采用慢病毒和逆转录病毒两种病毒转染系统建立的人的iPS细胞株实验操作流程Protocol

采用慢病毒和逆转录病毒两种病毒转染系统建立的人的iPS细胞株实验操作流程Protocol

 

IPSC Generation by Lentiviral System Protocol

 

Lentiviral Packaging

 

Medium

 

293FT medium: D-MEM/10 S, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.

 

293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.

 

293FT Cell Culture

 

Maintain 293FT cells in T75 flask with 293FT medium plus 500 ?g/ml Geneticin.

 

Reprogramming factors

 

Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4

 

Lentiviral Packaging (~10^5-10^7 particles/ml titer)

 

Materials Each T75 flask

 

293FT cells   10-15x106

 

MD.G (VSVG)  5 ?g

 

PsPAX2  10 ?g

 

PSIN vector (~10kb)  5 ?g

 

Superfect (Qiagen)  40 ?l

 

IMDM  400 ?l

 

293FT medium   10ml

 

1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.

 

2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).

 

3.  Add 10 ml of 293FT cell suspension to each T75 flask.

 

4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).

 

5.  Add corresponding amount of Superfect to the DNA solution, and mix with brief vortex. Briefly spin down to collect liquid at the bottom of the tube.

 

6.  Incubate the DNA/superfect mixture at R.T. for 10 min.

 

7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.

 

8.  Incubate the cells at 37?C O/N.

 

9.  Day 2: The next morning (9-10 am), remove the medium, and add 8 ml of 293FT complete medium (with sodium pyruvate) to each T75 flask.

 

10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.

 

11.  Centrifuge at 3000rpm for 5 min at 4?C to remove cell debris.

 

12.  Filter the supernatant through Millipore Millex-HV 0.45?M PVDF, Cat# SLHVR25LS).

 

13.  Use filtered virus directly or store in 800 ul aliquots in cryovials at -80°C until use. Lentivirus can be stored at -80°C for several months without loss lot of infectivity.

 

Preparation of human fibroblast cells (IMR-90)

 

1. Day 4 (~ 10am): when fibroblast cells have reached 80% confluence, aspirate medium, wash once with PBS, cover cells with 0.05% trypsin, incubate for 5 min at 37 ?C.

 

2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.

 

3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.

 

4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.

 

5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.

 

6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.

 

7. Incubate at 37 ?C, 5% CO2, for 6 h.

 

Lentiviral infection

 

1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.

 

2. Day 4 (~ 6pm) Add equal volume (800ul) supernatant of each factor ( Yu1, Yu2, Yu3) to fibroblast dish in the presence of 10ug/ml polybrene, incubate at 37 ?C, 5% CO2, for 2h.

 

3. Day 4 (~ 8pm) Add another 2ml fresh fibroblast medium and incubate at 37 ?C, 5% CO2 for overnight, then aspirate the virus medium and replace with 2ml fresh fibroblast medium.

 

4. Repeat transduction (including virus harvest) as described above.

 

5. A 3rd transduction may be necessary.

 

6. Day 8 (9~ 10am) Trypsinized fibroblast cells completely (4 days after 1st transduction), transfer to two 100-mm MEF dishes (3 x 10^6 MEF cells /dish).

 

7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.

 

8. Day 18 (9~ 10am) Change to conditional ES medium. Observe any colony formed and pick them up when they grow up.

 

IPSC Generation by Retroviral System Protocol

 

Retroviral Packaging

 

MEDIUM

 

293FT medium: D-MEM/10 S, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.

 

293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.

 

293FT CELL CULTURE

 

Maintain 293FT cells in T75 flask with 293FT medium plus 500 ?g/ml Geneticin.

 

REPROGRAMMING FACTORS

 

pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4

 

Transfection of 293 FT Cell with Lipofectamine 2000

 

For T-75 flask

 

Prepare 293 FT cell:

 

Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.

 

Observe cell dish before transfection. The density of the cell should be 80-90% confluent, and the cells should be evenly distributed and attached on the dish.

 

1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.

 

2.  For each T-75 flask, add 40 ul Lipofectamine 2000 transfection reagent, 600 ul of IMDM, and mix; incubate at room temperature for 5 min.

 

3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.

 

4. Mix and incubate at room temperature for 15 min.

 

5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.

 

6.  Incubate at 37 ?C, 5% CO2 for 48 h.

 

7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.

 

8.  Centrifuge at 3000rpm for 5 min at 4?C to remove cell debris.

 

9.  Filter the supernatant through Millipore Millex-HV 0.45?M PVDF, Cat# SLHVR25LS).

 

10.  Use filtered virus directly or store in 600 ul aliquots in cryovials at -80°C until use. Retrovirus can be stored at -80°C for several months without loss lot of infectivity.

