• 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 […]
  • Cells Weekly – June 28, 2015

    by Alexey Bersenev on June 29, 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!

    T-Cell-Expansion-Banner-550x80

    This week was a week of ISSCR 2015, so read all news from the conference here.

    1. The price of gene therapy trial failure
    A year ago, Celladon became the first gene therapy company, which received designation by US FDA “Breakthrough Therapy” for their MYDICAR platform in heart failure. It means very good data from Phase 1 trial and impressive efficacy in early Phase 2. Despite the recognition and all excitement, a year later, MYDICAR failed to deliver efficacy as result of Phase 2. The fate of the company was unclear.
    This week, the company warned their investor about potential termination – suspension of MYDICAR and other pre-clinical programs, sale/ merger of the company or liquidation:

    We are aggressively pursuing that course,” said Paul Cleveland, president and chief executive officer of Celladon. “If we are unable to identify a merger or sale that provides superior value to our shareholders, we will move forward with a liquidation and distribution of net cash to shareholders.”
    The Company also announced a second reduction in its workforce, with approximately half of the employees not previously notified of termination of employment being expected to depart in the third quarter.

    Very bad news for the field! Many lessons to learn…

    2. Heart peacemaker activity turned on by light
    The most interesting study from this week came from 2 Israeli scientists. They were able to modulate cardiac pacing in vivo, using optogenetic approach. AVV vector with light-sensitive protein transgene was injected directly into myocardium:

    This allowed optogenetic pacing of the hearts at different beating frequencies with blue-light illumination both in vivo and in isolated perfused hearts. Optical mapping confirmed that the source of the new pacemaker activity was the site of ChR2 transgene delivery. Notably, diffuse illumination of hearts where the ChR2 transgene was delivered to several ventricular sites resulted in electrical synchronization…

    This is fantastic! The author Lior Gepstein says:

    “Our work is the first to suggest a non-electrical approach to cardiac resynchronization therapy,” Gepstein said. “Before this, there have been a number of elegant gene therapy and cell therapy approaches for generating biological pacemakers that can pace the heart from a single spot. However it was impossible to use such approaches to activate the heart simultaneously from a number of sites for resynchronization therapy.”

    Read one more recent cool optogenetics study here.

    3. More on US Congress blockade of embryo editing
    Sara Reardon of Nature wrote very interesting piece on the recent move by US Congress to block embryo editing technologies on federal level:

    Its fiscal year 2016 spending bill for the US Food and Drug Administration (FDA) would prohibit the agency from spending money to evaluate research or clinical applications for such products.
    In an unusual twist, the bill — introduced on 17 June — would also direct the FDA to create a committee that includes religious experts to review a forthcoming report from the US Institute of Medicine (IOM).
    Privately funded research on editing the human germline remains legal in the United States.

    If this will happen… well, it will be too bad for US. Prohibition splash may affect in vitro and animal experiments and other than CRISPR tools for genome editing. Very reminiscent of embryonic stem cell paranoia. Did US benefit from prohibition of federal funding for embryonic stem cell research?

    “This step seems dumb — or ill-advised,” says Hank Greely, a bioethicist at Stanford University in California. It might also be premature because the FDA has not shown any indication that it would approve such research. And such a ban would not apply to the type of research that the Chinese scientists performed, because the embryos they used were not viable.

    4. Dynamics of heart regeneration
    A group of researchers from Karolinska Institute investigated a dynamics of heart cell numbers and turnover throughout human life:

    We report that the full complement of cardiomyocytes is established perinataly and remains stable over the human lifespan, whereas the numbers of both endothelial and mesenchymal cells increase substantially from birth to early adulthood. Analysis of the integration of nuclear bomb test-derived 14C revealed a high turnover rate of endothelial cells throughout life (>15% per year) and more limited renewal of mesenchymal cells (<4% per year in adulthood). Cardiomyocyte exchange is highest in early childhood and decreases gradually throughout life to <1% per year in adulthood…

    Yet another study, published in Nature, describes a possible candidate (in mice) for these <1% of cycling cardiomyocytes:

    …we identify a rare population of hypoxic cardiomyocytes that display characteristics of proliferative neonatal cardiomyocytes, such as smaller size, mononucleation and lower oxidative DNA damage. Notably, these hypoxic cardiomyocytes contributed widely to new cardiomyocyte formation in the adult heart.

