Isolation of neural precursor cells from skeletal muscle tissues and their differentiation into neuron-like cells

Skeletal muscle contains several precursor cells that generate muscle, bone, cartilage and blood cells. Although there are reports that skeletal muscle-derived cells can trans-differentiate into neural-lineage cells, methods for isolating precursor cells, and procedures for successful neural induction have not been fully established. Here, we show that the preplate cell isolation method, which separates cells based on their adhesion characteristics, permits separation of cells possessing neural precursor characteristics from other cells of skeletal muscle tissues. We term these isolated cells skeletal muscle-derived neural precursor cells (SMNPs). The isolated SMNPs constitutively expressed neural stem cell markers. In addition, we describe effective neural induction materials permitting the neuron-like cell differentiation of SMNPs. Treatment with retinoic acid or forskolin facilitated morphological changes in SMNPs; they differentiated into neuron-like cells that possessed specific neuronal markers. These results suggest that the preplate isolation method, and treatment with retinoic acid or forskolin, may provide vital assistance in the use of SMNPs in cell-based therapy of neuronal disease.

Long-term Effects of Antibiotic-coated Foley Catheter on Bacterial Biofilm Formations / 대한비뇨기과학회지

PURPOSE: A catheter-associated urinary tract infection, which frequently occurs in patients with an indwelling Foley catheter, can cause serious morbidity or mortality. Recently, antibiotic coated Foley catheters, to prevent catheter-associated urinary tract infections, have become commercially available. This study investigated the long-term effects of the use of antibiotic-coated Foley catheters on biofilm formations. MATERIALS AND METHODS: Silicone Foley or antibiotic-coated Foley catheters were indwelled in 72 patients with a neurogenic bladder. Each catheter was removed 1, 3, 5, 7, 14 and 28 days after insertion. The cell densities of the biofilm bacteria were evaluated by counting the number of colonies on plate cultures. The biofilm formations on the catheters were evaluated by scanning electron microscopy. The inner surface morphology of the catheter was imaged by field emission scanning electron microscopy (Philips-XL20SFEG), at 10kV, following gold sputtering for electrical conductance. Six catheters were studied in each group, and the means calculated for comparisons. RESULTS: Thick bacterial biofilms were observed on both the antibiotic- coated and silicone Foley catheters 7 days after insertion. There were no significant differences in the cell densities of the biofilm bacteria between the two types of catheter during days 7-28 after insertion (p<0.05). Two to three species of bacteria were isolated from the catheters in each patient; the most common species were Pseudomonas, Klebsiella, Serratia, Proteus species and Escherichia coli. CONCLUSIONS: The antibiotic-coated Foley catheters showed no preventive effects on the biofilm formations after 7 days of indwelling compared with the silicone Foley catheters. Our data suggest that the routine use of antibiotic-coated Foley catheters to prevent catheter-associated infection in patients with a neurogenic bladder is not reasonable. The emergence of resistance associated with antibiotic-coated catheters should be evaluated.

Preliminary Study of Tissue Engineered Bladder Regeneration with Poly (epsilon-caprolactone) (PCL) Sheet Seeded with Autologous Muscle-derived Stem Cell / 대한비뇨기과학회지

PURPOSE: To investigate the feasibility of using a poly (epsilon-caprolactone) (PCL) sheet seeded with autologous muscle-derived stem cells as a bladder substitute. MATERIALS AND METHODS: Muscle-derived stem cells were isolated from the gastrocnemius muscle of 9 female Sprague-Dawley rats using a preplate technique, and cultured on a 5x5mm PCL sheet. The sheets were implanted into the mesentery of the rats in an autologous manner. Three rats were sacrificed 2, 4 and 8 weeks after implantation, and the morphological changes were assessed by H&E and immunofluorescence staining including DAPI, myosin heavy chain (MHC) and choline acetyl transferase (CAT). RESULTS: All the rats survived for the scheduled time. A mild inflammatory reaction was observed around the PCL sheet in the postoperative 2-week specimen but this receded with time. Muscle cells on the sheet were observed over the experimental period. The 8-week specimen showed a moderate amount of muscle cells on the sheet, and MHC and CAT immunofluorescence staining showed a positive reaction. The muscle layer was not well organized. Angiogenesis was quite noticable between the sheet and the muscle cells on the 8-week specimen. CONCLUSIONS: A PCL sheet seeded with autologous muscle-derived stem cells showed skeletal muscle differentiation on the sheets 8 weeks after mesenteric implantation in an autologous manner. This suggests the feasibility of using a PCL sheet seeded with autologous muscle-derived stem cell as a bladder substitute.

