• Cryopreservation of mesenchymal stromal cells can attenuate clinical immune effects
    As Jacques Galipeau reported in conferences and in the paper, cryopreservation could negatively affect therapeutic “immunomodulatory value” of mesenchymal stromal cells (MSC). There was no independent confirmation of Galipeau’s findings, and many MSC product developers remained skeptical. This week, Katarina Le Blanc published a report, which supports Galipeau’s conclusions and provides more insight into potential clinical value of this phenomenon. Let me just say – this paper could change the field! Le Blanc concluded that freeze-thawed human MSC compared to […]
  • “Cancer bad luck” study from stem cell perspective

    by Alexey Bersenev on January 27, 2015 · 0 comments

    in cancer

    You may have heard a big buzz around the recent theoretical cancer study, which is trying to explain why probability of malignancies from some tissue much greater from the others. And the reason is a number of stem cell divisions in particular tissue. Study made very provocative claims and triggered huge wave of discussions about its methodology and interpretation. Significant part of discussions on the blogs was about statistical methods and interpretation. You can read some good posts here, here, here, here and here. It also caused very good discussion on PubMed Commons. However, it seem to me, not much attention was paid to author’s strategy to calculate the stem cells number and their divisions in every analyzed tissue. I’d like to bring your attention to few posts, which touched potential problems with stem cell frequency calculations in different tissues. I’m in agreement with these comments:

    from Knopfler Lab Stem Cell Blog:

    The most difficult issue is how to consistently and accurately calculate the number of lifetime stem cell divisions in a series of diverse tissues. This is extremely difficult and it’s not difficult to imagine such calculations being off by one or more orders of magnitude.
    Another factor is that not all cancers are going to originate with stem cells. Some tumors arise from progenitor cells or from differentiated cells that de-differentiate.
    Further, many organs have more than one type of stem or precursor-like population. How does one handle that in modeling?

    from the Signals blog:

    It is also worth mentioning that the majority of the data used in the estimation of the total lifetime stem cell divisions are largely derived from mouse models. In the current paper, the authors identify that mouse and human intestinal tissues are not equivocal (supplementary data); so then, why are mouse data used in the other calculations? The data that are of human origin are typically from in vitro (in a dish) or mathematical models. While animal, cellular, and computer modeling are acceptable for basic sciences research, applying those conclusions to whole-body human beings is a little difficult (and not good science).

    from PubPeer:

    “Acute myeloid leukemia” section, they described the reports claiming that the cell division cycles of the stem cells are 15, 17, 30, 57 days and the author of the current manuscript approximated it as 30 days. However, the reference number (38) is from the experiment of mouse, not human. For (37) I am not sure but as carcinogen BrdU was used, it should not have been a human experiment. Since the cell cycle of mouse is ~ 2 times faster than that of human, it violated the approximation with growth and is also be able to explain an apparent power law of the model. It is also notable that more than 3-fold differences in the reported cell cycles violate the approximation in this model.

    To illustrate how author’s calculations of stem cell number and divisions could be flawed, I’d like to cite “acute myeloid leukemia” part from supplementary materials:

    The lifetime incidence of acute myeloid leukemia is 0.41% (www.seer.cancer.gov) (3). There are a total of ~3·1012 blood cells and 7.5·1011 nucleated cells in the bone marrow (32). 0.18% of these normal bone marrow cells are CD34+ (33). The CD34+ are a heterogeneous population of cells and only about 10% of the CD34+ are hematopoietic stem cells (HSC), phenotypically defined as (Lin−CD34+CD38−CD90+CD45RA−) (34). The number of hematopoietic stem cells therefore equals 7.5·1011· 0.0018 · 0.1 = 1.35·108. Hematopoietic stem cells have ben estimated to divide every ~15 (35), 17 (36), 30 (37,38), 57 (39) days. Thus, we assume they divide every 30 days.

