Senescence promotes in vivo reprogramming through p16INK4a and IL-6

Cellular senescence is a damage response aimed to orchestrate tissue repair. We have recently reported that cellular senescence, through the paracrine release of interleukin-6 (IL6) and other soluble factors, strongly favors cellular reprogramming by Oct4, Sox2, Klf4, and c-Myc (OSKM) in nonsenescent cells. Indeed, activation of OSKM in mouse tissues triggers senescence in some cells and reprogramming in other cells, both processes occurring concomitantly and in close proximity. In this system, Ink4a/Arf-null tissues cannot undergo senescence, fail to produce IL6, and cannot reprogram efficiently; whereas p53-null tissues undergo extensive damage and senescence, produce high levels of IL6, and reprogram efficiently. Here, we have further explored the genetic determinants of in vivo reprogramming. We report that Ink4a, but not Arf, is necessary for OSKM-induced senescence and, thereby, for the paracrine stimulation of reprogramming. However, in the absence of p53, IL6 production and reprogramming become independent of Ink4a, as revealed by the analysis of Ink4a/Arf/p53 deficient mice. In the case of the cell cycle inhibitor p21, its protein levels are highly elevated upon OSKM activation in a p53-independent manner, and we show that p21-null tissues present increased levels of senescence, IL6, and reprogramming. We also report that Il6-mutant tissues are impaired in undergoing reprogramming, thus reinforcing the critical role of IL6 in reprogramming. Finally, young female mice present lower efficiency of in vivo reprogramming compared to male mice, and this gender difference disappears with aging, both observations being consistent with the known anti-inflammatory effect of estrogens. The current findings regarding the interplay between senescence and reprogramming may conceivably apply to other contexts of tissue damage.L.M. was recipient of an FPU contract from the Spanish Ministry of Education (MECD). Work in the laboratory of M.S. was funded by the CNIO and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (RETOS project), the European Research Council (ERC Advanced Grant), the European Union (RISK-IR project), and the Botin Foundation and Banco Santander (Santander Universities Global Division). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming

Aging; Epigenetic clocks; PluripotencyEnvelliment; Rellotges epigenètics; PluripotènciaEnvejecimiento; Relojes epigenéticos; PluripotenciaThe expression of the pluripotency factors OCT4, SOX2, KLF4, and MYC (OSKM) can convert somatic differentiated cells into pluripotent stem cells in a process known as reprogramming. Notably, partial and reversible reprogramming does not change cell identity but can reverse markers of aging in cells, improve the capacity of aged mice to repair tissue injuries, and extend longevity in progeroid mice. However, little is known about the mechanisms involved. Here, we have studied changes in the DNA methylome, transcriptome, and metabolome in naturally aged mice subject to a single period of transient OSKM expression. We found that this is sufficient to reverse DNA methylation changes that occur upon aging in the pancreas, liver, spleen, and blood. Similarly, we observed reversion of transcriptional changes, especially regarding biological processes known to change during aging. Finally, some serum metabolites and biomarkers altered with aging were also restored to young levels upon transient reprogramming. These observations indicate that a single period of OSKM expression can drive epigenetic, transcriptomic, and metabolomic changes toward a younger configuration in multiple tissues and in the serum

Natural killer cells act as an extrinsic barrier for <i>in vivo</i> reprogramming

The ectopic expression of transcription factors Oct4, Sox2, Klf4 and Myc (OSKM) enables reprogramming of differentiated cells into pluripotent embryonic stem cells. Methods based on partial and reversible in vivo reprogramming are a promising strategy for tissue regeneration and rejuvenation. However, little is known about the barriers that impair reprogramming in an in vivo context. We report that natural killer (NK) cells significantly limit reprogramming, both in vitro and in vivo. Cells and tissues at the intermediate states of reprogramming upregulate the expression of NK activating ligands, such as MULT1 and ICAM1. NK cells recognize and kill partially reprogrammed cells in a degranulation-dependent manner. Importantly, in vivo partial reprogramming is strongly reduced by adoptive transfer of NK cells, whereas it is significantly improved by depletion of NK cells. Notably, in the absence of NK cells, the pancreatic organoids derived from OSKM-expressing mice are remarkably large, suggesting the generation of cells with progenitor properties. We conclude that NK cells pose an important barrier for in vivo reprogramming, and this concept may apply to other contexts of transient cellular plasticity