    5. Cancer cells hijack normal niche via exosomes
    Very interesting study appeared this week in online version of Blood. Researchers demonstrated the mechanisms of hijacking normal stromal cells by cancer cells, on a leukemia model:

    We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts. As a result, stromal cells show enhanced proliferation, migration and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment.

    Tumor-released exosomes are bad guys!

    6. Model of neural connectivity in vitro
    For the first time, researchers were able to model connectivity, between 2 groups of iPS cell-derived neurons (neocortex and midbrain) in vitro. Read study here:

    We describe a technique for independently differentiating neocortical and mesencephalic dopaminergic (mDA) neurons from a single human pluripotent stem cell (hPSC) line, and subsequently allowing the two cell types to interact and form connections.

    Blogs coverage here and here.

    7. Apligraf versus Epifix for diabetic ulcers
    We are lacking of studies, which compare competing commercial cell regenerative products. One of such studies was recently published. Apligraf (made by Organogenesis) was compared with Epifix (MiMedx):

    Using a wound-care specific electronic medical record database we assessed real-world outcomes in 218 patients with 226 DFUs receiving treatment in 2014 at 99 wound care centers.
    Treatment with a bioengineered living cellular technology increased the probability of healing by 97% compared with a dehydrated amniotic membrane (hazard ratio = 1.97 [95% confidence interval 1.17, 3.33], p=0.01).

    From Organogenesis PR:

    Product efficacy in controlled clinical trials may not equate to effectiveness in general clinical practice, which is why these types of analyses are so valuable,” said lead study author Robert S. Kirsner..

    MiMedx did not have any PR…

    8. Adipose stromal vascular fraction as good as PRP for facial scars
    It was the most interesting clinical study of the week. Turns out that stromal vascular fraction (SVF)-enhanced fat grafting is much better that fat grafting only, but not better than platelet-rich-plasma (PRP)-enhanced fat grafting:

    In the patients treated with SVF-enhanced autologous fat grafts, we observed a 63% maintenance of contour restoring after 1 year compared with only 39% of the control group (n = 10) treated with centrifuged fat graft (P < 0.0001). In the patients treated with fat grafting and PRP, we observed a 69% maintenance of contour restoring after 1 year compared with that of the control group (n = 10).

    So, why bother with SVF? Seem like PRP can do it and it’s cheaper.

    9. How “stem cell business” can hurt patient needs
    Very interesting post appeared recently on California Stem Cell Report blog. Post covers old story about failed attempts to commercialize few technologies, developed by Stanford’s stem cell researcher Irving Weissman:

    “‘As long we have a health care system that puts profits before patients we will always be at the mercy of corporations looking to make profits. Any new innovation needs to be evidence-based and proven safe and effective no matter what money is to or is not to be made,’ she said.”
    Krieger wrote,
    “Now, a quarter-century after it was conceived, the technique is finally back in Weissman’s hands at Stanford — although Novartis still holds the patent.”
    She reported that Weissman hopes to take the potential therapy forward in a non-profit setting.

    Very interesting read!

    10. Tips for tissue enzymatic digestion
    Conversant Bio has a nice post about best practices for enzymatic tissue dissociation:

    Researchers sometimes struggle to optimize the conditions for tissue dissociation and so see variations in their data. Most controllable variations can be traced to these factors: the dissociation medium utilized, enzyme(s) selection, impurities in crude enzyme preps, enzyme concentration, temperature and incubation time. These are all important parameters that need to be defined and optimized for your specific research project in order to achieve both high yields and high cell viability.

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    ISSCR 2015 roundup

    by Alexey Bersenev on June 27, 2015 · 0 comments

    in conferences

    Annual meeting of the International Society for Stem Cell Research (ISSCR) is one of the most important events in stem cell field. Whole week I was hunting for the news from ISSCR 2015. Unfortunately, the coverage was not as good as in previous years (for example look at 2013). Nevertheless, I’ve learned a bit from discussions on a twitter – thanks to few individuals, who were tweeting and blogging! Here is roundup of the most important stem cell conference of the year and my own impressions of “hungry for info” observer.

    Blogs coverage
    Young stem cell researcher Heather Main debuted as a blogger on Knoepfler Lab Stem Cell Blog. She wrote 3 great posts, covering (mostly) plenary talks at ISSCR:
    Overview of Yamanaka Talk at #ISSCR2015
    Carla Kim & Hans Clevers talks on organoids at #ISSCR2015
    Review of Biotech/Translational Talks #ISSCR2015
    I hope Heather will continue to blog! I’d expect some #ISSCR2015 follow-up posts from Paul Knoepfler in next few days (so this post will be updated!)

