Thoughts about CRISPR mess and Big Prizes

by Alexey Bersenev on October 5, 2015 · 0 comments

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Despite many many speculative predictions, CRISPR-based genome editing did not win a Nobel Prize this year. Well, it’s just a matter of time. Usually, I don’t like predictions, but I put my bet for 2017. Another Nobel-worth discoveries for the next 5-10 years are CART cell immunotherapy, optogenetics and decell-recell organs.

Interestingly enough, it was recently discussed that CRISPR discovery may not win Nobel, because it’s a mess. It is hard to credit only 3 individuals for whole CRISPR/Cas9 editing discovery. That’s why we are witnessing a serious CRISPR IP battle right now. The recent excellent article in Wired magazine by @sarahzhang, brought up more potential CRISPR pioneers into the game – Virginijus Siksnys:

In May, they submitted a paper detailing exactly how Cas9 cuts DNA to the Proceedings of the National Academy of Sciences. Peer reviewers came back with questions and that back and forth took a few months—typical of peer review.

Here is where the more famous narrative intersects. That June, a month after Siksnys’ lab submitted their paper, Doudna and Charpentier’s paper came out in Science—with many of the same findings as Siksnys’. (The key difference is that Doudna and Charpentier’s paper shows that the two pieces of RNA that Cas9 needs to work can be fused into one chimeric segment.)

Science’s editors, who obviously saw something big on their hands, fast-tracked the paper’s review, and published it within a month of submission. The paper made a huge splash.

“Of course, we were disappointed,” says Siksnys. His paper came out in PNAS in September to less fanfare.

Well, Siksnys should be smarter and post manuscript as preprint! Sarah Zhang continues with a thought-provoking statement that all big discoveries, like CRISPR do not shoot from nothing, but rather built upon wealth of accumulated knowledge and many scientists contribution. Yes, it is a continuum. Indeed, today Doudna acknowledged “36 years history leading to CRISPR” discovery.

Siksnys, Doudna, Charpentier, and Zhang all cracked Crispr/Cas9 around the same time because they all built on the same research from yet other scientists who figured what Crispr actually is. The 2007 paper kicked off a race. “People were working on the Crispr system,” says Dana Carroll, a gene-editing expert at the University of Utah who was paid to write a technical analysis in support of Doudna’s patent. “They were kind of inching toward what the Doudna and Charpentier group finally demonstrated.” Doudna and Charpentier published first, by a hair.

She also said in the piece that scientific discovery is not just a discrete moment, but a negotiation. Scientific authority and past accomplishments, patents and commercialization strategies, institutional “PR machines”, pre-publication peer review, politics – all these and even more factors may play a role in negotiation about the credit for discovery.

If we look at bigger picture, we may ask: “Does Nobel Prize really matter for CRISPR?” Doudna and Charpentier were widely credited for CRISPR in the last 2 years and have gotten countless number of smaller prizes and awards. Isn’t it enough? Vinay Prasad in his recent New York Times article also looked at bigger picture and asked “Are prizes for medical research really good idea at all?”

Every recent recipient has undoubtedly deserved the honor. But that doesn’t mean that prizes for medical research are a good idea.

The Nobel, along with the Dickson, Lasker-DeBakey, Canada Gairdner and other major awards, honors the scientists who are usually in the least need of recognition and funding, which squeezes out opportunities for other scientists.

More important, by emphasizing the importance of scientific breakthroughs — serendipitous occurrences that rely on decades of research — these prizes play down, and diminish, the way that great medical advances build on one another.

Very well said! It made me think – What is the best reward for your discovery in biomedical science? And here is my answer – The best prize is to see a smile on patient face, cured by your contribution to the discovery! If your discovery led to cure of millions, you will always be acknowledged, credited and remembered by next generations. No need for big prizes, no need for patents!

I wish I can see the time when Doudna/Charpentier and Zhang will stop to fight for CRISPR priority and make all patents free to use and build upon discovery without any charge. Make it public good and you always be remembered.


Cells Weekly – October 4, 2015

by Alexey Bersenev on October 5, 2015 · 0 comments

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Cells Weekly is a digest of the most interesting news and events in stem cell research, cell therapy and regenerative medicine. Cells Weekly is posted every Sunday night!

1. Good news about eye stem cell trials
Beginning of long-awaited embryonic stem cell-based trial for macular degeneration in UK, made the biggest splash in the media this week. The trial is a part of London Project to Cure Blindness – 10-year old initiative of University College London. The trial is sponsored by Pharma giant Pfizer. The first patient was treated a month ago and doing well.

