Maria Alba Abad

Reprogramming in vivo produces teratomas and iPS cells with totipotency features

Reprogramming of adult cells to generate induced pluripotent stem cells (iPS cells) has opened new therapeutic opportunities; however, little is known about the possibility of in vivo reprogramming within tissues. Here we show that transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs, implying that full reprogramming can occur in vivo. Analyses of the stomach, intestine, pancreas and kidney reveal groups of dedifferentiated cells that express the pluripotency marker NANOG, indicative of in situ reprogramming. By bone marrow transplantation, we demonstrate that haematopoietic cells can also be reprogrammed in vivo. Notably, reprogrammable mice present circulating iPS cells in the blood and, at the transcriptome level, these in vivo generated iPS cells are closer to embryonic stem cells (ES cells) than standard in vitro generated iPS cells. Moreover, in vivo iPS cells efficiently contribute to the trophectoderm lineage, suggesting that they achieve a more plastic or primitive state than ES cells. Finally, intraperitoneal injection of in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic markers. We conclude that reprogramming in vivo is feasible and confers totipotency features absent in standard iPS or ES cells. These discoveries could be relevant for future applications of reprogramming in regenerative medicine.

Tissue damage and senescence provide critical signals for cellular reprogramming in vivo

For cell reprogramming, context matters Differentiated cells in a culture dish can assume a new identity when manipulated to express four transcription factors. This “reprogramming” process has sparked interest because conceivably it could be harnessed as a therapeutic strategy for tissue regeneration. Mosteiro et al. used a mouse model to study the signals that promote cell reprogramming in vivo. They found that the factors that trigger reprogramming in vitro do the same in vivo; however, they also inflict cell damage. The damaged cells enter a state of senescence and begin secreting certain factors that promote reprogramming, including an inflammatory cytokine called interleukin-6. Thus, in the physiological setting, cell senescence may create a tissue context that favors reprogramming of neighboring cells. Science, this issue p. 10.1126/science.aaf4445 In mice, senescent cells created by tissue damage induce reprogramming of neighboring cells, enhancing tissue repair. INTRODUCTION The ectopic expression of transcription factors OCT4, SOX2, KLF4, and cMYC (OSKM) enables reprogramming of adult differentiated cells into pluripotent cells, known as induced pluripotent stem cells (iPSCs), that are functionally equivalent to embryonic stem cells. Expression of OSKM in vivo leads to widespread cell dedifferentiation and reprogramming within tissues and eventually to the formation of teratomas (tumors arising from iPSCs). The molecular mechanisms operating during in vitro OSKM-driven reprogramming have been extensively characterized; however, little is known about in vivo reprogramming. RATIONALE The process of OSKM reprogramming is inefficient both in vitro and in vivo. A number of cell-intrinsic barriers have been identified in vitro, most of which are activated by cellular damage and are particularly prominent in aged cells. Mechanistically, these cell-intrinsic barriers for reprogramming are primarily mediated by the tumor suppressors p53, p16INK4a, and ARF (the latter two are encoded by the Ink4a/Arf gene locus). In this work, we have investigated the effect of these tumor suppressors, cellular damage, and aging on in vivo reprogramming. RESULTS We found that the expression of OSKM in vivo not only triggers reprogramming of some cells but also inflicts extensive damage on many other cells, driving them into a state known as cellular senescence. Senescent cells are characterized by their inability to proliferate and by their secretion of inflammatory cytokines. We have observed a positive correlation between senescence and OSKM-driven reprogramming. For example, tissues lacking p16INK4a/ARF do not undergo senescence, and their ability to reprogram is severely compromised. By contrast, in tissues lacking p53, damage is rampant; this leads to maximal levels of senescence, exacerbated cytokine production, and increased in vivo reprogramming. To explore the connection between senescence and reprogramming, we manipulated these processes in vivo through pharmacological interventions. In particular, an increase in senescence produced by palbociclib (a drug that functionally mimics p16INK4a) results in higher levels of reprogramming. Conversely, a reduction in senescence achieved by navitoclax (a proapoptotic drug with selectivity against senescent cells) leads to decreased in vivo reprogramming. We found that the cross-talk between senescence and reprogramming is mediated by the cytokine-rich microenvironment associated with senescent cells. This is based, among other evidence, on the observation that pharmacological inhibition of NFκB, a major driver of cytokine production, reduces in vivo reprogramming. Analysis of the