國田 智

The hepatitis E virus (HEV) is a causative agent of hepatitis E. HEV virions in circulating blood and culture media are quasi-enveloped, while those in feces are nonenveloped. The capsid (ORF2) protein associated with an enveloped HEV virion is reported to comprise the translation product of leucine 14/methionine 16 to 660 (C-terminal end). However, the nature of the ORF2 protein associated with fecal HEV remains unclear. In the present study, we compared the molecular size of the ORF2 protein among fecal HEV, cell-culture-generated HEV (HEVcc), and detergent-treated protease-digested HEVcc. The ORF2 proteins associated with fecal HEV were C-terminally truncated and showed the same size as those of the detergent-treated protease-digested HEVcc virions (60 kDa), in contrast to those of the HEVcc (68 kDa). The structure prediction of the ORF2 protein (in line with previous studies) demonstrated that the C-terminal region (54 amino acids) of an ORF2 protein is in flux, suggesting that proteases target this region. The nonenveloped nondigested HEV structure prediction indicates that the C-terminal region of the ORF2 protein moves to the surface of the virion and is unnecessary for HEV infection. Our findings clarify the maturation of nonenveloped HEV and will be useful for studies on the HEV lifecycle.

Rat hepatitis E virus (HEV) has been isolated from wild rats worldwide and the potential of zoonotic transmission has been documented. Escherichia coli (E. coli) is utilized as an effective system for producing HEV-like particles. However, the production of rat HEV ORF2 proteins in E. coli forming virus-like particles (VLPs) has not yet been reported. In this study, nine rat HEV ORF2 proteins of the ratELOMB-131L strain with truncated N- and C-termini (amino acids 339-594, 349-594, 351-594, 354-594, 357-594, 357-599, 357-604, 357-609, and 357-614 of ORF2 protein) were expressed in E. coli and the 357-614 protein self-assembled most efficiently. A bioanalyzer showed that the purified 357-614 protein has a molecular weight of 33.5 kDa and a purity of 93.2%. Electron microscopy revealed that the purified 33.5 kDa protein formed VLPs with a diameter of 21-52 (average 32) nm, and immunoelectron microscopy using an anti-rat HEV ORF2 monoclonal antibody (TA7014) indicated that the observed VLPs were derived from rat HEV ORF2. The VLPs attached to and entered the PLC/PRF/5 cells and blocked the neutralization of rat HEV by TA7014, suggesting that the VLPs possess the antigenic structure of infectious rat HEV particles. In addition, rat HEV VLPs showed high immunogenicity in mice. The present results would be useful for future studies on the development of VLP-based vaccines for HEV prevention in a rat model and for the prevention of rat HEV infection in humans.

The severe shortage of donor hearts hampered the cardiac transplantation to patients with advanced heart failure. Therefore, cardiac regenerative therapies are eagerly awaited as a substitution. Human induced pluripotent stem cells (hiPSCs) are realistic cell source for regenerative cardiomyocytes. The hiPSC-derived cardiomyocytes are highly expected to help the recovery of heart. Avoidance of teratoma formation and large-scale culture of cardiomyocytes are definitely necessary for clinical setting. The combination of pure cardiac spheroids and gelatin hydrogel succeeded to recover reduced ejection fraction. The feasible transplantation strategy including transplantation device for regenerative cardiomyocytes are established in this study.

BACKGROUND: Induced pluripotent stem cell (iPSC)‒based regenerative therapy is a promising strategy for cardiovascular disease treatment; however, the method is limited by the myocardial retention of grafted iPSCs. Thus, an injection protocol that efficiently introduces and retains human iPSC-derived cardiomyocytes (hiPSC-CMs) within the myocardium is urgently needed. The objective of the present study was to develop a method to improve the retention of hiPSCs in the myocardium for cardiac therapy. METHODS: We efficiently produced hiPSC-CM spheroids in 3-dimensional (3D) culture using microwell plates, and developed an injection device for optimal 3D distribution of the spheroids in the myocardial layer. Device biocompatibility was assessed with purified hiPSC-CM spheroids. Device effectiveness was evaluated in 10- to 15-month-old farm pigs (n = 15) and 5- to 24-month-old micro-minipigs (n = 20). The pigs were euthanized after injection, and tissues were harvested for retention and histologic analysis. RESULTS: We demonstrated an injection device for direct intramyocardial transplantation of hiPSC-CM spheroids from large-scale culture. The device had no detrimental effects on cell viability, spheroid shape, or size. Direct epicardial injection of spheroids mixed with gelatin hydrogel into beating porcine hearts using this device resulted in better distribution and retention of transplanted spheroids in a layer within the myocardium than did conventional needle injection procedures. CONCLUSIONS: The combination of the newly developed transplant device and spheroid formation promotes the retention of transplanted CMs. These findings support the clinical application of hiPSC-CM spheroid‒based cardiac regenerative therapy in patients with heart failure.

