Hi Pierre,

I observed cell death for a couple of days after electroporation of OSK (not of OSKML).

The MEFs that I used were passage#3. I don't expect them to endogenously express the reprogramming factors, do you?

That can be a problem only if I see massive cell death (major reduction in cell number) for OSKML (OCT4, SOX2, KLF4, N-MYC, and LIN28) cocktail, but I observe significant cell death only in the OSK (OCT4, SOX2, KLF4) cocktail. OSKML cells grow normally.

If you have a look at my question, you''ll see I had stated it before.

More details include:

MEFs were seeded with different densities: 12K, 18K, 18K

12-well plates

15 ug of each vector

Electroporation: 1 pulse, 250 mV, 0.01 ms

Culture conditions:

Protocol#1: 4d in standard fibroblast medium, and then switch the medium to mES medium (15% FBS+LIF).

Protocol#2: 2d in standard fibroblast medium, 4d in mES medium (15% FBS+LIF), and then switch the medium to KOSR-based mES medium (15% KOSR+LIF).

Note:

1) high death rate was observed in all OSK groups irrespective of culture condition.

2) What can I do to increase reprogramming efficiency for OSKML group (please see the primary question above).

Thanks in advance,

Sharif

Well, by low passage, I mean 3-5 passages.

By massive cell death, I mean when I see too many dead cells in the supernatant culture medium floating back and forth, so to speak, and also no significant increase in cell number as compared with the normal replication of MEFs in culture or with cell division I observe in the OSKNML group.

I have checked many papers regarding iPS generation in 12-well plates. The range of initial cell number in my case fits well with what I have seen in the literature.

I am not sure if my cells express Sox2 or Myc, but if this is the problem, I should have seen high death rate in OSKML treatment group too, right?

I have analyzed MEFs and they do not appear to express Oct4, Nanog, or Sox2.  They do however express Klf4. The efficiency of reprogramming with episomal constructs is extremely low.  For reprogramming, I typically use 2 million cells in a 10cm dish with 5-10ug of plasmid (in this case I usually get approximately 2-5 reprogrammed colonies per dish); most of the cells will die following electroporation (I use extremely high voltages).  Perhaps the issue is with the lack of c-Myc. c-Myc is a very important pro-proliferative/pro-survival factor. 

Hi Sharif,

you should optimize your DNA plasmid concentration Because high DNA Concentration have toxic effect during Electroporation .I use 2 million cells in a 10cm dish with 2-10ug of plasmids .

How did you prepare DNA plamids by your self or buy from company..?

Thanks Joseph for the good points.

I agree with you that MEFs do not express pluripotency factors, except for Klf4.

The problem is that I need to evaluate the effect of a kind of expensive agent on reprogramming, so it will cost too much if I induce iPS in 10-cm dishes.

Hi Maryam,

Thanks for your point. DNA concentration may be a reason.

The vectors are those generated and used by Yamanaka's group in Japan to produce human iPSCs. They deposited their vectors in Addgene and we purchased them.

Sharif,

You're welcome, not a problem.  Reprogramming can be bear at times.  And, indeed, 10cm plates will use considerable amounts of materials.  So, your objective is to assess the effect of your compound on reprogramming efficiency, yes?  The OSK vector yields no colonies, but the OSKML vector does.  This could suggest that c-Myc is not dispensable for reprogramming (at least in this case).  Given that OSKML does work for you, although at low efficiency, this could be considered your experimental baseline.  From here you can move forward.  Perform additional rounds of OSKML nucleofection and calculate a baseline efficiency of reprogramming.  Then perform additional nucleofections with OSKML and then add your various agents (compound X, Y, or Z); again, calculate the efficiency of these on iPS generation. This should answer your primary question.  Consider this scheme:

+ control: OSKM nucleofection  (% efficiency of reprogramming)

experimental A: OSKM nucleofection + compound A (% efficiency of reprogramming)

experimetnal B: OSKM nucleofection + compound B (% efficiency of reprogramming)

I would repeat these using your 12 well plate format until statistical significance is achieved.  You may also consider staining colonies in the 12 well plates for SSEA's to distinguish between partially and fully reprogrammed cells.  Keep in touch and let me know how it goes.  I think this approach should answer the questions that you are interested in.

Sincerely,

Joe 

Dear Joe,

Thank you very much. You're points are great to me. I was putting too much energy into using OSK cocktail to obtain iPSCs, but according to your experience, now it appears to me that it doesn't work as effective as I expected.

I appreciate the suitable work plan you suggested and will follow it (thanks!). Last time I did alkaline phosphatase staining to observe true iPSCs, do you think I need to stain for Ssea's too?

Sharif

Sharif,

Given that you are screening for reprogrammed colonies, AP activity should be more than sufficient for your purposes.  I suppose that staining each for stem cell specific antigens would be expensive and time consuming.  The iPS quality or differentiation capacity of colonies derived from your various conditions is something you could focus on later with in vitro SSEA expression and in vivo teratoma formation assays.  Let me know how your work turns out!

Best regards,

Joe 

Just a few additional points to the previous comments. It is an expected results that 3F without Myc will give you a significantly lower reprogramming efficiency. So your results are in line with that.

To perhaps help you increase efficiency and counteract your toxicity I suggest you a simple addition of inactivated MEFs when you notice the toxicity. The toxicity occurs through high expression of the reprogramming factors and that can be beneficial to your reprogramming efficiency. However, when the expression really kicks in, the cells are in a very fragile state and that is when extra support cells can help. Just add them into your medium. Don't add too many though, just enough to keep a close to confluent cell layer. Too high density will also reduce your efficiency.

Hi Sharif,

I think its worth to give sodium butyrate, VPA, AZA etc a go to enhance the reprogramming efficiency. A long list of these epigenetic modulators that have been proven beneficial for reprogramming can be easily found in many articles. 

this is one good example:

Mali P et al., 2010. Butyrate Greatly Enhances Derivation of Human Induced Pluripotent Stem Cells by Promoting Epigenetic Remodeling and the Expression of Pluripotency-Associated Genes. Stem cells. 

Hope it helps