Neurodegenerative diseases including Alzheimer's and Parkinson's disease are age-related disorders which severely impact quality of life and impose significant societal burdens. Cellular senescence is a critical factor in these disorders, contributing to their onset and progression by promoting permanent cell cycle arrest and reducing cellular function, affecting various types of cells in brain. Recent advancements in regenerative medicine have highlighted "R3" strategies-rejuvenation, regeneration, and replacement-as promising therapeutic approaches for neurodegeneration. This review aims to critically analyze the role of cellular senescence in neurodegenerative diseases and organizes therapeutic approaches within the R3 regenerative medicine paradigm. Specifically, we examine stem cell therapy, direct lineage reprogramming, and partial reprogramming in the context of R3, emphasizing how these interventions mitigate cellular senescence and counteracting aging-related neurodegeneration. Ultimately, this review seeks to provide insights into the complex interplay between cellular senescence and neurodegeneration while highlighting the promise of cell-based regenerative strategies to address these debilitating conditions.

Phenotypically unstable Schwann cell-like cells (SCLCs), derived from mesenchymal stem cells (MSCs) require intercellular contact-mediated cues for Schwann cell (SCs)-fate commitment. Although rat dorsal root ganglion (DRG) neurons provide contact-mediated signals for the conversion of SCLCs into fate-committed SCs, the use of animal cells is clinically unacceptable. To overcome this problem, we previously acquired human induced pluripotent stem cell-derived sensory neurons (hiPSC-dSNs) as surrogates of rat DRG neurons that committed rat bone marrow SCLCs to the SC fate. In this study, we explored whether hiPSC-dSNs could mimic rat DRG neuron effects to obtain fate-committed SCs from hBMSC-derived SCLCs.

hiPSCs were induced into hiPSC-dSNs using a specific chemical small molecule combination. hBMSCs were induced into hBMSC-derived SCLCs in a specific culture medium and then co-cultured with hiPSC-dSNs to generate SCs. The identity of hBMSC-derived SCs (hBMSC-dSCs) was examined by immunofluorescence, western bolt, electronic microscopy, and RNA-seq. Immunofluorescence was also used to detect the myelination capacity. Enzyme-linked immunosorbent assay and neurite outgrowth analysis were used to test the secretion of neurotrophic factors.

The hBMSC-dSCs exhibited bi-/tri-polar morphology of SCs and maintained the expression of the SC markers S100, p75NTR, p0, GFAP, and Sox10, even after withdrawing the glia-inducing factors or hiPSC-dSNs. Electronic microscopy and RNA-seq analysis provided evidence that hBMSC-dSCs were similar to the original human SCs in terms of their function and a variety of characteristics. Furthermore, these cells formed MBP-positive segments and secreted neurotrophic factors to facilitate the neurite outgrowth of Neuro2A.

These results demonstrated that phenotypically stable and functionally mature hBMSC-dSCs were generated efficiently via the co-culture of hiPSC-dSNs and hBMSC-derived SCLCs. Our findings may provide a promising protocol through which stable and fully developed hBMSC-dSCs can be used for transplantation to regenerate myelin sheath.

Dermal papilla cells (DPCs) are a crucial subset of mesenchymal cells in the skin responsible for regulating hair follicle development and growth, making them invaluable for cell-based therapies targeting hair loss. However, obtaining sufficient DPCs with potent hair-inducing abilities remains a persistent challenge. In this study, the Food and Drug Administration (FDA)-approved drug library was utilized to screen small molecules capable of reprogramming readily accessible human skin fibroblasts into functional DPCs. In the initial screening, five candidate small molecules were identified from a pool of 1,817 compounds, and the small molecule peficitinib was further identified by the further hair follicle regeneration experiments. Following peficitinib treatment, fibroblasts derived from primary human foreskin and scalp exhibited the capability to induce hair growth and possessed a molecular profile highly similar to that of primary DPCs. We refer to these cells as dermal papilla cell-like cells (DPC-LCs). Furthermore, transcriptome analysis showed that the wingless/integrated (Wnt) signaling pathway and the transforming growth factor β (TGF-β) signaling pathway, both of which play crucial roles in hair follicle morphogenesis, are upregulated and enriched in these DPC-LCs. These functional DPC-LCs offer a promising avenue for obtaining a plentiful supply of hair-inducing cells, thereby advancing the development of therapeutic strategies for hair loss treatment.

