Objective:To isolate, culture and identify human embryonic neural stem cells and to establish a practical passaging method.Method:The cerebral cortex cells were isolated from aborted embryos (11～13 weeks) by mechanic...Objective:To isolate, culture and identify human embryonic neural stem cells and to establish a practical passaging method.Method:The cerebral cortex cells were isolated from aborted embryos (11～13 weeks) by mechanical dissociation,and cultured in DMEM/F12 culture medium supplemented with N2 and growth factors for proliferation. Upon passaging, the neurospheres were pipetted gentlely to separate them into several cell masses and then grown in growth medium. The cells were grown in DMEM/F12 medium with serum (without growth factors) to induce differentiation. The stem cell, neuron, astrocyte and oligodendrocyte were identified by immunocytochemistry with antibodies to vimentin, MAP 2, GFAP and GalC, respectively. Results:The primary cells grew together and formed neurospheres at 5 th ～7 th day. They were all vimentin positive and could be passaged for at least 8 passages. After passaging, the cell masses grew up and formed new neurospheres rapidly.These cells could differentiated into MAP 2(+),GFAP(+) or GalC(+) cells.Conclusion:The neural stem cells from human embryonic cerebral cortex have the capacity of proliferation and multi-differentiation in vitro. The passaging methods we used in this experiment were practical and convenient.展开更多
Objective： To evaluate the dltterentlatlon ot human umbilical cord blood ceils into hepatocyte-like cells. Methods： Mononuclear cells （MNCs） derived from human umbilical cord blood were isolated using Ficoll. The ...Objective： To evaluate the dltterentlatlon ot human umbilical cord blood ceils into hepatocyte-like cells. Methods： Mononuclear cells （MNCs） derived from human umbilical cord blood were isolated using Ficoll. The experiment was derived into 3 categories： （1） MNCs co-cultured with 50 mg minced liver tissue separated by a trans-well membrane and then collected at 0 h, 24 h, 48 h and 72 h; （2） MNCs cultured along supplemented with 100 ml/L FBS, 100 μ/ml penicillin, 100 μg/ml streptomycin, 4.7 μg/ml linoleic acid, 1×ITS, 10^-4 mol/L L-Ascorbic acid 2-P and a combination of FGF4 （100 ng/ml） and HGF （20 ng/mL）. Cells were then collected at 0 d and 16 d to examine the expression profile of hepatocyte correlating markers; （3） 0.2-0.3 ml of MNCs with a cell density of 2×10^7/ml were transplanted into prepared recipient mice [n=12, injected with 0.4 ml/kg （20%） CCl4 and 150 ng/kg 5-fluorouracil （5-Fu） prior the transplant 24 h and 48 h, respectively] via injection through tail vein. Mice were sacrificed 4 weeks after transplantation. The hepatocyte correlating mRNAs and proteins were determined by RT-PCR, immunohistochemical analysis and immunoflurence technique. Results： （1） After 72 h, a number of glycogen positive stained cells were observed with MNCs co-cultured with damaged mouse liver tissues. The expression of hepatocyte markers, human albumin （ALB）, α-fetal protein （AFP） and human GATA4 mRNA and proteins were detected by RT-PCR and immunohistochemistry as well. For the confirmation, the DNA sequencing of PCR products was performed. In control groups, MNCs co-cuhured with normal mouse hepatocytes or MNCs cultured alone, all markers remained negative. （2） In growth factor supplemented culture system, MNCs developed into larger volume with richer cytoplasm and binucleation after 16 d. Positive expression of ALB, AFP, CK18 and CK19 mRNA were detected with RT-PCR, and ALB positive staining was observed by immunocytochemistry as well. In contrast, MNCs cultured without exogenous growth factors scarcely attached to the culture dish and ALB mRNA was not detected. （3） In transplantation experiment, both of ALB and AFP mRNA were detected by RT-PCR and HSA, PCNA and ALB positive staining were observed in the livers of recipient mice by immunocytochemistry. Conclusion： MNCs from human umbilical cord blood could convert into hepatocyte-like ceils in 3 different ways, indicating their potential use in the clinic applications for the treatment of human liver diseases.展开更多
