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Cellular reprogramming of somatic cells towards induced pluripotency is a multistep stochastic process mediated by the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM), which orchestrate global epigenetic and transcriptional changes. We performed a large-scale analysis of integrated ChIP-seq, ATAC-seq and RNA-seq data and revealed the spatiotemporal highly dynamic pattern of OSKM DNA binding during reprogramming. We found that OSKM show distinct temporal patterns of binding to different classes of pluripotency-related enhancers. Genes involved in reprogramming are regulated by the coordinated activity of multiple enhancers, which are sequentially bound by OSKM for strict transcriptional control. Based on these findings, we developed an unbiased approach to identify Reprogramming-Inducible Enhancers (RIEs), constructed enhancer-traps and isolated cells undergoing reprogramming in real time. We used a representative RIE taken from the Upp1 gene fused to Gfp and isolated cells at different time-points during reprogramming and found that they have unique developmental capacities as they are reprogrammed with high efficiency due to their distinct molecular signatures. In conclusion, our experiments have led to the development of an unbiased method to identify and isolate reprogrammable cells in real time by exploiting the functional dynamics of OSKM, which can be used as efficient reprogramming biomarkers. [ABSTRACT FROM AUTHOR]

In vitro-generated blastocyst-like structures are of great importance since they recapitulate specific features or processes of early embryogenesis, thus avoiding ethical concerns as well as increasing scalability and accessibility compared to the use of natural embryos. Here, we combine cell reprogramming and mechanical stimuli to create 3D spherical aggregates that are phenotypically similar to those of natural embryos. Specifically, dermal fibroblasts are reprogrammed, exploiting the miR-200 family property to induce a high plasticity state in somatic cells. Subsequently, miR-200-reprogrammed cells are either driven towards the trophectoderm (TR) lineage using an ad hoc induction protocol or encapsulated into polytetrafluoroethylene micro-bioreactors to maintain and promote pluripotency, generating inner cell mass (ICM)-like spheroids. The obtained TR-like cells and ICM-like spheroids are then co-cultured in the same micro-bioreactor and, subsequently, transferred to microwells to encourage blastoid formation. Notably, the above protocol was applied to fibroblasts obtained from young as well as aged donors, with results that highlighted miR-200′s ability to successfully reprogram young and aged cells with comparable blastoid rates, regardless of the donor's cell age. Overall, the approach here described represents a novel strategy for the creation of artificial blastoids to be used in the field of assisted reproduction technologies for the study of peri- and early post-implantation mechanisms. [ABSTRACT FROM AUTHOR]

Central nervous system diseases, particularly neurodegenerative disorders, pose significant challenges in medicine. These conditions, characterized by progressive neuronal loss, have remained largely incurable, exacting a heavy toll on individuals and society. In recent years, in vivo reprogramming using Yamanaka factors has emerged as a promising approach for central nervous system regeneration. This technique involves introducing transcription factors, such as Oct4, Sox2, Klf4, and c-Myc, into adult cells to induce their conversion into neurons. This review summarizes the current state of in vivo reprogramming research in the central nervous system, focusing on the use of Yamanaka factors. In vivo reprogramming using Yamanaka factors has shown promising results in several animal models of central nervous system diseases. Studies have demonstrated that this approach can promote the generation of new neurons, improve functional outcomes, and reduce scar formation. However, there are still several challenges that need to be addressed before this approach can be translated into clinical practice. These challenges include optimizing the efficiency of reprogramming, understanding the cell of origin for each transcription factor, and developing methods for reprogramming in non-subventricular zone areas. Further research is needed to overcome the remaining challenges, but this approach has the potential to revolutionize the way we treat central nervous system disorders. [ABSTRACT FROM AUTHOR]

Simple Summary: Cancer stem cells (CSCs) are thought to be involved in tumor initiation and recurrence, which makes them pivotal therapeutic targets. However, the molecular circuits behind CSC characteristics are not fully understood and the low number of CSCs is an obstacle for conducting tests that explore their properties. Thus, increasing the number of these cells via small molecule-mediated cellular reprogramming seems a valid alternative tool. Using the SORE6-GFP reporter system, based on SOX2/OCT4 activity and incorporated in gastric non-CSCs, we performed a high-throughput drug screen to identify small molecules capable of converting non-CSCs into CSCs. Our results show that ciclopirox olamine (CPX) is able to reprogram gastric cancer cells to a stem-like phenotype via SOX2 expression, reprogram cell metabolism, and induce senescence. Furthermore, these results suggest that the CSC phenotype is a resistance mechanism to CPX treatment, which is being considered as a repurposing drug for cancer treatment. Cancer stem cells (CSCs) are relevant therapeutic targets for cancer treatment. Still, the molecular circuits behind CSC characteristics are not fully understood. The low number of CSCs can sometimes be an obstacle to carrying out assays that explore their properties. Thus, increasing CSC numbers via small molecule-mediated cellular reprogramming appears to be a valid alternative tool. Using the SORE6-GFP reporter system embedded in gastric non-CSCs (SORE6−), we performed a high-throughput image-based drug screen with 1200 small molecules to identify compounds capable of converting SORE6− to SORE6+ (CSCs). Here, we report that the antifungal agent ciclopirox olamine (CPX), a potential candidate for drug repurposing in cancer treatment, is able to reprogram gastric non-CSCs into cancer stem-like cells via activation of SOX2 expression and increased expression of C-MYC, HIF-1α, KLF4, and HMGA1. This reprogramming depends on the CPX concentration and treatment duration. CPX can also induce cellular senescence and the metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis. We also disclose that the mechanism underlying the cellular reprogramming is similar to that of cobalt chloride (CoCl2), a hypoxia-mimetic agent. [ABSTRACT FROM AUTHOR]

