Soichi Takagi

Cell transplantation of adipose tissue-derived stem cells in combination with heparin attenuated acute liver failure in mice.

The effect of adipose tissue-derived stem cells (ASCs) in combination with heparin transplantation on acute liver failure mice with carbon tetrachloride (CCl4) injection was investigated. CCl4 is a well-known hepatotoxin and induces hepatic necrosis. Heparin did not affect the viability of ASCs for at least 24 h. The injection of heparin into the caudal tail vein decreased slightly the activities of the alanine aminotransferase (ALT), asparate aminotransferase (AST), and lactate dehydrogenase (LDH) in plasma. In the transplantation of ASCs (1 × 106 cells) group, there was a trend toward decreased activities of all markers. However, four out of six mice died of the lung infarction. In the transplantation of ASCs in combination with heparin group, there was also a trend toward decreased activities of all markers. In addition, all mice survived for at least the duration of the study period. In conclusion, the transplantation of ASCs in combination with heparin was thus found to effectively treat acute liver failure.

Phospholipase Cζ mRNA expression and its potency during spermatogenesis for activation of quail oocyte as a sperm factor

This study was conducted to investigate the role of a sperm‐borne compound in oocyte activation in special reference to the time when oocyte activation is required by testicular cells during spermatogenesis in quail. First, effects of a microinjection of quail sperm extract (SE) and quail phospholipase Cζ (PLCζ) cRNA into quail oocytes were assessed by observation of pronuclear formation and cytoplasmic segmentation, respectively. Secondly, the effects of a microinjection of round spermatids with or without PLCζ cRNA into quail oocytes were studied by observation of development. When the oocytes were injected with SE at 0.13 mg protein/ml, both pronuclear formation and cytoplasmic segmentation were optimally induced. However, pronuclear formation was blocked when SE was pretreated with heat or when the oocyte was pretreated with BAPTA (a Ca2+ chelator) before SE injection. On the other hand, when the oocytes were injected with PLCζ cRNA at 60 µg/ml, not only pronuclear formation but also cytoplasmic segmentation were optimally induced. However, PLCζ cRNA‐induced pronuclear formation was blocked by pretreatment with cycloheximide (an inhibitor of protein synthesis) or with BAPTA. Most interestingly, round spermatids alone cannot induce blastodermal development but microinjection of a round spermatid with PLCζ cRNA can induce development. In addition, RT‐PCR revealed that PLCζ mRNA is expressed in elongated spermatids and testicular sperm but not in round spermatids. It is concluded that PLCζ is a functional sperm factor for oocyte activation to initiate resumption of meiotic division in quail and its potency is acquired after elongated spermatid formation during the spermatogenesis. Mol. Reprod. Dev. 76: 1200–1207, 2009.

Fertilization and development of quail oocytes after intracytoplasmic sperm injection.

Abstract The present study was conducted to establish the intracytoplasmic sperm injection (ICSI) method for in vitro fertilization and development in quail. The efficiency of fertilization of oocytes was compared 1) between spontaneous and premature ovulation and 2) among testicular round spermatids, elongated spermatids, and immature and mature spermatozoa. The oocytes were injected with a single spermatozoon or spermatid and cultured for 24 h. Cell division was histologically observed with hematoxylin-eosin (HE) and a nucleus-specific fluorescent dye (DAPI). Five of 30 (16.6%) and 4 of 30 (13.3%) oocytes injected with mature sperm were fertilized in the spontaneous and induced ovulation group, respectively. Those embryos showed development at stages II–VII. Half the number (three of six) of the oocytes injected with testicular spermatozoa were fertilized and developed to stages IV–VII, and two of five oocytes injected with elongated spermatids were fertilized and developed to stage VI. All ooocytes injected with round spermatids were unfertilized. The results demonstrate that intracytoplasmic injection of a single sperm into quail oocyte can activate the oocyte and lead to fertilization. Oocytes prematurely ovulated are capable of fertilizing with mature sperm as are those spontaneously ovulated. In addition, the results suggest that the testicular round spermatids may not possess sufficient oocyte-activating potency but that the elongated spermatids and immature spermatozoa are competent to participate in fertilization and early embryonic development in quail.

