As you may know, Cell Therapy branch of FDA CBER has their own research lab, where they are working on mesenchymal stromal cells (MSC) characterization. A new study, which came up from CBER is investigating the issue of genetic stability of MSC. The findings from this study are somewhat surprising to me:

We found that chromosomal aberrations both exist and arise in culture-expanded MSCs and could be clonally propagated. However, in all cases, the aberrations diminished with extended culture, suggesting that they did not offer a replicative advantage…

I thought that clonal chromosomal/ genetic aberrations will only progress, lead to replicative advantage and tumorigenicity. Seem like it’s not always the case. As of today, most of MSC product developers do acknowledge the problem with genetic stability. However, seem like nobody knows for sure how to deal with it – how many cells with aneuploidy is enough for “NO GO” decision? Are these abnormalities harmful and could lead to tumorigenesis?

The authors detected chromosomal abnormalities in 6/10 MSC cultures, with value of abnormal cells up to 16%. In all donors, % of aberrant cells diminished (or even disappear in some donors) from 3rd to 7th or from 5th to 7th passages. However, due to low number of donors, this difference was not significant. The possible explanation of this prominent trend is replicative senescence and/or elimination of abnormal cells via apoptosis with prolonged culture adaptation to culture conditions. MSC subpopulations from 2/10 donors exhibited clonal karyotype abnormalities, which also diminished from 3rd to 5th passages and disappeared at 7th passage.

Couple of other interesting observations from the study: (1) There was a great variability of chromosomal aberrarions from donor to donor. (2) Frequency of chromosomal abnormalities does not increase with age (they checked 22-31 versus 39-41 years old donors). Interestingly, “young” donors had more aneuoploidy, but “older” donors had more translocations.

Take home messages from the study:

  1. Chromosomal abnormalities (including clonal) are exist and arise in human MSC culture
  2. Genetic stability of MSC is donor-dependent
  3. Karyotyping by SKY is good, robust and reliable method for screening of chromosomal aberrations in MSC cultures
  4. Passage 3 is good time checkpoint for “deciding the suitability of cells for further use”
  5. Genetic abnormalities exclusion limit for clinical use of MSC should be discussed and established.

The last message is especially important. The Cell Products Working Party as well as other groups have suggested such limits before. However, I don’t know if developers are following these recommendations and even karyotyping MSC products routinely.

Importantly, despite the trend for “loss of abnormal karyotypes with increasing passage” the study does not suggest to use MSC in advanced passages rather than in early (1-3) passages. The limitations of the study are (1) low number of donors and (2) absence of tumorigenicity testing.

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Cells Weekly – February 7, 2016

by Alexey Bersenev on February 8, 2016 · 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!


1. UK gives green light for CRISPR/Cas9 gene editing of human embryos in research
It was the most important event this week – a historic milestone! UK Human Fertilisation and Embryology Authority (HFEA) granted permission to Kathy Niakan (from Francis Crick Institute) to use CRISPR-based genome editing technique in studying development of normal human embryos. The reaction of scientists was mostly positive:

“It’s an important first. The HFEA has been a very thoughtful, deliberative body that has provided rational oversight of sensitive research areas, and this establishes a strong precedent for allowing this type of research to go forward,” says George Daley, a stem-cell biologist at Boston Children’s Hospital in Massachusetts.

Robin Lovell-Badge, a developmental biologist at the Crick institute, says that the HFEA’s decision will embolden other researchers who hope to edit the genomes of human embryos. He has heard from other UK scientists who are interested in pursuing embryo-editing research, he says, and expects that more applications will follow. In other countries, he says, the decision “will give scientists confidence to either apply to their national regulatory bodies, if they have them, or just to go ahead anyway”.

I’m feeling very happy about this decision!

@NatureNews polled public on a twitter about this decision – 62% agree with decision, 20% – disagree, 18% – not sure.

More coverage:
UK government agency approves editing genes in human embryos (STAT)
Kathy Niakan’s press conference (.mp3)

2. News on mitochondrial replacement therapy in US
US National Academies of Sciences had a meeting this week to discuss controversial mitochondrial replacement therapy (mitochondrial transfer or 3-donor IVF) and related risks and regulation. The panel issued a report, where recommended permissive regulation by FDA with some conditions.

In its report, the academy panel suggests limiting the tests of mitochondrial replacement to male embryos as a safety precaution. Male offspring would not be able to pass their modified mitochondria to future generations, because a child inherits its mitochondria from its mother.
The report also outlines several extra steps to monitor the safety of mitochondrial replacement. These include making every attempt to follow the children born as a result of the technique for years and sharing the resulting data with scientists and the public.
If mitochondrial replacement is proven safe in male offspring, it could be expanded to female embryos, the advisory panel said.

Unlike US, UK approved this procedure last year with no restriction on sex of embryos.

