|You are Only as Old as the Factors you are Reprogrammed By: Rejuvenating Senescent and Centenarian Human Cells by Reprogramming Through the Pluripotent State|
From Genes and Development
Although induced stem cell technology promises a source of patient-specific cells for cell replacement in diseased or dysfunctional tissues, multiple studies have shown that activation of cellular senescence pathways is a barrier to the reprogramming process and some studies have shown that cells differentiated from iPSCs show reduced functionality and early senescence (Feng et al and Liu et al). This suggests that currently utilised iPSC-derivation protocols may not achieve complete reprogramming of somatic cells to produce stem cells which share characteristics of youthful embryonic stem cells (ESCs). Further, this may have additional implications regarding the age of the cell-donor. Now, a study from the group of Jean-Marc Lemaitre at the Institute of Functional Genomics, Montpellier, France, published in Genes and Development has shown the ability of an optimized 6-factor reprogramming protocol to efficiently reverse these aging characteristics and allowing the derivation of fully pluripotent iPSCs with key characteristics of young cells (Lapasset et al).
The reprogramming experiments utilised 6-factors (OCT4, SOX2, KLF4, MYC, NANOG and LIN28 – OSKMNL) to reprogram human diploid fibroblasts from a 74 year-old donor, which were either proliferative (74P) or senescent (74S), induced by serial passaging. The senescent cells showed typical markers of senescent human cells; permanent cell cycle arrest, increased senescence-associated β-galactosidase (SA-β-Gal) activity, up-regulation of p16INK4A and p21CIP1, and formation of senescence-associated heterochromatin foci (SAHF), a state of chromatin associated with senescence (Narita et al). One week after reprogramming SAHF disappeared from the 74S cells, which then became proliferative at 18-20 days and yielded hESC-like colonies at 35-40 days, with an efficiency similar to that observed for reprogrammed 74P cells (0.06%). In 74P and 74S iPSCs, endogenous pluripotency-associated gene expression was reactivated, correlating to a reduction of promoter CpG methylation for OCT4 and NANOG, and these cells were also able to differentiate efficiently into endoderm (SMA), ectoderm (MAP2), and mesoderm (FOXA2) derivatives. When this was compared to 4-factor reprogramming (OSNL) in 74S cells, proliferation or hESC-like colony formation was not observed, and treatment with known chemical enhancers of reprogramming (VPA, 5-aza-dC, Wnt3A, or BIO) (Feng et al) failed to induce iPSC formation. This suggests that the combination of the six transcription factors (OSKMNL) is required for successful and efficient reprogramming that reverses cellular senescence to allow iPSC derivation, without any direct suppression of senescence effectors contributing as safeguards of the genome.
To explore whether high levels of p16INK4A and p21CIP1 in senescent cells affected reprogramming, iPSC formation from much older fibroblasts was examined. To this end, 92-, 94-, 96-, and 101-year-old donor fibroblasts were reprogrammed with the 6-factor mixture. This led to the generation of iPSCs with a similar efficiency and with similar characteristics to those observed from the senescent fibroblasts, and with levels of p16INK4A and p21CIP1protein reduced in all iPSCs to levels observed in hESCs. Telomerase function and telomere length are also correlated to senescence, and while current protocols generally result in an initial increase in telomere size in iPSCs compared with the parental differentiated cells, prematurely aged (shortened) telomeres appear to be a common feature of cultured iPSCs and their cell progeny (Suhr et al, Feng et al and Vaziri et al). 74S and 74P iPSCs however, had an increased mean size when compared to H9 hESCs, while telomeres from centenarians were also lengthened. Furthermore, all iPSCs lines could be cultured long term (>110 population doublings) without detectable decreases in telomere length or loss of self-renewal and pluripotency properties, as with H9 hESCs. Moreover, some iPSC clones showed telomeres that were longer than H9 hESCs, suggesting that OSKMNL reprogramming may allow for an increased proliferation capacity of re-differentiated cells.
Three iPSC clones (74P-H, 74S-F, and 96-1) were studied in detail and all cells had normal karotypes, formed well-defined teratomas and showed down-regulation of transgene expression. Transcriptional analysis of these lines found appropriate levels of pluripotency-associated gene expression compared to common hESCs and other 4 factor iPSCs, and upon non-supervised hierarchical clustering it was found that gene expression profiles of proliferative and senescent aged fibroblasts clustered together with embryonic and postnatal fibroblasts, indicating that they share a general common aging signature. Excitingly, the 6-factor iPSCs clustered with hESCs, separate from 4 factor iPSCs, and were found to express higher levels of genes involved in telomere metabolism and maintenance, telomerase activity and had longer telomeres. The focus then shifted to analysis of oxidative stress and mitochondrial dysfunction, two well described common features of senescence and aging (Passos et al and Moiseeva et al), and it was confirmed that 6-factor iPSCs reset these functions to an ESC-like state, as demonstrated by increased mitochondrial membrane potential to a level observed in hESCs, with similar number, distribution, and morphology and mitochondria between iPSCs and ESCs.
Whether or not iPSCs from senescent cells are able to produce differentiated cells with youthful characteristics was next addressed, and it was shown that fibroblasts derived from 74P, 74S, and 96 iPSCs did not enter prematurely into senescence, contrary to previous results (Feng et al and Liu et al). These fibroblasts had no SA-β-Gal activity after 10 PDs and no alterations in proliferative rate as compared to young proliferative fibroblasts. Importantly, the number of PDs to reach replicative senescence arrest was increased after reprogramming through the pluripotent state, with an estimated 50% of additional proliferation capacity compared with their parental fibroblasts for the 74P cells, while 74S and 96 iPSCs which were senescent or close to senescence were afforded another 60 PDs. Transcriptomic analysis of the cells differentiated from these iPSCs were shown to be similar to young proliferative embryonic fibroblasts derived from the H1 hESCs, compared to their parental counterparts which share a common aging signature, separating them from postnatal fibroblasts.
Overall these results suggest that 6 factor OSKMNL-mediated reprogramming allows for successful and efficient reprogramming which reverses cellular senescence to allow pluripotent iPSC derivation, avoiding the issue of cellular aging as a barrier to reprogramming, that reprogramming in this way can “erase” common markers of senescence and aging, and that this method imbues “old” cells with the key characteristics of “young” cells. This study is important, as while most reprogramming studies use embryonic fibroblasts or cells from relatively young donors, it is important to remember that most regenerative medicine applications arising from iPSC-technology will likely be targeted towards elderly patients showing tissue disease or dysfunction. Therefore the success of 6-factor reprogramming process may prove to be very important to the application of iPSC technology for these patients in the future.
Feng B, Ng JH, Heng JC, Ng HH.
Lapasset L, Milhavet O, Prieur A et al.
Liu GH, Barkho BZ, Ruiz S et al.
Moiseeva O, Bourdeau V, Roux A et al.
Narita M, Nunez S, Heard E et al.
Passos JF, Saretzki G, Ahmed S et al.
Suhr ST, Chang EA, Rodriguez RM et al.
Vaziri H, Chapman KB, Guigova A et al.