Mesenchymal stem cells(MSCs)have received significant attention in recent years due to their large potential for cell therapy.Indeed,they secrete a wide variety of immunomodulatory factors of interest for the treatmen...Mesenchymal stem cells(MSCs)have received significant attention in recent years due to their large potential for cell therapy.Indeed,they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases.MSCs can be extracted from multiple tissues of the human body.However,several factors may restrict their use for clinical applications:the requirement of invasive procedures for their isolation,their limited numbers,and their heterogeneity according to the tissue of origin or donor.In addition,MSCs often present early signs of replicative senescence limiting their expansion in vitro,and their therapeutic capacity in vivo.Due to the clinical potential of MSCs,a considerable number of methods to differentiate induced pluripotent stem cells(iPSCs)into MSCs have emerged.iPSCs represent a new reliable,unlimited source to generate MSCs(MSCs derived from iPSC,iMSCs)from homogeneous and well-characterized cell lines,which would relieve many of the above mentioned technical and biological limitations.Additionally,the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells.In this review,we analyze the main current protocols used to differentiate human iPSCs into MSCs,which we classify into five different categories:MSC Switch,Embryoid Body Formation,Specific Differentiation,Pathway Inhibitor,and Platelet Lysate.We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization.Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added.The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.展开更多
Objective:At present, human umbilical cord mesenchymal stem cells (hucMSC) have been widely used in basic research and clinical trials in treatment of many diseases, but how to obtain a large number and high quality o...Objective:At present, human umbilical cord mesenchymal stem cells (hucMSC) have been widely used in basic research and clinical trials in treatment of many diseases, but how to obtain a large number and high quality of stable and reliable mesenchymal stem cells for clinical application is still a scientific research needs to be resolved. Since each laboratory used different culture methods, the productivity of the different cultures may vary. Therefore, the purpose of this study is to optimize a stable and reliable hucMSC culture procedure. Methods: Mesenchymal stem cells derived from human umbilical cord were isolated and cultured in different culture medium. By comparing the morphology, passage, proliferation and using flow cytometry to detect the immunophenotypes of the cells, optimized cultural method was selected.Results: By comparison of the time of cells migrating out from the tissue, cell growth status, cell number and expression of CD90 andCD73 of the hucMSCs, we found that X-VIVO15+10% fetal bovine serum group, X-VIVO15+2% fetal bovine serum and 2% UltroserG group, and MSC basal medium+UltraGRO-Advanced group had an earlier migration time from the umbilical cord and shorter confluence time to cover 80%-90% of the cuturing flask. The Huc-MSCs cultured with MSC basal medium+2% UltraGRO-Advanced group revealed the fastest proliferation. The expression of CD90 and CD73 in Huc-MSCs cultured with MSC basal medium+2% UltraGRO-Advanced group were above 90%, while the expression of CD90 and CD73 in other groups were below 90%. The expression of CD34 and CD45 surface antigens were low in huc-MSCs from all the groups.Conclusion:In summary, MSC basal medium-UltraGRO-Advanced group was the optimal culture medium for human umbilical cord mesenchymal stem cells.展开更多
To establish and validate a method for cell irradiation in 96-well and 6-well plates using a linear accelerator, three irradiation methods(G0 B0 F40,G0 B1.5 F40, and G180 B1.5 F40) were designed to irradiate cell cult...To establish and validate a method for cell irradiation in 96-well and 6-well plates using a linear accelerator, three irradiation methods(G0 B0 F40,G0 B1.5 F40, and G180 B1.5 F40) were designed to irradiate cell culture plasticware simulated with RW3 slab phantom and polystyrene. The difference between the actual physical measured dose and the preset dose was compared among the three methods under the preparatory conditions of 2, 4, 6, 8, and 10 Gy. MDA-MB-231 cells were analyzed by using a cell proliferation assay and a clonogenic assay to verify the difference between the three cell irradiation methods on cell radiosensitivity. For