Differentiation and proliferation potencies of human bone tissue-derived mesenchymal stromal cells (hBT-MSCs) after long-term cryopreservation －Comparison among cells stored for 1, 5, 10, 15, and 20 years.

INTRODUCTION: We investigated bone differentiation and proliferation potencies of human bone tissue-derived mesenchymal stromal cells (hBT-MSCs) after long-term cryopreservation. We determined the presence of any morphological and characteristic changes due to freezing to identify issues that need to be solved for future clinical applications. SUBJECTS AND METHODS: A total of 15 samples of hBT-MSCs that had been cryopreserved for different lengths of time, ranging from one year to 20 years (n = 3 each), were thawed and recultivated after being collected from excess iliac cancellous bone specimens of patients who underwent secondary alveolar bone grafting for cleft lip and palate in our department. We determined viability by observing calcein/EthD-stained cells under a confocal microscope, and the cell proliferation experiment was performed for one week using the Water Soluble Tetrazolium salts (WST) assay method. A confocal microscope was also used to identify any excessively accumulated senescence-associated growth factor SA-ßgal. Differentiation potency was assessed in the following three groups: bone differentiation, adipocyte differentiation, and nondifferentiation induction. We examined bone/adipocyte differentiation potencies using Alizarin Red staining, Ca quantitation, and Oil Red staining after continuously culturing cells for four weeks. RESULTS: Viability test results indicated that the proportion of viable cells decreased as the number of years of cryopreservation increased. The cell proliferation experiment showed that cells cryopreserved for a shorter duration multiplied exponentially. In the aging test, cells cryopreserved for ≥5 years showed similar positive reactions independent of the number of years of cryopreservation. In the cell proliferation test, there was no statistically significant difference between the years of cryopreserving. We compared bone differentiation and adipocyte differentiation ability with the non-induction group, and the induction group was confirmed to have a statistical advantage. However, there was no significant difference in the induction group pertaining to different ages. CONCLUSIONS: Samples cryopreserved for 20 years remained competent in bone and adipocyte differentiation. However, their differentiation direction tended to skew to either bone or adipocyte differentiation. Our results suggest that freezing does not accelerate aging, and samples cryopreserved for a long time are useful in future clinical applications.

Development of an RT-LAMP Assay for the Rapid Detection of SFTS Virus.

Detection of severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) during the early phase of the disease is important for appropriate treatment, infection control, and prevention of further transmission. The reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a nucleic acid amplification method that amplifies the target sequence under isothermal conditions. Here, we developed an RT-LAMP with a novel primer/probe set targeting a conserved region of the SFTSV L segment after extraction of viral RNA (standard RT-LAMP). Both the Chinese and Japanese SFTSV strains, including various genotypes, were detected by the standard RT-LAMP. We also performed RT-LAMP using the same primer/probe set but without the viral RNA extraction step (called simplified RT-LAMP) and evaluated the diagnostic efficacy. The sensitivity and specificity of the simplified RT-LAMP were 84.9% (45/53) and 89.5% (2/19), respectively. The simplified RT-LAMP can detect SFTSV in human sera containing >103.5 copies/mL viral RNA. The two RT-LAMP positive but quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) negative samples were positive in the conventional RT-PCR, suggesting that there was no false positive reaction in the RT-LAMP. Both the standard and simplified RT-LAMP are useful for detecting the SFTSV genome in patients during the early phase of the disease.

Isolation of Severe Fever with Thrombocytopenia Syndrome Virus from Various Tick Species in Area with Human Severe Fever with Thrombocytopenia Syndrome Cases.

