• 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 – November 2014

    by Alexey Bersenev on November 25, 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:

    Mesenchymal stromal cells
    Comparison of immunomodulatory ability of umbilical cord MSCs from early and late passages (Mol Med Rep)
    Neural induction of umbilical cord matrix-derived MSCs (PLoS ONE)
    Method for generation of insulin-producing cells from bone marrow-derived MSC (J Diabetes Res)

    Cell labeling
    Using infrared fluorescent protein 1.4 for labeling of cardiac progenitors (PLoS ONE)

    Pluripotent stem cells
    Feeder-free generation of iPS cells from blood cells (Stem Cells TM)
    Method for detecting residual pluripotent stem cells in cell therapy product (PLoS ONE)
    Derivation of macrophage-like cells from mouse ES cells (PLoS ONE)
    Large-scale suspension culture for generation of pluripotent stem cell-derived cardiomyocytes (Stem Cells TM)

    Neural cells
    Efficient generation of neurons from human iPS cells (Stem Cells TM)
    Generation of embryonic spinal cord prototype in 3D culture (Stem Cell Rep)
    Cellular phenotype of Huntington disease in monkey iPS cells (Stem Cell Rep)
    Generation of schizophrenia neurons from iPS cells (Stem Cell Rep)

    Adipose stem cells
    Chondrogenic differentiation of human adipose‑derived stem cells using microcarriers (Mol Med Rep)
    Isolation of extracellular vesicles from adipose tissue mesenchymal stem cells (Front Immunol)

    Other stem cells
    Isolation of embryonic kidney stem cells (Stem Cell Rep)
    Identification of fetal lung stem cells (Stem Cell Rep)

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    Cells Weekly – November 23, 2014

    by Alexey Bersenev on November 23, 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!

    CD34+Banner_490x90

    1. CD34+ cells for myocardial infarction patients – results of clinical trial
    Press release of company NeoStem about preliminary results of Phase 2 cardiac cell therapy study, triggered lively discussions among professionals. I summarized the reasons for such discussions here. The investors “punished” NeoStem in the next day of news release (stock dropped -30%), because company did not provide explanation for changing primary endpoints on trial record. 4 days later, Neostem posted new clarifying press release, where provided explanation for changing study’s endpoints. However, it remains unclear why company did not share this information with investors in July (when endpoints were changed), but rather did it on the night of news release.

    2. Stem cell origin of fibrosis
    Harvard’s Humphreys lab described new kind of perivascular stem cells, which is a major contributor to organ fibrosis after injury. There cells have characteristics of mesenchymal stromal cells and express Gli-1 marker:

    Genetic lineage tracing analysis demonstrates that tissue-resident, but not circulating, Gli1+ cells proliferate after kidney, lung, liver, or heart injury to generate myofibroblasts. Genetic ablation of these cells substantially ameliorates kidney and heart fibrosis and preserves ejection fraction in a model of induced heart failure.

    So, stem cell are involved in many pathological processes and could be sometimes “bad guys”. Interesting part of work is therapeutic targeting of Gli1+ cells to stop/ prevent organ fibrosis. You can read more on potential commercialization here.

    3. Commercial release of bioprinted product for drug testing
    US-based company Organovo finally (it was long-awaited!) announced commercial release of liver tissue bioprinted product exVive3D:

    Organovo’s exVive3D Liver Models are bioprinted, living 3D human liver tissues consisting of primary human hepatocytes, stellate, and endothelial cell types, which are found in native human liver. The exVive3D Liver Models are created using Organovo’s proprietary 3D bioprinting technology that builds functional living tissues containing precise and reproducible architecture. The tissues are functional and stable for at least 42 days, which enables assessment of drug effects over study durations that well beyond those offered by industry-standard 2D liver cell culture systems.

    Well, good luck Organovo and let’s competition begin!

    4. Future perspectives of somatic cell nuclear transfer – interview with Dieter Egli
    This week, Paul Knoepfler interviewed Dieter Egli – a man behind the recent study about comparability of iPS and SCNT-derived stem cells. I find this interview very informative and productive. It touches many controversial topics. One excerpt:

    PK: What are your thoughts at this point as to the future of NT-ESC? Do you think they will have clinical relevance down the road as the basis for stem cell therapies or is that less likely now? In addition to possible translation to the clinic, is there more we can learn from NT-ESC and this kind of reprogramming?

