Senomorphic activity of a combination of niacinamide and hyaluronic acid: correlation with clinical improvement of skin aging.

Intrinsic and extrinsic factors, including lifestyle and sun exposure, can contribute to cell senescence, which impairs skin homeostasis, that may in turn lead to skin aging. Senescent cells have a specific secretome, called the senescence-associated secretory phenotype (SASP) that includes MMPs, CXCLs and S100A8/9. Reducing the SASP with senotherapeutics is a promising strategy to reduce skin aging. Here we evaluated the effect of a formula containing niacinamide and hyaluronic acid, which are known to limit senescence and skin aging. We conducted three different studies. (1) Ex vivo explants treated with the formula had more collagen and glycosaminoglycan. (2) In a clinical trial with forty-four women, two months of treatment improved fine lines, wrinkles, luminosity, smoothness, homogeneity, and plumpness. (3) In a third study on thirty women, we treated one arm for two months and took skin biopsies to study gene expression. 101 mRNAs and 13 miRNAs were differentially expressed. We observed a likely senomorphic effect, as there was a decrease in many SASP genes including MMP12 and CXCL9 and a significant downregulation of autocrine signaling genes: S100A8 and S100A9. These pharmaco-clinical results are the first to demonstrate the senomorphic properties of an effective anti-aging formula in skin.

Looking for the philosopher's stone: Emerging approaches to target the hallmarks of aging in the skin.

Senescence and epigenetic alterations are two important hallmarks of cellular aging. During aging, cells subjected to stress undergo many cycles of damage and repair before finally entering either apoptosis or senescence, a permanent state of cell cycle arrest. The first biomarkers of senescence to be identified were increased ß-galactosidase activity and induction of p16INK4a. Another feature of senescent cells is the senescence-associated secretory phenotype (SASP), a complex secretome containing more than 80 pro-inflammatory factors including metalloproteinases, growth factors, chemokines and cytokines. The secretome is regulated through a dynamic process involving a self-amplifying autocrine feedback loop and activation of the immune system. Senescent cells play positive and negative roles depending on the composition of their SASP and may participate in various processes including wound healing and tumour suppression, as well as cell regeneration, embryogenesis, tumorigenesis, inflammation and finally aging. The SASP is also a biomarker of age, biological aging and age-related diseases. Recent advances in anti-age research have shown that senescence can be now prevented or delayed by clearing the senescent cells or mitigating the effects of SASP factors, which can be achieved by a healthy lifestyle (exercise and diet), and senolytics and senomorphics, respectively. An alternative is tissue rejuvenation, which can be achieved by stimulating aged stem cells and reprogramming deprogrammed aged cells. These non-clinical findings will open up new avenues of clinical research into the development of treatments capable of preventing or treating age-related pathologies in humans.

Generation of patient-specific induced pluripotent stem cell lines with Type 2 Long QT Syndrome and the KCNH2 c.379C > T pathogenic variant.

Type 2 Long QT Syndrome (LQT2) is a rare genetic heart rhythm disorder causing life-threatening arrhythmias. We derived induced pluripotent stem cell (iPSC) lines from two patients with LQT2, aged 18 and 6, both carrying a heterozygous missense mutation on the 3rd and 11th exons of KCNH2. The iPSC lines exhibited normal genomes, expressed pluripotent markers, and differentiated into trilineage embryonic layers. These patient-specific iPSC lines provide a valuable model to study the molecular and functional impact of the hERG channel gene mutation in LQT2 and to develop personalized therapeutic approaches for this syndrome.

Senescence-Driven Inflammatory and Trophic Microenvironment Imprints Mesenchymal Stromal/Stem Cells in Osteoarthritic Patients.

