• Defining a stem cell product – working proposal and recommendations
    Last 2 months I was discussing a possible definition of “stem cell product“. I polled professionals and had few opinions exchange via email and LinkedIn. Today, I’m going to summarize this discussion and propose a working definition and recommendations. Why do we need to define it? 93% of polled professionals think that it is important issue to discuss. 7% think that there is no point to do it. Unfortunately, both sides did not elaborate publicly – why they think so. […]
  • VSEL in translation

    by Alexey Bersenev on April 15, 2014 · 0 comments

    in cell product,other adult stem cells

    Despite the controversy, surrounding VSEL cell biology and methodology, we are keep hearing news about its clinical translation. US-based company – NeoStem, which commercializes VSEL (VSEL is a trade mark of NeoStem), recently announced collaborations with Harvard/ MGH and NIH for VSEL translational studies. Company pumped > $4M into VSEL programs. About 70% of VSEL funding is dedicated to bone regeneration. Last year, NeoStem shared some exciting plans about use of VSEL in bone regeneration:

    Phase 2 investigation and first approved NIH clinical study of VSELs™ in humans. Enrollment for this study is expected to begin in 2014.

    The required preclinical data, cell manufacturing processes and clinical protocols necessary for submission of an IND to the FDA are in the final stages of preparation. The Company anticipates IND submission in late 2013 or early 2014.

    Half of year before this PR, CEO of NeoStem – Robin Smith said:

    … we hope to bring into human testing for boney defects in 2013

    Well, it did not happen in 2013. Will it happen in 2014? It’s not clear to me, how are they going to start Phase 2 trial without conducting Phase 1 safety study? Did they make a mistake in press release? By this time, they should have approved IND already, but I feel like the process is slowed down. In this post I’m going to analyze “clinical manufacturing readiness of VSEL cells” and highlight possible reasons for delay. This analysis is based on publicly available, published studies by Taichman’s group.

    Controversy and reproducibility issues
    In order to translate product to the clinic, research protocol should be: (1) highly reproducible, (2) robust, (3) scalable, (4) clinically grade. NeoStem bought VSEL-related IP few years ago from University of Louisville, made “VSEL trade mark” and offer Scientific Advisory Board position to Mariusz Ratajczak. Taking in account such involvement, it’s unclear to me why NeoStem did not make any public comment about ongoing VSEL controversy. However, last year, Robin Smith commented on VSEL controversy in interview:

    We acknowledge that the final chapter describing the full potential of VSELs is yet to be written, and that controversy exists at this time regarding how that final verse will read. However, we are proud of our efforts, and those of our collaborators, to get to the truth about these cells, and to apply rigorous scientific method to write the script.

    We don’t know if NeoStem ever attempted to reproduce original Ratajczak’s protocol for human VSELs. All published data so far indicate that NeoStem significantly modified original human VSEL protocol and even created their own. So, reproducibility of human VSEL cell isolation is inconclusive as of now. Smith commented:

    NeoStem and our collaborators have shown that VSELs are present in the bone marrow and other organs of humans, and can be mobilized from the bone marrow into the peripheral blood and collected by apheresis. It has been established that these human VSELs are comparable to those described by Dr. Ratajczak.

    But how much NeoStem’s VSELs are comparable to Ratajczak’s VSELs? And how much they are comparable to other stem cell populations in human bone marrow? In the last few articles Ratajczak emphasized:

    … we have always emphasized that, in contrast to their murine counterparts, much work still needs to be done to better characterize human VSELs.

    Recently, Ratajczak’s group published “the proper criteria” for identification of VSEL, citing cord blood as a source of human VSELs, not bone marrow. However, NeoStem is commercializing exclusively autologous bone marrow VSELs.

    So, (i) if much more work should be done in order to characterize human VSEL (especially from bone marrow) and (ii) if reproducibility of human VSEL protocol is unknown, are they ready for prime time?

