Reprogramming
Cellular reprogramming refers to the process of converting differentiated somatic cells into induced pluripotent stem cells (iPSCs) by erasing epigenetic marks and activating pluripotency genes, thereby reversing cellular differentiation to a stem cell-like state.[1][2] This technique was pioneered in 2006 by Shinya Yamanaka and colleagues, who identified four transcription factors—Oct4, Sox2, Klf4, and c-Myc (collectively known as OSKM)—sufficient to reprogram mouse embryonic fibroblasts into iPSCs capable of contributing to all cell lineages, including germline transmission.[3] Extended to human cells in 2007 using similar factors on dermal fibroblasts, the method enabled generation of patient-specific iPSCs for disease modeling and potential autologous therapies, circumventing ethical issues of embryonic stem cell derivation. Key mechanisms involve dynamic epigenetic remodeling, including DNA demethylation at pluripotency loci and histone modifications that facilitate a metastable intermediate state before stable pluripotency acquisition.[4][5] While transformative for regenerative medicine, with Yamanaka receiving the 2012 Nobel Prize in Physiology or Medicine, reprogramming faces challenges like low efficiency (typically 0.01-0.1%), incomplete epigenetic erasure leading to memory retention, and oncogenic risks from factors like c-Myc, prompting ongoing research into non-integrating delivery methods and partial reprogramming strategies to mitigate tumorigenesis while achieving rejuvenation.[6][7]Biological Contexts
Embryonic Development
In mammalian embryonic development, epigenetic reprogramming initiates immediately upon fertilization, erasing most DNA methylation marks from the parental genomes to establish totipotency in the zygote. This process involves a genome-wide reduction in 5-methylcytosine (5mC) levels, reaching a minimum at the blastocyst stage before de novo methylation during implantation.[8] The reprogramming is essential for zygotic genome activation (ZGA), which occurs around the 2- to 8-cell stage in mice and later in humans, enabling embryonic gene expression independent of maternal transcripts.[9] The demethylation exhibits asymmetry between parental genomes. In the paternal pronucleus, active demethylation predominates, driven by the TET3 enzyme that oxidizes 5mC to 5-hydroxymethylcytosine (5hmC), followed by dilution or further processing via base excision repair.[10] Conversely, the maternal pronucleus primarily undergoes passive demethylation through exclusion of maintenance methyltransferase DNMT1 during replication, leading to progressive loss over cell divisions.[11] Studies in mice demonstrate that paternal 5mC levels drop by approximately 80-90% within hours post-fertilization, while maternal levels decline more gradually.[12] Exceptions to global demethylation include differentially methylated regions (DMRs) at imprinted loci, which resist erasure to preserve parent-of-origin-specific expression patterns critical for development.[8] Disruption of this reprogramming, such as TET3 knockout in mice, impairs ZGA and embryonic viability, underscoring its causal role in early development.[13] Accompanying epigenetic changes involve histone variant exchanges, such as H3.3 incorporation, and chromatin opening to facilitate transcriptional priming.[14] In humans, single-cell epigenomic mapping reveals similar dynamics, with implications for assisted reproductive technologies where perturbations can lead to developmental abnormalities.[15]Learning and Memory
Learning and memory processes in the brain involve epigenetic modifications that dynamically alter gene expression patterns in neurons, effectively reprogramming the chromatin landscape to encode and stabilize experiences. DNA methylation, a key epigenetic mark, serves as a mechanism for long-term memory storage by influencing synaptic plasticity genes in regions such as the hippocampus and amygdala.[16] These changes occur rapidly following learning tasks, with demethylation activating genes required for memory consolidation and hypermethylation repressing others to prevent interference.[17] In contextual fear conditioning experiments with rodents, training induces specific DNA methylation alterations in plasticity-related genes like BDNF and Egr1, which correlate with memory acquisition and persistence up to 24 hours post-training.[17] Enzymes such as DNA methyltransferases (DNMTs), including DNMT3a, catalyze these methylation events in the hippocampus, where their inhibition impairs long-term memory formation without affecting short-term recall.[18] Conversely, ten-eleven translocation (TET) proteins facilitate active demethylation, enabling the expression of genes critical for engram stabilization in neuronal ensembles.