STUDY DESIGN: We established induced pluripotent stem cells (iPSCs) and neural stem/progenitor cells (NSPCs) from three newborns with spina bifida aperta (SBa) using clinically practical methods. PURPOSE: We aimed to develop stem cell lines derived from newborns with SBa for future therapeutic use. OVERVIEW OF LITERATURE: SBa is a common congenital spinal cord abnormality that causes defects in neurological and urological functions. Stem cell transplantation therapies are predicted to provide beneficial effects for patients with SBa. However, the availability of appropriate cell sources is inadequate for clinical use because of their limited accessibility and expandability, as well as ethical issues. METHODS: Fibroblast cultures were established from small fragments of skin obtained from newborns with SBa during SBa repair surgery. The cultured cells were transfected with episomal plasmid vectors encoding reprogramming factors necessary for generating iPSCs. These cells were then differentiated into NSPCs by chemical compound treatment, and NSPCs were expanded using neurosphere technology. RESULTS: We successfully generated iPSC lines from the neonatal dermal fibroblasts of three newborns with SBa. We confirmed that these lines exhibited the characteristics of human pluripotent stem cells. We successfully generated NSPCs from all SBa newborn-derived iPSCs with a combination of neural induction and neurosphere technology. CONCLUSIONS: We successfully generated iPSCs and iPSC-NSPCs from surgical samples obtained from newborns with SBa with the goal of future clinical use in patients with SBa.
Cells, Cultured ; Ethics ; Fibroblasts ; Humans ; Induced Pluripotent Stem Cells* ; Infant, Newborn* ; Meningomyelocele ; Plasmids ; Pluripotent Stem Cells ; Regenerative Medicine ; Skin ; Spina Bifida Cystica* ; Spinal Cord ; Spinal Dysraphism* ; Stem Cell Transplantation ; Stem Cells

The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.
Cells, Cultured ; DNA Transposable Elements ; Dental Pulp ; cytology ; Humans ; Induced Pluripotent Stem Cells ; cytology ; Nerve Tissue Proteins ; genetics ; Tooth, Deciduous ; cytology ; Transfection

The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.

This study aims at identifying how sports activity status, level of residual function, and independence in activities of daily living (ADL) affect the health-related quality of life (HRQOL) of persons with spinal cord injury (PSCI) who regularly participate in sports activities. Eighty-one male PSCI (21 persons with tetraplegia and 60 persons with paraplegia) who regularly participated in sports activities (wheelchair basketball or wheelchair twin basketball) were included in the present study. They were evaluated in terms of their characteristics, sports activity status, independence in ADL (Spinal Cord Independence Measure [SCIM]), and HRQOL (Medical Outcomes Study Short Form-36 [SF-36]). The age was a significant explanatory variable for physical functioning, role physical, and physical component summary (P < 0.05). On the other hand, the level of residual function was a significant explanatory variable for general health, vitality, mental health, and mental component summary (P < 0.05). The physical aspects of HRQOL of PSCI who regularly participate in sports activities were mainly affected by age, whereas the mental aspects were affected by the level of residual function.

Purpose: To investigate the factorial structure of physical fitness of male paraplegics with thoracic or lumbar spinal cord injury and to develop a battery of field tests for predicting their general physical fitness level.Methods: Fifty-three active male paraplegics with spinal cord injury (PSCI) (age range: 18-54; spinal cord injury level: T4 to L4) were examined. Thirteen feasible variables were selected using physical fitness components based on the International Committee for the Standardization of Physical Fitness Tests and previous PSCI studies. Factor analysis was applied to 14 variables; 13 of these involved physical fitness tests stratified by age to determine the factorial structure of physical fitness variables. Multiple regression analysis was performed to obtain a linear regression equation using a representative variable for each factor in the factorial structure as an independent variable. A first principal component score was obtained by principal component analysis using each variable as a dependent variable.Results: For factorial structure, wheelchair driving ability (3-minute shuttle run), body composition (sebum thickness), respiratory function (vital capacity) and shoulder joint extension force factors were obtained. The results of multiple regression analysis involved 5 variables (the 4 above-mentioned variables plus age); and the first principal component score of each subject from all variables provided a significant linear regression equation (r = 0.934, P ＜0.01) when the body composition factor was excluded.Conclusions: The representative measurement variables obtained from the factorial structure allowed for the development of a battery of field tests for predicting general physical fitness level of PSCI.

The purpose of this study was to investigate the gender differences in thermoregulatory responses during the recovery from prolonged exercise. Fourteen (7 males and 7 females) subjects cycled for 45 minutes at 55%VO₂max and then rested for 105 minutes in the hot and humid environment (30℃, 80% relative humidity) or cool environment (20℃, 60% relative humidity). Both males and females showed same variations in rectal temperature (Tre) during exercise and recovery periods. Total sweat volume and water intake were significantly larger in males than in females during recovery in the both environments. In the heat condition, mean skin temperature (Tsk) decreased immediately after the cessation of exercise in females but not in males (the recovery period (R) 5–15 min : p<0.01, R15–20 min : p<0.05). These results suggested there are gender-related differences in the mechanisms and also the process maintaining the balance of heat production and heat loss response during recovery from prolonged exercise. Moreover the heat might modify gender differences of heat loss response during recovery as an important factor.