    My favorite blogger David Kent covers ISSCR on the Signals blog. One post so far, but more is coming on Monday!
    ISSCR 2015 Stockholm Day 1: RM – learn from nature’s masters…

    That’s all from blogs that I was able to find. Not much :(

    Tweets
    The trend did no change – very few individuals with valuable tweets and a lot of valueless noise from companies and corporate accounts. No progress here. No unpublished information at all. From all tweet, which you can read via hashtag #ISSCR2015, I’d like to recommend a few individuals, who did a great job:
    @TheJenMoody
    @mincle
    @samanthazy
    @AndyLas
    @Dev_journal

    follow them!

    Clinical updates from companies
    A few stem cell companies released updated clinical trials information at ISSCR 2015. First of all, I’d like to highlight Ocata -
    Ocata Therapeutics PR
    They presented results of treatment of 31 patients with longest follow-up 4 years and … all of them had positive response! 100%! Even thought it was subjective, I’m blown away by this announcement!

    The next is StemCells Inc PR. Unfortunately no numbers here, but not all patients wit AMD improved, many were just stable. You can compare 2 abstracts here.

    Launches
    Fluidigm launched new “magic device” – Callisto™. Read more -
    New tech tool speeds up stem cell research
    Fluidigm Revolutionizes In Vitro Modeling for Stem Cell Researchers and Cell Biologists
    Well, not exactly the first launch… and not only for stem cell research…

    Gibco launched Essential 8 Flex Medium for pluripotent stem cells -
    New Pluripotent Stem Cell Media Eliminates Need for Daily Feeding
    Lonza launched 3 new research products – L7™ Reprogramming and Culture System/ “CytoSMART™ Live Cell Imaging System and “RAFT™: A Bio-Mimetic 3D Culture Method for PSC Hepatocyte

    Announcements
    ISSCR announced a draft of new Guidelines for Stem Cell Science and Clinical Translation and waiting for your comments!
    New OA journal was just announced by NPG (not on ISSCR but for ISSCR crowd) – npj Regenerative Medicine. Three letters at the beginning of the journal’s title makes it different from existent Regenerative Medicine, but it sounds funny to me.

    That’s all folks! Not much, but hope to get more updates in the next few days. Please feel free to add any links and news that I’ve missed!

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    Isolation of mesenchymal stromal cells (MSC) from tissues by single step (FACS or MACS) is a good alternative to commonly used adherence-based method. It allows to purify more homogeneous rapidly proliferating population of MSCs. However, it involves use of antibodies and special equipment (sorter). Approaches to sorting MSCs for clinical use are commercialized by some companies (ex: Mesoblast and Orbsen Therapeutics). Despite results of multiple studies, it remains unclear what the best marker or combination of markers for sorting of MSC from particular human tissue. Good marker for bone marrow MSC could not work well for adipose tissue or umbilical cord blood. The recent study, which appears online on Stem Cells and Development journal web-site, investigates these differences between bone marrow (BM) and adipose tissue (AT).

    Researchers checked the following markers: MSCA-1, SUSD2, CD271, CD34 and CD44. All populations were isolated by MACS without attempts to increase purity (double sort, depletion of CD45+). Both positive and negative fractions were tested for CFU-F clonogenicity post-MACS. I really like this approach, because it shows how these population are pure for particular marker.

    The authors demonstrated that BM MSC can be MACS-sorted by MSCA-1, SUSD2 and CD271 markers with very similar recoveries (0.6-07%). Overall, enrichment by CFU-F colony assay for these 3 markers was ~30 fold, compare to unfractioned marrow. All 3 fractions were highly proliferative in culture with MSCA-1 as a best performer. MSCA-1 demonstrated the highest enrichment ~50-fold on average with max 70-fold and expansion rate of 20×109 after 2 passages (expansion of unselected cells was 100 times less). Purities of MSC, isolated by MACS were lower in relation to described purities by FACS. For example, for CD271 only ~40-50%.

    Contrary to BM, the authors were not able to enrich MSC from AT by MSCA-1 and SUSD2. Interestingly, CD271 was good marker for MSC, isolated from lipoaspirates, but no colonies were observed from CD271-selected MSC from resected abdominal fat tissue. Another interesting finding is that only CD34+ was highly selective marker for AT-derived MSC, because was no colonies detected in residual CD34- fraction. Selection by any other marker resulted in detection of CFU-F colonies in both – positive and negative fractions. CD90 and CD146 were significantly enriched in CD34+ and CD271+ fraction of AT, but not in the same fractions of BM.