Engineering Liver Tissues Under the Kidney Capsule Site Provides Therapeutic Effects to Hemophilia B Mice

Recent advances in liver tissue engineering have encouraged further investigation into the evaluation of therapeutic benefits based on animal disease models. In the present study, liver tissues were engineered in coagulation factor IX knockout (FIX-KO) mice, a mouse model of hemophilia B, to determine if the tissue engineering approach would provide therapeutic benefits. Primary hepatocytes were isolated from the liver of wild-type mice and suspended in a mixture of culture medium and extracellular matrix components. The hepatocyte suspension was injected into the space under the bilateral kidney capsules of the FIX-KO mice to engineer liver tissues. The plasma FIX activities (FIX:C) of the untreated FIX-KO mice were undetectable at any time point. In contrast, the liver tissue engineered FIX-KO mice achieved 1.5–2.5% of plasma FIX activities (FIX:C) and this elevated FIX:C level persisted throughout the 90 day experimental period. Significant FIX mRNA expression levels were found in the engineered liver tissues at levels similar to the wild-type livers. The present study demonstrates that liver tissue engineering could provide therapeutic benefits in the treatment of hemophilia B.

Reconstruction of functional endometrium-like tissue in vitro and in vivo using cell sheet engineering.

Uterus is a female specific reproductive organ and plays critical roles in allowing embryo to grow. Therefore, the endometrial disorders lead to female infertility. Hence, the regeneration of endometrium allowing fertilized ovum to implant might be valuable in the field of fertility treatment. Recently, cell sheet engineering using a temperature-responsive culture dish has advanced in regenerative medicine. With this technology, endometrial cells were harvested as a contiguous cell sheet by reducing temperature. Firstly, mouse endometrial cell sheets were re-cultured for 3 days to evaluate the function. Histological analyses revealed that endometrial epithelial cell-specific cytokeratin 18 and female-specific hormone receptors, estrogen receptor β and progesterone receptor, were expressed. Furthermore, endometrial epithelial cells constructed epithelial layer at the apical side. Then, endometrial cell sheets from green-fluorescent-protein rat cells were transplanted onto the buttock muscle of nude rat for evaluating the function in vivo. Histological analyses showed that endometrial cell sheets reconstructed endometrium-like tissue, which was found to form uterus-specific endometrial glands having hormonal receptor to estrogen. In this study, endometrial cell sheets were speculated to contribute to the regeneration of functional endometrium as a new therapy.

Suitable reference genes for the analysis of direct hyperplasia in mice

The liver is capable of undergoing a proliferative growth, known as direct hyperplasia, in which the naïve liver increases in size due to stimulation with primary mitogens. To produce accurate gene expression data, housekeeping genes (HKGs) that are stably expressed need to be determined. In the present study, liver regeneration was promoted via the direct hyperplasia mode by inducing mice with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. Gene expression levels of nine commonly used HKGs were analyzed in the liver of different timing during the regeneration. The stability of gene expression was assessed using two different analysis programs, geNorm and NormFinder. Using these analyses, we identified that PPIA and RPL4 showed the most stable expression regardless of the status of the liver regeneration. In conclusion, the present study demonstrated that the use of PPIA and RPL4 were the most optimal in providing reliable normalization of gene expression when assessing liver regeneration attributed to direct hyperplasia.

Novel Method of Gene Transfer in Birds: Intracytoplasmic Sperm Injection for Green Fluorescent Protein Expression in Quail Blastoderms

This study was conducted to establish a new method of avian transgenesis by intracytoplasmic sperm injection (ICSI). First, we evaluated the fertilization ability of quail oocytes after microinjection of Triton X-100 (TX-100)-treated quail sperm with PLCZ cRNA. The quail oocytes were cultured for 24 h, and blastoderm development was examined by histological observation. The TX-100 treatment induced damage to the quail sperm membrane and interfered with fertilization of oocytes injected with sperm. On the other hand, when quail oocytes were injected with TX-100-treated sperm and PLCZ cRNA simultaneously, 43.5% (10/23) of the oocytes developed into blastoderms. This rate of development was comparable to that for oocytes injected with sperm without TX-100 treatment but with PLCZ cRNA (6 [42.9%] of 14). Second, we evaluated the rate of transduction of the enhanced green fluorescent protein (EGFP) gene in quail oocytes injected with TX-100-treated sperm and PLCZ cRNA. The EGFP expression was assessed by histological observation of fluorescence emission in the embryos. The intracytoplasmic injection of sperm without TX-100 treatment but with PLCZ cRNA and EGFP vector induced blastoderm development in 40% (4/10) of the oocytes, but those oocytes showed no fluorescence emission. In contrast, the intracytoplasmic injection of TX-100-treated sperm and PLCZ cRNA induced blastoderm development in 43.8% (7/16) of the oocytes, and, importantly, 85.7% (6/7) of oocytes showed fluorescence emission. In addition, PCR analysis detected GFP fragments in 50% (3/6) of GFP-expressing blastoderms. These results indicate that this ICSI method with additional treatments described herein may be the first step toward the production of transgenic birds.