[19] Histone modifications complement DNA methylation in this reprogramming, with acetylation promoting open chromatin for transcription during memory encoding, while deacetylation contributes to the maintenance phase.[20] Studies show that inhibiting histone deacetylases (HDACs) enhances memory retention, underscoring the reversible nature of these epigenetic programs that balance plasticity and stability.00433-8) In Drosophila models, the histone methyltransferase EHMT regulates an epigenetic program involving classic learning genes, linking chromatin dynamics to cognitive outcomes across species.[21] Recent advances indicate that partial cellular reprogramming of neurons can rejuvenate age-related declines in plasticity, restoring youthful epigenetic states and improving memory performance in aged mice through controlled expression of factors like OSK (Oct4, Sox2, Klf4).[22] This approach highlights reprogramming's potential to modulate learning circuits, though it primarily targets rejuvenation rather than basal memory formation. Overall, these mechanisms ensure that epigenetic reprogramming provides a molecular basis for enduring behavioral adaptations, with disruptions linked to disorders like Alzheimer's disease where aberrant methylation patterns impair memory.[23]Aging and Rejuvenation
Cellular reprogramming targets aging by reversing epigenetic alterations that accumulate over time, such as DNA methylation changes tracked by epigenetic clocks like the Horvath clock. Partial reprogramming, involving transient expression of Yamanaka factors (Oct4, Sox2, Klf4, and optionally c-Myc, or OSKM), resets these marks to a youthful state while preserving cellular identity, unlike full reprogramming to induced pluripotent stem cells (iPSCs) which erases differentiation.[24][6] This approach addresses causal epigenetic noise and loss of information proposed as drivers of aging phenotypes.01570-7) In mouse models, in vivo partial reprogramming has demonstrated functional rejuvenation. A 2020 study expressed OSKM in retinal ganglion cells of aged and glaucomatous mice, restoring youthful transcriptomic profiles, optic nerve regeneration, and vision, with treated mice regaining near-normal visual acuity within weeks.[25] Similarly, cyclic OSK expression (excluding c-Myc to reduce tumorigenicity) in progeroid Zmpste24-/- mice extended median lifespan by approximately 30%, improved body weight, fur condition, and reduced kyphosis, while lowering epigenetic age by over 50% in multiple tissues.[26] These effects correlated with decreased senescence markers and enhanced tissue repair, though full lifespan extension in wild-type mice remains under investigation. Chemical alternatives to genetic Yamanaka factors have accelerated rejuvenation protocols. In 2023, six chemical cocktails were identified that, applied for less than seven days to human fibroblasts, reversed transcriptomic age by 2-3 years per Horvath clock estimates, restoring youthful gene expression without compromising cell identity or inducing pluripotency.[27] In vivo, such partial reprogramming ameliorated age-related molecular changes in physiologically aging mice, including reduced inflammation and improved metabolic profiles.[28] A 2025 preprint reported a single-gene intervention, SB000, rivaling OSKM efficacy across germ layers in cellular assays, suggesting streamlined future therapies.[29] Challenges include risks of tumorigenesis from prolonged factor expression and incomplete reversal of all aging hallmarks, such as proteostasis decline.[30] While mouse data indicate causality between epigenetic reprogramming and phenotypic rejuvenation, human applications are preclinical, with epigenetic clock reversals observed in cellular models but not yet in vivo trials.[31] Ongoing research prioritizes safer, non-integrative delivery like gene therapy or small molecules to translate these findings.[32]Molecular Mechanisms
Epigenetic Modifications