The Temperature Differences among the Three Urethral Portions (Distal, Middle & Proximal) and Bladder in Incontinent Women

PURPOSE: We tried to find out an adequate sol-gel transition temperature of female urethra for the injection of thermosensitive polymer in incontinent patients. We measured the temperatures of three portions of female urethra and bladder. MATERIALS AND METHODS: Total of 53 female incontinent patients participated, excluding those with any kind of infection which could lead to an elevation of body temperature. The basal body temperatures were checked at the axilla, tympanic membrane and mouth. Temperatures of the proximal(U1), middle(U2), distal(U3) urethra and bladder(B) were measured by a digital thermometer under a lithotomy position. We divided our patients into 3 groups which were patients in follicular phase(F), luteal phase(L) and menopause(M). The temperature difference between the 4 portions of the urethra(D1; between U1 and U2, D2; between U2 and U3, D3: between U3 and B), was also analyzed. Statistics was done by the ANOVA of repeated measures, one-way ANOVA and Pearson correlation coefficient. RESULTS: The mean age of the patients was 48.1+/-10.7 years. The mean temperature of B, U1, U2, and U3 groups were 37.1+/-0.25 degreesC, 37.0+/-0.25 degreesC, 36.9+/-0.24 degreesC, and 36.7+/-0.25 degreesC. The mean temperature difference of D1, D2, and D3 were 0.2471+/-0.089 degreesC, 0.079+/-0.066 degreesC and 0.066+/-0.058 degreesC. The Pearson correlation coefficient of D1, D2 and D3 were 0.938, 0.965 and 0.970. This showed there was a constant temperature increase from distal urethra to bladder step by step. The number of patients in F, L and M groups were 25(47.2%), 10(18.9%) and 18(33.9%). There was no significant urethral temperature difference at each point(U1, U2, U3 and B) among these three groups. CONCLUSION: There was a constant temperature increase from distal urethra to bladder step by step. This is a baseline study for female urethra for future clinical study. We suggest that our data can be used as deciding the sol-gel transition temperature for thermosensitive polymer injection into incontinent female urethra.

The Isolation and Characterization of Muscle Derived Stem Cells from Gastrocnemius Muscle of Rats Using the Modified Preplate Method / 대한비뇨기과학회지

PURPOSE: This study attempted to characterize the muscle derived stem cells isolated from the primary cultured skeletal muscle of the rat gastrocnemius muscle; in addition, we modified the preplate method and then compared this to the original preplate method. MATERIALS AND METHODS: The hind limbs (gastrocnemius muscles) were removed from a 3-6 week olds SD-rat and the bone was dissected away. The muscle mass was finely minced and chopped using razor blades. In an original preplate method, the cells were dissociated using a triple enzyme mixture (collagenase XI, dipase and trypsin) for 1 hour at 37degreesC. The muscle cell extract was preplated on culture flasks as described by Dr. Qu (Qu et al., 1998). The pp1-pp4 cells were referred to as the early plate (EP) cells, and the pp5-pp6 cells were referred to as the late plate (LP) cells. When we modified the preplate method, the pp1-pp2 cells were called the early plate (EP) cells and the pp3-pp4 cells were called to late plate (LP) cells. The phenotypical characteristics of EP and LP cells were compared by immunostaining and FACS. RESULTS: In the original preplate methods, the early plate (EP) cells were mixed with myogenic cells (mostly fibroblasts, 90% desmin + cells). Yet in the modified preplate method, the muscle derived stem cells were determined to be CD34 (+ or -), CD45- and desmin- cells by immunohistochemical staining and FACS. CONCLUSIONS: In original methods, the LP cells exhibited stem cell properties (CD34+, less than 30%), and they were not from a hematogeous origin (CD45-), but rather, they were from a myogenic origin (desmin+). Yet in the modified preplate method, we purified the LP cells much earlier than the original method. The LP cells displayed CD34+(more than 50%), and CD45-; thus, we isolated more primitive (desmin-) cells.

