BACKGROUND： Midbrain-derived neural stem cells （mNSCs） can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson＇s disease. OBJECTIVE： ...BACKGROUND： Midbrain-derived neural stem cells （mNSCs） can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson＇s disease. OBJECTIVE： To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN, TIME AND SETTING： An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital （China） from March to November 2007. MATERIALS： Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, β-Ⅲ tubulin antibody, glial fibrillary acidic protein （GFAP） antibody and cyclic nucleotide 3＇-phosphohydrolase （CNPase） antibody were provided by Abcam; DMEM/F12 medium and N2 supplement were provided by Invitrogen; epidermal growth factor （EGF） and fibroblast growth factor-2 （FGF2） were provided by R＆D Systems. METHODS： The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/FI 2, 1% N： supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10ug/mL polylysine and induced to differentiate in the DMEM/F12 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES： The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for β Ⅲ tubulin, GFAP and CNPase. RESULTS： Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and β -Ⅲ-tubulin. CONCLUSION： Embryonic day 14 rat mNSCs can differentiate into neuron-like cells and glial cells following induction by EGF, FGF2 and N： additive.展开更多
Embryonic stem(ES) cells are isolated from theinner cell mass of a blastocyst, and are used for the generation of gene-modified animals. In mice, the transplantation of gene-modified ES cells into recipient blastocyst...Embryonic stem(ES) cells are isolated from theinner cell mass of a blastocyst, and are used for the generation of gene-modified animals. In mice, the transplantation of gene-modified ES cells into recipient blastocysts leads to the creation of gene-targeted mice such as knock-in and knock-out mice; these gene-targeted mice contribute greatly to scientific development. Although the rat is considered a useful laboratory animal alongside the mouse, fewer genemodified rats have been produced due to the lack of robust establishment methods for rat ES cells. A new method for establishing rat ES cells using signaling inhibitors was reported in 2008. By considering the characteristics of rat ES cells, recent research has made progress in improving conditions for the stable culture of rat ES cells in order to generate gene-modified rats efficiently. In this review, we summarize several advanced methods to maintain rat ES cells and generate gene-targeted rats.展开更多
Rats(Rattus norvegicus) have many advantages over mice in scientific studies,for example, they are more relevant to human in physiological and pharmacological responses.Therefore,rats are broadly used in experimental ...Rats(Rattus norvegicus) have many advantages over mice in scientific studies,for example, they are more relevant to human in physiological and pharmacological responses.Therefore,rats are broadly used in experimental studies.The recent breakthrough in the generation of rat embryonic stem cells(rESCs) opens the door to application of gene targeting to create models for the study of human diseases.In addition,the in vitro differentiation of rESCs into derivatives of three germ lines will serve as a powerful tool and resource for the investigation of mammalian development,cell function, tissue repair,and drug discovery.However, the distinct culture condition and signal inhibitor-depended maintenance of rESCs stand as a considerable challenge for its in vitro differentiation.To address it,we investigated whether rESCs are capable of forming terminal differentiated cardiomyocytes. We found that the embryoid bodies(EBs)-based method used in mouse ESC(mESC) differentiation failed to work in the cultivation of rESCs.We then modified the differentiation protocol and successfully developed an in vitro differentiation system to differentiate rESCs into three embryonic germ layers.By using this method,the rESCs form spontaneous beating cardiomyocytes with the properties similar to those derived from fetal rat hearts and mESCs.This unique cellular system will provide a new approach to study the early development and cardiac function as well as to perform pharmacological test and cell therapy study(Grants:the State Major Research Program of China(2009ZX09503-024,2010CB945603) and CAS(XDA01030000).展开更多