By December, doctors will know whether the woman, who has age-related macular degeneration, has regained her sight after a successful operation at Moorfields Eye Hospital in London last month. Over 18 months, 10 patients will undergo the treatment.

Yet another exciting news is one year follow-up from the single case iPS cell-based trial in Japan. No tumors, no disease progression:

“The swelling of the retina is going down and the reoccurrence of abnormal blood vessels, which cause the disease, cannot be seen,” said Yasuo Kurimoto, general manager for ophthalmology at the Institute of Biomedical Research and Innovation Hospital in Kobe.

“The patient’s eyesight [which was gradually weakening before the operation] has been maintained since the operation,” said Kurimoto, who performed the operation.

Takahashi said, “Amid international attention over whether the patient would develop cancer, we are glad we could confirm that she has not.”

2. Human MSC are doing better in serum-free media
@Thomas_Heathman and his colleagues from Loughborough University, published very interesting study on comparison of human mesenchymal stromal cell (MSC) expansion in FBS and serum-free media:

Expansion of BM-hMSCs in PRIME-XV SFM resulted in a significantly higher growth rate (P < 0.001) and increased consistency between donors compared with FBS-based culture.
PRIME-XV SFM has also shown increased consistency in BM-hMSC characteristics such as per cell metabolite utilization, in vitro colony-forming potential and osteogenic potential despite the higher number of population doublings.

3. New self-assembling material for tissue engineering
Researchers described a method for self-assembly of tubular structures from bioinspired protein/peptide system. No bioprinting required! From press release:

The method uses solutions of peptide and protein molecules that, upon touching each other, self-assemble to form a dynamic tissue at the point at which they meet. As the material assembles itself it can be easily guided to grow into complex shapes.

Self-assembled tubular structures supported growth of mesenchymal stromal and endothelial cells. The next step is in vivo experiments.

Our system provides a leap forward by enabling the fabrication of geometrically complex scaffolds solely by directed self-assembly and without the need for moulds or templates. In addition, hybrid structures that enable the use of proteins offer tunability and a higher level of scaffold complexity, versatility and functionality.

4. Failure in cancer stem cell targeting
Boston-based company Verastem, halted cancer stem cell targeting trial for futility. As claimed by a company their experimental drug VX-6063 is targeting cancer stem cells in mesothelioma. VX-6063 is a FAK pathway inhibitor, described by MIT Professor Robert Weinberg as an important player in epithelial-mesenchymal transition. Weinberg is on Verastem’s advisory board.

5. Leukemic stem cells arise from hematopoietic progenitors
In the recent study, published online in Cell Stem Cell, researchers demonstrated that leukemic stem cells arise from mutated progenitor cells rather than from normal hematopoietic stem cells:

Using C/EBPa KO mice with the MF9 and MOZ-TIF2 leukemia models, we demonstrate that block of the CMP-GMP transition completely abrogates MF9-induced LSC formation and AML, regardless of the origin being from HSCs or myeloid progenitors.
…we show that the absence of LSC formation and the failure of leukemia development are a result of the block in myeloid differentiation, rather than the absence of C/EBPa per se.

6. Importance of sorting T-cell subsets in generation of therapeutic CAR T-cells
Riddell’s group from Fred Hutchinson Cancer Research published very important study for CAR T-cell field. They demonstrated that sorting of T-cell subsets allows to generate the most therapeutically potent and uniform CAR T-cell product:

We show that CAR-T-cell products generated from defined T-cell subsets can provide uniform potency compared with products derived from unselected T-cells that vary in phenotypic composition. These findings have important implications for the formulation of T-cell products for adoptive therapies.

7. Hypoxic culture conditions differently affects bone marrow and adipose-derived MSC
Very interesting study was published this week in online version of Stem Cells. Hypoxic culture condition is currently widely used technique across labs. However, hypoxia could promote genomic instability. Researchers compared genomic stability of clinical-grade human mesenchymal stromal cells, derived from bone marrow and adipose tissue, expanded with platelet lysate in hypoxiic conditions:

We conclude that long-term cultures under 1% O2 were more suitable for BM-MSCs as suggested by improved genomic stability compared with ADSCs.

