Derivation And Cultivation Of Human Embryonic Stem Cells

ObjectiveDerivation of human embryonic stem(hES)cells and human germ(hEG) cells was first reported by Thomson nd Gearhart groups that these cells are capa-ble of unlimited self-renewal by symmetric division and pluripotency in 1998. At present,78 hES cell lines have been registered on the NIH registry,although only around 10 lines are available in sufficient quantity for analysis.Establish-ment of human embryonic stem cell lines were reported by Zhongsan University, Zhongnan University and Beijing University in our country.Human ES cells have become a powerful tool for in vitro investigation of de-velopmental processes at both cellular and organism levels,and offer tremendous potential for clinical application as an unlimited source of cells for transplanta-tion and tissue generation therapies.Culture of human embryonic stem cell de-pends on feeder cell.Usually,MEF was used as feeder ceils.As result,there is a risk of contamination by heterogeneity cell,protein and unknown;pathogen-ic microorganism in culture.They are not optimal in cultures aimed at cell trans-plantation in humans.We obtained frozen cleavage human embryos produced by in vitro ferlitilization for clinical purposes and 5-9 weeks postfertilization hu-man embryos(obtained as a result of therapeutic termination of pregnancy),af-ter obtaining written informed consent and approval by institutional review board of Shenyang obstetrics and gynecological hospital to establish human embryonic stem ceil lines.Primordial germ cells were plated on endometrium ceils as feed-er ceils and observe its growth characteristics.To establish a new human embry-onic stem cell lines from human cells will provide safeguard for clinical applica- tion.Methods1 Establishment and culture of hES cell linesFrozen-thawed cleavage stage human embryos,donated by couples under-going in vitro fertilization(IVF)treatment at Shenyang obstetrics and gynecolog-ical hospital,after they gave informed consent and after Ethics Committee ap-proval,were cultured to the blastocyst stage in G1.3 and G2.3 media (Vitrolife).The donors gave informed consent and Ethics Committee approval was obstained.ICM was isolated by immunosurgical method and plated on MEF feeder layer.ICM outgrowths were passaged to plates with fresh medium and new MEF by mechanical dissection.At early stage,for maintaining established stable hES cells,about 100 undifferentiated hES cells were mechanically isola-ted under a dissecting microscope and the resulting clumos propagated on a new feeder layer every 4-7days.Characterization and cytogenetic analysis of hES cellsFor alkaline phosphatase(AP)staining,hES ceils were washed with PBS, and NBT/BCIP solution(Roche Molecular Biochemicals)was added.The re-sulting color reaction was stopped with distilled water.For immunocytochemis-try,hES cells were washed with PBS and fixed in 4%paraformaldehyde at 4 C for 30 min.The surface specific markers of hES such as SSEA-3,SSEA-4, TRA-1-60,TRA-1-81 and OCT-4 were detected by immunofluorescence and confocal microscopy.For cytogenetic analysis,hES cells were incubated in hES culture medium with 0.1μg/ml colcemid for 3-4 hours,trypsinized,in-cubated in 0.1%sodium citrate at 37℃for 20 min,and fixed in methanol:ace-tic acid(3:1,v/v).After Giemsa staining,a cytogenetics specialist examined the karyotype of the hES cells at a resolution of 300 bands.Differentiation of hES cells in vivo and in vitroTo induce the formation of embryoid bodies(EBs),hES cell colonies were dissociated with collagenase and grown in suspension culture in the same hES culture medium but lacking bFGF.EBs were collected individually in drops of molten 1.5%low mehing point agarose in PBS and fixed in 4%PFA.EB sec-tions were tested by immunochemistry for muscle-specific actin(MSA),nes-tin,pan-cytokeratin.To examine in vivo differentiation,we performed histo-logical analyses on teratomas,hES colonies cultured for 5 days were harvested without feeder cells and injected into SCID-beige mice using a 25G needle. The mice were killed 10 weeks after injection,and the resulting teratomas were fixed with 4%PFA,embedded in paraffin,and cut in 10(m sections.The sec-tions were stained with HE and observed under a light microscope.2 A culture system using human endometrial fibroblasts as feeder cell allows production of human primodial germ cellsSpecimenEndometrium was obtained from the patients who were performed abdominal hysterectomy,and human fetuses at 5-9 weeks post conception were collected from the patients who terminate pregnancy at Shenyang obstetrics and gynecolog-ical hospital,they gave informed consent and Ethics Committee approvaled.Culture of human endometrial fibroblastsEndometrium tissue was washed by D-Hank's solution,and cut,and dis-sected by 0.1%collagenaseâ…£at room temperature,and blowed by Pasteur pi-pette.Add to medium,cell concentration was adjusted and cells were plated on culture flask.All cultures were maintained in 5%CO₂/95%humidity at 37℃.Treatment of feeder cellHuman endometrial fibroblasts were mitotically inactived by mitomycin C for 2-4 hours and changed fresh medium.Derived and cultured human primodial germ cell(PGCs)The Gonadal ridges and mesenteries were isolated from human fetus 5-9 weeks after fertilization and washed by PBS,and cut,and digested by 0.25% trypsin at room temperature,and were planted on to mitotically inactivated hu-man endometrial fibroblasts.Characterization and cytogenetic analysis of bEG cellsFor alkaline phosphatase(AP)staining,hEG cells were washed with PBS, and NBT/BCIP solution(Roche Molecular Biochemicals)was added.The re-sulting color reaction was stopped with distilled water.For immunocytochemis- try,hEG cells were washed with PBS and fixed in 4%paraformaldehyde at 4 C for 30 min.The surface specific markers of hEG such as SSEA-1,SSEA-3, SSEA-4,TRA-1-60,TRA-1-81 and OCT- 4 were detected by immuno-fluorescence and confocal microscopy.For cytogenetic analysis,hEG cells were incubated in hEG culture medium with 0.1μg/ml colcemid for 3-4h, trypsinized,incubated in 0.1%sodium citrate at 37℃for 20 min,and fixed in methanol:acetic acid(3:1,v/v).After Giemsa staining,a cytogenetics spe-cialist examined the karyotype of the hEG ceils at a resolutionof 300 bands.Differentiation of hEG cells in vivo and in vitroTo induce the formation of embryoid bodies(EBs),hEG cell colonies were dissociated with collagenase and grown in suspension culture in the same hEG culture medium but lacking bFGF.EBs were collected individually in drops of molten 1.5%low melting point agarose in PBS and fixed in 4%PFA.EB sec-tions were tested by immunochemistry for muscle-specific actin(MSA),nes-tin,pan-cytokeratin.To examine in vivo differentiation,we performed histo-logical analyses on teratomas,hES colonies cultured for 5 days were harvested without feeder cells and injected into athymic mouse using a 25G needle.The mice were killed 10 weeks after injection,and the resulting teratomas were fixed with 4%PFA,embedded in paraffin,and cut in 10(m sections.The sections were stained with HE and observed under a light microscope.Flow cytometryCultured hEG colonies were harvested with 0.25%trypsin,washed with PBS,and dissociated into single cell s with 0.5mM EDTA,The cells were fixed in 70%ethanol at 4℃for more than 1 hour,and washed thoroughly.To deter-mined DNA content,they were stained with 100(g/ml of propidium iodide,and examined by flow cytometry.Results.1 Derivation and cultivation of hES cellsWe obtained 9 blastocysts from frozen thawed human cleavage stage embry-os,and 7 ICM were isolated by immunosurgery,and established two human era- bryonic stem cell lines.These ceil lines were obtained from high- quailty blas-tocysts.Morphology of hES cellsâ… andâ…¢were cultured for 20 and 18 passages,respectively.Undifferenti-ated hES colonies were compact,fiat and round.â… andâ…¢had similar morpholo-gy.The undifferentiated hES cells had a high nucleus-to-cytoplasm ratio, one to three clearly visible nucleoli,and regular spacing between the cells.Karyotype analysisKaryotype analyses performed at various passages(6,13)revealed a nor-real karyotype(46,XX)in two cell lines.Detection of specific surface markersWe examined the expression of several hES cell specific surface markers by immunoflurescence.The hES ceils expressed SSEA-3,SSEA-4,TRA-1-60,TRA-1-81,OCT-4,while SSEA-1 was not detected.Differentiation of hES cells in vivo and in vitroAfter 4 days of culture in suspension,a basement membrane was formed on the outside of the EBs,which formed sphere-like structure.Immunohistochem-istry of EBs revealed expression markers of three germ layer,such as muscle-specific actin(MSA),nestin,pan-cytokeratin.SCID-beige mice were killed after injecting undifferentiated hES cells. Teratomas had formed in the right tests of all both mice injected with I and III. Pathological section showed mature teratomas and tissues derived from all three germ layers,including primitive neural epithelium,pigmented epithelium,and retina(ectoderm);cartilage,bone,and smooth muscle(mesoderm);and gas-trointestinal and glandular epithelium(endoderm).2 Dervitation and culture of hEG Growth of human endometrium stroma cellstroma cell present slightly width in center,to seem fusiform shape,or pol-ygon,and cell nucleus is orbicular-ovate,after lasting culture,fusiform cell can change longer spidle.They parallel each other to form fasciculation,human endometrium stroma cell may continue to grow for 4 weeks after treatmented with mitomyein C. Morphology of hEG cellsPGCs were obtained from mesentery more than gonad.A number of multi-layed clumps occurred when PGCs were culture in vitro for 7-10 days.It looks like bird nest.hEG cells had a high nucleus-to-cytoplasm ratio,more than two clearly visible nucleoli,resemble hES.PGCs have been cultured more than 4 months so far for 18 passages on human endometrial fibroblast feeder layer.Detection of specific surface markers and Karyotype analysisPGCs expressed several hES cell specific surface markers by immunoflures-cenee and still positive for plutipotent marker of embryonic stem cells such as SSEA-3,SSEA-4,TRA-1-60,TRA-1-81,OCT-4.Alkaline phos-phates(AKP)showed positive.These cells are rather stable in vitro and showed normal karyotype up to 14 passages.Differentiation of hEG cells in vivo and in vitroAthymic mouse were killed after injecting undifferentiated hEG cells.Tera-tomas had formed in the right tests of all both mice injected with hEG.Histologi-cal analysis showed mature teratomas and tissues derived from all three germ lay-ers,including sebiferous gland,fat,glandular organ,glandular organ,glandu-lar epithelium,squamous epithelium.After 4 days of culture in suspension,a basement membrane was formed on the outside of the EBs,which formed sphere-like structure.Immunohistochem-istry of EBs revealed expression markers of three germ layer,such as muscle-specific actin(MSA),nestin,pan-cytokeratin.Flow cytometryhEG cells were examined by flow cytometry with marker SSEA-4 antibod-y,and we found SSEA-4 positive cells reached 15.45%.Conclusions1.We established two human embryonic stem cell lines from frozen human cleavage stage embryos,and these cells had all characteristics of human embry-onic stem cells.2.This study indicates that estsblishment of hES cells from frozen-thawed blastocysts minimizes the ethical problem associated with the use of human em-bryos in research.Frozen-thawed embryos may be a source of hES.3.We used successfully human endometrial fibroblasts as feeder cell for derivation and continued undifferentiated growth of PGCS.4.Human primordial germ cells which grow on human endometrial fibro-blasts had all characteristics of human embryonic stem cells.