Objective To observe the migration and differentiation of the neural precursor cells （NPCs） that derived from murine embryonic stem cells （ESCs） when they were transplanted into amyloid β （Aβ）-treated rat hipp...Objective To observe the migration and differentiation of the neural precursor cells （NPCs） that derived from murine embryonic stem cells （ESCs） when they were transplanted into amyloid β （Aβ）-treated rat hippocampus. Methods MESPU35, a murine ESC cell line that express the enhanced green fluorescent protein （EGFP）, was induced differentiation into nestin-positive NPCs by modified serum-free methods. The Aβ plaques and the differentiation of the grafted cells were observed by immunofluorescent staining. Results Comparing 16 weeks with 4 weeks post-transplantation, the migration distance increased about 5 times; the rate of migratory NPCs differentiating into glial fibrillary acidic protein （GFAP）-positive cells kept rising from （30.41 ± 1.45）% to （49.25± 1.23）%, and the rate of NPCs differentiating into neurofilament 200 （NF200） positive cells increased from （16.68±0.95）% to （27.94± 1.21）%. Meanwhile, the GFAP-positive cells targeting to the ipsilateral side of Aβ plaques increased from 60.2% to 81.3 %, while the NF200-positive cells increased from 61.3% to 84.1%. The migration distance had significant positive linear correlations to the neuronal differentiation rate （r = 0.991） and to the astrocytic differentiation rate （r = 0.953）. Conclusion Engrafted NPCs migrate targetedly to the Aβ injection site and differentiate into neurons and astrocytes.展开更多
Rat is a valuable model for pharmacological and physiological studies. Germline-competent rat embryonic stem （rES） cell lines have been successfully established and the molecular networks maintaining the self-renewi...Rat is a valuable model for pharmacological and physiological studies. Germline-competent rat embryonic stem （rES） cell lines have been successfully established and the molecular networks maintaining the self-renewing, undifferentiated state of rES cells have also been well uncovered. However, little is known about the differentiation strategies and the underlying mechanisms of how these authentic rat pluripotent stem ceils give rise to specific cell types. The aim of this study is to investigate the neural differentiation capacity of rES cells. By means of a modified procedure based on previous publications - combination of mitogen-activated protein kinase （MAPK） and glycogen synthase kinase 3 （GSK3） inhibitors （two inhibitors, ＂2i＂） with feeder-conditioned medium, we successfully obtained high- quality rat embryoid bodies （rEBs） from rES cells and then differentiated them to tripotent neural progenitors. These rES cell-derived neural progenitor cells （rNPCs） were capable of self-renewing and giving rise to all three neural lineages, including astrocytes, oligo- dendrocytes, and neurons. Besides, these rES cell-derived neurons stained positive for y-aminobutyric acid （GABA） and tyrosine hydroxylase （TH）. In summary, we develop an experimental system for differentiating rES cells to tripotent neural progenitors, which may provide a powerful tool for pharmacological test and a valuable platform for studying the pathogenesis of many neurodegenerative disorders such as Parkinson＇s disease and the development of rat nervous system.展开更多
Knockout Brown Norway （BN） rat could be a useful disease model for human disorders,however,a failure to derive embryonic stem （ES） cells disturbs the further development of the model.In this study,we reported a ca...Knockout Brown Norway （BN） rat could be a useful disease model for human disorders,however,a failure to derive embryonic stem （ES） cells disturbs the further development of the model.In this study,we reported a case of successful derivation of the BN rat ES cells with the derivation efficiency comparable to that of Sprague Dawley （SD） rats.The BN rat ES cells expressed the key transcription factors,and were able to form embryonic bodies （EBs） when being differentiated in vitro.After injecting the BN rat ES cells into the SD rat blastocysts,high-contribution chimeric rats were generated and could survive to their adulthood.Our success in generating pluripotent rat ES cells will benefit the generation of the knockout rats in the future.展开更多