8. New methods and protocols:
Transplantation of vascular bone marrow niche cells to enhance hematopoiesis (Stem Cell Reports)
Printing of decellularized extracellular matrix bioink (Nat Commun)
iPS cell-derived neuronal cells cultured on hydrogels for detection of Botulinum neurotoxin (Sci Reports)
Crestospheres: A method for maintenance of neural crest stem cells (Stem Cell Reports)
New effective and faster method to decellularize and recellularize the kidney (Oncotarget)
Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells (Development)

9. Fresh reviews:
Enzymatic and non-enzymatic isolation of adipose tissue-derived cells (Cell Regen)
Clinical Trials with Mesenchymal Stem Cells: An Update (Cell Transplant)


STAP reproducibility report from RIKEN

by Alexey Bersenev on October 3, 2015 · 0 comments

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As you may know, more than a year ago RIKEN set internal STAP reproducibility project. It was led by Hitoshi Niwa – a co-author of Obokata on both STAP papers. The final report of RIKEN internal reproducibility effort was published this week as preprint in BioRXiv (read full text here). As you may guess, the verdict is NO – it is not reproducible.

I’d like to remind you that in March of 2014, RIKEN released detailed STAP protocol to boost confidence in reproducibility efforts. At the moment of protocol release, Niwa was interviewed by Nature News and said:

… Haruko Obokata, Hitoshi Niwa and Yoshiki Sasai, all of the RIKEN Centre for Developmental Biology in Kobe, say that despite its “seeming simplicity”, the method requires special care. But it is “absolutely reproducible”, Niwa told Nature News.

Today Niwa told Nature News that members of the team besides Obokata have replicated the bulk of the work and that others outside the laboratory have succeeded in the first crucial step, inducing Oct3/4 expression after the acid treatment.

As internal reproducibility project progressed, it became less clear, and, by the end of August of 2014 RIKEN released interim report, where acknowledged lack of STAP reproducibility. Niwa said in press conference:

No clear signs of the STAP phenomenon could be observed so far in 22 experiments.

So, the final RIKEN report, authored by Niwa alone, was published 5 days ago. By some reasons, it left unnoticed by the media. All original experiment in generation of acid-induced STAP cells was carefully repeated. It failed on every step!

Niwa’s study became #5 published STAP reproducibility report. The other 4 in chronological order: Lee, Endo, Daley and Matsuzaki.


Dear readers, today I’m happy to share my Q&A with Madeline Lancaster on organoids. Dr. Lancaster is a Group Leader at the MRC Laboratory of Molecular Biology, University of Cambridge (UK). She became well known as brain organoids expert after publication of the study from Jürgen Knoblich’s lab 2 years ago.

Dear Dr. Lancaster, for introduction, can you please tell us how did you get to stem cell research and why brain organoids?

Actually, it was something of an accident, which I suppose is how much of science starts out. During my PhD in the lab of Joseph Gleeson at UCSD, I saw a number of fellow researchers making use of 2D in vitro neural differentiation in the form of neural rosettes. I was very impressed with the capabilities of neural rosettes to more faithfully recapitulate early neurodevelopmental processes and so when I started my post-doc in the lab of Juergen Knoblich, we planned out a project making use of mouse neural rosettes. However, because I was so new to it, I hit a few stumbling blocks and had trouble getting the neuroepithelial cells to settle down in 2D and make nice rosettes. Instead, I ended up with 3D balls of neurepithelium that looked very interesting, so I developed them further by embedding in Matrigel. That was the birth of the brain organoid method essentially, and the natural step was then to develop it further with human stem cells.

Let’s talk about biology of tissue self-assembly. Is there any difference between cells self-assembly and self-organization? What are the major mechanisms behind of cells self-assembly into tissue? Do cells have some kind of “spatial memory” or ability to “sniff chemical signals” in suspension? How is cells self-assembly universal among different types of stem- and mature cells?

There is indeed a difference between self-assembly and self-organization, and the difference can be more subtle depending on the system, but generally it comes down to the energy needed. Self-assembly does not usually require added energy and happens spontaneously, as in the case of protein folding for example. However, higher intrinsic order that requires input, such as energy input, would be termed self-organisation. In the context of organoids, it is likely that both of these processes are at play. Although we have not specifically looked in organoids, other similar systems, such as reaggregates of embryonic tissues, have shown that cells can sort out according to differences in expression of cell surface adhesion proteins. This type of organisation is an example of self-assembly as it represents orderly arrangement of cells to a state of lowest energy simply due to their differential adhesion properties (the so-called Differential Adhesion Hypothesis). However, the generation of tissue architecture through a more concerted effort of stem cells to generate specific fates with different properties and their active positioning through processes such as spindle orientation, would, in my view, represent a process of self-organisation. This process is also at play in organoids, for example in the cerebral cortex where radial glial progenitors undergo oriented cell divisions to position more differentiated daughter cells basally.
Although organoids are in vitro and in suspension, within the tissue it is reasonable to assume that there are certain chemical signals, such as growth factor gradients, since the tissue is quite dense and would very likely maintain such a gradient despite being in suspension. However, this remains to be determined and I think organoids would provide a very interesting system in which to study the contribution of such growth factor gradients, versus more stochastic cell sorting out events, to tissue morphogenesis.