3. Update on Macchiarini’s saga
Famed regenerative medicine surgeon Paolo Macchiarini continues to struggle with allegations in research misconduct. This week, The Karolinska Institute, where Macchiarini has an appointment, started new investigation of potential ethical breaches. The trigger for investigation was new documentary about few patient stories and scandalous article in Vanity Fair. Later this week, Karolinska Institute says that they “lost confidence” in Macchiarini and will not renew his contract:

In an email to Science, KI spokesman Claes Keisu writes that Macchiarini has “overexploited Karolinska [Institute’s] brand in his work in Krasnodar. His activities there have undermined KI’s reputation and damaged the public’s trust in KI.” Discrepancies in Macchiarini’s CV contributed to the decision as well, Keisu writes.

Macchiarini did not immediately respond to a request for comment on KI’s decision. In an email to Science on Tuesday, he wrote that the TV series “was a gross misrepresentation of fact,” and that he couldn’t comment further at the moment. The university’s statement says that between now and November, the head of Macchiarini’s department will ensure that “Macchiarini uses his working hours to phase out the research he has conducted at KI” and that “the work of his research group is dismantled.”

In the light of Macchiarini’s investigation, Nobel Prize official Urban Lendhal has resigned. Reportedly, he advocated for Macchiarini’s hire by the Karolinska.

4. Elimination of senescent cell extends the lifespan of mice
Very impressive lifespan extension of mice was demonstrated in the study, published this week in Nature. Researchers from Mayo Clinic used transgene mouse model, which allowed them to eliminate senescent cell with p16(ink4a) phenotype by a drug. Using this approach, researchers were able to extend lifespan of mice up to 35%. The study yet to be reproduced, but the drug is commercialized already by Unity Biotechnology.

5. The first CRISPR genome editing company went public
US-based company Editas Medicine went public this week with $94.4M IPO.
In the light of CRISPR gene editing hype, it is a great opportunity to raise public money. Read Editas evaluation on Biotechr blog.

6. Cell culture media basics
All about cell culture media you can learn from the post on the Cell Culture Dish:

Cell culture is one of the most common and complex techniques used in the life sciences, and the media itself is a critical element for maintaining healthy, proliferating stem cells in culture. When you break it all down, all stem cell media essentially contain the same basic components: a basal medium, buffer system, glutamine, serum (or serum alternative), specific growth factors, and additional supplements. Here we look at a few of the main media components to better understand their importance and influence on cell cultures and how working with a highly pure and defined stem cell medium is key for regenerative medicine applications.

7. Impact of hypoxia on mesenchymal stromal cells regenerative function
Results of big preclinical study on primate model, which assessed impact of hypoxia on therapeutic potential of mesenchymal stromal cells (MSC), were recently published. This is very important study, because it assessed hypoxia versus ambient oxygen in cultured MSC on 49 monkeys for 9 months. The authors concluded that hypoxic conditioning of MSC worked significantly better:

HP improved the effectiveness of MSCs transplantation for the treatment of MI in nonhuman primates without increasing the occurrence of arrhythmogenic complications, which suggests that future clinical trials of HP-MSCs transplantation are warranted.

8. New methods and protocols:
Conversion of adult human peripheral blood mononuclear cells into induced neural stem cells (Stem Cell Res)
Xeno-free culture and cryopreservation of human adipose-derived MSC (Stem Cell TM)
Video bioinformatics for evaluation of process, quality and markers of pluripotent stem cells (PLoS ONE)
2D culture could be better for MSC than 3D (Stem Cells Int.)
Differentiation of human dental pulp MSC into dopaminergic neuron-like cells (J Korean Med Sci)
In vitro reconstruction of iPS cell-derived neuronal networks using microfabricated devices (PLoS ONE)
3D spheroid culture of umbilical cord tissue-derived MSC leads to enhanced paracrine function (Stem Cell Res Ther)


Last week, National Institute of Standards and Technology (NIST)/ IBBR hosted a meeting – CAR-T Biomanufacturing Symposium. Despite “CAR-T cell” in the title, many discussions, which took place during the meeting, were widely applicable to the whole field of cell therapy. As far as I know, it was the second conference, organized by NIST for cell therapy field. The first meeting – Strategies to Achieve Measurement Assurance for Cell Therapy Products took place last year.

You may ask – why NIST? Well, NIST is highly involved in development of standards and measurements for biomanufacturing industry. Cell therapy is relatively new, very fluid and rapidly evolving field, which currently is lacking standards in many aspects. In the last few years, NIST in collaboration with other organizations has started such cell therapy-related projects as cell counting, pluripotent stem cells and 3D tissue scaffolds. There are very new initiatives, supported by NIST, such as recently proposed Advancing Standards in Regenerative Medicine Act.