The breakthrough development of induced pluripotent stem cells(iPSCs)raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells.However,whether iPSC-derived functiona...The breakthrough development of induced pluripotent stem cells(iPSCs)raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells.However,whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear.In this study,we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells(NPCs)and analyzed their immunogenicity.Through co-culture with autogenous peripheral blood mononuclear cells(PBMCs),we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation.However,a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs.Furthermore,no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells(CD3+CD8 T cells,CD3+CD8+T cells or CD3 CD56+NK cells)by NPCs in both PBMC and T cell co-culture systems.These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants,and thus set a base for further preclinical evaluation of human iPSCs.展开更多
Due to the low number of collectable stem cells from single umbilical cord blood(UCB)unit,their initial uses were limited to pediatric therapies.Clinical applications of UCB hematopoietic stem and progenitor cells(HSP...Due to the low number of collectable stem cells from single umbilical cord blood(UCB)unit,their initial uses were limited to pediatric therapies.Clinical applications of UCB hematopoietic stem and progenitor cells(HSPCs)would become feasible if there were a culture method that can effectively expand HSPCs while maintaining their self-renewal capacity.In recent years,numerous attempts have been made to expand human UCB HSPCs in vitro.In this study,we report that caffeic acid phenethyl ester(CAPE),a small molecule from honeybee extract,can promote in vitro expansion of HSPCs.Treatment with CAPE increased the percentage of HSPCs in cultured mononuclear cells.Importantly,culture of CD34+HSPCs with CAPE resulted in a significant increase in total colony-forming units and high proliferative potential colony-forming units.Burst-forming unit-erythroid was the mostly affected colony type,which increased more than 3.7-fold in 1μg mL 1CAPE treatment group when compared to the controls.CAPE appears to induce HSPC expansion by upregulating the expression of SCF and HIF1-α.Our data suggest that CAPE may become a potent medium supplement for in vitro HSPC expansion.展开更多
Congenital human cytomegalovirus(HCMV) infection is a leading infectious cause of birth defects.Previous studies have reported birth defects with multiple organ maldevelopment in congenital HCMV-infected neonates. Mul...Congenital human cytomegalovirus(HCMV) infection is a leading infectious cause of birth defects.Previous studies have reported birth defects with multiple organ maldevelopment in congenital HCMV-infected neonates. Multipotent mesenchymal stromal cells(MSCs) are a group of stem/progenitor cells that are multi-potent and can self-renew, and they play a vital role in multiorgan formation. Whether MSCs are susceptible to HCMV infection is unclear. In this study, MSCs were isolated from Wharton's jelly of the human umbilical cord and identified by their plastic adherence, surface marker pattern, and differentiation capacity. Then, the MSCs were infected with the HCMV Towne strain, and infection status was assessed via determination of viral entry,replication initiation, viral protein expression, and infectious virion release using western blotting,immunofluorescence assays, and plaque forming assays. The results indicate that the isolated MSCs were fully permissive for HCMV infection and provide a preliminary basis for understanding the pathogenesis of HCMV infection in non-nervous system diseases, including multi-organ malformation during fetal development.展开更多
文摘Objective:To isolate, culture and identify human embryonic neural stem cells and to establish a practical passaging method.Method:The cerebral cortex cells were isolated from aborted embryos (11～13 weeks) by mechanical dissociation,and cultured in DMEM/F12 culture medium supplemented with N2 and growth factors for proliferation. Upon passaging, the neurospheres were pipetted gentlely to separate them into several cell masses and then grown in growth medium. The cells were grown in DMEM/F12 medium with serum (without growth factors) to induce differentiation. The stem cell, neuron, astrocyte and oligodendrocyte were identified by immunocytochemistry with antibodies to vimentin, MAP 2, GFAP and GalC, respectively. Results:The primary cells grew together and formed neurospheres at 5 th ～7 th day. They were all vimentin positive and could be passaged for at least 8 passages. After passaging, the cell masses grew up and formed new neurospheres rapidly.These cells could differentiated into MAP 2(+),GFAP(+) or GalC(+) cells.Conclusion:The neural stem cells from human embryonic cerebral cortex have the capacity of proliferation and multi-differentiation in vitro. The passaging methods we used in this experiment were practical and convenient.