Cell fate changes play a crucial role in the processes of natural development, disease progression, and the efficacy of therapeutic interventions. The definition of the various types of cell fate changes, including cell expansion, differentiation, transdifferentiation, dedifferentiation, reprogramming, and state transitions, represents a complex and evolving field of research known as cell lineage tracing. This review will systematically introduce the research history and progress in this field, which can be broadly divided into two parts: prospective tracing and retrospective tracing. The initial section encompasses an array of methodologies pertaining to isotope labeling, transient fluorescent tracers, non-fluorescent transient tracers, non-fluorescent genetic markers, fluorescent protein, genetic marker delivery, genetic recombination, exogenous DNA barcodes, CRISPR-Cas9 mediated DNA barcodes, and base editor-mediated DNA barcodes. The second part of the review covers genetic mosaicism, genomic DNA alteration, TCR/BCR, DNA methylation, and mitochondrial DNA mutation. In the final section, we will address the principal challenges and prospective avenues of enquiry in the field of cell lineage tracing, with a particular focus on the sequencing techniques and mathematical models pertinent to single-cell genetic lineage tracing, and the value of pursuing a more comprehensive investigation at both the spatial and temporal levels in the study of cell lineage tracing.

Direct reprogramming has garnered considerable attention due to its capacity to directly convert differentiated cells into desired cells. Fibroblasts are frequently employed in reprogramming studies due to their abundance and accessibility. However, they are also the key drivers in the progression of fibrosis, a pathological condition characterized by excessive extracellular matrix deposition and tissue scarring. Furthermore, the initial stage of reprogramming typically involves deactivating fibrotic pathways. Hence, direct reprogramming offers a valuable method to regenerate target cells for tissue repair while simultaneously reducing fibrotic tendencies. Understanding the link between reprogramming and fibrosis could help develop effective strategies to treat damaged tissue with a potential risk of fibrosis. This review summarizes the advances in direct reprogramming and reveals their anti-fibrosis effects in various organs such as the heart, liver, and skin. Furthermore, we dissect the mechanisms of reprogramming influenced by fibrotic molecules including TGF-β signaling, mechanical signaling, inflammation signaling, epigenetic modifiers, and metabolic regulators. Innovative methods for fibroblast reprogramming like small molecules, CRISPRa, modified mRNA, and the challenges of cellular heterogeneity and senescence faced by in vivo direct reprogramming, are also discussed.

The nervous system is the most complex system in the human body, and the normal development of the central nervous system (CNS) is essential for maintaining the healthy life activities of the individual. CNS development requires the orchestration of multiple internal or external or direct or indirect factors to regulate neural stem cell fate specification. Here, we provide a broad overview of the regulatory system of nerve cell fate decisions and discuss the latest technological approaches to achieve neural regeneration. Understanding the CNS development and regeneration mechanisms has shifted the paradigm from traditional experiments to high-throughput sequencing.

Despite tremendous advances in antiretroviral therapy (ART) against HIV-1 infections, no cure or vaccination is available. Therefore, discovering novel therapeutic strategies remains an urgent need. In that sense, miRNAs and miRNA therapeutics have moved intensively into the focus of recent HIV-1-related investigations. A strong reciprocal interdependence has been demonstrated between HIV-1 infection and changes of the intrinsic cellular miRNA milieu. This interrelationship may direct potential alterations of the host cells' environment beneficial for the virus or its suppression of replication. Whether this tightly balanced and controlled battle can be exploited therapeutically remains to be further addressed. In this context, the fluoroquinolone antibiotic Enoxacin has been demonstrated as a potent modulator of miRNA processing. Here, we test the hypothesis that this applies also to selected HIV-1-related miRNAs.

