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A Stem Cell Count Would Have Made It Better:
RoosterBio: Bringing Attention to Cell Changes During Stem Cell Expansion Culture

Entry #6
Wednesday, November 17, 2021

In this week’s entry, Asymmetrex congratulates human mesenchymal stem cell (hMSC) production company RoosterBio on its recent September 29 website blog titled, “What Is Population Doubling Level (PDL) & Why Is It Important for Cell Age?” The PDL, also called cumulative population doublings (CPD) in the historical literature and by Asymmetrex, is an important operational factor for primary mammalian cell cultures that is also an indicator of changes in their biological properties with increasing numbers of passages. The blog authors correctly inform that PDL or CPD is a better quantitative basis than the number of cell passages for comparing the culture experience of different cell preparations. Whereas PDL or CPD is like a state function for the natural history of a cell population in culture, passage number does not account for important culturing variables like transferred cell number and passage interval time. Evaluations based on PDL or CPD give a better indication of the relative biological properties of compared cultures.

Unfortunately, the RoosterBio blog commits the common simplification of using the metaphor of “cell age” to represent the well-known changes in the biological properties of primary human cell populations with increasing PDL. It is this feature, for which ideas on tissue stem cell counting would have made their blog post better.

It is now well-described that primary human tissue cell populations have a natural history during serial culturing that reflects a universal in vivo tissue cell kinetics turnover unit program. Tissue stem cells divide asymmetrically to produce committed progenitor cells. The progeny committed progenitor cells divide transiently until their lineages yield terminally-arrested cells. The exact in vivo tissue cell kinetics and differentiation are not maintained in culture, but invariably the fundamental cell kinetics program is. A priori, calling a process that continuously produces new young cells “aging” is an oxymoron. Though it is possible that the genomes of stem cells in the culture might “age” due to stable epigenetic and genetic changes, the more significant biological event is their loss due to dilution among the terminal lineages of their own progeny cells in culture.

Whether the effects of high PDL are called aging or tissue cell turnover unit evolution, RoosterBio is wise to pay attention to it; and they provide an important service to the stem cell medicine community by bringing more attention to it. However, for tissue stem cell manufacturing companies like RoosterBio, a better approach than limiting PDL is simply monitoring tissue stem cell fraction, which in current commercial manufacturing processes rapidly declines with increasing PDL…or CPD.

We contacted Jon Rowley, Founder and Chief Product Officer of RoosterBio, to get the company’s impressions on these ideas, but we received no response.

Next Review: December 1, Sartorius: How Can the Evaluation of Tissue Stem Cell Expansion Media Be Enhanced?

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A Stem Cell Count Would Have Made It Better:
Developing HSC Gene Editing Therapies for Sickle Cell Disease

Entry #5
Wednesday, November 3, 2021

This entry of Asymmetrex’s review of opportunities for improvement with KSC counting of therapeutic tissue stem cells focuses on a case in the emerging field of gene-edited stem cell therapies.  Because of its well-defined and simple genetic basis, sickle cell disease (SCD) is one of the early therapeutic targets for this new technological approach to curing debilitating illnesses that have an inherited genetic basis.

In a recent research article in Science Translational Medicine (Sci Transl Med. 2021 Jun 16;13(598):eabf2444. doi: 10.1126/scitranslmed.abf2444), Dr. Annalisa Lattanzi and co-authors reported their studies performed to support a future investigational new drug application for SCD gene editing clinical trials.  They quite successfully used cell reconstitution analyses in immunodeficient mice to evaluate and optimize CRISPR-Cas9 gene editing to correct the well-known single A to T change in the human beta-globin gene sequence that causes SCD.

These authors very appropriately referred to the mobilized CD34+-selected blood cells used for gene editing as hematopoietic stem and progenitor cells (“HSPCs”).  They were well aware that the hematopoietic stem cells (HSCs) are the critical cells for achieving successful gene editing therapies that have lifelong durability.  For this reason, their essential read-out for success in the study was long-term engraftment of gene-corrected HSCs (gcHSCs), which they demonstrated in multiple ways.

A major focus of the report was evaluating Cas9 variants with greater editing fidelity and modified culture conditions to achieve greater numbers of HSCs, towards obtaining higher rates of long-term engraftment with safer gene-edited HSCs.  The authors conducted these optimization analyses with only their final gcHSC engraftment results to assess their effectiveness.  That assessment required a minimum of 16 weeks in mice.  So, it was unable to evaluate effects of their optimizations at earlier stages in their development process that would have been informative.  A method for routine counting of HSCs at earlier stages would have improved their analyses in many respects.  For example, it would have allowed evaluation of more test conditions, some which might have proven better than the ones that were identified in their current study.