基金Supported by the Shenzhen Science & Technology Planning Program (No. 200204109, No. JH200505270412B)
文摘Objective： To evaluate the dltterentlatlon ot human umbilical cord blood ceils into hepatocyte-like cells. Methods： Mononuclear cells （MNCs） derived from human umbilical cord blood were isolated using Ficoll. The experiment was derived into 3 categories： （1） MNCs co-cultured with 50 mg minced liver tissue separated by a trans-well membrane and then collected at 0 h, 24 h, 48 h and 72 h; （2） MNCs cultured along supplemented with 100 ml/L FBS, 100 μ/ml penicillin, 100 μg/ml streptomycin, 4.7 μg/ml linoleic acid, 1×ITS, 10^-4 mol/L L-Ascorbic acid 2-P and a combination of FGF4 （100 ng/ml） and HGF （20 ng/mL）. Cells were then collected at 0 d and 16 d to examine the expression profile of hepatocyte correlating markers; （3） 0.2-0.3 ml of MNCs with a cell density of 2×10^7/ml were transplanted into prepared recipient mice [n=12, injected with 0.4 ml/kg （20%） CCl4 and 150 ng/kg 5-fluorouracil （5-Fu） prior the transplant 24 h and 48 h, respectively] via injection through tail vein. Mice were sacrificed 4 weeks after transplantation. The hepatocyte correlating mRNAs and proteins were determined by RT-PCR, immunohistochemical analysis and immunoflurence technique. Results： （1） After 72 h, a number of glycogen positive stained cells were observed with MNCs co-cultured with damaged mouse liver tissues. The expression of hepatocyte markers, human albumin （ALB）, α-fetal protein （AFP） and human GATA4 mRNA and proteins were detected by RT-PCR and immunohistochemistry as well. For the confirmation, the DNA sequencing of PCR products was performed. In control groups, MNCs co-cuhured with normal mouse hepatocytes or MNCs cultured alone, all markers remained negative. （2） In growth factor supplemented culture system, MNCs developed into larger volume with richer cytoplasm and binucleation after 16 d. Positive expression of ALB, AFP, CK18 and CK19 mRNA were detected with RT-PCR, and ALB positive staining was observed by immunocytochemistry as well. In contrast, MNCs cultured without exogenous growth factors scarcely attached to the culture dish and ALB mRNA was not detected. （3） In transplantation experiment, both of ALB and AFP mRNA were detected by RT-PCR and HSA, PCNA and ALB positive staining were observed in the livers of recipient mice by immunocytochemistry. Conclusion： MNCs from human umbilical cord blood could convert into hepatocyte-like ceils in 3 different ways, indicating their potential use in the clinic applications for the treatment of human liver diseases.
基金supported by the National Basic Research Program of China,Ministry of Science and Technology(2011CB965204,2012CB966802)the National Natural Science Foundation of China(31000402)+3 种基金the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA01020401,XDA-01020202)the Ministry of Science and Technology International Technology Cooperation Program(2012DFH30050)the National Science&Technology Major Special Project on Major New Drug Innovation(2011ZX09102-010-01)the Development and Technology Innovation for Equipment Functional Development Project of Chinese Academy of Sciences(yg2011082,yg2011083)
文摘The breakthrough development of induced pluripotent stem cells(iPSCs)raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells.However,whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear.In this study,we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells(NPCs)and analyzed their immunogenicity.Through co-culture with autogenous peripheral blood mononuclear cells(PBMCs),we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation.However,a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs.Furthermore,no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells(CD3+CD8 T cells,CD3+CD8+T cells or CD3 CD56+NK cells)by NPCs in both PBMC and T cell co-culture systems.These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants,and thus set a base for further preclinical evaluation of human iPSCs.
基金supported by the National High Technology Research and Development Program of China(2013AA020107)National Basic Research Program of China(2011CB964804)National Natural Science Foundation of China(31101040)
文摘Due to the low number of collectable stem cells from single umbilical cord blood(UCB)unit,their initial uses were limited to pediatric therapies.Clinical applications of UCB hematopoietic stem and progenitor cells(HSPCs)would become feasible if there were a culture method that can effectively expand HSPCs while maintaining their self-renewal capacity.In recent years,numerous attempts have been made to expand human UCB HSPCs in vitro.In this study,we report that caffeic acid phenethyl ester(CAPE),a small molecule from honeybee extract,can promote in vitro expansion of HSPCs.Treatment with CAPE increased the percentage of HSPCs in cultured mononuclear cells.Importantly,culture of CD34+HSPCs with CAPE resulted in a significant increase in total colony-forming units and high proliferative potential colony-forming units.Burst-forming unit-erythroid was the mostly affected colony type,which increased more than 3.7-fold in 1μg mL 1CAPE treatment group when compared to the controls.CAPE appears to induce HSPC expansion by upregulating the expression of SCF and HIF1-α.Our data suggest that CAPE may become a potent medium supplement for in vitro HSPC expansion.
基金supported by the National Science Foundation of China (81071350,81271850,and 31170155)the National Program on Key Basic Research Project (973 program 2011CB504804 and 2012CB519003)
文摘Congenital human cytomegalovirus(HCMV) infection is a leading infectious cause of birth defects.Previous studies have reported birth defects with multiple organ maldevelopment in congenital HCMV-infected neonates. Multipotent mesenchymal stromal cells(MSCs) are a group of stem/progenitor cells that are multi-potent and can self-renew, and they play a vital role in multiorgan formation. Whether MSCs are susceptible to HCMV infection is unclear. In this study, MSCs were isolated from Wharton's jelly of the human umbilical cord and identified by their plastic adherence, surface marker pattern, and differentiation capacity. Then, the MSCs were infected with the HCMV Towne strain, and infection status was assessed via determination of viral entry,replication initiation, viral protein expression, and infectious virion release using western blotting,immunofluorescence assays, and plaque forming assays. The results indicate that the isolated MSCs were fully permissive for HCMV infection and provide a preliminary basis for understanding the pathogenesis of HCMV infection in non-nervous system diseases, including multi-organ malformation during fetal development.