We studied the effect of Enoxacin on HIV-1 replication coupled with miRNA qRT-PCR analysis of HIV-1-related miRNAs in CEM-SS and MT-4 T-cells. The effects of miRNA mimic transfections combined with Enoxacin treatment on HIV-1 replication were assessed. Finally, we employed an in vitro DICER1 cleavage assay to study the effects of Enoxacin on a pro-HIV-1 miRNA hsa-miR-132 processing.

We established that Enoxacin, but not the structurally similar compound nalidixic acid, exhibits strong anti-HIV-1 effects in the T-cell line CEM-SS, but not MT-4. We provide experimental data that this effect of Enoxacin is partly attributed to the specific downregulation of mature hsa-miR-132-3p, but not other tested pro- or anti-HIV-1 miRNAs, which is likely due to affecting DICER1 processing.

Our findings show an anti-retroviral activity of Enoxacin at least in part by downregulation of hsa-miR-132-3p, which may be relevant for future antiviral therapeutic applications by modulation of the RNA interference pathway.

Temporal control of transgenes has advanced biomedical interventions, including in vivo reprogramming, often utilizing the doxycycline (Dox)-mediated Tet-ON system. Here, we developed the Dox-mediated Tet-ON or complementary Tet-OFF counterpart to thoroughly investigate spatial and temporal transgene regulation in adult tissues, revealing inherent limitations and unexpected capabilities of each system. In stark contrast with the Tet-ON system, which was effective only in particular tissues and cell types, primarily epithelial cells, the Tet-OFF system proved capable of gene induction across diverse cell types. Despite the drawback of the Tet-OFF system in inducibility and tunability identified in our study, we demonstrated that use of tetracycline (Tc) effectively addresses these issues, possibly through its pharmacologic properties. Our data suggest that the Tc-mediated Tet-OFF system not only enables more versatile control of transgene expression but also offers a more biocompatible alternative for in vivo applications such as tissue regeneration and organismal rejuvenation.

Direct cardiac reprogramming of fibroblasts into induced cardiomyocytes (iCMs) can be achieved by ectopic expression of cardiac transcription factors (TFs) via viral vectors. However, risks like genomic mutations, viral toxicity, and immune response limited its clinical application. Transactivation of endogenous TFs emerges as an alternative approach that may partially mitigate some of the risks. In this study, we utilized a modified CRISPRa/dCas9 strategy to transactivate endogenous reprogramming factors MEF2C, GATA4, and TBX5 (MGT) to induce iCMs from both mouse and human fibroblasts. We identified single-guide RNAs (sgRNAs) targeting promoters and enhancers of the TFs capable of activating various degrees of endogenous gene expression. CRISPRa-mediated Gata4 activation, combined with exogenous expression of Mef2c and Tbx5, successfully converted fibroblasts into iCMs. Despite extensive sgRNA screening, transactivation of Mef2c and Tbx5 via CRISPRa remained less effective, potentially due to de novo epigenetic barriers. While future work and refined technologies are needed to determine whether cardiac reprogramming could be achieved solely through CRISPRa activation of endogenous factors, our findings provide proof of concept that reliance on exogenous TFs for reprogramming can be reduced through CRISPRa-mediated activation of endogenous factors, particularly Gata4, offering a novel strategy to convert scar-forming fibroblasts into iCMs for regenerative purposes.

Tissue nanotransfection (TNT) topically delivers Etv2, Foxc2, and Fli1 (EFF) plasmids increasing vasculogenic fibroblasts (VF) and promoting vascularization in ischemic murine skin. Human dermal fibroblasts respond to EFF nanoelectroporation with elevated expression of endothelial genes in vitro, which is linked to increased ten-eleven translocase 1/2/3 (TET) expression. Single cell RNA sequencing dependent validation of VF induction reveals a TET-dependent transcript signature. TNTEFF also induces TET expression in vivo, and fibroblast-specific EFF overexpression leads to VF-transition, with TET-activation correlating with higher 5-hydroxymethylcytosine (5-hmC) levels in VF. VF emergence requires TET-dependent demethylation of endothelial genes in vivo, enhancing VF abundance and restoring perfusion in diabetic ischemic limbs. TNTEFF improves perfusion and wound closure in diabetic mice, while increasing VF in cultured human skin explants. Suppressed in diabetes, TET1/2/3 play a critical role in TNT-mediated VF formation which supports de novo blood vessel development to rescue diabetic ischemic tissue.

