Have no luck yet. This formula may work only for particular MEF cell lines. VPA (valproic acid) is too toxic for some lines. TTNPB does have a positive effect on increasing MEF reprogramming efficiency. There are potential problems of this approach: it is too labor-intensive and culturing cells for 1-2 months may increase genomic instability. I still prefer episomal approach.

Thanks for the information. VPA is certainly very toxic but it is required for openning up the DNA to improve reprogramming. Just put in an order for the kit, should be fun trying it out.. We have got our Episomes to work in reprogramming human senesence cells but the effciency was only 0.5% -beven when vitamin C was added.

Hi, Kenneth, please, also consider that such combination of the compounds is very case-sensitive. Definitely, you can get re-programming to stem cells for many cell types, but each cell type may require different combination of the molecules (concentration). Moreover, growth conditions is also very important (serum types, company etc). Please, also consider that vitamin C (ascorbic acid) as antioxidants inhibited re-programming at relatively high doses. Good luck!

Thank you for the advice Taras.

Hello Kenneth. We used TSA instead of VPA in human fibroblasts along with a different combination of 8 small molecules. VPA was very toxic as you mentioned. Our results were very promising and replicable so far. Maybe you can try small variants of Deng's combination to acheive the same thing in your lab. Like Thomson did with OSNL instead of OSKM.

Because when it comes to large numbers of small molecule combinations, you cannot know what will really happen. Interactions of pathways becomes more complicated each time you add another molecule into the game. Please let us know if you can replicate the Deng's experiment.

Dear Hakan, Thank you for suggesting the use of TSA instead of VPA. We are still waiting on the "Mouse iPSC Chemical Reprogramming Kit " to come from Selleckchem. The kit contains: Valproic acid, CHIR-99021, RepSox, Tranylcypromine (2-PCPA) HCl, Forskolin, TTNPB (Arotinoid Acid) and 3-Deazaneplanocin A (DZNeP) and very expensive. I am only 5% confident that it will work but still worth a try. Did you use similar small molecules in your fibroblast reprogramming experiments? - as you mentioned it was very promising and replicable. I will certianly keep everyone posted whether this experiement works!

Has anyone been thinking about the advantages and disadvantages of the various reprogramming methods? Do the resulting iPSCs vary depending on the method? I want to hear what people think.

Jeanne Loring

Yes; obviously it needs to be optimized for your cell line. Also, add Vitamin C - it decreases p53-p21 signaling which is a barrier to reprogramming, and also aids in demethylation of pluripotency markers.

Hi Jeanne. We tried lenti, episomal and sendai. If you ask about gene-reporter reported hiPSC -like something, they really might be different. However if we compare perfect hiPSC lines generated by each method let s say on passage 25-30 they are very similar.

Dear Kenneth, I'd like to give full combination, as soon as it's published which I hope it to happen in a short time. Even though my experiments ended 8 mounths ago, it has some similarities with Deng's molecules mentioned at the supplements of his paper. Because the mice and human pluripotency works on different ways, my combination is mainly different. But it contains Vit C, GSK3 inhibitors and TSA. It's still not perfect but gave me the same results three times, very effective and open to development. We had so little resources for the study so we couldn't screen thousands of small molecules like them (actually I could only pick 12). But I'm planning to continue the experiments. I'll give you an update as soon as I can. Meanwhile good luck with your molecules.

Dear Hakan, I look forward to reading your paper once it is published. Best of luck with the reviewers.

We haven't tried the small molecule method because either Sendai virus or episomes works so well. We have yet to detect a difference between iPSCs made by different methods using different human source cell types including fibroblasts, T cells, nasal epithelium and EBV-transformed B cells. The same is true for neuronal derivatives but our data is relatively sparse. We run Pluritest on all iPSCs.

Looks like “stimulus-trigged acquisition of pluripotency (STAP)" cell will probably surpass the use of the 7 small molecules to produce iPS cells!

Kenneth, I am trying to replicate STAP this week! I will let you know how it goes; it seems too good to be true - so fast and efficient.

Dear Marcela, my lab will also be replicating it too once the students come back from their Chinese New Year holidays. I will be using Oct4-GFP mouse fibroblasts. Please do let everyone know whether it works. Best of luck!

Dear kenneth and Marcela, please let me know if this methods can be reproduced. I would like to use this method as well.

Thanks and Best of Luck!

