Is conventional reprogramming too… conventional?

Transient naive reprogramming is capable of epigenetically correcting human induced pluripotent stem cells – should it be the preferred method?

Somatic cell reprogramming is resetting the epigenome of a somatic cell through cell fusion, somatic cell nuclear transfer (SCNT), and expression of the Yamanaka reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) [1]. Cells undergo significant reconfiguration when reprogrammed into human-induced pluripotent stem cells (hiPS cells). However, significant differences have been observed between human induced pluripotent stem cells (hiPS cells) and human embryonic stem (hES) cells. Previous studies have reported these two cells to be functionally and epigenetically different, so while hiPS cells were hailed as an effective replacement for hES cells, the seemingly random variation in the differentiation propensity of hiPSCs has frustrated researchers who were hoping to leverage their potential for disease modeling and cell replacement therapies.

Histone modifications and DNA methylation encode such epigenetic differences that are transmissible with differentiation. This limits the use of hiPS cells in cell therapies, drug screening, and disease modeling. Reprogramming brought about by somatic cell nuclear transfer (SCNT) has been reported to retain less epigenetic memory as compared with OKSM-reprogrammed cells. This suggests that epigenetic aberrations are not fundamental to reprogramming and can be reduced.

Longevity.Technology: Conventional reprogramming focuses on producing hiPS cells in a primed pluripotent state. However, recent research enables the reprogramming of somatic cells to a naive pluripotent state. Such reprogramming paradigms provide model systems that help to determine how epigenome resetting is affected by environments resembling different developmental states of pluripotency.

A new study published in Nature aimed to determine the origins, dynamics and mechanisms of epigenetic abnormalities in both primed and naive reprogramming to understand the process completely [2].

The results indicated that the largest CG DNA methylation occurred between 13 and 21 days for primed reprogramming while those for naive reprogramming occurred before day 13. Global CA methylation was found to increase within the first 5 days in naive reprogramming and after day 13 in primed reprogramming. Also, CG DNA methylation was found to change stepwise at regulatory elements in primed reprogramming while only one major change occurred for naive reprogramming. Moreover, elements whose methylation increased during reprogramming were found to be enriched with FOS, JUN and AP-1 motifs [2].

Aberrant CG DNA methylation was not observed during early reprogramming and begins to emerge only after day 13 of primed reprogramming which suggests that such aberrant methylation is a feature of primed and not naive reprogramming. Additionally, demethylation at imprinted loci was found to be more for cells that were cultured in naive conditions for longer durations.

The results also indicated that transient-naive-treatment (TNT) reprogramming was more effective in resetting the epigenome as compared to naive-to-primed (NTP) reprogramming in fibroblasts. Repressive chromatin domains that featured epigenetic memory were observed to be reset by TNT reprogramming. Epigenetic correction was also reported to be possible with primed–naive–primed (PNP) reprogramming.

“TNT reprogramming reorganizes chromatin architecture beyond what is achieved in conventional reprogramming,” says the team of researchers [2].

TNT reprogramming was also observed to maintain genomic imprinting. Epigenetic memory was also observed to persist through differentiation in primed-hiPS cells. Several differences were reported between isogenic primed-hiPS cells and hES cells including mesoderm markers, chromatin accessibility, genomic imprinting and transposable elements. However, in most cases TNT reformatting was found to be able to correct such differences, producing hiPS cells that closely resemble hES cells. Finally, TNT reprogramming was also reported to decrease epigenetic differences in primed-hiPS cells and improve their differentiation [2].

This study shows that the TNT reprogramming system is effective in resetting the epigenome. This makes it a powerful model system that enables the study of the epigenetic memory as well as mechanisms that maintain cell-of-origin heterochromatin.

As the paper authors put it,: “We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory [2].”

[1] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060150/
[2] https://www.nature.com/articles/s41586-023-06424-7