It seem to me that cell therapy developers are in agreement about potential benefits of collaboration in developing standards by NIST and other organizations. The most important reason for such collaboration was articulated by David Stroncek (NIH): “I do a lot daily in-process testing and product release testing with no controls – this is very bad practice!” (not because he is a bad, but because controls are not available). Talking of specifics, NIST is developing and would like to be involved in the following areas of cell therapy:
– enabling tools (devices calibration against standards)
– developing and publishing of protocols
reference materials
– analyticals (cell counting, cell characterization)
– bioprocessing (ancillary and raw materials, cell transportation and logistics)
– bioinformatics and modeling.

It is important to understand that NIST does not impose standards, they are discussed and accepted by consensus. The biggest impact for cell therapy could be standards in cell product characterization. Availability of reference material and ability to measure will help developers to understand how to characterize the cellular products.

Sadik Kassim (Novartis) presented very interesting data on variability in characterization of CAR T-cells by flow cytometry. He said: “Flow cytometry assays are the most variable assays in both – in-process and cell product release testing. Therefore, standardization of cell product analytics is usually related to flow cytometry standardization”. He thinks that automation of flow cytometry in clinical cell product characterization can provide potential cost-effective solution. Kassim, then discussed a problem with potency assays. All published CAR T-cell clinical studies cite only 2 types of potency assays – (1) level of interferon-gamma and (2) cytotoxicity by 51Cr release. Both potency assays are not very accurate and poorly differentiate responders versus non-responders. He said, that CAR T-cell products potency could not be captured in one dimension analytical readout. It is possible, that there may not be “one size fits all” potency assay for CAR T-cell products – it could be indication-specific or process-specific or effector cell-specific.

As expected, the potency assays was one of the hottest topics of this meeting. Somebody from NIST asked if they can develop and recommend few potency assays for the field. Victor Lu (FDA) responded that it will be very difficult to apply, because every cell product is usually unique. I think, it is still a good idea, because developer can look at the list of these potency assays, pick 1-2 and try to apply to specific product. It is better then nothing.

FDA was heavily present at the meeting and actively discussed hot topics. FDA has a great interest to standards development in cell therapy, because it will make their work easier – they will have something to compare with and something to reference. One important message from FDA folks was that they recognize variability between patients and challenges related to it. They would listen developers about all variables and advice. On the one hand, FDA wants to see potency assay with great correlation to clinical response, but, on the other hand, they understand how difficult it is to come up with such assay, considering specifics of alive cells as a medicine. So, try to do your best in developing potency to measure precisely biologic activity of your cells, but don’t kill yourself if it does not always correlate with clinical response. Another interesting remark from FDA was about potential collaboration between cell product developers. Each developer has their own dataset of assays (which FDA also has), but it is pretty much never shared. Seem like FDA encourages such sharing, since it could advance the field.

Throughout the meeting I felt like NIST folks are really really interested in cell therapy field. NIST people were very much engaged into all kinds of discussions and asked very good questions. It seem to me, they were trying to understand what is the best fit for them and what is the best area to get involved to help cell therapy developers. One good advice came from the panel – breaking up the whole field into small pieces and getting involved in some areas little-by-little will be more attractive approach. And the reason for this is a huge competition (CAR T-cell field is the best example) between companies, which are rushing to market and may see any sort of collaboration or consortia as distraction.

I was really enjoying the meeting and would highly recommend any other cell therapy meetings at NIST. Frank Gayle (NIST) said that currently in US there is a huge gap between innovation (R&D) and commercialization (mass manufacturing) and NIST sees its role in bridging this gap. I think, this is a great mission, because development of local manufacturing should be a national priority for every country.


Cells Weekly – January 31, 2016

by Alexey Bersenev on February 1, 2016 · 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!


1. Industry standards for regenerative medicine
Recently, new legislation was introduced to US Congress, entitled Advancing Standards in Regenerative Medicine Act. It was an initiative of Senator Baldwin. New bill will support of establishment “Standards Coordinating Body” for developing of standards.

The Secretary shall issue guidance, as appropriate, on how standards may be used in regulatory review for regenerative medicine and advanced therapies.

2. Generation of new synthetic logic gate receptors
A research group from UCSF, led by Wendell Lim, described new technique to manipulate of cell behavior through synthetic chimeric receptor. In the first paper, they introduced the concept of synNotch receptor. In the second paper, they used synNotch to “logically” switch-on CAR T-cells. From USCF press release:

“SynNotch receptors provide us with one of the most flexible ways we have to reprogram the behavior and function of almost any cell,” said Wendell A. Lim, PhD, chair and professor of cellular and molecular pharmacology at UCSF, and senior author of the two new papers. “One of the most exciting applications is in engineering therapeutic cells – we can now provide these cells with highly precise instructions about how to recognize and respond to disease.”