An update of our STAP experiments. We have treated Human umbilical Cord peri-vascular (HUPV) mesenchymal progenitor cells at PH 5.5 for 30 min. Our observation on the 3rd day , “the acid bath” HUPV cell behaviour was unremarkable compared with un-treated cells. They proliferate at the same rate as control cells and there was no increase in apoptosis. We have decided to continue culturing these cells over the weekend and then harvest the cells for real-time PCR - for stemness marker expression. We have also just harvested spleen cells from Oct4-GFP transgenic mice. The spleen cells will be “acid bath” tomorrow. We will observe them over 7 days for GFP expression. So far the results are negative but I hope I could be proved wrong!

Kenneth & Chan Jung, I also tried to generate STAP cells from mouse embryonic fibroblasts. I tried culturing them in suspension (low-attachment dishes without a substrate) +LIF+ACTH. After 7 days, I did not observe Oct4-GFP reactivation after exposure to pH 5.7 or pH 4.7. The cells that survive do appear a bit smaller (FSC on the Flow cytometer), but morphologically unremarkable. I am really hoping this is not another VSELs and MUSE story - where there is no consensus on their existence among the community due to low reproducibility. :-(

One of my colleague tried to generate STAP cells from Oct-4 GFP MEF, didn't get the GFP+ cells after 10days.

Neither CD45 human hematopoietic cells

Dear all,

Knoepfler's Blog: http://www.ipscell.com/stap-new-data/ is also providing results of people around the world trying to reproduce STAP. To date, all the results are negative. May I suugest if possible we upload some pictures of the STAP cells that we try to produce?

A Japanese blog suggesting Haruko Obokata papers maybe fraudulent.

http://blog.goo.ne.jp/lemon-stoism/e/008ac025ee1ccf4c694869f09b053ee7

Just did our first batch of acid bath Oct4-GFP splenocytes. After 6 days, we did not get any GFP positive cells.

Kenneth - now the Japanese blog has gotten press http://www.nature.com/news/acid-bath-stem-cell-study-under-investigation-1.14738?WT.ec_id=NEWS-20140218

Marcels

NB: Keep a tight control of the pH because it can change during the filtration/ sterilization process (going up by pH of 0.2) and check it again after the experiment. Also be careful of false-positive auto fluorescence due to induced cell death..

Update:

Sorted CD45+splenocytes from 1 week old Oct4-GFP mice. Will perform acid bath on the cells today. This is exactly what Haruko Obokata reported in her Nature paper. Want to detrmine whether her STAP process only work on CD45+splenocytes.

Just wonder whether using this cleanser will STAP and rejuvenate my skin?

Update:

Obokata has just published a more detail protocol of her STAP experiment on Nature Protocol Exchange: "Essential technical tips for STAP cell conversion culture from somatic cells" http://www.nature.com/protocolexchange/protocols/3013#/references.

Today, we have STAP our CD45+ male 1 week-old splenocytes exactly to her protocol.

We don't see much difference from this protocol with her published protocol in Nature journal.

Kenneth

Great to hear your updates!

Your SEBA approach really fascinating!! I am sure it will work in Scientifically!

Thanks to the hard work of my PHD students and Postdocs, we have nearly completed our STAP study. Below you will see some of our results. Enjoy!

More results

Wall Street Journal: Teruhiko Wakayama wants to retract the two STAP papers he co-authored in the Nature journal with Haruko Obokata. He cites there are “crucial mistakes” in the papers making them “no more credibility”.

http://online.wsj.com/article/BT-CO-20140310-703922.html

Looks like it is not the end of the STAP story!. Have been asked to try out the new protocol. Anyone else up to the challenge? Could join forces!

Hello Ken, thanks for sharing your results. Have you tried to passage the cells? If you did, how do they behave? - Kiran

 Does not work for me, I tried several times with mouse adult fibroblast, cells died by 7 days

Hi,

It looks like the gel electrophoresis in the supplementary Figure S8 E may be edited in the article Hou et al. 2013. Maybe I am wrong.

Could you check it, please?

Hi Farkas,  Go to pubpeer and you will see all sort of problems with this paper including the gel images you mentioned https://pubpeer.com/publications/23868920

Great, thanks!

I tried 3 times, the MEFs look terrible after all 7 compounds all added, give it up one year ago.

Dear Wenxiu,

Exactly, we tried using the 7 small molecules two years ago and gave up.  If it really works, then everybody will be using the protocol to produce their iPS cells by now.  Supprisng the study was published in Science

What a wast of time, money and effort!

Reference:  Pingping Hou et al,,"Pluripotent Stem Cells Induced from Mouse Somatic Cells by Small-Molecule Compounds" Science 341:651-654

These small molecules may work if intital cells have some kind of competence.

Nadine Hosny El Said

Another new paper was published (https://www.nature.com/articles/s41586-022-04593-5), but I'd love to know whether the Science article was really being repeated by anyone?

Liang Luo still trying, not much success though :/ they omitted a great deal of info from their protocols as it appears...