    CD34+ MSC with great clonogenicity were also purified from BM. Therefore, CD34 was the only universal MSC marker for both BM and AT. The study confirms previously described phenomenon – rapid disappearance of CD34+ expanded MSC culture. Enrichment of CD34+ cells was detected in CD271+ fraction for both – BM and AT. Unlike BM, only proliferation of CD34+ MSC was greater than plated unfractioned cells.

    One thing is not clear to me – why didn’t they deplete CD45+ before CD34 selection? How can they exclude contamination by hematopoietic cells? CD45+ coexpression was very high on all fractions, isolated from BM.

    Overall, it is very interesting study. It highlights significant differences in MSC markers in different tissues in situ. The paper also discusses technical aspects of human MSC isolation by MACS. This study is highly recommended to all “MSC folks”!

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    As you may know, I’m trying to capture all stem cell products available on a market worldwide. I realized that there are two groups of such products – the first is the products, approved by governmental regulatory agencies (usually considered drugs) and the second is so-called self-launched not approved cell/ tissue products. Today, I’d like to discuss some issues, related to second group of commercial “stem cell products”.

    Examples of self-launched cell/tissue products:

    Common features of these products:

    1. Self-launched by companies. Means there is no marketing approval/ authorization by government agency.
    2. Self-proclaimed as compliant with FDA regulations. But their self-proclaimed compliance is proved to be wrong (by FDA letters) or very questionable.
    3. Product launch on a market and claim of regulatory compliance happen without consulting with FDA.
    4. FDA cracked down on some of these products, claiming that these are in fact 351 products (drugs).
    5. Self-proclaimed as “stem cell products”. Usually means: “contains alive mesenchymal stem cells”.
    6. Almost exclusively used in orthopedics (so-called “orthobiologics”) and for skin repair.
    7. Big companies on orthopedics market love to have them in portfolio (Stryker, NuVasive, Arthrex).
    8. Post-marketing studies on efficacy and cost-effectiveness are very limited.

    Regulatory compliance
    As I noted above, self-proclaimed regulatory compliance of these product turns out to be a false. In the last 5 years FDA inspected these companies and issued a number of quite similar letters. Some of these letters:

    These letters indicate to non-compliant marketing of the products, which misclassified by companies as 361 HCT/P, instead of drugs (351). Interestingly, most of these products stayed on a market (AlloStem, Ovation/ BIO4), despite FDA’s warnings.

    Business model
    Despite FDA early letters (from Parcell and AlloSource in 2011), the companies continued to do the same thing over and over again in 2013 (Osiris) and in 2014 (RTI Surgical). Very recent deal between Stryker and Osiris on transformation of Ovation into BIO4 is an example of continuing trend. Why didn’t they learn? Did they miss those FDA letters? Did they consult with FDA on classification of their products? Do they actually care at all about FDA’s “current thinking”? NO, NO and NO! It indicates that self-launch and self-claims are working well for a profit. It seem to me, their regulatory compliance people did not do homework or were asked to follow the scheme:
    - don’t consult with FDA!
    - self-launch it with fancy label “stem cell”!
    - pitch it to surgeons – they like to be innovative and “push an envelope”!
    - enjoy a ride $$$ until FDA will crack down on you.
    Why this business model is still thriving? Well, first of all, FDA may never come to you (they have limited resources) if you don’t kill any patient. Second, if FDA will sniff you, it may take 1-2 years before audit, months for report, 1 year for responses and so on. Third, companies may get away with it by pleasing or negotiating with FDA (Osiris did it for Grafix) – changing marketing language (Biomet removed all osteogenic cells/ MSC-related language from description of Cellentra as advertised earlier in 2012) , narrowing down therapeutic indications, promise to do some post-marketing studies. Another possible model is divesting a product (Osiris sold Osteocel to NuVasive), partnership with big players (Osiris – Stryker deal).

    I’d like to give one more example of “changing a marketing language” about products in order to become “pseudo-compliant”. Nutech widely advertised their product “Nucel” as containing amniotic (stem) cells along with amniotic membrane (examples here and here). However, watching FDA cracking down on some 361 products and reading FDA updates (see the first update of 2012 from FDA Tissue Reference Group), they decided to tune up language and removed a mention of “cells” from their web site. Compliance is done? Very simple fix – remove word “cells” from everywhere!

