Induced pluripotent stem cell reprogramming: methodological evolution and challenges in clinical translation

Translational cell fate reprogramming and regenerative medicine enabled by microphysiological systems.

Recent advances in genetic engineering and in vivo reprogramming have opened transformative possibilities for controlling cell fate in tissue repair and regeneration. However, clinical translation remains constrained by the limited predictive value of animal models and traditional in vitro systems, which often fail to fully recapitulate human responses, including the physiological consequences of genetic manipulations. Emerging microphysiological systems, exemplified by three-dimensional organoids and organs-on-chips (OoCs) systems, help bridge this gap by recreating key aspects of human physiology while enabling precise bioengineering of the niche to modulate cell fate decisions and plasticity. Organoids derived from induced pluripotent stem cells, adult stem cells, primary tissues, or directly reprogrammed cells preserve the patient-specific genetic background, facilitating mechanistic studies of development and disease and the evaluation of gene correction and reprogramming strategies in a human-relevant context. Complementarily, OoC platforms provide regulated perfusion, tissue vascularization, mechanical forces, molecular gradients, and immune cell integration to promote tissue maturation, functional readouts, and quantitative assessment of therapeutic responses that are difficult to achieve in static cultures. In this review, we discuss how organoids and OoC-based platforms are being leveraged to study and enhance cell fate reprogramming, repair, and regeneration across multiple tissues. We highlight recent reports where these systems informed the design, optimization, and safety evaluation of gene and cell therapies. Finally, we outline current limitations, including scalability, standardization, and biomaterial constraints, and propose future directions for integrating organoids, OoC, and gene-modulation technologies to enable more predictive, personalized, and clinically translatable regenerative medicine.

AAV Vectors in Regenerative Medicine and Cellular Reprogramming: Potential, Pitfalls, and Specificity Constraints

The adeno-associated virus (AAV) has become the vector of choice for gene therapy and experimental gene delivery, owing to its non-pathogenic nature and ability to achieve persistent gene expression across diverse tissues. AAV has emerged as a key platform in cellular reprogramming and regenerative medicine, with applications spanning transcription factor delivery for in vivo lineage conversion and tissue repair across the CNS, heart, and musculoskeletal systems. However, significant limitations remain, particularly in the context of induced pluripotent stem cell (iPSC) engineering. We assess barriers to efficient iPSC transduction including receptor-dependent entry deficits and activation of p53-dependent DNA damage responses. Although AAV is widely described as non-integrating, evidence indicates that integration events occur in rapidly proliferating and actively reprogramming cells. Critically, we synthesize evidence that cell-type-specific promoters lose fidelity when paired with neurogenic transgene payloads, a cross-tissue problem not addressed in existing AAV reviews, and that published in vivo reprogramming efficiencies may be substantially confounded by promoter leakage in the absence of formal lineage tracing. These aspects, underrepresented in recent platform-level reviews, are specifically emphasized here as a resource for researchers designing rigorous AAV-based reprogramming and gene therapy strategies.

Modeling myeloid cell development in health and disease using induced pluripotent stem cells

The pioneering discovery by Yamanaka and colleagues enabling the reprogramming of terminally differentiated somatic cells into induced pluripotent stem cells (iPSCs) has opened transformative opportunities for disease modeling and regenerative medicine, particularly in the context of inherited monogenic disorders. Patient-specific iPSCs can be generated, expanded almost indefinitely, and differentiated into a broad spectrum of cell types, including hematopoietic stem and progenitor cells, mature myeloid cells, and leukemic cells. Despite important limitations – such as epigenetic memory, variable differentiation efficiency, and concerns regarding tumorigenicity – iPSCs have become an indispensable experimental platform for studying inherited hematological disorders and malignancies, providing a renewable and physiologically relevant source of cells for downstream analyses. Beyond their research applications, iPSC-derived blood cells are increasingly being explored in preclinical studies and early-phase clinical trials as potential therapeutic products. The advent of CRISPR/Cas9 genome editing, pioneered by Charpentier and Doudna, has further advanced iPSC-based models by enabling precise correction or introduction of disease-causing mutations and the generation of isogenic control lines. This approach facilitates detailed mechanistic studies of defective hematopoiesis, enables drug discovery and repurposing through in silico screening platforms – such as L1000CDS2 and the Connectivity Map – and supports preclinical therapeutic validation. In this review, we summarize key applications of iPSC technology in hemato-oncology, discuss its major advantages and current limitations, and highlight emerging directions, including scalable iPSC-derived blood cell therapies for inherited and acquired bone marrow failure syndromes and leukemia.

