Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission effici...Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission efficiency still limit the broad usage of this method in creating transgenic chickens. In this study, we implemented a simple strategy using modified lentiviral vectors targeted to chicken primordial germ cells(PGCs) to generate transgenic chickens. The lentiviral vectors were pseudotyped with a modified Sindbis virus envelope protein(termed M168) and conjugated with an antibody specific to PGC membrane proteins. We demonstrated that these optimized M168-pseudotyped lentiviral vectors conjugated with SSEA4 antibodies successfully targeted transduction of PGCs in vitro and in vivo. Compared with the control, 50.0%–66.7% of chicken embryos expressed green fluorescent protein(GFP) in gonads transduced by the M168-pseudotyped lentivirus. This improved the targeted transduction efficiency by 30.0%–46.7%. Efficient chimerism of exogenous genes was also observed. This targeting technology could improve the efficiency of germ line transmission and provide greater opportunities for transgenic poultry studies.展开更多
Umbilical cord blood(UCB)is an efficient and valuable source of hematopoietic stem cells(HSCs)for transplantation.In addition to HSCs it harbours low amounts of mesenchymal stem cells(MSCs).No single marker to identif...Umbilical cord blood(UCB)is an efficient and valuable source of hematopoietic stem cells(HSCs)for transplantation.In addition to HSCs it harbours low amounts of mesenchymal stem cells(MSCs).No single marker to identify cord blood-derived stem cells,or to indicate their multipotent phenotype,has been characterized so far.SSEA-3 and-4 are cell surface globoseries glycosphingolipid epitopes that are commonly used as markers for human embryonic stem cells,where SSEA-3 rapidly disappears when the cells start to differentiate.Lately SSEA-3 and-4 have also been observed in MSCs.As there is an ongoing discussion and variation of stem-cell markers between laboratories,we have now comprehensively characterized the expression of these epitopes in both the multipotent stem-cell types derived from UCB.We have performed complementary analysis using gene expression analysis,mass spectrometry and immunochemical methods,including both flow cytometry and immunofluoresence microscopy.SSEA-4,but not SSEA-3,was expressed on MSCs but absent from HSCs.Our findings indicate that SSEA-3 and/or-4 may not be optimal markers for multipotency in the case of stem cells derived from cord blood,as their expression may be altered by cell-culture conditions.展开更多
A big issue in stem cell research is to derive prospective totipotential stem cells. In this study, fMSC-SSEA-4 cells expressing SSEA-4 anti- gen were isolated from fetal marrow masenchymal stem cells (fMSCs) using im...A big issue in stem cell research is to derive prospective totipotential stem cells. In this study, fMSC-SSEA-4 cells expressing SSEA-4 anti- gen were isolated from fetal marrow masenchymal stem cells (fMSCs) using immunomagnetic bead sorting technique. The totipotent cells were identified and their biological characteristics were further stud- ied. The expression of Oct-4 and SSEA-4, carcino- genicity, and the ability to differentiation of fMSC- SSEA-4 cells were evaluated to verify the totipotent potential. fMSC-SSEA-4 cells were isolated suc- cessfully from fMSCs (2.5% among fMSCs), while no obvious differences were seen in morphology, growth curve, cell cycle and immunophenotype, Oct-4 and SSEA-4 expression between fMSC-SSEA-4 cells and fMSCs. fMSC-SSEA-4 cells showed normal diploid chromosome karyotype and no carcinoma was in- duced after inoculation into nude mice. fMSC- SSEA-4 cells could be induced to fat cells, osteo- genic cells and neuron-like cells in vitro with different induced factors. The results indicated that there may be a few totipotent cells among the fMSCs and it may offer the experimental basis for the further study and application of fMSCs.展开更多
基金the National Transgenic Breeding Project of China(2016ZX08009003006)National Natural Science Foundation of China(31672411)Discipline Innovative Engineering Plan(B12008)。
文摘Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission efficiency still limit the broad usage of this method in creating transgenic chickens. In this study, we implemented a simple strategy using modified lentiviral vectors targeted to chicken primordial germ cells(PGCs) to generate transgenic chickens. The lentiviral vectors were pseudotyped with a modified Sindbis virus envelope protein(termed M168) and conjugated with an antibody specific to PGC membrane proteins. We demonstrated that these optimized M168-pseudotyped lentiviral vectors conjugated with SSEA4 antibodies successfully targeted transduction of PGCs in vitro and in vivo. Compared with the control, 50.0%–66.7% of chicken embryos expressed green fluorescent protein(GFP) in gonads transduced by the M168-pseudotyped lentivirus. This improved the targeted transduction efficiency by 30.0%–46.7%. Efficient chimerism of exogenous genes was also observed. This targeting technology could improve the efficiency of germ line transmission and provide greater opportunities for transgenic poultry studies.
基金This work was supported by the Finnish Funding Agency for Technology and Innovation(TEKES)the EVO Medical Research Fund of Finnish Red Cross Blood Service and The Finnish Glycoscience Graduate School.
文摘Umbilical cord blood(UCB)is an efficient and valuable source of hematopoietic stem cells(HSCs)for transplantation.In addition to HSCs it harbours low amounts of mesenchymal stem cells(MSCs).No single marker to identify cord blood-derived stem cells,or to indicate their multipotent phenotype,has been characterized so far.SSEA-3 and-4 are cell surface globoseries glycosphingolipid epitopes that are commonly used as markers for human embryonic stem cells,where SSEA-3 rapidly disappears when the cells start to differentiate.Lately SSEA-3 and-4 have also been observed in MSCs.As there is an ongoing discussion and variation of stem-cell markers between laboratories,we have now comprehensively characterized the expression of these epitopes in both the multipotent stem-cell types derived from UCB.We have performed complementary analysis using gene expression analysis,mass spectrometry and immunochemical methods,including both flow cytometry and immunofluoresence microscopy.SSEA-4,but not SSEA-3,was expressed on MSCs but absent from HSCs.Our findings indicate that SSEA-3 and/or-4 may not be optimal markers for multipotency in the case of stem cells derived from cord blood,as their expression may be altered by cell-culture conditions.
文摘A big issue in stem cell research is to derive prospective totipotential stem cells. In this study, fMSC-SSEA-4 cells expressing SSEA-4 anti- gen were isolated from fetal marrow masenchymal stem cells (fMSCs) using immunomagnetic bead sorting technique. The totipotent cells were identified and their biological characteristics were further stud- ied. The expression of Oct-4 and SSEA-4, carcino- genicity, and the ability to differentiation of fMSC- SSEA-4 cells were evaluated to verify the totipotent potential. fMSC-SSEA-4 cells were isolated suc- cessfully from fMSCs (2.5% among fMSCs), while no obvious differences were seen in morphology, growth curve, cell cycle and immunophenotype, Oct-4 and SSEA-4 expression between fMSC-SSEA-4 cells and fMSCs. fMSC-SSEA-4 cells showed normal diploid chromosome karyotype and no carcinoma was in- duced after inoculation into nude mice. fMSC- SSEA-4 cells could be induced to fat cells, osteo- genic cells and neuron-like cells in vitro with different induced factors. The results indicated that there may be a few totipotent cells among the fMSCs and it may offer the experimental basis for the further study and application of fMSCs.