    Manufacturing steps and cost estimation
    Based on last studies, published by Taichman’s group, we can take a look at some piece of “clinical protocol” for VSEL manufacturing. Please note, that VSEL “manufacturing” was done by NeoStem staff in their lab, therefore it’s a “clinical prototype”. Human bone marrow VSELs isolation procedure includes:

    1. mobilization protocol (hrG-CSF, pre-apheresis cost ~$1.5-3k)
    2. apheresis (~$3k)
    3. elutriation (~$1k)
    4. positive selection by MACS for CD34+/CD133+ cells (in case of CliniMACS cost ~$2-5k)
    5. FACS sorting
    6. cryopreservation

    So, NeoStem VSEL protocol can end up with manufacturing cost ~$10-15k at least. If we look at main competitor of VSEL in clinical bone repair – mesenchymal stromal cells (MSC), we can estimate manufacturing cost as few hundred $ for allo- and maybe few thousand $ for auto- expanded MSCs. Interestingly, Taichman wrote:

    … there are potential limitations to the use of autologous MSCs in bone repair in humans because most preparatory protocols require the extensive expansion of MSC populations in vitro using animal-derived or recombinant growth factors as well as modulators of transcription and cell survival.

    My estimation does not support this claim.

    Questions to Taichman’s study
    The most important translational step for human VSEL was made with recently published study by Taichman’s group. Media called this report as “compelling evidence” for multipotent VSELs in human. I have some questions and concerns about interpretation of Taichman’s study.
    VSEL sorting (FACS):

    • There is no discrimination of aggregates (singlets versus doublets) on presented flow plots – it is extremely important for identification of tiny cell populations.
    • Too many dead cells – they showed a plot with 7AAD staining with 40% dead cells before FACS sorting – isn’t it too much?
    • Did MACS really work? They showed a plot with only 24% of CD34+/CD133+ (gated as alive Lin-) after positive selection for these 2 markers – it is way too low.
    • What was in Lin antibody cocktail? They didn’t indicate it clearly, but mentioned that cells were “gated for Lin- Gycophorin A (CD235a)- CD41-”. Were CD235 and CD41 included in Lin cocktail or used separately? What else was in Lin cocktail? It is very important!
    • Different groups employed different gating strategies for human VSEL – so, reproducibility is inconclusive.

    Multipotency interpretation:
    Multipotency of human VSEL was concluded, based on expression of genes, typical for 3 germ layers (osteocalcin, nestin, insulin), after addition of different inducers of differentiation. This is not a proper test for multipotency. Artifacts should be ruled out in vitro and function of VSEL-derived mature cells should be assessed. It is unclear whether those markers will be expressed without co-culture VSELs with C2C12 line. It is unclear to me why, on the one hand, VSEL made bone tissue after subcutaneous implantation, if there was no indication for their induction in vitro, but on the other hand, VSEL expressed markers of multiple tissue in bone defect site. Shouldn’t it be vice versa? Don’t we need only bone in bone defect site?
    Controls:

    • Analysis of normal blood for VSEL from unmobilized donors is not presented.
    • For 3 germ layers gene expression controls with VSEL, cultured in basal media (without inducers) should be used. It is known, that some cell populations can spontaneously over-express atypical markers in culture.
    • MSC, usolated from the same donors should be used as control for bone defect experiments in order to show advantage of VSELs.

    VSEL cells fresh or cultured?
    Unlike any other stem or progenitor cell population, VSEL cells do not proliferate in culture. In order to overcome this problem and test “multipotency” in vitro, Taichman’s group used C2C12 line (mouse myoblasts) to co-culture human VSEL. The most important clinical questions would be:

    1. How much VSELs is enough for therapeutic effect?
    2. Do VSEL need to be induced in vitro before administration?

    The first question can be tackled after safety trial, but the second question should be answered before. My understanding is that NeoStem will got for freshly isolated (not cultured) and cryopreserved VSELs.

    Scalability
    If we do the math, based on flow plots from Taichman’s study, we can estimate about 0.0002% of VSEL cells in positively selected (for CD34+/CD133+), elutriated, mobilizhed apheresis product. Taichman noticed, that they typically isolate ex vivo, I’d conclude that this technology is not scalable for now.

    My another big concern is related to quality control (QC) assays. If NeoStem will able to isolate 1M VSEL from 1 patient maximum, how many aliquotes of cells with how many cells can they retain for QC? Is it even doable?