The Isolation and Characterization of Muscle Derived Stem Cells from Gastrocnemius Muscle of Rats Using the Modified Preplate Method / 대한비뇨기과학회지

PURPOSE: This study attempted to characterize the muscle derived stem cells isolated from the primary cultured skeletal muscle of the rat gastrocnemius muscle; in addition, we modified the preplate method and then compared this to the original preplate method. MATERIALS AND METHODS: The hind limbs (gastrocnemius muscles) were removed from a 3-6 week olds SD-rat and the bone was dissected away. The muscle mass was finely minced and chopped using razor blades. In an original preplate method, the cells were dissociated using a triple enzyme mixture (collagenase XI, dipase and trypsin) for 1 hour at 37degreesC. The muscle cell extract was preplated on culture flasks as described by Dr. Qu (Qu et al., 1998). The pp1-pp4 cells were referred to as the early plate (EP) cells, and the pp5-pp6 cells were referred to as the late plate (LP) cells. When we modified the preplate method, the pp1-pp2 cells were called the early plate (EP) cells and the pp3-pp4 cells were called to late plate (LP) cells. The phenotypical characteristics of EP and LP cells were compared by immunostaining and FACS. RESULTS: In the original preplate methods, the early plate (EP) cells were mixed with myogenic cells (mostly fibroblasts, 90% desmin + cells). Yet in the modified preplate method, the muscle derived stem cells were determined to be CD34 (+ or -), CD45- and desmin- cells by immunohistochemical staining and FACS. CONCLUSIONS: In original methods, the LP cells exhibited stem cell properties (CD34+, less than 30%), and they were not from a hematogeous origin (CD45-), but rather, they were from a myogenic origin (desmin+). Yet in the modified preplate method, we purified the LP cells much earlier than the original method. The LP cells displayed CD34+(more than 50%), and CD45-; thus, we isolated more primitive (desmin-) cells.

Inhibition of Neurite Outgrowth by Stably Expressed Go alpha in F11 Cells / 대한해부학회지

Heterotrimeric G proteins mediate signals generated by neurotransmitters and hormones. Among G proteins, Go is found in a large quantity in brain and growth cone membranes of neurons. In spite of its abundance in neurons, the role of Go is not fully understood. In the previous study, we showed that transient expression of the alpha subunit of Go (alpha o) modulated neurite outgrowth in F11 cells. It is possible that transient transfection may cause transient accumulation of the protein, which itself may alter differentiation process in non-specific manner. In this study, we determined that modulation of neurite outgrowth by alpha o was specific by evaluating the effect of alpha o in stably transformed F11 cells. F11 cells stably expressing the wild type alpha o (alphao(wt)) and a constitutively active form of alpha o (alpha oQ205L) were established. In normal F11 cells and alpha o-stable cell lines, the neurite length was measured in the presence of dibutyryl cAMP. In normal F11 cells, the average length of neurites was 57.9+/-7.0 microgram. In alpha o(wt)- and alpha o(Q205L)-expressing cells, the average length were 34.4+/-5.1 microgram 30.5+/-3.6 microgram, respectively. Thus, stable expression of alpha o(wt) and alpha o(Q205L) caused a decrease in neurite outgrowth by 40.6%, 47.3% respectively. This result indicates that modulation of neurite by alpha o was specific to the function of alpha o but not due to accumulation of exogenous proteins.

