Establishment Of Nile Tilapia ES-like Cell Lines And Their Un-differentiated State Maintained By Leukemia Inhibitory Factor

Embryonic stem(ES)cells derived from early developing embryos have the potential for self-renewal and differentiation,which has put ES cells at the core of many studies in regenerative medicin,developmental biology and functional genomics.Consequently,ES cells have become the focus and hot topics in current life science research.However,prevention of spontaneous differentiation in vitro are the major challenges for ES cell development.Since the first ES cell lines were derived from the inner cell mass of preimplantation mouse embryos in 1981,different types of feeder cell monolayers,conditioned media supplemented with different growth factors such as leukemia inhibitory factor,have been explored to prevent ES cells differentiation.However,long-term stable ES cell lines have been only achieved or partially achieved in a limited number of species including human,pig,bovine,and monkey.Therefore,the maintenance of the undifferentiated state of ES cells and the conservation of its molecular mechanisms in different species need to be further studied.Work towards fish ES cells so far mainly has focused on zebrafish(Danio renio)and medaka(Oryzias latipes).Stable ES cell lines from medaka and zebrafish were successfully obtained by developing a feeder-free culture condition(including fish embryonic extraction and serum),and ES-like cells have also been derived from several marine fish species.These studies suggest that feeder layers are not indispensable for ES cell development in fish.Tilapia(Oreochromis niloticus)(in this paper,if not stated,tilapia is designated as Nile tilapia)is a worldwide farmed commercial fish species,which features fast growth,strong resistance to environment hardness and short spawning cycle(14 days).Thus,it is an ideal fish model organism for the research and application of ES cells.LIF,belonging to the interleukin-6(IL-6)cytokine family,is crucial for the proliferation,survival of multiple stem cells,such as primordial germ cells,neural stem cells,hematopoietic stem cells,mesenchymal stem cells.However,LIF is the most commonly used factor for the cultivation and proliferation of ES cells in a pluripotent state.Numerous studies reveal that the parallel LIF signaling through JAK/STAT3 and PI3 K pathways maintains the pluripotent state of mouse ES cells.In contrast,LIF/STAT3 signaling fails to maintain self-renewal of human ES cells.Consequently,elucidation the role of LIF in ES cells from other species has a positive role in revealing evolution of molecular mechanisms underlying cellular pluripotency.In view of these,this paper takes tilapia as the object to carry out the following two aspects: On the one hand,Nile tilapia ES cell lines(designed as TES1)was successfully established by using conditioned medium to isolate and culture blastomeres of Nile tilapia middle blastula embryos and further identified by pluripotency potential and differentiation assays such as expression of pluripotency genes,embryoid body and chimera formation.Meanwhile,the different components in the culture medium were detected and optimized by the detection of cell proliferation;On the other hand,the role of tilapia Lif protein(OnLif)in the proliferation,survival and differentiation of TES1 cells was intensive studied.The main findings are as follows: 1 Establishment of Nile tilapia embryonic stem-like cell lines1.1 Cell isolation and culture Three cell lines,designated as TES1-3,were initiated from blastomeres of Nile tilapia middle blastula embryos(MBE)in 28℃ by DMEM medium supplemented with Hepes,penicillin and streptomycin,fetal bovine serum(FBS),the non-protein supplement combination 5N(L-glutamine,Na-pyruvate,Na-selenite,non-essential amino acids and β-mercaptoethanol),human recombinant basic fibroblast growth factor(bFGF),Nile tilapia embryo extract(TEE)and Nile tilapia serum(TS).All of three cell lines showed ES like features.One representative line,TES1,showed colony-forming ability and phenotypic characteristics(e.g.,round or polygonal in shape,and rich in granules with relatively large nuclei and sparse cytoplasm)of ES cells over 200 days of culture with more than 59 passages under feeder-free condition.1.2 Pluripotency in vitro AP activity is extensively used as a valuable marker of putative ES cells.TES1 exhibited high alkaline phosphatase activity and expression of pluripotency genes including pou5f3,sox2,myc and klf4.Additionally,the Pou5f3 expression at the protein level was further confirmed by immunofluorescence assay.These results suggest that TES1 cells have pluripotency potential in vitro.1.3 Differentiation in vitro To test the EB formation ability of TES1 cells,the dissociated cells were cultivated in non-adhesive plastic dishes supplemented with RA.After 10 days of suspension culture supplemented with RA,the EBs have strong expression