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A Stem Cell Count Would Have Made It Better:
Overlooking the Cell Subtype Heterogeneity of Mesenchymal Stromal Cells Undermines Meta-Analyses of Clinical Trials for COVID-19 Associated Acute Respiratory Distress Syndrome

Entry #25
Wednesday, September 28, 2022

This week’s WHMIB forum considers a second meta-analysis of clinical trials of the treatment of COVID-19 associated acute respiratory distress syndrome (ARDS) with mesenchymal stromal cell (MSC) preparations.  This is the third WHMIB forum on clinical trials of MSCs for the treatment of COVID-19 associated ARDS.  WHMIB #4 reviewed an early single trial; and WHMIB #19 considered an earlier meta-analysis.

This forum’s choice of a June 27, 2022 publication in Stem Cells Translational Medicine, from Aidan M. Kirkham and co-authors, is particularly illustrative of the widespread misstep of ignoring the cell subtype-specific dosages in these trials.  Including this forum entry, 10 of the now 25 WHMIB forums focus on the commonly overlooked cell subtype heterogeneity of MSC treatment preparations (#4, #6, #9-10, #14, #19-21, #23).

This oversight makes the analysis of Kirkham and co-workers ironic, given the authors’ stated goal of yielding a more homogeneous meta-analysis by including only controlled clinical trials with a number of other required features for eligibility.  From their meta-analysis, refined in this manner, they find evidence of efficacious effects of MSC treatments that the individual trials lacked sufficient statistical power to detect.

The shortcoming of the meta-analysis was that the authors did not consider how the cell subtype heterogeneity of MSC treatment preparations undermine their analysis.  Table 2 in their report does list the known differences in MSC tissue source, whether fresh or frozen, total cell dose, extent of cell culture, and whether the International Society of Cell Therapy (ISCT) criteria for MSCs were met.  These reported differences guarantee that differences in the relative proportions of stem cells, committed progenitor cells, and terminally arrested cells in treatments also certainly differed among the studies, and possibly even among the patients in a given study.

Of course, the authors of a meta-analysis cannot do much about this important lack of definition in the studies they consider other than to note it and to factor it into the strength of their conclusions.  However, it is now possible for the field of MSC medicine to end this insidious problem in future trials by including cell subtype-specific dosage in their clinical trial design.  Asymmetrex’s kinetic stem cell (KSC) counting TORTOISE Test analysis provides this capability.

1. Aidan M Kirkham, Adrian J M Bailey, Madeline Monaghan, Risa Shorr, Manoj M Lalu, Dean A Fergusson, David S Allan, Updated Living Systematic Review and Meta-analysis of Controlled Trials of Mesenchymal Stromal Cells to Treat COVID-19: A Framework for Accelerated Synthesis of Trial Evidence for Rapid Approval—FASTER Approval, Stem Cells Translational Medicine, Volume 11, Issue 7, July 2022, Pages 675–687, https://doi.org/10.1093/stcltm/szac038.

We contacted the authors and invited them to share their responses to these ideas.  However, we did not receive a reply from them.  Perhaps readers will share their thoughts on this issue.

This will be the final WHMIB entry into this first 25-forum series.  Our readers are encouraged to continue to share comments of their thoughts and ideas on the first 25 topics presented.  We also welcome suggestions for future topics when the WHMIB forum resumes.

Your Comments Here

A Stem Cell Count Would Have Made It Better:
Specific Quantification of Hematopoietic Stem Cells for Improvement of Umbilical Cord Blood Hematopoietic Stem Cell Transplantation Therapy

Entry #24
Wednesday, September 14, 2022

This week’s WHMIB forum returns to an indication that clearly would benefit greatly from a convenient method for routine determination of specific stem cell dosage – umbilical cord blood (UCB) hematopoietic stem cell transplantation (HSCT) therapy.

There is now a 30-year history of clinical practice for UCB HSCT.  Its current primary indication is for hematopoietic rescue of children after high-dose chemotherapy for leukemia treatments.  Though reasonably effective, treatment for this indication is not ideal.  The number of HSCs in UCB is much lower than the number in other HSCT sources, including bone marrow and mobilized peripheral blood.  This deficiency contributes to significant later times to engraftment and much higher rates of UCB HSCT failure, approaching 20% of transplanted children.  It also currently precludes the use of UCB HSCT for adults because of their greater body mass, which necessitates an even higher total HSC dosage.

This week, the forum considers a review article published in advance on August 16 in Stem Cells Translational Medicine.  Dr. Zhongjie Sun and co-workers reviewed past clinical trials and preclinical studies that had the goal of increasing the effectiveness of UCB HSCT.  Though less potent, UCB has important advantages over other HSCT sources, which include greater availability, less strict HLA matching, and less graft versus host disease.  Therefore, solving the low potency problem would yield significant clinical benefits.

The authors reviewed studies applying two main conceptual strategies to increasing engraftment potency: 1) ex vivo expansion with the goal of increasing the dosage of HSCs; and 2) increasing the efficiency of in vivo homing of transplanted HSCs to engraftment niches.  The latest clinical trials based on these two approaches are discussed, as well present and future preclinical experimental research.

The authors use the correct descriptor, “hematopoietic stem and progenitor cells,” (“HPSCs”) to describe the cell populations evaluated in the reviewed trials and studies, even though the critical cells for long-term hematopoietic engraftment are HSCs.  Progenitor cells play an important role in providing important immune function prior to stable production of immune cells by engrafting transplanted HSCs.  In all the studies that the authors reviewed, this important dichotomy is hidden in the usage “HSPC,” because previously there was no method for determining the critical HSC dosage that determines long-term engraftment success.

A major challenge in the reviewed HSC expansion clinical trials was the need for a convenient and accurate method for monitoring specific changes in HSC fraction.  These studies monitored changes in CD34+ and CD133+ cell fractions, which include both HSCs and progenitor cells.  Because HSC are such a small fraction of total HSPCs, these studies were effectively blind to HSCs; and accordingly failed to define conditions for their expansion.  For future research with this focus, Asymmetrex’s rapid stem cell-counting calculators offer a clear benefit and advantage.

1. Zhongjie Sun, Bing Yao, Huangfan Xie, XunCheng Su, Clinical Progress and Preclinical Insights Into Umbilical Cord Blood Transplantation Improvement, Stem Cells Translational Medicine, 2022; szac056, https://doi.org/10.1093/stcltm/szac056/

We contacted the authors and invited them to share their responses to these ideas.  However, we did not receive a reply from them.  Perhaps readers will share their thoughts on this issue.

Next forum entry: September 28, “Overlooking the Cell Subtype Heterogeneity of Mesenchymal Stromal Cells Undermines Meta-Analyses of Clinical Trials for COVID-19 Associated Acute Respiratory Distress Syndrome”

A Stem Cell Count Would Have Made It Better:
Thinking About Cell Subtype Specificity for Future Dosing of Expanded Bone Marrow Mesenchymal Stem Cells Investigated for the Treatment of Lung Transplant Inflammation

Entry #23
Wednesday, August 31, 2022

The report selected for this entry of the WHMIB forum is one of the best examples so far of how stem cell medicine could be improved by quantification of the cell subtype-specific dosage of treatment preparations.

Dr. David Erasmus and colleagues at the Mayo Clinic published a recent report in the July 26 advanced access publication issue of Stem Cells Translational Medicine describing their results from a Phase 1b safety study, in which they observed the clinical course of patients with obstructive chronic lung allograft syndrome (o-CLAD) after intravenous infusion of an allogeneic, expanded, bone marrow-derived mesenchymal stem cell preparation.¹  The 13 patients enrolled in the study had developed moderate-to-severe o-CLAD after lung transplants.  Chronic rejection is the most important factor limiting survival after lung transplantation; and immune cell and other inflammatory mechanisms are postulated to be responsible for CLAD.

In an earlier smaller study, the authors investigated MSC treatment of CLAD because of reports of MSCs’ potential effects for down-modulating inflammation in other lung conditions like graft versus host disease.  Their earlier study suggested that lower doses of MSC treatments might be more effective for stabilizing or reducing CLAD.  The new study reported more recently was a follow-up evaluation of this possibility.

From the new study, the authors concluded that the “low-dose MSC” treatments were well tolerated and showed evidence of slowing CLAD progression in some patients.  However, they did not observe the previous “paradoxical inverse dose response at the lowest dose,” which was the motivation for the new study.