    DE: It is neither more nor less likely. There is no therapeutic application of reprogrammed stem cell lines yet. There are so many uncertainties that depend on biological, economical, and logistical factors, and on the patenting landscape, that it is hard to predict the outcome. With these many uncertainties, it would be risky to put all eggs into just one basket. I think the nuclear transfer field is going to have a strong presence and will remain a primary research focus of my lab.

    Also read some discussion on recent controversy in comment.

    5. Long-term persistence of in vivo reprogrammed cells
    Very interesting study was published this week in Nature Biotechnology. Extending and building upon of famous Melton’s direct reprogramming 2008 study, the authors tested long-term persistence of in vivo reprogrammed beta cells and their therapeutic value. Excerpt from Harvard Gazette:

    “The efficiency of reprogramming has always been an issue,” Zhou said. “Until now, the new cells have either dropped dramatically in number or disappeared completely,” he said, noting that since his work with Melton in 2008 there have been reports published in other programing systems that question whether the reprogrammed cells could be stable enough ultimately to be useful.

    “What we have demonstrated is that yes, the reprogrammed cells can be useful, and for that to happen you have to create a niche environment in which the cells can survive,” Zhou continued. “We have improved the reprogramming efficiency to a point where one can create a large enough number of the new cells that the new cells create their own niche environment.”

    6. Identification of “youth factors” to boost aged brain
    We’ve written about parabiosis model for stem cell and aging research. Recent study from Stanford’s Wyss-Coray lab made a big buzz. This week, Shelly Fan reported from Society for Neuroscience meeting about extension of this work:

    The key is to setup a robust system that screens for strong candidate youth factors with few mistakes. Scientists harvested the blood from three different models: normal mice aging, human aging and the mice parabiosis model. The idea is to use a technique called protein microarray, which assesses changes in the levels of thousands of proteins at the same time, and to look for overlapping candidate factors across all three models. By looking for factors that decrease with age, the researchers would be able to narrow the candidates down to a tenable amount for further research. After finding these potential “hits”, researchers can then directly deliver each candidate protein to aged mice and see if the treatment improves learning and memory.

    7. How aging affects mesenchymal stem cells
    Another group of researchers from Stanford U, demonstrated that pro-vascular/ angiogenic qualities of mesenchymal stromal cells (MSC) could be especially affected with age:

    We identify an age-related depletion of a subpopulation of MSCs characterized by a pro-vascular transcriptional profile. Supporting this finding, we demonstrate that aged MSCs are also significantly compromised in their ability to support vascular network formation in vitro and in vivo. Finally, aged MSCs are unable to rescue age-associated impairments in cutaneous wound healing.

    The results of this study could have direct implications to autologous MSC-based therapy.

    8. Lessons from Dendreon’s failure
    Dendreon – the first US company with approved immunocellular product on the market – filed recently for bankruptcy. Mark Curtis wrote a nice summary of some lessons we can learn from Dendreon’s failure. Highly recommended!

    The cost density of PROVENGE would not have been such a dilemma for Dendreon if physicians’ claims were being processed in a timely manner. Due to the novelty of the product, payers were slow to the mark and doctors, apparently, grew tired of waiting for reimbursement.

    Many, if not all, cell-based immunotherapies will have similar issues given their high cost and the nature of their administration to patients. So, efficient frameworks for the reimbursement of advanced cellular products will be required in the future if we are to expect physicians to prescribe these treatments.

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    bonus:

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    Serial transplantation assay was developed as a “gold standard” for testing of self-renewal of hematopoietic stem cells. However, it has been very difficult to apply the same assay to other adult stem cells. Because of some technical difficulties with serial transplantation for assessment of mesenchymal stromal cells (MSC), their “stemness” was questioned. I’ve written about MSC self-renewal assays here and here. Today, I’d like to bring to your attention an abstract, which will be presented at annual ASH meeting in 2 weeks.

