A lot of research is focused on the benefits of pluripotent stem cells. Yet before any experiments can be conducted, pluripotent stem cells must be harvested from a source and grown in a culture. This alone can be a challenge. To determine if the stem cells are indeed pluripotent, they must be tested. The teratoma assay is currently used to evaluate the pluripotency of cell lines. It has been criticized for its high cost and lengthy analysis time. An alternative method was recently developed by researchers at the Scripps Research Institute. Known as the PluriTest, this diagnostic tool reduces cost and expedites test results.
What Are Pluripotent Stem Cells?
Adult stem cells and embryonic stem cells are the two main types of stem cells. Adult stem cells, also known as somatic stem cells, are found in most types of tissue. They do not differentiate into other cell types, but are responsible for repairing damaged tissue by undergoing cell division and replacing dying cells. Embryonic stem cells are found in embryos, and they are able to differentiate into any type of cell, such as kidney cells, skin cells, or muscle cells. These cells will become part of one of the three germ layers. This includes the endoderm, mesoderm and ectoderm. A stem cell that can differentiate into any one of these layers is pluripotent.
It is possible to convert certain types of somatic cells into pluripotent stem cells through genetic engineering. Typically, genes from stem cells are introduced into somatic cells to induce differentiation. The resulting cells are called induced pluripotent stem cells.
Traditional Methods of Assessing Pluripotency
The established method for screening cell lines for pluripotency involves introducing stem cells into an animal model, usually mice, to observe if teratomas develop. Teratomas are encapsulated tumors that contain tissue from all three germ layers. The mice are usually immunodeficient, meaning their immune system is modified in some way, usually through a genetic mutation. The presence of teratomas in the mice confirms that the cell lines are pluripotent.
This method is referred to as the teratoma assay. In order for it to be useful, researchers need to prepare vials containing stem cells for testing. About 1,000,000 cells are required for each sample. Many laboratories require the sample to be tested for pathogens before being sent for the teratoma assay. Some labs will conduct a pathogen test at an additional cost. Usually, detailed information about the cell line needs to be provided to the lab, including method of genetic modification. Once the lab receives the samples, they will prepare enough cell lines for several injections. The cells are introduced into several mice either subcutaneously or intramuscularly. The progression of teratomas is monitored for six to eight weeks. Afterward, the lab creates a report outlining the results.
Advantages of the PluriTest
Molecular biologist Jeanne Loring, the principal investigator involved in the study of the PluriTest at Scripps Research Institute, contends the new pluripotent stem cell test is a vast improvement over the teratoma assay. The main advantage of the PluriTest over the teratoma assay is that it doesn’t require animal testing to determine if a specific cell line is pluripotent. Another advantage of the PluriTest is that it is immediately accessible to any researcher through an online website, and the results of most analyses (microarray data) takes about 10 minutes. This is considerably faster than the teratoma assay and is much more convenient since the researcher doesn’t have to send out samples or wait several weeks for the results.
How Does the PluriTest Work?
The PluriTest is a diagnostic test based on the DNA microarray. A microarray matches a DNA sequence with its complementary mRNA strand. It essentially identifies genes that are expressed in a cell. The researchers at Scripps have created a microarray database that contains all the genes in embryonic pluripotent stem cells, induced pluripotent stem cells and a few non-pluripotent cells. With this data, they created a molecular model for normal pluripotent stem cells.
Labs that want to determine if their cell lines are pluripotent would have to create a microarray and upload the data to the website https://www.pluritest.org. You need Microsoft Silverlight to access the site, and the file extension for uploaded data is .idat. In a relatively short time, the uploaded data is compared to the PluriTest microarray database, and the results of the analysis are displayed. It will include information about pluripotency and any abnormalities in the stem cells.
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4. “Microarrays: Chipping Away At the Mysteries of Science and Medicine.” NCBI. https://www.ncbi.nlm.nih.gov/About/primer/microarrays.html