**1. Introduction**

382 Advances in Hematopoietic Stem Cell Research

[134] Walters MC, Storb R, Patience M, et al. Impact of bone marrow transplantation for

sibling donor transplantation. *Blood.* Mar 15 2002; 99(6):1971-1977.

symptomatic sickle cell disease: an interim report. Multicenter investigation of bone marrow transplantation for sickle cell disease. *Blood.* Mar 15 2000; 95(6):1918-1924. [135] Weisdorf DJ, Anasetti C, Antin JH, et al. Allogeneic bone marrow transplantation for

chronic myelogenous leukemia: comparative analysis of unrelated versus matched

Potency is the quantitative measurement of biological activity of a product (European Medicines Agency (EMA), 2008). Potency provides assurance that production and manufacture demonstrate consistency and provides information on stability and performance of the product. It also allows correlation with the clinical response and can help avoid product failure or toxicity due to the improper dose of the product being administered. For biopharmaceutical products such as drugs, growth factors and cytokines, vaccines etc., measurement of potency to predict dose has been a routine procedure for many years. Cells, on the other hand, are complex living entities that are continuously in flux. The potency of cells can change depending on numerous physiological and external environmental factors. Yet, with the increased number of cellular therapeutic applications and clinical regimen involving numerous cell types, the need to reliably and reproducibly measure biological and functional activity to meet the requirements of potency and ensure patient safety is of increasing importance (EMA, 2008; U.S. Food and Drug Administration, (FDA), 2011).

Determining the potency of a stem cell therapeutic can be a daunting task, especially if knowledge of the system biology, physiology and regulation is limited. In contrast, the hematopoietic system has proven to be not only an excellent model for stem cell biology, but also a model system for proliferation and differentiation in different applications. One of these applications is stem cell transplantation, a procedure that had its origins during the 1950s, became a quantitative assay in mice in 1961 (Till & McCulloch), and a routine clinical procedure in the 1970s (Santos et al. 1972; Thomas et al. 1977; Santos, 1983) Since that time, the number of human bone marrow transplantations reached a peak in the late 1990s (National Marrow Donor Program (NMDP); Pasquini & Wang, 2010) and has been declining to be replaced by alternative stem cell sources derived from mobilized peripheral blood (Haas et al. 1990; Koerbling et al. 1990; Sohn et.al. 2002) and umbilical cord blood (Broxmeyer et al. 1989; Gluckman et al. 1989).

Regardless of the tissue source, a successful transplant of stem cells is dependent upon the ability of the transplanted stem cells to lodge or "seed" in the bone marrow and begin the process of proliferation to produce lineage-specific progenitor cells. These differentiate into functionally mature circulating neutrophils, platelets and erythroid cells, the number of

Hematopoietic Stem Cell Potency for Cellular Therapeutic Transplantation 385

in the initial stem cell culture and measurement step. The data obtained provides a degree of stem cell quality and potency assurance that has hitherto not been possible using the traditional methods of total nucleated cell count (TNC), viability and viable CD34+ counts, which provide no indication of stem cell functionality or growth. To illustrate the steps of the assay, a small number of mobilized peripheral blood samples are used to demonstrate the procedure for determining potency, quality and release criteria. A larger cohort of

The assay relies on two basic characteristics of stem cells, namely proliferation ability (quality) and potential (potency). It had been previously demonstrated that when hematopoietic stem cells were stimulated to proliferate in the presence of growth factors and cytokines, the intracellular ATP (iATP) concentration increased proportionately to the cell concentration plated (Rich & Hall, 2005; Rich, 2007). The steepness or slope of the cell dose response was dependent upon the primitiveness and proliferation potential of the cells being examined. Stem cells have a greater proliferation potential than lineagespecific progenitor cells (Botnick e al. 1979). It would therefore be expected that the slope of the cell dose response would be steeper for stem cells than progenitor cells. In other words, the steeper the slope of the cell dose response, the greater the proliferation potential and the greater the potency. This biological phenomenon was incorporated into an assay that first estimates the potency ratio for two stem cell populations of a sample compared to a reference standard of the same material. The information obtained from the initial culture step was then used in the second step to substantiate the correlation between stem cell potency and quality. Finally, stem cell potency and quality were combined to determine release criteria of a sample. This information is provided when the iATP is released after culture by lysis of the cells. The iATP acts as a limiting substrate for the most sensitive, non-radioactive signal detection system available. This is a luciferin/luciferase reaction that produces bioluminescence, which is measured as light in a plate luminometer (Rich, 2003). The procedure and results described in this communication lay the foundation for future studies of stem cell potency and clinical outcome that might improve the risk of graft failure (Picardi & Arcese, 2010; Querol et al.

Several cryopreserved, mobilized peripheral blood (mPB) samples from different donors were obtained from AllCells, Inc (Berkley, CA) in accordance with the company's Internal Review Board (IRB) approval. Vials of cryopreserved umbilical cord blood (UCB) samples were provided and released for research purposes by the University of Colorado Cord Blood Bank (ClinImmune, Inc) in Aurora, CO with approval by the respective Internal Review Board. Additional mPB and UCB cells were obtained from each source to use as

The establishment of RSs is an absolute requirement for performing a potency assay. For hematopoietic cell-based therapeutics the number of cells obtained from a single donor UCB unit, mPB procedure or bone marrow aspirate are limited. This poses severe restrictions on

umbilical cord blood samples is then used to show the applicability of the assay.