As seasoned clinical investigators, the authors appropriately state the small sample size of the study precludes conclusions regarding efficacy.  However, what they overlook is that the MSC preparations used for the first and second study may not be dose-equivalent for the critical effector cell subtypes.  The treatment preparations were expanded bone marrow cell populations from independent donors.  Donor-donor variation and variation in the expansion process will yield treatment preparations that differ in the fractions of stem cells, committed progenitor cells, and terminally-arrested cells.  If one of these cell-subtypes acts to prevent stabilization of CLAD, then a higher fraction of that subtype in the first study’s treatments could have resulted in a greater stabilization effect at lower total cell doses.

Since there are many other factors that might account for the differences observed by the authors, including statistical variation, their experience is an excellent opportunity to highlight how and why advancing from dosing based on total mononuclear cells to cell subtype-specific dosing would improve the design and interpretation of stem cell clinical trials.  Asymmetrex’s TORTOISE Test kinetic stem cell (KSC) counting technology offers this cell subtype-specific characterization for primary and expanded MSC-containing preparations.

1. David Brett Erasmus, Nisha Durand, Francisco A Alvarez, Tathagat Narula, David O Hodge, Abba C Zubair. Feasibility and Safety of Low-Dose Mesenchymal Stem Cell Infusion in Lung Transplant Recipients, Stem Cells Translational Medicine, 2022; szac051, https://doi.org/10.1093/stcltm/szac051

We contacted the authors and invited them to share their responses to these ideas.  However, we did not receive a reply from them.  Perhaps readers will share their thoughts on this issue.

Next forum entry: September 14, “Specific Quantification of Hematopoietic Stem Cells for Improvement of Umbilical Cord Blood Hematopoietic Stem Cell Transplantation Therapy”

A Stem Cell Count Would Have Made It Better:
Implementing Stem Cell-Specific Quantification to Improve the Therapeutic Potential of Cultivated Autologous Limbal Epithelial Cells for Corneal Stem Cell Therapies

Entry #22
Wednesday, August 17, 2022

After the first 21 WHMIB forums, readers might have the impression that the only tissue stem cells being investigated for medical therapies are HSCs and MSCs.  Though these are by far the predominant stem cell types pursued for stem cell and gene therapies, stem cells from other tissues are also of importance.  The best two examples of other stem cells of therapeutic interest are liver stem cells, to address organ shortages for liver transplantation, and limbal epithelial stem cells (LESCs) for treatments of cornea disorders and injuries.  This week’s forum will consider recent developments for the production of LESCs for cornea therapies.  No surprise, progress in this field of tissue stem cell medicine would also benefit from the implementation of methods to quantify stem cell-specific fraction and dosage, as has been recommended in this forum for HSC and MSC therapies.

In a July 2022 report in the American Journal of Ophthalmology, Dr. Ula Jurkunas, M.D. and co-authors provided a comprehensive of assessment of previous studies describing the use of both allogeneic and autologous LESCs for treatments for a variety of cases of limbal epithelial stem cell deficiency (LSCD).1  LSCD includes a varied array of disorders, diseases, and injuries that result in cornea dysfunction and blindness.  In the case of bilateral defects, allogeneic transplantation treatments with living-relative donor or cadaver sources of LESCs is the only solution, but their effectiveness is complicated by the need for chronic immunosuppression.  The authors’ focus was on autologous LESC transplantation in the setting of unilateral disorders.  However, though autologous transplantation does not have the problem of immune rejection, its requirement for a substantial amount of autologous cornea tissue has a significant risk of causing LSCD in the healthy donor eye.

The main aim of the authors’ study was to learn from previous reported clinical experiences with the purpose of developing improved standardized methods for GMP-grade manufacturing of cultivated autologous limbal epithelial cells (CALECs) for use in future transplantation clinical trials for LSCD.  Production of efficacious CALECs addresses the immune rejection problem of allogeneic transplants; and it would also require smaller amounts of autologous donor cornea tissue cells, reducing the risk of inducing LSCD in healthy donor eyes.

From their review of 34 total studies, the authors concluded that it was feasible to devise a GMP-manufacturing process that could become a quality source of CALECs for use in LSCD clinical trials in the U.S.  Beyond meeting clinical standards of defined, xenobiotic-free culture materials and safety standards of quality and sterility, the authors also wished to establish assays that quantified the “stem cell density” of CALEC production lots.  Unfortunately, neither of the two assays used for assessing the LESC fraction of CALEC preparations – colony forming efficiency (CFE) and intracellular ATP (iATP) – provides information on the specific fraction of tissue stem cells.

Using Asymmetrex®’s kinetic stem cell (KSC) counting technology, it is now possible to rapidly determine the specific fraction of LESC cells in CALEC preparations using only 72-hour cell culture data, which is much faster and more reproducible than 2 to 3-week CFE assays.  More importantly, KSC counting provides the LESC-specific fraction, which neither CFE nor iATP assays provide.  As the authors begin and end their report, the critical quality attribute for successful transplantation therapy for LSCD is the dosage of LESC cells in the CALEC treatment.

1. Jurkunas U., Johns L., and Armant, M. 2022. Cultivated autologous epithelial cell transplantation: New frontier in the treatment of limbal stem cell deficiency. Am J Ophthalmol 239: 244-258. doi: 10.1016/j.ajo.2022.03.015.

We contacted the authors and invited them to share their responses to these ideas.  However, we did not receive a reply from them.  Perhaps readers will share their thoughts on this issue.

Next forum entry: August 31, “Thinking About Cell Subtype Specificity for Future Dosing of Expanded Bone Marrow Mesenchymal Stem Cells Investigated for the Treatment of Lung Transplant Inflammation”

A Stem Cell Count Would Have Made It Better:
Considering the Clinical Significance of the Cellular Heterogeneity of MSC Preparations Used for Extracellular Vesicle Production and Treatment

Entry #21
Wednesday, August 3, 2022

This week’s WHMIB forum is taken from our increasingly common source of reports to review, the journal Stem Cells Translational Medicine.  Although an important contribution in its own right, the report considered in this week’s forum was chosen more because of its general topic than for its specific findings.

In their June 2022 SCTM report¹, Mayank Sharma and co-authors described their investigation of the treatment properties of extracellular vesicles (EVs) produced from two different sources of human mesenchymal stem cells (MSCs).  In a neonatal rat model of pediatric bronchopulmonary dysplasia (BPD) with complicating pulmonary hypertension (PH), they compared treatment effects of GMP-manufactured EVs from MSC cultures derived from human umbilical cord Wharton’s jelly and from human bone marrow.  Though mortality was not significantly altered by the EVs administered, many significant reductions were achieved in disease-related pathology and pathophysiology that would be beneficial if translatable to medical care for BPD-PH in the setting of premature births.

This report highlights how the presently significant void in thinking about the specific dosing of cell subtypes in MSC stem cell preparations continues to insidiously undermine stem cell medicine, as well as derivative new medical disciplines.  In the same way that the source MSC production preparations are erroneously used as if they were homogeneous stem cells, the field of EV medicine currently makes the error of using EV preparations – like the ones described by the authors of this report – as if they were derived from pure stem cells.

Given the now many WHMIB forums describing the cell-type heterogeneity of MSC preparations (e.g., see forums #10, #13, #14, and #20), it is easy to understand why EVs manufactured from them will have the same property.  The same causes of variation in the cell subtype composition for tissue stem cell preparations – donor variation, physiological variation, effects of culture expansion, biomanufacturing process variation – will also impact the heterogeneity of derivative EVs.  If the EVs produced from particular cell subtypes are the critical principles for therapeutic effects, treatment outcomes will vary according to differences in the dosages of the responsible cell subtypes in the source MSC preparation.

Using Asymmetrex’s kinetic stem cell (KSC) counting technology, it is now possible to quantify the cell subtype heterogeneity of biomanufactured MSCs and, thereby, produce EVs of known cell subtype composition.  Relating such critical dosage information to treatment outcome will provide greater statistical power in clinical trials for detecting treatment effectiveness and increasing the efficacy of subsequent treatment regimens.

