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”

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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

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:

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

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