日本理研（Riken）关于STAP验证试验的中期报告已于8月27日发布，结果是重复小保方晴子的STAP细胞制备方法的丹羽仁矢小组宣告试验失败。目前，仅有小保方晴子本人继续进行验证，结果尚不得而知。

令人心焦的是，小保方必须在今年11月底以前结束试验，无论成功与否，她都要将自己的结果交给Riken，然后以最终报告的形式向社会公布。可以说，余下的两个多月将是确认STAP是否存在的最后时刻！

就在这个火烧眉毛的节骨眼上，小保方的美国导师、Nature论文共同作者之一、哈佛大学医学院的Charles Vacanti及其实验室成员、另一位Nature论文共同作者小岛宏司于9月3日突然公布了一份新的实验方案，宣称成功制备STAP细胞的关键是在酸浴中添加过去从未提及的腺苷三磷酸（ATP）。

我估计在这份新方案发布之前，Vacanti应该早就知会了小保方。不管她以前是否知道它，这次必定已经测试过此方案。那么，Vacanti及小保方是否因为已经成功制备出STAP正在窃喜呢？我认为有这个可能，不然Vacanti也不会如此高调地公布一份可能让他丢脸的实验方案。

我不是研究干细胞诱导的，但我根据常识判断，能量缺乏时，AMP将升高，细胞会走向休眠；相反，能量充足时，ATP升高，细胞趋向分裂。因此，利用ATP促进干细胞分裂，并向多能细胞分化是有点道理的。

为什么Vacanti突然会冒出一个添加ATP的想法呢？是他们为了技术保密而故意隐瞒，还是他们自己都不记得当初是如何做的？从这份新方案的说明来看，应该是后一种情况。据他描述，最初在溶液中是加了ATP的，但发现加入ATP后pH值明显降低（pH3.0），于是他们就认为“酸浴”是获得STAP的必要条件。

然而，STAP（刺激触发的多能性获得）中的“刺激”（stimulus）应该是温和的，比如适度的针刺、轻微的机械压力等，而不能达到“胁迫”（stress）的程度，否则将抑制细胞分裂，当然就谈不上分化和发育了。因此，把刺激处理说成是stimulus是对的，而说成stress就不对了。

一般来说，营养缺乏、2,4-二硝基苯酚、一氧化氮、过氧化氢、雷帕霉素、白藜芦醇、二甲双胍等，相对于细胞而言都是不利的环境因素，是不可能促进干细胞分裂和分化的。至于弱酸性（pH5.0）是细胞分裂的激活剂还是抑制剂，恐怕弄清楚这一点才是成功的关键。

但愿小保方能借新法旗开得胜，就此逃过一劫，若是果真如此，那么就应了下面这句经典对子：“山重水复疑无路，柳暗花明又一村”或“踏破铁鞋无觅处，得来全不费工夫”！

附：

REVISED STAP CELL PROTOCOL.  09.03.14:

Below is a revised protocol to improve the chances of successfully creatng pluripotent STAP stem cells from mature somatc cells, not dependent on the source of cells. The protocol has been revised to refect improved techniques. As we learn more about the STAP cells, it is likely that new stresses will be identfied and that nutrients and chemicals will be developed which are much more effectve at creatng STAP cells.  

This protocol has been modified from what was originally published (and then retracted), and also from the previous protocol that we posted online on this site, for the following reasons:

1.  We made a significant mistake in our original declaraton that the protocol was “easy” to repeat. This was our belief at the time, but it turned out to be incorrect. Many of the steps described appear to be a function of the technique of the individual investgator. Consequently, the revised protocol below should increase the likelihood of success.  It combines the use of ATP as a supplemental source of energy with two very effectve stresses to achieve the desired result; that is, creation of pluripotent STAP cells.