of the differentiation gene nf200,actn2,hnf3 b and sox10 specific to the ectoderm,mesoderm,endoderm and neural crest.When the EBs were further induced for 1-2 weeks,several terminally differentiated cell types were observed: star-shaped cells,neuron-like cells with long processes and flat cells.Thus,TES1 has the potential of multi-directional differentiation in vitro.1.4 Differentiation in vivo TES1 cells were stained red with PKH26 and then introduced into Nile tilapia MBE.The resulting host embryos were examined up to fry under fluorescence microscope.Among the four groups,average 35 % and 13 % of the host blastula embryos(n=501)were PKH26-positive embryos and hatched chimeric fry,respectively.PKH26-positive chimeras were observed,which were found in one to several different areas,including the trunk,eye and fin.These results indicate that TES1 cells might contribute to the different body compartment development,suggesting differentiation potential of TES1 cells in vivo.1.5 Chromosome analysis The chromosome constitution of TES1(passages 40)cells was determined cytogenetically.Examination of more than 100 prometaphase and metaphases revealed that the majority of cells(over 70 %)possessed a diploid chromosome number(2n = 44).Moreover,the chromosomes exhibited a normal morphology.This suggests that TES1 cells have genetic stability during long-term culture.1.6 Effects of different components on proliferation of TES1 cells The growth responses of TES1(passages 28)cells were measured by CCK8 assay.TES1 cells had increased proliferation as the concentration of FBS increased(at the range of 0-15 %)increased,whilst almost no proliferation observed when FBS was at 0,1 and 20 %,suggesting strong dependence on the concentration of FBS.TEE and TS were higher mitogenic for TES1 cell growth when compared to medaka embryonic extract(MEE)and seabass serum(SS),respectively,suggesting that TES1 culture is dependent on species-specific factors in TEE and TS.Human bFGF at all indicated 5,10 and 20(ng/ml)is mitogenic for cell growth,but the most obvious effect on proliferation of TES1 cells was about 10(ng/ml).2 Effects of OnLif on TES1 cells proliferation,survival and pluripotency activity2.1 The proliferation of cells To define the effect of OnLif on the proliferation of cells,TES1 cells were cultured with 1,10,100 ng/ml OnLif for 24 h,48 h,72 h,96 h,respectively.The results of CCK-8 showed that TES1 cells show not significant difference in the presence of 1 ng/ml and 100 ng/ml OnLif(p > 0.05),but On Lif can dramatically promote the proliferation of TES1 cells after 48 h comparing with the control(p < 0.01).The proliferative activity of OnLif was further detected by EdU incorporation staining.Our datas indicate that On Lif can dramatically promote the proliferation of TES1 in a concentration-dependent manner.2.2 The survival of cells To test the predicted role of On Lif in preventing cell death,TES1 cells,seeded at low density,medium density and high density,treated in basal medium(absence of OnLif),conditioned medium(in presence of On Lif)or completed medium(TESM)for 48 h were compared for apoptosis.The results showed that significant cell apoptosis was found in low density and high density without On Lif,and no obvious apoptosis was observed in presence of On Lif and TESM.Despite the apoptotic effect of basal medium does not seem to change significantly compared to conditioned medium in medium density,cell apoptosis experiments showed that the proportion of AnnexinV-FITC/PI-positive cells was significantly higher in the presence of OnLif(22.8 %)than in control(8.9 %)(p < 0.01).Therefore,OnLif can maintain the survival of TES1 cells.2.3 The pluripotent activity of cells To define the effect of On Lif on the undifferentiated state of TES1 cells,cells were cultured with or without On Lif for 3 days and 5 days,respectively.RT-PCR analysis showed that the activation of differentiation genes(nf200,actn2,hnf3b)upon LIF withdrawal occurs simultaneously in parallel with a decrease in transcription of pluripotent genes pou5f3,sox2,myc and klf4.After 3 days and 5 days with On Lif,TES1 was still able to express these pluripotent genes,and the expression of the differentiation genes was not significant or not.Similar results were obtained by AP staining.Furthermore,this conclusion was confirmed by immunohistochemistry of Pou5f3 and transfection of its promoter vector pT2AL-Onpou5f3-EGFP to TES1 cells after treatments with or without OnLif.Accordingly,On Lif can maintain the undifferentiated state of TES1 cells.The tilapia is a world cultured fish.In this study,we successfully established tilapia embryonic stem cell lines and determined the role of leukemia inhibitory factor in the maintenance of fish embryonic stem cells for the first time.It provides a solution to the basic problems in the field of stem cell biology: the origin and evolution of molecular mechanisms underlying cellular pluripotency.