The intellectual foundations of the field reach back to classic experiments showing that the differentiated state of a cell is not permanently fixed. Nuclear transfer and cell fusion studies demonstrated that somatic cell identity could be reset, and the discovery that the transcription factor MyoD could convert mouse fibroblasts directly into myoblasts established the concept of direct reprogramming. The landmark identification by Takahashi and Yamanaka of four transcription factors capable of generating induced pluripotent stem cells, or iPSCs, then opened a path to generating nearly any cell type in the laboratory. Several iPSC-derived therapies have since received regulatory approval or entered clinical trials. Yet the review emphasizes that iPSC-based approaches face persistent challenges, including tumorigenesis risk, inefficient differentiation, and poor survival and integration of transplanted cells within host tissues.

Direct reprogramming, also called transdifferentiation, offers an alternative by converting one somatic cell type into another without passing through a pluripotent intermediate. Even this strategy, however, has struggled when performed in vitro. Cells reprogrammed in culture often remain incompletely or immaturely converted, show poor tissue homing after transplantation, and suffer low long-term survival. In vivo direct reprogramming flips the logic entirely: rather than making cells in a dish and delivering them, researchers deliver reprogramming factors directly to the injury site, converting tissue-resident cells in their native environment. The review attributes the superior maturity of in vivo reprogrammed cells to pro-developmental and regenerative cues within the tissue microenvironment, which help drive cells toward fully functional states and promote integration with surrounding tissue.

At the molecular level, direct reprogramming works by rewriting gene regulatory networks, the hierarchical webs of transcription factors, signaling pathways, non-coding RNAs, and chromatin remodelers that define cellular identity. Central to this process are pioneer factors, a class of transcription factors capable of binding compacted heterochromatin. During neuronal reprogramming of fibroblasts, for example, the pioneer factor Ascl1 binds neural-lineage targets regardless of chromatin accessibility, locally opens the chromatin, and recruits the master regulators Brn2 and Myt1l to establish a neuron-specific regulatory circuit. Pioneer factors begin by scanning chromatin through non-specific electrostatic interactions, then distort and unwrap DNA at specific binding sites, recruiting co-regulators that remodel histones and create a transcriptionally permissive state. Successful reprogramming culminates in activation of peripheral network components that govern terminal fate and maturity, such as the factors Mafa and Errγ, which drive glucose-responsive insulin secretion in reprogrammed pancreatic beta cells.

The reprogramming toolkit extends well beyond transcription factors. Small molecules can activate developmental signaling pathways and modify the epigenetic landscape; a cocktail of CHIR99021, SB431542, DAPT, and LDN193189, for instance, converted human fetal astrocytes into cortical neurons by suppressing glial programs and activating neural master regulators. MicroRNAs offer another route: a combination of miRNAs 1, 133, 208, and 499 reprogrammed cardiac fibroblasts into cardiomyocytes by reinforcing cardiac lineage programs while silencing fibroblast signatures. Short hairpin RNAs and antisense oligonucleotides against the RNA-binding protein PTB have converted astrocytes into neurons, with stereotactically injected PTB antisense generating dopaminergic neurons that alleviated Parkinsonian symptoms in mouse models. Even exosomes, tiny membrane vesicles released by cells, can carry reprogramming cargo; insulinoma-derived exosomes enriched in miR-127 and miR-709 converted exocrine cells into pancreatic beta-like cells in vitro.

Delivering these factors into living tissue remains one of the field’s central engineering problems, and the review systematically compares the options. Viral vectors dominate current practice. Adenoviruses carry large payloads and achieve high transduction of dividing and non-dividing cells, as demonstrated when delivery of the transcription factors Ngn3, Pdx1, and Mafa converted more than 20 percent of infected pancreatic exocrine cells into insulin-positive cells within ten days. Adeno-associated viruses persist episomally for months to years and have reprogrammed retinal Müller glia into rod photoreceptors and astrocytes into neurons at efficiencies near 20 percent. Lentiviruses integrate into the host genome, enabling sustained expression but raising insertional mutagenesis concerns. Non-viral alternatives include plasmids, which can carry very large cargoes with minimal immunogenicity but express transgenes only transiently, and nanoparticles, particularly lipid nanoparticles, which can be surface-engineered for tissue-specific targeting. In one striking example, neutrophil-membrane-coated mesoporous silicon nanoparticles carrying cardiac reprogramming miRNAs homed to injured heart tissue via fibrinogen-derived targeting peptides, improving cardiac function and reducing fibrosis after myocardial infarction in mice.