We invited the authors to consider this review of their report and provide a response, but did not receive a reply from them.  Perhaps some of our readers have thoughts to add…


A Stem Cell Count Would Have Made It Better:
Umbilical Cord Mesenchymal Stem Cell Treatment for COVID-19

Entry #4
Wednesday, October 20, 2021

Earlier this year, Dr. Camillo Ricordi and colleagues at the University of Miami Miller School of Medicine in Miami, Florida published an interesting stem cell clinical trial report in the journal Stem Cells Translational Medicine. Their report details the results of a double-blind, Phase 1/2a, randomized controlled trial to evaluate the safety and efficacy of banked human umbilical cord-derived mesenchymal stem cells (UC-MSCs) in the treatment of acute respiratory distress syndrome (ARDS) associated with COVID-19.

Although a small study with only 12 patients in the randomized control and treatment groups, the authors report the detection of statistically significant efficacy outcomes for two important clinical endpoints, patient survival (P = 0.015) and time to recovery (P = 0.03). Consistent with current concepts that observed beneficial effects of MSC preparations are due to reduction in detrimental inflammation, within 6 days of treatment, treated patients showed a significant decrease in the examined inflammatory cytokines.

The treating cells for the trial were allogeneic cells from a master bank of expanded UC-MSCs. It is this feature of the study for which tissue stem cell counting would have made it better. Visit Asymmetrex’s Stem Cell Counting Center to learn more.

Although the authors call their treatment “mesenchymal stem cells,” the actual dosage of stem cells is unknown to them and the master cell bank. Depending on the extent of cell expansion carried out to develop the master cell bank, there may be no stem cells in the treatment. From a practical clinical perspective, that situation would not pose a problem for future clinical trial and treatment development. However, if a remaining fraction of stem cells is responsible for the observed results, subsequent trials may fail to repeat the present results if that fraction is not maintained. Different cell lots from the master cell bank could vary significantly in their stem cell-specific fraction, and without stem cell counting the investigators will not know it. Beyond the future clinical trial success implications, knowing whether stem cells played any actual role in the reported observations is a crucial issue for understanding the clinical mechanism.

We invited the authors to consider this review of their report and provide a response. However, we did not receive a response from them. Perhaps, our readers will offer their comments.

Next Review: November 3, Developing HSC Gene Editing Therapies for Sickle Cell Disease


A Stem Cell Count Would Have Made It Better:
Miltenyi Biotec’s CliniMACS Prodigy® Platform for Hematopoietic Cell Engineering

Entry #3
Wednesday, October 6, 2021

On August 17, Asymmetrex attended an online webinar offered by Miltenyi Biotec on “Manufacturing of gene-engineered hematopoietic stem cells.” The webinar featured data comparing the viral transduction efficiency of CD34+-selected hematopoietic cells manufactured with Miltenyi’s automated CliniMACS Prodigy® platform to manually-produced similar cell preparations. By several important measures, the cells produced using the CliniMACS Prodigy® were superior. The results reported in recent Miltenyi Biotec company technical reports were referenced in the webinar.

Although the Miltenyi CliniMACS Prodigy® platform is a well-engineered system for the automated processing of human hematopoietic cell preparations, there is a major opportunity for it to become an even more powerful tool for its users who are working to develop safe and effective stem cell and gene therapies. Although the key focus of the platform is the isolation, expansion, and genetic engineering of HSCs, currently, the CliniMACS Prodigy® platform has no method for monitoring the relevant critical quality attribute, the HSC-specific fraction. Downstream treatment failings in many cases may be due to low HSC fraction in starting sources and further losses during processing that result in insufficient HSC dosage in final expanded and gene-engineered products. In particular, decline in HSC activity during in vitro cell culture is well known.

The current shortcoming with the CliniMACS Prodigy® platform is that it uses flow cytometry and colony forming unit (CFU) assays as indicators for HSC fraction, though it is well known (at least by experts) that neither of these methods is able to quantify the HSC-specific fraction or dosage of hematopoietic cell populations. During the webinar, as in Miltenyi Biotec’s technical literature, the mistake of referring to CD34+-selected cell fractions as “HSCs” was often made.

So, there is a great opportunity for the impact and success of Miltenyi Biotec’s CliniMACS Prodigy® platform to be made better by implementation of a supporting stem cell count technology. In this case, it would be HSC-specific counting to better optimize isolation, production, and gene engineering for achieving a final product certified for its HSC-specific dosage.

We invited officers of Miltenyi Biotec to consider this review of their report and provide a response. However, to date, we have not received a reply from them.

Next Review: October 20, Umbilical Cord Mesenchymal Stem Cell Treatment for COVID-19


A Stem Cell Count Would Have Made It Better:
Gamida Cell’s Omidubicel Expanded Umbilical Cord Blood Treatment

Entry #2
Wednesday, September 22, 2021

For the second entry, we consider the June 22, Blood report of Dr. Mitchell E. Horwitz and co-authors detailing stem cell biopharma company Gamida Cell’s recent results comparing transplant therapy with their expanded umbilical cord blood product, Omidubicel, to standard therapy using non-expanded umbilical cord blood.

