Stem Cell Assays

Serum-based versus serum-free clinical-grade culture of mesenchymal stromal cells – A poll

Alexey Bersenev — Fri, 18 May 2012 02:05:37 +0000

We’ve written a lot about the problem of serum use in clinical-grade cell culture. Today, I’d like to share more data and ask to express your opinion on this matter in our poll.

Use of serum and substitutes for ex vivo expansion of mesenchymal stromal cells (MSC) is a very hot topic. There are a lot of studies and protocols. Despite the fact that animal serum is not prohibited for clinical cultures, the development of commercial chemically-defined media for MSC is booming. Unfortunately, currently, there is no consensus on the optimal growth medium for MSC. Also, we are still lacking good comparative studies, which could analyze different kind of sera or serum versus artificial substitutes.

The considerations, which should be taken in account:

pathogen safety
MSC growth dynamics and quality
availability and cost

One of the recent studies has compared effects of different sera on adipose-derived MSC. They compared fetal calf serum (FCS), pretested fetal calf serum (FCS-Sp), human allogeneic serum (HS) or artificial serum substitute (AS):

We found that MSCs cultivated in the presence of different sera had similar global proteomic expression patterns, but comparisons of identified proteins revealed most differences in the MSCs cultivated with AS. Our results indicate that MSCs cultivated in the presence of FCS and HS display similar growth, differentiation, immunophenotypic and proteomic properties, while AS induces more profound changes in the physiology of MSCs, suggesting that further fundamental studies should be done before its introduction into clinical practice.

Sunghoon Jung comprehensively reviewed all pros and cons of defined serum-free media for clinical MSC culture. The authors currently doing a comparative study:

We have recently initiated a study to compare these commercial media along with other existing media. The media under investigation include Mesencult-XF (STEMCELL Technologies), StemPro MSC SFM Xeno-Free (Invitrogen), MSCGM-CD (Lonza), PPRF-msc6, and DMEM +10% FBS (Lonza). Our initial data show that the performance of the commercial hMSC media on the attachment and growth of primary BM-hMSCs is questionable and thus should be open to discussion.

It seem to me, that we still don’t have a good chemically-defined commercial serum substitutes/ media.

Now, I’d like to ask you: What is the best alternative to animal serum in clinical MSC expansion? Please vote in the poll:

For more information about humanized alternatives of animal serum, you can look at here.

PS: If you can not see a poll embedded in this post, follow the direct link.
PPS: If you represent a company, please refrain from obvious advertisement or promotion of your products in comments. Contact us about the promotion.

Alliance for Regenerative Medicine’s 2012 Report

Alexey Bersenev — Wed, 16 May 2012 03:55:43 +0000

Alliance for Regenerative Medicine has published 2012 Industry Report and made it available for the public.

It’s interesting and informative report. I’d encourage you to read it. A quick snapshot and some numbers from the report below:

Total number of RegenMed companies tracked: ~ 700 (similar to Proteus database);
65% companies located in US, 20% – in Europe, 15% – in other countries.

The most interesting part to me is representation of the value of cell therapy as a RegenMed sector. The Report is breaking down Regen for the following sectors:

Services and manufacturing – 9%
Tools and non-therapeutic products – 48%
Therapeutics (regenerative medicine) – 43%
The therapies composed of (1) cell and tissue-based therapies – 65%, (2) regenerative compounds and devices – 25% and (3) biopharmaceuticals – 10%.

So the total value of cell therapy of Regen industry is about 28%.
Interestingly, only about 25% of cell therapy companies are publicly traded. This is a critical issue, because many other reports make conclusions based on available data, listed by public companies. Such reports can not reflect the full picture and real situation in the RegenMed industry.

About commercial products:

There are approximately 300 cell and tissue-based therapeutics commercially available or in clinical development around the world, 55 of which are described and marketed as regenerative medicine products.

The top sellers cover mostly the following indications: orthopedics (38%), wounds/ non-cardiac ischemic (26%), skin (15%) and ocular (11%).

Finally, the trends for industry-sponsored cell therapy clinical trials:

From 2007 to 2012: a total number of trials up from 198 to 241; trials on Phase I – down from 77 to 52; Phase II trials – up from 89 to 148 and Phase II/III – up from 32 to 41

Alliance for Regenerative Medicine promotes legislative, regulatory and reimbursement initiatives on federal level. This is advocacy organization, which works closely with FDA and U.S. Congress. So, as professional organization, ARM has a power and authority to change current regulation and inject some federal money in regenerative medicine.

download report

**********************
RegenMed Digest on Stem Cell Assays is sponsored by Regenerative Medicine Jobs. Please visit Regenerative Medicine Jobs for recent position openings.