inflammatory cytokines produced by senescent cells, both in vivo and in vitro, led us to identify interleukin-6 (IL-6) as a critical secreted factor responsible for the ability of senescent cells to promote reprogramming. In support of this, blockade of IL-6 or its downstream kinase effector PIM potently reduced in vivo reprogramming. These observations can be recapitulated in vitro, where reprogramming efficiency is strongly enhanced by the presence of damaged cells or by the conditioned medium derived from damaged cells. Moreover, immunodepletion of IL-6 from the conditioned medium abolished reprogramming. Having established that senescence promotes reprogramming, we studied whether tissue injury leading to senescence has a positive effect on OSKM-driven reprogramming. In particular, we show that bleomycin-induced tissue damage strongly promotes reprogramming in the lung. Finally, aging, which is associated with higher levels of cellular senescence, also favors OSKM-driven reprogramming both in progeric and in physiologically aged mice. CONCLUSION The expression of OSKM in vivo triggers two different cellular outcomes: reprogramming in a small fraction of cells, and damage and senescence in many other cells. There is a strong positive association between these two processes, due to the fact that cellular senescence creates a tissue context that favors OSKM-driven reprogramming in neighboring cells. The positive effect of senescence on reprogramming is mediated by secreted factors, of which IL-6 is a key player. This also applies to tissue injury and aging, where there is an accumulation of senescent cells that send signals to surrounding cells to promote OSKM-driven dedifferentiation and reprogramming. A similar conceptual interplay may occur in physiological conditions, where damage-triggered senescence could induce cell dedifferentiation to promote tissue repair. Interplay between cellular senescence and OSKM-driven reprogramming. Expression of OSKM in vivo, apart from inducing the reprogramming of a small population of cells, also induces damage and senescence in many other cells. Senescent cells release factors that promote the reprogramming of neighboring cells, with IL-6 being a critical mediator. Tissue injury and aging, through the accumulation of senescent cells, favor in vivo reprogramming. Reprogramming of differentiated cells into pluripotent cells can occur in vivo, but the mechanisms involved remain to be elucidated. Senescence is a cellular response to damage, characterized by abundant production of cytokines and other secreted factors that, together with the recruitment of inflammatory cells, result in tissue remodeling. Here, we show that in vivo expression of the reprogramming factors OCT4, SOX2, KLF4, and cMYC (OSKM) in mice leads to senescence and reprogramming, both coexisting in close proximity. Genetic and pharmacological analyses indicate that OSKM-induced senescence requires the Ink4a/Arf locus and, through the production of the cytokine interleukin-6, creates a permissive tissue environment for in vivo reprogramming. Biological conditions linked to senescence, such as tissue injury or aging, favor in vivo reprogramming by OSKM. These observations may be relevant for tissue repair.

Neuronal Pentraxin 1 Contributes to the Neuronal Damage Evoked by Amyloid-β and Is Overexpressed in Dystrophic Neurites in Alzheimer's Brain

Accumulation of amyloid-β (Aβ) is thought to play a central role in the progressive loss of synapses, the neurite damage, and the neuronal death that are characteristic in brains affected by Alzheimers disease. However, the mechanisms through which Aβ produces such neurotoxicity remain unclear. Because Aβ depresses synaptic activity, we investigated whether the neurotoxicity of Aβ depends on the expression of NP1, a protein involved in excitatory synapse remodeling that has recently been shown to mediate neuronal death induced by reduction in neuronal activity in mature neurons. We found that treatment of cortical neurons in culture with Aβ produces a marked increase in NP1 protein that precedes apoptotic neurotoxicity. Silencing NP1 gene expression by RNA interference (short hairpin RNA for RNA interference) prevents the loss of synapses, the reduction in neurite outgrowth, and the apoptosis evoked by Aβ. Transgene overexpression of NP1 reproduced these neurotoxic effects of Aβ. Moreover, we found that NP1 was increased in dystrophic neurites of brains from patients with sporadic late-onset Alzheimers disease. Dual immunohistochemistry for NP1 and tau showed that NP1 colocalizes with tau deposits in dystrophic neurites. Furthermore, NP1 colocalized with SNAP-25 (synaptosomal-associated protein of 25 kDa) in the majority of dystrophic neurites surrounding amyloid deposits. NP1 was also increased in cell processes surrounding amyloid plaques in the cerebral cortex and hippocampus of APP/PS1 (mutant amyloid precursor protein/presenilin 1) transgenic mice. These findings show that NP1 is a key factor for the synapse loss, the neurite damage, and the apoptotic neuronal death evoked by Aβ and indicate that Aβ contributes to the pathology of Alzheimers disease by regulating NP1 expression.

