AIM： To explore the possibility of human umbilical cord mesenchymal stem cells（h UCMSCs）, human umbilical vein endothelial cells（h UVECs）, human dental pulp stem cells（h DPSCs） and human periodontal ligament st...AIM： To explore the possibility of human umbilical cord mesenchymal stem cells（h UCMSCs）, human umbilical vein endothelial cells（h UVECs）, human dental pulp stem cells（h DPSCs） and human periodontal ligament stem cells（h PDLSCs） serving as feeder cells in co-culture systems for the cultivation of limbal stem cells.METHODS： Different feeder layers were cultured in Dulbecco＇s modified Eagle＇s medium（DMEM）/F12 and were treated with mitomycin C. Rabbits limbal stem cells（LSCs） were co-cultured on h UCMSCs, h UVECs, h DPSCs, h PDLSCs and NIH-3T3, and then comparative analysis were made between each group to see their respective colony-forming efficiency（CFE） assay and immunofluorescence（IPO13,CK3/12）.RESULTS： The efficiency of the four type cells in supporting the LSCs morphology and its cellular differentiation was similar to that of NIH-3T3 fibroblasts as demonstrated by the immunostaining properties analysis, with each group exhibiting a similar strong expression pattern of IPO13, but lacking CK3 and CK12 expression in terms of immunostaining. But h UCMSCs, h DPSCs and h PDLSCs feeder layers were superior in promoting colony formation potential of cells when compared to h UVECs and feedercell-free culture.CONCLUSION： hUCMSCs, hDPSCs and hPDLSCs can be a suitable alternative to conventional mouse NIH-3T3 feeder cells, so that risk of zoonotic infection can be diminished.展开更多
A83-01 is a selective inhibitor of the TGF-β type I receptor ALK,which inhibits the TGF-β-induced epithelial-to-mesenchymal transition（EMT） via the inhibition of Smad2 phosphorylation.Previous studies have showed ...A83-01 is a selective inhibitor of the TGF-β type I receptor ALK,which inhibits the TGF-β-induced epithelial-to-mesenchymal transition（EMT） via the inhibition of Smad2 phosphorylation.Previous studies have showed that A83-01 promoted somatic cellular reprogramming significantly.Male germline stem cells（mGSCs）,as an alternative resource of pluripotent stem cells derived adult testis,have promising valuable in clinic medicine and regeneration,however,the derivation of mGSCs was complex and difficult.What the role A83-01 plays in promoting the proliferation of mGSCs is still unknown.In this study,combined with A83-01 and knockout serum replacement（KSR） medium,we obtained a relatively feeder-and serum-free system for mGSCs culturing in vitro and the optimal concentration of A83-01 was 0.25 μmol L-1.After continuous culturing,the proliferation efficiency of undifferentiated mGSCs and differentiation capacity of mGSC were examined as well.Results showed that,A83-01 dramatically increased the number of mGSCs and AP positive colonies,and the mitosis index according to the BrdU assay.A83-01 could also increase the expression of pluripotent markers including Oct4,Klf4,Nanog and c-Myc,analyzed byreal-time quantative PCR.mGSCs cultured in the optimal feeder-and serum-free system combined with A83-01 could form embryoid bodies（EBs）,which consisted of three embryonic layers detected by immunofluorescence and RT-PCR.Remarkably,the results demonstrated 0.25 μmol L-1A83-01 could promote the proliferation of mouse mGSC colonies and maintain their undifferentiated status under feeder-and serum-free systems.展开更多
Human embryonic stem cells （hESC） not only hold great promise for the treatment of degenerative diseases but also provide a valuable tool for developmental studies. However, the clinical applications of hESC are at ...Human embryonic stem cells （hESC） not only hold great promise for the treatment of degenerative diseases but also provide a valuable tool for developmental studies. However, the clinical applications of hESC are at present limited by xeno-contamination during the in vitro derivation and propagation of these cells. In this review, we summarize the current methodologies for the derivation and the propagation of hESC in conditions that will eventually enable the generation of clinical-grade cells for future therapeutic applications.展开更多
SNL cells(permanent line of irradiated mouse fibroblast cells), primary mice embryonic fibroblasts (PMEF) cells and primary chicken embryonic fibroblasts (PCEF) cells were respectively used as the feeder cells for chi...SNL cells(permanent line of irradiated mouse fibroblast cells), primary mice embryonic fibroblasts (PMEF) cells and primary chicken embryonic fibroblasts (PCEF) cells were respectively used as the feeder cells for chicken embryonic stem cell culture. The isolated blastoderm cells from the stage X embryos of chicken were cultured in Dulercco’s Modified Eagle Medium (DMEM) supplemented with leukemia inhibitory factor (LIF, 1 000 IU/ml), basic fibroblast growth factor (bFGF 10 ng/ml) and stem cell factor (SCF, 5 ng/ml). The alkaline phosphatase (AKP) test, differentiation experiment in vitro and chimeric chicken production were carried out. The resuts showed that culture on feeder layer of PMEF yielded high quality CES cell colonies. The shape of typical CES clone showed as follows: nested aggregation (clone) with clear edge and round surface as well as close arrangement within the clone. Strong positive AKP reactive cells were observed. On the other hand, the fourth passage CES cells could differentiate into various cells in the absence of feeder layer cells and LIF in vitro . The third and fourth passage cells were injected into the subgerminal cavity of recipient embryos at stage X. The manipulated embryos were incubated until hatching. Of 269 Hailan embryos injected with CES cells of Shouguang Chickens, 8 2% (22/269) survived to hatching, 3 feather chimeras had been produced, which suggests that an effective culture systems were established and it could promote the growth of CES cells and maintain them in an undifferentiated state .展开更多