    Clinical grade
    Because NeoStem claims start of VSEL trial this year, the protocol should be 100% clinically-grade. However, based on Taichman’s data, we cannot conclude clinical readiness. The biggest question is FACS sorting. As of today, the whole VSEL protocol based on FACS sorting. However, there is no clinical-grade FACS sorter, approved by FDA. Modification of existent instruments can, probably, justify requirements for phase 1 trial, but not more. The second, related issue is clinical-grade reagents – antibody for FACS sorting. In published studies, NeoStem used AutoMACS Pro for magnetic positive selection, but it is not clinical device.

    Concluding remark:
    I think, NeoStem should do more work for human VSEL cells isolation, characterization and tune up clinical manufacturing protocol. It will take some time. I think, at this point, human VSELs are not ready for clinical translation.

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    Cells Weekly – April 13, 2014

    by Alexey Bersenev on April 13, 2014 · 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!

    Cell Education update from this week – Regenerative Medicine Essentials – 5-day Summer Course at Wake Forest U. Please feel free to contribute to our crowdsourcing project!

    1. STAP news
    Week #10 of “STAP discovery” was all about Obokata’s first public appearance and press conference! Here is my pick of the news:
    Waseda U to probe all PhD theses for plagiarism
    Tweets from RIKEN press conference on STAP verification
    RIKEN plans to conduct STAP verification without Obokata
    Live updates from Obokata’s public press conference
    Live tweeting from Obokata’s press conference
    Obokata apologizes and appeals
    Obokata ready to “go anywhere” to help any scientist reproduce STAP
    RIKEN panel to prevent research misconduct
    Sasai has no doubts about the validity of the finding
    Yamanaka on STAP: “young researchers ‘immature,’ need education in ethics, but also more autonomy”

    2. Harvard stem cell scientist under fire
    This week was very bad for prominent Harvard’s stem cell scientist Piero Anversa. First, Circulation announced retraction of his controversial paper from 2012. The retraction followed from results of Brigham and Women’s Hospital investigation, which unveiled some compromised data:

    “This retraction is highly significant. In my 30 years in cardiovascular science I cannot recall a paper of similar prominence being retracted from Circulation,” Dr. Charles Murry… “This appears to settle the controversy about the rate of cell replacement in the human heart.”

    One possible reason for investigation is lack of reproducibility of original study and growing skepticism about its conclusion among other cardiovascular stem cell researchers.

    The second problem came with request of Harvard’s investigators to retract clinical trial report, published in Lancet in 2011. The trial was based on Anversa’s experimental data. As of now, the Lancet did not retract paper, due to ongoing investigation, but posted an “expression of concern“. Some quotes from Boston Globe:

    Adult stem cells were isolated from tissue taken from heart failure patients and later infused back into their hearts. The paper, which was a continuation of work by Anversa’s laboratory that had shown such cells could repair heart muscle in animals, reported improvements in some measures of heart function in people.
    “I continue to believe that the data show improved heart performance in most patients who participated in the study,” Anversa wrote in his e-mail.

    I am of course as anxious as anyone else to see the results of the ongoing investigation by the BWH,” Bolli wrote in an e-mail Friday. “The data in question were generated by Dr. Anversa’s lab independently of us in Louisville; we in Louisville have nothing to do with the issues cited in the Expression of Concern.”

    I’m pretty sure, now all “big Anversa’s papers” will be examined, including the one from NEJM.

    3. NIH closes stem cell program
    It was a very sad news. I was watching NIH stem cell projects with great interest, but apparently, it was not delivering a translational promise:

    Stem-cell researchers at the US National Institutes of Health (NIH) have been left frustrated and confused following the demise of the agency’s Center for Regenerative Medicine (CRM). The intramural programme’s director, stem-cell biologist Mahendra Rao, left the NIH, in Bethesda, Maryland, on 28 March, and the centre’s website was taken down on 4 April.

    Rao made decision after learning that NIH will fund only 1 out of 5 iPS cell clinical projects. Well, NIH will re-think and re-structure of its stem cell initiatives, but outcome is unknown. Rao already joined NYSCF.

    4. Direct reprogramming paper retracted from Cell
    One more big retraction this week – a paper about direct conversion of skin cells from Alzheimer’s patients into neurons. The retraction was requested by authors:

    Dr. Ryousuke Fujita, who was specifically and only responsible for the molecular analyses of Alzheimer’s-associated pathology, has acknowledged inappropriately manipulating image panels and data points, as well as misrepresenting the number of repeats performed…

    5. Growing vaginas and noses
    Antony Atala’s group published preliminary results of pilot clinical study, which assessed feasibility and function of tissue engineered autologous vaginas:

    Vaginal organs, engineered from the patient’s own cells and implanted, showed normal structural and functional variables with a follow-up of up to 8 years. These technologies could be useful in patients requiring vaginal reconstruction.