1. Mayank Sharma, Michael A Bellio, Merline Benny, Shathiyah Kulandavelu, Pingping Chen, Chawisa Janjindamai, Chenxu Han, Liming Chang, Shanique Sterling, Kevin Williams, Andreas Damianos, Sunil Batlahally, Kaitlyn Kelly, Daniela Aguilar-Caballero, Ronald Zambrano, Shaoyi Chen, Jian Huang, Shu Wu, Joshua M Hare, Augusto Schmidt, Aisha Khan, Karen Young, Mesenchymal Stem Cell-derived Extracellular Vesicles Prevent Experimental Bronchopulmonary Dysplasia Complicated By Pulmonary Hypertension, Stem Cells Translational Medicine, 2022;, szac041, https://doi.org/10.1093/stcltm/szac041.

We contacted the authors and invited them to share their responses to these ideas.  However, we did not receive a reply from them.  Perhaps readers will share their thoughts on this issue.

Next forum entry: August 17, “Implementing Stem Cell-Specific Quantification to Improve the Therapeutic Potential of Cultivated Autologous Limbal Epithelial Cells for Corneal Stem Cell Therapies”

A Stem Cell Count Would Have Made It Better:
Missed Opportunities in Cartilage Regeneration Treatments for Osteoarthritis Using Autologous Adipose-Derived Mesenchymal Stem Cells

Entry #20
Wednesday, July 20, 2022

One of the most challenging areas of regenerative medicine is cartilage repair for osteoarthritis (OA).  Cartilage has one of the lowest intrinsic rates of tissue cell renewal and is lost irreparably with aging and chronic joint wear.  The now increasing age demographics of many countries make osteoarthritic degeneration of the cartilage of the knees and hips a rapidly growing disability pandemic of debilitating immobility and chronic pain.  Patients seeking to avoid standard joint replacement surgery are a major patient population for private stem cell clinics.  The effectiveness of the treatments offered by these clinics, using autologous or perinatal mesenchymal stem cells, are largely unsubstantiated, creating many opportunities for both financial and medical exploitation of a highly vulnerable patient population.

This WHMIB forum takes a look at a recent report of a clinical study designed to evaluate the ability of autologous adipose-derived mesenchymal stem cells (ADMSCs) to increase cartilage regeneration in the setting of osteoarthritic knee joint repair surgery.  Scientific evidence of such effectiveness would not only impact the quality of care for surgical treatments for osteoarthritis, but it might also shed an important light on the likelihood of effective treatments in private stem cell clinics.

In their June 8 advanced access report in Stem Cells Translational Medicine, Dr. Jun-Ho Kim and co-authors detailed the results of a 2-year follow-up study investigating effects of intra-articular injection of autologous, culture-expanded, ADMSCs on cartilage regeneration after medial open-wedge high tibial osteotomy (MOWHTO) for knee osteoarthritis with varus malalignment.1  MOWHTO is used to correct knee joint misalignments that advance with age and accelerate osteoarthritic joint damage.  In this way, MOWHTO is deployed to delay the need for joint replacement.

The authors cite earlier reports of modest cartilage regeneration after MOWHTO.  The purpose of their randomized clinical trial was to evaluate whether this baseline cartilage repair could be improved by concomitant injection of patients’ autologous ADMSCs.  Although a small study, with only 13 patients in each treatment group, a modestly significant (p = 0.042) increase in cartilage regeneration, based on arthroscopic exam, was found to be associated with co-injection of ADMSCs; and, importantly, there were no significant increases in adverse effects.

Given the small numbers of patients involved in this study, stem cell-specific counting might have greatly improved the statistical confidence of the findings.  Four patients in the treatment group did not achieve complete cartilage regeneration.  The authors assumed that the critical dose of MSCs in these patients’ treatment was the same as the dose of the 9 patients who had complete regeneration.  Since the ADMSCs were harvested and cultured for three passages, they were certainly not “homogeneous MSCs” as referred to by the authors.  As described in earlier WHMIB forums (e.g., see forums #10, #13, and #14), the treatment preparations will differ in the fraction of stem cells, proliferating committed progenitor cells, and terminally-arrested cells.  If one of these cell subtypes is primarily responsible for the observed cartilage regeneration effects, then lower specific doses of them may account for the lesser responses of some treated patients.  By providing this important dose-specific data, kinetic stem cell (KSC) counting would have improved this important clinical investigation.

1. Jun-Ho Kim, Kang-Il Kim, Wan Keun Yoon, Sang-Jun Song, Wook Jin, Intra-articular Injection of Mesenchymal Stem Cells After High Tibial Osteotomy in Osteoarthritic Knee: Two-Year Follow-up of Randomized Control Trial, Stem Cells Translational Medicine, Volume 11, Issue 6, June 2022, Pages 572–585, https://doi.org/10.1093/stcltm/szac023.

We contacted the authors and invited them to share their responses to these ideas.  However, we did not receive a reply from them.  Perhaps readers will share their thoughts on this issue.

Next forum entry: August 3, “Considering the Clinical Significance of the Cellular Heterogeneity of MSC Preparations Used for Extracellular Vesicle Production and Treatment”

A Stem Cell Count Would Have Made It Better:
Accounting for Cell Subtype Heterogeneity in MSC Dosing: A Latest Example from a Meta-Analysis of Treatments for COVID-19 Acute Respiratory Distress Syndrome

Entry #19
Wednesday, July 6, 2022

Even before COVID-19 had reached its eventual high level of concern, there was much speculation in the stem cell medicine field that mesenchymal “stem” cells (MSCs) might provide therapeutic benefit to patients experiencing the newly-defined COVID-19 acute respiratory distress syndrome (ARDS).  Though the significant cellular heterogeneity of MSC preparations was being widely acknowledged at about the same time, – including proposals to limit their description to M”stromal”Cs or change their designation to “medicinal signaling cells” – their generally accepted anti-inflammation properties motivated their immediate evaluation as a potential treatment for inflammation associated with COVID-19 ARDS.

This WHMIB forum considers a recent report that assesses the broad outcome of the many MSC treatment studies and trials that have ensued during the COVID-19 crisis.  In a May 2022 report in Stem Cells Translational Medicine, titled “Efficacy and Safety of MSC Cell Therapies for Hospitalized Patients with COVID-19: A Systematic Review and Meta-Analysis,” Dr. Wenchun Qu, M.D., Ph.D. and co-authors detail the summary findings of their analysis of 34 varied investigations for MSC treatment of ARDS in hospitalized COVID-19 patients.  The analysis findings are based on the clinical results for 736 identified patients.1

Recognizing the shortcomings due to the heterogeneous nature of meta-analyses, the authors concluded that, on average, MSC treatment was associated with a significant reduction in the overall mortality of hospitalized COVID-19 patients, although specific effects on pulmonary pathophysiology were less pronounced.  The treatments were generally safe with no increases observed in adverse events and a significant reduction in serious adverse events.  In addition, half of the examined studies reported a reduction in inflammation biomarkers after MSC treatment, which is consistent with the hypotheses that the observed clinical benefits were due to anti-inflammation effects previously described for MSCs.

The remarkable feature of this report that was the basis for its selection as a WHMIB forum entry was the general use of total cell counts as the basis for dosing MSCs in all the studies examined.  Despite the well-described cell subtype heterogeneity MSC preparations, the described, as well as other current MSC clinical trials, continued to administer MSC treatment preparations as if they are homogenous for cell function, even though there is a clear recognition in the field that they are not.  A number of previous WHMIB forums have addressed this important issue directly (See WHMIB #4, #6, #9, #10-#14).

The MSC treatment preparations use in the 34 reviewed studies were isolated from a variety of allogeneic sources, including umbilical cord, bone marrow, adipose, menstrual blood, placenta, and heart.  These different tissue sources will differ in their cell subtype heterogeneity for stem cells, committed progenitor cells, and differentiated cells.  In addition, many of the preparations will have undergone cell culture expansion, which introduces further differences in cell subtype heterogeneity.

The cell subtype heterogeneity of treatment samples is an unaccounted variable in all the studies; and it is predicted to be a critical quality attribute for clinical outcome.  Accounting for the specific dosage of cell subtypes was not possible at the time that many of the evaluated studies were conducted.  If such specific dosage accounting were available, the COVID-19 MSC treatment studies would have been better informed.  Such specific dosage data is now available with Asymmetrex®’s kinetic stem cell (KSC) counting technologies.  The authors concluded that their new meta-analysis findings “support the urgent need for large, randomized double-blinded controlled trials to assess the safety and efficacy of MSC cell therapy for the treatment and prevention of severe COVID-19.”  The recommended trials will be even more informative if they include specific cell subtype dosing to account for effects due to the cell heterogeneity of treatment preparations.

