Recently, human blood-derived stem cells had been investigated on the ISS. Compared with cells cultivated on Earth, a reduced expression of Sox2, Oct3/4, Nanog, and E-cadherin was measured in space, together with an earlier osteogenic differentiation. 13 In addition, cancer cells have proven to re-differentiate after exposure to r-and s-μg. 10,14,15 Studies in μg demonstrated that cells exposed to gravitational unloading changed their growth behavior and started growing in a two-(2D) and threedimensional (3D) manner in space and on the rotating wall vessel (RWV), the RPM, CN, or on rotary cell culture systems (RCCS) ( Figure 1). 1,[16][17][18][19][20] Thus, it is possible to engineer scaffold-free and scaffold-containing 3D tissues such as preliminary vessels, cartilage, liver and bone pieces, and other organoids from different cell types using μg. 1 Currently, a large number of publications reported about the behavior of cancer cells in μg, but little information exists about the behavior of cancer stem cells (CSCs) in μg. Recently, investigations on colorectal CSCs were possible when HCT116 cells were exposed to a RCCS. Microgravity conditions provide a new method to study the biology of cancer stemness. 14 This concise review will focus on the current knowledge of the impact of μg on differentiation and cell growth in stem cells and CSCs.

| DEFINITION OF CSCs
CSCs represent a subset of cells within the heterogeneous bulk of liquid and solid tumors. 21 The term CSC was introduced for the first time by Dr John E. Dick and colleagues more than 25 years ago in their revolutionary study of certain kinds of human leukemia. 22 CSCs share features with somatic stem cells, and therefore they possess characteristics of asymmetric division, self-renewal, quiescence, and differentiation. 23 In addition, these cells own the ability to replicate the parental tumor upon transplantation into a host as shown by Dick et al. 22 CSCs also play a pivotal role in tumor pharmacological resistance due to upregulation of drug efflux transporters and DNA repair components. 21,23 Besides leukemia, CSCs have been identified in numerous tumor types, including those of the brain, breast, prostate, kidney, lung, and colon. 23,24 Accumulating evidence suggests mutations and epigenetic alterations in some tumor cells as main drivers leading to the establishment of CSCs. 25 Microenvironment-driven selection has also been implicated in the emergency of resistant cells with stem-like characteristics. 26 As CSCs are highly resistant to current therapeutic approaches, and therefore the main reason for cancer relapse, these cells are considered the prime therapeutic target for cancer treatment. 25,[27][28][29] F I G U R E 1 Platforms and devices used for stem cell research under real and simulated microgravity conditions. A, The International Space Station enables long-term cell culture in real microgravity. B, Custom-made random positioning machine (RPM) for the cultivation of mammalian cells under simulated microgravity conditions. 20 C, The rotating wall vessel (RWV) is another type of microgravity simulator suitable for suspension cell cultures or adherent cells on microcarrier beads

Significance statement
Microgravity provides a unique environment for cell culture and has been shown to induce cellular changes and processes that could not be achieved under normal gravitational conditions. Over the past years, it has therefore gained increasing importance in different research fields such as cancer research, where microgravity may help understanding and suppressing tumor metastasis, or tissue engineering, where it induces the scaffold-free formation of three-dimensional multicellular spheroids. This review will give a concise overview of the current knowledge on the effects of microgravity on stem cells and cancer stem cells, and will highlight novel therapeutic options derived from it.

| CSCs EXPOSED TO MICROGRAVITY
Little is known about the behavior of CSCs exposed to μg conditions, but cancer researchers have started to work on this important topic.
Tumor cell heterogeneity and the presence of CSCs are responsible for a poor outcome of cancer patients and that metastatic breast cancer remains currently incurable, despite the progress in their detection and treatment. 30 Breast CSCs have been identified as CD44 high , CD24 low , or aldehyde dehydrogenase positive (ALDH + ). 31,32 Breast, thyroid, and prostate cancer cells differentiate and show various morphological alterations, change their growth behavior when grown in space or under s-μg. [33][34][35][36] The cancer cells differentiate into two phenotypes in the μg environment. One part grows adherently on the bottom of the cell culture flasks and the other one assembled to 3D spheroids. 33,34,36,37  Focused research on CSCs exposed to μg is published for various types of tumors.