each preset dose, the difference between the actual measured dose and the preset dose was the lowest for Method G0 B1.5 F40, the second lowest for Method G180 B1.5 F40, and the maximum for Method GOB0 F40. The ranges of the differences were-0.28 to 0.02%,-2.17 to-1.80%, and-4.92 to-4.55%, and 0.31 to-0.12%,-3.42 to-2.86%, and-7.31 to-6.92%,respectively, for 96-well and 6-well plates. The cell culture experiments proved that Method G0 B1.5 F40 was an accurate, effective, simple, and practical irradiation method. The most accurate and effective cell irradiation method should always be used, as it will reduce dose differences and instability factors and provide improved accuracy and comparability for laboratories researching cellular radiosensitivity.展开更多
AIM: Activated pancreatic stellate cells (PSCs) have been implicated in the pathogenesis of pancreatic fibrosis and inflammation. Primary PSCs can be subcultured only several times because of their limited growth pote...AIM: Activated pancreatic stellate cells (PSCs) have been implicated in the pathogenesis of pancreatic fibrosis and inflammation. Primary PSCs can be subcultured only several times because of their limited growth potential. A continuous cell line may therefore be valuable in studying molecular mechanisms of these pancreatic disorders. The aim of this study was to establish a cell line of rat PSCs by spontaneous immortalization.METHODS: PSCs were isolated from the pancreas of male Wistar rats, and conventional subcultivation was performed repeatedly. Telomerase activity was measured using the telomere repeat amplification protocol. Activation of transcription factors was assessed by electrophoretic mobility shift assay.Activation of mitogen-activated protein (MAP) kinases was examined by Western blotting using anti-phosphospecific antibodies. Expression of cytokine-induced neutrophil chemoattractant-1 was determined by enzyme immunoassay.RESULTS: Conventional subcultivation yielded actively growing cells. One clone was obtained after limiting dilution,and designated as SIPS. This cell line has been passaged repeatedly more than 2 years, and is thus likely immortalized.SIPS cells retained morphological characteristics of primary,culture-activated PSCs. SIPS expressed α-smooth muscle actin, glial acidic fibrillary protein, vimentin, desmin, type Ⅰ collagen, fibronectin, and prolyl hydroxylases. Telomerase activity and p53 expression were negative. Proliferation of SIPS cells was serum-dependent, and stimulated with platelet-derived growth factor-BB through the activation of extracellular signal-regulated kinase. Interleukin-1β activated nuclear factor-κB, activator protein-1, and MAP kinases.Interleukin-1β induced cytokine-induced neutrophil chemoattractant-1 expression through the activation of nuclear factor-κB and MAP kinases.CONCLUSION: SIPS cells can be useful for in vitro studies of cell biology and signal transduction of PSCs.展开更多
文摘Mesenchymal stem cells(MSCs)have received significant attention in recent years due to their large potential for cell therapy.Indeed,they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases.MSCs can be extracted from multiple tissues of the human body.However,several factors may restrict their use for clinical applications:the requirement of invasive procedures for their isolation,their limited numbers,and their heterogeneity according to the tissue of origin or donor.In addition,MSCs often present early signs of replicative senescence limiting their expansion in vitro,and their therapeutic capacity in vivo.Due to the clinical potential of MSCs,a considerable number of methods to differentiate induced pluripotent stem cells(iPSCs)into MSCs have emerged.iPSCs represent a new reliable,unlimited source to generate MSCs(MSCs derived from iPSC,iMSCs)from homogeneous and well-characterized cell lines,which would relieve many of the above mentioned technical and biological limitations.Additionally,the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells.In this review,we analyze the main current protocols used to differentiate human iPSCs into MSCs,which we classify into five different categories:MSC Switch,Embryoid Body Formation,Specific Differentiation,Pathway Inhibitor,and Platelet Lysate.We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization.Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added.The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.
基金National Natural Science Foundation(:81570157)Shandong Province Medical and Health Technology Development Project(2013ws0291).