2010) as well as safety and efficacy for the patient.

**2. Materials and methods** 

internal reference standards. **2.2 Reference standards (RS)** 

**2.1 Cells** 

which provides information on the time at which engraftment took place. Proper lymphohematopoietic reconstitution occurs much later. The ability of the stem cells to engraft is dependent upon two primary factors. The first is the status and condition of the patient. The second is the proliferation ability and potential of the stem cells prior to being transplanted.

Proliferation ability is equivalent to the proliferation status of the stem cells at the time of testing. This parameter defines stem cell "quality". Proliferation potential, on the other hand, is the capacity or potential of the stem cells to proliferate. For a continuously proliferating system such as lympho-hematopoiesis, stem cell potential decreases from the most primitive to the most mature stem cells. Thus, the more primitive a stem cell, the greater its proliferation potential and therefore its potency. It follows that the primary goal of stem cell transplantation is to provide the patient with stem cells that exhibit varying degrees of proliferation potential or potency. In this way, the patient can be endowed with stem cells that provide both short- (Charbord, 1994; Civin et al. 1996; Leung et al. 1999; Zubair et al. 2006) and long-term (Civin et al. 1996; Leung et al. 1999; Zubair et al. 2006; Duggan et al. 2000) engraftment and reconstitution.

A product that is "balanced" to provide the correct amount of short- and long-term stem cell engraftment and reconstitution would be the ideal situation. Present technology is not, however, capable of measuring or delivering a "balanced" stem cell product. In many cases, the donor stem cell product is skewed towards a greater proportion of mature rather than primitive stem cells or visa versa. However, it is possible to quantitatively measure both stem cell quality and potency of representative stem cell populations to provide a reasonably good approximation of the overall quality and potency of the stem cell product. These parameters would then predict the potential of the stem cells to engraft and reconstitute the system.

In 2009, the U.S. Food and Drug Administration (FDA, 2009) designated umbilical cord blood as a drug because, when transplanted into a patient, it results in systemic effects. The consequence of this designation has meant that virtually every aspect from cord blood collection to transplantation must be validated and documented according to regulatory requirements. Included in this process are the tests and assays to monitor the procedures and characterize the product prior to use. Besides histocompatability testing, the most important parameter that should be measured just prior to the stem cell product being used is potency. The FDA guidance on potency for cellular therapeutic products specifically describes the regulations that define a potency assay as compliant (FDA, 2011). A potency assay must provide quantitative data demonstrating the biological activity of all "active ingredients" specific to the product. In the case of a stem cell product, the "active ingredients" are the stem cell themselves. The results must meet pre-defined acceptance and/or rejection criteria so that the test results provide information as to whether the product can be released for use. In addition, the assay(s) must include reference materials, standards and controls, since without these, the necessary validation parameters (accuracy, sensitivity, specificity, precision and robustness) cannot be measured and documented.

The present communication describes an *in vitro* assay that measures stem cell potency and quality and helps define release criteria for hematopoietic products derived from mobilized peripheral blood, umbilical cord blood or bone marrow. The assay was designed to comply with regulatory requirements. In the 3-step process, all of the data required is accumulated in the initial stem cell culture and measurement step. The data obtained provides a degree of stem cell quality and potency assurance that has hitherto not been possible using the traditional methods of total nucleated cell count (TNC), viability and viable CD34+ counts, which provide no indication of stem cell functionality or growth. To illustrate the steps of the assay, a small number of mobilized peripheral blood samples are used to demonstrate the procedure for determining potency, quality and release criteria. A larger cohort of umbilical cord blood samples is then used to show the applicability of the assay.

The assay relies on two basic characteristics of stem cells, namely proliferation ability (quality) and potential (potency). It had been previously demonstrated that when hematopoietic stem cells were stimulated to proliferate in the presence of growth factors and cytokines, the intracellular ATP (iATP) concentration increased proportionately to the cell concentration plated (Rich & Hall, 2005; Rich, 2007). The steepness or slope of the cell dose response was dependent upon the primitiveness and proliferation potential of the cells being examined. Stem cells have a greater proliferation potential than lineagespecific progenitor cells (Botnick e al. 1979). It would therefore be expected that the slope of the cell dose response would be steeper for stem cells than progenitor cells. In other words, the steeper the slope of the cell dose response, the greater the proliferation potential and the greater the potency. This biological phenomenon was incorporated into an assay that first estimates the potency ratio for two stem cell populations of a sample compared to a reference standard of the same material. The information obtained from the initial culture step was then used in the second step to substantiate the correlation between stem cell potency and quality. Finally, stem cell potency and quality were combined to determine release criteria of a sample. This information is provided when the iATP is released after culture by lysis of the cells. The iATP acts as a limiting substrate for the most sensitive, non-radioactive signal detection system available. This is a luciferin/luciferase reaction that produces bioluminescence, which is measured as light in a plate luminometer (Rich, 2003). The procedure and results described in this communication lay the foundation for future studies of stem cell potency and clinical outcome that might improve the risk of graft failure (Picardi & Arcese, 2010; Querol et al. 2010) as well as safety and efficacy for the patient.