| Lung cancer
As in most other tumors, lung CSCs exhibit an increased expression of cell surface markers CD44 and CD133. Alternatively, lung CSCs may be purified using functional assays like ALDH +8 and side population (SP) assays. 39 Several studies have investigated the influence of reduced gravity on lung cancer cell behavior. [40][41][42] However, only few studies, to our knowledge, have reported an impact of s-μg on lung CSCs. One prominent example is the study by Pisanu et al in which lung CSCs, enriched from the stable non-small cell lung cancer cell line H460, were subjected to s-μg obtained by means of an RPM. 15 The study provides evidence that lung CSCs are committed to selective differentiation when subjected to reduced gravity.
Furthermore, the study also reported increased apoptosis of CSCs incubated on the RPM compared to 1g controls. Collectively, these data suggest that lung CSCs, similar to somatic stem cells, are rescued from their quiescent state and lose their stemness default state when cultured in μg.
Consistent with this observation, a decrease in ALDH levels as well as downregulation of Nanog and Oct4 genes were observed. Intriguingly, the μg-induced traits were stably attained and conserved by CSCs when cells were returned to 1g conditions. 15

| Gastrointestinal tumors
The gastrointestinal tract comprises stomach, colon, small intestine, rectum, and pancreas. During embryogenesis, these organs develop from stem cells and even in adults relevant stem or progenitor cells are persisting. 43 They are capable to keep up the integrity of these organs, but may also be the origin of cancer development. 44 Therefore, stem cell behavior is intensively investigated by a number of methods including exposure to s-or r-μg. 45 However, little is known about the μg-dependent behavior of stem cells of the gastrointestinal tract. Whether the downregulation of the tropomyosin 1 gene (Tpm1) in stem cells 46 is responsible for downregulated Tpm1 gene in gastrocnemius muscle of mice observed after a 12-day lasting spaceflight remains to be proven. 47  The subsequent investigation of the organoids revealed that the ratio of stem cells influenced the growth rate of the colon carcinoma cells. 49 Stem cells are also present within adult human and mice pancreas cell populations. 50,51 If pancreatic cells are singularized and cultured within a hanging drop culture, they form pseudoislets with improved physiological properties. 52 The result could be explained by the hypothesis that 3D spheroid formation favors the growth of progenitors, as it has been shown by Samuelson and Gerber, 53 who successfully induced differentiation of mouse pancreatic progenitor cells by culturing them in a RWV bioreactor. Furthermore, if human pancreatic islets were predispersed and cocultured with stem cell such as amniotic epithelial cells (AECs) on a rotational cell culture system, islet:AEC aggregates were formed with improved insulin secretory capacity. 54

| Osteosarcoma and other tumors
Kelly et al investigated human osteosarcoma cells (SAOS-2 cells) exposed to the NASA-developed hydrofocusing bioreactor (HFB) and to the RCCS. 55 The authors showed that CSCs were stimulated to proliferate when they are cultured in μg conditions produced by the HFB. In addition, μg sensitized CSCs to chemotherapeutic agents. 55 Moreover, the authors investigated various cell types on the HFB such as osteosarcoma cells (SAOS-2), prostate cancer cells, lung cancer cells, and melanoma cells and demonstrated that HFB exposure increased CD133-positive cell growth from various cell lines compared with the RCCS vessel and to normal gravity control. 55

| Use of stem cells for cartilage tissue engineering
Considering the overall development of progressively aging societies, the impact of cartilage damage and accompanying disorders such as osteoarthritis, rheumatoid arthritis, or intervertebral disk damage is steadily growing. One main problem of the treatment of cartilage lesions is the poor regenerative capacity of this tissue due to its avascularity and relatively low cell density, which is further augmented by the fact that the surviving chondrocytes shift their metabolic activity toward predominantly degenerative processes. 56 For applications in regenerative medicine, it is therefore of high interest to circumvent these problems and to stimulate cartilage repair. One approach for this is the use of stem cells (Table 1)

