Asymmetrex Technology to Count Stem Cells

For more than a half-century, stem cell science and stem cell medicine have struggled with the need for a technology to count stem cells. This deficiency has limited progress in many disciplines, with one of the most important being the ability to determine stem cell treatment doses. In 2016, Asymmetrex announced the invention of a method to count adult tissue stem cells accurately. The method relies on the asymmetry in the growth patterns of stem cells and progenitor cells during culture. Yet, despite validations of the new technology, few stem cell scientists or physicians have adopted it so far. 

The stem cell counting problem

The perennial problem with counting tissue stem cells is well known among experts. At the root of the problem are the biological properties unique to stem cells: Stem cells have very low concentration in tissues of the body; stem cells divide to produce both more stem cells but also committed progenitor cells; and it is difficult to distinguish the stem cells from the committed progenitor cells.^1-4

First, the concentration of stem cells in human tissues ranges from 1 in 1000 to less than 1 in 10,000. Methods that seek to isolate stem cells by growing them out in tissue cultures (explant methods) always produce a heterogeneous cell mixture, they are never pure stem cells. Even the most stem cell-enriched cultures only achieve a final stem cell concentration of a few percent.

Second, early committed progenitor cells are the immediate products of stem cell renewal by division. Unlike stem cells, progenitor cells cannot renew themselves indefinitely. Progenitor cells are committed to multiplying themselves for a limited number of divisions, before their lineages end with mature tissue cells that perform specific organ and tissue functions.⁵

Finally, until now there has been no practical method to distinguish stem cells from early committed progenitor cells. For example, all of the so-called “stem cell biomarkers” that are used to count stem cells (e.g., CD34, CD133, CD90) are expressed on the cell surface of both stem cells and committed progenitor cells.^1,4  Since committed progenitor cells outnumber stem cells by as much as 100 to 1, they obscure all attempts to count stem cells by using these biomarkers. Another example of a measure that counts both stem and committed progenitor cells is the widely used colony-forming unit (CFU) assay⁶, which cannot discern whether a cell colony originated from a stem cell or from an early committed progenitor cell.

Asymmetrex solution  to stem cell counting

In September 2016, Asymmetrex announced its AlphaSTEM Test™ technology that answers the crucial question of how to quantify tissue stem cells independent of committed progenitor cells. The underlying biological principle for the method is the asymmetric kinetics of stem cell self-renewal.^7-9

During asymmetric self-renewal, stem cells divide continuously to produce equal numbers of new stem cells and committed progenitor cells. As a result, the stem cell number remains relatively constant, while generation after generation of committed progenitor cells are produced, and the committed progenitor cells continue dividing until they produce non-dividing mature cells.⁵  Tissue stem cells in cell cultures retain these asymmetric self-renewal kinetics to a large extent.¹⁰

Because of the unique kinetics of stem cell renewal during serial cell cultures, it is possible to define an algorithm that discovers the number of tissue stem cells based on changes in total cell number with culture. Asymmetrex partnered with AlphaSTAR Corporation to develop the AlphaSTEM Test™ computer software, which applies the algorithm to yield the tissue stem cell number at any time during serial culture. This achievement is the first method for counting adult tissue stem cells.

The AlphaSTEM Test™ compares favorably to the previous gold standard for counting hematopoietic stem cells, the limiting-dilution SCID-mouse repopulating cell assay.¹¹ Both assays rely on statistical modeling, but the AlphaSTEM Test™ has the advantages of simplicity, low expense, greatly increased throughput, robustness, and application for other types of stem cells.

Onward to adoption

Asymmetrex has reported numerous validations of the AlphaSTEM Test™.^7-9  The technology has been used to count stem cells from various human tissues, including liver and lung, to count hematopoietic stem cells from bone marrow and umbilical cord blood, and to count mesenchymal stem cells from bone marrow, umbilical cord tissue, and amniotic membrane. The technology is able to quantify stem cell fractions ranging from a few percent to as low as 1 in 10,000. Additional validations have explored the effects of stem cell-toxic agents and stem cell-activating agents.