Senescent cells promote progressive tissue degeneration through the establishment of a combined inflammatory and trophic microenvironment. The cellular senescence state has therefore emerged as a central driving mechanism of numerous age-related diseases, including osteoarthritis (OA), the most common rheumatic disease. Senescence hallmarks are detectable in chondrocytes, synoviocytes and sub-chondral bone cells. This study investigates how the senescence-driven microenvironment could impact the cell fate of resident osteoarticular mesenchymal stromal/stem cells (MSCs) that are hence contributing to OA disease progression. For that purpose, we performed a comparative gene expression analysis of MSCs isolated from healthy donors that were in vitro chronically exposed either to interferon-gamma (IFN-γ) or Transforming Growth Factor beta 1 (TGFß1), two archetypical factors produced by senescent cells. Both treatments reduced MSC self-renewal capacities by upregulating different senescence-driven cycle-dependent kinase inhibitors. Furthermore, a common set of differentially expressed genes was identified in both treated MSCs that was also found enriched in MSCs isolated from OA patients. These findings highlight an imprinting of OA MSCs by the senescent joint microenvironment that changes their matrisome gene expression. Altogether, this research gives new insights into OA etiology and points to new innovative therapeutic opportunities to treat OA patients.

Reduction of senescence-associated secretory phenotype and exosome-shuttled miRNAs by Haritaki fruit extract in senescent dermal fibroblasts.

OBJECTIVE: Skin ageing is linked to the accumulation of senescent cells and a "senescence-associated secretory phenotype" (SASP). SASP factors include chemokines, cytokines, and small extracellular vesicles (EVs) containing miRNAs. We characterized SASP profile markers in normal human dermal fibroblasts (HDFs) and evaluated the effect of Haritaki fruit extract on these senescence markers. METHODS: Senescence was induced in HDFs by ionizing radiation (X ray), followed by 14 days of culture. Parallel incubations included fibroblasts treated for 12 days with 10 or 100 µg/mL Haritaki (a standardized extract of Terminalia chebula fruit). Senescence was assessed on Day 14 according to cell morphology, ß-galactosidase activity, RT-qPCR measurement of SASP genes, as well as semi-quantitative (RT-qPCR) expression of miRNAs contained in EVs isolated from the medium. The size and distribution of EVs were measured by Nanoparticle Tracking Analysis. RESULTS: Human dermal fibroblasts exhibited a senescent phenotype 14 days after ionizing-radiation, demonstrated by a flattened and irregular shape, increased ß-galactosidase activity and over-expression of SASP genes. CSF3, CXCL1, IL1ß, IL6 and IL8 genes were increased by 1492%, 1041%, 343%, 478%, 2960% and 293%, respectively. The cell cycle inhibitor, CDKN1A, was increased by 357%, while COL1A1, was decreased by 56% and MMP1 was increased by 293%. NTA analysis of the EVs size distribution indicated a mix of exosomes (45-100 nm) and microvesicles (100-405 nm). miRNA expression in EVs was increased in senescent fibroblasts. miR 29a-3p, miR 30a-3p, miR 34a-5p, miR 24a-3p and miR 186-5p were increased in senescent HDF by 4.17-, 2.43-, 1.17-, 2.01, 12.5-fold, respectively. Incubation of senescent fibroblasts with Haritaki extract strongly decreased SASP mRNA levels and miRNA expression in EVs. CONCLUSION: Haritaki strongly reduced SASP expression and EV-shuttled miRNAs in senescent fibroblasts. These results indicate that Haritaki has strong senomorphic properties and may be a promising ingredient for the development of new anti-ageing dermo-cosmetic products by inhibiting deleterious effects of senescent cells.