I wonder how suspension of cells should be “treated” to trigger and maintain self-assembly? Is addition of extracellular matrix (ECM) sufficient or something else should be done?

It really depends on the organoid in question. It seems that some tissues require the addition of ECM in order to promote organisation, while others do not. However, I don’t think the ECM itself triggers self-assembly, as much of the early organisation that occurs in organoids, for example during the embryoid body phase, occurs before the addition of ECM. I think ECM is required later to support and trigger certain morphogenetic events, particularly epithelial budding in gut and in brain organoids. Timing really seems to be key in all steps though. It seems that if we can simply provide the right environment at the right time, the tissue is capable of intrinsic organisation and self-assembly. And my guess is that this is actually not far off from what is happening in an embryo. If you think about it, an embryo also develops due to intrinsic organisational cues.

There are many articles in the media about success and advances in generation of organoids from stem cells, but I’d like to focus on technical challenges. Can you please talk about methodological issues in organoid techniques in general and in brain organoids in particular?

In general, organoids develop quite stochastically since there is a lack of external body axes and other organisational cues to help pattern the tissue. Therefore, this leads to quite significant variability between organoids and between preparations of organoids. I think this applies to essentially all current organoid methodologies and certainly to a great extent to brain organoids. However, when we focus on specific brain regions, then you find a stereotypic organisation and developmental program that is quite reliable from organoid to organoid. Nonetheless, I think any technical readout making use of organoids, requires a large n in order to be confident in the findings.

What is a progress in generation of bigger vascularized organoids? How long organoids could be maitain in culture with adequate nutrients supply and vasculature?

To my knowledge, no one has successfully created fully in vitro vascularised organoids. This is a huge hurdle and one that the field of tissue engineering has been working on for many years. I am not an expert in engineering approaches to generate such in vitro vascularised constructs, but my feeling is that this hurdle will require a coming together of these fields to successful achieve in vitro vascularised organoids. Without vasculature, of course organoids are limited in their development. This can be overcome by, for example, breaking apart the organoids and “splitting” them, as in the case of gut organoids. Alternatively, organoids can be ectopically transplanted into a host organism for vascularisation and blood supply, as has been done for liver buds, for example. But in general, the full complexity of an organ cannot be recreated in a dish without achieving vascularisation and so I think this will be a major focus of future methods.

Is it possible to create desired brain region by changing spatial configuration/ polarity or chemical signals? Would it be possible to make from one “common” organoid or source cells/ organoids should be pre-selected from the mix?

Generating specific individual brain regions is certainly possible and has been pioneered by the late Yoshiki Sasai, who successfully generated the first brain regions in 3D. These included cortex, cerebellum, pituitary, and perhaps most well known are optic cup organoids. These different brain regions are possible to generate in isolation by providing certain combinations of growth factors at specific time points. For example, the addition of FGF19 gives rise to cerebellar tissue in 3D culture.

What is your take on potential ethical issues, related to creation of “mature” complex (with connectivity) and sized human brain organoids?

I don’t claim to be an expert in bioethics, but there is a solid foundation of research into network formation in model organisms that indicates that neural networks cannot reach maturity without both input, such as sensory input, and output. Thus, without both sensory input and output, such as the ability to interact with the surroundings, organoids will not form mature “thinking” networks. I think we are a very long way off from being able to create such a construct for organoids that would allow both input and output, so my feeling is that this will not be an issue in the immediate future. However, it may be possible in the more distant future and then it will be necessary to revisit the issue at that time.

We are all curious, of course, about possibility of translation organoids into medical applications. Can you please talk about such “translational landmarks” as efficiency, reproducibility and scalability of organoids? One particular question is about Matrigel – is there any alternative industry-grade defined ECM for organoids?

Organoids from a variety of organs have enormous potential for therapeutic applications. However, the reproducibility is still perhaps the largest hurdle. We can attempt to overcome this simply by using a large n, and this seems to work quite well for detecting phenotypes. Therefore, I hope it would also be successful in detecting drug effects such as in drug screening assays, but that remains to be shown. With regards to Matrigel, there are alternatives, but since it’s not completely clear what Matrigel is entirely made of, it has been difficult to generate a wholly synthetic alternative. I imagine that once animal studies show therapeutic potential of transplanted organoids, there will very likely be a number of scientists working on this problem.