Epigenetic modifications encompass heritable changes in gene expression without altering the underlying DNA sequence, primarily through DNA methylation, histone post-translational modifications, and chromatin remodeling, which collectively govern cellular reprogramming by modulating chromatin accessibility and transcriptional programs.[4] In induced pluripotency, these modifications enable the transition from somatic to pluripotent states by erasing lineage-specific marks and reinstating embryonic-like epigenetic landscapes.[33] DNA methylation, characterized by the addition of methyl groups to cytosine residues predominantly at CpG dinucleotides, serves as a key repressive mechanism in differentiated cells, silencing pluripotency genes such as Nanog and Oct4 through promoter hypermethylation.[33] During reprogramming with Yamanaka factors (Oct4, Sox2, Klf4, c-Myc), demethylation occurs primarily in late stages, aligning with the acquisition of embryonic stem cell-like identity, where TET family enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), facilitating passive loss via replication-dependent dilution or active base excision repair.[34] [4] This process is rate-limiting, with incomplete demethylation at pluripotency loci acting as a barrier; supplementation with vitamin C enhances TET activity and reprogramming efficiency by up to tenfold in some protocols.[4] Hypermethylation gains, conversely, accumulate gradually at non-pluripotent sites, stabilizing the new identity.[34] Histone modifications provide dynamic regulation, with activating marks like H3K4 methylation deposited early by reprogramming factors to prime enhancers and promoters for transcription, often preceding gene activation.[33] Repressive marks, including H3K9me3 in heterochromatin and H3K27me3 mediated by Polycomb repressive complex 2 (PRC2), pose significant barriers; their removal via demethylases such as UTX (for H3K27me3) or inhibition of methyltransferases like SUV39H1 (for H3K9me3) accelerates reprogramming kinetics and boosts colony formation rates.[4] [35] Bivalent chromatin domains, bearing both H3K4me3 and H3K27me3, resolve during reprogramming to activate developmental regulators while repressing somatic genes.[33] Histone deacetylase inhibitors, like valproic acid, further enhance accessibility by promoting acetylation, increasing iPSC generation efficiency.[35] In developmental contexts, such as zygotic genome activation, global epigenetic erasure involves rapid TET3-dependent demethylation post-fertilization, mirroring mechanisms in somatic cell nuclear transfer where Tet3 deficiency impairs reprogramming.[4] Additional layers, including non-coding RNAs (e.g., miR-302 cluster targeting repressors) and ATP-dependent remodelers, cooperate to dismantle somatic chromatin architecture.[4] Persistent epigenetic memory from donors, such as incomplete heterochromatin dissolution, underlies inefficiencies in cloning and iPSC derivation, underscoring the need for targeted modulation.[4]Key Enzymes and Factors
The core transcription factors driving induced pluripotent stem cell (iPSC) reprogramming, known as the Yamanaka factors, consist of Oct4 (Pou5f1), Sox2, Klf4, and c-Myc (OSKM). These factors, when ectopically expressed in somatic cells, initiate a cascade that overrides lineage-specific gene expression and reactivates pluripotency networks. Oct4 and Sox2 form a heterodimer that binds enhancer regions of pluripotency genes, while Klf4 and c-Myc facilitate chromatin opening and proliferation, respectively.[36][37] Epigenetic enzymes play crucial roles in erasing somatic memory during reprogramming. Ten-eleven translocation (TET) enzymes, particularly TET1 and TET2, catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), promoting active DNA demethylation at pluripotency loci. This process cooperates with OSKM factors to enable gene reactivation, as TETs are recruited to target sites by pluripotency transcription factors like Nanog.[5][38] DNA methyltransferases (DNMTs), including DNMT1 for maintenance methylation and DNMT3A/B for de novo methylation, oppose reprogramming by preserving somatic methylation patterns. Successful reprogramming requires downregulation or inhibition of DNMT activity, often achieved through TET-mediated demethylation or replication-dependent dilution. Inhibition of DNMTs enhances reprogramming efficiency, underscoring their barrier role.[38][39] Other factors, such as histone deacetylases (HDACs), contribute by modulating chromatin accessibility, with HDAC inhibitors boosting OSKM-induced reprogramming. However, the primary drivers remain the OSKM transcription factors and TET-DNMT axis for epigenetic reconfiguration.[40]Transcriptional Regulation
