• 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 […]
  • Recently I’ve been reading a post on investor’s web-site Seeking Alpha, entitled “Asterias Biotherapeutics’ Stem Cell Program Is Dead On Arrival“. It made me think – Aren’t most (if not all) divested cell products are “dead on arrival”? And if yes, what can we learn from it?

    Let’s look at the biggest shots in cell products divestment of the last 3 years:

      Now, I’d like to highlight some common features of these divested products:

      1. They were divested for a reason. The most frequent reason, mentioned in press releases is financial and strategic – too expensive to handle and changing the company’s focus. However, I truly believe that single most important untold reason is therapeutic under-performance.
      2. None of them were stellar therapeutically. Everyone was hoping for dramatic (significant, impressive, nearly curative – pick the right term) therapeutic improvements, but reality was very different.
      3. All of them were sort of pioneering. Read – first generation. Read – obsolete.
      4. All of them have very very long history of development (20+ years for Prochymal and Provenge)
      5. Most of them had a history of bumpy regulatory approval paths (FDA versus Geron/ Osiris/ Dendreon).

      So, there is nothing more important than therapeutic boost! If 3 out 5 of Geron’s trial patients would walk away from wheel chair in couple of months after “magic injection”, investors would “shower them by gold” and company would not drop the program. But none of 5 patients reported notable improvements. In cell therapy today our expectations set so high – we are looking for amazing therapeutic performance! Who in clear mind today would drop potentially curative product candidate with response rate 90% (yes, I’m talking about CART)? But if therapeutic effects are marginal (Provenge) or moderate (Prochymal) or response rate below 50%, companies may divest without looking back.

      If product cannot deliver great therapeutic performance, it should deliver great economics benefit. In other words – save a lot of money performing therapeutically as good as competitor. In order to achieve economics benefit, the cost of manufacturing should be low, logistics should be very simple and therapeutic effect should last very long. This is not the case for divested products, mentioned above. Manufacturing of Provenge is expensive and logistics is very complex. Prochymal required multiple infusions (up to 10 in GVHD), lasting short time.

      The last point, I’d like to make here is rapid obsolescence of pioneering cell products. In the current speed of technological innovations, long-term cell product development will collide with unavoidable obsolescence. Provenge and Prochymal are typical examples of such collision. They were stalled in development and at the moment of arrival they were already obsolete – dead on arrival. So, why “beat a dead horse” and try to resuscitate it? This field is fluid, let it go!

      So, what can we learn from examples of divestment? I’d put a few things for discussion:

      1. In the field with high demand for dramatic therapeutic boost, the products which able to deliver it should be picked and cherished.
      2. In such dynamic highly technological and innovative field as cell therapy, divestment of products is normal.
      3. There are only 2 real significant reasons for divestment of cell products – therapeutic under-performance and unjustified cost.
      4. If you’re spending 10/15+ years for cell product development you’re running into the risk of technological obsolescence.
      5. Frequently, there is no reason to pick up divested products and try to ask “dead horse to race”. Let it go, kill it. Learn from it and develop new amazing product!

      For those of you, who picked up divested products and trying to resuscitate it – good luck guys! I’d not be in your place.

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    As you may heard Yesterday (read good coverage by Nature and Science and the Scientist), David Glass from Novartis has challenged a significance of highly profiled “rejuvenating factor” – GDF11. In particular, the results of his study showed that some conclusions from famous Amy Wagers paper, may be wrong. Contrary to Wagers, Glass demonstrated that GDF11 actually increase with age and negatively impact muscle regeneration. In the heart of controversy – antibody!

    First, Glass repeated Wager’s experiment, using the same antibody. He demonstrated that the antibody was non-specific and cross-reacted with GDF11 analog – myostatin. Using specific antibody, researchers got different from Wagers group results. Wagers comment on Glass findings:

    Harvard stem cell biologist Amy Wagers, who led much of the original work, says the Novartis data on GDF11 levels are not persuasive. And although they “appear to conflict with” her group’s, “we are actually very excited to see the Novartis data,” she says. “We remain convinced that at least one form of GDF11 declines in blood with age and that maintaining GDF11 levels in an appropriate physiological range is essential for muscle health.”

    Wagers sticks by her data, noting that her group’s Science paper also found a drop in GDF11 with age using a different antibody that distinguished GDF11 from myostatin. And she says the Glass team’s injury experiment cannot be compared to hers because they used young animals and a dose of GDF11 three times higher. (Glass did this in part because he did not see any effect in old mice at the dose Wagers used.) The signaling pathway in which GDF11 lies “is notoriously dose-sensitive,” and low and high doses can have different or opposite effects, she says. Moreover, she says, the Novartis team’s muscle regeneration test was not comparable to hers—the Harvard team made the injury by freezing tissue, which is less likely than a toxin to kill muscle stem cells needed for regeneration.

    Wagers says new data from her group will show that “there is a very compelling biological explanation for the apparent discrepancies.”