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5'-nucleotidase subclass I and purine-specific 5'-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5'-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes.
Animals ; Babesia/*immunology ; Babesiosis/blood/immunology/parasitology/*veterinary ; Cell Differentiation/immunology ; Dog Diseases/*blood/immunology/*parasitology ; Dogs ; Electrophoresis, Polyacrylamide Gel ; Erythrocytes/*immunology ; Reticulocytes/*immunology

During exercise at high temperatures, body temperature increases impairing exercise performance and resulting in heat illnesses. Water ingestion during exercise is a simple and practical strategy to prevent hyperthermia. In the present study, we examined the effects of water ingestion interval on thermoregulatory responses during exercise in a hot, humid environment (32t, 80% relative humidity) . Eight male university students performed a 60-min cycling exercise (60% of the maximal O₂ uptake) under four separate conditions; no drinking (ND), water ingestion (mineral water) at 5 (D5), 15 (D15), and 30 (D30) min intervals. The total volume of water ingestion (TWI) was identical during D5, D15, and D30, and equal to the amount of fluid lost in sweat during ND. TWI was divided equally by the number of drinking times in each experiment. During exercise, both rectal and mean skin temperature were lower in D5 than those in the other conditions (p<0.05) . There was no significant difference in total sweat loss between the four conditions, however, evaporative sweat loss and sweat efficiency (evaporative sweat loss total sweat loss) were significantly (p<0.05) higher in D5 than those in the other conditions. These results suggest that the shorter water ingestion interval increases evaporative sweating and attenuates higher body temperature during exercise in a hot, humid environment.

Although left anterior descending coronary artery (LAD) grafting with a left internal thoracic artery (ITA) on a beating heart via a small left anterior thoracotomy (LAST) has become widely accepted, significant limitations exist due to the limited surgeon experience, smallness of exposure, thus making harvesting of the ITA, visualization of the surgical field and anastomosis quite difficult. Patients often have significant pain and wound complications postoperatively. A lower mini-sternotomy approach in 4 patients was performed from December 1998 through January 1999. Results: The length of mini-sternotomy incision is 7 to 14cm. These operations were accomplished without morbidity or mortality. No patients required intraoperative conversion to conventional bypass. Postoperative angiography showed patency of graft without stenosis of the anastomosis in all 4 patients. The patients did not complain of significant pain and their postoperative hospital stay was 5 to 11 days. The lower mini-sternotomy approach or“xyphoid” approach proposed by Benetti seems to be an excellent novel approach giving the freedom of extension of the incision if needed with satisfactory exposure for left ITA harvest and access to LAD as well as the distal RCA, and causes less postoperative incisional pain.

To clarify changes in body temperature during endurance exercise in patients with spinal cord injury (SCI), we measured tympanic temperature (Tty) and skin temperature in the head, arm, chest, thigh, shin and calf in 5 patients with SCI (T6-T 12) and 7 normal controls during 30 minutes arm cranking exercise (20 watts) from 10 minutes before the initiation of exercise until 10 minutes after the termination of exercise in an artificial climate room at a temperature of about 25°C with a relative humidity of about 50%. The Tty in the SCI group was lower than that in the control group from 10 minutes before the initiation of exercise to 10 minutes after the termination of exercise with a significant difference only at the initiation of exercise. The difference in Tty slightly decreased with continuation of exercise. The Tty in the SCI group at rest was 36.05-37.15°C. Four patients in this group showed a decrease of 0.04-0.12°C in the early stage and an increase of 0.66°C±0.19 (mean±SD) at the end of exercise over the value at the initiation of exercise.
The skin temperature was lower in the SCI group than in the control group in all sites excluding the arm. Significant differences were observed in the head in the early stage of exercise and after exercise, in the chest from 10 minutes before the initiation of exercise to 5 minutes after the termination of exercise, in the thigh from 10 minutes before the initiation of exercise to 10 minutes after the termination of exercise, in the shin 10 minutes and 5 minutes before the initiation of exercise, and in the calf from before to 15 minutes after the initiation of exercise. In the SCI group, marked individual differences were observed in the skin temperatures in the thigh, shin, and calf, suggesting specificity of the skin temperature response in and near the paralysis area.
Results in Tty in this study suggested no heat retention in the SCI patients. Therefore, the risk for heat disorders seems to be low during moderate or mild exercise under moderate temperature environment at a temperature of about 25°C with a relative humidity of about 50% even when the skin temperature is low, and thermolysis is not marked.