    First, hematopoietic stem cells within CD34+ is defined population, however there are other HSC populations, which are CD34-. Second, they picked Weissman’s group 2011 study, to define phenotype of human HSC. It’s not very clear why this study was picked, if more recently Dick’s group showed that human HSCs could be either CD90+ or CD90-. Why don’t use the most recent and accurate study? Third, all cited studies, which calculate HSC divisions rate, were done on mice. How authors can extrapolate it to human? They took 4 numbers from mouse studies and simply calculated average (15+17+30+57)/4 = 30. Assumption = mouse + mouse + mouse + mouse/4 = human. The authors did bad work with literature. They can find very nice 2011 simulation study by Sandra Catlin, which estimate a rate of human HSC replication as 1 time in 40 weeks. Now compare: every 30 days versus every 40 weeks.

    This is just an example from AML. If you look at other tissues, you can find comments about the same issues. For example, a comment from PubPeer about testicular cancer:

    The estimation for the number of stem cell divisions is, in my opinion, highly speculative.

    Looking at how testicular stem cell divisions were calculated (my particular field of knowledge), there are so many assumptions being made, that I wouldn’t be surprised if the calculation was off by many orders of magnitude. I wouldn’t be surprised if the calculations for other tissues was subject to similar margins of error.

    Some of the dubious assumptions:
    1) A-single spermatogonia are the stem cells of the mouse, which is contested; it is even unclear whether there is a fixed, deterministic pool of stem cells present.
    2) Human spermatogenesis is essentially a scaled-up version of murine spermatogenesis, for which there is no data;
    3)The number of lifetime stem cell divisions can be deduced from the number of total sperm produced in a lifetime

    Overall, the authors are making a number of assumptions, which are not reflecting reality:

    1. Data on tissue adult stem cell number in human are available and valid. Not true! There are very limited data on number of defined stem cell populations in adult human tissues. Most of these studies are hypothetical and their validity is unknown. That’s why, for example, the authors were not able to find good studies on such frequent cancers as breast and prostate.
    2. There is only one well defined stem cell population in human adult tissues. Not true! There are different stem cell populations within tissues. For example, for hematopoietic tissue I cited a few above. Markers for some of them are overlapping, for some are unique. The question – which one to pick for calculation?
    3. There is only one valid study, describing well defined adult stem cell population. Not true! There are many studies from different groups, most of them are conflicting! The question – which one to pick?
    4. Mouse = human. Not true! Stem cell calculations for mouse cannot be used for math models in human.
    5. Only divisions of stem cells responsible for carcinogenesis. Not true! It is well known that progenitor cells are more rapidly dividing then adult stem cells. Due to rapid division history, some progenitor populations in some tissue are more prone to accumulation of mutations and carcinogenesis. Both differentiated cells and progenitor cells could acquire self-renewal via mutations and become cancer stem cells. Most adult stem cells are quiescent. Stem cell quiescence could be dynamic and functions as a “safe harbor” from exhaustion and “acquisition of multiple mutations”.

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    Cells Weekly – January 25, 2015

    by Alexey Bersenev on January 26, 2015 · 0 comments

    in 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!

    CD34+Banner_490x90

    1. Stem cell scientist responds to criticism from post-publication peer review site
    I’ve mentioned many times a great post-publication peer review platform PubPeer. Recently, after STAP scandal, stem cell topic was heated on PubPeer again. One of highly criticized stem cell scientists was Jacob Hanna. He recently responded to criticism from PubPeer. Interestingly, his response was not just a comment on site, but well written lengthy explanations to discussions about few papers. You can find it here. He also responded to critical piece, posted on pre-print not peer-reviewed repository bioRxIV.
    This case sets one more great example of how post-publication peer review could work to science self-correction. He admitted mistakes and he is willing to discuss it and correct it! I couldn’t be more happy about such new tool as PubPeer, which makes science much better!

