Generation of Induced Pluripotent Stem Cells from Lymphoblastoid Cell Lines by Electroporation of Episomal Vectors

Background and Objectives@#Lymphoblastoid cell lines (LCLs) deposited from disease-affected individuals could be a valuable donor cell source for generating disease-specific induced pluripotent stem cells (iPSCs). However, generation of iPSCs from the LCLs is still challenging, as yet no effective gene delivery strategy has been developed. @*Methods@#and Results: Here, we reveal an effective gene delivery method specifically for LCLs. We found that LCLs appear to be refractory toward retroviral and lentiviral transduction. Consequently, lentiviral and retroviral transduction of OCT4, SOX2, KFL4 and c-MYC into LCLs does not elicit iPSC colony formation. Interestingly, however we found that transfection of oriP/EBNA-1-based episomal vectors by electroporation is an efficient gene delivery system into LCLs, enabling iPSC generation from LCLs. These iPSCs expressed pluripotency makers (OCT4, NANOG, SSEA4, SALL4) and could form embryoid bodies. @*Conclusions@#Our data show that electroporation is an effective gene delivery method with which LCLs can be efficiently reprogrammed into iPSCs.

Robust and Reproducible Generation of Induced Neural Stem Cells from Human Somatic Cells by Defined Factors

Background and Objectives@#Recent studies have described direct reprogramming of mouse and human somatic cells into induced neural stem cells (iNSCs) using various combinations of transcription factors. Although iNSC technology holds a great potential for clinical applications, the low conversion efficiency and limited reproducibility of iNSC generation hinder its further translation into the clinic, strongly suggesting the necessity of highly reproducible method for human iNSCs (hiNSCs). Thus, in orderto develop a highly efficient and reproducible protocol for hiNSC generation, we revisited the reprogramming potentials of previously reported hiNSC reprogramming cocktails by comparing the reprogramming efficiency of distinct factor combinations including ours. @*Methods@#We introduced distinct factor combinations, OSKM (OCT4+SOX2+KLF4+C-MYC), OCT4 alone, SOX2 alone, SOX2+HMGA2, BRN4+SKM+SV40LT (BSKMLT), SKLT, SMLT, and SKMLT and performed comparative analysis of reprogramming potentials of distinct factor combinations in hiNSC generation. @*Results@#Here we show that ectopic expression of five reprogramming factors, BSKMLT leads the robust hiNSC generation (>80 folds enhanced efficiency) from human somatic cells compared with previously described factor combinations. With our combination, we were able to observe hiNSC conversion within 7 days of transduction. Throughout further optimization steps, we found that both BRN4 and KLF4 are not essential for hiNSC conversion. @*Conclusions@#Our factor combination could robustly and reproducibly generate hiNSCs from human somatic cells with distinct origins. Therefore, our novel reprogramming strategy might serve as a useful tool for hiNSC-based clinical application.

Evaluating the Clinical Symptoms of Neonates with Suspected Dysphagia

OBJECTIVE: To evaluate the prevalence of dysphagia in neonates who showed abnormal findings on videofluoroscopic swallowing studies (VFSSs), and to compare the accuracy of the clinical evaluation with the VFSS results. METHOD: A clinical investigation of 142 neonates admitted to a neonatal intensive care unit was carried out to evaluate the presence of low O2 saturation (<80%), symptoms of cyanosis, coughing and/or vomiting, nasal regurgitation, drooling saliva, voice change and crying during feeding. VFSSs were performed on the neonates who had at least one of these clinical abnormalities. RESULTS: Of the 142 patients, 37 (26.1%) had at least one of the clinical symptoms of dysphagia. Twenty two of 37 (59.5%) showed abnormal findings on the VFSS. The patients exhibiting cyanosis and coughing during feeding had a higher incidence of aspiration (11 of 11, 100%) on the VFSSs than did the patients with other symptoms: cyanosis (3 of 13, 30.8%), cyanosis with vomiting (0 of 2, 0.0%), coughing without cyanosis (2 of 5, 40.0%), voice change (2 of 2, 100%) and nasal regurgitation (1 of 3, 33.3%). CONCLUSION: The prevalence of laryngeal penetration or subglottic aspiration among those neonates who were clinically suspected of dysphagia was 59.5%. Coughing with cyanosis during feeding was the best predictor of both these abnormalities.

Biomechanical Factors Associated with Plantar Fasciitis in Non-obese Patients / 대한스포츠의학회지

The purpose of this study was to identify the biomechanical factors that correlate with plantar fasciitis in non-obese patients whose body mass index were below 25 kg/m2. The subjects were non-obese patients who were diagnosed as plantar fasciitis by clinical appearance, physical examination, and ultrasonographic findings (n=48), and non-obese control persons without clinical diagnosis of plantar fasciitis (n=30). The two groups were compared on fat pad thickness, ankle dorsiflexion range of motion (ROM), resting calcaneal stance position (RCSP), incidence of calcaneal spur, and calcaneal pitch. The results showed that, there were statistically significant differences between two groups in ankle dorsiflexion ROM, RCSP, and calcaneal pitch (p<0.05). Multiple logistic regression analysis showed ankle dorsiflexion ROM and RCSP strongly correlated with presence of plantar fasciitis as independent predictors (p<0.05). In conclusion, reduced ankle dorsiflexion ROM and negative RCSP (valgus tendency in rear foot) may be the biomechanical factors associated with plantar fasciitis in non-obese patients.