Immunotherapy can lead to long-term survival for some cancer patients, yet generalized success has been hampered by insufficient antigen presentation and exclusion of immunogenic cells from the tumor microenvironment. Here, we developed an approach to reprogram tumor cells in vivo by adenoviral delivery of the transcription factors PU.1, IRF8, and BATF3, which enabled them to present antigens as type 1 conventional dendritic cells. Reprogrammed tumor cells remodeled their tumor microenvironment, recruited, and expanded polyclonal cytotoxic T cells; induced tumor regressions; and established long-term systemic immunity in multiple mouse melanoma models. In human tumor spheroids and xenografts, reprogramming to immunogenic dendritic-like cells progressed independently of immunosuppression, which usually limits immunotherapy. Our study paves the way for human clinical trials of in vivo immune cell reprogramming for cancer immunotherapy.

During the last decade, artificial intelligence (AI) has increasingly been applied in biophysics and related fields, including cellular engineering and reprogramming, offering novel approaches to understand, manipulate, and control cellular function. The potential of AI lies in its ability to analyze complex datasets and generate predictive models. AI algorithms can process large amounts of data from single-cell genomics and multiomic technologies, allowing researchers to gain mechanistic insights into the control of cell identity and function. By integrating and interpreting these complex datasets, AI can help identify key molecular events and regulatory pathways involved in cellular reprogramming. This knowledge can inform the design of precision engineering strategies, such as the development of new transcription factor and signaling molecule cocktails, to manipulate cell identity and drive authentic cell fate across lineage boundaries. Furthermore, when used in combination with computational methods, AI can accelerate and improve the analysis and understanding of the intricate relationships between genes, proteins, and cellular processes. In this review article, we explore the current state of AI applications in biophysics with a specific focus on cellular engineering and reprogramming. Then, we showcase a couple of recent applications where we combined machine learning with experimental and computational techniques. Finally, we briefly discuss the challenges and prospects of AI in cellular engineering and reprogramming, emphasizing the potential of these technologies to revolutionize our ability to engineer cells for a variety of applications, from disease modeling and drug discovery to regenerative medicine and biomanufacturing.

The limited regenerative capacity of damaged neurons in adult mammals severely restricts neural repair. Although stem cell transplantation is promising, its clinical application remains challenging. Direct reprogramming, which utilizes cell plasticity to regenerate neurons, is an emerging alternative approach.

We utilized primary postnatal cortical astrocytes for reprogramming induced neurons (iNs) through the viral-mediated overexpression of the transcription factors Ngn2 and Pax6 (NP). Fluorescence-activated cell sorting (FACS) was used to enrich successfully transfected cells, followed by single-cell RNA sequencing (scRNA-seq) using the 10 × Genomics platform for comprehensive transcriptomic analysis.

The scRNA-seq revealed that NP overexpression led to the differentiation of astrocytes into iNs, with percentages of 36% and 39.3% on days 4 and 7 posttransduction, respectively. CytoTRACE predicted the developmental sequence, identifying astrocytes as the reprogramming starting point. Trajectory analysis depicted the dynamic changes in gene expression during the astrocyte-to-iN transition.

This study elucidates the molecular dynamics underlying astrocyte reprogramming into iNs, revealing key genes and pathways involved in this process. Our research contributes novel insights into the molecular mechanisms of NP-mediated reprogramming, suggesting avenues for optimizing the efficiency of the reprogramming process.

