Readers of this blog will recognize that I have been very excited about induced pluripotent stem cells (iPSCs). iPSCs are made from adult cells. When particular genes are introduced into adult cells, these cells de-differentiate into embryonic-like cells and a fraction of them become so similar to embryonic stem cells that they are called induced pluripotent stem cells. This re-programming is the induced by the introduced genes drives the cells into this embryonic state. Because this technique can make stem cells without destroying human embryos, they are an attractive alternative to embryonic stem cells (ESCs).
The traditional manner in which iPSCs are made makes use of viruses that insert their viral DNA into the genome of the host cell. Reprogramming uses these viruses to overexpress four different genes (c-Myc, Oct4, Klf4, and Sox2) into the genomes of host cells, and these cause the cells to dedifferentiate into an embryonic state. One of the problems with this technique is that is generates cells that have foreign DNA inserted into the genome in random places. These random insertions can produce mutations if they insert into a protein-coding region. Therefore, researchers have used safer techniques that are not as efficient, but do not leave foreign DNA in the host cells.
Unfortunately, several studies have shown that the process by which iPSCs are made and cultured actually introduces mutations into the cells. Five different studies have established that the reprogramming process and subsequent culture of iPSCs in vitro often induces genetic (DNA sequence) and epigenetic (chromosome structure) abnormalities in these cells (Hussein, S. M. et al. Nature 471, 58–62 (2011); Gore, A. et al. Nature 471, 63–67 (2011); Lister, R. et al. Nature 471, 68–73 (2011); Mayshar, Y. et al. Cell Stem Cell 7, 521–531 (2010); Laurent, L. C. et al. Cell Stem Cell 8, 106–118 (2011). The Hussein paper examined copy number variation (CNV) across the genome during iPSC generation. CNV refers to duplications of various portions of chromosomes. Duplications represent a form of gross chromosomal mutation. Duplications tend to occur in cancer cells or other types of cells that have experience significant stresses like ionizing radiation, ultraviolet radiation, drugs that break DNA or other types of major stresses. The paper by Gore and colleagues searched for point mutations (single base changes) in iPSCs by using genome-wide sequencing of protein-coding regions. Lister and co-workers examined DNA methylation, which is a chemical modification of DNA bases (an epigenetic mark) across the genomes of ESCs and iPSCs at the single-base level. In addition to these studies, Mayshar et al have examined changes in chromosome numbers in iPSCs. All these data, with comparisons of CNV in ESCs and iPSCs by Laurent and colleagues have lead to the conclusion that reprogramming and subsequent expansion of iPSCs in culture lead to the accumulation of diverse abnormalities at the chromosomal, subchromosomal and single-base levels.
A new paper in Cell Stem Cell, however, that utilized sophisticated new techniques to analyze the genomic DNA of iPSCs shows that these deep fears might be unwarranted. Kristin Baldwin, associate professor at The Scripps Research Institute’s Dorris Neuroscience Center, said, “We’ve shown that the standard reprogramming method can generate induced pluripotent stem cells that have very few DNA structural mutations, which are often linked to dangerous cell changes such as tumorigenesis,”
Researchers at Scripps Research and the University of Virginia used the latest chromosomal error-mapping methods to determine how many mutations are actually introduced by iPSC induction. The new methods include a high-resolution version of a DNA-error-finding technique known as paired-end mapping, and an advanced algorithm called “Hydra,” for handling the mapping data.
To generate iPSCs, the team followed the standard, four-gene reprogramming procedure, but in order to minimize other potential sources of mutations they selected host cells that were not decades old as in the other papers. Instead they selected relatively error-free fibroblast cells from fetal mice. These fibroblasts were only kept in lab dishes for a very brief time before reprogramming. When the team analyzed these iPSCs they used two different strategies to distinguish which mutations were present in rare donor fibroblast cells and which were newly acquired during reprogramming. Their advanced techniques also allowed them to find more kinds of mutations, across a wider range of the genome. However, instead of finding more mutations, they found almost none. “We sequenced three iPSC lines at very high resolution, and were surprised to find that very few changes to the chromosomal sequence had appeared during reprogramming,” says Michael J. Boland, a research associate in Baldwin’s lab.
Each of the iPSC lines contained only a single mutation that may have originated from the reprogramming process. Mutations inherited from the donor fibroblast cell were present in one pair of lines, and a second line “inherited” none. The researchers noted the complete absence of new mutations caused by mobilization of retrovirus-like sequences that burrowed into the mammalian genome long ago, can become active again in certain cell types. Because these retrovirus-like sequences can actually jump from one location in the genome to another new location, they can cause severe mutations that even include breakage of chromosomes and loss or duplication of chromosomal material. Fortunately, all cells have ways to suppress such “retroelements,” but the suppression mechanisms in normal cells are different from those in stem cells. Therefore, the researchers worried that retroelements would be allowed to escape suppression during the transition to a stem cell state. While no previous surveys of iPSCs could detect these mutations, the study showed that despite very sensitive detection of controls, no retroelements had become active during reprogramming.
In order to establish that they had in fact made iPSCs, they implanted their reprogrammed cell lines into mouse embryos, and they made live, fertile mice. Baldwin added, “The mice generated from these cells have survived to a normal lab-mouse lifespan without obvious diseases that might arise from new DNA mutations.”
The Baldwin lab now is trying to determine if a similar reprogramming method could also yield relatively error-free human iPSCs. Baldwin concluded, “If our results with these mouse cells are applicable to human cells, then selecting better donor cells and using more sensitive genome-survey techniques should allow us to identify reprogramming methods that can produce human iPSCs that will be safer or more useful for therapies than current lines.”