Facile cell sheet manipulation and transplantation by using in situ gelation method

Cell sheets harvested from temperature-responsive cell culture dishes (TRDs) has attracted considerable attention as effective tools for reconstructing the lost functions of tissues and organs in the regenerative medicine field. However, because of their thinness, handling problems sometimes arise when transferring cell sheets from a TRD to a target surface. In this study, we developed a facile cell transfer method referred to as in situ gelation by using both gelatin hydrogel and a support membrane. Gelation and low-temperature processes were simultaneously performed on TRD. Confluent cultured cells were efficiently harvested from TRD in less than 5 min by decreasing the incubation temperature to 20°C. Harvested cells were found to maintain their cell viability, extracellular matrix, and original shape, thus allowing transfer of the cells to another surface with a short incubation time at 37°C. This method is applicable for various cell types regardless of the formation of tight cell-cell junctions. In addition, because of the high flexibility of the gelatin-coated membrane, cells were efficiently transferred to the surface of a mouse subcutis and liver. When compared with conventional cell sheet manipulation methods, the interaction between the cell surface and membrane was reinforced by the uniformly formed gelatin gel layer without using a special device. Therefore, the in situ gelation method is a promising technique for cell sheet-based tissue engineering and regenerative medicine.

Cell shape regulation based on hepatocyte sheet engineering technologies.

The de novo engineering of a uniform hepatocyte sheet in vitro is considered as a novel approach for liver-directed therapeutics. Hepatocytes can be cultured on a temperature-responsive culture dishes coated with poly(N-isopropylacrylamide) (PIPAAm). Following multiple days of culturing, the hepatocytes can be easily harvested as a uniform sheet by decreasing temperature from 37°C to 20°C. By modifying the sheet harvesting protocol, we have noticed that two different forms of the hepatocyte sheets, “extended” and “shrinking,” were obtained. This study describes the methods for harvesting the two different forms of sheets, and their cellular structure and hepatocyte-specific functions. To obtain an “extended sheet” form, a cluster of hepatocytes covered with a support membrane was harvested by the temperature reduction. For the “shrinking sheet” form, the hepatocyte sheet was floated after reducing the culture temperature, and the floating process allowed the sheet to shrink spontaneously. Histological analysis revealed that the hepatocytes in the extended sheet form were predominantly flat, whereas the shrinking sheet contained cuboidal shaped hepatocytes. The preservation of hepatocyte-specific ultrastructures was confirmed in both types of sheets. To investigate hepatocyte-specific functionality, the harvested hepatocyte sheets were recultured on Matrigel-coated dishes. Assessment of protein production levels and chemical metabolizing activities showed the similar functionalities for each form. In contrast, the recalculation of these values per sheet versus per square centimeter of sheet surface demonstrated that the function of the shrinking sheet was significantly higher than that of the extended sheets. This study demonstrated that the hepatocyte sheets created on the PIPAAm dish could spontaneously shrink in size, but retain their hepatocyte functionality. This type of hepatocyte sheet could be utilized for the engineering of liver tissue in limited areas that are unable to give adequate transplant space.

Transplantation of cancerous cell sheets effectively generates tumour-bearing model mice.

Tumour‐bearing mice were created by transplanting cancerous cell sheets onto the subcutaneous tissue of the dorsal region, using luciferase gene‐transfected mammary gland adenocarcinoma cells, 4T1‐luc2, to investigate the tumourigenicity of the cell sheet relative to a conventional injection of cell suspension. Contiguous breast cancerous cell sheets were harvested from temperature‐responsive culture dishes by reducing the temperature from 37 °C to 20 °C; the sheets were then transplanted onto the dorsal side of the mouse subcutaneous tissue, using a chitin‐based supporting membrane. Cell suspensions obtained by trypsin digestion were subcutaneously injected into the dorsal region of mice. The tumour growth of the transplanted cancer cells was evaluated by the tumour volume and by the bioluminescence from luciferase‐gene transfected cancer cells, using an in vivo imaging system. The cell sheet method improved the 4 T1‐luc2 engraftment efficiency in living mouse tissues at the initial stage by 13‐fold compared with that from injecting cell suspensions. On day 14 after the transplantation, the tumour formation at the transplanted area of cell sheet‐transplanted mice also accelerated, and the mean tumour volume became 1116 mm3, which was 10 times larger than that in cell suspension‐transplanted mice. The cell sheets engrafted on the recipient tissues efficiently due to the preserved extracellular matrix on their basal sides, such that cancer cells were supplied with sufficient oxygen and nutrients from the host tissues to develop tumour tissues. Therefore, cancerous cell sheet‐based transplantation is a promising method for efficiently creating cancer‐bearing mice. Copyright