OBJECTIF: Le vieillissement cutané est lié à l'accumulation de cellules sénescentes et à un « phénotype sécrétoire associé à la sénescence ¼ (SASP). Le SASP est constitué de chimiokines, cytokines et de petites vésicules extracellulaires (VE) contenant des miARN. Nous avons caractérisé les marqueurs du SASP dans des fibroblastes dermiques humains normaux (HDF) et évalué l'effet d'un extrait de fruit d'Haritaki sur ces marqueurs de la sénescence. MÉTHODES: La sénescence a été induite dans les HDF par des rayonnements ionisants (rayons X), suivis de 14 jours de culture. Parallèlement, des HDF ont été traités pendant 12 jours avec 10 ou 100 µg/mL d'Haritaki (un extrait standardisé de fruit de Terminalia chebula). La sénescence a été évaluée au jour 14 en fonction de la morphologie cellulaire, de l'activité ß-galactosidase, de la mesure des gènes du SASP par RT-PCR, ainsi que de l'expression semi-quantitative (RT-qPCR) des miARN contenus dans les VE isolées du milieu. La taille et la distribution des VE ont été mesurées par Nanoparticle Tracking Analysis (NTA). RÉSULTATS: Les HDF ont présenté un phénotype sénescent 14 jours après le rayonnement ionisant, en effet, ils avaient une forme aplatie et irrégulière, une activité ß-galactosidase accrue et une surexpression des gènes du SASP. Les ARNm de CSF3, CXCL1, IL1ß, IL6 et IL8 ont été augmentés de 1492%, 1041%, 343%, 478%, 2960% et 293%, respectivement. L'inhibiteur du cycle cellulaire, CDKN1A, a été augmenté de 357%, tandis que le COL1A1 a diminué de 56% et la MMP1 a augmenté de 293%. L'analyse NTA de la distribution de taille des VE a montré un mélange d'exosomes (45-100 nm) et de microvésicules (100-405 nm). L'expression des miARN dans les VE a augmenté dans les fibroblastes sénescents. Les miR 29a-3p, miR 30a-3p, miR 34a-5p, miR 24a-3p et miR 186-5p ont été augmentés dans le HDF sénescent de, respectivement, 4,17-, 2,43-, 1,17-, 2,01 et 12,5- fois. L'incubation de fibroblastes sénescents avec l'extrait de Haritaki a fortement diminué les niveaux d'ARNm du SASP et l'expression de miARN dans les VE. CONCLUSION: L'extrait d'Haritaki a fortement réduit l'expression du SASP et de miARN contenus dans les VE des fibroblastes sénescents. Ces résultats indiquent que Haritaki possède de fortes propriétés sénomorphiques et pourrait être un ingrédient prometteur pour le développement de nouveaux produits dermo-cosmétiques anti-âge en inhibant les effets délétères des cellules sénescentes.

Emerging Therapeutic Approaches to Target the Dark Side of Senescent Cells: New Hopes to Treat Aging as a Disease and to Delay Age-Related Pathologies.

Life expectancy has drastically increased over the last few decades worldwide, with important social and medical burdens and costs. To stay healthy longer and to avoid chronic disease have become essential issues. Organismal aging is a complex process that involves progressive destruction of tissue functionality and loss of regenerative capacity. One of the most important aging hallmarks is cellular senescence, which is a stable state of cell cycle arrest that occurs in response to cumulated cell stresses and damages. Cellular senescence is a physiological mechanism that has both beneficial and detrimental consequences. Senescence limits tumorigenesis, lifelong tissue damage, and is involved in different biological processes, such as morphogenesis, regeneration, and wound healing. However, in the elderly, senescent cells increasingly accumulate in several organs and secrete a combination of senescence associated factors, contributing to the development of various age-related diseases, including cancer. Several studies have revealed major molecular pathways controlling the senescent phenotype, as well as the ones regulating its interactions with the immune system. Attenuating the senescence-associated secretory phenotype (SASP) or eliminating senescent cells have emerged as attractive strategies aiming to reverse or delay the onset of aging diseases. Here, we review current senotherapies designed to suppress the deleterious effect of SASP by senomorphics or to selectively kill senescent cells by "senolytics" or by immune system-based approaches. These recent investigations are promising as radical new controls of aging pathologies and associated multimorbidities.

A single short reprogramming early in life initiates and propagates an epigenetically related mechanism improving fitness and promoting an increased healthy lifespan.

Recent advances in cell reprogramming showed that OSKM induction is able to improve cell physiology in vitro and in vivo. Here, we show that a single short reprogramming induction is sufficient to prevent musculoskeletal functions deterioration of mice, when applied in early life. In addition, in old age, treated mice have improved tissue structures in kidney, spleen, skin, and lung, with an increased lifespan of 15% associated with organ-specific differential age-related DNA methylation signatures rejuvenated by the treatment. Altogether, our results indicate that a single short reprogramming early in life might initiate and propagate an epigenetically related mechanism to promote a healthy lifespan.

