A graphic of two stem cells colored pink and blue splitting from each other, with binary code shown in the background.
In hiPSCs, researchers developed a strategy to prevent epigenetic aberrations caused by reprogramming.
© iStock, ArtemisDiana

Human embryonic stem cells (ESCs), derived from the inner cell mass of the blastocyst before its attachment to the uterus, can differentiate into multiple cell types. Due to the ethical debate surrounding their use in research, scientists have developed strategies to generate a different type of pluripotent cell—induced pluripotent stem cells (iPSCs). By inducing the expression of four specific transcription factors, researchers reprogram differentiated cells to generate iPSCs that can give rise to different cell types.

“You could take skin cells from somebody with a particular genetic disorder, and [from these cells] in a dish, generate human iPSCs that can then be differentiated into the relevant cell types for that disorder,” said Ryan Lister, an expert on human iPSCs from the University of Western Australia. Researchers can culture these differentiated cells to study disease models, screen for drugs to repair mutations, or remedy cellular dysfunctions in patient cells.  

In addition to their pluripotency, iPSCs and ESCs are similar in other ways, such as their morphology and pluripotency marker expression.2,3 Yet, they differ significantly from each other functionally and epigenetically, which limits iPSCs’ potential use in research.4,5 Researchers previously discovered that iPSCs retain memory of the cells that they came from in the form of epigenetic marks, such as DNA methylation and histone modifications, and they tend to differentiate into cells that are closely related to that original cell type.6 ESCs do not have this problem and they differ from iPSCs in their methylation states, but it is unknown how these epigenetic differences emerge in iPSCs. In their recent Nature article, a group of researchers led by Lister and Jose Polo, an expert on epigenetics from the University of Adelaide, explored how these epigenetic changes emerge in iPSCs during reprogramming, which led them to develop a strategy that could prevent these epigenetic anomalies.7

To determine when epigenetic changes occur during reprogramming, the researchers used two different culture conditions to turn human fibroblasts into iPSCs. These conditions produced cells that were either in a highly methylated “primed” state or in a “naïve” state with low methylation. Naïve and primed pluripotent stem cells differ in their growth characteristics and their ability to give rise to different somatic cell types. As the reprogramming went on, Lister and Polo’s team isolated batches of cells every few days. They then profiled their DNA methylation status over time using whole-genome bisulfite sequencing. They found that DNA methylation appeared earlier in the naïve iPSCs, whereas in the primed iPSCs, the epigenetic aberrations emerged midway through reprogramming.

To see if these epigenetic abnormalities could be avoided, Lister and Polo developed a new protocol called transient-native-treatment (TNT), where after a week of reprogramming they cultured fibroblasts in naïve conditions for 5 days to allow demethylation to occur and then switched to regular primed media to complete the transition to iPSCs. The researchers found that the iPSCs generated this way were not only morphologically and molecularly similar to ESCs, but also had minimal epigenetic aberrations and did not overexpress transposable elements, which are capable of jumping in the genome and causing genetic mutations. The TNT reprogramming also produced iPSCs with the ability to differentiate into a plethora of cell types, including endoderm, cortical neurons, skeletal muscle cells, and lung epithelial cells. Notably, they could differentiate into neural stem cells with an efficiency similar to ESCs. 

The researchers also found that the epigenetic marks in iPSCs mostly existed in large genomic regions attached to the nuclear membrane. For proper reprogramming, these regions should move away from the membrane, but a subset of these regions remain attached and do not get corrected under the traditional reprogramming protocol. “When we put them through the [TNT], we hypothesize that these subsets get away from the nuclear envelope [and correctly reset],” Lister explained. He believes that this is why his team produced iPSCs that were similar to ESCs. 

“One important step in this study is that they characterized the problems with induced stem cells and found a solution. But the results will need to be replicated,” said Gabriele Stocco, an epigeneticist from the University of Trieste, who was not involved with this work. It also remains to be seen whether TNT reprogramming can correct epigenetic aberrations in various cell types. With further study, the researchers think that this protocol will be applicable in many therapeutic applications and could be useful for others studying epigenetic memory. 


  1. Zakrzewski W, et al. Stem cells: past, present, and future. Stem Cell Res Ther. 2019;10(1):68.
  2. Okita K, et al. Generation of germline-competent induced pluripotent stem cells. Nature. 2007;448(7151):313-317.
  3. Wernig M, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 2007;448(7151):318-324.
  4. Polo JM, et al. Cell type of origin influences the molecular and functional properties of mouse-induced pluripotent stem cells. Nat Biotechnol. 2010;28(8):848-855.
  5. Kim K, et al. Epigenetic memory in induced pluripotent stem cells. Nature. 2010;467(7313):285-290.
  6. Nukaya D, et al. Preferential gene expression and epigenetic memory of induced pluripotent stem cells derived from mouse pancreas. Genes Cells. 2015;20(5):367-381
  7. Buckberry S, et al. Transient naive reprogramming corrects hiPS cells functionally and epigenetically. Nature. 2023;620(7975):863-872