POT1 and Damage Response Malfunction Trigger Acquisition of Somatic Activating Mutations in the VEGF Pathway in Cardiac Angiosarcomas

Background: Mutations in the POT1 gene explain abnormally long telomeres and multiple tumors including cardiac angiosarcomas (CAS). However, the link between long telomeres and tumorigenesis is poorly understood. Methods and Results: Here, we have studied the somatic landscape of 3 different angiosarcoma patients with mutations in the POT1 gene to further investigate this tumorigenesis process. In addition, the genetic landscape of 7 CAS patients without mutations in the POT1 gene has been studied. Patients with CAS and nonfunctional POT1 did not repress ATR (ataxia telangiectasia RAD3-related)-dependent DNA damage signaling and showed a constitutive increase of cell cycle arrest and somatic activating mutations in the VEGF (vascular endothelial growth factor)/angiogenesis pathway (KDR gene). The same observation was made in POT1 mutation carriers with tumors different from CAS and also in CAS patients without mutations in the POT1 gene but with mutations in other genes involved in DNA damage signaling. Conclusions: Inhibition of POT1 function and damage-response malfunction activated DNA damage signaling and increased cell cycle arrest as well as interfered with apoptosis, which would permit acquisition of somatic mutations in the VEGF/angiogenesis pathway that drives tumor formation. Therapies based on the inhibition of damage signaling in asymptomatic carriers may diminish defects on cell cycle arrest and thus prevent the apoptosis deregulation that leads to the acquisition of driver mutations

Cellular reprogramming in the adult organism and its interplay with tissue damage