Generation of catecholaminergic polymorphic ventricular tachycardia patient-specific induced pluripotent stem cell line.

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a genetic disorder characterized by ventricular tachycardia, that can cause the heart to stop beating leading to death. The prevalence is 1/10.000 and in approximately 60% of cases, the syndrome can be due to a mutation of the cardiac ryanodine receptor gene (RyR2). We derived an induced pluripotent stem cell (iPSC) line from an 11-year-old patient blood-cells, carrying a heterozygous missense mutation on the 8th exon of the RyR2 N-terminal part. This reprogramed CPVT line displayed normal karyotype, expressed pluripotent markers and had a capacity to differentiate in trilineage embryonic layers.

RNA-Based Strategies for Cell Reprogramming toward Pluripotency.

Cell therapy approaches to treat a wide range of pathologies have greatly benefited from cell reprogramming techniques that allow the conversion of a somatic cell into a pluripotent cell. Many technological developments have been made since the initial major discovery of this biological process. Recently reprogramming methods based on the use of RNA have emerged and seem very promising. Thus, in this review we will focus on presenting the interest of such methods for cell reprogramming but also how these RNA-based strategies can be extended to eventually lead to medical applications to improve healthspan and longevity.

Long non-coding RNA exploration for mesenchymal stem cell characterisation.

BACKGROUND: The development of RNA sequencing (RNAseq) and the corresponding emergence of public datasets have created new avenues of transcriptional marker search. The long non-coding RNAs (lncRNAs) constitute an emerging class of transcripts with a potential for high tissue specificity and function. Therefore, we tested the biomarker potential of lncRNAs on Mesenchymal Stem Cells (MSCs), a complex type of adult multipotent stem cells of diverse tissue origins, that is frequently used in clinics but which is lacking extensive characterization. RESULTS: We developed a dedicated bioinformatics pipeline for the purpose of building a cell-specific catalogue of unannotated lncRNAs. The pipeline performs ab initio transcript identification, pseudoalignment and uses new methodologies such as a specific k-mer approach for naive quantification of expression in numerous RNAseq data. We next applied it on MSCs, and our pipeline was able to highlight novel lncRNAs with high cell specificity. Furthermore, with original and efficient approaches for functional prediction, we demonstrated that each candidate represents one specific state of MSCs biology. CONCLUSIONS: We showed that our approach can be employed to harness lncRNAs as cell markers. More specifically, our results suggest different candidates as potential actors in MSCs biology and propose promising directions for future experimental investigations.

Reprogramming: Emerging Strategies to Rejuvenate Aging Cells and Tissues.

Aging is associated with a progressive and functional decline of all tissues and a striking increase in many "age-related diseases". Although aging has long been considered an inevitable process, strategies to delay and potentially even reverse the aging process have recently been developed. Here, we review emerging rejuvenation strategies that are based on reprogramming toward pluripotency. Some of these approaches may eventually lead to medical applications to improve healthspan and longevity.

Establishment of a collection of human pluripotent stem cell lines (iPSC) from mesenchymal stem cells (MSC) from three healthy elderly donors.

The study of molecular mechanism driving osteoarticular diseases like osteoarthritis or osteoporosis is impaired by the low accessibility to mesenchymal stem cells (MSC) from healthy donors (HD) for differential multi-omics analysis. Advances in cell reprogramming have, however, provided both a new source of human cells for laboratory research and a strategy to erase epigenetic marks involved in cell identity and the development of diseases. To unravel the pathological signatures on the MSC at the origin of cellular drifts during the formation of bone and cartilage, we previously developed iPSC from MSC of osteoarthritis donors. Here we present the derivation of three iPSCs from healthy age matched donors to model the disease and further identify (epi)genomic signatures of the pathology.

