STAP細胞の再現に香港のグループが成功か?

香港のグループがSTAP細胞の再現に成功か?

私がKnoepfler Lab Stem Cell Blogに投稿した疑問点や小保方さん、丹羽さんにメールした内容に対する回答が、Vacantiが公開したSTAP細胞の作製法の中に記載されていた。やはり、Key issueの一つはtrituration procedureであった。

( I appreciate very much if you answer my question below. I sent the following e-mail to Dr Obokata. In your published today’s protocol, you did not describe the trituration procedures in detail. Is it not important?)

Protocol for generating STAP cells from mature somatic cells

https://research.bwhanesthesia.org/site_assets/51520d191eea6679ce000001/cterm/Refined_STAP_protocol-9a685fc86fec5ca857ad58ae75462d07.pdf

https://research.bwhanesthesia.org/research-groups/cterm/stap-cell-protocol

A5. As a final extremely important step in the trituration process, make two fire polished pipettes with very small orifices as follows: Heat the standard 9” glass pipette over a Bunsen burner and then pull and stretch the distal (melting) end of the pipette, until the lumen collapses and the tip breaks off, leaving a closed, pointed glass tip. Wait until the pipette cools, and then break off the closed distal tip until a very small lumen is now identifiable. Repeat this process with the second pipette, but break the tip off a little more proximally, creating a slightly larger distal lumen. The larger lumen should be about 100-150 microns in diameter, while the other pipette should have a smaller lumen of about 50-70 microns. Now triturate the cell suspension through the pipette with the larger lumen for 10 minutes. Follow this with trituration through the pipette 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 fire polished pipette of the smaller bore. This is a very important step. Do not skip this step, or take a shortcut.Again, remember to precoat each pipette with media. Also, during trituration, try to avoid aspirating air and creating bubbles or foam in the cell suspension.

 

その方法に基づいて、香港の研究グループがSTAP細胞の再現に一部成功した模様だ。しかし、数回以上の再確認が必要である。

 

Blogger Reports STAP Success

A stem-cell researcher claims to have reproduced stimulus-triggered acquisition of pluripotency by following a revised protocol posted online last week.

http://www.the-scientist.com/?articles.view/articleNo/39601/title/Blogger-Reports-STAP-Success/

Last week, Chinese University of Hong Kong’s Kenneth Lee said he would blog his group’s attempts to replicate the controversial stimulus-triggered acquisition of pluripotency (STAP) technique described in two January Nature papers using a refined protocol that Charles Vacanti of Harvard Medical School and Brigham & Women’s Hospital had posted online. Vacanti, one of the authors on the original STAP publications, has vigorously defended the technique even though other labs have failed to replicate the results. “The data and conclusions are honest and valid,” he told reporters in early March.

 

Lee now claims he has succeeded at reproducing STAP using Vacanti’s protocol—well, sort of. In posts at ResearchGate, Lee presented confocal and qPCR analyses from the transgenic fibroblasts his team mechanically triturated and bathed in acid over a period of three days, per the latest instructions. Only one day into this procedure, Lee’s team noticed that lots of stressed-out cells were dying. “We expect more and more necrotic cells will form during culture,” Lee wrote on March 28, adding: “The third day of culture is the critical period as reported by Vacanti.”

 

Today (April 1), Lee posted his team’s day three qPCR analysis, which showed that the trituration-only treated cells expressed higher levels of the pluripotency regulator Oct4 than did the trituration plus acid-bathed cells or the controls. “I am shocked and amazed by the qPCR results for the 3 day-old control and STAP cultures,” he wrote.

 

But other commenters at ResearchGate have questioned whether autofluorescence may be skewing Lee’s STAP results. And the irony of Lee posting such positive news today was not lost on Sergey Kiselev from the Russian Academy of Sciences’ Vavilov Institute of General Genetics, who—citing an earlier misconduct investigation surrounding STAP—wrote: “Great joke for April Fools! . . . Go on, Ken, please.”

 

Correction (April 1): An earlier version of this post stated the cells were titrated when in fact they were triturated. The Scientist regrets the error. (引用終了)

 

上記の記事の引用元:

ResearchGateからの引用(一部編集,誤字訂正)

Kenneth Ka-Ho Lee · The Chinese University of Hong Kong

Reviewer

24th March 2014 7:30pm Hong Kong time.

 

We have sufficient lung fibroblasts from 5 day old postnatal Pou5f1-GFP mice for STAPing. These cells are passage 2. Tomorrow, we will make the pipettes for robust trituration and perform the “acid bath” processes.

 

Kenneth Ka-Ho Lee · The Chinese University of Hong Kong

Reviewer

25th March 2014 10:30pm Hong Kong time.