Cellular Therapies Manufacturing and Clinical Trials – CCRM Workshop

Alexey Bersenev — Tue, 15 May 2012 01:39:54 +0000

Canadian Centre for Commercialization of Regenerative Medicine (CCRM) is organizing the first Workshop for cell therapy manufacturing and clinical trials. This is a great opportunity to learn how to develop and advance cellular therapies into the clinic.

This workshop combines lectures, case studies and demonstrations to facilitate an appreciation of the multiple aspects of conducting clinical trials in Canada including preclinical studies, quality control and regulatory approval, as well as basic cellular manufacturing principles.

For more information, please follow the link.

PS: If you would like to promote your conference, meeting or training, please contact us!

Stem Cell Autopsy – a collection of clinical cases

Alexey Bersenev — Sat, 12 May 2012 03:16:46 +0000

More than 2 years ago I’ve started to collect a clinical cases of pathology (autopsy/ biopsy), from the patients who underwent cell therapy procedures. Initially I intended to cover only “stem cell therapy”, but later extended to all “cell therapies”. My analysis is limited to the cases, published in medical literature. My aim is evaluation of significance of such findings for professionals:

I believe that information from pathological findings will allow us to gain knowledge and avoid possible complications (sometimes fatal) in designing new clinical trials.

After some silence, the Stem Cell Autopsy is back! The reason for such refreshment is a new report, which includes the analysis of 18 autopsy cases after mesenchymal stromal cell infusions.

Intravenous infusion of matched allogeneic MSC does not cause severe complications, such as tumor or ectopic tissue formation due to rapid clearance from the recipient. More likely, allogeneic MSC act as “medicinal cells” shortly after infusion and do not engraft in the tissues. The persistence of donor’s MSC in recipient does not correlate with treatment efficacy.

I think, this is, so far, the best publication of 2012 in “stem cell therapy” field.

Dear readers, I’d like to ask you to contribute to this collection. Send me links to all autopsy cases that you know and we will discuss it!

Clinical cell processing news – part 1

Alexey Bersenev — Thu, 10 May 2012 02:03:43 +0000

In the new series of posts I’d like to review recent advances in clinical cell processing. In this series I’ll overview a literature and focus on some particular techniques.

1. Production and process of platelet lysate for MSC expansion (Cytotherapy) open access!
Fetal bovine serum replacement by platelet lysate (PL)

PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC.

2. Processing of mobilized blood products by counterflow centrifugal elutriation (Stem Cells TM) free full text upon registration

Our data demonstrate that, using standard elutriation procedures, >99% of red blood cells and platelets were removed from apheresis products with high recoveries of total white blood cells and enrichment of CD34+ cells in two of five fractions.
The processes were highly automated and closed from receipt of the apheresis product through formulation of target-enriched cell fractions. Thus, elutriation is a feasible method for the initial manipulations associated with primary blood cell therapy products and supports cGMP and current good tissue practice-compliant cell processing.

3. Sepax validation for bone marrow processing in cardiac cell therapy trial (Transfusion)

Although the Sepax procedure takes only 90 minutes, the total time for processing was approximately 7 hours. Contributing to this were incoming testing and device preparation, release testing, patient randomization, and product delivery.

4. Improvement of CD34+ cell enrichment from cord blood using CliniMACS (Cytotherapy)
Comparison of standard tubing set (TS) versus the larger capacity tubing set (LS):

… LS provided higher post-selection viability and more efficient recovery. In this case, a lower maximum TNC specification of TS was not predictive of better performance. The same may hold for smaller scale enrichment of other cell types with the CliniMACS instrument.

5. Validation of Sepax for adipose tissue processing (Tissue Engineering: Part C)

As compared with the manual process, automation resulted in a 62% higher isolation yield, with 2.6±1.2×10⁵ nucleated cells per mL of liposuction, and a 24% higher frequency of clonogenic progenitors. The variability in the isolation yield and clonogenicity across different preparations was reduced by 18% and 50%, respectively.

Please let me know what do you think about this series.

Trainings and courses in regenerative medicine

Alexey Bersenev — Tue, 08 May 2012 03:24:45 +0000

We’ve written about educational programs at universities and it was one of the most popular our posts. Professionals and young people have a great interest to education and career development in regenerative medicine. Today, we will focus on Master’s degree programs, summer schools and short courses and trainings.

