• 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 […]
  • Methods and Protocols Digest – October 2014

    by Alexey Bersenev on October 27, 2014 · 0 comments

    in methods,protocols

    Methods and Protocols Digest is a collection of links to the methodological articles, freely available online. Here you can also share your lab protocol with community!

    FEATURED:

    General lab
    Commercial antibodies and their validation (F1000 Research)
    Why do you have low cell number after FACS sorting? (Bitsize Bio)
    Tips for a Happily Functioning Tissue Culture Room (Bitsize Bio)
    10 Things Smart Scientists Do Before Sorting Cells (Excyte)

    Pluripotent stem cells
    Animal components-free human embryonic stem cell culture – review (Stem Cells TM)
    Generation of lung epithelium from murine pluripotent stem cells in 3D culture (Stem Cells Dev)
    Testing spinner flask for iPS cell culture (PLoS ONE)
    Generation of human chondroprogenitors from embryonic stem cells for cartilage repair (Stem Cells TM)
    Efficient generation of retinal pigment epithelium from human pluripotent stem cells (Stem Cells TM)

    Neural cells
    Generation of adult limbal neurosphere cells (PLoS ONE)
    Derivation of mature neurons from human embryonic stem cells (F100 Research)
    Neurotrophic requirements of human pluripotent stem cell-derived motor neurons (PLoS ONE)

    Endothelial cells
    High density dot culture for bone marrow-derived endothelial progenitors (PLoS ONE)

    Mesenchymal stromal cells
    Umbilical cord Wharton’s jelly MSC repeated culture system (PLoS ONE)

    Tissue engineering
    Generation of human iPS cell-engineered cardiac tissue sheets (Sci Reports)

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    Cells Weekly – October 26, 2014

    by Alexey Bersenev on October 26, 2014 · 1 comment

    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!

    This week, I was covering some news from FDA. First, FDA released clarification of “same surgical procedure” definition. Second, I started a new series of posts – Letters from Regulators. In the first post of the series I attempted to analyze recent untitled letter from FDA to orthopedic company. I’d like to encourage you to discuss these regulatory issues with me!

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    1. Analysis of recent Melton’s study on generation of beta-like cells
    Juan Dominguez-Bendala – a diabetes researcher from University of Miami wrote very interesting opinion piece about recent hype around Doug Melton’s study. He compares two recent “high profile” studies (Melton’s and Kieffer’s) and commercial technology from ViaCyte, which is entering clinical trials. His conclusions:

    Both of these protocols try to address a concern that still hovers over the Viacyte approach, namely, whether hESc-derived progenitors will mature in a diabetic HUMAN host as they do in a mouse (we will know this soon enough). However, the Kieffer protocol still falls short (some degree of maturation still has to happen within the host, since it takes 40 days for the cells to achieve competence); and the Melton one does not even attempt to reverse already established diabetes. In short, there are still a lot of questions to answer, and room for improvement.

    Highly recommend to read!

    2. Refutation of Betatrophin significance
    To continue “Melton’s topic”, a group of researchers from Regeneron Pharmaceuticals was not able to reproduce famous betatrophin 2013 study. The authors of “betatrophin refutation study” came up with opposite conclusions:

    These data indicate that ANGPTL8 does not play a role in controlling beta cell growth, nor can it be given to induce such expansion.

    Paul Knoepfler covered this study here, indicating that Melton admitted his mistake. As of now, there is no news on possible retraction of 2013 paper. Great comment from Jeanne Loring:

    If we make a mistake, it is our responsibility as scientists to remove that mistake from the literature, so that no one bases their further research on it or invests precious resources trying to develop it further. I hope that Doug retracts the paper. In regenerative medicine, we need to hold ourselves to a higher standard.

    3. A buzz around paralyzed man case
    Mass media was over-hyping a clinical case about dramatic recovery of paralyzed man after cell therapy. Famous sci blogger PZ Myers summarized story on his blog. However, he described wrong study – 3 cases report, published by the same group in 2013. The mistake was pointed in comments, but PZ did not respond and did not update the post. The correct study is describing single case in great details. I’d recommend you to read the original article (freely available). A single case is not a reason for buzz and hype. Case reports are usually “cherry picking” and overall efficacy pretty much always diluted in controlled clinical trials. There are hundreds (to few thousands) cases of olfactory ensheathing cell transplantation for neurological conditions, described in literature. Some studies also have a “miracle cases” alike Fidyka’s case.

