Stem Cell Culture in Microgravity and Its Application in Cell-Based Therapy

Introduction

The International Space Station (ISS) was established more than 15 years ago, and a lot of space experiments are being performed in ISS [1 –4]. Regarding life sciences, scientists have been interested in the anatomical or physiological changes that occur in an astronaut's body during space flight to understand the mechanisms underlying these microgravity-induced changes and develop strategies against space adaptation syndrome [5]. Several studies have demonstrated the dynamic changes at the cellular or molecular level during space flight [1,2,6 –10]. This has resulted in not only achieving a better understanding of the microgravity phenomenon but also its application for diverse objectives. For instance, space experiments have been used to monitor protein crystallization in microgravity [3]. Pietsch et al. [4] conducted a space experiment called “The SPHEROIDS project” in SpaceX CRS-8 ISS space mission to determine the impact of microgravity on the formation of three-dimensional (3D) structures, and revealed a scaffold-free formation of spheroids in space. A number of studies have shown changes in the characteristic features of cells cultured in microgravity [1,2,8 –12]. Most notably, one of the research revealed that gene expression related to neural development is upregulated in space [2]. Because our primary focus has been on the application of the microgravity environment in cell-based therapy for central nervous system (CNS) diseases, we found these data particularly interesting. In this study, we provide an overview of the current understanding about various aspects of changes that occur during space flight or in simulated microgravity environment, and discuss our recent data of the application of microgravity environment in the cell-based therapy for CNS diseases.

Effects of Space Flight on Somatic Cells and Stem Cells

It is well known that microgravity during space flight leads to physiological and anatomical changes in an astronaut's body, such as bone atrophy [5,7,13 –16], muscle atrophy [5,14 –17], and cardiovascular deconditioning [5,16,18 –22]. Along with physiological and anatomical changes in tissues and organs, dynamic changes at the cellular and molecular levels have also been reported during space flight [1,2,6 –10]. Lorenzi et al. [23] cultured hamster kidney cells (HaK) in their first International Microgravity Mission (IML-1) in Space laboratory. While they concluded that microgravity had no effect on cell growth and metabolism of HaK, later studies have clearly demonstrated dynamic changes in the structure and function of various cell types cultured under microgravity conditions [1,2,6 –11]. Meloni et al. [1] observed the cytoskeletal structure of human monocyte cells during space flight and showed that the exposure of monocyte cells to microgravity affected the distribution of the different filaments and reduced the fluorescence intensity of F-actin fibers. Monticone et al. [2] demonstrated interesting results obtained from experiments performed under the KUBIK aboard space mission ISS 12S from March 30 to April 8, 2006 (experiment “Stroma-2”). In their study, mouse bone marrow-derived mesenchymal stem cells (MSCs) were cultured in KUBIK for 200 h and stored until landing. Gene expression profiling showed that space flight-exposed mouse MSCs had higher expression of genes involved in neural development, neuron morphogenesis, and transmission of nerve impulse and synapse than those of MSCs cultured on the ground [2]. These studies showed that space flight results in cellular, anatomical, physiological, and functional changes. Furthermore, Casey et al. [12] stated that changes in gene expression during space flight are affected by epigenetic regulation, such as chromatin structure and DNA methylation. However, the mechanism underlying these microgravity-induced changes remains unknown. Further studies related to cellular changes due to space flight and their mechanisms are needed.

Stem Cell Culture in Simulated Microgravity

Many cell culture experiments have been performed under simulated microgravity on the ground as well as under true microgravity (space flight) to understand various changes associated with microgravity. For simulated microgravity experiments, various cell culture devices have been developed. We have developed a multidirectional gravity control device “Gravite^®” (Space Bio-Laboratories, Co., Ltd.) for simulating microgravity and hypergravity culture conditions. By the controlled rotation of two axes, this device minimizes the cumulative gravity vector at the center of the device, generating an average of 10⁻³ g over time. We cultured several different cell types obtained from various species in simulated microgravity and demonstrated that microgravity suppresses the differentiation of human osteoblasts [24], human MSCs [25], mouse MSCs [26], mouse embryonic stem (ES) cells [27], rat MSCs [28], and rat myoblasts [29,30]. We recently demonstrated that simulated microgravity attenuates myoblast differentiation by controlling DNA methylation by using Gravite [30]. Other groups have also published their experimental results regarding suppressing cell differentiation, which are consistent with our data. For instance, Chen et al. [31] and Hu et al. [32] demonstrated that microgravity inhibits the osteogenic differentiation of MSCs and rat osteoblasts. Chen et al. [31] illustrated mechanisms underlying the suppression of osteogenic differentiation by inhibiting PDZ-binding motif (TAZ), which acts as an important regulator of osteogenesis. Although several studies have reported the advantages of microgravity in promoting cell differentiation [33 –35], differences in cell type or culture devices might affect the consistency of the results.