Speaking of using organoids for Pharma R&D, what do you think is advantage of organoids over other sources of 3D tissues, such as bioprinted, defined iPS/ES-derived lines, and organ-on-a-chip? Our 2014 poll showed that organoids are not yet ready for prime time (see –

In general, the organoid field is in need of some proof of principle work showing that organoids, of any sort, can provide a platform for therapeutic applications such as drug discovery. My guess is that a number of companies are beginning to dip their toes into the organoid field, but once one takes the plunge and shows that a drug has actually been successfully developed with the help of organoids, then I think the pharmaceutical industry as a whole will jump on this technology.

What are the perspectives for using of organoids for cell- or organ replacement therapy? What indications and organoid types, in your opinion, are more promising and closer to clinical trials?

Gut organoids are certainly the closest to cell therapy approaches, since intestinal organoids have already been shown to successfully engraft when transplanted into the mouse intestine. I could imagine the most immediate need would be for liver, kidney and retinal organoids. Brain organoids are not as likely to be useful for whole organ transplants for obvious reasons, but they may provide a good way for directed differentiation of particular neural types that could then be transplanted. For example, spinal cord might be useful for victims of spinal cord injury.

You just started your own lab. I believe it’s very exciting time for you! Any research plans would you share with our readers? What advice would you give to young folks who are entering the field and are trying to make right career decision?

Yes, it’s truly an amazing time in my career. I think I’ve never had so much fun than I am right now with setting up the lab! It is a bit hectic though and I feel like I never have enough time or hands to do everything I would like to do. But that’s alright and I think probably one piece of advice I would give to another young soon-to-be group leader would be to not try to do too much and to instead focus on one or two key questions. For me, that’s brain size regulation. My lab is interested in using organoids to study developmental processes that regulate brain size determination, both in evolutionary terms but also in relation to neurodevelopmental disorders that affect brain size. I hope that by going down this path, we can learn something about what makes our brains unique and what goes wrong in the case of certain neurodevelopmental disorders like microcephaly.

Dr. Lancaster, I’d like to thank you for your time and for opportunity to talk! Good luck with your new lab!


Cells Weekly – September 27, 2015

by Alexey Bersenev September 27, 2015 notes

Cells Weekly is a digest of the most interesting news and events in stem cell research, cell therapy and regenerative medicine. Cells Weekly is posted every Sunday night! 1. New improved CRISPR-based genome editing technique A week before announcement of Nobel prizes. Excitement about potential Nobel for CRISPR genome editing is in the air. Meantime, […]

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A protocol for manufacturing of GMP-compliant iPS cell lines

by Alexey Bersenev September 26, 2015 cell product

Yesterday, Stem Cell Reports published “must read” paper, which describes manufacturing of GMP-grade iPS cell line for potential clinical use. We saw a few very similar paper titles in in the past, but this one is special. Here is why: This is the first (as claimed by authors) fully GMP-compliant manufacturing protocol for generation of […]

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End of STAP story?

by Alexey Bersenev September 24, 2015 notes

Guess what? STAP cell phenomenon is not reproducible! STAP cells do not exist! Oh really? What a news, ha ha! Yes, this is “yet another STAP post”. You probably have heard today about 2 “STAP reproducibility” papers, published in Nature (paper 1/ paper 2), and all that buzz around it. From massive media coverage, read […]

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Cells Weekly – September 20, 2015

by Alexey Bersenev September 20, 2015 notes

Cells Weekly is a digest of the most interesting news and events in stem cell research, cell therapy and regenerative medicine. Cells Weekly is posted every Sunday night! 1. The first commercial “stem cell drug” approved in Japan Two days ago, Australian company Mesoblast jointly with Japanese company JCR Pharmaceuticals announced “full approval” of Temcell […]

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Introducing Cell & Gene Therapy Insights

by Alexey Bersenev September 18, 2015 notes

Dear readers, I’m happy to tell you that new journal – Cell & Gene Therapy Insights was officially launched! To my knowledge, this is the first OPEN ACCESS journal in cell therapy field! You have to register to read everything for free. Content is licensed as CC 4.0. I’m very excited, because it is not […]

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The first iPS cell clinical trial insights

by Alexey Bersenev September 17, 2015 cell product

“For an hour on Friday 12 September, Masayo Takahashi sat alone, calmly reflecting on the decade of research that had led up to this moment”. (David Cyranoski, Nature doi:10.1038/516311a)   A year ago, the first historic transplantation of iPS cell-based product took place in Japan. Recently, the trial was halted due to RIKEN’s decision on […]

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