 

Today, we made the glass pipettes for triturating the fibroblasts before acid treatment. Two sets of pipettes were made; one with 150µm and other set with 75 µm. We also performed pilot studies with the pipettes – since the bore size is very small. We tried using a Pipetman and a mouth piece to determine which is the better method. It was decided a 5ml Pipetman was the better choice because it was exhausting trying to titrate by mouth.

 

Kenneth Ka-Ho Lee · The Chinese University of Hong Kong

Reviewer

26th March 2014 5:30pm Hong Kong time.

 

We have attempted to generate STAP cells using Vacanti’s newly released protocol.

 

What we did today:

 

Lung fibroblasts (passage 2) were trypsinized, pelleted and washed with HBSS.

 

~0.5×106 cells were then resuspended in “Sphere Media” (DMEM/F-12, 1%PS, 1xB27, 1,000U/mL LIF). -ve control.

 

6×106 cells were triturated. Briefly, the cells were suspended in 2mL of HBSS (cell conc. = ~0.8x106cells/mL). Using glass pipettes, pre-coated with chilled HBSS, with different lumen sizes for trituration: 5 minutes with standard 9” glass pipette. 10 minutes through larger lumen (124μm). Then 15 minutes through small lumen (61μm). During the process, no air bubbles were allowed to form.

 

After that, additional HBSS was added drop-wise and air bubbles avoided, and made to final volume of 20mL. The cells were pelleted by centrifugation at 1,200 rpm for 5 minutes.

 

~0.5×106 cells were then resuspended in “Sphere Media” (DMEM/F-12, 1%PS, 1xB27, 1,000U/mL LIF). “Trituration only and no acid treatment” –ve Control

 

~1.1×106 cells were then re-suspended in HBSS at pH 5.4 at room temperature, at concentration of 2×106 cells/mL. The pH rise to around pH5.8. The pH of the cell suspension was then titrated to pH5.67 with diluted HCl in HBSS, immediately before sealing a conical tube for incubation.

 

The acid bath and non-acid bath cell suspensions were incubated at 37°C for 25 minutes, followed by centrifugation at 1,200rpm for 5 minutes.

 

Finally, cells were re-suspended in “sphere media” and seeded on non-adhesive culture dishes.

 

Note:

 

1. Non-adhesive 35-mm culture dishes were used. (SPL, cat#:11035)

2. Seeding density for producing the STAP cells was ~1.2×105 cells/cm2

3. No EGF, bFGF or heparin was added in the ‘Sphere Media’ à Obokata’s original recipe should be able to support and maintain cell reprogramming

 

Kenneth Ka-Ho Lee · The Chinese University of Hong Kong

Reviewer

 

Hi Sra,

 

Unfortunately this site does not accommodate superscript or Greek alphabets.

 

It should be 0.8 X 10 to the power 6 or 0.8 million cells.   Thank you for pointing this out.

 

The same also apply for ~1.2×105 cells/cm2 which should be 1.2 X 10 to the power 5 and the 2 after cm

 

Kenneth Ka-Ho Lee · The Chinese University of Hong Kong

Reviewer

27th March 2014 11:00pm Hong Kong

 

Dear All,

 

It is now 1day after our transgenic (Oct4-GFP) fibroblasts have been mechanically titrated and acid bathed. Below you will find the results of our confocal analysis. Enjoy!

 

Kenneth Ka-Ho Lee · The Chinese University of Hong Kong

Reviewer

 

Dear Jeanne,

 

Yip, I am very sure because the cell indicated by the green arrow in Figure C and C’ has a slight green grow but the DAPI staining revealed the nucleus is not intact i.e. a dying or dead cells.

 

Anyway, time will tell as we expect more and more necrotic cells will form during culture. The third day of culture is the critical period as reported by Vacanti.

 

We will validate all our results and conclusions by qPCR.

 

Kenneth Ka-Ho Lee · The Chinese University of Hong Kong

Reviewer

28th March 2014 8:00pm Hong Kong

 

Dear All,

 

It is now 2 days after our Oct4-GFP fibroblasts have been mechanically titrated and acid bathed. Below you will find the results of our confocal analysis. Enjoy!

Our manipulation and observations today:

 

Trituration was performed twice, same as day 1. 0.5mL of ‘Sphere Media’ was added to each of the 35mm dishes, as described in Vacanti Lab’s protocol.

 

Although no statistics was cited by Vacanti, the cell density decreased drastically. We estimated that there was a 50% decrease in cell number. In the original paper reported in Nature, such decrease in cell count was reported for day 2, which is in line with our current experiment. Day 3 will be critical as this was the time Oct4-GFP expression was reported for STAP cells. If we find that the cell number decreased even more drastically in our cultures, we will harvest some of the cultures and use them directly for qPCR analysis.