Recent research has reevaluated the traditional view of cancer's linear progression and recurrence by introducing cellular reprogramming a process in which cancer cells can their state under certain conditions. This change is driven by a combination of genetic and epigenetic factors, with pivotal roles played by key genes, and pathways, notably Wnt and Notch. The complexity of cancer's behavior is further influenced by factors such as the epithelial-mesenchymal transition (EMT) and therapy-induced stress, both of which are significant contributors to cancer recurrence. In this context bibliometric analysis emerges as a crucial tool for evaluating the impacts and trends within scientific literature. Our study utilized bibliometrics to analysis the role of cellular reprogramming oncology over the past two decades, highlighting its potential to improve cancer treatment outcomes. In conducting this analysis, we searched for literature search on cellular reprogramming (CR) in the Web of Science database, covering the years 2002-2022. We employed visualization tools like Citespace, VOSviewer, and Bibliometrix to analyze the collected data resulting in a dataset of 3102 articles. The United States and China emerged as leading contributors to this field, with the University of Texas MD Anderson Cancer Center being the most prolific institution. Menendez was the most influential scholar in this research domain. Cancers was the journal with the most publications on this subject. The most local-cited document was the article titled "Hallmarks of Cancer: The Next Generation". A comprehensive analysis has been conducted based on keywords and cited references. In recent years, the research emphasis has shifted to "extracellular vesicles," "cancer therapy," and "cellular plasticity". Therefore, this analysis uses bibliometrics to chart cutting-edge progress in cancer's cellular reprogramming, aiding experts to quickly understand and innovate in this crucial area.

The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease treatments. Clustered regularly interspaced short palindromic repeats (CRISPR) activator (CRISPRa) is a powerful tool that can conveniently and efficiently activate the expression of multiple endogenous genes simultaneously, providing a new strategy for cell fate determination. The main purpose of this study is to explore the feasibility of applying CRISPRa for hepatocyte reprogramming and its application in the treatment of mouse liver fibrosis.

The differentiation of mouse embryonic fibroblasts (MEFs) into functional induced hepatocyte-like cells (iHeps) was achieved by utilizing the CRISPRa synergistic activation mediator (SAM) system, which drove the combined expression of three endogenous transcription factors- Gata4, Foxa3 , and Hnf1a -or alternatively, the expression of two transcription factors, Gata4 and Foxa3 . In vivo , we injected adeno-associated virus serotype 6 (AAV6) carrying the CRISPRa SAM system into liver fibrotic Col1a1-CreER ; Cas9fl/fl mice, effectively activating the expression of endogenous Gata4 and Foxa3 in fibroblasts. The endogenous transcriptional activation of genes was confirmed using real-time quantitative polymerase chain reaction (RT-qPCR) and RNA-seq, and the morphology and characteristics of the induced hepatocytes were observed through microscopy. The level of hepatocyte reprogramming in vivo is detected by immunofluorescence staining, while the improvement of liver fibrosis is evaluated through Sirius red staining, alpha-smooth muscle actin (α-SMA) immunofluorescence staining, and blood alanine aminotransferase (ALT) examination.

Activation of only two factors, Gata4 and Foxa3 , via CRISPRa was sufficient to successfully induce the transformation of MEFs into iHeps. These iHeps could be expanded in vitro and displayed functional characteristics similar to those of mature hepatocytes, such as drug metabolism and glycogen storage. Additionally, AAV6-based delivery of the CRISPRa SAM system effectively induced the hepatic reprogramming from fibroblasts in mice with live fibrosis. After 8 weeks of induction, the reprogrammed hepatocytes comprised 0.87% of the total hepatocyte population in the mice, significantly reducing liver fibrosis.

CRISPRa-induced hepatocyte reprogramming may be a promising strategy for generating functional hepatocytes and treating liver fibrosis caused by hepatic diseases.

The manipulation of cell identity by reprograming holds immense potential in regenerative medicine, but is often limited by the inefficient acquisition of fully functional cells. This problem can potentially be resolved by better understanding the reprogramming process using in vivo genetic models, which are currently scarce. Here we report that both enterocytes (ECs) and enteroendocrine cells (EEs) in adult Drosophila midgut show a surprising degree of cell plasticity. Depleting the transcription factor Tramtrack in the differentiated ECs can initiate Prospero-mediated cell transdifferentiation, leading to EE-like cells. On the other hand, depletion of Prospero in the differentiated EEs can lead to the loss of EE-specific transcription programs and the gain of intestinal progenitor cell identity, allowing cell cycle re-entry or differentiation into ECs. We find that intestinal progenitor cells, ECs, and EEs have a similar chromatin accessibility profile, supporting the concept that cell plasticity is enabled by pre-existing chromatin accessibility with switchable transcription programs. Further genetic analysis with this system reveals that the NuRD chromatin remodeling complex, cell lineage confliction, and age act as barriers to EC-to-EE transdifferentiation. The establishment of this genetically tractable in vivo model should facilitate mechanistic investigation of cell plasticity at the molecular and genetic level.