Relationship between the Incidence of Vocal Cord Palsy and Aspiration Risk in Acute Ischemic Stroke Patients

OBJECTIVE: To investigate the incidence of vocal cord palsy (VCP) in acute ischemic stroke patients and its influence on aspiration risk. METHOD: Fifty patients with first-ever acute stroke were enrolled. The mean age was 68.3 years and there were 21 men and 29 women. Based on clinical and neuroimaging findings, their stroke subtype was categorized into cortical/ subcortical (Group A), lateral medulla (Group B) and other brainstem (Group C). We examined them by using flexible fiberoptic rhinolaryngoscope and videofluroscopic swallowing study (VFSS) within 2 weeks after stroke onset. The Penetration - Aspiration Scale (PAS) was used to score each VFSS. RESULTS: Among the 50 patients, VCP was found in 15 (30%): 15.8% of group A, 100% of group B and 40% of group C. VCP was contralateral to the brain lesion in group A and ipsilateral in 85.7% of group B. Aspiration risk was found in 38% of the all patients and 53% of VCP had aspiration risk. No differences in the incidence of aspiration risk were noted according to VCP (chi-square=2.138, p=0.144). CONCLUSION: There was no relationship between VCP and aspiration risk in acute ischemic stroke patients. Although VCP is a known risk factor for aspiration, other factors are important in determining an effective swallowing.

Evaluation of the Extraction Method for the Cytotoxicity Testing of Latex Gloves

In this study, the cytotoxicity of medical latex gloves to cultured L-929 cells was determined using various extraction conditions. According to the extraction time and temperature, three types of extraction conditions were used: 1) 24 h at 37 degrees C; 2) 72 h at 37 degrees C; 3) 72 h at 50 degrees C. Also, four different extraction vehicles were used, namely, distilled water (DW), 9 g/l sodium chloride (saline) in DW, and culture media with or without serum. Under the above-mentioned conditions, the samples were extracted and then 2-fold serially diluted in the concentration range 3.13 - 50%. When extracted with either DW or saline for 24 h or 72 h at 37 degrees C, only 50% diluted samples showed distinct cytotoxicity to L-929 cells. Moreover, no cytotoxic potentials were observed when gloves were extracted with DW or saline at 50 degrees C for 72 h. Cytotoxicity was markedly greater when gloves were extracted with culture medium, irrespective of the presence of serum in the medium. These results suggest that optimal extraction conditions should be established for the cytotoxicity evaluations of biomaterials and medical devices.

In Vitro Bioassay of Endotoxin Using Fluorescein as a pH Indicator in a Macrophage Cell Culture System

Based on the biological activity of endotoxin, we propose a possible new method for detecting endotoxin using a pH- indication system of macrophage culture media. After RAW 264.7 macrophage cells were treated with lipopolysaccharide (LPS), the addition of fluorescein to the LPS-treated media reproductively reduced its absorption and emission spectra (it was a dose-dependent reduction). The advantages of this LPS- detection method were compared with the Limulus Amebocyte Lysate (LAL) test by using purified bacterial LPS (Salmonella minnessota, Escherichia coli, and Pseudomonas aeruginosa). Additionally, the absorption and fluorescence intensity of fluorescein, following treatment of RAW 264.7 cells with a high concentration of Staphylococcus aureus (Gram-positive, lysed bacteria), could not generally be detected by the LAL test, but they were found to be reduced, in a dose-response relationship, with this new system. The macrophage culture system-method might be a good supplement to the LAL assay for detection of LPS, Gram-negative and Gram-positive bacteria.

The Effects of Recombinant Human BMP-7, Prepared from a COS-7 Expression System, on the Proliferation and Differentiation of Rat Newborn Calvarial Osteoblasts

A family of proteins, the bone morphogenetic proteins (BMPs), which promote osteoblast differentiation and bone mineralization, have recently been identified. One, BMP-7, has shown the ability to induce cartilage and bone formation processes. In this report, the possibility that other cell lines, to CHO cells, may also be available as host cells for the expression of hBMP-7 was validated. Recombinant human BMP (rhBMP) -7 was produced in COS-7 cells, as a processed mature disulfide-linked homodimer, with an apparent molecular weight of 36, 000. Examination of the expressions of the markers characteristic of osteoblast phenotypes showed that the rhBMP-7 specifically stimulated the inductions of alkaline phosphatase (ALP) (5-fold increase at 100 ng of rhBMP-7/ml), parathyroid hormone (PTH) -mediated intracellular cAMP production (4-fold increase at 100 ng of rhBMP-7/ml) and osteocalcin synthesis (5-fold increase at 100 ng of rhBMP-7/ml). In summary, the in vitro mineralization assay results provide evidence that the rhBMP-7 peptide, produced by COS-7 expression system, possesses intact biological activity. A similar pattern of biological activity was observed for the BMP-7 in COS-7 cells compared to the corresponding CHO cell expression system. Thus, these findings can be experimentally utilized for the production of rhBMPs for in vitro or in vivo studies.