基金Supported by the Project Plan of Science and Technology Assistance in Xinjiang Autonomous Region(No.201491171)
文摘AIM： To explore the possibility of human umbilical cord mesenchymal stem cells（h UCMSCs）, human umbilical vein endothelial cells（h UVECs）, human dental pulp stem cells（h DPSCs） and human periodontal ligament stem cells（h PDLSCs） serving as feeder cells in co-culture systems for the cultivation of limbal stem cells.METHODS： Different feeder layers were cultured in Dulbecco＇s modified Eagle＇s medium（DMEM）/F12 and were treated with mitomycin C. Rabbits limbal stem cells（LSCs） were co-cultured on h UCMSCs, h UVECs, h DPSCs, h PDLSCs and NIH-3T3, and then comparative analysis were made between each group to see their respective colony-forming efficiency（CFE） assay and immunofluorescence（IPO13,CK3/12）.RESULTS： The efficiency of the four type cells in supporting the LSCs morphology and its cellular differentiation was similar to that of NIH-3T3 fibroblasts as demonstrated by the immunostaining properties analysis, with each group exhibiting a similar strong expression pattern of IPO13, but lacking CK3 and CK12 expression in terms of immunostaining. But h UCMSCs, h DPSCs and h PDLSCs feeder layers were superior in promoting colony formation potential of cells when compared to h UVECs and feedercell-free culture.CONCLUSION： hUCMSCs, hDPSCs and hPDLSCs can be a suitable alternative to conventional mouse NIH-3T3 feeder cells, so that risk of zoonotic infection can be diminished.
基金supported by grants from the National Natural Science Foundation of China, China (30972097, 31272518)the Program for New Century Excellent Talents in University, China (NCET-09-0654)the Fundamental Research Funds for the Central Universities, China (QN2011012)
文摘A83-01 is a selective inhibitor of the TGF-β type I receptor ALK,which inhibits the TGF-β-induced epithelial-to-mesenchymal transition（EMT） via the inhibition of Smad2 phosphorylation.Previous studies have showed that A83-01 promoted somatic cellular reprogramming significantly.Male germline stem cells（mGSCs）,as an alternative resource of pluripotent stem cells derived adult testis,have promising valuable in clinic medicine and regeneration,however,the derivation of mGSCs was complex and difficult.What the role A83-01 plays in promoting the proliferation of mGSCs is still unknown.In this study,combined with A83-01 and knockout serum replacement（KSR） medium,we obtained a relatively feeder-and serum-free system for mGSCs culturing in vitro and the optimal concentration of A83-01 was 0.25 μmol L-1.After continuous culturing,the proliferation efficiency of undifferentiated mGSCs and differentiation capacity of mGSC were examined as well.Results showed that,A83-01 dramatically increased the number of mGSCs and AP positive colonies,and the mitosis index according to the BrdU assay.A83-01 could also increase the expression of pluripotent markers including Oct4,Klf4,Nanog and c-Myc,analyzed byreal-time quantative PCR.mGSCs cultured in the optimal feeder-and serum-free system combined with A83-01 could form embryoid bodies（EBs）,which consisted of three embryonic layers detected by immunofluorescence and RT-PCR.Remarkably,the results demonstrated 0.25 μmol L-1A83-01 could promote the proliferation of mouse mGSC colonies and maintain their undifferentiated status under feeder-and serum-free systems.
文摘Human embryonic stem cells （hESC） not only hold great promise for the treatment of degenerative diseases but also provide a valuable tool for developmental studies. However, the clinical applications of hESC are at present limited by xeno-contamination during the in vitro derivation and propagation of these cells. In this review, we summarize the current methodologies for the derivation and the propagation of hESC in conditions that will eventually enable the generation of clinical-grade cells for future therapeutic applications.
文摘SNL cells(permanent line of irradiated mouse fibroblast cells), primary mice embryonic fibroblasts (PMEF) cells and primary chicken embryonic fibroblasts (PCEF) cells were respectively used as the feeder cells for chicken embryonic stem cell culture. The isolated blastoderm cells from the stage X embryos of chicken were cultured in Dulercco’s Modified Eagle Medium (DMEM) supplemented with leukemia inhibitory factor (LIF, 1 000 IU/ml), basic fibroblast growth factor (bFGF 10 ng/ml) and stem cell factor (SCF, 5 ng/ml). The alkaline phosphatase (AKP) test, differentiation experiment in vitro and chimeric chicken production were carried out. The resuts showed that culture on feeder layer of PMEF yielded high quality CES cell colonies. The shape of typical CES clone showed as follows: nested aggregation (clone) with clear edge and round surface as well as close arrangement within the clone. Strong positive AKP reactive cells were observed. On the other hand, the fourth passage CES cells could differentiate into various cells in the absence of feeder layer cells and LIF in vitro . The third and fourth passage cells were injected into the subgerminal cavity of recipient embryos at stage X. The manipulated embryos were incubated until hatching. Of 269 Hailan embryos injected with CES cells of Shouguang Chickens, 8 2% (22/269) survived to hatching, 3 feather chimeras had been produced, which suggests that an effective culture systems were established and it could promote the growth of CES cells and maintain them in an undifferentiated state .