1. Wenchun Qu, et al., Efficacy and Safety of MSC Cell Therapies for Hospitalized Patients with COVID-19: A Systematic Review and Meta-Analysis, Stem Cells Translational Medicine, 2022;, szac032, https://doi.org/10.1093/stcltm/szac032.

We contacted the authors and invited them to share their responses to these ideas.  However, we did not receive a reply from them.  Perhaps readers will share their thoughts on this issue.

Next forum entry: July 20, “Missed Opportunities in Cartilage Regeneration Treatments for Osteoarthritis Using Autologous Adipose-Derived Mesenchymal Stem Cells”

A Stem Cell Count Would Have Made It Better:
Identifying Effective Donor HSC Samples for Making Humanized Mice: Another
Resignation by the Stem Cell Supply Industry That Is No Longer Necessary

Entry #18
Wednesday, June 22, 2022

This WHMIB forum features two reports separated by nine years, one published in July 2011 and the other in May 2020. Both reports focus on the challenges that variation in the effectiveness of hematopoietic stem cell (HSC) donor samples presents for the production of humanized mice. Currently, companies that supply humanized mice also manufacture and supply the human HSC sources that are used to produce the mice. These papers were selected for the WHMIB forum because, although in the past the industry has resigned itself to being unable to address this problem, with the introduction of rapid kinetic stem cell (KSC) counting algorithms, it no longer needs to do so.

Humanized mice are a valuable research tool for investigating properties of the human hematopoietic system and the human immune system; and they are now essential pre-clinical models for developing new medical therapies to treat disorders and diseases of these systems.The first report from Dr. Julie Lang, Ph.D. and co-workers describes their work to evaluate multiple in vitro factors for their ability to increase successful development of humanized mice using CD34⁺ umbilical cord blood cells.¹They reasoned that achieving an overall higher HSC engraftment efficiency would somewhat mitigate the problem of the variation in HSC donor quality. Their insightful investigation defined improvements that have now been incorporated into some production procedures (e.g., short-term culture), but donor variability continues to be a significant problem.

The later report from Dr. Lia Walcher, Ph.D. and co-authors compared the humanized mice HSC-engraftment efficiency of human CD34⁺ umbilical cord blood donors and CD34⁺ adult mobilized peripheral blood donors.² Though umbilical cord blood donors were 19-fold more effective as a group in this comparison, they still exhibited high donor variability, with a 52% coefficient of variation. As is typical, the same cord blood and adult blood samples were indistinguishable by the current industry-standard assays for quantifying hematopoietic stem/progenitor cells, including CD34⁺ count, mononuclear cell viability, and colony forming unit (CFU).

Given that HSCs are a major critical quality attribute (CQA) for efficient production of humanized mice, their specific fraction in donor samples will identify the more effective samples. Asymmetrex’s new rapid-counting RABBIT Count algorithms provide the HSC-specific fraction of a donor sample based on 72-hour conventional cell culture count data. With this short time requirement, the technology can be readily integrated into current humanized mice production workflows that include a short-term culture period as reported by Lang and co-workers in their 2011 report.¹

Immediate deployment of the rapid HSC-counting algorithm technology will accelerate the work of investigators, like the authors of the featured reports, to identify other CQAs by allowing them to evaluate samples that do not vary significantly in their HSC fraction. Companies developing humanized mice for the stem cell research and stem cell medicine communities could immediately realize increases in production efficiency and reductions in production costs. Each of these coming adoptions will help to open a more important door to bringing rapid HSC-counting algorithms into needed use for benefiting patients in stem cell clinical trials and stem cell medicine.

1. Lang, J., Weiss, N., Freed, B. M., Torres, R. M., & Pelanda, R. (2011). Generation of hematopoietic humanized mice in the newborn BALB/c-Rag2null Il2rγnull mouse model: a multivariable optimization approach. Clinical immunology (Orlando, Fla.), 140(1), 102–116. https://doi.org/10.1016/j.clim.2011.04.002.

1. Walcher, L., Hilger, N., Wege, A. K., Lange, F., Tretbar, U. S., Blaudszun, A. R., & Fricke, S. (2020). Humanized mouse model: Hematopoietic stemcell transplantation and tracking using short tandem repeat technology. Immunity, inflammation and disease, 8(3), 363–370. https://doi.org/10.1002/iid3.317.

We contacted the authors of both reports and invited them to share their responses to these ideas. However, we did not receive a reply from them. Perhaps readers will share their thoughts on this issue. In particular, it would be great for the forum to hear from readers with experience producing and using HSC-humanized mice.

Next forum entry: July 6, “Accounting for Cell Subtype Heterogeneity in MSC Dosing: A Latest Example from a Meta-Analysis of Treatments for COVID-19 Acute Respiratory Distress Syndrome”

A Stem Cell Count Would Have Made It Better:
Improving Cryopreservation and Freeze-Thawing for Stem Cell Therapies and other Common Cellular Therapeutics with Cell-Subtype Specificity

Entry #17
Wednesday, June 8, 2022

This WHMIB forum features another perspective on standardizing cell and tissue biomanufacturing processes and their regenerative medicine and cell therapy applications.  The focus is on cell cryopreservation, an essential procedure at all important stages of regenerative medicine and cell therapy development, including cell product biomanufacturing, cell product storage, cell product transport, and cell product treatment administration.

In a timely perspective recently published in Current Stem Cell Reports, a group of co-authors, led by Dr. Guido Moll, Ph.D. and Dr. Raghavan Chinnadurai, Ph.D., reviewed past and current biomanufacturing and clinical practices for freezing and thawing of cell therapy products.  The authors concluded that past experience indicates that cell cryopreservation may potentially be an “Achilles’ heel” for achieving safe and efficacious cell therapy trials and future routine use.1

Because they constitute the majority of clinical trials and approved therapies, reports of the use of freeze-thawed hematopoietic stem cell (HSC) products and freeze-thawed mesenchymal stromal/stem cell (MSC) products were the main focus of discussion.  Overall past research and clinical experience indicates that HSC transplantation (HSCT) therapies tolerate routinely used freezing and thawing procedures very well.  Even so, the authors observed that “no universal methodology is in practice to date”.  In contrast to HSCT therapies, the authors described a diverse array of many reported functional effects of freeze-thawing of MSC preparations that can compromise their safety and efficacy.  They reviewed reports of technology innovation attempted to address this issue that may have substantially limited the clinical efficacy of MSC therapies in the past, but a generally applicable solution is still an unmet need.

This critical perspective on problems that cryopreservation presents for HSC and MSC therapeutics was selected for a WHMIB forum, because it illustrates that all evaluations of the effects and quality of freeze-thawing processes is based on the total cell count.  In particular, the possibility of different effects occurring for functionally important subtypes of cells in these preparations – i.e., stem cells, committed progenitor cells, and mature differentiated cells – has been completely overlooked for many years.  Even though detrimental effects of freeze-thawing have not been significant for current HSC therapies, knowing about specific effects on HSCs could be beneficial during the development of therapeutic products with fewer HSCs or more fragile ones (e.g., HSC-targeted gene therapies).  Based on the authors’ review of the many examples of unexplained effects of freeze-thawing on MSC preparations and other common cellular therapeutics (e.g., T-reg, T-eff, CAR-T, and iPSCs), delineating specific effects on the different subtypes of cells present could be highly informative about the responsible mechanisms.

At the time when the reviewed and discussed studies were conducted, no method was available for quantifying the effects of freezing and thawing on the different cell subpopulations in HSC and MSC preparations.  Now, Asymmetrex’s kinetic stem cell (KSC) counting technologies make this advance possible.  The KSC counting computational simulation software can delineate the specific viability and the specific cell cycling time of each different cell subtype with and without, before and after, freezing and thawing procedures.  This capability provides a new, highly relevant, quantitative basis for developing, optimizing, and validating cryopreservation and freeze-thaw procedures for tissue stem cell biomanufacturing and regenerative medicine.

1. Cottle, C., Porter, A.P., Lipat, A. et al. Impact of Cryopreservation and Freeze-Thawing on Therapeutic Properties of Mesenchymal Stromal/Stem Cells and Other Common Cellular Therapeutics. Curr Stem Cell Rep 8, 72–92 (2022). https://doi.org/10.1007/s40778-022-00212-1.