Generation of a human induced pluripotent stem cell line (iPSC) from peripheral blood mononuclear cells of a patient with a myasthenic syndrome due to mutation in COLQ.

Congenital myasthenic syndromes (CMS) are a class of inherited disorders affecting the neuromuscular junction, a synapse whose activity is essential for movement. CMS with acetylcholinesterase (AChE) deficiency are caused by mutations in COLQ, a collagen that anchors AChE in the synapse. To study the pathophysiological mechanisms of the disease in human cells, we have generated iPSC from a patient's Peripheral Blood Mononuclear cells (PBMC) by reprogramming these cells using a non-integrative method using Sendai viruses bearing the four Yamanaka factors Oct3/4, Sox2, Klf4, and L-Myc.

Generation of three Duchenne Muscular Dystrophy patient-specific induced pluripotent stem cell lines DMD_YoTaz_PhyMedEXp, DMD_RaPer_PhyMedEXp, DMD_OuMen_PhyMedEXp (INSRMi008-A, INSRMi009-A and INSRMi010-A).

Duchenne Muscular Dystrophy (DMD) is a X-linked degenerative pathology with a prevalence of 1/3600-6000 boys due to the absence of functional dystrophin in muscles. This muscular disease leads to skeletal muscle damages, respiratory failure and in the later stages dilated cardiomyopathy (DCM) leading to heart failure. We generated iPSC lines from three different DMD patients carrying respectively deletions of exons 1, 52 and 55 in the dystrophin gene. The reprogrammed iPSC lines showed expression of pluripotent markers, capacity to differentiate in trilineage embryonic layers and a normal karyotype.

iPSC reprogramming of fibroblasts from a patient with a Rothmund-Thomson syndrome RTS.

Rothmund-Thomson Syndrome (RTS) is a rare autosomal recessive disease that manifests several clinical features of accelerated aging. These findings include atrophic skin and pigment changes, alopecia, osteopenia, cataracts, and an increased incidence of cancer for patients. Mutations in RECQL4 gene are responsible for cases of RTS. RECQL4 belongs to the RECQ DNA helicase family which has been shown to participate in many aspects of DNA metabolism. To be able to study the cellular defects related to the pathology, we derived an induced pluripotent cell line from RTS patient fibroblasts, with the ability to re-differentiate into the three embryonic germ layers.

Generation of human pluripotent stem cell lines (iPSCs) from mesenchymal stem cells (MSCs) from three elderly patients with osteoarthritis.

Mesenchymal stem cells (MSCs) are a unique population of adult stem cells that can differentiate into many cell types. As such, MSCs represent an interesting source of stem cells for use in the clinical treatment of a variety of disorders involving tissue regeneration. It is therefore crucial to investigate further, whether MSCs from patients with bone or cartilage diseases are able to provide iPSCs lines with efficient differentiation ability into MSC derivatives. For this purpose, we derived 3 stable iPSC lines from the MSCs of 3 elderly patients with osteoarthritis (OA) able to re-differentiate into MSC to make bone, cartilage and adipose tissue.

4D Genome Rewiring during Oncogene-Induced and Replicative Senescence.

To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intra- versus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. This OIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression. We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging can identify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.

Recurrent Genetic Abnormalities in Human Pluripotent Stem Cells: Definition and Routine Detection in Culture Supernatant by Targeted Droplet Digital PCR.

Genomic integrity of human pluripotent stem cells (hPSCs) is essential for research and clinical applications. However, genetic abnormalities can accumulate during hPSC generation and routine culture and following gene editing. Their occurrence should be regularly monitored, but the current assays to assess hPSC genomic integrity are not fully suitable for such regular screening. To address this issue, we first carried out a large meta-analysis of all hPSC genetic abnormalities reported in more than 100 publications and identified 738 recurrent genetic abnormalities (i.e., overlapping abnormalities found in at least five distinct scientific publications). We then developed a test based on the droplet digital PCR technology that can potentially detect more than 90% of these hPSC recurrent genetic abnormalities in DNA extracted from culture supernatant samples. This test can be used to routinely screen genomic integrity in hPSCs.