When synNotch-equipped T cells were injected into the mice, the T cells did not attack tumors expressing only the antigen targeted by the CAR, because the synNotch activation necessary for the CAR to be present did not occur. If both antigens were present, however, synNotch activation prompted the expression of the CAR receptor, which then recognized the second antigen and launched the T cell’s killing program.

3. A decade of iPS cell discovery
Cell journal has a nice feature, dedicated to 10 years of Yamanaka’s first iPS cell publication. First, they put great editorial – A Cell-ebration of Induced Pluripotency. Second, an iPS Timeline poster. Finally, paperclip podcast. As they said, more reviews are coming.

4. Advance in encapsulation of stem cell-derived beta cells
A study, published this week in Nature Medicine, was portray by mass media as “breakthrough” and “diabetes cure”. Well, for studies backed up by “big name” scientists from MIT and Harvard, this reaction is very typical. Encapsulation of human embryonic stem cell-derived beta-cells allowed to achieve long-term control of glycemia in diabetic immunocompetent mice. From STAT news:

The beta cells performed “every bit as good as the body’s own cells,” said Melton, a co-author on the Nature Medicine paper.
The transplanted cells controlled glucose levels in the mice without immune-suppressing drugs, the researchers reported. And when the scientists removed the capsules after almost six months, the cells were still cranking out insulin and there was little sign of an immune response to the capsules.

5. Surprising gene editing patent by Cellctis
This is a year old news, but it captured my attention very recently.
You all have heard about ongoing CRISPR patent battle between University of Berkley and Broad Institute. But, recent article in BioPharm Reporter discusses potential repercussions of the patent, which was awarded about a year ago to French company Cellectis and Harvard University. This is a broad patent, which covers gene editing techniques, based on chimeric endonucleases. The patent could cover variety of gene editing methods, including ZFN, TALENs, CRISPR and mega-TALE. BioPharm Reporter interviewed some experts and companies, which could be affected.

6. Patient sues stem cell clinic in US
Paul Knoepfler wrote about very interesting lawsuit case, where US Stem Cell Clinic (Florida) is being sued by their patient:

Importantly, neither U.S. Stem Cell, Inc. (USSC) and U.S. Stem Cell Clinic (USCC; owned by USSC) nor the individuals mentioned in the lawsuit have been found guilty of anything and this case is still pending so it is impossible to know for certain what did or did not happen.

7. New methods and protocols:
CRISPR/Cas9 editing of retinitis pigmentosa in patient-derived iPS cells (Sci Rep)
Engraftment and long-term safety of enteric neural crest cells transplanted into mouse gut (PLoS ONE)
Serum provokes gene expression variability in mouse ES cell culture (Cell Rep)
Generation of trunk neural crest from human pluripotent stem cells (Sci Rep)
Cell type of origin minimally affects gene expression in iPSCs (PLoS Genet)


Standardization of apheresis collections for consistent cell product manufacturing

by Alexey Bersenev January 30, 2016 cell product

With success and increasing interest to immunocellular therapies, apheresis became the most frequent incoming raw material for product manufacturing.  Everyone, who is involved in day-to-day processing of immunocellular therapy products, very familiar with a big variability of  apheresis collections. But, until now, professionals have not discussed openly manufacturing risks and potential solutions, related to variability […]

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Cells Weekly – January 24, 2016

by Alexey Bersenev January 25, 2016 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. CRISPR- “Lander saga” continues on social media As you know about a week ago, Director of Broad Institute, geneticist Eric Lander, published a review in Cell […]

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Lecture: Jacques Galipeau – Industrial MSC product failure analysis

by Alexey Bersenev January 23, 2016 lectures

Jacques Galipeau is a Professor at Emory University, Director of Emory Personalized Immunotherapy Center. Readers of this blog may remember Jacques Galipeau as the author of the first-of-its-kind analysis “why mesenchymal stromal cells did not work in industry trials“. He is prominent translational MSC researcher and an investigator in clinical trials. His talks and published […]

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Mogrify – a new computational tool for direct reprogramming

by Alexey Bersenev January 21, 2016 direct reprogramming

This week, international group of researchers described a new algorithm for identification of transcription factors, used in direct cell reprogramming. This is freely available computational tool, called Mogrify. This tool will allow to save a lot of time in finding the right transcription factor for conversion of one cell type to another. Before it was […]

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Cell/ gene therapy industry 2016 – expectations for market approvals

by Alexey Bersenev January 21, 2016 RegenMed digest

Last 3 years were not very successful for approvals of cell/ gene therapy products on the market. From zero (in 2014) to three (in 2013 and 2015) product approvals per year – very typical for the industry. I hope, this year will be a beginning of the change. In this post I’m trying to summarize […]

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Cells Weekly – January 18, 2016

by Alexey Bersenev January 18, 2016 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. CRISPR patents war is heating up This week there were a lot of news and emotional discussions on social media about interference between Doudna’s (U of […]

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