Mechanisms of cell reprogramming by pluripotency-related transcription factors or nuclear transfer seem to be mediated by similar pathways, and the study of the contribution of OCT4 and SOX2 in both processes may help elucidate the mechanisms responsible for pluripotency. Bovine fibroblasts expressing exogenous OCT4 or SOX2, or both, were analyzed regarding the expression of pluripotency factors and imprinted genes H19 and IGF2R, and used for in vitro reprogramming. The expression of the H19 gene was increased in the control sorted group, and putative iPSC-like cells were obtained when cells were not submitted to cell sorting. When sorted cells expressing OCT4, SOX2, or none (control) were used as donor cells for somatic cell nuclear transfer, fusion rates were 60.0% vs. 64.95% and 70.53% vs. 67.24% for SOX2 vs. control and OCT4 vs. control groups, respectively; cleavage rates were 66.66% vs. 81.68% and 86.47% vs. 85.18%, respectively; blastocyst rates were 33.05% vs. 44.15% and 52.06% vs. 44.78%, respectively. These results show that the production of embryos by NT resulted in similar rates of in vitro developmental competence compared to control cells regardless of different profiles of pluripotency-related gene expression presented by donor cells; however, induced reprogramming was compromised after cell sorting. [ABSTRACT FROM AUTHOR]

Generating specialized cell types via cellular transcription factor (TF)-mediated reprogramming has gained high interest in regenerative medicine due to its therapeutic potential to repair tissues and organs damaged by diseases or trauma. Organ dysfunction or improper tissue functioning might be restored by producing functional cells via direct reprogramming, also known as transdifferentiation. Regeneration by converting the identity of available cells in vivo to the desired cell fate could be a strategy for future cell replacement therapies. However, the generation of specific cell types via reprogramming is often restricted due to cell fate-safeguarding mechanisms that limit or even block the reprogramming of the starting cell type. Nevertheless, efficient reprogramming to generate homogeneous cell populations with the required cell type's proper molecular and functional identity is critical. Incomplete reprogramming will lack therapeutic potential and can be detrimental as partially reprogrammed cells may acquire undesired properties and develop into tumors. Identifying and evaluating molecular barriers will improve reprogramming efficiency to reliably establish the target cell identity. In this review, we summarize how using the nematode C. elegans as an in vivo model organism identified molecular barriers of TF-mediated reprogramming. Notably, many identified molecular factors have a high degree of conservation and were subsequently shown to block TF-induced reprogramming of mammalian cells. [ABSTRACT FROM AUTHOR]

Cardiovascular disease is a globally prevalent disease with far-reaching medical and socio-economic consequences. Although improvements in treatment pathways and revascularisation therapies have slowed disease progression, contemporary management fails to modulate the underlying atherosclerotic process and sustainably replace damaged arterial tissue. Direct cellular reprogramming is a rapidly evolving and innovative tissue regenerative approach that holds promise to restore functional vasculature and restore blood perfusion. The approach utilises cell plasticity to directly convert somatic cells to another cell fate without a pluripotent stage. In this narrative literature review, we comprehensively analyse and compare direct reprogramming protocols to generate endothelial cells, vascular smooth muscle cells and vascular progenitors. Specifically, we carefully examine the reprogramming factors, their molecular mechanisms, conversion efficacies and therapeutic benefits for each induced vascular cell. Attention is given to the application of these novel approaches with tissue engineered vascular grafts as a therapeutic and disease-modelling platform for cardiovascular diseases. We conclude with a discussion on the ethics of direct reprogramming, its current challenges, and future perspectives. [ABSTRACT FROM AUTHOR]

The importance of redox systems as fundamental elements in biology is now widely recognized across diverse fields, from ecology to cellular biology. Their connection to metabolism is particularly significant, as it plays a critical role in energy regulation and distribution within organisms. Over recent decades, metabolism has emerged as a relevant focus in studies of biological regulation, especially following its recognition as a hallmark of cancer. This shift has broadened cancer research beyond strictly genetic perspectives. The interaction between metabolism and redox systems in carcinogenesis involves the regulation of essential metabolic pathways, such as glycolysis and the Krebs cycle, as well as the involvement of redox-active components like specific amino acids and cofactors. The feedback mechanisms linking redox systems and metabolism in cancer highlight the development of redox patterns that enhance the flexibility and adaptability of tumor processes, influencing larger-scale biological phenomena such as circadian rhythms and epigenetics.

