Multiplexing Using PrimeTime® qPCR Assays
MultiStem® cells, an adult adherent stem cell product
Dr Bart Vaes joined ReGenesys in 2010 and began work on a project that continued characterizing a primitive mesenchymal stem cell variant. Specifically, his work focuses on cultivating cells called MultiStem cells (Athersys, Inc) under serum-free conditions (Figure 1). MultiStem cells are derived from adult bone marrow and are based on a stem cell type called MAPC (multipotent adult progenitor cell) originally discovered by Catherine Verfaillie .
Figure 1. Differentiation of MultiStem® cells into alkaline-phosphatase-positive osteoblasts (blue) and lipid-accumulating adipocytes (red).
Advantages over mesenchymal stem cells
MultiStem cultures can differentiate into cells of all 3 germ layers so they have greater differentiation capability than typical mesenchymal stem cells. In addition, these cells have greater expansion capability. This latter factor is particularly important for stem cell use in clinical application as treatments require a large cell dose—as many as 200 million cells. MultiStem cultures can easily be expanded through 60–70 doublings while mesenchymal cells have a maximum of around 30–35 doublings. This large expansion capability allows the production of >100,000 clinical doses from one donor, which means the MultiStem cells have the potential to be used as an off-the-shelf product to treat several diseases. In addition to increased expansion, MultiStem cells have the advantage of being immune privileged and can, therefore, be used in allogeneic therapies where the cells can come from any individual, which means that matching MultiStem cells with the recipient is not necessary. For this reason, these cells can be used in clinical settings where immune suppression is critical, as in graft versus host disease (GVHD).
Before they create the MultiStem cultures, Dr Vaes’s team needs a way to fully characterize the stem cells they receive from donors to determine if the cells are appropriate for clinical treatments. Culturing and expanding the cells takes a lot of time, money, and consumables, so verification of cell quality prior to this process is very important.
Currently 2 MultiStem cell projects are in FDA-approved, phase II clinical trials where the cells are being tested for treatment of acute myocardial infarction and inflammatory bowel disease. A phase I clinical trial is ongoing to evaluate MultiStem cell administration to prevent graft versus host disease during bone marrow transplantations. Projects are also underway to examine MultiStem cells in treatment of stroke and for solid organ transplant support.
Characterizing donor stem cells
Before they create the MultiStem cells, Dr Vaes’s team needs a way to fully characterize the stem cells they receive from donors to determine if the cells are appropriate for clinical treatments. Culturing and expanding the cells takes a lot of time, money, and consumables, so verification of cell quality prior to this process is very important. The group is developing a protocol to verify donor cell quality by identifying specific stem cell biomarkers using qPCR to quickly analyze the gene expression of each sample. For instance, differentiation markers are being tested to validate multipotentiality of MultiStem cells (Figure 2).
Multiplex qPCR: stem cell differentiation
|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, Dr Vaes uses microarray and DNA methylation analysis to find new candidate genes and then validates those results using qPCR. The team is running multiplex qPCR assays using PrimeTime® qPCR Assays from IDT. They need to analyze at least 3 genes per reaction and multiplexed assays allow them to analyze more genes in less time. A side benefit is a reduction in reagent volume, thus lowering the expense per reaction.
Using IDT PrimeTime qPCR Assays and cells from several donors, Dr Vaes tested 6 stem cell housekeeping genes and was able to identify the most stable gene for the cell type. The team is currently developing more expansive multiplex experiments with the housekeeping genes they have identified. They plan to combine an Assay for one housekeeping gene with Assays for the stem cell differentiation biomarkers so that, in one multiplexed PCR, they will be able to relate expression of the biomarker(s) to the housekeeping gene, resulting in a more efficient cell quality screen.
Much of the work is still in progress; the research team has high-priority plans to develop more gene combinations using additional PrimeTime qPCR Assays for new gene candidates as they continue to characterize and select stem cell biomarkers. They will compare the bone marrow cells to mesenchymal cells from the same donor to verify cell quality.