Master’s degrees:
MSc program in regenerative medicine: clinical and industrial delivery at the University of Edinburgh (1-year, UK)
MSc Stem Cell and Regenerative Medicine at the University of Sheffield (UK)
Tissue Engineering for Regenerative Medicine MRes at the University of Manchester (UK)
Master of Science Degree in Stem Cell Biology at the University of Minnesota (US)

For junior researchers:
CIHR Training Program in Regenerative Medicine (Canada)
Summer School for Regenerative Medicine at Utrecht University (2 weeks, Netherlands)
Summer Internship Program at Harvard Stem Cell Institute (2 months, US)
Summer Scholars Program in Regenerative Medicine at the Wake Forest Institute for Regenerative Medicine (10 weeks, US)
Multiple iPS/ES training courses, aggregated on ISSCR web-site

For business professionals and cell therapy trialists:
Cellular Therapies Manufacturing workshops and GMP-training at the CCRM (Canada)
PharmiCell Cell Product GMP Manufacturing Training Service (Germany):

Pharmicell offers hands on training courses for manufacturing and quality control of adult human stem cells. The hands on course is accompanied by theoretical lectures and seminars on regulations, stem cells and practical solutions including qualification, validation, clean room monitoring, analytics and SOP development.

Annual Business Educational Course: Regenerative Medicine (US).

I’d like to note, that currently we have a lot of opportunities for research training in pluripotent stem cells culture. Unfortunately, there are no trainings in adult or neonatal stem cell isolation, culture and assays. Also, we are missing trainings in clinical cell processing and manufacturing, GMP and stem cell labs management.

If you know more information about trainings, courses and workshops, especially of those which we are missing, please send us a note or comment. If you would like to promote or give detailed information about your training for regenerative medicine and cell therapy professionals and students, please contact us.

**********************
RegenMed Digest on Stem Cell Assays is sponsored by Regenerative Medicine Jobs. Please visit Regenerative Medicine Jobs for recent position openings.

Till & McCulloch Meetings 2012 – Reflections on Caplan’s view of mesenchymal stem cells

Alexey Bersenev — Mon, 07 May 2012 02:19:41 +0000

Arnold Caplan is a scientist, who coined a term “mesenchymal stem cells” in 1991 for the type of bone marrow cells, previously known as “stromal stem cells”. This term became widely adopted. He gave a very interesting talk during plenary session of Till & McCulloch Meetings. I’ll try to summarize his current view on “Mesenchymal Stem Cells” (MSC) biology and clinical applications.

Caplan said that a term “Mesenchymal Stem Cells” got him in trouble later, because not everyone believed in their “stemness”. More than a year ago he proposed to use an alternative term “Medicinal Signaling Cells” instead of “Mesenchymal Stem Cells”. According our recent poll, professionals didn’t adapt yet a new MSC’s name.

Caplan believes that MSC are progeny of pericytes – the cells, attached to blood vessel’s wall in all tissues and organs. So, they are not stem cells, but progenitors. Ironically, nobody call pericytes as “stem cells”, even though they are MSC’s ancestors. Importantly, he thinks that cell definition should be functional – based on biological role in vivo. Right after blood vessel injury, pericytes give MSC progeny. MSC get activated in inflammatory environment and express their “medicinal” functions – immunomodulatory and trophic. So, MSC’s biological function has nothing to do with “stemness”. He noted: “We should focus on what these cells can do medicinally instead of what they can differentiate to”. Also, Caplan thinks that MSC have nothing to do with stroma. So, no to “stem” no to “stromal” in definition. As he said: “Turn your “stem cell brains” off and think!”

I’d argue, that in normal condition, bone marrow MSC have a stromal/ structural role as hematopoietic stem cell niche. Unfortunately, Caplan completely ignored this very basic MSC function, described by Alexander Friedenstein and Joseph Chertkov more than 30 years ago. I don’t think we can define and name a cell type, based only on their function in pathological conditions. Should it be two different names – “stromal” for normal condition and “medicinal” (activated stromal) for pathology? It is very confusing!

Caplan views MSCs as a “drugstores”, which can produce 10-20 biologically active substances. He thinks that FDA standards should not be applied to “live drugstores”. He said: “We can’t use Pharma logic in regenerative medicine”. I was thinking – why every MSC-based therapy developer trying to show “tri-lineage differentiation assay” in preclinical protocol and put it in product characterization? Based on medicinal signaling cell logic, it doesn’t make any sense. Almost all clinical trials in regenerative medicine currently assess medicinal functions of MSC, which have nothing to do with “stemness”. So, probably just one good potency assay is enough for product characterization. Ironically, everyone call it “stem cell therapy”.