Severe fever with thrombocytopenia syndrome (SFTS), caused by Dabie bandavirus, generally called SFTS virus (SFTSV), is an emerging zoonosis in East Asia. In Japan, 50-100 cases of SFTS have been reported each year since the first case was reported in 2013. SFTS is a tick-borne infectious disease, and SFTSV has been isolated from ticks in China and South Korea. Haemaphysalis longicornis and Amblyomma testudinarium are considered the primary vectors in Japan. However, the other tick species seldom feeding on humans might also play an important role in maintaining the virus in nature. In this study, we collected ticks on vegetation around the location where two SFTS patients were estimated to have been infected in Miyazaki Prefecture, Japan, isolated live SFTSV, and performed a phylogenetic analysis. A total of 257 ticks were collected, and SFTSV RNA was detected in 19.5% (9/46) of tick pools. A total of 10 infectious SFTSVs were successfully isolated from A. testudinarium, Haemaphysalis flava, Haemaphysalis formosensis, Haemaphysalis hystricis, and Haemaphysalis megaspinosa. Furthermore, the whole viral sequences isolated from ticks were highly homologous to sequences isolated from SFTS patients in the same sampling area in the past. These results suggest that SFTSVs are maintained in these tick species in the sampling area and sporadically transmitted to humans. Surveillance of SFTSV in ticks provides important information about the risk of incidental transmission to humans.

Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Virus in Small-Animal Veterinarians and Nurses in the Japanese Prefecture with the Highest Case Load.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is the causative agent of SFTS, an emerging tick-borne disease in East Asia, and is maintained in enzootic cycles involving ticks and a range of wild animal hosts. Direct transmission of SFTSV from cats and dogs to humans has been identified in Japan, suggesting that veterinarians and veterinary nurses involved in small-animal practice are at occupational risk of SFTSV infection. To characterize this risk, we performed a sero-epidemiological survey in small-animal-practice workers and healthy blood donors in Miyazaki prefecture, which is the prefecture with the highest per capita number of recorded cases of SFTS in Japan. Three small-animal-practice workers were identified as seropositive by ELISA, but one had a negative neutralization-test result and so was finally determined to be seronegative, giving a seropositive rate of 2.2% (2 of 90), which was significantly higher than that in healthy blood donors (0%, 0 of 1000; p < 0.05). The seroprevalence identified here in small-animal-practice workers was slightly higher than that previously reported in other high-risk workers engaged in agriculture and forestry in Japan. Thus, enhancement of small-animal-practice workers' awareness of biosafety at animal hospitals is necessary for control of SFTSV.

Expression Status of Serine Protease 27: A Prognostic Marker for Esophageal Squamous Cell Carcinoma Treated with Preoperative Chemotherapy/Chemoradiotherapy.

BACKGROUND: A previous study conducted a transcriptome analysis of paired normal and esophageal squamous cell carcinoma (ESCC) tissue samples. The results showed that the expression of serine protease 27 (PRSS27) was perturbed in tumor samples. Hence, this retrospective study aimed to validate the prognostic significance of PRSS27 in patients with preoperative treatment for ESCC. METHODS: We enrolled 86 patients who received preoperative treatment before esophagectomy for ESCC. The expression of PRSS27 in resected ESCC and biopsy tissue samples obtained before preoperative treatment was evaluated via immunostaining, and its relationship with clinicopathological features and prognosis was analyzed. RESULTS: In normal esophageal mucosa tissue samples, PRSS27 was expressed in the cytoplasm of spinous cells in the suprabasal layer and basal cells in the basal layer. Of 64 resected ESCC tissue samples, 35 (54.7%) expressed PRSS27 and 29 (45.3%) did not. Moreover, ectopic nuclear expression of PRSS27 was observed. Based on multivariate analysis, PRSS27 expression in resected tumor samples was a predictor of poor prognosis. In cases in which PRSS27 expression was observed in biopsy samples, patients with PRSS27-negative resected tumors had a better postoperative prognosis than those with PRSS27-positive resected tumors. CONCLUSIONS: PRSS27 expression in resected ESCC tissue samples is a poor prognostic factor in ESCC patients with preoperative treatment. Furthermore, conversion of PRSS27 expression from positive in biopsy samples to negative in resected tumor samples is a predictor of good prognosis in these patients. Hence, PRSS27 status is an effective tool for decision making regarding adjuvant treatment in ESCC patients.

Correction to: RNA-sequencing reveals positional memory of multipotent mesenchymal stromal cells from oral and maxillofacial tissue transcriptomes.