    So, whose antibody is better? Antibody is the biggest possible reason for lack of reproducibility in biomedical research. Conclusions of many “hot” stem cell papers, solely based on use of antibodies. However, most researchers seem to be not bothered by comparative validation of antibodies quality. Why don’t researchers talk to each other? Write reviews, rank antibodies and freely share results of titrations/ validations! After all, Wagers and Glass go to the same bar to drink (theirs institutions just “across the road”), why not discuss antibodies before conduct experiments? Yet another issue is considerations for multiple isoforms or analogs of studied factor.

    Coming back to reproducibility of the muscle injury experiment. I noticed that different rGDF11 were used (from PeproTech vs. R&D) by both Wagers and Glass groups. Even though Wagers said that injury models were different (btw, supplement to Wagers paper says cardiotoxin was also used for muscle injury), GDF11 administration and time points of experiments were the same. Would muscle injury model dramatically change conclusions? I doubt it.

    Ok, we got one more interesting controversy here. The future excitement about “rejuvenating factor GDF11″ now will depend on new data from Wagers lab. It seem to me, that controversy could be easily resolved if researchers would agree to run parallel experiments with the same reagents. See examples of it here and here.

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    Cells Weekly – May 17, 2015

    by Alexey Bersenev on May 17, 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!

    Blog update:
    Summer schools and courses – bioprinting and regenerative medicine! Read more on Cell Education page!

    T-Cell-Expansion-Banner-550x80

    1. BigPharma giant is ditching stem cell therapy
    As a result of two failures of Athersys in Phase 2 trials, their partner Pfizer terminates its investment in stem cell therapy:

    Received notification from Pfizer Inc. that, following an internal portfolio review, it no longer intended to invest in the cell therapy program for ulcerative colitis and would terminate the 2009 license agreement between us, effective July 2015

    Bad news for the field!

    2. Prothromobogenic activity of mesenchymal stromal cells
    Highlight of the week – very interesting study on prevention of procoagulant activity of MSC. The authors identified tissue factor (triggers coagulation cascade) on the surface of MSC and tested their prothromobogenic properties in vitro and in myocardial infarction model. Importantly, they showed that co-administration of heparin can fix the problem:

    Heparin reduced MSC-associated thrombosis incorporating platelets and VWF within the microvasculature. Heparin-assisted therapeutic MSC delivery also reduced apoptosis in the infarct border zone at 24 hours, significantly improved infarct size, left ventricular ejection fraction, LV volumes, wall motion and attenuated histologic evidence of scar formation at six weeks post AMI. Heparin alone or heparin-assisted fibroblast control cell delivery had no such effect.

    3. Effective controlled elimination of therapeutic cells after administration
    So-called safety switches, most of which based on suicide genes, are aimed to eliminate “wayward therapeutic cells” after administration. The typical example of it is T-cell therapy for malignancies – on the one hand it could reconstitute immunity and protect from viral infections after hematopoietic cell transplant, but on the other hand it could cause GVHD or cytokine release syndrome. This week, results of the clinical trial, assessing caspase 9 suicide gene modified T-cells, were published in Blood:

    Four patients received a single AP1903 dose. CID infusion eliminated 85-95% of circulating CD3+CD19+ T cells within 30 minutes, with no recurrence of GvHD within 90 days. In one patient, symptoms and signs of GvHD-associated cytokine release syndrome (CRS-hyperpyrexia, high levels of pro-inflammatory cytokines and rash) resolved within 2 hours of AP1903 infusion.

    30 minutes and all cells are gone! Impressive! The same approach could be used for pluripotent stem cell-based therapies.

    4. Interview with Hans Clevers
    Hans Clevers is a pioneer in developing of gastro-intestinal organoids techniques. Based on his study, published last week, Nature did an interview with him:

    Organoids model the key component of the tumour but they lack some important elements. We want to combine organoids with other elements to make more-complete tools. For instance, we would like to introduce the immune system so that we can study the effects of the fantastic new immunotherapy drugs. We think that we can build it up in a reductionist way — take lymphocytes isolated from a tumour, bring these together with cancer organoids derived from the same tumour and watch what happens. And maybe we can also put microorganisms in these organoids.

    5. Difference between bone marrow- and adipose tissue-derived MSCs
    Democratizing Cell Technologies blog has a very interesting post (even a study!) about differences between bone marrow and adipose tissue-derived MSCs:

    Our results underscore the need to characterize each population of hMSC, regardless of tissue source, and use their unique functional properties to choose the cell population best suited to each therapy or application. While the results presented here demonstrate differences between hAD- and hBM-MSC manufactured using RoosterBio expansion media and our protocols, it’s important to note that differences may, at least in part, be due to inherent donor-to-donor variability in addition to the tissue source.