Placentation disorders, including severe preeclampsia and fetal growth restriction, have their origins in early pregnancy, whereas symptoms typically present later on. To investigate the pathogenesis of these diseases, there is a need for a reliable in vitro model system of early placenta development with known pregnancy outcomes. Therefore, we optimized the generation of human induced trophoblast stem cells (iTSCs) from term umbilical cord, enabling non-invasive collection of patient-derived material immediately after birth. Using a direct reprogramming approach previously described for dermal fibroblasts, we investigated the effects of three supplements (A-485, BMP4, and EPZ-6438) to assess their potential to enhance iTSC induction. The generated iTSCs fulfilled the criteria for bona fide first-trimester trophoblasts and exhibited key functional capacities, including long-term self-renewal, differentiation into hormone-producing syncytiotrophoblasts and invasive extravillous trophoblasts, and the formation of organoids. Furthermore, transcriptomic analysis revealed high similarity between the generated iTSCs and trophoblast stem cells derived from first-trimester placental tissue. The supplements did not improve the generation of iTSCs. In conclusion, we successfully generated bona fide iTSCs from term umbilical cord using a direct reprogramming approach, providing a robust and clinically relevant model to study early placentation mechanisms in patient-derived trophoblasts.

Heart disease, including myocardial infarction (MI), remains a leading cause of morbidity and mortality worldwide, necessitating the development of more effective regenerative therapies. Direct reprogramming of cardiomyocyte-like cells from resident fibroblasts offers a promising avenue for myocardial regeneration, but its efficiency and consistency in generating functional cardiomyocytes remain limited. Alternatively, reprogramming induced cardiac progenitor cells (iCPCs) could generate essential cardiac lineages, but existing methods often involve complex procedures. These limitations underscore the need for advanced mechanistic insights and refined reprogramming strategies to improve reparative outcomes in the heart. Partial cellular fate transitions, while still a relatively less well-defined area and primarily explored in longevity and neurobiology, hold remarkable promise for cardiac repair. It enables the reprogramming or rejuvenation of resident cardiac cells into a stem or progenitor-like state with enhanced cardiogenic potential, generating the reparative lineages necessary for comprehensive myocardial recovery while reducing safety risks. As an emerging strategy, partial cellular fate transitions play a pivotal role in reversing myocardial infarction damage and offer substantial potential for therapeutic innovation. This review will summarize current advances in these areas, including recent findings involving two transcription factors that critically regulate stemness and cardiogenesis. It will also explore considerations for further refining these approaches to enhance their therapeutic potential and safety.

Treatment of full-thickness articular cartilage defects with exposure of subchondral bone often seen in osteoarthritic conditions has long been a great challenge, especially with a focus on the feasibility of in situ cartilage regeneration through minimally invasive procedures. Osteoblasts that situate in the subchondral bone plate may be considered a potentially vital endogenous source of cells for cartilage resurfacing through direct reprogramming into chondrocytes. Microarray-based gene expression profiles were generated to compare tissue-specific transcripts between subchondral bone and cartilage of mice and to assess age-dependent differences of chondrocytes as well. On osteoblast cell lines established from mouse proximal tibial subchondral bone, sequential screening by co-transduction of transcription factor (TF) genes that distinguish chondrocytes from osteoblasts reveals a shortlist of potential reprogramming factors exhibiting combined effects in inducing chondrogenesis of subchondral bone osteoblasts. A further combinatorial approach unexpectedly identified two 3-TF combinations containing Sox9 and Sox5 that exhibit differences in reprogramming propensity with the third TF c-Myc or Plagl1, which appeared to direct the converted chondrocytes toward either a superficial or a deeper zone phenotype. Thus, our approach demonstrates the possibility of converting osteoblasts into two major chondrocyte subpopulations with two combinations of three genes (Sox9, Sox5, and c-Myc or Plagl1). The findings may have important implications for developing novel in situ regeneration strategies for the reconstruction of full-thickness cartilage defects. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (iPSCs) have been considered an essential tool in stem cell research due to their potential to develop new therapies and technologies and answer essential questions about mammalian early development. An important step in generating iPSCs is selecting their precursor cell type, influencing the reprogramming efficiency and maintenance in culture. In this study, we aim to characterize bovine mesenchymal cells from adipose tissue (bAdMSCs) and fetal fibroblasts (bFFs) and to compare the reprogramming efficiency of these cells when induced to pluripotency. The cells were characterized by immunostaining (CD90, SSEA1, SSEA3, and SSEA4), induced differentiation in vitro, proliferation rates, and were subjected to cell reprogramming using the murine OSKM transcription factors. The bFFs presented morphological changes resembling pluripotent cells after reprogramming and culture with different supplementation, and putative iPSCs were characterized by immunostaining (OCT4, SOX2, NANOG, and AP). In the present study, we demonstrated that cell line origin and cellular proliferation rate are determining factors for reprogramming cells into pluripotency. The generation of biPSCs is a valuable tool to improve both translational medicine and animal production and to study the different supplements required to maintain the pluripotency of bovine cells in vitro. [ABSTRACT FROM AUTHOR]