An amendment to this paper has been published and can be accessed via the original article.

RNA-sequencing reveals positional memory of multipotent mesenchymal stromal cells from oral and maxillofacial tissue transcriptomes.

BACKGROUND: Multipotent mesenchymal stromal cells (MSCs) can be isolated from numerous tissues and are attractive candidates for therapeutic clinical applications due to their immunomodulatory and pro-regenerative capacity. Although the minimum criteria for defining MSCs have been defined, their characteristics are known to vary depending on their tissue of origin. RESULTS: We isolated and characterized human MSCs from three different bones (ilium (I-MSCs), maxilla (Mx-MSCs) and mandible (Md-MSCs)) and proceeded with next generation RNA-sequencing. Furthermore, to investigate the gene expression profiles among other cell types, we obtained RNA-seq data of human embryonic stem cells (ESCs) and several types of MSCs (periodontal ligament-derived MSCs, bone marrow-derived MSCs, and ESCs-derived MSCs) from the Sequence Reads Archive and analyzed the transcriptome profile. We found that MSCs derived from tissues of the maxillofacial region, such as the jaw bone and periodontal ligament, were HOX-negative, while those derived from other tissues were HOX-positive. We also identified that MSX1, LHX8, and BARX1, an essential regulator of craniofacial development, were strongly expressed in maxillofacial tissue-derived MSCs. Although MSCs may be divided into two distinct groups, the cells originated from over the neck or not, on the basis of differences in gene expression profile, the expression patterns of all CD antigen genes were similar among different type of MSCs, except for ESCs. CONCLUSIONS: Our findings suggest that MSCs from different anatomical locations, despite meeting general characterization criteria, have remarkable differences in gene expression and positional memory. Although stromal cells from different anatomical sources are generally categorized as MSCs, their differentiation potential and biological functions vary. We suggested that MSCs may retain an original tissue memory about the developmental process, including gene expression profiles. This could have an important impact when choosing an appropriate cell source for regenerative therapy using MSCs.

An in vitro long-term study of cryopreserved umbilical cord blood-derived platelet-rich plasma containing growth factors-PDGF-BB, TGF-ß, and VEGF.

PURPOSE: Umbilical cord blood-derived platelet-rich plasma (UCB-PRP) containing growth factors has attracted attention as a biomaterial useful for regenerative medicine. The osteoblastic differentiation of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) can be induced by UCB-PRP. MATERIALS AND METHODS: Nine samples of UC and UCB were used to conduct an in vitro study that determined the contents of three growth factors (i.e., platelet-derived growth factor, transforming growth factor ß-1, and vascular endothelial growth factor) and that examined, by staining with Alizarin red, their ability to induce the osteoblastic differentiation of UC-MSCs at the baseline, 3 months, and 3 years of cryopreservation. RESULTS: The contents of growth factors in cryopreserved UCB-PRP were markedly elevated compared to those found in UCB at baseline. The samples of UCB that were added with cryopreserved UCB-PRP and those with bone morphogenetic protein-2 were stained granularly with Alizarin red, thus indicating the presence of calcium. The samples of UCB that were not added with UCB-PRP were not stained with Alizarin red. The above-mentioned contents and ability were maintained at 3 years of cryopreservation. Cryopreserved UCB-PRP possibly and advantageously induced the osteoblastic differentiation of UC-MSCs. CONCLUSION: The potential clinical application of cryopreserved UCB-PRP to regenerative medicine was suggested.

Development of three-layered rumen escapable capsules for cattle.