    4. Interview with Shinya Yamanaka
    A blogger Paul Knoepfler interviewed Nobel Prize 2012 winner Shinya Yamanaka. The interview focused on clinical translation of iPS cell-based technologies:

    There are two major clinical applications of iPSCs, namely regenerative medicine and drug discovery. CiRA has a number of researchers working on either or both. For regenerative medicine, Prof. Koji Eto at CiRA is working on generating platelets via iPSCs, and we expect this will also proceed to clinical research in a few years. Besides work at CiRA, a team at Keio University has a plan to conduct clinical research on patients with acute spinal cord injury in four to five years, while Osaka University and Keio University hope to transplant iPSC-derived cardiac myocytes into patients with heart diseases within a few years. CiRA is collaborating with these teams as well.

    5. More on new RegenMed legislation in Japan
    Lee Buckler was interviewed by The LifeSciences Report on details and potential significance of recent Japanese law, which allows accelerated approval of commercial RegenMed products.

    I think most companies in the regenerative medicine industry have a tremendous desire to have a Japanese strategy right now. The problem is that a lot of companies in this sector don’t have enough bandwidth, or capital, to execute on that.

    6. Videos from Stem Cell Meeting on the Mesa 2014
    Alliance for Regenerative Medicine uploaded video recordings from annual Stem Cell Meeting on the Mesa. 11 panel discussions and 49 company presentations available on their YouTube channel. You can also watch it here.

    7. Current status and perspectives of commercial animal cloning
    Very good overview of commercialization of animal cloning was posted by Bloomberg. This is the best overview of the topic that I’ve ever read. Highly recommended!

    Rare breeds, dogs cloned for devoted owners, and specialized working dogs (for police and bomb-sniffing work, as well as cancer detection) will be a part of that business, he says, but only a small one. “Dogs are the entry point,” he says. The far larger and more important focus will be cloning cows to help China deal with a growing appetite for beef.

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    bonus:

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    I’m starting new series – Letters from regulators. In this series I’ll analyze issues with regulation of cellular products, resulted in letters or other actions from regulatory agencies. I hope this series will:

    • help to understand current regulation of cellular products better;
    • navigate cell product developers in correct regulatory path;
    • help to learn and avoid mistakes in early cell product development;
    • identify deficiencies in current regulatory system.

    Please give your feedback and suggest recent interesting cases and letters for analysis!

    Today, I’ll go through recent “untitled letter” from FDA, regarding map3 Chips Allograft developed by RTI Surgical.

    Introduction:
    To many of you, it seems obvious that any cells, which underwent extensive manipulations, such as ex vivo expansion, should be regulated “as drugs”. Today’s case, involved Multipotent Adult Progenitor Cells (MAPC) – allogeneic bone marrow cells, commercialized by US-based company Athersys as product “MultiStem”, since 2003. RTI Surgical licensed MAPC from Athersys and implemented it in their new product “map3 Chips Allograft” (map3). This product is composed of allogeneic accellular bone matrix and MAPC, prepared (thawed) and mixed at point-of-care. As far as I know from literature and patents on MACP – these cells are pure “cell culture product”. There is no protocol and description of the properties of freshly isolated MAPC. Therefore, MAPC are always cultured (expanded ex vivo). Based on many FDA letters and famous case Regenerative Sciences versus FDA, the agency views any “ex vivo expanded or cultured” cell product as a drug.

    Your map3™ Chips Allograft cultured cell product is a human cell, tissue, or cellular or tissue­based product (HCT/P) as defined in 21 CFR 1271.3(d), and is subject to regulation under 21 CFR Part 1271, issued under authority of section 361 ofthe Public Health Service Act (PHS Act (42 U.S.C. 264]). HCT/Ps that do not meet all of the criteria in 21 CFR 1271.10(a), are subject to additional regulation, including appropriate premarket review.