Cellular plasticity can occur naturally in an organism and is considered an adapting mechanism during the developmental stage. However, abnormal cellular plasticity is observed in different diseased conditions, including cancer. Cancer cell plasticity triggers the stimuli of epithelial-mesenchymal transition (EMT), abnormal epigenetic changes, expression of stem cell factors and implicated signaling pathways, etc., and helps in the maintenance of CSC phenotype. Conversely, CSC maintains the cancer cell plasticity, EMT, and epigenetic plasticity. EMT contributes to increased cell migration and greater diversity within tumors, while epigenetic changes, stem cell factors (OCT4, NANOG, and SOX2), and various signaling pathways allow cancer cells to maintain various phenotypes, giving rise to intra- and inter-tumoral heterogeneity. The intricate relationships between cancer cell plasticity and stem cell factors help the tumor cells adopt drug-tolerant states, evade senescence, and successfully acquire drug resistance with treatment dismissal. Inhibiting molecules/signaling pathways involved in promoting CSCs, cellular plasticity, EMT, and epigenetic plasticity might be helpful for successful cancer therapy management. This review discussed the role of cellular plasticity, EMT, and stem cell factors in tumor initiation, progression, reprogramming, and therapy resistance. Finally, we discussed how the intervention in this axis will help better manage cancers and improve patient survivability.

Aging and age-associated disease are a major medical and societal burden in need of effective treatments. Cellular reprogramming is a biological process capable of modulating cell fate and cellular age. Harnessing the rejuvenating benefits without altering cell identity via partial cellular reprogramming has emerged as a novel translational strategy with therapeutic potential and strong commercial interests. Here, we explore the aging-related benefits of partial cellular reprogramming while examining limitations and future directions for the field.

In contrast to prior findings that have illustrated the conversion of non-neuronal cells into functional neurons through the specific targeting of polypyrimidine tract-binding protein 1 (PTBP1), accumulated evidence suggests the impracticality of inducing neuronal transdifferentiation through suppressing PTBP1 expression in pathological circumstances. Therefore, the present study explored the effect of knocking down PTBP1 under physiological conditions on the transdifferentiation of mouse hippocampal neuron HT22 cells and mouse astrocyte (MA) cells. A total of 20 µM negative control small interfering (si)RNA and siRNA targeting PTBP1 were transfected into HT22 and MA cells using Lipo8000™ for 3 and 5 days, respectively. The expression of early neuronal marker βIII-Tubulin and mature neuronal markers NeuN and microtubule-associated protein 2 (MAP2) were detected using western blotting. In addition, βIII-tubulin, NeuN and MAP2 were labeled with immunofluorescence staining to evaluate neuronal cell differentiation in response to PTBP1 downregulation. Under physiological conditions, no significant changes in the expression of βIII-Tubulin, NeuN and MAP2 were found after 3 and 5 days of knockdown of PTBP1 protein in both HT22 and MA cells. In addition, the immunofluorescence staining results showed no apparent transdifferentiation in maker levels and morphology. The results suggested that the knockdown of PTBP1 failed to induce neuronal differentiation under physiological conditions.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by continuous and selective degeneration or death of dopamine neurons in the midbrain, leading to dysfunction of the nigrostriatal neural circuits. Current clinical treatments for PD include drug treatment and surgery, which provide short-term relief of symptoms but are associated with many side effects and cannot reverse the progression of PD. Pluripotent/multipotent stem cells possess a self-renewal capacity and the potential to differentiate into dopaminergic neurons. Transplantation of pluripotent/multipotent stem cells or dopaminergic neurons derived from these cells is a promising strategy for the complete repair of damaged neural circuits in PD. This article reviews and summarizes the current preclinical/clinical treatments for PD, their efficacies, and the advantages/disadvantages of various stem cells, including pluripotent and multipotent stem cells, to provide a detailed overview of how these cells can be applied in the treatment of PD, as well as the challenges and bottlenecks that need to be overcome in future translational studies.

