Possible issues in cell preparation for cardiac cell therapy trials There are multiple clinical trials, assessing the efficacy of autologous bone marrow mononuclear cells in acute myocardial infarction. With a recent results of the TIME study, all 3 clinical trials (FOCUS, LateTIME and TIME), supported by US Federal Government and coordinated by Cardiovascular Cell Therapy Research Network (CCTRN), considered as failed. Everyone today is discussing the possible reasons of failures. It could be trial design, end points, statistical analysis, cell source and cell product preparation. Maybe cells simply don’t work! [...]
In the previous post we defined adipose-derived: stromal vascular fraction (SVF) versus stromal (stem) cells and highlighted difference between them. Today we will look at SVF more precisely and define its composition.
SVF is freshly isolated heterogeneous cell fraction, which could be derived from native adipose tissue or liposuction aspirates. SVF could be derived from both – the fatty and fluid portions of liposuction aspirates after enzymatic digestion. If SVF derived from fatty portion, it’s also called PLA (processed lipoaspirates) cells.
Basically, SVF is what remained in the pellet after removal blood and fat components. It is very crude and heterogeneous mix of multiple cell populations with different degree of maturity and function. I draw a scheme of SVF isolation and composition:
Based on method of adipose tissue processing, cellular composition of SVF can vary significantly. Most sources indicate that adipose-derived stromal (stem) cells represent up to 10% (2-10%) of SVF. Endothelial cells (mature and progenitors) could represents anything from 7% up to ~30% of SVF. Depending on processing, fibroblasts could represent up to 50% of SVF (Cytori data, presented at ISCT 2010). CD34+ cells are present at large number and could compose up to 63% of SVF.
It has also been described that the SVF is composed of 11% CD2+ cells, 18% CD11a+ cells, 29% CD14+ cells, 49% CD31+ cells, 57% CD45+ cells, and 60% CD90+ cells (referring to ASCs and endothelial cells) [source]. Others detected a different composition of the SVF (nearly 11% CD14+ cells, ~2% CD31+ cells, ~7% CD34+, ~9% CD45+ cells, ~29% CD90+, and ~47% 146+ cells) [source].
The SVF of human adipose tissue contained: endothelial progenitors, (15.4 ± 4.8)% (mean ± standard error), pericytes (2.0 ± 1.1)%, CD146+/CD34+ transitional cells 0.5 ± 0.3, and SA-ASC (59.0 ± 10.0)% of non-hematopoietic (CD45−/CD14−/CD33−/glycophorin A−) singlet cells.
To summarize:
SVF composed of many mature, progenitor and stem cell types. Depending on adipose tissue processing method, the composition of SVF and relative values of each cell population can vary significantly. Adipose-derived stromal (stem) cells represent 2-10% of SVF.
*************** This post is a part of series Breaking Down Fat. In this series we will talk about identification, characterization and clinical processing of potentially therapeutic cell populations from adipose tissue. We started this series in response to the growing trend of wide (mostly uncontrolled) clinical use adipose-derived cells and some controversies/ misconceptions in the field.If you would like to contribute to this series or become a sponsor, please contact us!
The success came through minor technical tweaks. The researchers used inactivated Sendai virus (known to induce fusion of cells) to unite the egg and body cells, and an electric jolt to activate embryo development. When their first attempts produced six blastocysts but no stable cell lines, they added caffeine, which protects the egg from premature activation.
Now we have one more way to make autologous pluripotent cells for therapies! There were some warnings in the mass media about potential human “cloning hysteria”, based on results of this study. However, Mitalipov said:
“The embryos we produce this way did not lead to pregnancy in monkeys,” he says. “We think there is something in the manipulations to make them that make a successful pregnancy impossible.”
Overall, this paper is fascinating and a huge development, but this is a double-edged sword too.
…the elephant in the room for this paper is the potential for future reproductive human cloning.
I think, that the potential problem of human reproductive cloning is definitely overblown! Obviously, this is the most exciting and significant news in stem cell research of this year so far! What more interesting is a debate about medical potential of pluripotent stem cells, derived from iPS cells versus SCNT. I think, generation of therapeutic cells via SCNT is absolutely worthy to pursue! It has some advantages compare to iPS cells.
But Stojkovic, like others, awaits the results of head-to-head comparisons between iPS and SCNT cells. Some research has shown that iPS cells are not completely reprogrammed and that stem cells derived from SCNT are more like embryonic stem cells derived from in vitro fertilization. Mitalipov and Tachibana are now conducting a study to compare iPS cells and SCNT cells derived from the same donor cell. “These results,” says Daley, “will be fascinating.”
I’ve collected some links and opinion about this great news in storify:
Cells Weekly! is a digest of the most interesting news and events in stem cell research, cell therapy and regenerative medicine. We pick and post it every Sunday. Follow us!