    A group of researchers from University of Lund (Sweden) were trying to answer the question: “Are MSCs stem cells?” They purified CD271+ MSC (linneg/CD45neg/CD271pos/PDGFRlow/neg) by FACS sorting from human bone marrow and performed in vitro and in vivo self-renewal assays. At the first step, the authors utilized clonogenic cell culture, performing classic CFU-F and recently described mesensphere assays. There was no difference between CFU-F and mesensphere-derived clonogenic MSCs in serial replating assay. The second step was a transplantation of MSC, derives from clonogenic cultures. Interestingly, only mesensphere-derived MSC performed well in serial transplantation assay in NSG mice:

    After 8 weeks, implants were harvested, human cells were FACS-isolated (CD90 and CD105 expression), and re-assayed under CFU-F and sphere conditions. Whereas in-vivo self-renewal of CFU-F could not be shown (111.5 ± 36 –fold decrease in total CFU-F numbers after primary transplantation, n=3), sphere self-renewal was clearly demonstrated by increased numbers of spheres after primary as well as secondary transplantation (1.13 ± 0.05 and 2.06 ± 0.26 –fold, respectively, n=3), which is remarkable given the fact that the number of recovered human cells is underestimated due to the isolation approach. Here, confirming GFP-marking experiments are ongoing.

    In primary and secondary transplant, they also tested differentiation capacity. Even though, there is no data on differentiation in abstract, the authors conclude that CD271+ mesensphere-derived MSC passed self-renewal test and, therefore, they are true stem cells.

    I have many questions about some details of the study (ex: why isolate CD90+ and CD105+ cells from primary recipient, but not CD271+?), but abstract is abstract. What I really like is methodological approach to test self-renewal of human MSCs. Combining of clonigenic in vitro assay with serial transplant seem like good idea. It reminiscent of Bianco’s assay, but still different. Two other things I learned: (1) CD271+ is good marker of human MSC and (2) mesensphere culture is very different from CFU-F and allow to maintain/ enrich true “stem MSCs”.

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    Cells Weekly – November 16, 2014

    by Alexey Bersenev on November 16, 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!

    CD34+Banner_490x90

    1. More on Doug Melton and betatrophin
    In the last month, Harvard’s Professor Doug Melton was all over the news. And there is a good reason for it – many people are interested in his research and willing to discuss it. My favorite blog post and discussion of the week was about Melton’s research. Retraction Watch posted some of his comments about recent “betatrophin refutation” story:

    That’s how science progresses sometimes. You make a claim, based on evidence. You then reinterpret it, you do more experiements, and you’re wiser for it in the end.

    Melton about anonymous discussions on PubPeer:

    It’s a complicated and evolving science. There are new papers coming out that I think are going to be very interesting to the community, so I’ve decided it’s not really helpful to talk to people who write comments like that, so I won’t be providing any rejoinder.

    I’d encourage you to read the whole discussion (20 comments on Retraction Watch and 17 comments on PubPeer), because it’s not just about betatrophon, but about science in general.

    2. Discussion on role of Nanog in pluripotency
    To continue on PubPeer discussions – two recent discussions (discussion 1/ discussion 2) of Nanog studies by Austin Smith group are heating up! Austing Smith replied to anonymous in discussion 1, but not in discussion 2.
    Very interesting discussions! So far fits in Melton’s paradigm “That’s how science progresses sometimes”.

    3. Long-term survival of neural stem cells in ALS trial
    This week, US-based company NeuralStem published very interesting study about postmortem findings in ALS patients after transplantation of investigational stem cell product. I wrote a post about it here:

    The question is that whether detection of donor DNA equivalent of cell survival? The authors said YES, and, most likely they are right. However, we cannot exclude horizontal transfer of donor’s DNA with apoptotic bodies, described before as phenomenon of chimerism.

    The biggest outstanding question remain: “Is donor cell long-term engraftment linked to therapeutic benefit”? It’s unclear from the pathology studies, but company believes that cell survival and efficacy are linked.

    4. Gene therapy for eye disease granted Breakthrough designation by FDA
    Gene therapy company Spark Therapeutics was granted Breakthrough designation by FDA. From press release (.pdf). The company is enrolling in pivotal Phase 3 trial for treatment of night blindness.
    This is the second Breakthrough Designation of gene therapy trials by FDA. The first was Celladon in April of 2014. Both companies is a good example of using therapeutic genes in regenerative medicine. FDA’s Breakthrough Designation means impressive results in early clinical studies and possibly could lead to accelerated approval on a market.