    The chronology of events:

    1. TRI Surgical licensed MAPC technology from Athersys in 2010
    2. RTI wanted to launch product in 2012, but started to report first cases of map3 implantation only in August 2013 and officially announced release to the market in 2014.
    3. In October 2014 RTI got untitled letter from FDA, which indicates that map3 should be regulated as “drug” under section 351(i) of the PHS Act.
    4. Company responded that in development of map3 they “properly considered the relevant regulatory questions“, map3 web site was shut down.
    5. The company has a history of some facility-related issues, which were brought by FDA’s inspection in 2012 (resulted with 483 letter) and resolved.

    Summary of FDA letter:

    1. FDA says that map3 product is not qualified as HCT/P and should be regulated as a “drug” with IND, clinical trials and BLA.
    2. FDA’s reasons are the following: MAPCs are allogeneic cells, more than minimally manipulated (cultured), depend on metabolic effects for their function.
    3. FDA says that company “in not performing all of the necessary donor testing” for infectious diseases.

    Some questions for discussion:

    1. Is MAPC product licensed from Athersys freshly isolated or cultured? FDA wrote in the letter “cultured”.
    2. Is RTI manufacturing MAPC themselves or Athersys/ its contractor is acting as manufacturer?
    3. Did RTI register map3 with FDA as HCT/P before launching on the market?
    4. Did RTI tell Athersys about their intention to market MAPC-containing product without registration with FDA as HCT/P? How did Athersys can let it go with knowledge that all MAPC products are regulated as “drugs” (351)?

    Conclusion:
    I think, RTI made big mistake on management level by launching map3 as “tissue product” (HCT/P). The company should consult and register map3 with FDA in development process before market launch. As consequence of this FDA letter, both RTI and Athersys will be affected financially. Companies can also lose credibility and trust from investors. This letter can also affect similar products, such as Osteocel Plus by NuVasive, Trinity ELITE by Orthofix, AlloStem by AlloSource, Cellentra by Biomet.

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    Today, FDA released a new guidance for industry document, which clarifies “same surgical procedure” definition. Please note that this is a draft document, which open for public comments. What is importance of this guidance and why FDA would clarify it?

    As outlined in document’s background section, “same surgical procedure” is an exception from the regulation of cell products “as drugs” (351) and HCT/P with requirement of “FDA registration” (361). You can read more about it in section 1271.15 of CFR 21. Basically, if you transfer patient’s own cells from one place to another withing “same procedure”, you don’t need to deal with FDA, since it’s equal to “practice of medicine” as “surgery”. As FDA put it, 3 criteria must be met:

    a. Remove and implant the HCT/Ps into the same individual from whom they were removed (autologous use);
    b. Implant the HCT/Ps within the same surgical procedure; and
    c. The HCT/Ps remain “such HCT/Ps;” they are in their original form.4 The communicable disease risks, as well as safety risks, generally would be no different from those typically associated with surgery.

    But, devil in the details… And the biggest twist to me is in the phrase “HCT/Ps remain “such HCT/Ps”". Turns out that criteria of HCT/P similarity in this case are even narrower than definition of “minimal manipulation”:

    … even manufacturing steps considered minimal manipulation within § 1271.10(a), will typically cause the HCT/P to no longer be “such HCT/P” under §1271.15(b), unless the HCT/P is only rinsed, cleaned, sized, or shaped.

    So, to me the biggest question here – Is centrifugation falling into “rinsing, cleansing, sizing or shaping” the tissue? I don’t think so. But it could be debated. So, if centrifugation is “out of game”, pretty much none of currently used cell suspensions could be used under “same surgical procedure” exception. Of course, I’m talking about the most discussed (i) adipose tissue-derived stromal vascular fraction (SVF) and (ii) bone marrow mononuclear cells (BM MNC) – 2 types of cell products, which FDA continues to ignore in citations as examples. Isolation of both SVF and BM MNC (even in case of MNC enrichment = bone marrow concentrate) requires centrifugation – no matter what device you are using. A quote:

    Processing of the autologous HCT/P raises safety concerns, such as contamination and cross-contamination, beyond those typically associated with surgery.

    Here under processing they mean more than just “rinsing, cleansing, sizing and shaping” of tissue. It is not very clear to me how centrifugation will possess higher safety concerns (particularly, FDA cares about risk of microbial contamination) than rinsing cells in saline solution in the bag? How to measure which safety risks are higher?

