ABSTRACT
BACKGROUND: Human skeletal muscle derived myoendothelial cells (MECs) are located in the vascular wall and co-express the markers of muscle stem cells and vascular endothelial cells (CD56+CD34+CD144+CD45-). Studies have shown that MECs are similar to mesenchymal stem cells, express the surface markers of mesenchymal stem cells and have the potential of multidirectional differentiation. OBJECTIVE: To establish an in vitro culture system for human umbilical cord blood CD34+ cells with MECs as trophoblastic layer, and to evaluate the in vitro supporting effect of human skeletal muscle MECs on hematopoietic stem/progenitor cells by measuring the changes in the number, immunophenotype and colony forming ability of CD34+ cells before and after culture. METHODS: There were three groups in the experiment. In experimental group, human umbilical cord blood CD34+ cells were co-cultured with MECs as the nourishing layer; in control group, human umbilical cord blood CD34+ cells were co-cultured with bone marrow mesenchymal stem cells as the nourishing layer; and in blank control group, human umbilical cord blood CD34+ cells were cultured alone without the nourishing layer. The main outcome measures, including the number of human umbilical cord blood CD34+ cells, immunophenotype of blood cells and colony formation ability of hematopoietic stem/progenitor cells were analyzed and compared at 1, 2, and 4 weeks after co-culture. No detection was conducted at 5 weeks due to the lack of survived cells. RESULTS AND CONCLUSION: (1) The number of human umbilical cord blood CD34+ cells increased by MECs as the nourishing layer compared with bone marrow mesenchymal stem cells as the nourishing layer at 1, 2, and 4 weeks; however, there was no significant difference between the two groups (P > 0.05). (2) The cell immunophenotype by flow cytometry analysis indicated that only in the 2nd week, the expression of CD34+CD33- in human umbilical cord blood CD34+ cells in the control group was significantly higher in the experimental group (P 0.05). (3) The colony formation capacity of hematopoietic stem/progenitor cells showed no significant difference between the experimental and control groups at 1, 2, and 4 weeks (P > 0.05). (4) Due to the non-nourishing layer culture system, the number of human umbilical cord blood CD34+ cells decreased significantly in the 1st week, and no cells survived in the 2nd week. Therefore, blood cell immunophenotype and colony analysis could not be performed. (5) To conclude, human skeletal muscle MECs as trophoblasts are the same as human bone marrow mesenchymal stem cells, which have a hematopoietic support in vitro.
ABSTRACT
BACKGROUND: Perivascular cells have been shown to be the precursor cells of mesenchymal stem cells, which regulate the behavior of hematopoietic stem cells and support hematopoiesis through cell-to-cell contact or paracrine effects. Hematopoietic support of human skeletal muscle-derived pericytes/perivascular cells (hMD-PCs) remains to be studied. OBJECTIVE: To identify the biological characteristics of hMD-PCs isolated from human skeletal muscle and to study their supporting effect on umbilical cord blood CD34+ cells in vitro. METHODS: (1) hMD-PCs with phenotype CD146+ CD56-CD34-CD144-CD45- were sorted from human skeletal muscle by enzymatic digestion and multiparameter fluorescence-activated cell sorting, and their biological characteristics were identified. (2) The in vitro culture system of umbilical cord blood CD34+ cells co-cultured with human CD146+ hMD-PCs (experimental group) or with human bone marrow mesenchymal stem cells (positive control group) was established. After 1, 2 and 4 weeks of co-culture, the number of cells, the colony formation ability and immunophenotype were measured and statistically analyzed. RESULTS AND CONCLUSION: (1) CD146+ hMD-PCs were sorted by multiparameter fluorescence-activated cell sorting and the purity was (91.5±1.85)% (n=5). CD146+ hMD-PCs expressed mesenchymal surface markers CD73, CD90, CD105, CD44, and did not express hematopoietic cell and endothelial cell markers CD45, CD34, and CD31. After induced culture, CD146+ hMD-PCs could differentiate into osteoblasts, chondrogenesis, adipocytes and myoblasts. (2) There were no significant differences in the cell number, colony f ormation ability or immunophenotype (CD45+, CD34+ CD33-, CD14+, CD10+/CD19+) between experimental and positive control groups (P > 0.05, n=6). The number of cells in the blank control group without feeder was significantly decreased at 1 week of culture, and there was almost no cell survival at 2 weeks of culture. (3) In summary, CD146+ hMD-PCs, like human bone marrow mesenchymal stem cells, have hematopoietic support capacity in vitro.