Soft tissue sarcoma (STS) comprise a large group of mesenchymal malignant tumors with heterogeneous cellular morphology, proliferative index, genetic lesions and, more importantly, clinical features. Full elucidation of this wide diversity remains a central question to improve their therapeutic management and the identity of cell(s)-of-origin from which these tumors arise is part of this enigma. Cellular reprogramming allows transitions of a mature cell between phenotypes, or identities, and represents one key driver of tumoral heterogeneity. Here, we discuss how cellular reprogramming mediated by driver genes in STS can profoundly reshape the molecular and morphological features of a transformed cell and lead to erroneous interpretation of its cell-of-origin. This review questions the fact that the epigenetic context in which a genetic alteration arises has to be taken into account as a key determinant of STS tumor initiation and progression. Retracing the cancer-initiating cell and its clonal evolution, notably via epigenetic approach, appears as a key lever for understanding the origin of these tumors and improving their clinical management. [ABSTRACT FROM AUTHOR]

Retinal neurodegeneration (RN), an early marker of diabetic retinopathy (DR), is closely associated with Müller glia cells (MGs) in diabetic subjects. MGs play a pivotal role in maintaining retinal homeostasis, integrity, and metabolic support and respond to diabetic stress. In lower vertebrates, MGs have a strong regenerative response and can completely repair the retina after injuries. However, this ability diminishes as organisms become more complex. The aim of this study was to investigate the gliotic response and reprogramming potential of the human Müller cell line MIO-M1 cultured in normoglycemic (5 mM glucose, NG) and hyperglycemic (25 mM glucose, HG) conditions and then exposed to sustained high-glucose and glucose fluctuation (GF) treatments to mimic the human diabetic conditions. The results showed that NG MIO-M1 cells exhibited a dynamic activation to sustained high-glucose and GF treatments by increasing GFAP and Vimentin expression together, indicative of gliotic response. Increased expression of SHH and SOX2 were also observed, foreshadowing reprogramming potential. Conversely, HG MIO-M1 cells showed increased levels of the indexes reported above and adaptation/desensitization to sustained high-glucose and GF treatments. These findings indicate that MIO-M1 cells exhibit a differential response under various glucose treatments, which is dependent on the metabolic environment. The in vitro model used in this study, based on a well-established cell line, enables the exploration of how these responses occur in a controlled, reproducible system and the identification of strategies to promote neurogenesis over neurodegeneration. These findings contribute to the understanding of MGs responses under diabetic conditions, which may have implications for future therapeutic approaches to diabetes-associated retinal neurodegeneration.

Metabolic reprogramming of immune cells within the tumor microenvironment (TME) plays a pivotal role in shaping tumor progression and responses to therapy. The intricate interplay between tumor cells and immune cells within this ecosystem influences their metabolic landscapes, thereby modulating the immune evasion tactics employed by tumors and the efficacy of immunotherapeutic interventions. This review delves into the metabolic reprogramming that occurs in tumor cells and a spectrum of immune cells, including T cells, macrophages, dendritic cells, and myeloid-derived suppressor cells (MDSCs), within the TME. The metabolic shifts in these cell types span alterations in glucose, lipid, and amino acid metabolism. Such metabolic reconfigurations can profoundly influence immune cell function and the mechanisms by which tumors evade immune surveillance. Gaining a comprehensive understanding of the metabolic reprogramming of immune cells in the TME is essential for devising novel cancer therapeutic strategies. By targeting the metabolic states of immune cells, it is possible to augment their anti-tumor activities, presenting new opportunities for immunotherapeutic approaches. These strategies hold promise for enhancing treatment outcomes and circumventing the emergence of drug resistance.

Rotator cuff tears (RCT) are the most common cause of shoulder pain among adults. "Rotator cuff" refers to the four muscles that cover the shoulder joint: supraspinatus, infraspinatus, subscapularis, and teres minor. These muscles help maintain the rotational movement and stability of the shoulder joint. RCT is a condition in which one or more of these four muscles become ruptured or damaged, causing pain in the arms and shoulders. RCT results from degenerative changes caused by chronic inflammation of the tendons and consequent tendon tissue defects. This phenomenon occurs because of the exhaustion of endogenous tendon stem cells. Tendon regeneration requires rejuvenation of these endogenous tendon stem/progenitor cells (TSPCs) prior to their growth phase. TSPCs exhibit clonogenicity, multipotency, and self-renewal properties; they express classical stem cell markers and genes associated with the tendon lineage. However, specific markers for TSPC are yet to be identified. In this review, we introduce novel TSPC markers and discuss various strategies for TSPC reprogramming. With further research, TSPC reprogramming technology could be adapted to treat age-related degenerative diseases, providing a new strategy for regenerative medicine.