    Interestingly, HCT/P could be stored for days (!) and still be used as part of “same surgical procedure”, without FDA oversight. So, you are not limited by time of procedure as long as it’s “the same one”. But in this case, refrigerator should be in the same hospital.

    Another puzzle to me is definition of “surgical procedure” itself. Is puncture of blood vessel a surgical procedure per FDA? What if I’ll make 1 cm incision to insert catheter into artery to deliver cells into heart or brain and then put 1 suture at the end? Sounds like “surgical” to me, since involves incision, but how is it different from puncture? FDA is not explicit about it.

    Overall, this is very interesting document. It clarifies something, but still leaves some questions. As of now, it looks like FDA does not give any chances to adipose SVF and BM MNC to qualify for “same surgical procedure” exception. I’d encourage you to read it, discuss here and comment for FDA. I may send comments to FDA myself and ask to clarify “centrifugation” and bring SVF and BM MNC as examples.

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    Clinical cell processing news – part 5, 2014

    by Alexey Bersenev October 21, 2014 clinical lab

    Clinical Cell Processing News is a series about new protocols, products and techniques for clinical-grade cell processing and manufacturing. Cell processing devices, cultureware, bioreactors, GMP-grade reagents, cell separation techniques. This series is posted every 2 months. FEATURED: Protocol for manufacturing of gene-modified T-memory stem cells (Human Gene Therapy Methods) … we show the robust clinical-scale production of human peripheral blood T-cells with an early memory phenotype that express a MART-1-specific TCR. By combining selection and stimulation using anti-CD3/CD28 beads for […]

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    Cells Weekly – October 19, 2014

    by Alexey Bersenev October 19, 2014 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. First results of embryonic stem cell trial told by Robert Lanza Stem cell feed this week was dominated by news about results of ACT’s trial. Here is a video, where Robert Lanza explains published results: Also read: ACT Interim Clinical Results Are Outstanding by Irv Arons. Encouraging New Paper on ACT […]

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    Products in the pipeline – Aastrom’s Ixmyelocel-T

    by Alexey Bersenev October 17, 2014 cell product

    This is second post of the series Products in the Pipeline. In this series I’ll provide publicly available information about therapeutic products – candidates for commercialization by cell therapy industry companies. Please leave your feedback and propose cell products to cover! Today I’m going to analyze Ixmyelocel-T manufactured by Aastrom Product candidate name: Ixmyelocel-T Developer: Aastrom Biosciences (Vericel Corporation) History of development: Company founded in 1989. Product in development >10 years. Phase 1 studies started ~2005, Phase 2 in 2007 […]

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    First results of embryonic stem cell trials

    by Alexey Bersenev October 14, 2014 cell product

    Today – October 14 of 2014 is a very important day in “stem cell history”. US-based company Advanced Cell Technology (ACT) released results of the clinical trials, where embryonic stem cell-derived product was tested in patients with macular degeneration. The first ever peer-reviewed results of FDA-approved clinical trials, involved use of embryonic stem cells (ESC), published online in Lancet (keep renewing your browser window until Lancet will put abstract in). You can download article here. This is a big milestone […]

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    Cells Weekly – October 12, 2014

    by Alexey Bersenev October 12, 2014 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. Stem Cell Person of the Year 2014 Paul Knoepfler runs annual contest Stem Cell Person of the Year. Please cast your vote! I noticed that most nominee are keep doing a lot of good things for stem cell field persistently from year to year. So, in order to pick the best […]

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    Using molecular beacons for isolation of human osteogenic progenitors from adipose SVF

    by Alexey Bersenev October 9, 2014 adipose

    Molecular beacons (MB) is a new technique for cell isolation, based on capture of genetic (intracellular or intranuclear) markers. MB have been used in research for isolation of embryonic stem cells and cardiomyocytes since 2011. I’m dreaming about using MB for clinical cell isolation. Recently, a group of researchers from Brown University published a study, which makes one step closer to clinical application of MB. The authors used MB, which captures mRNA of alkaline phosphatase (ALPL) in osteogenic progenitors, derived […]

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