Moreover, human MSCs cultured in simulated microgravity (MSCs-MG) showed marked proliferation compared with those cultured in normal gravity condition in our study [25]. Furthermore, cardiac progenitors derived from human pluripotent stem cells showed enhanced proliferation in simulated microgravity culture [34]. Kawahara et al. [27] reported that simulated microgravity allows leukemia inhibitory factor (LIF) and animal-derived material-free culture methods for mouse ES cells, and suggested the possibility of simulated microgravity culture as a novel and effective means for stem cell culture. Interestingly, simulated microgravity culture allows 3D culturing of cells [36]. Li et al. [37] also suggested that 3D simulated microgravity culture contributes to tissue engineering by improving the proliferation and differentiation ability of human dental pulp stem cells.

Thus, microgravity culture is not only a useful tool for understanding the changes occurring during space flight but also an optimal stem cell culture system. With the increasing popularity of cell culture in microgravity, National Aeronautics Space Administration established the Microgravity Simulator Facility (MSF) at Kennedy Space Center (KSC) to support research conducted by visiting scientists using various simulated microgravity devices. With the same objective, Gravite^® that we developed has already been introduced in MSF at KSC, and is being utilized by many scientists.

Cell-Based Therapy Using MSCs for CNS Diseases

Despite the development of innovative treatments, such as tissue plasminogen activator administration [38] and endovascular treatment [39] for patients with acute ischemic stroke and novel drugs for patients with spinal cord injury [40], it is still difficult to achieve dramatic recovery in patients with CNS diseases. Cell-based therapy has been gaining attention as a novel and effective therapeutic strategy for CNS diseases. MSC transplantation has been shown to promote the recovery of motor and somatosensory function in animal models for stroke [41 –45], spinal cord injury [46,47], and degenerative diseases such as amyotrophic lateral sclerosis [48]. Clinical trials for MSC transplantation have been conducted in ischemic stroke patients [49,50]. Lee et al. [49] evaluated the long-term safety and efficacy of intravenous autologous MSC transplantation for ischemic stroke patients, and suggested that intravenous MSC transplantation was safe during long-term follow-up for up to 5 years. The feasibility and safety of autologous MSCs cultured in autologous human serum have also been demonstrated, and phase III clinical trials to evaluate the efficacy of MSC transplantation for ischemic stroke patients are currently underway [50].

MSCs promote the functional recovery of target tissues after transplantation in several different ways. Homing and neural differentiation of transplanted MSCs at the lesion site is a principal recovery mechanism [51]. Chen et al. showed that bone marrow MSC transplantation along with the brain-derived neurotrophic factor (BDNF) into the ischemic boundary zone of rat brain enhances the differentiation of MSCs and improves the functional recovery after middle cerebral artery occlusion [51]. However, the differentiation potential of MSCs in vivo is still unclear because very few transplanted MSCs are detected at the lesion site [52]. Transplanted MSCs also modulate the inflammatory response to aid in functional recovery [53]. In addition, paracrine signaling mediated by growth factors and chemokines released by transplanted MSCs, such as neurotrophins 1 or 2, BDNF, grail cell line-derived neurotrophic factor, and vascular endothelial growth factor, promote functional recovery in CNS diseases [52 –54].

In fact, Neirinckx et al. [55] showed that MSCs were able to secrete a lot of growth factors and chemokines, and MSC transplantation modified the inflammatory reaction at the lesion site in acute spinal cord injury model mice.

Application of Microgravity in Cell-Based Therapy

Several studies have provided evidence for microgravity-induced changes in cell cultures [1,2,8 –12]. In particular, the space experiment Stroma-2 revealed changes in the expression of genes involved in neural development [2]. Based on these findings, we hypothesized that MSC cultures maintained in microgravity may be used for cell-based therapy in CNS diseases. We showed, for the first time, a critically high therapeutic effect of mouse MSCs-MG [26]. In this study, brain-injured mice were transplanted with MSCs cultured in normal gravity (MSCs-1G) or MSCs-MG. MSCs-MG expressed C–X–C chemokine receptor type 4 (CXCR4), an important gene for cell homing to the injured site, and exhibited greater migration and survival potential in the lesion area. Mice transplanted with MSCs-MG showed greater motor functional recovery than those transplanted with MSCs-1G. Similarly, Mitsuhara et al. [28] reported therapeutic effects of MSCs-MG in a rat model of spinal cord injury. The expression of apoptosis-inhibiting factor at the lesion site and motor functional recovery were higher in MSCs-MG-transplanted rats than in MSCs-1G-transplanted ones. Although detailed treatment mechanisms remained unclear, these studies revealed that simulated microgravity culture could enhance therapeutic effects of MSCs for CNS diseases. We recently demonstrated the therapeutic properties of human MSCs-MG against apoptosis and inflammation in the mouse model of acute injury [56]. The human MSCs-MGs showed significantly higher expression of hepatocyte growth factor (HGF) and transforming growth factor-beta (TGFb) than MSCs-1G in vitro [56]; both HGF and TGFb are known to play important roles in anti-inflammation and antiapoptosis [57 –61]. Moreover, the expression of inflammation and apoptosis-related proteins at the brain injury site was significantly suppressed in the mice transplanted with MSCs-MG, suggesting that MSCs-MG transplantation has a high potential to reduce inflammation and apoptosis following acute injury. From our stem cell transplantation experiments using MSCs-MG, it seems that simulating microgravity would be a useful culture environment to enhance therapeutic effects of MSCs using cell-based therapy for CNS disease.