1. Circulating factor able to “rejuvenate the heart”
For a decade, Harvard’s investigator Amy Wagers was looking for factors circulating in the blood and able to rejuvenate aged organs and tissues. Her studies, methodologically based on conjunction of circulation young and old animals, finally brought some results. For the first time, Wagers and Richard Lee’s labs were able to identify a factor, circulating in blood of young animals – Growth Differentiation Factor 11 (GDF-11), which reversed age-related heart hypertrophy.
Obviously, this is just one factor from many. Taking in account rapidly increasing aged population, this study can provide a platform for future identification of other “rejuvenating” factors and have significant impact in regenerative medicine.
2. Japan proposed a rapid track for stem cell-based therapies regulation Nature reported on new proposal for regulation of stem cell therapies in Japan:
A retooling of Japan’s drug authorization framework, on its way to becoming law, could produce the world’s fastest approval process specifically designed for regenerative medicine.
Rather than using phased clinical trials, companies will have to demonstrate efficacy in pilot studies of as few as ten patients in one study, if the change is dramatic enough, or a few hundred when improvement is more marginal.
With the bar for regenerative therapies dramatically lowered by requiring only limited safety and efficacy data—and essentially doing away with the need for high-powered phase 3 trials—the amendments’ architects say it will be possible to get a stem cell treatment to the market in just three years, rather than the typical six or more.
We will watch how this proposal will be accepted and implemented in Japan.
On December 13, 2012, concerned about the safety of the increasing number of individuals with ALS receiving adipose-derived stem cells at Precision StemCell, I contacted the FDA’s Center for Biologics Evaluation and Research and urged FDA officials to investigate the clinic. I took that step because it appeared to me that Dr. Williams was violating federal regulations, putting stem cell recipients at risk of harm, and charging for interventions that had not first been tested for safety and therapeutic efficacy. Dr. Williams’ administration of stem cells to a minor also played a role in my decision to send a letter to the FDA.
Will FDA investigate this case. More likely YES. Has FDA a muscle to capture and inspect all clinics like that in US? More likely NO.
4. More on Italian stem cell therapy regulation scandal
We’re following the recent scandalous case of deregulation of stem cell therapy in Italy. This topic is highly discussed all over online professional communities for the last 2-3 weeks. This week, physician-scientist and popular blogger David Gorski posted here and here about Italian case:
One point that the scientists didn’t really nail, and that’s the issue of informed consent. One notes that Vannoni’s stem cell quackery has no evidence for it published in the peer-reviewed biomedical literature nor any compelling clinical trial results. Consequently, if claims are being made for this treatment it is impossible to give informed consent because there is no evidence upon which to base even a rough estimate of the chances for success are weighed against the risks of the treatment. Even worse, we don’t even know that these are really stem cells. Seriously. As the scientists point out, there is no transparency, and if there’s an area of clinical research where transparency is essential, it’s stem cell research.
His post generated nearly 200 comments from both blogs! I’d highly recommend you to read his post and comments here and here.
The overall gestalt is that iPS cells are unlikely to be immunogenic so that’s really not a big concern today, but people do remain concerned, first of all, about epigenetic issues such as epigenetic blemishes in iPS cells including memories ,and secondly most people are still convinced that iPS cells have a handful of mutations.
Very interesting and importnat information!
6. Skin into the heart – more on cellular alchemy
Professor Jalees Rehman in his recent post discusses the conversion of fibroblasts into functional heart cells:
Where does this whole body of work leave us? One major finding seems to be fairly solid. Fibroblasts can be converted into beating heart cells. The efficiency of conversion and the quality of the generated heart cells – from mouse or human fibroblasts – still needs to be optimized. Even though the idea of cellular alchemy sounds fascinating, there are many additional obstacles that need to be overcome before such therapies could ever be tested in humans. The method to introduce these genes into the fibroblasts used viruses which permanently integrate into the DNA of the fibroblast and could cause genetic anomalies in the fibroblasts. It is unlikely that such viruses could be used in patients.
Because of huge interest in using adipose-derived cells as “therapies”, it is important to know what is a cellular composition of this tissue. You may frequently hear a statement about fat tissue as a “richest source of stem cells” in our body. What kind of stem cells exist in adipose tissue? Can we define them?
We can roughly break down fat tissue on mature adipocytes, blood and everything else. The latter frequently called “stromal vascular fraction” (SVF). SVF is enriched for multiple progenitor cell populations and stem cells. SVF was isolated for the first time by Rodbell in 1964 using proteolytic enzymes and centrifugation. The “multipotent potential” of stem cells from human fat-derived SVF-like population was characterized by Zuk in 2001. Zuk called these cells “PLA cells”, because of starting material – processed lipoaspirates.