A new rumen escapable tool is presented for cattle in prospect of developing medical treatment or supplementing trace elements for disease prevention. This tool consists of a three-layered capsule that dissolves in the lower digestive tract, but not in the rumen. The capsule was manufactured by capsule-forming techniques through the use of liquid surface tension. This method does not involve high-temperature treatment, so the capsule can contain not only lipophilic substances but also hydrophilic or heat-sensitive substances. Furthermore, the capsule has a specific gravity of 1.3 and diameter of 6.0 mm, which were previously shown to be appropriate to avoid rumination. The objective of this study was to confirm the effectiveness of the capsule pertinent to rumen escaping. In order to validate rumen escape, capsules containing 30 g of water-soluble vitamin (thiamine hydrochloride) per head were administered to four lactating cows assigned in a crossover trial. In the group administered encapsulated thiamine hydrochloride, blood thiamine levels increased from 12.4 ± 1.03 ng/ml before administration to 54.8 ± 2.21 ng/ml at 6 hr following administration, whereas the level remained at 13.3 ± 2.05 ng/ml in the control group administered via aqueous solution. This indicates that the three-layered capsules passed through the rumen and dissolved in the lower digestive tract, thus functioning as a rumen escapable tool.

Free flap transfer to preserve main arterial flow in early reconstruction of open fracture in the lower extremity.

The selection of recipient vessels is crucial when reconstructing traumatized lower extremities using a free flap. When the dorsalis pedis artery and/or posterior tibial artery cannot be palpated, we utilize computed tomography angiography to verify the site of vascular injury prior to performing free flap transfer. For vascular anastomosis, we fundamentally perform end-to-side anastomosis or flow-through anastomosis to preserve the main arterial flow. In addition, in open fracture of the lower extremity, we utilize the anterolateral thigh flap for moderate soft tissue defects and the latissimus dorsi musculocutaneous flap for extensive soft tissue defects. The free flaps used in these two techniques are long and include a large-caliber pedicle, and reconstruction can be performed with either the anterior or posterior tibial artery. The preparation of recipient vessels is easier during the acute phase early after injury, when there is no influence of scarring. A free flap allows flow-through anastomosis and is thus optimal for open fracture of the lower extremity that requires simultaneous reconstruction of main vessel injury and soft tissue defect from the middle to distal thirds of the lower extremity.

Basolateral secretion of Wnt5a in polarized epithelial cells is required for apical lumen formation.

Wnt5a regulates planar cell polarity in epithelial cells, but it remains to be determined whether Wnt5a and its receptors are sorted apically or basolaterally, and how Wnt5a signaling is involved in apical and basolateral polarization. We found that Wnt5a was secreted basolaterally in polarized kidney epithelial cells. The basolateral secretion of Wnt5a required Wntless (Wls), clathrin and adaptor protein 1 (AP-1). Wnt5a receptors were also localized to the basolateral membranes, but their sorting did not require Wls. Wnt5a-induced signaling was stimulated more efficiently at the basolateral side than the apical side of epithelial cells. Knockdown of Wnt5a delayed apical lumen formation of the epithelial cyst, and these phenotypes were rescued by wild-type Wnt5a, but not by a Wnt5a mutant that is secreted apically. Although apoptosis was not required for apical lumen formation in a wild-type cyst, apoptosis was necessary for eliminating luminal cells in a Wnt5a-depleted cyst. These results suggest that Wnt5a and its receptors are sorted to their correct destination by different mechanisms and that the basolateral secretion of Wnt5a is necessary for apical lumen formation in the epithelial cyst.

Immediate reconstruction after removal of nasal prosthesis using diced cartilage wrapped with temporal fascia.

Various materials are used for nasal augmentations. Silicone is the most prevalent because it is durable and facilitates sculpturing. However, the unfortunate patient who presents with complication of the nasal implants and wants to remove them is vexed with a significant resultant cosmetic defect if the implant is removed. However, the patients who have some troubles after augmentation by the implants tend to hate the use of the prosthesis again. Ideally, immediate reconstruction would be offered to the patient, sparing him/her the deformity left by removal of the implant. We treated 16 patients who had undergone immediate nasal reconstruction after removal of foreign body. We reconstructed nasal deformity by diced cartilage wrapped with temporal fascia. The cartilage harvested from the ear concha was finally diced into 1- to 2-mm cubes. A bag was made from deep or superficial temporal fascia, and diced cartilage cubes were placed in the bag, which was grafted onto the nasal dorsum. This procedure had several advantages including getting natural contouring and enough volume and absence of foreign body reaction. It was also soft to the touch compared with prosthesis. The fascia could support the thin dorsum skin. The nasal augmentation effect of this procedure was comparable with that of prosthesis methods. It had lower risks for infection and exposure and provided more psychologic comfort. The nasal deformities were successfully reconstructed using diced cartilage wrapped with temporal fascia. We believe that this is the good method for the immediate nasal reconstruction after the removal of foreign body.