ABSTRACT
O presente estudo tem como objetivos: a) verificar a concordância entre os métodos da impedância bioelétrica (BIA) e da absortometria radiológica de dupla energia (DEXA), para a estimativa da massa muscular esquelética (MME); b) analisar o poder preditivo das variáveis antropométricas e de BIA para predição da MME em idosos. Foram avaliados 60 homens idosos (61 a 80 anos), residentes na região Sul do Brasil. Mensuraram-se as variáveis antropométricas (massa corporal e estatura), as variáveis de resistência e hidratação dos tecidos livres de gordura foram medidas pela técnica da BIA tetrapolar (Biodinamics - BF-310), realizou-se também um scan de corpo inteiro através da DEXA (LUNAR PRODIGY DF + 14319 Radiation e software 7.52.002 DPX-L). A diferença entre os métodos foi verificada pelo teste t, análise dos resíduos e o coeficiente de correlação. O valor preditivo das variáveis antropométricas e de BIA foi verificado pela regressão Linear Múltipla. Observou-se que a BIA superestimou em média 0,6 kg (dp= 1,59) a MME, quando comparada com a DEXA, contudo não houve diferença estatística (p<0,05). Foi observada uma forte relação entre os métodos (r=0,90; p<0,01). A análise de regressão demonstrou que a variável EST²/R explica 86 por cento da variação da MME, quando ajustada para massa corporal e idade e esta relação é independente das variáveis de gordura corporal, hidratação dos tecidos livres de gordura e IMC. Assim, nota-se que o método da BIA, aqui testado, é válido para a estimativa da MME em homens idosos e seus valores podem ser melhor preditos pelo modelo de regressão proposto a partir da medida de EST²/R ajustada para a massa corporal e idade.
The aim of the present study was twofold: a) to determine the agreement between bioelectrical impedance analysis (BIA) and dual energy X-ray absorptiometry (DEXA) for the estimation of skeletal muscle mass (SMM), and b) to analyze the predictive power of anthropometric variables and BIA for the prediction of SMM in the elderly. Sixty elderly men (61 to 80 years) from the southern region of Brazil were studied. Anthropometric variables (body weight and height) were measured, the resistance and hydration of fat-free tissues variables were determined by tetrapolar BIA (BF-310, Biodynamics). A whole body DEXA scan was also performed (Lunar Prodigy DF + 14319 Radiation and 7.52.002 DPX-L software). Differences between methods were analyzed using the t-test, analysis of residues and correlation coefficient. The predictive value of the anthropometric variables and BIA was evaluated by multiple linear regression. BIA overestimated SMM on average by 0.60 kg (sd=1.59) when compared to DEXA, however, no statistical difference was observed (p>0.05). There was a strong correlation between methods (r=0.90; p<0.01). Regression analysis demonstrated that the Ht²/R variable explained 86 percent of the variation in SMM when adjusted for body weight and age, and this relationship did not depend on body fat, hydration of fat-free tissues or BMI. Thus, BIA as tested here is a valid method for the estimation of SMM in elderly men and its values can be best predicted using the regression model proposed, which included Ht²/R adjusted for body weight and age.
Subject(s)
Humans , Female , Middle Aged , Aging , Body Composition , Densitometry , Electric Impedance , Muscle, SkeletalABSTRACT
BACKGROUND: Intravenous immunoglobulin (IVIg) has been widely used in the management of patients with various autoimmune neurological diseases, however, its action mechanisms have not fully been elucidated yet. This study focused on the effects of IVIg on the production of interleukin-6 (IL-6), one of major proinflammatory cytokine, using a human skeletal muscle cell line (HM4). METHODS: After HM4 cells were cultured in Dulbecco's modified eagle's medium (DMEM) containing 5% fetal bovine serum for 24 h, the culture medium was changed with serum-free media. TNF-alpha (tumor necrosis factor-alpha, 100 ng/mL) and IVIg (5 mg/mL) were treated alone or in combination and cultured for various time. RT-PCR and ELISA kit were employed for mRNA expression and secretion of IL-6, respectively. RESULTS: Treatment with TNF-alpha or/and IVIg significantly induced IL-6 mRNA expression (p<0.001). Although IL-6 production was markedly increased by TNF-alpha (p<0.001), IVIg treatment alone or in combination with TNF-alpha had no effect on the production of IL-6 except at 6 h after the treatment. CONCLUSIONS: IVIg seems not to have a significant effect on IL-6 production as an action mechanism of its immunomodulatory capabilities, at least in the HM4 cell line.
Subject(s)
Humans , Cell Line , Culture Media, Serum-Free , Enzyme-Linked Immunosorbent Assay , Immunoglobulins , Immunoglobulins, Intravenous , Interleukin-6 , Muscle, Skeletal , Necrosis , RNA, Messenger , Tumor Necrosis Factor-alphaABSTRACT
BACKGROUND: Muscle is a target of immunological injury in several muscle diseases, such as idiopathic inflammatory myopathy. However, it is also a target for gene therapy. Therefore, it is important to understand the immunological capabilities of muscle cells. To assess as to whether muscle cells are actively involved in the inflamed muscle tissue, a human skeletal muscle cell line was tested for the expression of several cytokines and chemokine at the mRNA level. METHODS: A human skeletal muscle cell line (SKM14) had been developed by a retroviral vector encoding v-myc transfection into a 12-week-old human fetal skeletal muscle tissue characterized by the immunostaining of several musclespecific markers. Human skeletal myoblasts of this cell line were tested for their capacity to express different cytokines (IL-1beta, -6, -10, -12, -15, and TNF-alpha) and chemokine (IL-8) mRNA levels at the basal state and in the presence of TNF-alpha(10 ng/ml). RESULTS: The SKM14 cell line was confirmed to be able to express various cytokines constitutively (IL-6, -8, -12, -15, and TNF-alpha) and in the presence of TNF-alpha(IL-1beta, -6, -8, -10, -12, -15, and TNF-alpha). CONCLUSIONS: Our results suggest that muscle cells may play a role as immunocompetent cells.