    In the same day, Lancet released Ivan Martin’s study on autologous nasal reconstruction:

    Autologous nasal cartilage tissues can be engineered and clinically used for functional restoration of alar lobules. Engineered cartilage should now be assessed for other challenging facial reconstructions.

    Great news for clinical tissue engineering!

    6. Cell lines or patient samples?
    Nice post on the Signals blog outlines pros and cons of using available cell lines or primary patient samples for cell culture:

    Obviously patient samples must be the way to go, right? They come right from the patient, and are used right away, so they are super-relevant to the original tumour. Except, you need a consenting cancer patient to donate their tumour. And the cells need to not die before you use them. And you need enough cells to test things multiple times, never mind optimize. Forget about them surviving a freeze-thaw cycle…

    Highly recommended, especially to cancer researchers!

    7. First gene therapy got “Breakthrough Therapy” status in US
    FDA granted “Breakthrough Therapies” designation to the first gene therapy candidate – Mydicar by Celladon.
    Good sign for investors in gene therapy!

    8. Are MSCs ready for stroke clinical trials?
    YES – says meta-analysis, published this week in Neurology. 44 of 46 experimental studies were “positive” for efficacy. I wonder if they look at differences in MSCs preparations.

    ***************************************
    bonus:

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    This is fresh issue of the series “Experimental bone marrow transplantation 101“. In this series we talk about basics of whole bone marrow and hematopoietic cells transplantation models to study hematopoietic stem cells and blood formation in normal conditions and malignancies.

    Why serial BMT?
    The purpose of serial bone marrow transplantation (BMT) is to test the self-renewal capability of HSC or/and their aging and functional exhaustion.

    Recipients and experimental scheme
    Recipient mice should be identical throughout all series of transplant. Conditioning (usually lethal irradiation) should be the same in each series. The optimal age for recipients is 8-12 weeks.
    In BMT models, donor-derived cells are traceable throughout all rounds of serial transplantation if you use the same recipient mice.

    Donors
    Donor mice are previously transplanted (underwent at least primary transplant) and (usually) genetically different from recipients. Donors should be at the final readout time point, because bone marrow harvested from euthanized animals. Donor-recipient could be one-to-one or pooled. One-to-one transplant is more precise method, but may could require more animals for statistics. Try to harvest bone marrow from the same type of bones throughout experiment.
    On the day of transplant, detection of donor HSC engraftment in donor’s BM is highly recommended. For example, you can harvest 3 leg bones for flow cytometry and one bone (femur) save in the media for transplant.

    Cell dose and number of rounds
    Your cell input in the primary transplant could be any that justify your experimental goals. For the second and subsequent rounds there are some recommended doses. For whole marrow transplantation you have to input a sufficient number of primary HSC, capable of self-renewing for at least 2-3 rounds with significant readable output. For normal wild type mouse, anything between 1 to 10 million (M) total BM cells will be good. Typically, 2M or 5M of unseparated BM cells are used. Usually 3-4 rounds is enough to see a difference between control and test groups. Many labs do only 2 rounds to see if cells are capable to self-renew at all.

    Keep in mind that transferring 5 – 10M BM cells from normal wild type mice will require at least 4 rounds of serial transplant in order to see significant self-renewal decline and HSC exhaustion. So, you can adjust the cell dose, based on the timeframe of your experiment. For example, If each round takes 4-6 months, and you want to see a difference sooner than later, you may consider transferring 2M of unseparated BM cells each round of transplantation instead of 5M.

    Time planning and readout
    The conclusion about donor HSC performance should be based on positively repopulated primary recipients. At least 16 weeks post-transplant is recommended for valid readout of blood repopulation and bone marrow engraftment by donor HSC. Therefore, each round of serial transplant will take at least 16 weeks. However, there is no unified opinion on intervals between rounds, starting from the second. For example, you can transplant with an interval of 2 months between transplant from round 2 to 5, with valuable readouts (16 weeks) at 1st and 5th transplants.

