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 Blothttp://www.ipscell.com/stap-new-data/
STAP NEW DATA
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 pagePlease if possible email me (knoepfler@ucdavis.edu) actual data (e.g. photomicrographs, etc) and I’ll include it. I’m encouraging people to include their full names, but not requiring itUpdate on 2/17/14Nine reports so far…

Dr. Pierre Debs 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 Blot2. Repeat of the above is underway3. Spleen from 35week Oct4-IRESGFP mouse ( GFP knocked in to endogenous Pou locus, on day 4 as it standsIF 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 pictureSide by side with VSELS I have in the incubator, you can´t tell the different visuallyI will post pictures when I have a complete data setWe also tried MEFs and my own lymphocytes, but these experiments we used regulare iPS/Lif media while waiting for B27―–these experiments produced no oct4Update 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. BummerSTAP 1st Try V2

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)PH STAP test

Yoshiyuki Seki on 2/13/14 wrote:

We used mouse embryonic fibroblast derived from Nanog-EGFP TgNow 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 cultureNanog GFP STAP

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 treatmentSasha wrote on 2/12/14

We have tried to generate STAP cells from mouse embryonic fibroblasts, mouse adult neural stem cells and mouse embryonic neural stem cells. We did not observe Oct4-GFP reactivation from either cell type after the exposure to pH5.7, pH 4.7 and pH3.2 at different cell densities (including 100,000 cells/ml described in the paper). We have also tried gelatin- and laminin-coated tissue culture plates as well as low-attachment dishes without a substrate. All with no Oct4-GFP reactivation after 7 days of culture (assessed by flow cytometry). B27 and LIF were used.STAP stem cell attempt flow
Subhash Kulkarni 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. Subhash Kulkarni PhD
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:

The pictures shown in the NewScientist article resemble mouse es cells… and human es cells in its primed form don’t look anything like that; maybe they use naive conditions, but somehow I doubt itAlso, I’m one week in with my own pilot test with human neonatal fibroblast, and the combination of acidic shock and serum free media makes the cells to form aggregate, but a live staining indicates that they are not Tra-1-60 positive… and other than that; the culture looks pretty boring, no MET or anything interesting going on. It’s still possible that they need more time, but based what on what I’ve seen so far I’ll put a big red flag on this one. Picture at left is control pH 7.0 and picture at right is pH 5.6. Ruben Alvarez Rodriguez, PhD, The Salk.stap try pH 5.6STAP try pH 7.0
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.