    2. Hematopoietic cell transplantation in multiple sclerosis – results of clinical trial
    Results of clinical trial, assessing auto- hematopoietic cells in patients with multiple sclerosis, were published this week. The authors analyzed outcome of 145 patients, with median follow-up of 2 years. Disability scale significantly improved in about half of the patients. As it was shown before, this risky cell therapy works well in relapsing remitting form of multiple sclerosis, but useless in secondary progressive (neurodegenerative) form.

    3. Overview of parabiosis model in aging research
    Nature posted a big article on history of parabiosis as a model in aging research. It nicely overviews and citing all major research groups, which use this model to study aging. The article also mentions the first clinical trial, which aims to assess impact of “young blood” on cognitive function of patients with Alzheimer’s disease:

    The initial Alkahest study is expected to conclude by the end of this year, and the company plans to initiate further studies testing young plasma in the treatment of different types of dementia and age-related conditions.

    4. Impact of FDA regulation on cord blood banks
    Very interesting article about FDA regulation of cord blood transplantation and banks you can find in the last issue of Parent’s Guide to Cord Blood Foundation newsletter. Mahendra Rao overviews:

    Ironically, while the FDA is now regulating unrelated cord blood transplants as a drug, unrelated bone marrow transplants are continuing to operate outside this regulation. Obviously this is creating a disproportionate burden on the providers of cord blood transplants. Moreover, public cord blood banks can only release transplants for the specific indications that have already received FDA approval; any transplants for novel indications can only be performed under a clinical trial (ie: after the trial sponsor files an IND application).
    The new FDA regulations increase the financial burdens on public cord blood banks, limit their practices, and necessitate a re-examination of their business models. The public cord blood banks have a larger cost burden than providers of bone marrow transplants and have to limit the use of their samples.

    5. Adipose MSC survive cryopreservation better
    Interesting post about impact of cryopreservatiton on different sources of mesenchymal stromal cells (MSC) you can find on SamplingScience blog:

    The researchers examined the effect of cryopreservation on mesenchymal (i.e. non-hematopoietic) stem cells originating from rat adipose, bone marrow and dental pulp. They isolated those tissues from six weeks old Wistar Han rats and cryopreserved cells grown from these tissues using a simple freezing protocol and 10% dimethylsulfoxide as cryoprotectant. Subsequently, the scientists compared viability and expression of a number of genes and surface markers from cryopreserved cells with cells that had not been frozen.

    6. Building organs on chips – profile of Don Ingber
    I’d recommend you the article about scientific path of Don Ingber – director of Wyss Institute. He and his institution is pioneering a new promosing technology – organs on chips.

    7. New methods and protocols
    New mouse model for human hematopoietic stem cell engraftment does not require irradiation (Stem Cell Reports)
    Asymmetric parental genome engineering by Cas9 (Sci Reports)
    Generation of chimeras from mouse ES and iPS cells through injection or coculture of embryos (Stem Cells Int)
    Osteoinduction by controlled release of BMP-2 from bioprinted construct (PLoS ONE)
    Computational analysis of pluripotency in human and mouse stem cells (Sci Reports)
    Direct reprogramming of human adult somatic cells into neural stem cells by HMGA2/let-7b (Cell Reports)
    Impact of AAV vector design on hepatotoxicity in gene therapy (JCI)

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    A new study by Jana Luetzkendorf, published in the last issue of Cytotherapy, argues against potential negative impact of cryopreservation on characteristics of human mesenchymal stromal cells (MSC). Previously, two studies – by Galipeau’s and Le Blanc’s groups, demonstrated potential alteration of immunomodulatory properties of MSC. Later, Copland and Galipeau published more mechanistically detailed study. These 3 studies have triggered lively discussions among professionals about potential negative impact of cryopreservation on MSC function.