From the recent therapeutic trends, Caplan is really excited by antibacterial function of MSCs. These cells can make natural antibiotic-like substances (LL37) in contact with bacteria. His data confirm and complement of studies by Matthay’s group. He also impressed by use of MSC in veterinary medicine. He credited Vet-Stem, which have treated more than 5000 horses for tendons injuries with 75% of cure rate. Finally, as a founder of Osiris, he noted: “Osiris data on Crohn’s disease is spectacular!”

Caplan concluded that we have to accept the paradigm shift from MSC characterization in vitro (tri-lineage differentiation) to medicinal function, which is irrelevant to concept of “stemness”.

2012 Till & McCulloch stem cell meeting – clinical translation

Alexey Bersenev — Wed, 02 May 2012 11:29:18 +0000

I’m currently attending Till & McCulloch meeting in Montreal. It has been a great conference with fantastic opportunity to network. I’m planning to cover some exciting, in my opinion, topics in few upcoming posts. Today, I’d like to summarize and share some of the talks on clinical translation of stem cell therapies.

There were a lot of great “translational talks”, capturing the problem from different perspectives. Tania Bubela from U of Alberta has indicated that unrealistic public expectations is a major non-scientific challenge in clinical translation. Her team have identified a large discrepancy between public expectations (measured by mass media coverage) and real picture (publications, clinical trials). The problem is that if these expectations are not addressed, support for stem cell research and translation could be seriously affected.

Yet another challenge, noted by many speakers, is regulatory framework. Tania Bubela has proposed that in order to solve discrepancy between public expectations and delivery of real stem cell therapy, we need to have rational conversation with regulators. By answering my question about who and how should do that, she pointed to professional network organizations, such as Canadian Stem Cell Network, which could play an important role.

Despite the non-scientific challenges (over-hyped public expectations and regulation), we are still frequently lacking a good science and evidence in pre-clinical stages. Brian Kwon has delivered an excellent talk on readiness for clinical trials in spinal cord injury. He asked: “Are we ready for real thing?”. His systematic analysis indicated that, in fact, we are not ready. Most of therapeutic goals in research are reportedly being achieved, but models are not relevant and reproducibility is lacking. For example, preclinical work on Geron’s trial was never independently reproduced, never done on large animals, yielded a little therapeutic benefit and approval was based entirely on single paper. Taking in account the huge cost of the trials and a length of 10-15 years, we shouldn’t think of it as a casual thing that we can easily try and see what happen. Each criteria for pre-clinical testing, proposed by professionals, should be rigorously assessed and checked. He said: “Is the science good enough? – stop kidding yourself!”

Tim’s Caulfield team has done very interesting work on influence of “external factors” on stem cell researchers. They surveyed members of international stem cell research communities and found that the pressure for clinical translation was moderate in 64%, intense – in 24% and non-existent in 12%. Emphasis on economic benefit of stem cell research was indicated as “little” by 44% of researchers, big – 40% and none – 14%.

One of the general themes of the conference was the agreement on pivotal role of collaboration in clinical translation. Collaboration on all possible levels! Bartha Knoppers called for solidarity in stem cell research and creation of “Data Commons”. Traceability and identifiability are becoming extremely important criteria of a progress in stem cell research. During the commercialization session, Greg Bonfiglio said: “Think about your competitors as potential collaborators”. He thinks that collaboration is a future key element in successful commercialization of regenerative medicine. Bongfilio also emphasizes the role of academic-based translational centers, which could serve as “bioincubators” for the training and early stages of cell product development. One of the examples of such center is Canadian CCRM.

Also read:
View from the floor 1: Till & McCulloch Meetings
View from the floor 2: Till & McCulloch Meetings
twitter: #TMM2012

Do we need to assess genomic stability on culture expanded cells for therapy?

Alexey Bersenev — Sat, 28 Apr 2012 03:33:23 +0000

I came across of very interesting opinion piece, written by Darwin Prockop in Stem Cells journal. He comments on the study about genomic instability of human adult stem cells, published last year and discussed here.

The authors of that study have concluded:

…validating the genomic stability of stem cells of all types in culture is crucial …for their safe implementation in cell therapy.

We highlighted this study and were in favor of development specific assays for genomic stability of cell products.

Prockop argues that there are no surprises here. We been here before and such tests could have very limited value in cell therapy. He gave a historical example, where in the 1960′s vaccine manufacturers were required to test cells for karyotype and tumorigenicity in vivo:

Concern about the chromosomal stability of cells in culture arose over 40 years ago when Wi-38 human fibroblasts were first introduced by Hayflick et al. [2] as substrates for viruses to develop vaccines. Manufacturers of vaccines were required to ensure that the cells were free, not only of any adventitious agents, but also of what were then referred to as “any neoplastic properties”.

