In a recent study published in the Aging Journal, researchers identified chemical drug combinations that could reverse cellular aging.
Study: Chemically induced reprogramming to reverse cellular aging. Image Credit: AnusornNakdee/Shutterstock.com
Background
Loss of epigenetic information is a characteristic of cellular aging in eukaryotes, resulting in changes in gene expression, loss of cellular identity, mitochondrial malfunction, inflammation, and cellular senescence, which contribute to aging and age-related illnesses.
Studies have reported that epigenetic loss can be restored. The authors of the present study previously demonstrated that ectopically induced transcription factors, Octamer-binding transcription factor 4 (OCT-4), sex-determining region Y-box 2 (SOX2), and Kruppel-like factor 4 (KLF-4) (collectively known as OSK factors) among mammals can reverse aging by restoring youthful patterns of deoxyribonucleic acid (DNA) methylation, transcriptomic profiles, and tissue functioning without cell identity loss.
About the study
In the present study, researchers devised high-throughput cellular assays that can differentiate between young, older, and aged cells of the body, including transcriptomic aging clocks and real-time quantitative nucleocytoplasmic compartmentalization (NCC) assays, to identify compounds that can reverse the aging process without genomic alterations.
The NCC system was developed to identify small molecules that reverse the effects of aging and senescence. The system was designed to be efficient, allowing for high-throughput analysis of cellular health and youthful gene expression patterns.
The NCC reporter system was introduced into human fibroblasts from a 22-year-old donor, and experiments were conducted to monitor age-associated alterations in nuclear permeability.
Passaging 40.0 times resulted in senescent fibroblasts with no growth over 14 days, morphological alterations typical of aged cells, and a rise in cell-cycle regulator p21 (CDKN1A) transcripts.
The fibroblasts were treated with doxycycline to activate the OSK system, and the NCC assay underwent testing to determine whether the system could identify the impact of genomically-regulated epigenetic age reversing.
The method could detect the consequences of a genetically induced epigenetic reversal of age using lentivirus transduction and gene ontology (GO) analysis. The team compiled a list of compounds that effectively converted human and mouse somatic cells into chemically induced pluripotent stem cells (CiPSCs) and assessed them using the NCC assay.
The researchers used VC6TF and C6NYSA as basal reprogramming cocktails and supplemented them with boosters [sodium butyrate, alpha-ketoglutarate (α-KG), and basic fibroblast growth factor (bFGF)] to increase iPSC efficiency.
The boosters' effects on VC6TF (cocktail 1 or C1, valproic acid, CHIR-99021, E-616452, tranylcypromine, and forskolin) were evaluated, and inhibitors of chromatin remodeling factors were incorporated to investigate rejuvenation barriers and drivers. Immunofluorescence was performed to assess the expression of pluripotency-related genes following all cocktail treatments.
The study assessed the genetic expression patterns of chemically treated cells compared to aged human-origin cells and OSK(MYC)-induced murine and human iPSC.
Gene set enrichment analysis (GSEA) was performed to identify pathways related to the differences and similarities between chemical therapies, aging signatures, and the OSK(M)-induced iPSCs using the Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome pathways, and HALLMARK genomic databases.
Ribonucleic acid (RNA) sequencing (to analyze transcriptomic profiles), signature association, transcriptomic clock, and iPSC profiling analyses were performed.
The team investigated molecules participating in the early stages of human CiPSC generation, including C1 and C6NYSA (C4, CHIR-99021, Y-27632, E-616452, TTNPB, and ABT-869 as basal cocktails for cellular reprogramming.
Results
The team identified six chemical cocktails that can restore a youthful genome-wide transcript profile and reverse transcriptomic age in less than a week without compromising cellular identity to a similar extent as OSK overexpression.
The researchers discovered that OSK expression in cells, such as murine and human fibroblasts, may significantly repair the epigenetic environment and patterns of gene expression of aged cells.
Of the 20 most upregulated genes associated with aging markers such as development, transportation, and localization disruption, 11 were restored to normal levels by OSK activity.
Senescence induced minor but significant alterations in the messenger ribonucleic acid (mRNA) levels of cell cycle genes, including p21. Senescence enriched for downregulated genes in several cell cycle-related activities, and 19 out of the 20 most altered genes returned to normal limits following OSK activity, demonstrating that OSK induction largely counteracted age-related alterations caused by senescence.
The VC6TF baseline cocktail was the most successful at recovering NCC quality, a critical indicator of age reversal, among the 80 cocktails examined in the NCC experiment.
The six cocktails statistically improved compartmentalization in senescent cells, both in correlation analysis and imaging of NCC signaling. All six reprogramming cocktails also significantly decreased the estimated chronological age of NCC senescent cells by several years.
Cocktails obtained from mice C1-3 demonstrated more consistent and profound anti-aging impacts that affected the cell transcriptome than cocktails derived from humans (C4-6).
The anti-aging properties of chemical cocktails, particularly those derived from mice, were linked to an upregulation of respiration-related pathways such as mitochondrial translation and oxidative phosphorylation, as well as the reduction of hypoxia and numerous inflammation-related pathways, including interferon (IFN) and Janus kinase/signal transducers and activators of transcription (JAK-STAT) signaling.
Conclusion
Based on the study findings, genetics and chemical means can achieve rejuvenation by age reversal without erasing cell identity.
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