Six considerations when evaluating functionality of hematopoietic stem and progenitor cells in cell therapy products

22 Mar 2023

Written by STEMCELL Technologies

Key questions to consider when designing potency assays

There are unique challenges when it comes to developing cell therapy products, including ensuring the reproducibility and consistency of product lots. These specific concerns arise from the inherent variability of the starting material, which may come from multiple patients or donors. Before using these cells as therapeutics, the quality – including both purity and potency – of the final product must be established. This, in turn, requires reliable assays.

Two of the most common assays performed to measure the functionality of hematopoietic stem and progenitor cells (HSPCs) are an in vivo mouse model engraftment study and the in vitro colony-forming unit (CFU) assay. Both assays have their advantages and disadvantages. In this article, you will find answers to six key questions that you may consider while designing assays to evaluate the functionality of HSPCs in your cell therapy workflow.

1. What are the available sources of HSPCs?

The three main sources of primary CD34⁺ cells are: cord blood (CB), bone marrow (BM), and mobilized peripheral blood (mPB). Choosing the cell source is strongly dependent on the end goal of the experiment. Research has shown that CB engraftment can be 5- to 10-fold higher per CD34^⁺ input cell when compared to BM [1]. For this reason, BM requires 5- to 10-fold more cells for positive engraftment in non-obese diabetic/immunodeficient (NOD/SCID) mouse models.

2. What is the CFU assay and how is it set up?

The CFU assay is a functional assay that is used as a readout for HSPCs. It was first described in 1979 by Metcalf et al. [2] and was shown to be a clonal assay that can directly measure the growth and frequency of functionally viable HSPCs, as well as their ability to differentiate into mature cells of each specific lineage found in normal PB. The assay enables quantification of the number of stem and progenitor cells within any blood source based on this biological ability.

The setup for the CFU assay requires the addition of BM, CB, PB or mPB, MNCs, or purified or expanded CD34⁺ cells into a semi-solid methylcellulose-based medium (e.g., MethoCult™) that allows the clonal growth of a single progenitor cell that remains spatially isolated from other colonies within the culture. This spatial separation allows the colonies to be separately identified and counted based on the mature cell progeny generated by a single HSPC.

The CFU assay allows the classification of each HSPC colony based on mature cell morphology and determines the frequency of progenitors for each specific lineage, assuming that an optimal progenitor-specific cytokine cocktail is included in the culture medium. Many years of research have shown that the CFU assay is a quantitative assay that is able to identify the frequency and lineage potential of functionally viable HSPCs. Indeed, results from the CFU assay correlate strongly with the engraftment potential of HSPCs in patients. Furthermore, this assay is compatible with automated scoring and standardized cell counting and classification, increasing the assay’s robustness.

You can learn the finer details of performing the CFU assay by registering for STEMCELL’s on-demand course.

3. How do you evaluate the lineage differentiation of HSPCs in semi-solid cultures?

One way to determine the quality of a hematopoietic cell therapy product is by evaluating the multilineage differentiation potential of HSPCs. This can be assessed by both in vitro assays, including the CFU, long-term culture initiating cell (LTC-IC), and lymphopoiesis assays, and in vivo by evaluating long-term engraftment in the bone marrow of NOD/SCID mice.

When cultures are maintained in a suitable semi-solid matrix (such as MethoCult™) supplemented with appropriate cytokines and supplements, individual progenitor cells called colony-forming units (CFUs) proliferate and differentiate to form discrete cell clusters or colonies with distinct morphology. Colonies derived from different types of progenitor cells are classified and counted based on the number and types of mature cells they contain using morphological and phenotypic criteria.

The CFU assay is most commonly used to detect multipotential and lineage-restricted progenitors of the erythroid, granulocytic, and macrophage lineages. Erythroid progenitor-derived colonies( burst-forming unit-erythroid; BFU-E and colony-forming unit-erythroid; CFU-E) will consist of small, red-colored erythroblast cells that are distinctively different when observed using the 10x objective of an inverted microscope from myeloid colonies, (colony forming unit granulocyte, erythrocyte, monocyte, megakaryocyte; CFU-GEMM) which consist of larger, clear and shiny granulocytes, monocytes, and macrophages.