Interestingly, these requirements caused a lot of confusions, because of inconsistency in results of such tests.

Most importantly, there were no differences in growth characteristics or in tumorigenicity in mice between the cultures that did or did not contain t(7:12).

So, the historical attempts to implement genomic stability and tumorigenicity assays in biotechnology, left us with uncertainty:

Where does this leave us in evaluating the potential dangers of therapies with cells that are expanded in culture?
What are the appropriate tests? Given that most human cancer cells do not produce tumors in immune deficient mice, the assay for tumorigenicity using nude mice has unacceptably low sensitivity.

He proposed that the most simple and reliable test today is a senescence of cultured cells:

Do the cells senesce in culture? If they do, they are unlikely to produce tumors or malignancies in patients. Unlikely in the sense that there is a low probability, but not a certainty, that the expanded cultures do not contain a few cells that have acquired an oncogenic mutation. In contrast, cells that are immortal in culture carry a serious risk. Unfortunately, at present there is no strategy for eliminating this risk.

Examples:

Embryonic stem cells and induced pluripotent cells do not pass the test of senescence in culture. Therefore the risk/benefit ratio is likely to be high. In contrast, the adult stem cells referred to as mesenchymal stem cells or mesenchymal stromal cells (MSCs) currently in use in a large number of clinical trials reproducibly senesce in culture.

Prockop comments intensively on this issue. His opinion is very interesting and definitely should be taken in consideration. I’d agree, that for routine cell products manufacturing, tumorigenicity in vivo test doesn’t hold any future. It should be done in pre-clinical phase, before starting a trial. But for routine cell manufacturing a few assays should be developed. Simple, cheap and reliable assays. For example, a chip for the most nasty mutation, which always gives tumors in mice. Karyotype, senescence – all good. Also, different cell products will require different tests for genomic stability. It will depend on starting cell type – mature versus progenitor versus stem cell, number of passages and population doublings in culture, condition treated and so on. What do you think?

Are mesenchymal stromal cells actually fibroblasts?

Alexey Bersenev — Fri, 27 Apr 2012 03:53:21 +0000

Peiman Hematti in his recent opinion piece, has argued that until now we don’t know any distinct differences between cultured mesenchymal stromal cells (MSC) and fibroblasts.

He noted, that since the Friedenstein’s pioneering work, nobody was able to address the problem of MSC identity. In fact, such characteristics of MSC and fibroblasts as morphology, phenotype, gene expression profile, immunologic properties, tissue repair mechanisms are remarkably similar or even indistinguishable.

… there are no distinct differences in culture-derivation methodology, morphology, cell-surface marker expression pattern, differentiation potential and gene expression signature that consistently and unequivocally distinguish ex vivo culture-expanded ‘MSC’ from ‘fibroblasts’.

Some examples of “mistaken and confused identity”, that he gave in the article:
Surface markers and tri-lineage differentiation:
Multilineage differentiation potential of human dermal skin-derived fibroblasts
MSC versus pericytes versus fibroblasts
Dermal fibroblasts versus fat-derived MSC

Immunologic properties:
Antiproliferative effect
Immunosupression of T-cells: 1, 2

Interestingly, he pointed out some clinical questions, such as:

If MSC are indeed fibroblasts, and fibroblasts are responsible for fibrosis, should we be concerned about the potential for fibrogenic effects of MSC given to treat inflammatory conditions? The answer is probably no, because so far there is no evidence to suggest that MSC engrafts long-term after infusion, despite clinical benefits observed (40). Nevertheless, some patients receiving MSC in a clinical trial for treatment of refractory chronic graft-versus-host disease (GvHD) developed bronchiolitis obliterans; although causality between receiving MSC and the development of this fibrotic lung disease is not certain.

He concluded:

Forty years after the first description of bone marrow fibroblasts by Friedenstein, and despite the introduction of the more popular term ‘MSC’, little has changed about the way we derive and culture these cells. There is no accepted single cell-surface marker for their isolation and, because they are ex vivo culture-expanded from a small population of cells, there is still no sure way to trace them back to their in vivo counterparts. Our efforts to name these fibroblast-looking cells in culture can be likened to the old Indian tale of six men in a dark room asked to describe an elephant.

Very interesting opinion piece. Highly recommended!
Also read: Mesenchymal stem cells: the fibroblasts’ new clothes?