This is a crowdsource page for people who want to post their findings on their attempts to validate the STAP stem cell ( ＳＴＡＰ細胞) method. I’m going to color successful or even moderately encouraging reports green and failures/discouraging results in red. *For those with longer attention spans, see some additional important considerations for this crowdsourcing effort at the bottom of this page.
Please if possible email me ([email protected]) actual data (e.g. photomicrographs, etc) and I’ll include it. I’m encouraging people to include their full names, but not requiring it.
Update on 3/24/14.
Elevent reports so far…
Felix wrote on 3/24/14
|We have been repeating the experiment with MEFs from OG2 mice. We indeed observed a significant increase of GFP positive population 4 days after acid (pH5.7) treatment, however almost all GFP positive cells are also showing strong red/blue fluorescence under the microscope, indicating the presence of intense cellular autofluorescence after treatment.|
Ray and Sandy wrote on 2/19/14:
We used MEF and treated in HBSS at normal pH or titrated to pH5.7 with HCl as described in the manuscript in suspension culture (~5×10^5 cells in 2mL DMEM/F12 media + 2% B27 + LIF). On day 1, some of the MEF formed small clumps in suspension, but this happened with and without low pH treatment. On day 7, the cells were plated down onto slides using cytospin, fixed/permeabilized and stained for Oct4. Cells in all conditions appeared Oct4 negative (results attached). Please note that the culture medium was not changed during the 7 day incubation period as it was not mentioned in the manuscript.
Dr. P wrote on 2/17/14 about a mixed bag of results:
STAP Experiments we have tried:
1. Rat Spleens from 6 month rats, LSM separated lymphocytes, treated exactly as published, cultured in DMEM/F12,2%B27,Lif. Oct4 detected after 7 days by western Blot.
2. Repeat of the above is underway.
3. Spleen from 35week Oct4-IRESGFP mouse ( GFP knocked in to endogenous Pou locus, on day 4 as it stands.
IF you don´t sort the Oct4-GFP spleenocytes, you will get GFP positive cells, but very few at the beginning of the culture. If after 7 days I see a significant increase in GFP, I will sort them, and take picture.
Side by side with VSELS I have in the incubator, you can´t tell the different visually.
I will post pictures when I have a complete data set.
We also tried MEFs and my own lymphocytes, but these experiments we used regulare iPS/Lif media while waiting for B27—–these experiments produced no oct4.
Update from Yoshiyuki on 2/13/14. Suspension culture (100,000 cells/ml) pH5.7, incubation time 25 min. See images for various conditions below. Note from Paul–Yoshiyuki now reports this green signal is determined to be autofluorescence. Bummer.
Andres wrote on 2/13/14 regarding a STAP experiment in progress at 8 days out using human fetal fibroblasts. See images below. Spheres generated (left), but seem fibroblastic in nature as do cells migrating out of the spheres once plated down either on geltrex (middle) or on feeders (right).
Yoshiyuki Seki on 2/13/14 wrote:
|We used mouse embryonic fibroblast derived from Nanog-EGFP Tg.
Now we are performing resuspended culture in B27 + LIF or serum + LIF for 4 days. In B27 + LIF medium, we can’t detect GFP-positive, while we can detect weak-GFP positive cells in serum + LIF. However, we observe many dead cells in GFP-positive clump. Therefore it might be difficult to expand clump of GFP positive cells in adherence culture.
Hong wrote on 2/12/14
|I have try the pH=5.62 DMEM Media to treat Mouse ES cell (2nd passage) for 25min, centrifuge 1000rpm/5min, raise them in B27 and Lif Media for 6 days to detect the endogenous mOct4(RT-PCR) but nothing no matter with/without treatment.|
Sasha wrote on 2/12/14
SK wrote on 2/12/14
|Try (1) Neonatal whole blood = failure
Try (2) Adult whole blood from three animals = failure
Try (3) Adult spleen cells from three animals = in progress. at day 2 most cells dead, some cells aggregate.biggest problem i keep running into is to keep the pH the same over 30 minutes. The more cells die in the low pH conditions, the more rapidly does the pH change. B27 and LIF were used. SK
Elliott Schwartz wrote on 2/11/14:
|A post-doc in my lab tried the acid treatment with human fibroblasts plated in mTeSR. 2 abnormal-looking groups of cells were picked, but nothing seemed to happen to them afterwards. I’d say try #1 was a failure|
Ruben Rodriguez wrote on 2/11/14:
Ethan said on 2/10/14:
|It has been more than a week now, have yourself or someone you know of been able to reproduce the mouse STAP cells. We tried, didn’t work for the first time.|
* Note (from Paul) that the results posted here are not necessarily endorsed by me or reflective of my own views. I would suggest that blog readers not take any one result too seriously and rather look for patterns. Also, I wonder…could there be a bit of a bias in this crowdsourcing toward negative results because those who get positive results, assuming there are positive results in the mix, may be more inclined to try to publish it in a journal versus in this domain? I don’t know.