Aging is an important risk factor for tumorigenesis. Metabolic reprogramming is a hallmark of both aging and tumor initiation. However, the manner in which the crosstalk between aging and metabolic reprogramming affects the tumor microenvironment (TME) to promote tumorigenesis was poorly explored. We utilized a computational approach proposed by our previous work, MMP 3 C (Modeling Metabolic Plasticity by Pathway Pairwise Comparison), to characterize aging-related metabolic plasticity events using pan-cancer bulk RNA-seq data. Our analysis revealed a high degree of metabolically organized heterogeneity across 17 aging-related cancer types. In particular, a higher degree of several energy generation pathways, i.e., glycolysis and impaired oxidative phosphorylation, was observed in older patients. Similar phenomena were also found via single-cell RNA-seq analysis. Furthermore, those energy generation pathways were found to be weakened in activated T cells and macrophages, whereas they increased in exhausted T cells, immunosuppressive macrophages, and Tregs in older patients. It was suggested that aging-induced metabolic switches alter glucose utilization, thereby influencing immune function and resulting in the remodeling of the TME. This work offers new insights into the associations between tumor metabolism and the TME mediated by aging, linking with novel strategies for cancer therapy.

Glioblastoma (GBM) is an aggressive and highly malignant primary brain tumor characterized by rapid growth and a poor prognosis for patients. Despite advancements in treatment, the median survival time for GBM patients remains low. One of the crucial challenges in understanding and treating GBMs involves its remarkable cellular heterogeneity and adaptability. Central to the survival and proliferation of GBM cells is their ability to undergo metabolic reprogramming. Metabolic reprogramming is a process that allows cancer cells to alter their metabolism to meet the increased demands of rapid growth and to survive in the often oxygen- and nutrient-deficient tumor microenvironment. These changes in metabolism include the Warburg effect, alterations in several key metabolic pathways including glutamine metabolism, fatty acid synthesis, and the tricarboxylic acid (TCA) cycle, increased uptake and utilization of glutamine, and more. Despite the complexity and adaptability of GBM metabolism, a deeper understanding of its metabolic reprogramming offers hope for developing more effective therapeutic interventions against GBMs.

The high mortality in the global population due to chronic diseases highlights the urgency to identify effective alternative therapies. Regenerative medicine provides promising new approaches for this purpose, particularly in the use of induced pluripotent stem cells (iPSCs). The aim of the work is to establish a new pluripotency cell line obtained for the first time by reprogramming human gingival mesenchymal stem cells (hGMSCs) by a non-integrating method. The hGMSC-derived iPS line characterization is performed through morphological analysis with optical and electron scanning microscopy and through the pluripotency markers expression evaluation in cytofluorimetry, immunofluorescence, and RT-PCR. To confirm the pluripotency of new hGMSC-derived iPS, the formation of embryoid bodies (EBs), as an alternative to the teratoma formation test, is studied in morphological analysis and through three germ layers' markers' expression in immunofluorescence and RT-PCR. At the end, a comparative study between parental hGMSCs and derived iPS cells is performed also for the extracellular vesicles (EVs) and their miRNA content. The new hGMSC-derived iPS line demonstrated to be pluripotent in all aspects, thus representing an innovative dynamic platform for personalized tissue regeneration.

Astrocytes play a pivotal role in maintaining brain energy homeostasis, supporting neuronal function through glycolysis and lipid metabolism. This review explores the metabolic intricacies of astrocytes in both physiological and pathological conditions, highlighting their adaptive plasticity and diverse functions. Under normal conditions, astrocytes modulate synaptic activity, recycle neurotransmitters, and maintain the blood-brain barrier, ensuring a balanced energy supply and protection against oxidative stress. However, in response to central nervous system pathologies such as neurotrauma, stroke, infections, and neurodegenerative diseases like Alzheimer's and Huntington's disease, astrocytes undergo significant morphological, molecular, and metabolic changes. Reactive astrocytes upregulate glycolysis and fatty acid oxidation to meet increased energy demands, which can be protective in acute settings but may exacerbate chronic inflammation and disease progression. This review emphasizes the need for advanced molecular, genetic, and physiological tools to further understand astrocyte heterogeneity and their metabolic reprogramming in disease states.