We contacted the authors and invited them to share their responses to these ideas.  Here is the response they shared with us:

“Cryopreservation and freeze-thawing are crucial processes in the production of commercially viable cell therapy approaches, e.g., to allow for the intermediate and long-term storage and shipping of manufactured cell batches and quality testing of cell lots before clinical release.  Similar applies for the correct enumeration of thawed cells for correct dosing in clinical trials.  Unfortunately, the translation from preclinical proof-of-concept studies to larger clinical trials has indicated that these processes may potentially present an “Achilles’ heel” to optimal cell product performance.  Thus, many cell therapy manufacturers now invest into research and new technologies to optimize these crucial processes to yield the optimal outcome with their unique products.  In particular, a better understanding on how different subpopulations are affected could be of importance to best preserve the optimal functionality of the cell population of interest in the therapeutic approach.”

Next forum entry: June 22, “Identifying Effective Donor HSC Samples for Making Humanized Mice: Another Resignation by the Stem Cell Supply Industry That Is No Longer Necessary”

A Stem Cell Count Would Have Made It Better:
The Dosage of Stem Cells in Hematopoietic Stem Cell Transplants in Real-World Clinical Treatments No Longer Needs to Be an Unmeasured Quantity

Entry #16
Wednesday, May 25, 2022

This WHMIB forum entry continues consideration of clinical studies evaluating the effectiveness of nonmyeloablative hematopoietic stem cell transplant (HSCT) therapy for multiple sclerosis.  The selected report for review and discussion details the investigation of “real-world” effects of autologous treatments with mobilized HSCTs on slowing or preventing the progression of relapsing-remitting multiple sclerosis (RRMS) and newly diagnosed secondary progressing multiple sclerosis (SPMS).

In a follow-up to their 2019 JAMA report of a multi-site study reviewed previously (WHMIB #15), Dr. Richard K. Burt, M.D. and co-workers described, in the October 2021 online issue of the Journal of Neurology, a 16 year single-site study of the effectiveness of nonmyeloablative HSCT for the treatment of 414 patients with RRMS and 93 patients with newly diagnosed SPMS.  Adding general confirmation to the results reported in the 2019 JAMA article, in this longer real-world evaluation, single-dose, conventional, autologous nonmyeloablative HSCT was effective in reducing and preventing progression of RRMS during the 5 year period of follow-up.  The effectiveness for similar treatment of newly diagnosed SPMS was not as impressive, but limited efficacious benefits were also observed.

The reason for selecting this report as a WHMIB topic is the remarkable absence of any mention of the dosage of HSCs received by treated patients.  The therapeutic strategy is non-ablative treatment of patients’ hematopoietic systems with the aim of eliminating disease-causing immune cells, followed by hematopoietic system restoration with mobilized autologous HSCs.  This well-established clinical practice is so highly effective for hematopoietic system recovery that the specific dosage of rescuing HSCs is neither considered nor evaluated.

Yet, this real-world practice, used so commonly throughout HSCT medicine, is shortsighted and misses opportunities for better treatment outcomes and better management of scarce clinical resources.  Certainly, even with the current very effective therapy, not all patients had the same degree of clinical response.  Whereas differences in clinical response may not be due to insufficient dosage of engrafting HSCs, they could easily be due to differences in the fractions of other cell types, which vary in the mobilized apheresis blood cell preparations used for treatment.  Defining the HSC-specific dosage would also reciprocally define the fraction of progenitor and differentiated cells, whose levels could determine overall clinical responses.

Advancing all real-world and clinical trial HSCT therapies to intentional use of HSC-specific dosage will improve HSCT medicine.  There are many discoveries and improvements waiting to be made.  Asymmetrex’s kinetic stem cell (KSC) counting technologies make this advance possible now for the first time.

We contacted the authors and invited them to share their responses to these ideas, but we did not hear back from them.  We look forward to impressions and thoughts from readers!

Next forum entry: April 8, “Improving Cryopreservation and Freeze-Thawing for Stem Cell Therapies and other Common Cellular Therapeutics with Cell-Subtype Specificity”

A Stem Cell Count Would Have Made It Better:
Treatment of Multiple Sclerosis with Nonmyeloablative Hematopoietic Stem Cell Transplantation: An Example of the Current Practice of Blind Dosing for Stem Cells in Clinical Trials

Entry #15
Wednesday, May 11, 2022

This WHMIB forum entry considers another stem cell treatment for multiple sclerosis.  In this case, the reviewed clinical trial evaluated the effectiveness of nonmyeloablative hematopoietic stem cell transplant (HSCT) therapy for slowing or preventing the progression of relapsing-remitting multiple sclerosis (RRMS).

In their 2019 JAMA report, Dr. Richard K. Burt, M.D. and co-workers described their multi-site, open-label, randomized clinical trial comparing progression outcomes of RRMS patients treated with disease-modifying therapy (DMT) to those of RRMS patients receiving autologous, nonmyeloablative HSCT after an immune ablation regimen to reduce or eliminate disease-causing, auto-reactive lymphocytes.

In this preliminary study, HSCT therapy was very effective for preventing and reducing the progression of RRMS during the 5 year period of patient follow-up.  Whereas disease progression occurred in 34 of the 55 patients in the DMT group, only 3 cases of progression occurred among the 55-patient HSCT therapy group.  In addition, during the first year of the study, the mean disability scores for the HSCT group decreased significantly, while they increased for the DMT group (p < 0.001).

For their HSCT treatments, Burt and co-workers used standard clinical practice for mobilizing HSCs, collecting them by apheresis, and administering them to trial subjects.  In the trial protocol supplement provided with their report, they state what has become the accepted resignation of HSCT medicine:

“The majority of mononuclear cells collected by peripheral blood apheresis (or bone marrow harvest) are immune cells such as lymphocytes and monocytes not HSCs.  While the true identity of human HSCs remains elusive, purified CD34+ or AC133+ hematopoietic progenitor cells are sufficient for hematopoietic reconstitution.  In general, a minimum number of 2 x 106 CD34+ cells/kg recipient weight will ensure engraftment.”

Achieving this well-established effective dosage of 2 x 106 CD34+ cells/kg can require as many as three apheresis sessions over a several-day period.  Though this regimen has a high rate of successful restoration of patient’s hematopoietic system, the actual dosage of the responsible HSCs is still not known – not in general and not for individually dosed patients.

Given the high degree of effectiveness of HSCT treatments dosed by their CD34+ cell count (>96%), the actual HSC dosage given may often be excessive.  As was discussed in the recent WHMIB #12 entry (See below.) for the umbilical cord blood, the CD34+ count correlates poorly with a wide variation in HSC count defined by multi-marker flow cytometry, which is also not exclusive for HSCs.  By providing an accurate measure of the HSC-specific dosage of HSCT transplant treatments, kinetic stem cell (KSC) counting technology could both reduce the amount of apheresis required for both autologous and allogeneic HSCT therapy.  In the case of allogeneic treatments, determination of the HSC-specific dosage could also allow scarce donor samples to be used to treat more patients.

We contacted the authors and invited them to share their responses to this review.  We did not receive a response from them.  We look forward to impressions and thoughts from readers!

Next forum entry: May 25, “The Dosage of Stem Cells in Hematopoietic Stem Cell Transplants in Real-World Clinical Treatments No Longer Needs to Be an Unmeasured Quantity”

A Stem Cell Count Would Have Made It Better:
Making Mesenchymal Stem Cell Treatments for Progressive Multiple Sclerosis Better with Cell Type-Specific Dosing

Entry #14
Wednesday, April 27, 2022

The most recent WHMIB forums have reviewed reports that illustrate the natural cell heterogeneity of tissue stem cell preparations and how it impacts the effectiveness and safety of medical treatments that use them (See Entries 9-13).  Although the forums have considered other important forms of heterogeneity (e.g., cell size, biomarker expression, and hemocompatibility), the primary focus remains on the heterogeneity of cell types, which determines the stem cell-specific dosage of treatment preparations.

This 14th WHMIB forum returns to the primary focus on the importance of stem cell medicine advancing to tissue stem cell-specific dosing to improve stem cell clinical trials and approved stem cell treatments.  The example selected for this week’s forum is a recent brief report in Stem Cell Translational Medicine titled, “Effects of Mesenchymal Stem Cell Transplantation on Cerebrospinal Fluid Biomarkers in Progressive Multiple Sclerosis.  In this February 2022 report, Dr. Panayiota Petrou and co-authors describe the results of their double-blind randomized Phase II clinical trial evaluating the effects of intrathecal or intravenous autologous bone marrow-derived mesenchymal stem cells (MSCs) on clinical indicators of the status and prognosis of progressive multiple sclerosis (MS).

