A three-dimensional (3D) clinostat is a device for generating multidirectional G force, resulting in an environment with an average of 10(3) G. Here we report that human mesenchymal stem cells (hMSCs) cultured in a 3D-clinostat (group CL) showed marked proliferation (13-fold in a week) compared with cells cultured under normal conditions of 1 G (group C) (4-fold in a week). Flow cytometry revealed a 6-fold increase in the number of hMSCs double-positive for CD44/CD29 or CD90/CD29 in group CL after 7 days in culture, compared with group C. Telomere length remained the same in cells from both groups during culturing. Group C cells showed increasing expression levels of type II collagen and aggrecan over the culture period, whereas group CL cells showed a decrease to undetectable levels. Pellets of hMSCs from each group were explanted into cartilagedefective mice. The transplants from group CL formed hyaline cartilage after 7 days, whereas the transplants from group C formed only noncartilage tissue containing a small number of cells. These results show that hMSCs cultured in a 3D-clinostat possess the strong proliferative characteristic of stem cells and retain their ability to differentiate into hyaline cartilage after transplantation. On the contrary, cells cultured in a 1-G environment do not maintain these features. Simulated microgravity may thus provide an environment to successfully expand stem cell populations in vitro without culture supplements that can adversely affect stem cell-derived transplantations. This method has significant potential for regenerative medicine and developmental biology.

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The functional disorders caused by central nervous system (CNS) diseases, such as ischemic stroke, are clinically incurable and current treatments have limited effects. Previous studies suggested that cell-based therapy using mesenchymal stem cells (MSCs) exerts therapeutic effects for ischemic stroke. In addition, the characteristics of MSCs may depend on their sources. Among the derived tissues of MSCs, we have focused on cranial bones originating from the neural crest. We previously demonstrated that the neurogenic potential of human cranial bone-derived MSCs (cMSCs) was higher than that of human iliac bone-derived MSCs. Therefore, we presumed that cMSCs have a higher therapeutic potential for CNS diseases. However, the therapeutic effects of cMSCs have not yet been elucidated in detail. In the present study, we aimed to demonstrate the therapeutic effects of transplantation with rat cranial bone-derived MSCs (rcMSCs) in ischemic stroke model rats. The mRNA expression of brain-derived neurotrophic factor and nerve growth factor was significantly stronger in rcMSCs than in rat bone marrow-derived MSCs (rbMSCs). Ischemic stroke model rats in the rcMSC transplantation group showed better functional recovery than those in the no transplantation and rbMSC transplantation groups. Furthermore, in the in vitro study, the conditioned medium of rcMSCs significantly suppressed the death of neuroblastoma × glioma hybrid cells (NG108-15) exposed to oxidative and inflammatory stresses. These results suggest that cMSCs have potential as a candidate cell-based therapy for CNS diseases.

Recently, regenerative medicine with bone marrow stromal cells (BMSCs) has gained significant attention for the treatment of central nervous system diseases. Here, we investigated the activity of BMSCs under simulated microgravity conditions. Mouse BMSCs (mBMSCs) were isolated from C57BL/6 mice and harvested in 1G condition. Subjects were divided into 4 groups: cultured under simulated microgravity and 1G condition in growth medium and neural differentiation medium. After 7 days of culture, the mBMSCs were used for morphological analysis, reverse transcription (RT)-polymerase chain reaction, immunostaining analysis, and grafting. Neural-induced mBMSCs cultured under 1G conditions exhibited neural differentiation, whereas those cultured under simulated microgravity did not. Moreover, under simulated microgravity conditions, mBMSCs could be cultured in an undifferentiated state. Next, we intravenously injected cells into a mouse model of cerebral contusion. Graft mBMSCs cultured under simulated microgravity exhibited greater survival in the damaged region, and the motor function of the grafted mice improved significantly. mBMSCs cultured under simulated microgravity expressed CXCR4 on their cell membrane. Our study indicates that culturing cells under simulated microgravity enhances their survival rate by maintaining an undifferentiated state of cells, making this a potentially attractive method for culturing donor cells to be used in grafting.

Objective: We investigated whether neural stem cells (NSC) with transgenic expression of human nerve growth factor (hNGF) transplanted into the brain could offer a therapeutic option for the treatment of Alzheimer’s disease (AD). Methods: We infused okadaic acid into rat lateral ventricles to establish a chronic AD animal model. In addition, NSC were stably transduced with hNGF and enhanced green fluorescent protein (eGFP) genes (NSC-hNGF-eGFP) by using a recombination adeno-associated virus serotype 2 (rAAV2) vector. These genetically modified stem cells were grafted into the cerebral cortex of AD rats. Results: AD model rats showed significant damage in learning and memory function, with the formation of senile plaques and neurofibrillary tangles in the cerebral cortex. The transferred hNGF gene conferred stable and high levels of protein expression in NSC in vitro. Moreover, the NSC-hNGF-eGFP, but not the NSC, survived, integrating into the host brain and enhancing cognitive performance after transplantation. Conclusion: The injection of okadaic acid into rat lateral ventricles constitutes a promising animal model for investigating selective aspects of AD. rAAV2-mediated hNGF delivery can render long-term and stable transduction of hNGF in NSC. NSC-hNGF-eGFP transplantation may offer a viable therapeutic approach for treatment of AD.