The therapeutic results assembled in the review span nearly every major organ system. In the brain, delivery of the transcription factors Ascl1, Lmx1a, and Nurr1 converted striatal glial cells into functional neurons that integrated into host circuits within three months. AAV-mediated suppression of PTB converted astrocytes into neurons with efficiencies rising from roughly 20 percent at three weeks to 80 percent at twelve weeks, replenishing lost dopaminergic neurons and restoring striatal dopamine in Parkinson’s disease models. In the heart, retroviral delivery of Gata4, Mef2c, and Tbx5 generated induced cardiomyocytes that developed mature sarcomeres, fired action potentials, and electrically coupled with endogenous tissue, improving cardiac function after infarction. A seven-molecule chemical cocktail administered orally and intraperitoneally achieved lower but measurable conversion with functional recovery. In the pancreas, reprogrammed beta cells secreted insulin and improved glucose tolerance in diabetic mice, while in adipose tissue, exosome-driven conversion of inflammatory macrophages and browning of white fat improved insulin sensitivity and energy expenditure in obese mice.

Despite these preclinical successes, the review is unflinching about why in vivo direct reprogramming has yet to enter a single clinical trial. Reprogramming kinetics and efficiency in living tissue lag far behind culture conditions: a small-molecule cocktail that converted 94 percent of neonatal astrocytes into neurons in vitro achieved only about 11 percent in adult mouse brain, and in vitro-optimized doses proved insufficient in vivo, sometimes requiring tripled concentrations. The complex three-dimensional tissue microenvironment, with its biophysical cues such as matrix stiffness and shear stress, and its biochemical milieu of injury-induced inflammatory signals, reactive oxygen species, and cellular senescence, can either promote or antagonize reprogramming in ways that two-dimensional cultures cannot predict. Safety concerns compound the problem: reprogramming factors such as c-Myc carry oncogenic potential, integrating vectors risk insertional mutagenesis, transient progenitor-like states may mimic cancer phenotypes, and non-target cells sharing lineage history with targets may be inadvertently converted. Partially reprogrammed cells that revert or drift into undefined states represent a further unresolved risk.

The path forward, the authors argue, will require a convergence of emerging technologies. CRISPR-based transcriptional activators offer programmable activation of lineage-specific gene networks, as shown when AAV-delivered CRISPR repression of Nrl converted rod photoreceptors into cone-like cells in a retinitis pigmentosa model. Artificial intelligence and machine learning models trained on single-cell multi-omics data can predict gene perturbation effects, identify bifurcation points where reprogramming trajectories succeed or fail, and screen for novel factor combinations. Organ-on-chip platforms that replicate tissue-specific stiffness, fluid flow, and electrical properties may finally allow preclinical optimization of dose and exposure time under realistic three-dimensional conditions. Stimuli-responsive delivery systems, including microneedle patches, hydrogels, and electromagnetic-field-activated gene switches, promise controlled and targeted release. Before first-in-human trials, the review concludes, the field must validate outcomes in large animal models, establish GMP-compliant manufacturing, define patient stratification criteria, and develop regulatory frameworks that evaluate the integrated platform of factors and delivery vehicles as a single therapeutic entity. If those challenges can be met, in vivo direct reprogramming could move regenerative medicine beyond repair toward genuine rejuvenation of organs currently considered untreatable.

Subject of Research: In vivo direct reprogramming of tissue-resident cells for regenerative therapy

Article Title: In Vivo Direct Reprogramming: Current Progress and Future Prospects from Mechanisms to Therapeutic Application

Article References: Singh, R. D., Calvoli, M., & Kim, K. K. (2026). In Vivo Direct Reprogramming: Current Progress and Future Prospects from Mechanisms to Therapeutic Application. Advanced Science, Article e77370. https://doi.org/10.1002/advs.77370

Image Credits: AI Generated

DOI: 10.1002/advs.77370

Keywords: direct reprogramming, in vivo reprogramming, regenerative medicine, transcription factors, pioneer factors, gene regulatory networks, iPSCs, cardiac regeneration, neuronal reprogramming, viral vectors, lipid nanoparticles, CRISPR