Transcriptional regulation in cellular reprogramming centers on the ectopic expression of core transcription factors, notably the Yamanaka factors—Oct4, Sox2, Klf4, and c-Myc (OSKM)—discovered by Shinya Yamanaka's team in 2006, which reprogram somatic cells to induced pluripotency by overriding somatic gene networks and establishing a pluripotent transcriptional program.[41] These factors collaborate to repress lineage-specific enhancers while activating pluripotency-associated enhancers and promoters, initiating a self-reinforcing endogenous regulatory circuit involving genes like Nanog and Sall4.[42] OSK (Oct4, Sox2, Klf4) predominantly target enhancers, recruiting and redirecting somatic transcription factors such as AP-1 and C/EBP away from their native sites, whereas c-Myc exerts broader effects at promoters to facilitate proliferation and global transcriptional amplification.[42][5] The process unfolds in temporally distinct phases marked by dynamic gene expression shifts. Early reprogramming features stochastic binding of OSK to somatic and transient enhancers, driving mesenchymal-to-epithelial transition (MET) via downregulation of mesenchymal genes (e.g., Snai1, Zeb1) and upregulation of epithelial markers (e.g., E-cadherin), which is essential for progression but constitutes a barrier in fibroblasts.[5] Intermediate stages involve partial activation of early pluripotency genes like Utf1 and Esrrb, with chromatin opening at select loci; late phases transition to deterministic activation of the full pluripotency network, including core factors like endogenous Oct4 and Nanog, stabilizing the reprogrammed state.[42] Transcriptional pausing at promoters of pluripotency genes represents a key rate-limiting step, where OSKM recruit P-TEFb to release paused RNA polymerase II, enabling efficient elongation and gene activation.[43] Pioneer activity of Oct4 and Sox2 enables initial access to compacted chromatin, often in cooperation with Klf4 to enhance long-range chromatin interactions and topological domain connectivity, thereby facilitating enhancer-promoter looping for target gene activation.[5] Dosage of these factors critically modulates outcomes: suboptimal levels increase heterogeneity and inefficiency, while balanced ratios—such as equimolar OSKM—optimize enhancer occupancy and reduce stochastic barriers, as evidenced by single-cell analyses showing dose-dependent trajectories in reprogramming intermediates.[44][45] Despite advances, residual somatic transcriptional memory persists in iPSCs, influencing differentiation bias and underscoring incomplete erasure of original identities.[5] Overall, transcriptional reprogramming integrates pioneer binding, network rewiring, and pause release to achieve cell fate conversion, with efficiencies typically below 1% in standard protocols due to these regulatory hurdles.[5]Historical Development
Early Experiments in Nuclear Transfer and Fusion
The earliest experiments in nuclear transfer, precursors to modern somatic cell nuclear transfer (SCNT), were conducted by Robert Briggs and Thomas J. King in 1952 using embryos of the frog Rana pipiens. They transplanted nuclei from blastula-stage donor cells into enucleated eggs, achieving development to tadpole stages, though success rates were low and further progression to fertile adults was not observed.[46] These experiments demonstrated that early embryonic nuclei could direct development but highlighted challenges with differentiated nuclei, including incomplete reprogramming due to nuclear-cytoplasmic incompatibilities.[46] John B. Gurdon advanced these techniques in the late 1950s and early 1960s using Xenopus laevis eggs, which allowed ultraviolet irradiation for enucleation without mechanical damage. In 1962, Gurdon successfully cloned tadpoles and, through serial nuclear transfers, produced fertile adult frogs from nuclei of differentiated intestinal epithelial cells of feeding tadpoles.[47] Out of approximately 270 reconstructions with differentiated nuclei, only about 1-2% developed into normal frogs, underscoring the inefficiency but confirming that somatic nuclei retain totipotency when exposed to egg cytoplasm, which actively reprograms chromatin structure and gene expression.[48] This work established the principle of nuclear reprogramming, showing that differentiation does not involve irreversible genetic loss but reversible epigenetic changes.[49] Parallel early experiments in cell fusion, pioneered by Henry Harris and colleagues in the mid-1960s, provided complementary evidence for reprogramming. In 1965, Harris and J. F. Watkins used inactivated Sendai virus to fuse human and mouse cells, creating stable hybrid lines that exhibited selective gene activation and extinction, such as the reactivation of inactive X chromosomes.[50] These heterokaryons revealed cytoplasmic factors capable of influencing nuclear gene expression across species barriers, with embryonic or pluripotent cell cytoplasms dominating to suppress differentiated traits.[51] By the early 1970s, fusions between somatic cells and embryonal carcinoma cells demonstrated rapid reprogramming, including pluripotency marker expression and tumorigenic potential in hybrids, further illustrating the plasticity of somatic genomes.[52] Such findings supported the existence of trans-acting reprogramming factors, paving the way for understanding epigenetic dominance in hybrid cells.[53]