    Therapeutic efficacy
    There are very little good published clinical data available about efficacy of these products. If we look at orthobiologics, things are not so positive as one may think. For example, the recent report on efficacy of Osteocel (NuVasive) in matched cohort spinal fusion, was negative:

    This is the first non-industry sponsored study to analyze a matched cohort assessing the one-year arthrodesis rates associated with a non-structural MSC allograft in one and two-level ACDF procedures. There were no statistically significant differences in fusion rates between the two cohorts.

    The recent overview (highly recommended!) of commercially available cellular bone matrices in spinal surgery, came up with the following conclusions:

    Although CBMs appear to be safe for use as bone graft substitutes, their efficacy in spinal fusion surgery remains highly inconclusive. Large, nonindustry sponsored studies evaluating the efficacy of CBMs are required. Without results from such studies, surgeons must be made aware of the potential pitfalls of CBMs in spinal fusion surgery. With the currently available data, there is insufficient evidence to support the use of CBMs as bone graft substitutes in spinal fusion surgery.

    The situation with 361 “stem cell products” for skin defects is much better. For example, recent report about Grafix efficacy in diabetic foot ulcers.

    For physicians:
    I hope this post will help physicians to learn something about not approved cell/ tissue products, advertised as “stem cell products”. Please be informed and protect yourself! Try to be innovative, but equip yourself with a knowledge about the product! Do not always trust advertisement! Finally, very good advice you can also find here:

    Ask your friendly company representative to provide you with a letter from the company in which they state that the product on offer was discussed with the FDA and the FDA said that they agreed that the product was Section 361 compliant. If you really want to make them sweat, ask them if they have had a response from the Tissue Reference Group.

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    Cells Weekly – June 21, 2015

    by Alexey Bersenev June 21, 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. Genome editing-related issues – hearing in the Congress of USA Yes, the CRISPR debates have reach the Congress of US! Prominent researchers – Jennifer Doudna, Elizabeth McNally, Jeffrey Kahn and Victor Dzau testified on gene editing progress, risks and ethics at Congressional Hearing. Paul Knoepfler did nice coverage of the event: […]

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    Gadgets Review: Organ-on-chip companies

    by Alexey Bersenev June 18, 2015 gadgets

    Post updated on June 21, 2015 Organ-on-a-chip/ body-on-a-chip is a hottest topic in biomedical research. Despite infancy of the field and many translational challenges, we are witnessing a race for commercialization of these technologies. I’m sure you’ve heard multiple news about “academic institution X developed organ-on-a-chip Y and spinout company Z”. Today, I’m reviewing some of these companies. I was trying to focus on companies, which are actually making organ-on-chip “ready to go” (with cells in it), but not just […]

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    Cells Weekly – June 14, 2015

    by Alexey Bersenev June 15, 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. Australia to close a loophole in regulation of autologous stem cell therapy Australian regulatory agency – Therapeutic Goods Administration (TGA) proposed a new regulation of autologous stem cell therapies at the beginning of this year and posted it for public comments. As a response to proposal, the agency received 80 submissions […]

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    Impact of antibiotics on function of human cells in culture

    by Alexey Bersenev June 10, 2015 cell culture

    Recently, I came across very interesting study, which assessed impact of antibiotics on differentiation of human adipose-derived stem cells. Antibiotics is still commonly used ancillary material in clinical cell cultures. Interestingly, the authors noted that despite wide use of antibiotics in human cell culture, their impact on cell physiology function remains unknown: We accomplished a very extensive literature review and, surprisingly, only found a very small number of reports on this topic, and none of them in human. They tested […]

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    Cells Weekly – June 7, 2015

    by Alexey Bersenev June 7, 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! Don’t forget to apply for summer schools and courses! 1. New achievements in organ/ tissue decellularization – recellularization A few recent studies, demonstrated new advances in tissue decellularization as a basis for whole organ engineering. The first highly publicized study came from Harald Ott’s lab. For the first time, Ott’s lab decellularized […]

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    miRNA molecular switches for purification of pluripotent stem cell-derived progenitors

    by Alexey Bersenev June 4, 2015 methods

    Recently, Japanese researchers described a new methodology for purification of iPS/ES cell-derived tissue-specific progenitors by genetic markers. Unlike previous molecular beacon-based approaches, the authors designed so-called miRNA switches – synthetic modified mRNAs, which target tissue-specific miRNAs. First, they screen multiple cardiomyocyte-specific miRNAs and picked 3, which were able to purify target cell population with high efficiency. miR-208a was the best performer with purity >95% on different stages of cardiomyocytes differentiation in culture. The beauty of this study is in designing […]

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