A new method for establishing ES cell lines from 129/ter、C57BL/6J mice was set up which was characterized by the murine embryonic fibroblast cell(MEF) feeder, the medium of rat heart cell-conditioned medium(RH-CM)for...A new method for establishing ES cell lines from 129/ter、C57BL/6J mice was set up which was characterized by the murine embryonic fibroblast cell(MEF) feeder, the medium of rat heart cell-conditioned medium(RH-CM)for ES cells, and the consecutive digestion by the digestion liquid containing 1% serum. Every group of improved experiments was done with a control of routine method. The results showed that, compared with routine method, the improved way increased the ratio of ES cell lines of 129/ter mice from 11.8% to 33.3%, and of C57BL/6J from 3.7% to 13.3%. The difference is distinct. The passage culture of ES cells showed that, compared with medium added LIF, RH-CM not only inhibited the differentiation of murine ES cells, maintained its dipoild karyotype, but also promote its adherence growth. This kind of culture condition not only maintained the ES cells in an undifferentiated state and their normal dipoild karyotype, but also a series of other characteristics of totipotent embryonic stem cells during extended culture period.展开更多
基金the National Natural Science Foundation of China,No.30672151
文摘BACKGROUND： Midbrain-derived neural stem cells （mNSCs） can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson＇s disease. OBJECTIVE： To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN, TIME AND SETTING： An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital （China） from March to November 2007. MATERIALS： Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, β-Ⅲ tubulin antibody, glial fibrillary acidic protein （GFAP） antibody and cyclic nucleotide 3＇-phosphohydrolase （CNPase） antibody were provided by Abcam; DMEM/F12 medium and N2 supplement were provided by Invitrogen; epidermal growth factor （EGF） and fibroblast growth factor-2 （FGF2） were provided by R＆D Systems. METHODS： The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/FI 2, 1% N： supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10ug/mL polylysine and induced to differentiate in the DMEM/F12 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES： The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for β Ⅲ tubulin, GFAP and CNPase. RESULTS： Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and β -Ⅲ-tubulin. CONCLUSION： Embryonic day 14 rat mNSCs can differentiate into neuron-like cells and glial cells following induction by EGF, FGF2 and N： additive.
文摘Embryonic stem(ES) cells are isolated from theinner cell mass of a blastocyst, and are used for the generation of gene-modified animals. In mice, the transplantation of gene-modified ES cells into recipient blastocysts leads to the creation of gene-targeted mice such as knock-in and knock-out mice; these gene-targeted mice contribute greatly to scientific development. Although the rat is considered a useful laboratory animal alongside the mouse, fewer genemodified rats have been produced due to the lack of robust establishment methods for rat ES cells. A new method for establishing rat ES cells using signaling inhibitors was reported in 2008. By considering the characteristics of rat ES cells, recent research has made progress in improving conditions for the stable culture of rat ES cells in order to generate gene-modified rats efficiently. In this review, we summarize several advanced methods to maintain rat ES cells and generate gene-targeted rats.
文摘Rats(Rattus norvegicus) have many advantages over mice in scientific studies,for example, they are more relevant to human in physiological and pharmacological responses.Therefore,rats are broadly used in experimental studies.The recent breakthrough in the generation of rat embryonic stem cells(rESCs) opens the door to application of gene targeting to create models for the study of human diseases.In addition,the in vitro differentiation of rESCs into derivatives of three germ lines will serve as a powerful tool and resource for the investigation of mammalian development,cell function, tissue repair,and drug discovery.However, the distinct culture condition and signal inhibitor-depended maintenance of rESCs stand as a considerable challenge for its in vitro differentiation.To address it,we investigated whether rESCs are capable of forming terminal differentiated cardiomyocytes. We found that the embryoid bodies(EBs)-based method used in mouse ESC(mESC) differentiation failed to work in the cultivation of rESCs.We then modified the differentiation protocol and successfully developed an in vitro differentiation system to differentiate rESCs into three embryonic germ layers.By using this method,the rESCs form spontaneous beating cardiomyocytes with the properties similar to those derived from fetal rat hearts and mESCs.This unique cellular system will provide a new approach to study the early development and cardiac function as well as to perform pharmacological test and cell therapy study(Grants:the State Major Research Program of China(2009ZX09503-024,2010CB945603) and CAS(XDA01030000).
基金This work was supported by the National Natural Science Foundation of China （No. 30571770）.