Acute inflammation is a rapid and dynamic process involving the recruitment and activation of multiple cell types in a coordinated and precise manner. Here, we investigate the origin and transcriptional reprogramming of monocytes using a model of acute inflammation, zymosan-induced peritonitis. Monocyte trafficking and adoptive transfer experiments confirmed that monocytes undergo rapid phenotypic change as they exit the blood and give rise to monocyte-derived macrophages that persist during the resolution of inflammation. Single-cell transcriptomics revealed significant heterogeneity within the surface marker-defined CD11b + Ly6G - Ly6C hi monocyte populations within the blood and at the site of inflammation. We show that two major transcriptional reprogramming events occur during the initial six hours of Ly6C hi monocyte mobilisation, one in the blood priming monocytes for migration and a second at the site of inflammation. Pathway analysis revealed an important role for oxidative phosphorylation (OxPhos) during both these reprogramming events. Experimentally, we demonstrate that OxPhos via the intact mitochondrial electron transport chain is essential for murine and human monocyte chemotaxis. Moreover, OxPhos is needed for monocyte-to-macrophage differentiation and macrophage M(IL-4) polarisation. These new findings from transcriptional profiling open up the possibility that shifting monocyte metabolic capacity towards OxPhos could facilitate enhanced macrophage M2-like polarisation to aid inflammation resolution and tissue repair.

The risk of aberrant growth of induced pluripotent stem cell (iPSC)-derived cells in response to DNA damage is a potential concern as the tumor suppressor genes TP53 and CDKN2A are transiently inactivated during reprogramming. Herein, we evaluate the integrity of cellular senescence pathways and DNA double-strand break (DSB) repair in Sendai virus reprogrammed iPSC-derived human fibroblasts (i-HF) compared to their parental skin fibroblasts (HF). Using transcriptomics analysis and a variety of functional assays, we show that the capacity of i-HF to enter senescence and repair DSB is not compromised after damage induced by ionizing radiation (IR) or the overexpression of H-RAS V12 . Still, i-HF lines are transcriptionally different from their parental lines, showing enhanced metabolic activity and higher expression of p53-related effector genes. As a result, i-HF lines generally exhibit increased sensitivity to various stresses, have an elevated senescence-associated secretory phenotype (SASP), and cannot be immortalized unless p53 expression is knocked down. In conclusion, while our results suggest that i-HF are not at a greater risk of transformation, their overall hyperactivation of senescence pathways may impede their function as a cell therapy product.

The reprogramming of somatic cells to pluripotent stem cells has immense potential for use in regenerating or redeveloping tissues for transplantation, and the future application of this method is one of the most important research topics in regenerative medicine. These cells are generated from normal cells, adult stem cells, or neoplastic cancer cells. They express embryonic stem cell markers, such as OCT4, SOX2, and NANOG, and can differentiate into all tissue types in adults, both in vitro and in vivo. However, tumorigenicity, immunogenicity, and heterogeneity of cell populations may hamper the use of this method in medical therapeutics. The risk of cancer formation is dependent on mutations of these stemness genes during the transformation of pluripotent stem cells to cancer cells and on the alteration of the microenvironments of stem cell niches at genetic and epigenetic levels. Recent reports have shown that the generation of induced pluripotent stem cells (iPSCs) derived from human fibroblasts could be induced using chemicals, which is a safe, easy, and clinical-grade manufacturing strategy for modifying the cell fate of human cells required for regeneration therapies. This strategy is one of the future routes for the clinical application of reprogramming therapy. Therefore, this review highlights the recent progress in research focused on decreasing the tumorigenic risk of iPSCs or iPSC-derived organoids and increasing the safety of iPSC cell preparation and their application for therapeutic benefits.

Osteoarthritis (OA) is a degenerative disease of the hyaline articular cartilage. This disease is progressive and may lead to disability. Researchers proposed many regenerative approaches to treat osteoarthritis, including stem cells. Trans-differentiation of a fully differentiated cell state directly into another different differentiated cell state avoids the disadvantages of fully reprogramming cells to induced pluripotent stem cells (iPSCs) in terms of faster reprogramming of the needed cells. Trans-differentiation also reduces the risk of tumor formation by avoiding the iPSC state. OSKM factors (Oct4, Sox2, Klf4, and cMyc) accompanied by the JAK-STAT pathway inhibition, followed by the introduction of specific differentiation factors, directly reprogrammed mouse embryonic fibroblasts to chondroblasts. Our results showed the absence of intermediate induced pluripotent stem cell formation. The resulting aggregates showed clear hyaline and hypertrophic cartilage. Tumor formation was absent in sub-cutaneous capsules transplanted in SCID mice. [ABSTRACT FROM AUTHOR]

Geminiviruses are plant-infecting viruses characterized by a single-stranded DNA (ssDNA) genome. Geminivirus-derived proteins are multifunctional and effective regulators in modulating the host cellular processes resulting in successful infection. Virus-host interactions result in changes in host gene expression patterns, reprogram plant signaling controls, disrupt central cellular metabolic pathways, impair plant's defense system, and effectively evade RNA silencing response leading to host susceptibility. This review summarizes what is known about the cellular processes in the continuing tug of war between geminiviruses and their plant hosts at the molecular level. In addition, implications for engineered resistance to geminivirus infection in the context of a greater understanding of the molecular processes are also discussed. Finally, the prospect of employing geminivirus-based vectors in plant genome engineering and the emergence of powerful genome editing tools to confer geminivirus resistance are highlighted to complete the perspective on geminivirus-plant molecular interactions. [ABSTRACT FROM AUTHOR]