 

Preparation of human fibroblast cells (IMR-90)

 

1.  Day 3 (~ 10am): when fibroblast cells have reached 80% confluence, aspirate medium, wash once with PBS, cover cells with 0.05% trypsin, incubate for 5 min at 37 ?C.

 

2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.

 

3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.

 

4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.

 

5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.

 

6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.

 

7.  Incubate at 37 ?C, 5% CO2, for 6 h.

 

Retroviral infection

 

1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.

 

2.  Day 3 (~ 6pm) Add equal volume (600ul) supernatant of each factor ( OCT3/4, SOX2, c-MYC, KLF4) to fibroblast dish in the presence of 10ug/ml polybrene, incubate at 37 ?C, 5% CO2, for 2h.

 

3.  Day 3 (~ 8pm) Add another 2ml fresh fibroblast medium and incubate at 37 ?C, 5% CO2 for overnight, then aspirate the virus medium and replace with 2ml fresh fibroblast medium.

 

4.  Repeat transduction (including virus harvest) as described above.

 

5.  A 3rd transduction may be necessary.

 

6.  Day 7 (9~10am) Trypsinized fibroblast cells completely (4 days after 1st transduction), transfer to two 100-mm MEF dishes (3 x 10^6 MEF cells /dish).

 

7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.

 

8.  Day 17 (9~10am) Change to conditional ES medium. Observe any colony formed and pick them up when they grow up.

 

 

 

问题与讨论

 

问题1：unconditional human ES medium 和conditional ES medium的差别？

 

unconditional human ES medium 在feeder cell(如MEF)上培养20-24小时后收集起来过滤便成为了conditional ES medium，加入bFGF后便可用于直接培养hES细胞了。

 

问题2：参考文献

 

参考文献是那些CNS的paper，两篇nat.protocol，根据体会修改而成的，现在也成为美国某个实验室的操作指南了。

 

问题3：是否需要浓缩？两种包装效率？质粒比例？

 

采用这种方法不经浓缩的病毒悬液一般都能达到5*10^6-10^7左右，这对于做iPS已经足够了。经超速离心浓缩后病毒低度是可以提高几十倍，但是对于初学者，多出的那些步骤会增加病毒泄露、丢失、污染等风险。看你做iPS的目的究竟是什么，如果仅仅是为了建立一个平台，一个真正的克隆就足矣了，要那么多干嘛。权衡利弊，我建议初学者暂时不要浓缩病毒。在我实验中，每个因子病毒的量用到500ul就足够了。因为我用逆转录病毒是四个因子，慢病毒是六个因子，不具有可比性，而且我更注重产生克隆后真正iPS克隆所占的比例，就我个人实验结果来看，四因子得到iPS的比例只有20%，而6因子的比例可超过90%。至于包装质粒的比例， protocol里面都有描述。

 

问题4：protocol中retro收病毒上清的时间是转染后48小时，而lenti是68-72小时；在其他protocl好像一些是48和72小时各收一次。请问收病毒上清最佳时间点retro和lenti在哪里？是否做过先用retro(lenti)感染，然后再用lenti（retro）感染？效果如何？

 

其实病毒上清是可以在48和72h左右各收集一次的，但是这又牵涉到一个问题，就是你收集上清再加入新鲜培基的时候，被感染后的包装细胞（293FT）是非常脆弱的，你稍有不慎就会造成细胞大片脱壁死亡，因此第二次的病毒上清滴度会很低，另外如前面一样的问题，两次收集也会增加风险，因此，只要做得好，初学者收一次上清基本上够做10次左右的感染了，没必要多次收集。关于第二个问题的回答是斩钉截铁的，多次反复感染有助于提高重编程效率。

 

问题5：有个问题我没看明白：几种因子的病毒同时包装并且一起感染么？

 

分开包装再加到一起感染。

 

问题6：克隆问题：我也正在做，好不容易把这几个克隆都拿到手了，不知道在克隆nanog过程中，是否遇到麻烦，我在中间花了很多的时间，至今无法搞清是克隆的问题，还是基因本身的问题，所以想问问各位同仁是否有同样的问题，如果有，可以一起讨论

 

就我个人而言，还没能成功用Oct4，Sox2，Nanog，Lin28将成体成纤维细胞重编程为iPS细胞（据我所知，目前除了威斯康辛，世界上其他牛组也没有相关报道），所以细心者发现在慢病毒那部分，其实我用的是六个因子。Nanog和Lin28对于细胞重编程不是必需的，但是它们一起可以显著增加重编程效率（特别是提高所出现克隆中真正iPS克隆的比率） ，而且可以提高某些方向的分化效率（比如神经），目前我们的工作正在Plos One修回。关于Nanog的载体，Addgene是有卖的，我用过，没有问题。