文摘Objective:At present, human umbilical cord mesenchymal stem cells (hucMSC) have been widely used in basic research and clinical trials in treatment of many diseases, but how to obtain a large number and high quality of stable and reliable mesenchymal stem cells for clinical application is still a scientific research needs to be resolved. Since each laboratory used different culture methods, the productivity of the different cultures may vary. Therefore, the purpose of this study is to optimize a stable and reliable hucMSC culture procedure. Methods: Mesenchymal stem cells derived from human umbilical cord were isolated and cultured in different culture medium. By comparing the morphology, passage, proliferation and using flow cytometry to detect the immunophenotypes of the cells, optimized cultural method was selected.Results: By comparison of the time of cells migrating out from the tissue, cell growth status, cell number and expression of CD90 andCD73 of the hucMSCs, we found that X-VIVO15+10% fetal bovine serum group, X-VIVO15+2% fetal bovine serum and 2% UltroserG group, and MSC basal medium+UltraGRO-Advanced group had an earlier migration time from the umbilical cord and shorter confluence time to cover 80%-90% of the cuturing flask. The Huc-MSCs cultured with MSC basal medium+2% UltraGRO-Advanced group revealed the fastest proliferation. The expression of CD90 and CD73 in Huc-MSCs cultured with MSC basal medium+2% UltraGRO-Advanced group were above 90%, while the expression of CD90 and CD73 in other groups were below 90%. The expression of CD34 and CD45 surface antigens were low in huc-MSCs from all the groups.Conclusion:In summary, MSC basal medium-UltraGRO-Advanced group was the optimal culture medium for human umbilical cord mesenchymal stem cells.
基金supported by the Hospital Personnel Climbing Plan of the Tenth People's Hospital Affiliated to Tongji University
文摘To establish and validate a method for cell irradiation in 96-well and 6-well plates using a linear accelerator, three irradiation methods(G0 B0 F40,G0 B1.5 F40, and G180 B1.5 F40) were designed to irradiate cell culture plasticware simulated with RW3 slab phantom and polystyrene. The difference between the actual physical measured dose and the preset dose was compared among the three methods under the preparatory conditions of 2, 4, 6, 8, and 10 Gy. MDA-MB-231 cells were analyzed by using a cell proliferation assay and a clonogenic assay to verify the difference between the three cell irradiation methods on cell radiosensitivity. For each preset dose, the difference between the actual measured dose and the preset dose was the lowest for Method G0 B1.5 F40, the second lowest for Method G180 B1.5 F40, and the maximum for Method GOB0 F40. The ranges of the differences were-0.28 to 0.02%,-2.17 to-1.80%, and-4.92 to-4.55%, and 0.31 to-0.12%,-3.42 to-2.86%, and-7.31 to-6.92%,respectively, for 96-well and 6-well plates. The cell culture experiments proved that Method G0 B1.5 F40 was an accurate, effective, simple, and practical irradiation method. The most accurate and effective cell irradiation method should always be used, as it will reduce dose differences and instability factors and provide improved accuracy and comparability for laboratories researching cellular radiosensitivity.
基金Grant-in-Aid for Encouragement of Young Scientists from Japan Society for the Promotion of Science(to A.M.)Pancreas Research Foundation of Japan(to A.M.)
文摘AIM: Activated pancreatic stellate cells (PSCs) have been implicated in the pathogenesis of pancreatic fibrosis and inflammation. Primary PSCs can be subcultured only several times because of their limited growth potential. A continuous cell line may therefore be valuable in studying molecular mechanisms of these pancreatic disorders. The aim of this study was to establish a cell line of rat PSCs by spontaneous immortalization.METHODS: PSCs were isolated from the pancreas of male Wistar rats, and conventional subcultivation was performed repeatedly. Telomerase activity was measured using the telomere repeat amplification protocol. Activation of transcription factors was assessed by electrophoretic mobility shift assay.Activation of mitogen-activated protein (MAP) kinases was examined by Western blotting using anti-phosphospecific antibodies. Expression of cytokine-induced neutrophil chemoattractant-1 was determined by enzyme immunoassay.RESULTS: Conventional subcultivation yielded actively growing cells. One clone was obtained after limiting dilution,and designated as SIPS. This cell line has been passaged repeatedly more than 2 years, and is thus likely immortalized.SIPS cells retained morphological characteristics of primary,culture-activated PSCs. SIPS expressed α-smooth muscle actin, glial acidic fibrillary protein, vimentin, desmin, type Ⅰ collagen, fibronectin, and prolyl hydroxylases. Telomerase activity and p53 expression were negative. Proliferation of SIPS cells was serum-dependent, and stimulated with platelet-derived growth factor-BB through the activation of extracellular signal-regulated kinase. Interleukin-1β activated nuclear factor-κB, activator protein-1, and MAP kinases.Interleukin-1β induced cytokine-induced neutrophil chemoattractant-1 expression through the activation of nuclear factor-κB and MAP kinases.CONCLUSION: SIPS cells can be useful for in vitro studies of cell biology and signal transduction of PSCs.