    5. Therapeutic value of mononuclear cells secretome in experimental stroke
    The most frequently used material in regenerative medicine trials – bone marrow mononuclear cells, usually do not engraft long-term, but cause some transient therapeutic effect. New study, posted in F1000Research, unveils potential mechanisms of such therapeutic benefit in stroke model:

    Administration of rat as well as human apoptotic MNC-secretomes significantly reduced ischemic lesion volumes by 36% and 37%, respectively. Neurological examinations revealed improvement after stroke in both treatment groups. Co-incubation of human astrocytes, Schwann cells and neurons with hMNCapo sec resulted in activation of several signaling cascades associated with the regulation of cytoprotective gene products and enhanced neuronal sprouting in vitro. Analysis of neurotrophic factors in hMNCapo sec and rat plasma revealed high levels of brain derived neurotrophic factor (BDNF).
    Our data indicate that apoptotic MNC-secretomes elicit neuroprotective effects on rats that have undergone ischemic stroke.

    6. Current status of cell therapy in sickle cell disease
    Nature posted very good piece on stem cell transplantation in sickle cell disease:

    The first hint that a milder approach might work came from examining the blood of children whose bone marrow had been destroyed in the usual way before the transplant. In a small proportion of cases, donor cells were found living alongside native cells that had managed to survive. Having as few as 11% of the donor cells in this ‘mixed chimaera’ state was sufficient to overcome the disease, and there was no evidence of graft-versus-host attack or immune rejection. The reasons for this mutual tolerance of host and donor cells remain unclear…

    7. Explanation of repair versus regeneration
    HEART blog gives very good short and illustrated explanation of difference between repair and regeneration.

    8. California stem cell experiment – 10 years on
    In 2004 California made a huge move and started unique government backed-up $3 billion stem cell program. David Jensen evaluates CIRM performance in the recent article. David is blogging tirelessly all about CIRM on California Stem Cell Report. I’d highly recommend you to read his evaluation and analysis on a blog!

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    Letters from regulators: Misbranding and adulteration of PRP device

    by Alexey Bersenev November 13, 2014 notes

    This is a second post of the 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. Today, i’d like to look at untitled letter by FDA to RegenLab America Inc. regarding use of Platelet-Rich-Plasma (PRP) making device RegenKit®THT. The letter was issued by April 19, 2013. The case is not recent, but very demonstrative. Background: There is a number of companies (mostly in cosmetic surgery […]

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    Cells Weekly – November 9, 2014

    by Alexey Bersenev November 9, 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. Comparison of somatic cell nuclear transfer and iPS cell technologies The method of the year of 2013 – somatic cell nuclear transfer (SCNT) – got into discussions this week. A group of researchers, led by Dieter Egli from NYSCF, published head-to-head comparison study of iPS versus SCNT-derived cells. Contrary to Mitalipov’s […]

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    Not Lost in Translation: Preclinical cell product development – analysis of FDA’s IND submissions

    by Alexey Bersenev November 7, 2014 cell product

    Earlier this year FDA published the first analysis of trends in IND (Investigational New Drug application) submissions to Center for Biologics Evaluation and Research (CBER)/ Office of Cellular, Tissue and Gene Therapies (OCTGT). Recently, the second trends analysis paper was released. This analysis is dedicated to preclinical cell product development. This is again a great job by FDA’s fellows and recognition of significance of cell therapy/ regenerative medicine field. Before I go through the paper, I’d like to share a […]

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    Defining of organoids

    by Alexey Bersenev November 4, 2014 cell culture

    Organoid is a buzz word today. Everyone is talking about organoids in the mass media and biomedical literature. But what are organoids? How can we define it? In simple terms organoid is miniature organ in vitro, but scientifically, definition could be more complex. I compiled a list of common properties, which can be used in defining of organoids. Organotypic culture. Organoid is cell/ tissue culture method, which is aimed to mimic organ structure and function. The other types of organotypic […]

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    Cells Weekly – November 2, 2014

    by Alexey Bersenev November 2, 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. Single unit versus double unit cord blood transplantation in pediatrics It was the most important and interesting clinical study of the week. Results of Phase 3 multicenter controlled trial, assessing single versus double unit cord blood transplantation in pediatric hematology-oncology, were published in NEJM. I remember the optimism about early preliminary […]

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    A call for testing of senescence in clinical MSC culture

    by Alexey Bersenev October 30, 2014 cell product

    I’ve written about potential significance of mesenchymal stromal cell (MSC) senescence here and here. As Luc Sensebé said: “Old cells will never work!” Yes, senescence can screw up the potency of your MSC-based product. It seem like MSC developers recognize this problem now, but what is resolution? I was wondering if any MSC product developer came up with clinically-relevant senescence assay, and I’ve asked this question on ISCT LinkedIn group. I did not get any reply. Recent study, published online […]

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