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 10-02-2017Cell identity can be genetically manipulated in such a way that differentiated cells can achieve full pluripotency in a process known as cellular reprogramming. This process has been thoroughly investigated in vitro, however, little is known regarding the effects of the activation of the reprogramming factors Oct4, Sox2, Klf4 and c-Myc or OSKM in an adult organism. To shed some light on in vivo reprogramming, we generated reprogrammable mouse lines, or i4F for inducible-four factors, which contain a doxycycline-inducible OSKM transgene. Upon activation of OSKM, epithelial cells from pancreas, stomach and intestine, suffered a process of dedifferentiation, characterized by a progressive loss of their identity, and ultimately reached full pluripotency, thereby demonstrating that cellular reprogramming is feasible in an adult organism. Moreover, i4F mice also presented circulating iPSCs in the bloodstream, which could be isolated and cultured in vitro. These in vivo-generated iPSCs, contrary to ESCs or standard in vitro-generated iPSCs, shared some transcriptional similarities to the cells of the morula or blastomeres and presented an increased ability to differentiate into extraembryonic tissues both in vitro and in vivo. Remarkably, i4F mice and in vivo iPSCs had the ability to generate ‘embryonic-like’ structures, which suggests that in vivo reprogramming leads to the acquisition of a more plastic pluripotent state that is closer to totipotency. On the other hand, we also observed that the in vivo activation of OSKM, apart from inducing reprogramming within tissues, also produces cellular damage, which in turn results in cellular senescence. These two OSKM-induced processes, reprogramming and senescence, coexist within the same tissue environment and a positive correlation was found between them. Pharmacological manipulations showed that senescent cells promote cellular reprogramming through secreted factors, being IL6 a key mediator of this crosstalk. Analyzing the role of the tumor suppressor genes Ink4a/Arf and p53 on these processes, we observed that the induction of senescence by OSKM and the secretion of IL6 require a functional Ink4a/Arf locus, as tissues deficient for Ink4a/Arf did not undergo senescence and their reprogramming ability was severely compromised. On the contrary, tissues lacking p53 suffered intense cellular damage and senescence leading to increased reprogramming. All these observations can be recapitulated in vitro. Importantly, biological contexts of increased damage and senescence, such as tissue injury or aging, promote cellular reprogramming in vivo. Altogether, these results demonstrate that cell autonomous barriers for reprogramming, such as Ink4a/Arf, damage and aging, play additional and dominant cell-extrinsic roles in promoting cellular reprogramming and this could have important implications for tissue repair and regeneration processes. !La identidad celular puede ser manipulada genéticamente de tal modo que células diferenciadas pueden adquirir características de pluripotencia, proceso conocido como reprogramación celular. Dicho proceso ha sido ampliamente investigado in vitro, sin embargo apenas se conocen los efectos que puede tener en un organismo adulto la activación de los genes de reprogramación: Oct4, Sox2, Klf4 and c-Myc o OSKM. Con este propósito, hemos generado líneas de ratón reprogramables, o i4F de ‘cuatro factores inducibles’, que contienen el transgén OSKM inducible por doxiciclina. La expresión de OSKM provoca un proceso de desdiferenciación en páncreas, intestino y estómago, donde las células epiteliales pierden progresivamente su identidad, adquiriendo finalmente la pluripotencia total, lo que demuestra que la reprogramación celular es factible en un organismo adulto. Adicionalmente, los ratones i4F presentan iPSCs en el torrente sanguíneo, que pueden ser aisladas y cultivadas in vitro. Las iPSCs generadas in vivo, a diferencia de las ESCs o de las iPSCs estándar generadas in vitro, comparten algunos rasgos transcripcionales con las células en el estado de mórula o blastómeros y presentan una mayor capacidad de diferenciación a tejidos extraembrionarios tanto in vitro como in vivo. Además, los ratones i4F y las iPSCs in vivo tienen la capacidad de generar estructuras ‘pseudoembrionarias’, lo que sugiere que la reprogramación in vivo permite la adquisición de un estadio de pluripotencia con una mayor plasticidad, más cercano a la totipotencia. Por otro lado, hemos observado que la activación de OSKM in vivo, además de inducir reprogramación en los tejidos, también produce daño celular que a su vez se traduce en senescencia celular. Los dos procesos inducidos por OSKM, reprogramación y senescencia, coexisten en un mismo tejido y existe una correlación positiva entre ambos, ya que hemos observado mediante manipulaciones farmacológicas que las células senescentes promueven la reprogramación mediante la secreción de factores, y entre ellos hemos visto que IL6 juega un papel fundamental. Estudiando el papel de los supresores tumorales Ink4a/Arf y p53 sobre dichos procesos, hemos visto que inducción de senescencia por OSKM y la secreción de IL6 requieren de un locus Ink4a/Arf funcional, ya que en ausencia de dichos genes, los tejidos no tienen senescencia y su capacidad de reprogramación se ve comprometida. Por el contrario, tejidos sin p53 sufren un daño y senescencia intensos, lo que conlleva a una mayor reprogramación. Todas estas observaciones pueden recapitularse in vitro. Finalmente, situaciones caracterizadas por una acumulación de células dañadas y senescentes, como el daño tisular o el envejecimiento, promueven la reprogramación in vivo. Estos resultados demuestran que barreras para la reprogramación intrínsecas a la célula, como Ink4a/Arf, el daño o el envejecimiento, tienen papeles extrínsecos adicionales y dominantes que promueven la reprogramación celular, lo que podría tener implicaciones importantes para procesos de reparación de tejidos y regeneración

Metabolic decisions in development and disease.

The intimate relationships between cell fate and metabolism have long been recognized, but a mechanistic understanding of how metabolic pathways are dynamically regulated during development and disease, how they interact with signalling pathways, and how they affect differential gene expression is only emerging now. We summarize the key findings and the major themes that emerged from the virtual Keystone Symposium 'Metabolic Decisions in Development and Disease' held in March 2021.JvdA is a Wellcome Clinical Research Career Development Fellow (219615/Z/19/Z) and receives support from the Medical Research Council Mitochondrial Biology Unit (MC_UU_00015/10)