Growth inhibition by the tumor suppressor p33ING1 in immortalized and primary cells: involvement of two silencing domains and effect of Ras.

ABSTRACT ING1 was identified as an inhibitor of growth and has been described as a tumor suppressor. Furthermore, the expression of ING1 is induced in senescent cells and antisense ING1 extends the proliferative life span of primary human fibroblasts. Cooperation of p33ING1 with p53 has been suggested to be an important function of ING1 in cell cycle control. Intriguingly, it has been shown that p33ING1 is associated with histone acetylation as well as with histone deacetylation function. Here we show that p33ING1 is a potent transcriptional silencer in various cell types. However, the silencing function is independent of the presence of p53. By use of deletion mutants two potent autonomous and transferable silencing domains were identified, but no evidence of an activation domain was found. The amino (N)-terminal silencing domain is sensitive to the histone deacetylase inhibitor trichostatin A (TSA) whereas the carboxy-terminal silencing function is resistant to TSA, suggesting that p33ING1 confers gene silencing through both HDAC-dependent and -independent mechanisms. Interestingly, the presence of oncogenic Ras, which is able to induce premature senescence, increases the p33ING1-mediated silencing function. Moreover, ING1-mediated silencing was reduced by coexpressing dominant-negative Ras or by treatment with the mitogen-activated protein kinase inhibitor PD98059 but not by treatment with SB203580, an inhibitor of the p38 pathway. In addition, we show that both silencing domains of ING1 are involved in cell cycle control, as measured by inhibition of colony formation of immortalized cells and by thymidine incorporation of primary human diploid fibroblasts (HDF). Interestingly, p33ING1 expression induces features of cellular senescence in HDFs.

Structural basis for microtubule recognition by the human kinetochore Ska complex

The ability of kinetochores (KTs) to maintain stable attachments to dynamic microtubule structures (‘straight’ during microtubule polymerization and ‘curved’ during microtubule depolymerization) is an essential requirement for accurate chromosome segregation. Here we show that the kinetochore-associated Ska complex interacts with tubulin monomers via the carboxy-terminal winged-helix domain of Ska1, providing the structural basis for the ability to bind both straight and curved microtubule structures. This contrasts with the Ndc80 complex, which binds straight microtubules by recognizing the dimeric interface of tubulin. The Ska1 microtubule-binding domain interacts with tubulins using multiple contact sites that allow the Ska complex to bind microtubules in multiple modes. Disrupting either the flexibility or the tubulin contact sites of the Ska1 microtubule-binding domain perturbs normal mitotic progression, explaining the critical role of the Ska complex in maintaining a firm grip on dynamic microtubules.

HIF-1α is neuroprotective during the early phases of mild hypoxia in rat cortical neurons

Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that plays a key role in regulating the adaptive response to hypoxia. HIF-1α is stabilised during hypoxia and, after dimerisation with hypoxia-inducible factor 1β (HIF-1β), triggers the expression of various genes involved in cell cycle control and energy metabolism associated with cell survival. However, HIF-1α also regulates the expression of proapoptotic genes. The aim of this study was to ascertain the influence of HIF-1α on neurotoxicity evoked by hypoxia in rat cortical neurons. We found that mild hypoxia induces time-dependent neuronal death involving free radical production, mitochondrial depolarisation, cytochrome c release and caspase-3 activation. Lentivirus-mediated HIF-1α knockdown markedly strengthened all of these effects during the initial 24h of hypoxia, which suggests that HIF-1α plays a neuroprotective role in hypoxia-mediated neuronal death. After this initial period, the protective actions of HIF-1α disappeared over the course of the hypoxia-mediated HIF-1α stabilisation. Moreover, lentiviral-mediated overexpression of HIF-1α increased lactate dehydrogenase (LDH) A, one of the target genes for HIF-1α, but did not show protective actions on hypoxia-mediated neuronal death, indicating that the level of endogenous HIF-1α stabilisation achieved during hypoxia was already the maximum required for HIF-1α transcription activities. These results indicate that HIF-1α is neuroprotective in the early phases of hypoxia.

The tumor suppressor ING1 contributes to epigenetic control of cellular senescence

Cellular senescence is an effective tumor‐suppressive mechanism that causes a stable proliferative arrest in cells with potentially oncogenic alterations. Here, we have investigated the role of the p33ING1 tumor suppressor in the regulation of cellular senescence in human primary fibroblasts. We show that p33ING1 triggers a senescent phenotype in a p53‐dependent fashion. Also, endogenous p33ING1 protein accumulates in chromatin in oncogene‐senescent fibroblasts and its silencing by RNA interference impairs senescence triggered by oncogenes. Notably, the ability to induce senescence is lost in a mutant version of p33ING1 present in human tumors. Using specific point mutants, we further show that recognition of the chromatin mark H3K4me3 is essential for induction of senescence by p33ING1. Finally, we demonstrate that ING1‐induced senescence is associated to a specific genetic signature with a strong representation of chemokine and cytokine signaling factors, which significantly overlaps with that of oncogene‐induced senescence. In summary, our results identify ING1 as a critical epigenetic regulator of cellular senescence in human fibroblasts and highlight its role in control of gene expression in the context of this tumor‐protective response.

Reprogramming activity of NANOGP8, a NANOG family member widely expressed in cancer

NANOG is a key transcription factor for pluripotency in embryonic stem cells. The analysis of NANOG in human cells is confounded by the presence of multiple and highly similar paralogs. In particular, there are three paralogs encoding full-length proteins, namely, NANOG1, NANOG2 and NANOGP8, and at least eight additional paralogs that do not encode full-length NANOG proteins. Here, we have examined NANOG family expression in human embryonic stem cells (hESCs) and in human cancer cell lines using a multi-NANOG PCR that amplifies the three functional paralogs and most of the non-functional ones. As anticipated, we found that hESCs express large amounts of NANOG1 and, interestingly, they also express NANOG2. In contrast, most human cancer cells tested express NANOGP8 and the non-coding paralogs NANOGP4 and NANOGP5. Notably, in some cancer cell lines, the NANOG protein levels produced by NANOGP8 are comparable to those produced by NANOG1 in pluripotent cells. Finally, we show that NANOGP8 is as active as NANOG1 in the reprogramming of human and murine fibroblasts into induced pluripotent stem cells. These results show that cancer-associated NANOGP8 can contribute to promote de-differentiation and/or cellular plasticity.

Ing1 Mediates p53 Accumulation and Chromatin Modification in Response to Oncogenic Stress

ING proteins are putative tumor suppressor proteins linked to the p53 pathway and to the chromatin modification machinery. Here we have analyzed the role of the products of the murine Ing1 locus in cellular tumor-protective responses, using mouse primary fibroblasts where the Ing1 locus has been inactivated by the integration of a βgeo cassette. We show that Ing1-deficient mouse embryonic fibroblasts display a defective senescence-like antiproliferative response against oncogenic Ras, affecting several senescence-specific markers. This phenotype is accompanied by a reduced accumulation of p53, which can be explained by the reduced basal p53 protein stability in the Ing1-deficient background. Ing1 deficiency also results in defects in the appearance of heterochromatic marks upon expression of oncogenic Ras, suggestive of impaired heterochromatin formation during oncogene-induced senescence. Our results support an important role for the Ing1 locus in protection against oncogenic stress in vivo, both as a mediator of p53 activation and as a regulator of chromatin remodeling processes.

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

Summary 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.