2.  In reviewing our original approach, and the rationale exposing the cells to a low pH, it was recalled that we had originally experimented with the additon of ATP as an external energy source in hope of improving the viability of the cells and the spheres generated. At that time,it was noted that the additon of ATP not only resulted in better sphere formation, but was also associated with a marked decrease in the pH of the soluton to which the mature cells were exposed. Consequently, we started to focus on the effect of low pH alone as a stress to induce the creation of STAP cells, without evaluating other potential beneficial effects of ATP.  In recent months, our lab decided to re explore the utlity of a low pH soluton containing ATP in generatng STAP cells. We found that while pH alone resulted in the generaton of STAP cells, the use of a low pH soluton containing ATP, dramatcally increased the efcacy of this conversion.

When this acidic ATP soluton was used in combinaton with mechanical trituraton of mature cells, the results were even more profound. Consequently, our protocol has been now modifed as described below to utlize these fndings to increase the efcacy of generatng STAP cells.

The revised protocol seems to be much more efectve for generatng STAP cells in our lab, regardless of the cell type being studied. It combines the additon of ATP as a supplemental energy source, with elements of the most effective approaches originally published and later retracted in the article, (Obokata et. al., Stmulus triggered fate conversion of somatc cells into pluripotency. Nature 505. 641-647, 2014).

It is very important that each step be performed precisely as described. The protocol will vary slightly if you are startng with tissue rather than a cell suspension. It is especially important to triturate the cell suspension in the acidic ATP soluton for a minimum of 30 minutes, until the suspension can be easily triturated up and down the reduced bore pipetes of the smallest orifices. We first describe the protocol when starting with a suspension of cells, and then describe additional steps necessary when starting with a soft tissue.

Generatng STAP cells from a suspension of mature somatic cells:

A1.  First, make a stock solution of ATP, 200 mM, (which will be added to HBSS in the next step), by adding 110.22 mg of ATP powder (Adenosine 51 Triphosphate Disodium Salt Hydrate - Sigma A2383) to each 1 mL of water (MilliQ water).  The pH of the resulting ATP soluton solution is about 3.0.

A2. Place 5 mL of HBSS Hanks Balanced Saline Soluton (HBSS Ca+Mg+ Free: Gibco 14170-112) into a 15 mL tube. Place a clean pH sensor into the HBSS. Titrate in the ATP stock soluton, drop by drop, into the HBSS until the desired pH of 5.0 is obtained (roughly a few hundred microliters).  Mix the soluton regularly to ensure that the measurement is accurate. The final acidic HBSS soluton is has an ATP concentraton of about 3-5 mM.  This will be used below in Step A5.

A3.  Add the live somatic cells to be treated, as a cell suspension to a centrifuge tube, and then centrifuge at 1200 rpm for 5 minutes. Note: Trypsin-EDTA, 0.05 % (Gibco: 25300-054) can be added to the tssue culture dish containing cells, for 3-5 minutes, to release adherent cells to be added to the centrifuge tube.

A4.  Aspirate the supernatant down to the cell pellet.

A5. Resuspend the resulting pellet at a concentraton of 1 million cells/ml in the low pH, Hanks Balanced Saline Soluton with ATP, (made above in Step 1A) in a 50 ml tube. Note: We recommend working with a volume of 2-3ml of the cell suspension in a 50ml tube.

A6. Precoat a standard 9" glass pipete with media (so the cells do not stick to the pipete - we use: (Fisher brand 9" Disposable Pasteur Pipetes: 13-678-20D). Triturate the cell suspension in and out of the pipete for 5 minutes to dissociate cell aggregates and any associated debris. This can be done with a fair amount of force.

A7. As a final extremely important step in the trituration process, make two fire polished pipetes with very small orifices as follows: Heat the standard 9" glass pipete over a Bunsen burner and then pull and stretch the distal (meltng) end of the pipete, until the lumen collapses and the tip breaks of, leaving a closed, pointed glass tip. Wait until the pipete cools, and then break of the closed distal tip until a very small lumen is now identifable. Repeat this process with the second pipete, but break the tip of a little more proximally, creating a slightly larger distal lumen.  The larger lumen should be about 100-150 microns in diameter, while the other pipete should have a smaller lumen of about 50-70 microns.  Now triturate the cell suspension through the pipete with the larger lumen for 10 minutes. Follow this with trituraton through the pipete having the smaller lumen (50-70 microns) for an additional 15 minutes. Continue to triturate the suspension until it passes easily up and down the free polished pipete of the smaller bore. This is a very important step. Do not skip this step, or take a shortcut. Again,remember to precoat a each pipete with media. Also, during trituraton, try to avoid aspiratng air and creatng bubbles or foam in the cell suspension.

