Stem cells that are grown in the lab are recognized to acquire mutations, but whether these mutations are especially numerous or risky remains unclear. Mutations acquired within stem cell culture, it is feared, would complicate initiatives to deploy stem cells in regenerative medicine. At least, lab-grown stem cells may need to be screened for deleterious mutations, with special attention devoted to vulnerable portions of the genome or flaws that could lead to dire consequences, such as malignancy.
To characterize the mutations that may occur among stem cells in vitro , scientists have been introducing gene-sequencing tests. For example , in a latest study, scientists based at Harvard have determined that will human pluripotent stem cells are prone to develop mutations within the TP53 gene, which ordinarily helps suppress cancer. The particular mutated versions of the TP53 found by the Harvard group, however , tend to drive cancer development.
Information on this work appeared April 26 in the journal Character, in an article entitled, “ Human Pluripotent Stem Cellular material Recurrently Acquire and Expand Dominant Negative P53 Variations. ” This article describes how the Harvard team sequenced the particular protein-coding genes of 140 human embryonic stem cellular (hES) cell lines— 26 of which were developed just for therapeutic purposes using Good Manufacturing Practices, a quality manage standard set by regulatory agencies in multiple nations. The remaining 114 human pluripotent stem cell lines had been listed on the NIH registry of human pluripotent stem cellular material. This gene-sequencing exercise was followed by computational work that will allowed the scientists to identify mutations present in a subset of cells in each cell line.
“[We] identified five unrelated hES cellular lines that carried six mutations in the TP53 gene that encodes the tumor suppressor P53, ” wrote the article’ s writers. “ The TP53 variations we observed are dominant negative and are the variations most commonly seen in human cancers. We found that the TP53 mutant allelic fraction improved with passage number under standard culture conditions, recommending that the P53 mutations confer selective advantage. ”
The scientists also mined published RNA sequencing data from 117 human pluripotent stem cell outlines, and observed another nine TP53 mutations, all leading to coding changes in the DNA-binding domain of P53. “ Within three lines, ” the authors of the Nature document detailed, “ the allelic fraction exceeded 50%, recommending additional selective advantage resulting from the loss of heterozygosity at the TP53 locus. ”
These findings suggest that cell lines should be screened designed for mutations at various stages of development as well as instantly before transplantation.
“Our results underscore the advantages of the field of regenerative medicine to proceed with care, inch said the study’s co-corresponding author Kevin Eggan, Ph level. D. “[They] indicate that an additional number of quality control checks should be implemented during the production associated with stem cells and their downstream use in developing treatments. Fortunately, these genetic checks can be readily performed along with precise, sensitive, and increasingly inexpensive sequencing methods. inch
“Cells in the lab, like cells in your body, acquire mutations all the time, ” added Steve McCarroll, Ph level. D., co-corresponding author. “Mutations in most genes have small impact on the larger tissue or cell line. But cellular material with a pro-growth mutation can outcompete other cells, turn out to be very numerous, and ‘take over’ a tissue. All of us found that this process of clonal selection— the basis of malignancy formation in the body— is also routinely happening in laboratories. ”
Although the Harvard scientists expected to discover some mutations in stem cell lines, they were amazed to find that about 5% of the stem cell ranges they analyzed had acquired mutations the TP53 gene, which encodes the tumor suppressor protein P53.
Nicknamed the “guardian of the genome, ” P53 controls cell growth and cell death. People who inherit p53 mutations develop a rare disorder called Li-Fraumeni Symptoms, which confers a near 100% risk of establishing cancer in a wide range of tissue types.
The particular mutations that the researchers observed are “dominant negative” variations, meaning, when present on even one copy associated with P53, they are able to compromise the function of the normal proteins, whose components are made from both gene copies. The exact same major negative mutations are among the most commonly observed mutations within human cancers.
The researchers performed a classy set of DNA analyses to rule out the possibility that these variations had been inherited rather than acquired as the cells grew within the lab. In subsequent experiments, the Harvard scientists discovered that P53 mutant cells outperformed and outcompeted nonmutant cells in the lab dish. In other words, a culture using a million healthy cells and one P53 mutant cell, mentioned Dr . Eggan, could quickly become a culture of just mutant cells.
“The spectrum of cells at risk for transformation when harboring a P53 veränderung include many of those that we would like to target for repair along with regenerative medicine using human pluripotent stem cells, inch noted Dr . Eggan. Those organs include the pancreas, mind, blood, bone, skin, liver, and lungs.
However , Drs. Eggan and McCarroll emphasized that since this phenomenon has been found, inexpensive gene-sequencing tests enables researchers to identify and remove from the production line cellular cultures with concerning mutations that might prove dangerous right after transplantation.
The researchers point out in their document that screening approaches to identify these P53 mutations yet others that confer cancer risk already exist and are utilized in cancer diagnostics. In fact , in an ongoing clinical trial which is transplanting cells derived from induced pluripotent stem cells, or even iPSCs, gene sequencing is used to ensure the transplanted cell items are free of dangerous mutations.