Pigs with X-linked severe combined immunodeficiency (X-SCID) caused by a mutation of the interleukin-2 receptor gamma chain gene (IL2RG) are of value for a wide range of studies. However, they do not survive longer than 8 weeks because of their susceptibility to infections. To allow longer survival of X-SCID pigs, the animals must be born and reared under germ-free conditions. Here, we established an efficient system for piglet derivation by hysterectomy and used it to obtain and maintain a germ-free X-SCID pig. In four trials using pregnant wild-type pigs, 66% of piglets after hysterectomy started spontaneous breathing (range of 20-100% per litter). The resuscitation rate was found to negatively correlate with elapsed time from the uterus excision to piglet derivation (r=-0.97, P<0.05). Therefore, it is critical to deliver piglets within 5 min to achieve a high resuscitation rate (82% estimated from regression analysis). In a fifth trial with an IL2RG+/- pig, four piglets were delivered within 4.2 min of uterus excision and three were alive (75%). One of the live born piglets was genotypically and phenotypically determined to be X-SCID and was reared for 12 weeks. The X-SCID piglet was free from both bacteria and fungi at all time points tested by microbial culture and grew without any abnormal signs or symptoms. This study showed successful production and rearing of germ-free pigs, enabling experiments involving long-term follow-up of X-SCID pigs.

Rat hepatitis E virus (ratHEV) genome has four open reading frames (ORFs: ORF1, ORF2, ORF3 and ORF4). The functions of ORF3 and ORF4 are unknown. An infectious cDNA clone (pUC-ratELOMB-131L_wt, wt) and its derivatives including ORF3-defective (ΔORF3) and ORF4-defective (ΔORF4) mutants, were constructed and their full-length RNA transcripts transfected into PLC/PRF/5 cells. ΔORF3 replicated as efficiently as wt in cells. However, ≤1/1000 of the number of progenies were detectable in the culture supernatant of ΔORF3-infected cells compared with wt-infected cells. ORF4 protein was not detectable in ratHEV-infected cells or in the liver tissues of ratHEV-infected rats. No marked differences were noted between wt and ΔORF4 regarding the viral replication and protein expression. ORF3 mutants with proline-to-leucine mutations at amino acids (aa) 93, 96 and/or 98 in ORF3 were constructed and transfected into PLC/PRF/5 cells. Wt and an ORF3 mutant with leucine at aa 98 (ORF3-L98) replicated efficiently (density 1.15-1.16 g/cm3), while ORF3-L93 + L96 exhibited a decreased viral release and banded at 1.26-1.27 g/cm3, similar to ΔORF3. In conclusion, the ORF3 protein, especially its proline residues at aa 93 and 96, is essential for the release of membrane-associated ratHEV particles, and ORF4 is unnecessary for the replication of ratHEV.

Center for Development of Advanced Medical Technology (CDAMTec) in Jichi Medical University was established in 2009. It is the first educational research facility specialized for medical research and training using swine in Japan. Preclinical studies on large animals are essential prior to clinical trials to develop regenerative medical products and medical equipment. We have continued comprehensively considering using miniature swine for experiments to develop advanced medical technologies and train physicians with advanced clinical abilities, while paying attention to animal welfare. The center plays a pioneering role in this field by accumulating know-how such as (1) Construction and effective utilization of research facilities, (2) Procurement of quality animal resources, (3) Education and training of technical staff, (4) Establishment of support system for physicians and researchers. We now open up widely these expertise and foundation for medical research and training not only within our university but also outside the university, so as to move faster to practical use of advanced medical technology and contribute to human health and welfare.

Eight murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the C-terminal 15-amino-acid portion of the ORF3 protein of rat hepatitis E virus (ratHEV) were produced and characterized. Immunofluorescence assays using the anti-ratHEV ORF3 MAbs revealed the accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmids or inoculated with cell-culture-generated ratHEV strains. Anti-ORF3 MAbs could capture ratHEV particles in culture supernatant and serum following treatment with 0.5 % deoxycholate, but not those without prior detergent treatment or fecal ratHEV particles. Following treatment with 0.5 % deoxycholate and 0.5 % trypsin, the buoyant density of ratHEV particles in culture supernatant with ORF3 protein on the surface shifted from 1.15 g/cm3 to 1.26 g/cm3 in a sucrose gradient; the resulting particles were capturable by an anti-ORF2 MAb but not by an anti-ORF3 MAb. This indicates that the ORF3 protein (at least its C-terminal portion) is incorporated into the enveloped ratHEV virions released from infected cells but that it is not found in the virions in the feces, supporting the hypothesis that the ratHEV ORF3 protein is associated with the egress of virions from infected cells, similar to human HEV, despite the fact that the ratHEV ORF3 protein lacks a PSAP amino acid motif.

As the phenotype of a given single-gene mutation in mice is modulated by the genetic background of the inbred strain, embryonic stem (ES) cells derived from various inbred mouse strains are required to produce gene-targeted mice without the need for backcrossing and for detailed analysis of gene function in vivo. Here, we performed a comparative investigation of the effects of three culture conditions, LIF + KSR/ES medium described previously, High LIF + KSR/ES medium and iSTEM + LIF medium containing three inhibitors of glycogen synthase kinase 3, mitogen-activated protein kinase kinase, and fibroblast growth factor receptor signaling (3i), on the establishment of germline-competent ES cells derived from strains BALB/c and NZB mice. The results indicated that LIF + KSR/ES medium was permissive for the derivation of ES cells from NZB mice, which contribute to the somatic lineage in vivo, but not to the germline lineage. In contrast, ES cells that contribute to the makeup of chimeric mice were not propagated from blastocysts of BALB/c mice. Both germline and somatic competency were improved by increased LIF concentration in cultures of BALB/c ES cells, although we fa