Pediatric cord blood transplants are just one example of the potential applications for this technology. Currently, an estimated 11% to 18% of cord blood transplants in the United States fail to engraft, and this is suspected to result from low counts of hematopoietic stem cells in the cord blood unit.^12,13

At present, the AlphaSTEM Test™ is available as a contract service. AlphaSTEM Test™ can be applied to a testing segment of the stem cell therapy, to infer the stem cell dose of the entire unit. If the stem cell therapy goes through a manufacturing process that involves serial expansions of cultures, the software can provide stem cell count data at each step of the production process. This knowledge enables the manufacturer to optimize the process design. Asymmetrex continuously invites collaboration with stakeholders in the stem cell industry, including stem cell transplant centers, stem cell therapy companies, and academic stem cell labs. The sooner this stem cell counting technology is widely adopted, the sooner we can advance stem cell dosing in the practice of medicine.

James L. Sherley, MD PhD, is the founder and director of Massachusetts stem cell biotechnology company Asymmetrex, LLC. Asymmetrex develops and markets technologies for advancing stem cell medicine, including the first-in-kind technology for specific counting of adult tissue stem cells. This technology is also applied to design optimized procedures for more effective manufacturing of therapeutic adult tissue stem cells at greatly reduced cost. Dr. Sherley is a graduate of Harvard College, with a BA degree in biology, and the Johns Hopkins University School of Medicine, earning joint MD and PhD degrees. Prior to founding Asymmetrex, he held academic research appointments at the Fox Chase Cancer Center, Massachusetts Institute of Technology, and Boston Biomedical Research Institute. Dr. Sherley’s professional awards include Pew Biomedical Research Scholar, Ellison Medical Foundation Senior Scholar in Aging Research, and NIH Director’s Pioneer Award.

References

1. Ivanovic Z. (2010) Hematopoietic Stem Cells in Research and Clinical Applications: The “CD34 issue,” World J Stem Cells 2, 18-23.
2. Sherley, J. L. (2018) Achieving Dose Standardization for the Stem Cell Clinical Trials Industry, Operations 10:03
3. Sherley, J. L. (2018) The Stem Cell Emperor’s New Clothes, Analysis, Nov. 6
4. Sherley, J. L. (2018) Dose Determination for Stem Cell Medicine: A Need Whose Time Has Come, in Perinatal Stem Cells: Research and Therapy, eds. A. Atala, C. Cetrulo, K. Cetrulo, S. V. Murphy, and R. Taghizadeh, Elsevier, (Amsterdam).
5. Potten, C. S. and Morris, R. J. (1988) Epithelial Stem Cells In Vivo, J. Cell Sci. Suppl. 10, 45-62.
6. Rich, I. N. (2015) Improving Quality and Potency Testing for Umbilical Cord Blood: A New Perspective, Stem Cells Trans. Med. 4, 967-973.
7. Sherley, J. L. (2017)  Methods for Determining the Effects of an Agent on Tissue Stem Cells, U.S. Patent No. 9,733,236.
8. Sherley, J. L. (2018) Stem Cell Therapy: Resolving the Mismatch, Pharmaceutical Manufacturing, March, 38-41.
9. Dutton, R., Abdi, F., Minnetyan, L. and Sherley, J. L. (2018) A Computational Technology for Counting of Adult Tissue Stem Cells, submitted.
10. Paré, J.-F. and Sherley, J. L. (2006) Biological Principles for Ex Vivo Adult Stem Cell Expansion, in Current Topics in Developmental Biology, ed. G. Schatten, Elsevier, Inc. (San Diego), Vol. 73, 141-171.
11. Ziegler, B. L. et al. (1999) KDR receptor: A Key Marker Defining Hematopoietic Stem Cells, Science 285, 1553-1558; DOI: 10.1126/science.285.5433.1553.
12. Olsson, R. et al. (2013) Graft Failure in the Modern Era of Allogeneic Hematopoietic SCT, BMT 48, 537-543.
13. Wagner, J. et al. (2014) One-Unit versus Two-Unit Cord-Blood Transplantation for Hematologic Cancers. N Engl J Med  371:1685-1694.