Keiji Itaka

Treatment of Bone Defects by Transplantation of Genetically Modified Mesenchymal Stem Cell Spheroids

Cell transplantation is promising for regenerative medicine. A combination of a three-dimensional spheroid culture system with gene transfection was developed to enhance the therapeutic effects of mesenchymal stem cell (MSC) transplantation. The spheroid cell culture system is based on micropatterned substrates composed of a regular array of 100-μm-diameter cell-adhesion areas coated with a temperature-responsive polymer, poly (N-isopropylacrylamide-co-methacrylic acid), which allows for spheroid detachment by simply cooling the plates. In this study, MSC spheroids were transfected with plasmid DNA encoding runt-related transcription factor 2 (Runx2) and tested for their ability to enhance bone regeneration. In vitro analyses revealed that osteogenic differentiation of the MSCs was enhanced by forming spheroids and was further promoted by Runx2 expression. The enhanced osteogenic differentiation was maintained under pathological conditions, such as hypoxia and inflammation. Transplanting Runx2-transfected MSC spheroids into bone defects on rat femurs induced significantly faster bone regeneration compared with nontransfected MSC spheroids or genetically modified MSCs from conventional monolayer culture. MSC migration into the bone defect area was enhanced by upregulation of cell-migration-related genes. In conclusion, genetically modified MSC spheroids are effective for enhancing bone regeneration, providing a promising option for cell transplantation therapy in the fields of regenerative medicine.

Prolonged engraftment of transplanted hepatocytes in the liver by transient pro-survival factor supplementation using ex vivo mRNA transfection

ABSTRACT Cell transplantation therapy needs engraftment efficiency improvement of transplanted cells to the host tissues. Ex vivo transfection of a pro‐survival gene to transplanted cells is a possible solution; however prolonged expression and/or genomic integration of the gene can be cancer promoting. To supply pro‐survival protein only when it is needed, we used mRNA transfection, which exhibits transient protein expression profiles without the risk of genomic integration. Ex vivo transfection of mRNA encoding Bcl‐2, a pro‐survival factor, led to enhanced hepatocyte engraftment in both of normal and diseased mouse liver, effectively supporting liver function in a model of chronic hepatitis. The transplanted hepatocytes maintained their viability and function in the liver for at least one month, though Bcl‐2 expression from mRNA was sustained for just a few days. Mechanism analyses suggest that Bcl‐2 inhibits Kupffer cell‐mediated hepatocyte clearance, which occurs within 2days after transplantation. Within 2days, hepatocytes migrated to the liver parenchyma, presumably a suitable place for the hepatocytes to survive without Bcl‐2 expression. Thus, the duration of Bcl‐2 expression from mRNA was sufficient to achieve prolonged engraftment. Ex vivo mRNA transfection allows supply of pro‐survival factors to transplanted cells with minimal safety concerns accompanying prolonged expression, providing an effective platform to improve engraftment efficiency in cell transplantation therapy. HIGHLIGHTSCell transplantation therapy needs engraftment efficiency improvement.Pro‐survival mRNA transfection is a safe option to improve engraftment efficiency.Ex vivo Bcl‐2 mRNA transfection to hepatocytes improved their engraftment to liver.Transient Bcl‐2 expression from mRNA allowed long‐term hepatocyte engraftment.Bcl‐2‐mRNA‐transfected hepatocytes improved liver function in mice with hepatitis.

Designing immunostimulatory double stranded messenger RNA with maintained translational activity through hybridization with poly A sequences for effective vaccination

Messenger (m)RNA vaccines require a safe and potent immunostimulatory adjuvant. In this study, we introduced immunostimulatory properties directly into mRNA molecules by hybridizing them with complementary RNA to create highly immunogenic double stranded (ds)RNAs. These dsRNA formulations, comprised entirely of RNA, are expected to be safe and highly efficient due to antigen expression and immunostimulation occurring simultaneously in the same antigen presenting cells. In this strategy, design of dsRNA is important. Indeed, hybridization using full-length antisense (as)RNA drastically reduced translational efficiency. In contrast, by limiting the hybridized portion to the mRNA poly A region, efficient translation and intense immunostimulation was simultaneously obtained. The immune response to the poly U-hybridized mRNAs (mRNA:pU) was mediated through Toll-like receptor (TLR)-3 and retinoic acid-inducible gene (RIG)-I. We also demonstrated that mRNA:pU activation of mouse and human dendritic cells was significantly more effective than activation using single stranded mRNA. Inxa0vivo mouse immunization experiments using ovalbumin showed that mRNA:pU significantly enhanced the intensity of specific cellular and humoral immune responses, compared to single stranded mRNA. Our novel mRNA:pU formulation can be delivered using a variety of mRNA carriers depending on the purpose and delivery route, providing a versatile platform for improving mRNA vaccine efficiency.

Combined CatWalk Index: an improved method to measure mouse motor function using the automated gait analysis system

ObjectiveMeasuring motor function in mice is important for studying models of spinal cord injury (SCI) or other diseases. Several methods exist based on visual observation of mice moving in an open field. Though these methods require very little equipment, observers must be trained, and the possibility of human error or subjectivity cannot be eliminated. The Noldus CatWalk XT Automated Gait Analysis system assesses mouse motor function by taking high-resolution videos of the mice, with specialized software to measure several aspects of the animal’s gait. This instrument reduces the possibility of human error, but it is not always clear what data is important for assessing motor function. This study used data collected during mouse SCI experiments to create a simple mathematical model that combines the data collected by the CatWalk system into a single score, the Combined CatWalk Index or CCI.ResultsThe CCI system produces similar results to the Basso Mouse Scale or the CatWalk’s Step Sequence Regularity Index. However, the CCI has a significantly smaller coefficient of variation than either other method. Additionally, CCI scoring shows slightly better correlation with impact force. The CCI system is likely to be a useful tool forxa0SCI research.