Overexpression of neurogenin1 induces neurite outgrowth in F11 neuroblastoma cells

Neurogenin1 (Ngn1) is a basic helix-loop-helix (bHLH) transcription factor expressed in neuronal precursors in the developing nervous system. The function of Ngn1 in neurogenesis has been shown in various aspects. In this study, we investigated the neurogenic potential of Ngn1 using neuroblastoma cell line, F11, which could be induced to differentiate into neurons in the presence of cAMP. To investigate the expression of Ngn1, expression vectors for the full-length and the C- terminal deletion mutant of Ngn1 were constructed and their transactivation potential was verified using reporter gene containing the E-box sequence. Overexpression of the full-length Ngn1 induced neurite outgrowth in F11 cells in the absence of cAMP. A C-terminal deletion mutant, Ngn1(1-197), inhibited neurite outgrowth induced by cAMP in F11 cells. These results indicate that the Ngn1 plays an important role in differentiation of neuroblastoma cells and the C terminus of Ngn1 is essential for the efficient differentiation.

Inhibition of BETA2/NeuroD by Id2

Id (Inhibitor of Differentiation) proteins belong to a family of transcriptional modulators that are characterized by a helix loop helix (HLH) region but lack the basic amino acid domain. Id proteins are known to interact with basic helix-loop-helix (bHLH) transcription factors and function as their negative regulators. The negative role of Id proteins has been well demonstrated in muscle development and some in neuronal cells. In this study, we investigated the effect of Id on the function of BETA2/NeuroD, a bHLH transcription factor responsible for neuron and endocrine cell specific gene expression. cDNAs of several Id isoforms were isolated by yeast two-hybrid system using the bHLH domain of E47, a ubiquitous bHLH partner as a bait. Id proteins expressed in COS M6 cells, were found in both cytosolic and nuclear fractions. Electrophoretic mobility shift assay showed that coexpression of Id2 proteins inhibited BETA2/ NeuroD binding to its target sequence, E-box. Id2 inhibited E-box mediated gene expression in a dose dependent manner in BETA2/NeuroD expressing HIT cells. Id coexpressed with BETA2/NeuroD in HeLa cells, inhibited the stimulatory activity of BETA2/NeuroD. These results suggest that Id proteins may negatively regulate tissue specific gene expression induced by BETA2/NeuroD in neuroendocrine cells and the inhibitory role of Id proteins during differentiation may be conserved in various tissues.

Inhibition of Neurite Outgrowth by Overexpression of Goa / 대한해부학회지

G proteins mediate signal transductions generated by neurotransmitters and hormones. Among G proteins, Go is found in a large quantity in brain, but its precise role in the nervous tissue is not fully understood. In addition, Go is one of the major proteins in growth cone membranes, which implies an important role of Go in the regulation of axon outgrowth. In this study, we attempted to determine the role of Go in axon outgrowth. We overexpressed the a subunit of Go (ao) in F11 neuroblatoma cells and examined the effect of ao on the neurite outgrowth. In F11 cells, dibutyryl cAMP increased neurite outgrowth remarkably upto 0.1 mM in a concentration dependent manner, but in a less degree at higher concentration. In the presence of 0.5 mM dibutyryl cAMP, the differentiation of F11 cells was almost saturated and the cells exhibited a typical neuronal morphology. Overexpression of ao caused a reduction of neurite outgrowth by 77.4% in length while increasing the number of neurites by 2.2 fold. The average neurite length was 38.9+/-12.5 mm in the ao-overexpressing F11 cells but 172.3+/-25.9 mm in the untransfected cells The total number of nurites per cell was 5.6+/-0.4 in the ao-overexpressing cells but 2.5 0.2 in the untransfected cells. This result suggests that Go may play an important role in growth cone collapse during neuronal cell differentiation.