Gamida Cell’s work in umbilical cord blood expansion is often misstated by reporters as expansion of hematopoietic stem cells. For long-term, durable recovery of the bone marrow of children treated with high-dose chemotherapy for leukemia, hematopoietic stem cells are the critical blood cell type. Expanding their number in umbilical cord blood transplantation units is a longstanding holy grail of umbilical cord blood transplantation medicine. The authors of the new report correctly wrote that their Omidubicel is expanded hematopoietic progenitor cells, which may include expanded hematopoietic stem cells. However, Gamida Cell has not reported the dosage of hematopoietic stem cells in Omidubicel, which are always a much smaller fraction than committed progenitor cells.

The results reported for this new phase III clinical trial have inspired much excitement in the umbilical cord blood transplantation medicine community. In the trial, compared to standard transplantation therapy with non-expanded umbilical cord blood cells, treatment with Omidubicel showed faster rates of neutrophil engraftment and platelet recovery with reduced transplant-related complications.

Though certainly meriting attention, the report’s observed short-term improvements in transplantation effectiveness do not predict long term improvements in hematopoietic stem cell transplantation effectiveness. To date, Gamida Cell has not reported increased long-term engraftment by Omidubicel. The company’s current cell denominators for such analyses, which are total mononuclear cells and CD34+ cells, lack sufficient specificity for the level of statistical power needed to detect the long-term effects of hematopoietic stem cells. A stem cell count would have made it better.  Visit Asymmetrex’s Stem Cell Counting Center to learn more.

Theoretically, Gamida Cell could determine the hematopoietic stem cell-fraction by SCID mouse repopulation cell assays. However, this method has already proven to be too cumbersome, too expensive, and too protracted to be practical for the volume of hematopoietic stem cell dosage determinations required. In contrast, Asymmetrex’s KSC counting technology provides a practical solution.

We invited the authors and officers of Gamida Cell to consider this review of their report and provide a response. So, far they have not engaged with a response. Perhaps readers will!

Next Review: October 6, Miltenyi Biotec’s CliniMACS Prodigy® Platform for Hematopoietic Cell Engineering


A Stem Cell Count Would Have Made It Better

Inaugural Entry #1
Wednesday, September 8, 2021

As the inaugural entry for Asymmetrex’s new informational “A Stem Cell Count Would Have Made It Better,” page, we consider the July 27, Stem Cells Translational Medicine report of Dr. Beata Świątkowska-Flis and co-authors (https://stemcellsjournals.onlinelibrary.wiley.com/doi/epdf/10.1002/sctm.21-0027). Their report was also featured online by news platforms like Medical Xpress (https://medicalxpress.com/news/2021-07-stem-cell-treatments-alleviate-muscular.html). The report describes the authors’ observations of improvements in the signs and symptoms of patients with several different muscular dystrophies after treatment with expanded “umbilical cord-derived mesenchymal stem cells (UC-MSCs).” The authors do not use stem cell-specific dosage for their treatments. Here, we consider how a stem cell count would have made their study better.

The authors follow the common practice of using their expanded UC-MSC preparations as if they were homogeneous stem cells. They are not; and the stem cell-specific fraction varies with the extent of culture, characteristically decreasing with passage number. In addition, though it is not possible to discern from the report if patients in the study were treated with different lots of UC-MSCs (e.g., produced from umbilical cords from different donors), if they were, then their stem cell-specific dose would vary.

Because a stem cell-specific count was not used, the study could not detect treatment outcomes related to tissue stem cells versus the other two main types of cells present in treatment preparations. Those are non-stem committed progenitor cells and terminally-arrested cells. Equally important, the study missed the opportunity for greater statistical power to detect stem cell-specific treatment effects. Simply put, many of the treated patients showed poorer responses or no responses. If these non-responsive patients were found to have on average a lower specific dosage of any of the three types of cells injected, and of stem cells in particular, such information would be a major advance to better understanding the basis for the observed differences in treatment outcomes. Moreover, it would better inform the design and development of the next generation of stem cell medicine clinical trials that the authors envision. A stem cell count would have made it better. Visit Asymmetrex’s Stem Cell Counting Center to learn more.

We invited corresponding author, Dr. Beata Świątkowska-Flis, to consider this review and provide a response from the authors. They were non-responders, too.

Next review: September 22, Gamida Cell’s Omidubicel Trial

We are pleased to share in the discussion comments received from readers:

  • I totally agree with your comments. This seems to be the same process seen with most new discoveries. The information is very new and everyone wants to get that information on the new process or material as quickly as possible. To satisfy that desire the early users are anxious to get articles and presentations out there often regardless of appropriate study protocols for the use of the new material in order to be recognized as the first users and therefore the "experts" in the new process. And as you pointed out these flawed studies, although quickly and easily accomplished, provide either inadequate information or worse, misinformation. In the long run it seems that this delays acceptance of the new item by the more cautious as well as by regulatory agencies, requires better studies to be done that should have been done in the first place and probably damages the reputation of those who publish or present these poorly designed studies. Thank you for pointing this out but I wonder when and if we will learn that taking a little more time with well designed studies will actually get the new item in to general use faster?

    Jack Coleman, M.D. September 8
AsymmetrexA Stem Cell Count Would Have Made It Better