4. What to consider when using HSPCs for cell therapy applications?

Before using HSPCs for therapy, the quality must be defined through both purity and potency.

Purity is defined as the proportion of desired cells and is expressed as a percentage of the total cellular component of the product. This is usually assessed through fluorescence-activated cell sorting (FACS), which provides information about the cell population based on the light scattering and fluorescent characteristics of the cells.

Potency identifies the capacity of the product to give a desired result. For HSPCs, this is the ability to engraft into recipients and to proliferate and differentiate into mature blood cells. Quantitative in vitro assays that measure the capability of cells to form CFUs upon culture in semi-solid medium can aid in the assessment of HSPC product potency.

According to the FDA, viability, which is defined only as the number of healthy cells in a sample, is generally not accepted as the only measurement of efficacy; therefore, quantifying potency through CFU growth is recommended.

A successful potency assay must demonstrate product activity. In the case of hematopoietic cell therapy products the activity pertains to the functionality of the cells and the long-term engraftment of the cell product. Engraftment following HSPC transplantation is typically considered to be successful in humans when the numbers of neutrophils and platelets in the circulation have recovered to a sufficient level that the patient is considered ‘transfusion independent’; typically, this is defined as >500 neutrophils and >50,000 platelets per μL of blood [3]. The best assay would be one in which engraftment is evaluated; for human HSPCs, this can be tested using the NOD/SCID mouse transplantation assay. However, routine in vivo studies for potency assays are very time-consuming, expensive, and difficult to validate.

Since the CFU assay significantly alleviates the time and cost issues associated with the in vivo mouse transplantation assay and has been shown to correlate well with in vivo engraftment in patients [4], it is recommended for evaluating the quality and potency of HSPCs used in cell therapy.

5. Does the CFU assay meet the FDA’s requirements for a potency and stability assay suitable for cell therapy products?

The FDA recommends that potency assays comply with applicable regulations. Some of these regulations suggest that cell therapy products should:

demonstrate product activity
have a quantitative readout
indicate product stability and consistency
be validated

To learn more about the utility of the CFU assay in the development and manufacturing of hematopoietic cell therapy products, watch this webinar on Determining the Potency and Stability of Hematopoietic Cells by STEMCELL expert Jackie Damen.

6. How do iPSC-derived HSCs compare to bone marrow (BM)-derived HSCs and mobilized peripheral blood (mPB) HSCs in terms of the CFU assay?

The CD34⁺ cells derived from pluripotent stem cell (PSC) cultures are not functionally the same as the CD34⁺ cells derived from adult sources. Depending on the stage of embryogenesis from which the cells arise, their functional potential can be different. For example, lymphopoiesis is not required at primitive stages of development.

Depending on the method used to derive HSPCs from PSC cultures, the total CFU frequency and lineage progenitor frequency can be very different. Most research has shown that the frequency of CFU/CD34⁺ input is lower for PSC-derived CD34⁺ cells (compared to both BM and mPB sources) and not all lineages are represented. In addition, the resulting colonies are typically smaller with fewer cells, suggesting PSC-derived CD34⁺ cells have lower proliferation potential in CFU cultures.

Further Reading from STEMCELL Technologies:

References:

Wang JC, Doedens M, Dick JE et al. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood. 89(11), 3919-3924 (1997).
Metcalf D, Johnson GR, Mandel TE et al. Colony formation in agar by multipotential hematopoietic cells. J. Cell. Physiol. 98(2), 401-420 (1979).
Yahng SA, Lee JW, Kim Y et al. New proposed guidelines for early identification of successful myeloid and erythroid engraftment in hematopoietic stem cell transplantation. J Clin Lab Anal. 28(6), 469-477 (2014).
Page KM, Zhang L, Mendizabal A et al. Total colony-forming units are a strong, independent predictor of neutrophil and platelet engraftment after unrelated umbilical cord blood transplantation: a single-center analysis of 435 cord blood transplants. Biol Blood Marrow Transplant. 17(9), 1362-1374 (2011).

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The opinions expressed in this interview are those of the interviewee and do not necessarily reflect the views of RegMedNet or Future Science Group.

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