In the last decade there were a lot of confusions about nomenclature of stem cells, derived from adipose tissue. Some professionals called SVF as “stem cells”, but some use this term only for the cells, propagated in culture. Until now, you can find a multiple terms for adipose tissue-derived stem cells, used as synonyms in literature. For example:
adipose-derived adult stem (ADAS) cells;
adipose-derived adult stromal cells;
adipose-derived stromal cells (ADSC);
adipose stromal cells (ASC);
adipose mesenchymal stem cells (AdMSC);
preadipocytes;
processed lipoaspirate (PLA) cells;
adipose-derived stromal/stem cells (ASCs);
lipoblast;
pericyte
International Federation for Adipose Therapeutics and Science (IFATS) has a leading role in establishing nomenclature and standards for using adipose-derived cells in medicine. About 6 years ago, IFATS recommended to use a term “adipose-derived stem cells” (ASCs) “ to identify the isolated, plastic-adherent, multipotent cell population“. However, IFATS later statementdoes not recommend to use term “stem cells”, but use “adipose tissue-derived stromal cells (ASCs)” instead to characterize culture-propagated cells. Therefore, despite IFATS recommendation, there is no clarity in literature on using term “stem cells” for adipose-derived cells, propagated in culture:
Recognizing the validity of the term “stem cell” may be questioned; it is accepted that some investigators will use the acronym to mean “adipose-derived stromal cells.”
Unlike cultured adipose stromal cells, the consensus on using of term “stem cells” to describe SVF, has been reached. Taking in account of high heterogeneity, the term “stem cells” should not be applied to SVF cells, freshly isolated from adipose tissue. The difference between SVF and ASCs is significant! Unlike SVF, ACSs relatively homogenous, plastic adherent population and serially passaged in culture.
Since, cultured SVF cells remarkably similar to bone marrow-derived mesenchymal stromal cells (MSC), the most professionals consider ASCs as an equivalent of MSC. However, other stem cell populations were described in adipose tissue, such as pericytes (considered as equivalent of MSC by some researchers), supra-adventitial adipose stromal cells and hematopoietic stem cells.
To summarize: Stromal Vascular Fraction (SVF) – freshly isolated heterogeneous cell fraction, isolated from native adipose tissue or liposuction aspirates. Adipose-derived stem cells = adipose-derived stromal cells (ASCs) = adipose-derived MSC – homogeneous, plastic adherent cell population, derived from SVF and propagated in culture.
*************** This post is a part of series Breaking Down Fat. In this series we will talk about identification, characterization and clinical processing of potentially therapeutic cell populations from adipose tissue. We started this series in response to the growing trend of wide (mostly uncontrolled) clinical use adipose-derived cells and some controversies/ misconceptions in the field.If you would like to contribute to this series or become a sponsor, please contact us!
Clinical Cell Processing News series overviews new protocols, products and techniques for clinical-grade cell processing and manufacturing. Cell processing devices, cultureware, bioreactors, GMP-grade reagents, cell separation techniques. Follow us! 1. Validation of dry-thawing device for hematopoietic cell products (Transfusion): Progenitor cell viability and function are preserved with this dry-thawing system. The time to hematopoietic engraftment of patients after transplantation is comparable to those infused with progenitor cells thawed with the water bath technique. 2. Validation of automated system for cord [...]
Welcome to Cells Weekly! We’re trying to navigate you in the ocean of information by picking the most interesting news and events in stem cell research, cell therapy and regenerative medicine! 1. Transplantation of tissue engineered trachea in US – Hannah Warren’s case This was a biggest mass media buzz this week. An international team, led by famous Paolo Macchiarini, has performed the first tissue engineered trachea transplant in US. You can learn more from press-conference: This case is really [...]
We’ve written a lot about assessment of tumorigenicity risk in mesenchymal stromal cell (MSC)-based products. Recently, recommendations of the Cell Products Working Party and the Committee for Advanced Therapies (from meeting held in October 2011) have been reviewed and published online in Cytotherapy. This is very interesting and useful review, which summarizes current experience and position of regulatory agency on MSC products risk assessment. Some conclusions and recommendations from this meeting: Due to cancer cell lines contamination issues, it is [...]
This is third post of our series “Not Lost in Translation“. Please feel free to contribute by a post or comment! ********************** The necessity and significance of animal models in cell therapy Cell therapy is a field of medicine, which highly dependent and simply can not succeed without animal models. The necessity of animal testing is very high and only in some rare cases it could be replaced by in vitro assays. Besides proof-of-principle experiments (aimed to demonstrate that cells [...]
Cells Weekly is a digest of the most interesting news and events in stem cell research, cell therapy and regenerative medicine. 1. Edogenous hormone boosts proliferation of insulin-producing cells It was the most significant scientific paper of the week. Harvard’s Doug Melton’s lab discovered a betatrophin – activator of pancreatic β cell replication. Learn more about discovery from this video: This study is a good example of successful screening for new endogenous regulators of cell proliferation. It also shows that [...]
The minimal criteria for defining of human mesenchymal stromal cells (MSC), recommended by International Society for Cellular Therapy (ISCT) in 2006, have been critically evaluated and considered as outdated. One of concerns is that criteria does not reflect the function, which tested in clinical trials. As a response to this criticism, the MSC Committee of ISCT has released a working proposal for a standardized approach to assess immunological function of MSC, explored in clinical trials. The aim of this proposal [...]