Enhanced expression of cystine/glutamate transporter in the lung caused by the oxidative-stress-inducing agent paraquat.

In mammalian cultured cells, the activity of a cystine/glutamate transporter, designated System xc(-), has been shown to be essential for maintaining intracellular glutathione levels and the extracellular cystine/cysteine redox balance. The substrate-specific subunit of this transporter, xCT, is strongly induced by various stimuli, including oxidative stress, which suggests that xCT is one of the adaptive cellular defense systems against these types of stress. Embryonic fibroblasts from xCT-deficient mice fail to survive unless a cysteine precursor, N-acetylcysteine, is present. However, it is unclear whether xCT has similar functions in vivo because xCT-deficient mice are apparently normal. In this study, we investigated the phenotype of the xCT-deficient mice under paraquat-induced oxidative stress. At a paraquat dose of 45mg/kg, the survival rate of the xCT-deficient mice was significantly lower than that of the wild-type mice. Under this condition, total glutathione (the reduced form of glutathione (GSH)+the oxidized form of GSH) levels in the lungs of the xCT-deficient mice were lower than those in the lungs of the wild-type mice. Histopathological examinations showed that paraquat administration worsened the alveolar structure of the xCT-deficient mice compared with the wild-type mice. After paraquat treatment, obvious 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal reactivity was detected in the lungs of the xCT-deficient mice. Although xCT expression was slightly detectable in the lungs of the normal wild-type mice, paraquat administration induced xCT mRNA expression in the lung. Constitutive expression of xCT mRNA was detected in alveolar macrophages isolated from the pulmonary lavage fluid of the wild-type mice, and paraquat administration strongly enhanced xCT mRNA expression in these cells. GSH levels in bronchoalveolar lavage fluid were significantly higher in the paraquat-treated wild-type mice than in the paraquat-treated xCT-deficient mice. These results suggest that xCT contributes to the maintenance of glutathione levels in lungs and the glutathione redox state as a protective system against paraquat toxicity in vivo.

Regulation of neurotrophin-3 gene transcription by Sp3 and Sp4 in neurons.

Neurotrophin-3 (NT-3), a neurotrophin member, plays crucial roles in neuronal development, function and plasticity. Previous studies have demonstrated that NT-3 gene transcription is driven by alternative promoters A and B, located upstream of exons 1A (EIA) and 1B (EIB), respectively. However, the transcription factors and DNA elements that drive NT-3 gene transcription remain to be identified. Here, we analysed the promoter region of the NT-3 gene and found that an NT-3 transcript containing EIB is predominantly expressed in cortical neurons which preferentially utilize promoter B, and two tandemly repeated GC-boxes, located between -100 and -60 base pairs within promoter B, are required for the transcription. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that both specificity protein (Sp)3 and Sp4 were able to bind to the Sp1 binding sequences within the GC boxes. Expression of dominant-negative Sp3 and Sp4 small interfering RNA in cortical neurons reduced the activity of the NT-3 gene promoter. Over-expression of Sp1 family members, especially Sp4, resulted in an increase of the NT-3 gene promoter. These findings indicate that the NT-3 gene is a target gene for Sp4 that is abundantly expressed in the brain.

Reconstruction of the umbilicus using a reverse fan-shaped flap.

The goal of the reconstruction for umbilical absence is to obtain a natural three-dimensional appearance of the umbilicus with minimal operative scarring. This paper presents two cases of umbilical reconstruction using a reverse fan-shaped flap. In both cases, the umbilicus was lost during surgical procedures on the abdominal wall when the patients were newborns. We performed this technique in both cases. This technique is simple and safe. With this technique, a permanent umbilical depth and ring can be obtained without any complications.