This sixth Counting Impact review considers a report that continues the ongoing examination of the CD34 count as the basis for the dosage of cells used for hematopoietic stem cell transplantation (HSCT) after blood stem cell-ablative high-dose treatments for cancers.  In the October 31 issue of Blood Cancer Journal, Oren Pasvolsky and his co-workers report an evaluation for the case of HSCT with patients’ own blood cells after they received high-dose chemotherapy for multiple myeloma. (1)

They found that patients who received a lower CD34 cell dosage had significantly poorer cancer survival than patients receiving a dosage above a higher threshold.  However, within either group of patients, their individual CD34 cell dosage was not predictive of the progression of their cancer or their overall survival.  So, the cause of the difference in treatment effectiveness related to the CD34 count dosage threshold was not revealed by this study; and it continues to be an important mystery whose solution could improve cancer treatment.

Although the CD34 count is used throughout blood stem cell research and HSCT medicine, it is not a differential HSC count.  In fact, the CD34 count is widely known to greatly overestimate the number HSCs, because it does not delineate the HSCs in a blood sample from the more abundant progenitor cells.  Progenitor cells cannot restore hematopoietic cell production like HSCs.

In the Pasvolsky et al. study, only one patient (in the high CD34 cell dosage group) received a transplant that failed to be restorative.  However, the restoration of patients receiving the lower CD34 cell dosage took longer.  Given these treatment features, it is of interest whether the differential HSC count of HSCT treatments is a better predictor of both restoration rates and cancer survival, because the CD34 cell count is quite a poor indicator of the differential HSC count. (2,3)

1. Pasvolsky, O., Marcoux, C., Milton, D.R. et al. Optimal infused CD34⁺cell dose in multiple myeloma patients undergoing upfront autologous hematopoietic stem cell transplantation. Blood Cancer J.14, 189 (2024). https://doi.org/10.1038/s41408-024-01165-w.

 2. Mantri S, Reinisch A, Dejene BT, Lyell DJ, DiGiusto DL, Agarwal-Hashmi R, Majeti R, Weinberg KI, Porteus MH. CD34 expression does not correlate with immunophenotypic stem cell or progenitor content in human cord blood products. Blood Adv. 2020 Nov 10;4(21):5357-5361. doi: 10.1182/bloodadvances.2020002891. PMID: 33136125; PMCID: PMC7656928.

3. Sherley JL, Daley MP, Dutton RA. Validation of Kinetic Stem Cell (KSC) counting algorithms for rapid quantification of human hematopoietic stem cells. J Stem Cell Ther Transplant. 2022; 6: 029-037.

A recent intriguing report by Severin Ruoss and colleagues at the University of California – San Diego, published in the July 12, 2024 issue of Stem Cell Advances (1), provides a compelling motivation for future studies to evaluate the utility of kinetic stem cell (KSC) counting for defining effective tissue stem cell treatment dosages.

The new study reports comparative analyses of preparations of two related types of donor tissue cell preparations commonly used for stem cell research and for stem cell treatments.  Measures of cell phenotypes, cell gene expression, and cell protein content were compared between the two preparations isolated from the same donor.  For statistical power in the study, comparison analyses from a total of 21 male and female donors were aggregated.

The compared tissue cell preparations were uncultured bone marrow aspirate concentrates (BMAC) and adipose-derived stromal vascular fractions (ADSVF).  Throughout fields of stem cell research and stem cell medicine, these donor tissue cell preparations are widely regarded as containing functionally similar effector “stem cells.”  However, the authors concluded that their direct comparison study “challenges the prevalent notion that there is one therapeutic cell type present in both tissues.”

Although the two tissue preparation types did not align significantly for any of the measured cell properties, there was one feature they shared.  Neither yielded a confidently identified stem cell population based on existing cell marker expression profiles; and both were lacking in cells with marker profiles currently used to define “stem cells” in these preparations after cell culture expansion.

The authors propose that their results can enable future studies with the aim of delineating subpopulations of BMAC cells and ADSVF cells whose dosages will be confident indicators of beneficial treatment outcomes.  Conceptually, this proposal is certainly reasonable.  But the prospect of discovering such cells and implementing their dosing with current highly complex and uncertain methods, like single-cell RNA sequencing analyses, is quite daunting.

This insightful new report is another excellent indication of the potential impact of future studies that relate cell subtype populations delineated and quantified by KSC counting to treatment outcomes for BMAC and ADSVF.  KSC counting defines stem cell, committed progenitor cell, and arrested differentiated cell populations in mesenchymal tissue cell preparations.  The simple evaluation of how the specific doses of these distinct cell kinetics subtypes relates to treatment outcomes would certainly be an important complement to the complex studies envisioned by the authors; and it might independently identify a critical determinant of treatment outcomes.

1. Severin Ruoss et al., Comparative single-cell transcriptional and proteomic atlas of clinical-grade injectable mesenchymal source tissues. Sci. Adv. 10, eadn2831(2024). DOI:10.1126/sciadv.adn2831.

This COUNTING IMPACT forum considers a recent meta-analysis of clinical trials of the treatment of COVID-19 associated acute respiratory distress syndrome (ARDS) with mesenchymal stromal cell (MSC) preparations.

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.

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.  CONTACT US to have your comments posted here.

This COUNTING IMPACT forum addresses 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 forum considers a review article published in advance on August 16, 2022 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 HSCs 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; szaco56, 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.  CONTACT US to have your comments posted here.

The report selected for this COUNTING IMPACT forum entry of 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, 2022 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. CONTACT US to have your comments posted here.