Various factors are known to contribute to the diversity of human induced pluripotent stem cells (hiPSCs). Among these are the donor's genetic background and family history, the somatic cell source, the iPSC reprogramming method, and the culture system of choice. Moreover, variability is seen even in iPSC clones, generated in a single reprogramming event, where the donor, somatic cell type, and reprogramming platform are the same. The diversity seen in iPSC lines often translates to epigenetic differences, as well as to differences in the expansion rate, iPSC line culture robustness, and their ability to differentiate into specific cell types. As such, the diversity of iPSCs presents a hurdle to standardizing iPSC-based cell therapy manufacturing. In this review, we will expand on the various factors that impact iPSC diversity and the strategies and tools that could be taken by the industry to overcome the differences amongst various iPSC lines, therefore enabling robust and reproducible iPSC-based cell therapy manufacturing processes.

Acute myeloid leukemia (AML) is the most common and incurable leukemia subtype. Despite extensive research into the disease's intricate molecular mechanisms, effective treatments or expanded diagnostic or prognostic markers for AML have not yet been identified. The morphological, immunophenotypic, cytogenetic, biomolecular, and clinical characteristics of AML patients are extensive and complex. Leukemia stem cells (LSCs) consist of hematopoietic stem cells (HSCs) and cancer cells transformed by a complex, finely-tuned interaction that causes the complexity of AML. Microenvironmental regulation of LSCs dormancy and the diagnostic and therapeutic implications for identifying and targeting LSCs due to their significance in the pathogenesis of AML are discussed in this review. It is essential to perceive the relationship between the niche for LSCs and HSCs, which together cause the progression of AML. Notably, methylation is a well-known epigenetic change that is significant in AML, and our data also reveal that microRNAs are a unique factor for LSCs. Multiple-targeted approaches to reduce the risk of epigenetic factors, such as the administration of natural compounds for the elimination of local LSCs, may prevent potentially fatal relapses. Furthermore, the survival analysis of overlapping genes revealed that specific targets had significant effects on the survival and prognosis of patients. We predict that the multiple-targeted effects of herbal products on epigenetic modification are governed by different mechanisms in AML and could prevent potentially fatal relapses. Thus, these strategies can facilitate the incorporation of herbal medicine and natural compounds into the advanced drug discovery and development processes achievable with Network Pharmacology research.

Several oncogenic viruses are associated with approximately 20% of human cancers. Experimental models are crucial for studying the pathogenicity and biological aspects of oncogenic viruses and their potential mechanisms in tumorigenesis. Current cell models have considerable limitations such as: their low yield, genetic and epigenetic modification, and reduction in tumor heterogeneity during long propagation. Cancer cell lines are limited and not appropriate for studying the viral life cycle, for example, natural viral life cycles of HPV and EBV, and their persistence and latency in epithelial cells are poorly understood, since these processes are highly related to epithelial differentiation. Therefore, there is an urgent need of reliable human physiological cell models to study viral life cycle and cancer initiation. Conditional cell reprogramming (CCR) is a rapid and robust cell culture system, where the cells can be established from minimally invasive or noninvasive specimens and their lineage functions preserved during the long-term culture. These CR cells retain their ability to differentiate at air-liquid interface (ALI). Here, we recapitulated the applications of CR and ALI approaches in modeling host-virus interactions and viral-mediated tumorigenesis.

Cellular metabolism (or energetics) and epigenetics are tightly coupled cellular processes. It is arguable that of all the described cancer hallmarks, dysregulated cellular energetics and epigenetics are the most tightly coregulated. Cellular metabolic states regulate and drive epigenetic changes while also being capable of influencing, if not driving, epigenetic reprogramming. Conversely, epigenetic changes can drive altered and compensatory metabolic states. Cancer cells meticulously modify and control each of these two linked cellular processes in order to maintain their tumorigenic potential and capacity. This review aims to explore the interplay between these two processes and discuss how each affects the other, driving and enhancing tumorigenic states in certain contexts.

Cardiac diseases are the foremost cause of morbidity and mortality worldwide. The heart has limited regenerative potential; therefore, lost cardiac tissue cannot be replenished after cardiac injury. Conventional therapies are unable to restore functional cardiac tissue. In recent decades, much attention has been paid to regenerative medicine to overcome this issue. Direct reprogramming is a promising therapeutic approach in regenerative cardiac medicine that has the potential to provide in situ cardiac regeneration. It consists of direct cell fate conversion of one cell type into another, avoiding transition through an intermediary pluripotent state. In injured cardiac tissue, this strategy directs transdifferentiation of resident non-myocyte cells (NMCs) into mature functional cardiac cells that help to restore the native tissue. Over the years, developments in reprogramming methods have suggested that regulation of several intrinsic factors in NMCs can help to achieve in situ direct cardiac reprogramming. Among NMCs, endogenous cardiac fibroblasts have been studied for their potential to be directly reprogrammed into both induced cardiomyocytes and induced cardiac progenitor cells, while pericytes can transdifferentiate towards endothelial cells and smooth muscle cells. This strategy has been indicated to improve heart function and reduce fibrosis after cardiac injury in preclinical models. This review summarizes the recent updates and progress in direct cardiac reprogramming of resident NMCs for in situ cardiac regeneration.