    The “main feature” of Luetzkendorf’s study is GMP manufacturing of MSC in xenogen-free conditions. This is the first study, which assess impact of cryopreservation on GMP-grade MSC, manufactured in xeno-free conditions. They proposed that the use of animal serum (FBS) is a potential explanation for “negative results” of 3 studies, mentioned above. It could be very interesting hypothesis to test, indeed. Unfortunately, it was not tested in the study, because they did not have FBS control. So, this explanation remain speculative.

    The authors showed that cryopreservation does not alter the following characteristics of GMP-grade MSC: viability, surface phenotype, tri-lineage differentiation, immunomodulating potential and immunogenicity. I’d like to note that immune- assays were done in vitro, so we don’t know how it will be reflected in vivo. Ideally, it would be great to check biodistribution and therapeutic effects of fresh versus cryo- MSC in normal and “diseased” mice.

    What important in this discussion is figure out a reason why there are some differences between groups. In order to do so, one may reproduce Galipeau’s experiments first – culture and freeze MSC in the same way and perform the same tests in vitro (IFN-γ induction of IDO) and in vivo (biodistribution). Then, start to change conditions – remove FBS, manufacture in GMP and so on. Importantly, comparison should be done on the same passage or PDL number.

    Since Luetzkendorf’s study was performend on low passages (3-4) and PDL 6.4-14 with 20-27 days in culture and cell number max 180 millions, it is not really reflecting of industrial manufacturing of allo- MSCs. Alterations of immunomodulatory or other MSC functions (which could not be detected in some in vitro assays) more likely to be expected in higher expansion rates. It will be great to compare how cryopreservation will affect GMP-grade MSC at 10, 20 and 40 PDL with or without FBS.

    Overall, I think, we still far from the definitive answer. It could be more complex than we think. A lot of different factors could play role: cell culture conditions (FBS vs platelet lysate, 10 vs 40 PDL, normoxia vs hypoxia…), specificity of potency assays (inhibition of T-cells vs. IFN-γ/IDO), underlying disease (MSC from donor #3 – older AML patient – had impaired expansion and viability in Luetzkendorf’s study). Taking in account this uncertainty, the right thing to do is to focus on your particular trial – test cryopreservation impact on MSC from your patients, based on developed good potency assay.

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    How to support StemCellAssays

    by Alexey Bersenev on January 19, 2015 · 1 comment

    in notes

    Dear readers, Today I’d like to highlight how important your feedback and how to support this blog. StemCellAssays exists only because you keep reading it and giving a feedback. The main reason for me to keep going with a blog is growing number of readers and growing demand for high quality reliable information in the field. Since 2009, when blog was co-founded by me and William Gunn, we had a number of people who contributed to the project in different ways – guest authors, editors, web-designers, advertisers and sponsors. That’s why I like to say “we” and “us”. StemCellAssays was created as community project and I’m trying very hard to keep it this way. The sense of community, however, is forming in discussions and in participation. I’m very very hopeful about increasing “community participation” in the future.

    Now, I’d like to highlight a number of ways to help and support us:

    Give us a feedback
    Feedback is the most important factor in making StemCellAssays better. It will help us to (1) understand the demand for information, (2) filter relevant information together, correct mistakes and make it reliable, (3) improve the blog (content- and design-wise), (4) learn new things about the field and keep it up-to-date.
    How to do it – (1) write a comment under the post, (2) use contact form for any question or suggestion, (3) send a link, suggestion and comment via twitter, (4) send me email or direct message on LinkedIn/ twitter if you are in my contacts.

    Spread the word about us
    If you like our content, why don’t spread the word? Help us to gain more readers and potential contributors!
    How to do it – (1) tell your colleague next to your lab bench/ office space about us, (2) email/ forward our posts to your colleagues, (3) use information from our blog (with a link to us) in your presentations, (4) print a post and hand it over to your peer.

