Review of Partial Reprogramming

In the 1960s, John Gurdon's lab was able to induce the normal development of enucleated frog egg cells injected with the nucleus of aged, fully differentiated somatic cells (Gurdon, 1962). This was significant evidence that aged cells contain all the DNA information necessary to be young, and a loss of genomic integrity is not the cause of aging. The paradigm then shifted to a loss of epigenetic integrity being causative of aging, and the search was begun for the rejuvenation factors in the ooplasm. In 2006, four germ-line ‘reprogramming factors’ (Oct4, Sox2, Klf4, and c-Myc, abbreviated as OSKM) were shown to be sufficient to restore pluripotency in some old differentiated cells (Takahashi and Yamanaka, 2006).

As early as 2010, it was speculated that the rejuvenation and the de-differentiation of this induced pluripotency could be separated (Singh and Zacouto, 2010), and 'partial reprogramming' protocols were developed with the hope of rejuvenating tissues without losing their identity. Partial reprogramming protocols inject samples with a short burst of reprogramming factors either once or periodically, rather than injecting the reprogramming factors continuously.

Continuous injection of reprogramming factors eventually leads to permanent loss of cellular identitiy. Hypothesize a point of no return.

All partial reprogramming regimens so far have not been able to fully separate de-differentiation and rejuvenation. That is, all rejuvenation regimens with reprogramming factors first produce a temporary period of de-differentiation and rejuvenation, followed by a period of re-differentiation and sustained rejuvenation. It is unclear if rejuvenation and re-differentiation occur in series or in parallel. There have been no successful in vivo partial reprogramming protocols on wild-type animals, but there is increasing evidence that reprogramming itself does not harm the animal. Reprogramming protocols that successfully attained rejuvenation in vitro were tissue-specific and organism-specific. Some results are discussed as follows:

Examples of successful single short burst in vitro protocols:

(1) Roux et al. in 2022 used a lentiviral system to transiently express OSKM reprogramming factors for 3 days in aged adipogenic and mesenchymal stem cells. De-differentiation occurred during the 3 days of OSKM expression, but cells then re-differentiated when OSKM was stopped. Some features of youthful gene expression were maintained up to 10 days after OSKM reprogramming was stopped.

(2) [Human] Gill et al. in 2022 induced OSKM factors for 10, 13, 15, and 17 days in aged human dermal fibroblasts. De-differentiation was noted morphologically during reprogramming. Re-differentiation and sustained rejuvenation were observed by some metrics. Reprogramming of 10 and 13 days produced better rejuvenation than 15 and 17 days.

Examples of successful periodic short burst in vitro protocols:

Examples of successful periodic short burst in vivo protocols:

(1) [progeriod mice] Ocampo et al. in 2016 showed that in vivo cyclical OSKM expression (2 days ‘on’, 5 days ‘off’) regime in a mouse LAKI premature aging model can rejuvenate mice to some degree and extend lifespan.

(2) [WT-mice] Rodríguez-Matellán et al. in 2020 showed that in vivo, cyclical (3 days ‘on’, 4 days ‘off’) regime in 6-month-old wild-type mice, continuing for 4 months, rejuvenate some age-related characteristics and did not increase mortality.

(3) [WT-mice] Browder et al. in 2022 showed cyclic OSKM expression (2 days ‘on’, 5 days

‘off’) in 15-month-old mice for 7 months showed some rejuvenation and did not increase mortality.

Questions

(1) There is some question as to whether aging is the result of the passive accumulation of damage or an active self-destruction program. What are the implications of the success of somatic cell nuclear transfer and partial reprogramming to the question of programmed aging? For example, if partial reprogramming protocols are successful at reversing aging in vivo, would this imply that the aging cannot be the result of an accumulation of damage that is external to cells? Also, is there any evidence that partial reprogramming affects biological clocks that the body uses for other things, such as the timing of puberty?

(2) All partial reprogramming regimens so far have not been able to fully separate de-differentiation and rejuvenation. The development of various organisms is much more similar than the aging of those organisms. Is there a study that you know of that compares the epigenetic changes induced by reprogramming on various organisms? It could be possible to conclude that similar changes induced by reprogramming various organisms are the changes that induce de-differentiation, whereas the rest are responsible for rejuvenation.

I found particularly interesting the question "Do age reprogrammed cells re-age at the rate of young cells or do they age more rapidly?"

Evidence points to the fact that they do age fast, so it is not turning back the clock?

reprogramming in plants

mechanism of the reprogramming factors

forming colony-like structures typical of reprogramming intermediates.

This revealed a reduction in the mean transcription age of the cells of ∼20-30 years on days 10 to 13 of However, reprogramming for days 15 and 17 did

not reduce transcription age further. A similar picture emerged from

an epigenomic analysis, revealing a reduction in epigenetic age

(eAge) of ∼30 years after 13 days of reprogramming, with days 15

and 17 eliciting a less pronounced reduction in eAge. These data

indicate that there may be an optimum degree of de-differentiation,

beyond which return to the original somatic identity does not

rejuvenate cells further (Fig. 2; compare trajectory A with B). Based

on this study, it was suggested that there are (undefined) cellular

stresses when OSKM-mediated reprogramming is stopped after day

13 (on days 15 and 17) and this reduces the extent to which

transiently reprogrammed fibroblasts are rejuvenated (Gill et al.,

2022).