Harnessing Endogenous Plasticity Rather than Reprogramming of Mature Cells Will Advance Regenerative Medicine, Cancer Treatment and Rejuvenation.

Integrating delivery systems and microenvironmental cues to accelerate clinical translation of cardiac reprogramming

Direct cardiac reprogramming, which converts cardiac fibroblasts into functional cardiomyocytes, has emerged as a promising regenerative strategy for heart failure. Preclinical studies have demonstrated its potential to mitigate adverse remodeling; however, critical challenges remain in delivery precision, reprogramming efficiency, and scalability for clinical translation. Here, we review innovations in delivery systems and microenvironmental engineering aimed at overcoming these barriers. Integration-free viral vectors (e.g. Sendai virus and adeno-associated virus 5) and non-viral platforms (e.g. nanoparticles and modified ribonucleic acid) have shown improved targeting of cardiac fibroblasts and enabled transient reprogramming. In parallel, microenvironmental modulation—such as immune regulation, biomechanical tuning of substrate stiffness, and the use of 3D-bioprinted matrices—has enhanced the survival and functionality of reprogrammed cells. Recent studies emphasize the need to balance genomic safety with delivery efficiency. While viral systems can achieve sustained factor expression, non-viral approaches minimize integration-associated risks. Large-animal models have further demonstrated the role of immune modulation and tissue-specific hydrogels in improving therapeutic retention. Advances in pericardial delivery systems and optimized manufacturing protocols now pave the way for future clinical translation. Nonetheless, unresolved issues—such as the long-term genomic stability of reprogrammed human cells and immune compatibility—still warrant rigorous investigation. By integrating targeted delivery, microenvironmental engineering, and scalable production, direct reprogramming strategies could redefine heart failure therapy. Collaborative efforts to address residual scientific and technical hurdles will determine their potential to benefit the 64 million patients affected globally.

Revisiting Pluripotency Acquisition: A Unified Framework for Reprogramming Strategies

The traditional hierarchical view of totipotent cells generating diverse lineages of terminally differentiated cells was challenged by the discovery of induced pluripotent stem cells (iPSCs). This breakthrough demonstrated that the ectopic expression of four transcription factors, Oct4, Sox2, Klf4, and c‐Myc (OSKM), can reprogram somatic cells to a pluripotent state. Since then, studies have expanded into diverse reprogramming strategies employing different cell types, factor combinations, delivery methods, and microenvironmental cues, resulting in the generation of diverse pluripotent and multipotent states. iPSCs modeled as canonical pluripotent reprogramming are defined by a core transcriptional network, the capacity for tri‐lineage differentiation, and a permissive epigenetic landscape. However, growing evidence has revealed other reprogramming trajectories, including direct lineage reprogramming (or transdifferentiation), atypical pluripotent reprogramming, and noncanonical pluripotent reprogramming—exemplified as bacterial protein‐mediated multipotency. Here, we summarize emerging insights of canonical and alternative pluripotent reprogramming strategies to propose a more nuanced framework that views pluripotency as a spectrum of molecular and functional states. These unconventional states often exhibit incomplete erasure of somatic identity, altered regulatory networks, and restricted lineage potential, underscoring the plastic and context‐dependent nature of pluripotency. This review aims to reconceptualize fundamental perspectives on the acquisition of pluripotency in mammalian fibroblasts based on recent advances. We delineate the criteria for direct lineage reprogramming, atypical pluripotent reprogramming and noncanonical pluripotent reprogramming, explore novel approaches like bacterial ribosome‐mediated cell fate conversion. This study integrates these strategies into a unified resource to enhance conceptual clarity and establishes a platform for advancing regenerative medicine.