| Stem cells for the engineering of bone-like tissues
It is well known that μg influences calcium, sodium, and bone metabolism of humans in space. Bone loss and spaceflight-induced osteopenia are problematic issues for long-term space missions and future space exploration. 68 Bone loss and radiation are the most substantial health risks of astronauts and should be managed by efficient countermeasures. 68 Despite this knowledge from space travelers, it is also well acknowledged that culturing of stem cells and bone cells in s-μg, using the RWV, the RCCS bioreactor, or the RPM, is a suitable technique for bone tissue engineering purposes [69][70][71] and has been reviewed earlier (Table 1). 1,3 3D cell culture environments were useful to enhance osteoblast differentiation and to engineer osseous-like tissues from human preosteoblastic cells and human mesenchymal preosteoblastic cells ( Figure 2). 69,70 The RWV bioreactor was used and patented by Clarke et al to grow mineralized 3D bone constructs. 72 Another study demonstrated that after a 2-week culture of fetal osteoblasts (hFOB 1.19 cell line) on an RPM, MCSs could be engineered presenting a bone-specific morphology. The hFOB 1.19 cells are well-characterized, stable osteoprogenitor and a well-known model for normal osteoblast differentiation. 71 These bone tissues may be used to study the mechanisms behind spaceflight-related bone loss or other bone diseases such as osteonecrosis or bone injuries. 71 A recent study demonstrated the osteogenic potential of rat bone marrow-derived MSCs (BMSCs) in vitro. 73 Encapsulated BMSCs differentiated into osteoblastic cells and had formed bone-like tissue under osteogenic μg bioreactor conditions. 73 Human blood-derived stem cells (BDSCs) were investigated recently in space on the ISS during the Italian VITA mission. Osteoblastic differentiation was induced by rapamycin. 13 Rapamycin influenced the transcriptional activation of BDSCs toward osteogenic differentiation via elevated GATA4 and Sox17. Both factors modulate downstream transcription factors (like Runx2), critical for bone formation. 13 Another study reported on the behavior of human BMSCs exposed to s-μg. The authors found an inhibition of proliferation and differentiation toward osteoblasts, but an increase in adipogenesis. 74 s-μg also selected highly tumorigenic cells for survival. 74 In addition, MSCs cultured on the SJ-10 recoverable scientific satellite revealed an increase in the p38 MAPK activity and a de-repression of AKT activity. The satellite flight conditions inhibited osteogenic differentiation and promoted adipogenic differentiation, even under osteogenic induction conditions. 75 Bone MSCs exposed to μg revealed alterations in regulation or functioning of the actin cytoskeleton which may cause the inhibited osteogenesis. 76 The depolymerization of actin inhibited the osteogenic differentiation of the bone MSCs through impeding nuclear aggregation of the transcriptional coactivator with PDZ-binding motif. 76    and proliferative potential. 84 RNA-sequencing revealed that 2635 genes were differentially expressed among μg-exposed and control samples, including genes involved in mitochondrial metabolism. 12

| Heart tissue
Biofabrication of heart tissue is a hot topic in tissue engineering. The usefulness of tissue engineered cardiac tissue is dependent among others on the differentiation status of cardiomyocytes (CMs) in vitro, on the contractility, and electrophysiological characteristics. 86 As the growth of CMs is in part regulated by mechanical loading, the idea suggests itself that μg can lead to growth changes as well. A recent study on the ISS investigated human-induced pluripotent stem cellderived CMs. 10 The authors demonstrated that long-term cell culture (5.5 weeks) of advanced, highly specialized cell types such as human CMs is possible aboard the ISS.
Microgravity has proven to alter the CM behavior and thus can help to promote myocardial differentiation of induced pluripotent stem cells. 87,88 A combination of 3D culture and s-μg created by an RPM can be used to efficiently generate highly enriched CMs. 89 Dissociated cardiac muscle cells cultured on 3D scaffolds in RCCS formed engineered constructs with structural and functional features resembling those of native cardiac tissue. 90 Another study reported that simulated μg provided by a 2D CN modulated the differentiation processes of embryonic stem cells. 46 This ability can be a driving element for biofabrication methods.
In view of future space exploration plans, it is of high interest to test the effects of simulated Mars and Moon gravity using a special RPM manufactured by ADS, Leiden, and NL on the tissue engineering of heart tissue. 91

| Stem cells for skin repair
Different resident skin stem cell pools contribute to the maintenance and repair of the various epidermal tissues of the skin. Their development to differentiated skin cells is regulated by various factors. 92 There are a few publications, which suggest that μg may have an influence on this process. Exposing murine embryonic stem cells to parabolic flights changed 14 genes, which are involved in skin development. 93 Human epidermal stem cells (hEpSCs) isolated from children's foreskin were successfully propagated in vitro, while hEpSCs marker and proliferative capacity are maintained. Subsequently, they were seeded in a rotary bioreactor together with microcarrier beads for 1 day, which allowed the cells to attach to the surface of the beads.
Then, the sample was divided into two groups, including a rotation culture group in an RCCS and a static culture group on six-well cell culture plates. The cells in a rotary bioreactor were prone to accumulate on the microcarrier beads forming 3D aggregates, whereas the cells in six-well cell culture plates appeared as monolayer sheet structure on the surface of microcarrier beads. In the rotation culture group, there were a large number of cells aggregated over microcarriers beads forming 3D tissue-like epidermis structure. 94 Fibroblasts, an important type of skin cells, could be derived from adipose-derived stem cells (ADSCs) exposed to μg. They were isolated from fat specimens of patients. Afterward, they were cultured on a 2D CN and under 1g conditions. Comparing the cells after 7 days of incubation showed that the connective tissue growth factor-induced fibroblastic differentiation of ADSCs can be modulated by exposure to s-μg. Most notably, the expression of CD44 was reduced while the production of collagen III was enhanced. 95