AAVvector-mediated in vivo reprogramming into pluripotency

In vivo reprogramming of somatic cells into induced pluripotent stem cells (iPSC) holds vast potential for basic research and regenerative medicine. However, it remains hampered by a need for vectors to express reprogramming factors (Oct-3/4, Klf4, Sox2, c-Myc; OKSM) in selected organs. Here, we report OKSM delivery vectors based on pseudotyped Adeno-associated virus (AAV). Using the AAV-DJ capsid, we could robustly reprogram mouse embryonic fibroblasts with low vector doses. Swapping to AAV8 permitted to efficiently reprogram somatic cells in adult mice by intravenous vector delivery, evidenced by hepatic or extra-hepatic teratomas and iPSC in the blood. Notably, we accomplished full in vivo reprogramming without c-Myc. Most iPSC generated in vitro or in vivo showed transcriptionally silent, intronic or intergenic vector integration, likely reflecting the increased host genome accessibility during reprogramming. Our approach crucially advances in vivo reprogramming technology, and concurrently facilitates investigations into the mechanisms and consequences of AAV persistence.We kindly acknowledge support of this work by the German Research Foundation (DFG, EXC81 (Cluster of Excellence CellNetworks) to E.S., E.W. and D.G.; SFB1129 (Collaborative Research Center 1129, TP2) to D.G. and H.L.; and TRR179 (Transregional Collaborative Research Center 179, TP18) to D.G.). E.S. and D.G. acknowledge further support by the Helmholtz Initiative for Synthetic Biology. E.S. is grateful to the Heidelberg University Graduate Academy for a PhD completion grant to the Heidelberg Biosciences International Graduate School (HBIGS) at Heidelberg University for support and to the Spanish Association Against Cancer (AECC) for a postdoctoral fellowship. L.M. was a recipient of a FPU contract from the Spanish Ministry of Education (MECD). M.A. is grateful for support from the Ministry of Economy (MINECO, SAF2015-69413- R). Work in the laboratory of M.S. (CNIO) was funded by the CNIO, and by grants from the MECD cofunded by the European Regional Development Fund (SAF project) and from the European Research Council (ERC Advanced Grant). M.S. (NCT) acknowledges support by grants NCT3.0_2015.13 ImmunOmics and NCT3.0_2015.2 SPL/RP. E.S. and D.G. thank A. Grewenig for providing MEF, A. Schambach for providing the lentiviral OKSM expression plasmid and viral stocks derived thereof, K. Börner and D. Gadella for fluorophore-containing plasmids, and E. Kienle for providing the AAV tyrosine mutants. We moreover thank various members of the Abad, Schmidt, Serrano and Grimm groups for critical reading of the manuscript.S

Common Telomere Changes during In Vivo Reprogramming and Early Stages of Tumorigenesis

Reprogramming of differentiated cells into induced pluripotent stem cells has been recently achieved in vivo in mice. Telomeres are essential for chromosomal stability and determine organismal life span as well as cancer growth. Here, we study whether tissue dedifferentiation induced by in vivo reprogramming involves changes at telomeres. We find telomerase-dependent telomere elongation in the reprogrammed areas. Notably, we found highly upregulated expression of the TRF1 telomere protein in the reprogrammed areas, which was independent of telomere length. Moreover, TRF1 inhibition reduced in vivo reprogramming efficiency. Importantly, we extend the finding of TRF1 upregulation to pathological tissue dedifferentiation associated with neoplasias, in particular during pancreatic acinar-to-ductal metaplasia, a process that involves transdifferentiation of adult acinar cells into ductal-like cells due to K-Ras oncogene expression. These findings place telomeres as important players in cellular plasticity both during in vivo reprogramming and in pathological conditions associated with increased plasticity, such as cancer.We are indebted to R. Serrano for expert mouse colony manage- ment and the Comparative Pathology Unit at CNIO for technical assistance. We are grateful to Dr. Ana Losada for providing us with SA1 antibody. We thank Ana Carolina Moises da Silva for her assistance in setting-up RNA-FISH experiments. Work in the laboratory of M.A.B. is funded by the Spanish Ministry of Economy and Competiveness (PLAN RETOS SAF2013-45111-R) and by the Fundacion Botın.S

Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming.

The expression of the pluripotency factors OCT4, SOX2, KLF4, and MYC (OSKM) can convert somatic differentiated cells into pluripotent stem cells in a process known as reprogramming. Notably, partial and reversible reprogramming does not change cell identity but can reverse markers of aging in cells, improve the capacity of aged mice to repair tissue injuries, and extend longevity in progeroid mice. However, little is known about the mechanisms involved. Here, we have studied changes in the DNA methylome, transcriptome, and metabolome in naturally aged mice subject to a single period of transient OSKM expression. We found that this is sufficient to reverse DNA methylation changes that occur upon aging in the pancreas, liver, spleen, and blood. Similarly, we observed reversion of transcriptional changes, especially regarding biological processes known to change during aging. Finally, some serum metabolites and biomarkers altered with aging were also restored to young levels upon transient reprogramming. These observations indicate that a single period of OSKM expression can drive epigenetic, transcriptomic, and metabolomic changes toward a younger configuration in multiple tissues and in the serum