Scientists at the U.S. Food and Drug Administration (FDA) showed that the high replication rate of induced pluripotent stems cells (iPSCs) likely contributes to the formation of breaks in DNA routinely seen in these cells.
Undifferentiated iPSCs are not used directly; however, they can be coaxed to make any cell types, such as nerve, liver, and heart muscle cells. They are derived in the laboratory by treating adult cells with special factors that cause them to revert to an undifferentiated state. These cells have many potential uses in regenerative medicine, but it is important to determine that they are safe for use in humans. Scientists have shown that iPSCs have DNA damage. Some of this comes from the cells used to make them, but some of it is newly occurring. DNA damage can lead to tumor formation, so understanding the sources of the damage in iPSCs is important to their medical use.
The FDA finding that the high replication rate of iPSCs contributes to breaks in the DNA is important to understanding how to safely use these cells, which have therapeutic potential for repairing or replacing diseased or damaged tissues and organs. The DNA breaks are usually repaired by specific cellular processes in a process called DNA repair. However, the accumulation of breaks in the cells after they are converted to iPSCs increases the likelihood that some of them might not be repaired.
Therefore, it is important for scientists to be able to detect DNA breaks in iPSCs and understand why they occur. Such information could help both researchers and FDA reviewers better understand the importance of such breaks, especially as they could affect the safety and efficacy of therapeutic stem cells made from them.
To get a “snapshot” of the extent of DNA breakage that occurs in replicating iPSCs, the FDA scientists tracked the appearance of a tell-tale chemical change of a protein in the chromatin of iPSCs that occurs following such a break. Chromatin is the complex formed by DNA tightly coiling around proteins, which enables the genetic material to be packed into a single cell. Following a break in the DNA, one of the chromatin proteins, called H2AX becomes modified into a form called gamma-H2AX. This occurs whether the break occurs in both strands of the double-stranded DNA molecule or in only one of the strands.
To monitor the extent of gamma-H2AX production, the scientists produced iPSCs by converting fibroblasts into iPSCs. Fibroblasts are mature cells found in skin. The FDA team then treated the newly made iPSCs with growth factors that caused them to differentiate into more specialized cells called multipotent stromal cells (MSCs). MSCs are a type of stem cell that is being studied by numerous laboratories for their potential in treating a variety of diseases. Fibroblasts and MSCs divide less rapidly than the undifferentiated iPSCs.
The study found that iPSCs have elevated levels of gamma-H2AX compared to both fibroblasts and MSCs. Moreover, this higher level of gamma-H2AX was accompanied by increased levels of two chemicals: 5‐ethynyl‐2′‐deoxyuridine (EdU), which is incorporated into newly synthesized DNA, and Replication Protein A (RPA), a protein that appears during DNA replication. This was further evidence that the rapid rate of replication in iPSCs was associated with the increased level of breaks in DNA.
High Basal Levels of gamma-H2AX in Human Induced Pluripotent Stem Cells Are Linked to Replication‐Associated DNA Damage and Repair
STEM CELLS 2018; doi: 10.1002/stem.2861
Haritha Vallabhaneni1, Patrick J. Lynch2, Guibin Chen3, Kyeyoon Park4, Yangtengyu Liu3,5, Rachel Goehe1, Barbara S. Mallon4, Manfred Boehm3, and Deborah A. Hursh1
1Division of Cellular and Gene Therapy, Office of Tissue and Advanced Therapies, Center for Biologics Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD, 20993; USA, 2Division of Biotechnology Review and Research II, Office of Pharmaceutical Quality, Center for Drugs Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD, 20993; USA, 3Laboratory of Cardiovascular Regenerative Medicine, National Heart, Lung and Blood Institute, 4 Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892; USA
5 Current address: Department of Rheumatology and Immunology, Xiangya Hospital, Central South University, Changsha, China
Corresponding Author: Deborah A. Hursh, Ph.D, Division of Cellular and Gene Therapy, Office of Tissue and Advanced Therapies, Center for Biologics Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD, 20993; USA, Deborah.firstname.lastname@example.org; 5