The ultimate goal of Dr Vaes’s work is to culture MultiStem cells in conditions that are serum-free and xeno-free (without animal proteins). Serum is variable and not well-characterized which makes it problematic for use in clinical products. Additionally, it may carry animal-related diseases, like prion diseases, which would prevent cell use in a clinical setting applied to humans. Finally, using large amounts of serum as required for clinical production comes with ethical issues surrounding the number of animals needed to supply the serum. In contrast, serum-free products can be better validated and have the additional advantage of being free of animal proteins and the need for animal donors.
The team has been successful at getting expansion of clinical doses without serum. They have also been able to isolate these cells from bone marrow without serum. They must now test whether the cells still have multipotentiality and are immune suppressive, and verify that the gene profile does not change when serum is removed. As with the quality control, the team is using qPCR to verify the gene expression in the absence of serum. They have seen some slight changes in gene expression without serum but do not yet know if those changes are clinically important. The next step will be to optimize the work flow and begin experiments to determine if the serum-free cultured cells are still active in biological assays.
Getting started with PrimeTime qPCR Assays
Dr Vaes contacted IDT with accession numbers for his genes of interest. IDT scientists then helped him with qPCR assay design for a multiplex format. They also worked with Dr Vaes to determine the best dye choices for assay probes. Dr Vaes says, “The process was convenient for us. The emails were very helpful, the conclusions were clear, and the right people were involved. The service was very good and we got the right products immediately. All of the Assays worked and we were able to get good results with no duplex formation. We are very happy with the PrimeTime qPCR Assays.”
Researcher profile Dr Bart Vaes received his PhD in 2007 from Radboud University Nijmegen, The Netherlands, where he studied mesenchymal stem cells with a focus on microarray analysis of differentiating osteoblasts. During his two postdoctoral fellowships at Baylor College of Medicine, Houston TX, and Wageningen University, The Netherlands, he studied transcriptomics and bone research as well as the effect of nutritional factors on mesenchymal stem cell differentiation. In January of 2010, Dr Vaes began his work as a senior scientist at ReGenesys, a subsidiary of Athersys, where he supervises the MultiStem cell project. ReGenesys is located in Leuven, Belgium, and has 10 people including Dr Jef Pinxteren (Head of R&D), Dr Bart Vaes (Senior Scientist), 4 scientists, and 3 research associates. Dr Vaes is on the right end of the back row.
1. Jiang Y, Jahagirdar BN, et al. (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature, 418(6893):41–49.
Relevant publicationsVaes B, Van't Hof W, et al. (2012) Application of MultiStem® Allogeneic Cells for Immunomodulatory Therapy: Clinical Progress and Pre-Clinical Challenges in Prophylaxis for Graft Versus Host Disease. Front Immunol, 3:345. doi: 10.3389/immu.2012.00345.
Roobrouck VD, Clavel C, et al. (2011) Differentiation potential of human postnatal MSC, MAB and MAPC reflected in their transcriptome and partially influenced by the culture conditions. Stem Cells, 29(5)871–882. doi: 10.1002/stem633.
Busch SA, Hamilton JA, et al. (2011) Multipotent adult progenitor cells prevent macrophage-mediated axonal dieback and promote regrowth after spinal cord injury. J Neurosci, 31(3):944–953.
Mays RW, Borlongan CV, et al. (2010) Development of an allogeneic adherent stem cell therapy for treatment of ischemic stroke. J Exp Stroke Transl Med, 3(1):34–46.
Kovacsovics-Bankowski M, Streeter PR, et al. (2009) Clinical scale expanded adult pluripotent stem cells prevent graft-versus-host disease. Cell Immunol, 255:55–60.
Kovacsovics-Bankowski M, Mauch K, et al. (2008) Preclinical safety testing supporting clinical use of allogeneic multipotent adult progenitor cells. Cytotherapy, 10(7):730–742.
Author: Jaime Sabel is a Scientific Writer at IDT
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