The Vacanti Lab at Brigham and Women’s Hospital/Harvard Medical School has now posted online their own version of the STAP cell production protocol.
The thing that first struck me most strongly about this published protocol is that it is not the same protocol reported in the Nature papers and it also is not the same as the recently posted RIKEN STAP protocol.
What the heck?
Given that the two STAP papers were just published 7 weeks in Nature, how can there already be so many different protocols coming from one collaborative team? I don’t know, but it seems puzzling to me.
The Vacanti Lab Protocol in part indicates that this new protocol is in essence a “best of both worlds” kind of methodology:
“The protocol below is a combination of the two most effective approaches described in our Jan 31, 2014 article published in Nature (Obokata et al., Stimulus Triggered Fate Conversion of Somatic Cells into Pluripotency. Nature 505. 641-647, 2014).”
Again, as with the RIKEN protocol, people attempting to make STAP cells with this new method are cautioned numerous times about crucial details to the protocol that if not performed correctly could derail the process. Even in the introduction to the protocol the authors (whoever they are) write “It is very important that each step be performed precisely as described.”
It seems worth quoting Dr. Vacanti from my interview with him on this blog in the first days that the STAP papers came out along with my questions #4 and #5 for him:
4. Do you think other labs will have a relatively straightforward, easy time to make STAP cells too now? Have any other independent labs indicated to you that they’ve now already done this successfully?
Vacanti: Yes. I believe they will. It is easy. I suspect that it has not yet been repeated in 2 days.
5. Are there some methodological subtleties to making STAP cells? Would you be willing to make available a full, step-by-step, day-by-day detailed Vacanti lab protocol with notes, etc. on making STAP cells for the global stem cell community?
Vacanti: No subtleties. We studied 7 stresses. We focused on one effective stress, low pH. I suspect that many more effective stresses will be described. If not, we would be delighted to give a “how to”. But that is essentially what the paper is.
This new method seems to be the Vacanti Lab “how to”, but to me at least there sure seem to be subtleties.
This new Vacanti Lab protocol involves combining in sequential steps both of two key stressing steps for cells: trituration and acid treatment. The main difference from previous protocols seems to be the implication that both of these stressors in combination are recommended. Further the trituration is divided into 3 steps: cells going through a standard Pasteur Pipette, cells then going through a pipette with a 100-150 micron lumen and then finally through a pipette with a smaller lumen of 50-70 microns.
Since the average cell diameter is about 10 microns, I’m not quite sure what happens to cells as they go through these pipette lumens, and how it would be connected to reprogramming.
Presumably many cells are mechanically destroyed during this process by literally crashing into the edge of the lumen or squeezing through in clusters, others are mechanically damaged, and perhaps a minority survive just having been subject to pressure and sheer stress.
It seems to me like a very imprecise, crude process of cellular battery. The recommended fire polishing process including breaking the end seems subject to great variation, although using such customized pipettes for other applications is not unusual in biomedical science experiments.
After trituration, then comes the acid treatment that is literally referred to by the protocol authors as an “acid bath”, and placement in “sphere media”, which is changed regularly. There the protocol ends abruptly.
Overall takes on the new protocol?
I would say that to my eye this protocol seems far less detailed than the online RIKEN one. For example at step A7, acid treatment, there are no details given as to the actual exact cell # to be used and the volume of low pH buffer to be used is not stated. It just says 2 million cells/ml in a general sense. Isn’t the total volume here important particularly since addition of cells raises the pH? The greater the total volume the lower the impact of the cells on the overall pH, right?
What is also notable about this protocol more generally is the large number of other seemingly important things that are not mentioned in it.
There is no mention of efficiency, validation, expected results, the kind of starting cells, the species of cells, the % cell death expected at any given step and how this is assessed, sorting procedures before or afterwards, and a whole lot more. Puzzlingly, the protocol in addition has no listed authors and is not signed so I am just inferring that this is coming from the Vacanti Lab.
Maybe they can post a new version that includes more detail later.
Will this new protocol help other labs make STAP cells? One can hope, but I remain fairly skeptical that these procedures would specifically create totipotent stem cells.
Any other reactions?