The report details the exciting treatment outcome that, 6 months after its administration, a single intrathecal injection of MSCs significantly reduced the levels of neurofilament light chains (NF-L) in the cerebrospinal fluid (CSF) of patients with progressive MS.  CSF NF-L are established clinical indicators of neurodegeneration in MS.  These newest findings add to earlier evidence that autologous bone marrow-derived MSC treatments have potential to reduce or prevent the progression of MS.

There is an important consideration for which a stem cell count would have made this study better.  Of the 15 patients who received intrathecal MSCs, 11 showed reduced CSF NF-L levels.  However, only 6 of the eleven showing a decrease had a decrease that was greater than 50%.  The remaining 4 treated patients maintained essentially the same levels (2 patients) or showed significant increases (2 patients).  Among the possible causes for the differences in treatment outcomes are differences in the dosage of the responsible cell type in the MSC treatment preparations.

Like many in the field, the authors of this study assumed that each patient’s autologous MSC treatment preparation had the same potency based on being homogenous for the expression of biomarkers like CD90 and CD73.  However, as considered in many earlier entries in this WHMIB forum, the use of these biomarkers obscures the natural heterogeneity of these tissue cell preparations for three natural tissue cell types – stem cells, committed progenitor cells, and terminally-arrested cells – any one of which might be responsible for the observed treatment effects.

The relative fractions of stem cells, committed progenitor cells, and terminally-arrested cells in MSC treatment preparations will certainly differ from patient to patient.  Each treatment sample was independently harvested, selected by plastic adherence, cultured for at least three passages, cryopreserved, and thawed before administration.  Each of these procedure steps is a source of processing variability that will impact the final cell type distributions in addition to the natural biological variability of the isolated tissue cells.

The single statistically significant result of the study – for reduced CSF NF-L levels after intrathecal injection – had a modest level of confidence, P = 0.026.  However, the therapeutic effect measured might increase substantially, if the basis for comparison were not total injected cell dose, but instead the specific dose of the responsible cell type.  The observed decrease in CSF NF-L levels could be caused by only one of the cell types in MSC treatments (e.g., the stem cells), which also varies significantly from patient to patient.  Asymmetrex’s TORTOISE TestTM technology provides the different fractions of tissue stem cells, committed progenitor cells, and arrested cells in tissue cell treatment samples.  Employing such technologies for future tissue stem cell clinical trials and approved treatments would make both much better in progress and quality.

We contacted the authors and invited them to share their responses to this review.  We did not receive a response from them.  We look for to impressions and thoughts from readers!

Next forum entry: May 11, “Treatment of Multiple Sclerosis with Nonmyeloablative Hematopoietic Stem Cell Transplantation: An Example of the Current Practice of Blind Dosing for Stem Cells in Clinical Trials”

A Stem Cell Count Would Have Made It Better:
Better Characterization: The Solution to the Natural Heterogeneity of Mesenchymal Stromal/Stem Cell (MSC) Treatments

Entry #13
Wednesday, April 13, 2022

This week’s entry for the WHMIB forum’s current focus on the natural heterogeneity of tissue stem cell preparations takes a somewhat different angle on the issue.  Today, we consider an insightful report from Dr. Guido Moll, Ph.D. and colleagues published very recently (February, 2022) in Stem Cells Translational Medicine.  The report, titled “Improved MSC Minimal Criteria to Maximize Patient Safety: A Call to Embrace Tissue Factor and Hemocompatibility Assessment of MSC Products,” addresses an important type of cell heterogeneity issue concerning mesenchymal stromal/stem cell (MSC) products and treatments that poses an increasing risk of a specific type of adverse events for patients that can lead to disability and even death, blood clots upon MSC infusion.

Moll and co-authors discuss thromboembolism and blood-incompatibility complications that occur in some patients as a result of the expression of highly-procoagulant tissue factor (TF/CD142) and other factors that affect the hemocompatibility of MSC products.  They raise the concern that, in the past decade, rapidly rising numbers of diverse sources and clinical applications of MSCs have put more and more patients at risk of unsafe treatments and adverse treatment outcomes.  This is mainly due to lack of stringent characterization and regulation of these increasingly diversified therapeutics.

The MSC heterogeneity, for which these authors propose a solution, is the variable and often unevaluated hemocompatibility of intravascular MSC treatment preparations.  The authors’ proposed solution makes perfectly good sense.  In their report, after establishing that hemocompatibility is an important factor for safety and effectiveness, they identify and propose practical methods for characterizing the hemocompatibility of MSC treatments before they are administered to patients!

This important report by Moll and co-authors caught the eye of the WHMIB forum because it models so well what also needs to be done to advance MSC medicine to the better characterization of treatments with their stem cell-specific dosage.  Referring to the standard panel of flow cytometry biomarkers currently used to characterize MSC preparations, the authors wrote:

“While useful, this panel of surface markers does not address either the therapeutic MoA [mode of action] of MSCs or identify potential risks or even heterogeneity of the product.”

For these authors, the focus of the risk is hemocompatibility, but for the entire field the risk because of unknown stem cell-specific dosage is ineffective treatments.  There is also a good-sense and practical solution for this problem.  It is called kinetic stem cell (KSC) counting.

We contacted the authors and invited them to share their thoughts.  Here’s what lead author Dr. Guido Moll shared:

“MSC therapeutics have profound immunomodulatory and regenerative properties that are of great interest as treatment for a multitude of clinical indications with unmet medical need.  Importantly, MSC therapeutics have increasingly diversified in the past decade, entailing many different types of tissue sources and manufacturing protocols, but also modes of clinical application/ delivery to patients.

The correct dosing, enumeration, molecular and functional characterization of MSCs in the primary tissue starting material and clinical products derived thereof is crucial for consistent and reproducible manufacturing of therapeutic MSC products.  Any new technology that improves consistent manufacturing of safe and effective cellular therapeutics is greatly appreciated to bring the field forward.”

We encourage readers to share their thoughts, too!

Next forum entry: April 27, “Making Mesenchymal Stem Cell Treatments for Progressive Multiple Sclerosis Better with Cell Type-Specific Dosing”

A Stem Cell Count Would Have Made It Better:
Immunophenotyping HSCs Out of the Cell Heterogeneity of Umbilical Cord Blood CD34+ Hematopoietic Cell Preparations

Entry #12
Wednesday, March 30, 2022

An attuned colleague brought to our attention this entry for the recent focus on the challenges that natural tissue cell heterogeneity imposes on the quantification of therapeutic tissue stem cells.  This review is also a reminder that the widespread treatment of the CD34+ count, as a specific measure of dosage of hematopoietic stem cells (HSCs), continues to be misinformed and misguided.  As noted in the three previous related WHMIB entries for both human and animal mesenchymal stem cell (MSC) preparations, presently available respective flow cytometry biomarkers like CD90, CD73, and now considered CD34 do not quantify the specific dosage of tissue stem cells.

In their November 2020 Blood Advances report, titled “CD34 expression does not correlate with immunophenotypic stem cell or progenitor content in human cord blood products,” Sruthi Mantri and colleagues investigated the flow cytometric immunophenotypes of CD34+-selected cell fractions from 50 samples of donor umbilical cord blood.  They used the best available immunophenotype for specific identification of HSCs (Lin– CD34+ CD38– CD45RA– CD90+) to evaluate inter-donor variation and relationship to the CD34+ cell fraction.  Their study reveals a high degree for donor-to-donor variation in the HSC fraction defined by this immunophenotype.  By this measure, the HSC fraction of CD34+ cell preparations ranged from 0.1% to 17.3% with a median of 6.7%.  Another important observation was the lack of significant correlation between this cell fraction of umbilical cord blood collections and their CD34+ cell fraction.

The conclusion of Mantri and co-authors from their study is the same as the theme of this Asymmetrex information forum.  A stem cell count would make umbilical cord blood HSC transplantation medicine better for patients.  Currently, cord blood units are initially selected for transplantation treatment based on the total number of blood cells collected.  After cryopreservation, small analysis samples are thawed and evaluated for their CD34+ cell fraction and colony forming unit (CFU) activity.  The report of Mantri et al. explains clearly the well-known observation that about 20% of cord blood transplant units are discovered, too late for the treated children, to have insufficient HSCs for successful treatment.  The study also indicates that many cord blood units are going unused because the currently applied clinical approval tests (i.e., total cell count, CD34+ cell fraction, and CFU assay) do not provide a specific count for HSCs.