The monoamine neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has important functions both in the neural system and during embryonic development in mammals. In this study, we set out to investigate whether and how endogenous serotonin affects reprogramming to pluripotency. As serotonin is synthesized from tryptophan by the rate limiting enzymes tryptophan hydroxylase-1 and -2 (TPH1 and TPH2), we have assessed the reprogramming of TPH1- and/or TPH2-deficient mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells (iPSCs). The reprogramming of the double mutant MEFs showed a dramatic increase in the efficiency of iPSC generation. In contrast, ectopic expression of TPH2 alone or in conjunction with TPH1 reverted the rate of reprogramming of the double mutant MEFs to the wild-type level and besides, TPH2 overexpression significantly suppressed reprogramming of wild-type MEFs. Our data thus suggest a negative role of serotonin biosynthesis in the reprogramming of somatic cells to a pluripotent state.

Central nervous system (CNS) repair after injury or disease remains an unresolved problem in neurobiology research and an unmet medical need. Directly reprogramming or converting astrocytes to neurons (AtN) in adult animals has been investigated as a potential strategy to facilitate brain and spinal cord recovery and advance fundamental biology. Conceptually, AtN strategies rely on forced expression or repression of lineage-specific transcription factors to make endogenous astrocytes become "induced neurons" (iNs), presumably without re-entering any pluripotent or multipotent states. The AtN-derived cells have been reported to manifest certain neuronal functions in vivo. However, this approach has raised many new questions and alternative explanations regarding the biological features of the end products (e.g., iNs versus neuron-like cells, neural functional changes, etc.), developmental biology underpinnings, and neurobiological essentials. For this paper per se, we proposed to draw an unconventional distinction between direct cell conversion and direct cell reprogramming, relative to somatic nuclear transfer, based on the experimental methods utilized to initiate the transformation process, aiming to promote a more in-depth mechanistic exploration. Moreover, we have summarized the current tactics employed for AtN induction, comparisons between the bench endeavors concerning outcome tangibility, and discussion of the issues of published AtN protocols. Lastly, the urgency to clearly define/devise the theoretical frameworks, cell biological bases, and bench specifics to experimentally validate primary data of AtN studies was highlighted.

G-protein-coupled receptors (GPCRs) are the largest family of cell surface receptors. They modulate key physiological functions and are required in diverse developmental processes including embryogenesis, but their role in pluripotency maintenance and acquisition during the reprogramming towards hiPSCs draws little attention. Meanwhile, it is known that more than 106 GPCRs are overexpressed in human pluripotent stem cells (hPSCs). Previously, to identify novel effectors of reprogramming, we performed a high-throughput RNA interference (RNAi) screening assay and identified adhesion GPCR, GPR123, as a potential reprogramming effector. Its role has not been explored before. Herein, by employing GPR123 RNAi we addressed the role of GPR123 for hPSCs. The suppression of GPR123 in hPSCs leads to the loss of pluripotency and differentiation, impacted colony morphology, accumulation of cells at the G2 phase of the cell cycle, and absence of the scratch closure. Application of the GPR123 RNAi at the initiation stage of reprogramming leads to a decrease in the percentage of the "true" hiPSC colonies, a drop in E-cadherin expression, a decrease in the percentage of NANOG+ nuclei, and the absence of actin cytoskeleton remodeling. Together this leads to the absence of the alkaline-phosphatase-positive hiPSCs colonies on the 18th day of the reprogramming process. Overall, these data indicate for the first time the essential role of GPR123 in the maintenance and acquisition of pluripotency.

Cellular metabolism governs the signaling that supports physiological mechanisms and homeostasis in an individual, including neuronal transmission, wound healing, and circadian clock manipulation. Various factors have been linked to abnormal metabolic reprogramming, including gene mutations, epigenetic modifications, altered protein epitopes, and their involvement in the development of disease, including cancer. The presence of multiple distinct hallmarks and the resulting cellular reprogramming process have gradually revealed that these metabolism-related molecules may be able to be used to track or prevent the progression of cancer. Consequently, translational medicines have been developed using metabolic substrates, precursors, and other products depending on their biochemical mechanism of action. It is important to note that these metabolic analogs can also be used for imaging and therapeutic purposes in addition to competing for metabolic functions. In particular, due to their isotopic labeling, these compounds may also be used to localize and visualize tumor cells after uptake. In this review, the current development status, applicability, and limitations of compounds targeting metabolic reprogramming are described, as well as the imaging platforms that are most suitable for each compound and the types of cancer to which they are most appropriate.