    Become an author
    We are always looking for highly enthusiastic experts in the field of stem cell research/ cell therapy/ regenerative medicine! We would like to invite you to contribute to our community by becoming an author. My position is that content could be high quality and reliable only if it’s written by dedicated expert. I have to admit, I’m not an expert in all topics that I’m writing about here. So, I’m always hungry for expert’s opinion.
    How to do it – If you’re know very well particular relevant topic and have an interest in writing something about it, please let me know. I’m always looking for authors! If you would write more than few posts, I may grant you access to administration and you can upload posts yourself. Since I’m getting more into cell therapy manufacturing and translation, I’d like to find contributing experts in: (1) basic stem cell research (with good knowledge of cell/ molecular biology) – especially pluripotent stem cells, (2) biomaterials/ bioprinting/ tissue engineering, (3) all aspects of cell culture (especially media and supplements) and cryopreservation, (4) gene transfer and genome editing.

    Share on social media
    If you like what you read, please share it with your peers by simple clicking on social media icons at the beginning of the post. Also, share information from the blog on social media or email it if you use RSS reader. I’d like to emphasize that one of the simplest but most “impactful” ways to support us is to retweet!

    Donate
    Good and effective way to support your favorite source of information – donate some money!
    How to do it – click on a button:




    How I’d use donated money – (1) invest in better site design and functionality, (2) pay experts to write a critical opinions essays, (3) attend conferences and cover it on a blog/ twitter.

    Advertise with us
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    You can support particular series on our blog as a sponsor!

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    Cells Weekly – January 18, 2015

    by Alexey Bersenev January 18, 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 “tissue fitness gene” impacts aging and regeneration Cell competition for the niche and “cellular fitness” is an important mechanism in tissue regeneration, aging and carcinogenesis. New gene, which is responsible for elimination of unfit cells, called Azot, was recently described in Drosophila by Eduardo Moreno’s group: We find that fitness-based […]

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    Breaking down fat: Comparison of devices for SVF isolation

    by Alexey Bersenev January 14, 2015 adipose

    For the routine clinical use of adipose tissue-derived stromal vascular fraction (SVF), automated commercial processing devices should be considered. These devices allow to save a lot of time, money, decrease patient-to-patient and technician-to-technician variability, improve: safety of the procedure and quality of final SVF product. There is a variety of SVF devices worldwide in different stages of development and regulatory status. If you are trying to choose the best SVF processing device, you should consider the following paramenters: Device availability […]

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    Cells Weekly – January 11, 2015

    by Alexey Bersenev January 11, 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. Advance in tissue engineering of small intestine Researchers from Childrens Hospital Los Angeles created tissue engineered functional mouse and human small intestine. They modified previous techniques and based their method on organoid culture. Human organ was transplanted into immunodeficient mice and harvested 4 weeks after for analysis. Morphological studies confirmed digestive […]

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    Lecture: Jordan Miller – 3D printing of vascularized tissue

    by Alexey Bersenev January 10, 2015 lectures

    Jordan Miller is a Professor at the Rice University. His lab is specializing on creation of artificial vascular networks for tissue biofabrication. His recent essay – The Billion Cell Construct made some buzz and facilitate discussion on challenges for organ biofabrication. The video was posted by NIH 3D Print Exchange.

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    More on tissue clearing techniques

    by Alexey Bersenev January 7, 2015 methods

    I’ve written about CLARITY tissue clearing techniques last year. What I did not mention is that there are so many of other tissue clearing techniques right now. The recent advances in these techniques allow to visualize spacial relationships between cell networks inside of “unsectioned” whole organ or even whole body. This is entirely different new approach to understanding normal development and pathology on cellular and tissue level in 3D. That’s why there is a great interest to it among researchers. […]

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    Cells Weekly – January 4, 2015

    by Alexey Bersenev January 4, 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. Results of the year 2014 In the last couple of weeks I’ve written many posts, summarizing events of 2014. If you missed it earlier, here is the list: People who mattered in 2014 2014 Year in review: Methods 2014 Year in review: Stem Cell Discoveries RegenMed, stem cell and cell therapy […]

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