文摘Objective To observe the migration and differentiation of the neural precursor cells （NPCs） that derived from murine embryonic stem cells （ESCs） when they were transplanted into amyloid β （Aβ）-treated rat hippocampus. Methods MESPU35, a murine ESC cell line that express the enhanced green fluorescent protein （EGFP）, was induced differentiation into nestin-positive NPCs by modified serum-free methods. The Aβ plaques and the differentiation of the grafted cells were observed by immunofluorescent staining. Results Comparing 16 weeks with 4 weeks post-transplantation, the migration distance increased about 5 times; the rate of migratory NPCs differentiating into glial fibrillary acidic protein （GFAP）-positive cells kept rising from （30.41 ± 1.45）% to （49.25± 1.23）%, and the rate of NPCs differentiating into neurofilament 200 （NF200） positive cells increased from （16.68±0.95）% to （27.94± 1.21）%. Meanwhile, the GFAP-positive cells targeting to the ipsilateral side of Aβ plaques increased from 60.2% to 81.3 %, while the NF200-positive cells increased from 61.3% to 84.1%. The migration distance had significant positive linear correlations to the neuronal differentiation rate （r = 0.991） and to the astrocytic differentiation rate （r = 0.953）. Conclusion Engrafted NPCs migrate targetedly to the Aβ injection site and differentiate into neurons and astrocytes.
基金supported in part by the grants from the National Basic Research Program of China(No.2012CB966501 to X.Z.and 2011CB965300 to L.W.)the"Strategic Priority Research Program"of the Chinese Academy of Sciences(No. XDA01020100 to Q.Z.)
文摘Rat is a valuable model for pharmacological and physiological studies. Germline-competent rat embryonic stem （rES） cell lines have been successfully established and the molecular networks maintaining the self-renewing, undifferentiated state of rES cells have also been well uncovered. However, little is known about the differentiation strategies and the underlying mechanisms of how these authentic rat pluripotent stem ceils give rise to specific cell types. The aim of this study is to investigate the neural differentiation capacity of rES cells. By means of a modified procedure based on previous publications - combination of mitogen-activated protein kinase （MAPK） and glycogen synthase kinase 3 （GSK3） inhibitors （two inhibitors, ＂2i＂） with feeder-conditioned medium, we successfully obtained high- quality rat embryoid bodies （rEBs） from rES cells and then differentiated them to tripotent neural progenitors. These rES cell-derived neural progenitor cells （rNPCs） were capable of self-renewing and giving rise to all three neural lineages, including astrocytes, oligo- dendrocytes, and neurons. Besides, these rES cell-derived neurons stained positive for y-aminobutyric acid （GABA） and tyrosine hydroxylase （TH）. In summary, we develop an experimental system for differentiating rES cells to tripotent neural progenitors, which may provide a powerful tool for pharmacological test and a valuable platform for studying the pathogenesis of many neurodegenerative disorders such as Parkinson＇s disease and the development of rat nervous system.
基金supported in part by the grants from the Chinese National Basic Research Program (No 2007CB948001)the National High Technology R&D Program of China (No 2006AA02A101)Integrated Project funded by the Sixth Framework Programme (FP6) of the European Union (Contract:LSHG-CT-2005-019015)
文摘Knockout Brown Norway （BN） rat could be a useful disease model for human disorders,however,a failure to derive embryonic stem （ES） cells disturbs the further development of the model.In this study,we reported a case of successful derivation of the BN rat ES cells with the derivation efficiency comparable to that of Sprague Dawley （SD） rats.The BN rat ES cells expressed the key transcription factors,and were able to form embryonic bodies （EBs） when being differentiated in vitro.After injecting the BN rat ES cells into the SD rat blastocysts,high-contribution chimeric rats were generated and could survive to their adulthood.Our success in generating pluripotent rat ES cells will benefit the generation of the knockout rats in the future.
文摘A new method for establishing ES cell lines from 129/ter、C57BL/6J mice was set up which was characterized by the murine embryonic fibroblast cell(MEF) feeder, the medium of rat heart cell-conditioned medium(RH-CM)for ES cells, and the consecutive digestion by the digestion liquid containing 1% serum. Every group of improved experiments was done with a control of routine method. The results showed that, compared with routine method, the improved way increased the ratio of ES cell lines of 129/ter mice from 11.8% to 33.3%, and of C57BL/6J from 3.7% to 13.3%. The difference is distinct. The passage culture of ES cells showed that, compared with medium added LIF, RH-CM not only inhibited the differentiation of murine ES cells, maintained its dipoild karyotype, but also promote its adherence growth. This kind of culture condition not only maintained the ES cells in an undifferentiated state and their normal dipoild karyotype, but also a series of other characteristics of totipotent embryonic stem cells during extended culture period.