 

Kenneth Ka-Ho Lee · The Chinese University of Hong Kong

Reviewer

30th March 2014 8:00pm Hong Kong (been so busy even got the year wrong in all previous posts!! Really embarrassing).

 

Weekend, but we still managed to get access to the confocal microscope. Below you will see the results of the 3 day-old Control and STAP cultures using Vacanti’s Protocol. Enjoy!

 

Paul Knoepfler · University of California, Davis

 

Thanks, Ken. For reporting your studies here. This is extremely helpful.

 

Kenneth Ka-Ho Lee · The Chinese University of Hong Kong

Reviewer

 

Dear All,

 

I am shocked and amazed by the qPCR results for the 3 day-old control and STAP cultures. Totally speechless!

 

ENJOY!

 

The STAP Saga continues………..

 

Kenneth Ka-Ho Lee · The Chinese University of Hong Kong

Reviewer

 

Dear All,

 

This not an April Fool trick!. The negative control came up “positive” while the acid bath came up “negative”. Looks like mechanical trituration could induce STAP cells!?!?!? However, this the first time we did this experiment and will require several more rounds to validate.

 

Julien Maruotti · Johns Hopkins Medicine

 

Dear Dr. Lee,

 

Sometimes stressed cells can express a wide range of genes, not necessarily connected to a given phenotype. Although it is interesting to note that SOX2 is not up-regulated in the trituration only sample, contrary to OCT4 and NANOG, maybe other markers -not necessarily related to pluripotency- would show similar profile of up-regulation. Do you plan to also check additional genes?

Anyway it looks very intriguing.

 

Paul Knoepfler · University of California, Davis

 

Let’s see how this develops, but I remain skeptical that this is a specific induced pluripotency-related event related to trituration and that what you are seeing here is STAP cells. I hope I’m wrong and it is something real on the STAP front, but I doubt it. Thanks again, Ken, for all the hard work that your lab is doing!

Paul

 

Andrés M Bratt-Leal · The Scripps Research Institute

 

Hi Dr. Lee,

 

Dividing by a number close to zero can lead to large increases in fold change even without significant gene expression. Can you post a comparison to pluripotent stem cells or the fold change compared to your housekeeping gene in the untreated and treated samples?

 

Thanks,

Andres

 

Kenneth Ka-Ho Lee · The Chinese University of Hong Kong

Reviewer

 

Dear All,

Mechanical trituration is not hard to do. Can some other labs please try and see whether using Vacanti’s protocol of just mechanical trituration could induce expression of the stemness markers that I just mentioned?

 

Potentially, expression of these pluripotent markers could be the bi-product of un-regulated gene expression by the dying or stressed cells. I agree 100% with Paul Knoepfler’s comments.

 

I am not claiming that “STAP” cells exist – only presenting the results of our research as it is – which is open to interpretation. Please, don’t Hype up this data!

 

http://www.researchgate.net/publication/259984904_Stimulus-triggered_fate_conversion_of_somatic_cells_into_pluripotency/reviews/103

ResearchGate

 

 

 

http://blog.with2.net/link.php/36571 ブログランキングに登録しています。

marugametorao について

神経内科専門医 neurologist
カテゴリー: ニュースと政治, 神経内科医, 神経学, 医学, 医学教育, 挑戦 パーマリンク

STAP細胞の再現に香港のグループが成功か? への2件のフィードバック

  1. marugametorao より:

    Lee教授は、「個人的にはSTAP細胞は存在するとは思わない、これ以上この実験を遂行することはマンパワーと研究資金の浪費となるであろう」とResearchGateで述べている。

    Kenneth Ka-Ho Lee · 40.99 · 291.53 · The Chinese University of Hong Kong
    Reviewer

    Thank you all for your excellent suggestions on improving the data. Personally, I don’t think STAP cells exist and it will be a waste of manpower and research funding to carry on with this experiment any further.

    I think this live-blogging is a good and bad idea:

    Good in the sense that it will stimulate interest in the scientific community, so helping to draw younger scientists into the stem cell field.

    Bad in the sense that you don’t have the time to think and carefully assess the data properly – as everyone expect you to post the results as soon as it is generated. E.g. In my case a 10 folds increase in Oct4 and Nanog expression is definitely not sufficient and we need to see at least 100 folds as in my previous post (see above Figure3 for iPSCs). And for Sox2 it is only at 2 fold increase – which is even worst.

    I will no longer blog on this page any more. I want to get back to doing
    my own science interest.

    Anyone want to collaborate with me to look at the function of the BRE gene which is a component of the BRCA1 and BRISC complex?

  2. ピンバック: STAP細胞:私自身のコツ、レシピのようなものはある。 | ニューロドクター乱夢随想録

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