Mantri and co-authors recommend that cord blood transplantation medicine institute their flow cytometry HSC immunophenotype for routine determination of the specific dosage of HSCs in cord blood units at the time of their collection before cryopreservation.  Asymmetrex also endorses this vision of implementation of this long overdue improvement in HSC transplantation medicine.  But one has to ask, “Given that the immunophenotyping technology has been available for decades, why wasn’t it implemented, as now recommended, years earlier?”

There are several reasons that may account for this long-standing poor state of affairs that the authors did not discuss in their brief report.  The first reason is that although their immunophenotype is the closest one defined to date for specifying human HSCs, it still is not ideal.  Past flow sorting analyses have estimated that perhaps only 1 in 5 cells with this immunophenotype are HSCs.  Even so, this degree of specificity is a huge improvement over the current methods used as indicators of cord blood unit potency.

Two other reasons have probably been the greater barriers to implementation of such an obviously important improvement for cord blood transplantation medical practice.  These are technical difficulty and cost.  Providing on-site, rapid, quantitative, multi-color flow cytometric immunophenotyping of whole or CD34+-fractionated cord blood requires expensive instruments and reagents and highly-trained technical personnel.

The latter two reasons are aspects where Asymmetrex’s kinetic stem cell (KSC) counting could make this clinical situation a whole lot better.  Asymmetrex’s rapid-counting Rabbit CountTM algorithms can determine the HSC-specific fraction of unfractionated cord blood from only a few days of inexpensive cell culture.  We are hopeful that this review of the excellent report of Mantri and co-workers may foster collaborations to evaluate how KSC counts of cord blood unit HSCs are related to their “HSC” immunophenotype.

We contacted the authors to invite them to share their responses.  We did not receive a reply from them.  We invite readers to share their own responses…

Next forum entry: April 13, “Better Characterization: The Solution to the Natural Heterogeneity of Mesenchymal Stromal/Stem (MSC) Cell Treatments”

A Stem Cell Count Would Have Made It Better:
An Irony in an Analysis of the Cell Size Heterogeneity of Canine Adipose-Derived Mesenchymal Stem Cell Preparations

Entry #11
Wednesday, March 16, 2022

When today’s entry was brought to our attention, we were excited to learn of an original scientific report about an investigation of how the tissue cell heterogeneity of mesenchymal stem cell (MStemC)-containing tissue cell preparations related to their functional capabilities.  However, our excitement was soon replaced with the realization of a more impressive irony of the report.

In a 2021 report in Tissue Engineering: Part C, titled “Microfluidic Separation of Canine Adipose-Derived Mesenchymal Stromal Cells” (“Ad-MSCs”), Dr. Zhuoming Liu and co-workers from the FDA and the Massachusetts Institute of Technology present an intriguing, comprehensive analysis of cells in Ad-MSC preparations isolated from beagles.  They show that discrete subpopulations of cells of different size have different functional properties that are commonly used to characterize MStemC preparations for stem cell therapies.  The stated motivation for the study was to investigate one of many possible types of Ad-MSC cellular heterogeneity that might contribute to the significant and unpredictable variability in clinical outcomes with such cellular treatments.

The report describes the use of a microfluidic sorting technique to isolate subpopulations for canine Ad-MSCs with three different respective mean diameters.  The authors establish clearly that differences in mean cell diameter are associated with differences in cell proliferation rates, senescence development rates, differentiation kinetics, and gene expression, depending on whether fetal bovine serum is supplemented.  A highly noteworthy demonstration is that the cell size-defined subpopulations, though functionally distinct for these important properties, are not distinguished by “MSC biomarkers” that are commonly used as quality measures for these tissue cell preparations.

Though conducted with canine tissue cells, the reported findings are quite relevant to the experience of human clinical studies with human Ad-MSC preparations.  Asymmetrex recently published a perspective highlighting the importance of firsts in veterinary stem cell medicine research and practice for accelerating progress in improving human stem cell medicine.

The unexpected irony of this report is that, although the authors were focused on defining important elements of cellular heterogeneity that influence functional properties important for effective stem cell therapy, they did not consider the heterogeneity of the stem cell-specific fraction of their Ad-MSC preparations.  They made the common error of treating these preparations as if all cells present were stem cells.  Even though they conservatively referred to their cells as Ad-MstromalCs, they discussed the three fractionated subpopulations of different size as if each were itself a homogeneous population of stem cells.

Their research is an excellent example of a case in which a stem cell-specific count would have made it better in a highly informative manner.  In addition to quantifying the number of tissue stem cells in their unfractionated donor preparations, they could have discovered how renewing tissue stem cells fractionated with respect to their three different size populations.  Asymmetrex’s kinetic stem cell (KSC) counting TORTOISE TestTM software also has the ability to define changes in the subfractions of stem cells, committed progenitor cells, and terminally-arrested differentiated cells during culture passaging.  Certainly, the fraction of tissue stem cells must be one of the more important elements of cellular heterogeneity that is a determinant of the outcome of stem cell treatments.

We contacted the authors and invited them to share their responses.  We did not receive a reply from them.  We invite readers to share their own responses…

Next forum entry: March 30, “Immunophenotyping HSCs Out of the Cell Heterogeneity of Umbilical Cord Blood CD34+ Hematopoietic Cell Preparations”

A Stem Cell Count Would Have Made It Better:
Flow Cytometric Characterization of “Adipose-Derived Stem Cells” in the Stromal Vascular Fraction from Breast Cancer Patients

Entry #10
Wednesday, March 2, 2022

When this report, published in 2022, was brought to our attention by a colleague, it fit very well within this forum’s current focus on the intrinsic cell heterogeneity of tissue stem cell-containing clinical preparations.  Of particular relevance, the presented studies provide a unique look at how cell heterogeneity defined by flow cytometry cell surface biomarkers evolves with expansion culture.

In their research article in Stem Cells International, titled “Extensive Characterization of Mesenchymal Stem Cell Marker Expression on Freshly Isolated and In Vitro Expanded Human Adipose-Derived Stem Cells from Breast Cancer Patients,” Dr. Premrutai Thitilertdecha, Ph.D. and co-authors described their use of flow cytometry to investigate characteristics of targeted cell types in the stromal vascular fraction (SVF) of liposuction isolations from breast cancer patients undergoing breast reconstruction surgery.

Using flow cytometry analysis, the authors defined four cell subpopulations of interest within the SVF.  These were designated as endothelial progenitor cells (EPCs), pericytes, mesenchymal stem cells (MSCs), and adipose-derived stem cells (ADSCs).  The ADSCs constituted about 60% of SVF cells, outnumbering MSCs by at least 10-fold.  The authors reviewed earlier reports that inclusion of the SVF with fat implants improved the cosmetic quality and durability of breast reconstructions.  Their investigation had the purpose of laying a foundation for future attempts to define properties of freshly isolated or expanded ADSCs that would provide better reconstructive surgery outcomes.

After only three culture passages, the flow cytometric distinction between ADSCs and MSCs was lost, with the majority of cells showing a MSC-like phenotype.  Unfortunately, the authors provided neither the number of population doublings (PDLs; See entry #6, Nov. 17, 2021) nor the data that would allow its determination for the purpose of comparison to other reports of changes in MSCs during expansion culture.  This transition in flow cytometric phenotype may reflect an in vitro lineage relationship between MSCs and ADSCs.  The authors reported that after three passages, the cells were able to undergo multipotent adipogenic, osteogenic, and chondrogenic differentiation.  However, disappointingly, they did not compare the differentiation potency of the uncultured cells.

Investigations of this type would be more informative, if they also included independent, specific quantification of the fraction of renewing tissue stem cells throughout their analyses.  Relating multi-marker flow cytometric cell subsets to the tissue stem cell-specific fraction would better delineate the tissue restoration properties of subtyped cell populations, which is the vision of the investigations.  Kinetic stem cell (KSC) counting can provide this capability.

We contacted the authors and invited them to share their responses and thoughts.  We did not receive a reply from them.  We hope readers will offer their insights and thoughts on the important issues the authors’ work raise for improving tissue stem cell science and medicine.

