Use of qPCR to Detect Biomarkers for Assessing Stem Cells

In the human body, undifferentiated stem cells are the raw material used to repopulate tissues and organs in response to accidental cell death or normal cell turnover. The ability of undifferentiated stem cells to adopt a variety of cell fates offers great potential for use in treating a range of human diseases such as cancer, heart disease, and spinal cord injury.

Specifically, stem cells can be encouraged to differentiate into a specific variety of cell by altering the cocktail of influencing factors to which they are exposed. For example, mesenchymal stem cells have the potential to form bone, cartilage, fat, tendon, muscle, and marrow stroma after exposure to different combinations of molecules, including insulin and transforming growth factor (TGF).1

Within the adult body, there are several pools of undifferentiated stem cells, which could be harvested from an individual, chemically triggered to adopt a certain cell fate, and then used to treat the same person providing “personalized” therapy. The current challenge is to optimize the collection, growth, differentiation, and reintroduction of stem cells into the affected adult in order to make the technique attractive for widespread use in the clinic.

Overcoming the limits of mesenchymal stem cells

Figure 1 - Differentiation of MultiStem cells into alkaline-phosphatase-positive osteoblasts (blue) and lipid-accumulating adipocytes (red).

Although mesenchymal cells have wide potential for use in clinical therapy, other cell types have recently been receiving significant attention due to their ability to improve certain aspects of stem cell growth, maintenance, and differentiation. Dr. Bart Vaes, a senior scientist at ReGenesys (Heverlee, Belgium), leads a research group working to characterize a primitive mesenchymal stem cell variant known as MultiStem® cells (Athersys, Inc., Cleveland, OH). MultiStem cells (Figure 1) are derived from adult bone marrow and are based on the multipotent adult progenitor cell (MAPC) stem cell type originally discovered by Catherine Verfaillie.2

Unlike typical mesenchymal stem cells, multipotent adult progenitor cells have the capacity to differentiate into cells characteristic of all three germ layers. In addition, MultiStem cells can undergo approximately twice as many cell divisions as mesenchymal cells (70 vs 35 rounds of division) before they lose the ability to replicate, producing significantly more cells per culture. To put this into perspective, 35 more divisions theoretically produce up to 35 billion times more cells (235). This factor is particularly important for stem cell use in clinical applications, since each treatment requires a large cell dose, often including as many as 200 million living cells. The large expansion capability of the cell type allows the production of more than 100,000 clinical doses from one donor, which means they have the potential to be used as an off-the-shelf product to treat multiple diseases from a single source of cells.

MultiStem cells also have the advantage of being immune privileged. They can therefore be used in allogeneic therapies without the need for a match between the donor and the recipient, providing greater flexibility. For this reason, these cells can be used in situations where immune suppression is critical, as in graft versus host disease.

Using biomarkers to characterize MultiStem cells

Due to the benefits afforded by MultiStem cells, Dr. Vaes and his colleagues have been working toward further developing the cell line for reproducible and accurate use in clinical therapy. To help meet this goal, it is necessary to develop a system for characterizing the cells obtained from donors in order to ensure that they are of the correct cell type, retain multipotency, and are appropriate for use in clinical treatments. The early assessment of cell identity and usefulness is essential, since the subsequent culture and expansion of the selected cells requires a lot of time, money, and consumables. The approach in Dr. Vaes’s laboratory revolves around the identification and assay of specific biomarkers, using each as a readout of viability, cell type, and differentiation capacity.

Figure 2 - Multiplex qPCR analysis of MultiStem cells differentiation. In a single qPCR reaction, three differentiation markers (genes A, B, and C) were analyzed. By comparing control cells with differentiating cells, multiplex qPCR is an efficient method to qualify the differentiation process.

To identify these markers, the group uses microarray and DNA methylation analyses to find new candidate genes before validating their results using the quantitative polymerase chain reaction (qPCR). Once suitable biomarkers have been identified, these can be used to quickly and accurately characterize donor cell cultures by analyzing the relevant gene expression profile of each sample using qPCR. In order to increase the throughput, speed, and insight offered by the approach, Dr. Vaes and colleagues have turned to the PrimeTime® qPCR Assays from Integrated DNA Technologies (IDT) (Coralville, IA), which can be used to carry out multiplex experiments investigating the expression levels of multiple genes at the same time. For instance, several differentiation markers are being tested that can be used to validate the multipotent capacity of the cells harvested (Figure 2).

In another study, the group used qPCR assays and cells from several donors to test six stem cell housekeeping genes, enabling them to identify the most stable gene for the cell type. This is important because the accurate quantification of biomarker gene expression levels hinges on comparing these results with the expression of a stable internal reference gene. Having successfully identified such a gene, the team is currently developing more expansive multiplex qPCR experiments that will allow them to screen multiple biomarkers with a housekeeping reference gene, all in a single assay, ultimately increasing accuracy and efficiency.

Much of this work is still in progress; the research team has high-priority plans to develop more gene combinations using additional assays for new gene candidates as they continue to characterize and select stem cell biomarkers. Using these assays, they will compare bone marrow cells and mesenchymal cells from the same donor in order to reliably verify cell quality.

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