Ideal Freezing Curve Can Avoid the Damage by Latent Heat of Fusion During Freezing / 대한흉부외과학회지

BACKGROUND: Liquid nitrogen freezing techniques have already met with widespread success in biology and medicine as a means of long-term storage for cells and tissues. The use of cryoprotectants such as glycerol and dimethylsulphoxide to prevent ice crystal formation, with carefully controlled rates of freezing and thawing, allows both structure and viability to be retained almost indefinitely. Cryopreservation of various tissues has various controlled rates of freezing. MATERIAL AND METHOD: To find the optimal freezing curve and the chamber temperature, we approached the thermodynamic calculation of tissues in two ways. One is the direct calculation method. We should know the thermophysical characteristics of all components, latent heat of fusion, area, density and volume, etc. This kind of calculation is so sophisticated and some variables may not be determined. The other is the indirect calculation method. We performed the tissue freezing with already used freezing curve and we observed the actualfreezing curve of that tissue. And we modified the freezing curve with several steps of calculation, polynomial regression analysis, time constant calculation, thermal response calculation and inverse calculation of chamber temperature. RESULT: We applied that freezing program on mesenchymal stem cell, chondrocyte, and osteoblast. The tissue temperature decreased according to the ideal freezing curve without temperature rising. We did not find any differences in survival. The reason is postulated to be that freezing material is too small and contains cellular components. We expect the significant difference in cellular viability if the freezing curve is applied on a large scale of tissues. CONCLUSION: This program would be helpful in finding the chamber temperature for the ideal freezing curve easily.

A bone replaceable artificial bone substitute: morphological and physiochemical characterizations

A composite material consisting of carbonate apatite (CAp) and type I atelocollagen (AtCol) (88/12 in wt/wt%) was designed for use as an artificial bone substitute. CAp was synthesized at 58 degrees C by a solution-precipitation method and then heated at either 980 degrees C or 1,200 degrees C. In this study, type I AtCol was purified from bovine tail skins. A CAp-AtCol mixture was prepared by centirfugation and condensed into composite rods or disks. The scanning electron-microscopic (SEM) characterization indicated that the CAp synthesized at 58 degrees C displayed a crystallinity similar to that of natural bone and had a high porosity (mean pore size: about 3-10 microns in diameter). SEM also revealed that the CAp heated at 980 degrees C was more porous than that sintered at 1,200 degrees C, and the 1,200 degrees C-heated particles were more uniformly encapsulated by the AtCol fibers than the 980 degrees C-heated ones. A Fourier transformed-infrared spectroscopic analysis showed that the bands characteristic of carbonate ions were clearly observed in the 58 degrees C-synthesized CAp. To enhance the intramolecular cross-linking between the collagen molecules, CAp-AtCol composites were irradiated by ultraviolet (UV) ray (wave length 254 nm) for 4 hours or vacuum-dried at 150 degrees C for 2 hours. Compared to the non cross-linked composites, the UV-irradiated or dehydrothermally cross-linked composites showed significantly (p < 0.05) low collagen degradation and swelling ratio. Preliminary mechanical data demonstrated that the compressive strengths of the CAp-AtCol composites were higher than the values reported for bone.

Viability and enzymatic activity of cryopreserved porcine heart valve

Fibroblast viability of a natural tissue valve for replacing a defective heart valve through allograft or xenograft has been suggested to affect its clinical durability. In this study, the cell viability and enzymatic activity of porcine heart valve leaflets were examined in regard to concerning to the preservation process [variable warm ischemic time (WIT), cold ischemic time (CIT), and cryopreservation]. Porcine heart enblocs were obtained and valve dissection was performed after 2, 12, 24, or 36 hours, in respective groups A, B, C, and D, as WIT. Each group was stored for 24 hours as CIT and cryopreserved. Leaflets were dissected from a valved conduit after each process, and cell viability and enzymatic activity in the leaflet were investigated using trypan blue staining and API ZYM kits. WIT extension significantly decreased fibroblast viability (p < 0.05, 92.25 +/- 2.7% at 2 hours, 84.9 +/- 6.7% at 12 hours, 57.0 +/- 10.2% at 24 hours, 55.9 +/- 7.9% at 36 hours), while CIT for 24 hours was also influenced significantly (p < 0.05), whereas cryopreservation demonstrated no effect on cellular viability. In enzyme activity observation, several enzymes related to lipid or nucleotide degradation (esterase, esterase lipase, particularly phosphatase, phosphohydrolase) were remarkably changed following the valve-fabrication process. After 24 hours CIT, these enzymatic activities in groups B, C and D significantly increased, but the activities decreased after cryopreservation. Particularly, both the viability and enzymatic activity showed remarkable changes after CIT in group B (WIT = 12 hours). These results suggest that WIT is more important than CIT in maintaining viability of the valve, and that completing all the cryopreservation process within 12 hours after acquisition is recommended.