Next forum entry: March 16, “An Irony in an Analysis of the Cell Size Heterogeneity of Canine Adipose-Derived Mesenchymal Stem Cell Preparations”

A Stem Cell Count Would Have Made It Better:
A Critical Perspective on the Consequences of the Natural Heterogeneity of Tissue Stem Cell Preparations: The Case for Skeletal Stem Cells

Entry #9
Wednesday, February 16, 2022

The choice for the first entry of 2022 is a remarkably comprehensive perspective that addresses the fundamental need and challenge in tissue stem cell science and medicine for which Asymmetrex’s kinetic tissue stem cell (KSC) counting technology was conceived.  In their 2019 review article published in Frontiers in Cell and Developmental Biology, titled “A Revised Perspective of Skeletal Stem Cell Biology,” Dr. Thomas H. Ambrosi, Ph.D. and co-authors provide a rich history and cogent critical analysis of how the natural cell heterogeneity of tissue stem cell preparations has led to a corresponding problematic heterogeneity in how tissue stem cell preparations are perceived and represented.

From the vantage point of their specific focus on skeletal stem cells (SSCs), which have a historical and biological entwinement with more widely studied “mesenchymal stem” and “mesenchymal stromal” cell preparations, the authors illuminate the basis for the problems of identifying and quantifying tissue stem cells in tissue cell preparations.  The essential biological problem is natural, intrinsic tissue cell heterogeneity with tissue stem cells being a minor or rare fraction; and the crucial technical challenge is the lack of biomarkers that distinguish asymmetrically renewing tissue stem cells (“true stem cells”) from their committed progenitor progeny cells, which outnumber stem cells significantly in vivo and in vitro even in stem cell-enriched preparations.

Every new student, as well as every experienced investigator, in stem cell science and medicine should read this perspective.  It edifies understanding of the fundamental properties of tissue stem cells; and it cautions about the detrimental consequences to stem cell research and medicine when these fundamentals are unappreciated, oversimplified, or disregarded, as, unfortunately, they often are.  This perspective provides an outstanding service to the stem cell research, industry, and medicine communities; and more scholarly assessments like it of the current state of such important scientific ideas and technologies are needed.

We contacted the authors and invited them to share additional thoughts, but we did not hear from them.  We hope readers may have more to say.

Next forum entry: March 2, “Flow Cytometric Characterization of ‘Adipose-Derived Stem Cells’”

A Stem Cell Count Would Have Made It Better:
Tissue Stem Cell-Specific Fraction and Dosage: A Needed Standardization for Stem Cell Medicine

Entry #8
Wednesday, December 15, 2021

For the final entry of 2021, we consider an exciting, excellent recent special issue of the journal Stem Cells Translational Medicine. Professor Terry Lappin, Ph.D., from Queen’s University in Belfast, Northern Ireland and Professor Tao Cheng, M.D., from the Chinese Academy of Medical Sciences and Peking Union Medical College in Tianjin, People’s Republic of China, co-edited the November 2021 Special Issue: Standardization of Stem Cells and Stem Cell-Derived Products.

Prof. Lappin and Prof. Cheng titled their co-authored introductory editorial for the special issue, “An urgent need for standardization of stem cells and stem-cell derived products towards clinical applications.” They identify the lack of stem cell standardization and guidelines for many stem cell therapies as the “bottleneck” responsible for the disappointingly limited translation of the many recent discoveries on stem cells into effective clinical practice.

The co-editors relate that the special issue was inspired by the proceedings of the November 2020 seventh International Forum on Stem Cells held in Tianjin. The outgrowth of this inspiration is the assembly of eight excellent concise reviews of recent progress in studies to translate advances in stem cell biology into effective clinical applications, from the special perspective of development of reliable standardization for preclinical assessment of stem cell therapeutic products.

Seven out of the eight concise reviews in the special issue address the development of clinical applications with tissue stem cells (i.e., mesenchymal stem cells, hematopoietic stem cells, and endothelial stem cells). None of these reviews explicitly highlight the absence of quantifying the specific fraction or specific dosage of the discussed tissue stem cells. However, the need for this specific standardization lurks in the shadows of all of the described studies. This special issue would have been even better if the universal need for standards for quantifying and certifying the specific fraction and specific dosage of therapeutic tissue stem cells were addressed directly.

We contacted the co-editors for their responses to this recommendation towards improving future discussions of needed stem cell standardizations. Professor Terry Lappin graciously replied in a November 30 email message that,

“Professor Tao Cheng and I have consulted with the Managing Editor and Editor-in-Chief of STEM CELLS Translational Medicine. They point to the importance of refraining from an editorial response in relation to a commercial product.”

Perhaps, the editors overlooked that Asymmetrex provides its tissue stem cell counting technology free to the stem cell research community and industry on its company website.

Next Review: Begins February 16 in 2022!

~ Best wishes for a joyous holiday season and a Happy New Year! ~

A Stem Cell Count Would Have Made It Better:
Sartorius: How Can the Evaluation of Tissue Stem Cell Expansion Media Be Enhanced?

Entry #7
Wednesday, December 1, 2021

In this week’s review entry, Asymmetrex takes a look at another important company in human tissue stem cell supply space, but from a different perspective than RoosterBio. Whereas RoosterBio’s primary market focus is supplying expanded human mesenchymal stem cells (hMSCs), Sartorius now markets cell culture medium products for use in the expansion of stem cells, including hMSCs, for both clinical applications and research. Sartorius recently increased its position in this market with its recent acquisition of cell culture media producer Biological Industries, Ltd.

Sartorius is representative of a number of prominent companies that produce and market cell culture medium products for the growth and expansion of therapeutic tissue stem cells, including hMSCs, human pluripotent stem cells (hPSCs), and hematopoietic stem cells (HSCs). Although it is generally well recognized that such products for HSCs provide limited if any HSC expansion capability, the same shortcoming is not the impression for hMSCs media. However, contradicting this prevalent attitude, current functional assays for hMSC-associated activities are well-described for declining after 5-10 passages (PDLs unstated!) in any commercial culture medium.

The difference in attitudes and beliefs about the expansion of HSCs and hMSCs is due to a difference in the expression kinetics of their respective biomarkers by the committed progenitor cells (CPCs) and differentiating cells they produce in culture. In the case of hematopoietic cells, biomarkers like CD34 and CD133, which are expressed by both HSCs and CPCs, decline rapidly in CPCs and their differentiating progeny. However, in contrast, markers like CD73 and CD90, which are also expressed by both MSCs and their progeny CPCs, continue to be expressed in MSC-produced CPCs. So, late passaged cultures of hMSCs can have high fractions of CD73-positive and CD90-positive cells, despite waning levels of MSC function.

Sartorius and its newly acquired hMSC culture medium products are being highlighted in this entry because of its recent e-book announced in the October 22, 2021 issue of RegMedNet’s online Regeneration Weekly newsletter. The e-book introduced Sartorius’s hMSC culture media products and provided attendees with many excellent examples of their effectiveness for culture of mesenchymal tissue cells from several different human tissue sources. It is noteworthy that Sartorius scientists keep their analyses to no more than 5 passages.

Sartorius and other suppliers of cell culture medium products for culturing and maintaining tissue stem cells, like HSCs, hPSCs and hMSCs, would benefit from evaluating the effects of their products on the tissue stem cell-specific fraction during culture. So, would the users of their products as well.

We contacted Maya Rotman, the corresponding author for the application report in Sartorius’s webinar e-book, to get the company’s impressions. Here is their response:

Dear Asymmetrex,

Thank you for focusing on Sartorius’s publication on Reg.Med.Net e-Book, about hMSCs. As you mentioned, Sartorius offers cell culture medium products for maintenance, expansion, and differentiation, as well as cryopreservation, of many types of cells, such as MSCs, iPSCs and immune cells.

Sartorius emphasizes all products go through strict QC tests, and the cells are examined for many characteristics to ensure accurate performance reports. Our R&D and QC labs are up to date with the most advanced performance assays for all our products, and we make a point of delivering reliable and evidence-based results to our customers.

It is reasonable to assume that using advanced mathematical evaluation methods, such as Asymmetrex promotes, for specific counting of therapeutic tissue stem cells could be beneficial for identifying stem cell-specific fractions in later passages, exceeding the first 5-10.

Maya Rotman, MSc
Product Management Organization Support and Content Writer
Sartorius

Next Review: December 15, Tissue Stem Cell-Specific Fraction and Dosage: A Needed Standardization for Stem Cell Medicine

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?

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: