Optimized Culture Of Human Embryonic Stem Cells And Differientiation Into Neural Stem Cells

This experiment aimed to set the optimized culture system of two Chinese humanembryonic stem cell lines and induced the purified embryonic stem cells to neural stemcells. During the research we explored some experiment conditions and techniques related.Partâ… Immortalized Human Adult Fibroblasts As Feeder Layer toSupport the Long-Term Culture of Human Embryonic stem cellsObjective Replace mouse embryonic fibroblasts by immortalized human adult fibroblastsas feeder layer to support the long-term culture of two Chinese human embryonic stem celllines. Removed the cells of animal origin.Methods Two human embryonic stem cells lines were cultured on HAFi feeder layer indifferent densities.The growth informations were detected and drew the growth curve ofHAFi and hESC on it. Compared the influences of 0.05% trypsin, Collagenaseâ…£andmechanical method in cell passages. HESC obtained from HAFi feeder layer wereidentified on their fundamental characteristics. Flow cytometry was used to detect theundifferentiated cell rates.Cells cultured with MEF feeder layer as control. Results 1.Two Chinese human embryonic stem cell lines cultured on HAFi feeder layerwere similar with those on MEF feeder layer as morphology was concerned and keep thefundamental characteristics: AKP, TRA-1-60,TRA-1-81and OCT-4 were all positive.Karyotypes kept Normal.2. Differentiated rated of two cell lines cultured on HAFi feeder layer were similarwith those on MEF feeder layer. The results of SSEA-4 positive cells of Flow cytometrywere: 90.467%±0.907 and 92.1%±0.82. Proliferations of cells were similar too and couldbe passaged every 7 days.3. Different densities of HAFi feeder layer could influence the growth anddifferentiated rates of the two Chinese human embryonic stem cell lines. Density of6×10⁵/mL which could keep lower differentiated rate and higher proliferate rate was theperfect density. The density used here was higher than those reported abroad.4. Collagenaseâ…£was the best way to passage hESC cultured on HAFi feeder layer.It was easy to operated and difficult to cause pollution, influences on cell growth waslower.The shortage of this way was that feeder cells could be passaged together with hESCConclusion HAFi could replace MEF as feeder layer to support the long-term culture oftwo Chinese human embryonic stem cell lines. This could remove the cells of animal originand heterogenetic pollution in the culture system. It also reduced the culture of primarycells and solved the instability caused by different batches of primary cells.This part alsosuggested that two Chinese human embryonic stem cell lines were different with those setby the west. Partâ…¡Foundation and Identification of Feeder-free Culture Systemfor Human Embryonic Stem CellsObjective To set the feeder-free culture systems for two Chinese human embryonic stemcell lines and purified the stem cells.Methods Take matrigel as extracelluar matrix. MEF-CM and HAFi-CM which wascollected from feeder-layers and unconditional medium supplied with TGFβ₁, noggin anddifferent concentrations of bFGF and also FT, FTN, FTNI, TNI which were multiple factoraffiliations were used respectively as culture medium. The growth information in differentfeeder-free culture systems were detected. Flow cytometry was used to detect theundifferentiated rates. The passage cycles of cells were calculated.Then we evaluated thecontribution of different feeder-free culture systems and factors; Compared MEF-CMobtained from MEF feeder cells in different densities and different ways for passage; HESCobtained in different feeder-free culture system were identified on their fundamentalcharacteristics.Results 1 .MEF-CM, FM160, FM250 and FT, FTN, FTNI medium combined with matrigelcould support the long term culture of two Chinese human embryonic stem cell lines.2. Densities between 6×10⁵/mL- 12×10⁵/mL were suitable to get MEF-CM.3. Mechanical way could be used in transfer steps from feeder-layer to feeder-freesystem and Collagenaseâ…£was suitable for the long term passage4. Undifferentiated rates of flow cytometry: MEF-CM＞FTNI≈FTN＞FT＞FM160≈FM250＞TNI5. Passage cycle : TNI＞FTN＞FT＞FM160=FTNI＞FM250＞MEF-CM6. Two Chinese human embryonic stem cell lines could keep all the fundamentalcharacteristics in feeder-free culture system.Conclusion We set feeder-free Culture system for two Chinese human embryonic stem celllines. FTNI was the optimal unconditional medium factor affiliation suitable for two Chinese human embryonic stem cell lines which haven't been reported abroad. It suppliedthe feasible way to purify hESC and convenient the use in lab and clinical applications inthe future.Partâ…¢Induce purified human embryonic stem cells to neural stemcellsObjective To explore an efficient approach to induce purified human embryonic stem cellsto neural stem cellsMethods Two dfifferent way were used: EB way and direct induce way. For EB wayconsiderable EBs were got first and then these EBs were dispersed and induced withinduced medium in adherent culture. Direct induce: purified human embryonic stem cellswere digested in proper cell aggregates and then be suspended and cultured with inducedmedium.After another culture 16-20 days in adherent cells which were digested with0.05% trypsin. Go on cultured with neural amplificated medium.Results 1. EBs could be got from purified human embryonic stem cells efficiently.2. Nestin positive cells of EB way in flow cytometry was 51.7%±1.70 and88.83%±3.40 of direct induce way.3. GFAP and MAP2 were expressed when neural stem cell derived using both twoways differentiated.Conclusion EB way was a cheap induced approach but need breakthrough on techniques.Induced medium composed of NeuroBasal Medium supplied with bFGF, noggin, B27, N2,ITS+1 was efficient and little time consumed induce approach when corporate withmatrigel and could be generalizated.