| Use of stem cells for the engineering of liver tissue
The liver is the largest inner organ and exhibits numerous unique biochemical functions. More than 500 physiological functions of the liver are reported. 96,97 The liver is involved in metabolic processes, in biochemical synthesis as well as in detoxification processes. Although it shows a high capacity of regeneration, diseases of the liver such as cirrhosis, hepatitis, and hepatocellular carcinomas account for about 2 million deaths per year worldwide. 98 Exposure of the liver to toxicants such as alcohol due to alcohol misuse or infection with hepatitis C and B viruses and other nonalcoholic liver diseases 99 may lead to liver cirrhosis, which is responsible for about 1.03 million deaths per year. 100 Up to now, orthologous liver transplantation is the only treatment option available for patients suffering from such severe liver diseases. However, there is a drastic shortage of donor organs, 98,101,102 so alternative methods for treating end-stage liver disease and liver regeneration should be explored. 10 μg/mL insulin, 5 μg/mL transferrin, and 5 ng/mL selenium. 104 Interestingly, in human hepatic and biliary tree stem/progenitor cells s-μg was shown to promote the generation of 3D spheroids and to stimulate the glycolytic metabolism, but also to favor maintenance of stemness and not the differentiation toward hepatocytes. 105

| General research on embryonic, pluripotent and MSCs
In general, studies on embryonic stem cells exposed to μg suggested that μg inhibits the stem cells' differentiation and retains their selfrenewal markers. 110 Its inhibitory effect could be due to downregulation of a number of genes in embryonic stem cells. 46  it was shown that r-μg prevented terminal differentiation of mESCs and promoted cell survival. 113 Studies on murine-induced pluripotent stem cells on the same mission seemed to suggest that r-μg promoted the expression of the pluripotency marker Oct4, therefore retaining stemness and resulting in a more dynamic behavior compared with ground controls. 114 When cultured under s-μg in rotary bioreactors in a differentiation medium, mESC-derived embryonic bodies showed a distinct differentiation pattern, which diverged from that observed in mESCs cultivated in spinner flasks using the same medium. While s-μg yielded more sca-1 + progenitors, spinner flasks generated more c-Kit + progenitors. 115 Using mESCs, Lei et al have shown that RCCS cultivation allows for a more controlled production of embryonic bodies with more uniform sizes and more regular shapes than those obtained from static culture techniques. They also reported that s-μg enhanced mesendoderm differentiation, whereas neuroectodermal differentiation was reduced; an effect the authors suggest was mediated by the Wnt/β-catenin pathway. 116,117 Human adipose tissue-derived MSCs (ADMSCs) cultured in s-μg in standard laboratory incubators alter their proliferation and differentiation. The ADMSCs were exposed for 14 days to an RPM and cultures under two oxygen concentrations: 5% and 20%. The authors showed that gene expression in RPM-exposed cells is differently modulated depending on the oxygen concentration. They also demonstrated that simulated μg influences the cytoskeleton, whereas oxygen is a key player, influencing the degree of these alterations. 118

| CONCLUSION AND FUTURE PERSPECTIVES
Exposing stem cells to μg conditions increased the current knowledge about tissue engineering of various tissues such as cartilage, bone, vasculature, heart, and liver tissue (Table 1, Figure 2). In addition, stem  15 whereas human colorectal cancer cell HCT116 exposed to the RCCS showed an elevated but staggered autophagic flux and increased stemness, including CD133/CD44 dual positive cells. 48 CSC generation from different cancer cell types exposed to μg was successful and μg simulation will be an excellent method to enrich the CD133-positive CSCs from various cancer cell lines. 55 This relatively small subset of cancer cells exhibits a strong capability of selfrenewal, is essentially resistant to chemotherapy, highly metastatic, and believed to be a key factor in tumor survival and progression. 119 Therefore, tests to evaluate the efficacy of chemotherapeutic drugs on cancer cells and CSCs in vitro prior to their use in the clinic may be a novel therapy option for cancer patients. The identification of new target structures involved in CSC proliferation and differentiation may furthermore lead to the development of novel tumor-suppressing drugs. Because of differing results obtained when using RPM, RCCS, or HFB devices, more research on different cancer types is necessary to study the biology of cancer stemness.
When comparing results from r-and s-μg, it should not be forgotten that there are some fundamental differences between the two conditions. Cell culture in r-μg in space takes place in an essentially forcefree environment without any perturbations of the culture medium except for the naturally occurring diffusion of nutrients and cellular waste products due to local concentration gradients. Cells on an s-μg device such as an RPM, RWV, RCSS, or CN on the other hand experience residual acceleration depending on their distance to the center of rotation, shear forces, and a constant mixture of the cell culture medium. 18 Although these effects are not necessarily detrimental, they introduce additional factors and variations over the course of the cell culture procedure, which will eventually lead to deviations between results from r-and s-μg experiments. The 14-day ESA-SPHEROIDS space mission investigated endothelial cells in space and revealed a scaffold-free formation of spheroids and TS in space. 8,120 Spheroid and TS formation can also be found when endothelial cells were exposed

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.