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    In order to resolve (at least in part) inter-labs variability in mesenchymal stromal cell (MSC) preparations, Darwin Prockop’s group is proposing to include “in-process” data in research publications:

    … we recently developed a simplified table to record in-process data on the preparation of each master bank and the characteristics of MSCs expanded from a working bank prepared from the same master bank…

    The proposed table includes donor’s ID; data from each passage (plating concentration, number of cells in harvest, number of days for expansion); population doublings; differentiation potential at P2; phenotype at P2. The authors believe that sharing of such data will allow to compare MSC quality between publications from different labs and understand differences in results.

    Prockop’s NIH-sponsored center prepares and distributes MSC reference material (master cell bank) for researchers for many years:

    … we have been distributing the MSCs from the master banks to more than 250 laboratories in this country and abroad for the past 10 years. We have devoted considerable time and effort to standardizing the protocols for preparing the master banks.

    We have provided investigators who received MSCs from our master banks with detailed protocols for expanding the cells from master banks (referred to as P1 cells) to generate working banks. We have also provided them with specification sheets summarizing the data obtained in our own laboratory on the characteristics of MSCs expanded from the same master bank.

    This is a good practice to distribute reference material. However, even if reference material was used, in-process data are not published. The authors suggest to include such data in supplemental material of the publication. I think, these kind of “MSC passport” could be useful for researchers. What do you think?

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    Cells Weekly – April 6, 2014

    by Alexey Bersenev April 6, 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! If you need a degree and looking for advancing your career in stem cell research, tissue engineering, cell therapy, RegenMed – visit Cell Education page! This is crowdsourcing project with frequent updates! 1. STAP news Week #9 of “STAP discovery” was extremely hot! See my pick of the news below: Results of […]

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    Not Lost in Translation: Standards in cell therapy

    by Alexey Bersenev April 3, 2014 cell product

    This is a sixth post of the series “Not Lost in Translation“. If you’re trying to develop a cellular product and just entering the field of cell therapy, you should be aware of existent standards. Why is it important? Knowing standards in your field allows to: guide and accelerate product development meet expectations of regulators set high quality specifications Even though, cell therapy filed relatively new, there are numerous related standards. Unfortunately, many professionals are unaware about organizations and standards […]

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    Cells for Pharma R&D – results of a poll

    by Alexey Bersenev April 1, 2014 notes

    A couple of months ago, we’ve asked you about the most promising cell-based technology for Pharma R&D (drug screening/ discovery and toxicology). Today, I’m closing this poll and sharing results. We got 30 votes and 2 comments (overall 32 participants). 50% voted for iPS/ ES cells option. Bioprinted tissues got 27%. See all results below: We got 2 comments: 1. adult cell therapy 2. all or none of the above: the issue is that each class of drugs is likely […]

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    Cells Weekly – March 30, 2014

    by Alexey Bersenev March 30, 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! While ago we’ve asked you to vote in our poll “Cells on Drugs” – the best cell-based method for Pharma R&D. If you missed it and would like to participate, please vote here. Next week, we will close the poll and post results. Stay tuned! Tomorrow (Monday, March 31), FDA is organizing […]

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    Tracking divisional history of hematopoietic stem cells

    by Alexey Bersenev March 29, 2014 hematopoietic

    One of the interesting and unresolved questions in stem cell biology is how divisional history of adult stem cells correlates with their function in homeostasis. Some studies have demonstrated, that hematopoietic stem cells (HSC), once activated and divided, can come back to quiescent (dormant) state and retain function. However, this HSC homeostatic model was recently challenged by Kateri Moore’s group study, published in Stem Cell Reports. We study the function of HSC mostly in pathological conditions, namely – transplantation model […]

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    The best corporate blogs

    by Alexey Bersenev March 26, 2014 notes

    I’m reading a lot of blogs every day about stem cells, cell therapy, regenerative medicine and science in general. Corporate blogs (which created and maintained by companies) are not very well represented in “stem cell/ cell therapy blogosphere”, but there are a few. Most of them, of course, heavily promoting their products in the posts. But some, give additional interesting general information about the field and recent discoveries. Here, I’ve picked 5 blogs, which are, in my opinion, worth a […]

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