A8. Add normal HBSS (containing no ATP) to the suspension to a total volume of 20ml, centrifuge at 1200rpm for 5 minutes, and then aspirate the supernatant.

A9. Resuspend the resulting pellet in 5ml of what we term “sphere media”(DMEM/F12 with 1% Antbiotc and 2 % B27 Gibco 12587-010) and place at a concentraton of 10^5 cells/ml, within a non-adherent tissue culture dish in the presence of the following supplements: b-FGF (20ng/ml), EGF (20ng/ml), heparin (0.2%, Stem Cell Technologies 07980). LIF (1000U) should be added if the cells are murine).

Note: Supplements such as bFGF, EGF and heparin may not be necessary.Important: Afer the cells are placed in tssue culture dishes, they should be gently pipeted using a 5ml pipete, twice/day for 2 minutes, for the first week, to discourage them from attaching to the botom of the dishes. This is important to generate good sphere formaton. Add sphere media containing the supplements described every other day. (Add 1ml/day to a 10cm culture dish, or 0.5ml/day to a 6cm dish.)

Generatng STAP cells when startng with soft tissues that contain many RBCs.

B1. Place the excised, washed sterile organ tissue into an 60mm petri dish containing 50-500μl of collagenase, depending on the size of the tissue. Add a sufficient volume of the collagenase to wet the entre tssue.

Note:Diferent types of collagenase or other enzymes are better for diggestion of different organ tissues. (The spleen may not need to be exposed to any digestive enzymes.)

B2. Mince and scrape the tissue for 10 minutes using scalpels and scissors to increase surface area that is exposed to the collagenase, until the tissue appears to become gelatenous in consistency.

B3. Add additonal collagenase to the dish to make the total volume = 0.5ml, and place in an incubator/shaker for 30 minutes at 37°C at 90rpm.

B4. Add 1.5ml of the low pH HBSS/ATP soluton (see step A2) to the dish (yielding a total volume of 2.0ml) and then aspirate the entire contents via a 5ml pipete and place into a 50ml tube.

B5. Now proceed to triturate as previously described above (step A6-A7) when starting with mature somatic cells.

B6. Afer trituraton is completed (through step A5 when using a culture dish of mature somatic cells), add 3ml of HBSS, yielding a volume of 5ml, to the 15ml tube and then slowly add 5ml of Lympholyte to the bottom of the tube to create a good bilayer.

Note: It is important to add this as described to create a bilayer and avoid mixing of the two solutons.

B7. Centrifuge this tube at 1500g for 10 min. Rotate the tube 180° and recentrifuge at 1500g for an additional 10 min. This will cause the erythrocytes to form a pellet at the bottom of the tube.

B8. Using a standard 9” glass pipete, aspirate the cell suspensions layer between HBSS and Lympholyte and place in a new 50 ml tube.

B9. Add HBSS to the suspension to a total volume of 20ml of HBSS and then thoroughly mix the suspension by pipeting via a 5ml pipete for 1 minute.

B10. Centrifuge the solution at 1,200rpm for 5 minutes and aspirate the supernatant.

B11. To fnish the process, proceed with Steps A5 – A9, above.

The above protocol seems to be the most efectve protocol “du jour” based on our most recent studies. In time, as more is learned about the STAP cell phenomenon, we are certain that the approach will continue to be modified to effectively utlize new stresses and chemical environments to create pluripotent STAP stem cells from mature somatic cells.

We have developed this most recent protocol to address concerns that to date, to our knowledge, other groups have been able to generate STAP cells using our previously published or posted protocols. While we are confident that the original protocols published, will work if performed with meticulous atention to detail, we have tried to develop a protocol based on new information, that should be much more effectve in demonstrating a phenomenon in which we have absolute confidence.

Charles A. Vacant, MD, and Koji Kojima, MD, PhD

September 3, 2014