    6. New methods and protocols:
    Injectable hydrogel improves thesSurvival and integration of stem cell in retina and brain (Stem Cell Reports)
    Osteogenic embryoid body-derived devitalized material induces bone formation in vivo (Sci Reports)
    Decell cartilage for osteochondral tissue engineering (PLoS ONE)
    Decell and recell lungs in dynamic suspension culture (PLoS ONE)
    CRISPR/Cas9-mediated generation of knockin hPSC reporter cell lines (Cell Reports)
    Decell – recell of whole liver (J Clin Exp Hepatol)
    Consensus comparative analysis of human ES-derived cardiomyocytes (PLoS ONE)
    Scalable 96-well plate based iPSC culture using robotics (JoVE)
    Isolation of human adult olfactory sphere cells as a cell source of neural progenitors (Stem Cell Res)
    Systems mapping for hematopoietic progenitor cell heterogeneity (PLoS ONE)
    Urine-derived stem cells for bone regeneration (PLoS ONE)

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    Whole organ bioengineering by recellularization of decellularized matrix is a new promising technology, which aims to address the problem of organs shortage in transplantation. Because bioengineered organ is maintained in culture as a whole rather than cell suspension, one may ask about daily cell viability assessment in these settings. Harald Ott’s group from Harvard, recently proposed to use colorimetric Resazurin assay for testing cell viability of bioengineered organs ex vivo.

    A reliable colorimetric or fluorometric assay for repetitive quantification of viable cell numbers in large-size, three-dimensional bioengineered organ constructs needs to meet the following criteria. First, the diffusion of substrate dye and its metabolized product into and out of bioengineered constructs needs to be efficient, which ensures that the sampling from culture medium is representative of the composition of substrate dye and its metabolized product inside the constructs. Second, the reagents need to have minimal cytotoxicity during the assay period and need to be efficiently washed out after a measurement, thus enabling repetitive measurement during the entire culture period of bioengineered organ constructs.

    Resazurin is metabolized by live cells and can be detected by fluorescent product-metabolite in the culture medium. Reduction of resazurin with time is proportional to cell number changes. For assay validation, it should be compared with standard direct cell counting after tissue dissociation. For 3D tissue engineered constructs, the authors proposed resazurin reduction perfusion assay:

    … first, the resazurin-based PrestoBlue reagent with low cytotoxicity, efficient metabolism and diffusion allows repetitive cell number quantification; second, resazurin delivery by means of perfusion through the organs’ own vascular bed greatly decreases diffusion distances allowing efficient and homogeneous distribution of the colorimetric and fluorometric reagent throughout tissue-engineered constructs.

    Perfusion allowed good equilibration of dye in the organ. Researchers compared this method on 2 different endothelial cell types, seeded on rat decell lung biomatrix.

    Viability estimates in the study were very close to histological assessment. The authors think that organ perfusion method could be used for other reagents. Overall, this method allows long-term precise daily estimation of cell viability in 3D cultured tissues/ organs, including large volume bioreactors.

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    Cells Weekly – May 10, 2015

    by Alexey Bersenev May 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. Cancer organoids biobank for personalized therapy The study, published this week in Cell, offers a new approach for development of highly personalized cancer treatment. Hans Clevers group described a method of concurrent derivation of normal and malignant organoids from the patients with colorectal carcinoma. Importantly, “organoids are amenable to high-throughput drug […]

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    Breaking down fat: Quality control and release criteria for SVF-based products

    by Alexey Bersenev May 9, 2015 adipose

    Since FDA views adipose-derived stromal vascular fraction (SVF) as a “drug”, it is important to discuss quality control testing. Even if you will operate under different jurisdiction, where SVF is not a drug, you want to make sure that quality of fat tissue processing device output will not possess any risks to your patients. So, before get started with patients, you better validate the device by measuring quality its output (SVF). Under FDA jurisdiction, you should establish quality control testing […]

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    Cells Weekly – May 3, 2015

    by Alexey Bersenev May 3, 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. More on human germline gene modification Seem like it will be discussion of the year! Professional organizations called for moratorium! Ethics splits scientific community. And, yes – “new Asilomar” is not probably a solution. Here is my pick of the coverage from this week: Embryo editing sparks epic debate (Nature News) […]

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    Dissecting effects of bone marrow mononuclear cell populations in cardiac cell therapy

    by Alexey Bersenev April 30, 2015 cell product

    There are many ongoing cardiac cell therapy trials right now. Most of them assessing different bone marrow cell types. Total bone marrow mononuclear cell fraction (MNC), sorted fresh CD34+ or CD133+ cells and cultured mesenchymal stromal cells (MSC) are evaluated for therapeutic efficacy after injection into affected myocardium. The biggest question remain here – what is the best bone marrow-derived cell type for cardiac diseases therapy? As of today, nobody can figure it out. Historically, many trials with total autologous […]

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    Cells Weekly – April 26, 2015

    by Alexey Bersenev April 26, 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. Opinions on human germline genome editing The discussion of the week is genome modification of human germline. A new wave of debate was triggered by recent study, which demonstrated possibility of genome editing on human embryos. Actually Nature News pulled the trigger, but not paper itself. Here I collected some interesting […]

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    Genetic editing of human embryos

    by Alexey Bersenev April 22, 2015 gene transfer

    As you may have heard today from Nature News, Chinese researchers recently reported the first data on using CRISPR/Cas9 genome editing technique in human embryos. You can read study here (open access). The authors used “defective” tripronuclear zygotes, left over from IVF procedures. The study is short, but with a clear message – CRISPR/Cas9 human germline editing did not work the way it supposed to: low efficiency of editing (~ 52%); off-target genotoxicity; unwanted mutagenesis; genetic mosaicism. To me, the […]

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