Diabetic wound healing presents a significant clinical challenge due to the interplay of systemic metabolic disturbances and local inflammation, which hinder the healing process. Macrophages undergo a phenotypic shift from M1 to M2 during wound healing, a transition pivotal for effective tissue repair. However, in diabetic wounds, the microenvironment disrupts this phenotypic polarization, perpetuating inflammation, and impeding healing. Reprograming macrophages to restore their M2 phenotype offers a potential avenue for modulating the wound immune microenvironment and promoting healing. This review elucidates the mechanisms underlying impaired macrophage polarization toward the M2 phenotype in diabetic wounds and discusses novel strategies, including epigenetic and metabolic interventions, to promote macrophage conversion to M2. Hydrogels, with their hydrated 3D cross-linked structure, closely resemble the physiological extracellular matrix and offer advantageous properties such as biocompatibility, tunability, and versatility. These characteristics make hydrogels promising candidates for developing immunomodulatory materials aimed at addressing diabetic wounds. Understanding the role of hydrogels in immunotherapy, particularly in the context of macrophage reprograming, is essential for the development of advanced wound care solutions. This review also highlights recent advancements in immunotherapeutic hydrogels as a step toward precise and effective treatments for diabetic wounds.

Enhanced regenerative therapeutic strategies are required to treat intractable ischemic heart disease.

Since the discovery of putative endothelial progenitor cells (EPCs) in 1997, many studies have focused on their extraction, ex vivo processing, and autotransplantation under ischemic conditions. Nonetheless, numerous randomized clinical trials involving thousands of patients have yielded only marginal treatment effects, highlighting the need for advances regarding insufficient dosage and complex ex vivo processing. The prevailing paradigm of cellular differentiation highlights the potential of direct cellular reprogramming, which paves the way for in situ reprogramming. In situ reprogramming holds the promise of significantly enhancing current therapeutic strategies, yet its success hinges on the precise targeting of candidate cells for reprogramming. In this context, the spleen emerges as a pivotal "in situ reprogramming hub," owing to its dual function as both a principal site for nanoparticle distribution and a significant reservoir of putative EPCs. The in situ reprogramming of splenic EPCs offers a potential solution to overcome critical challenges, including the aforementioned insufficient dosage and complex ex vivo processing.

This review explores the latest advancements in EPC therapy and in situ reprogramming, spotlighting a pioneering study that integrates those two strategies with a specific focus on the spleen. Such an innovative approach will potentially herald a new era of regenerative therapy for ischemic heart disease.

Direct reprogramming of somatic cells into functional cells still faces major limitations in terms of efficiency and achieving functional maturity of the reprogramed cells. While different approaches have been developed commonly based on exploiting biochemical signals, introducing appropriate mechanical cues that stimulate the reprogramming process is rarely reported. In this study, collagen-coated polyacrylamide (PAM) hydrogels with stiffness close to that of collagenous bone (40 kPa) were adopted to augment the direct reprogramming process of mouse fibroblasts to osteoblastic-like cells. The results suggested that culturing cells on a hydrogel substrate enhanced the overexpression of osteogenic transcription factors using nonviral vectors and improved the yield of osteoblast-like cells. Particularly, a synergistic effect on achieving osteogenic functionality has been observed for the mechanical cues and overexpression of transcriptional factors, leading to enhanced osteogenic transformation and production of bone mineral matrix. Animal experiments suggested that reprogramed cells generated on matrix hydrogels accelerated bone regeneration and stimulated ectopic osteogenesis. Mechanism analysis suggested the critical involvement of actomyosin contraction and mechanical signal-mediated pathways like the RhoA-ROCK pathway, leading to a synergistic effect on the key transcriptional processes, including chromatin remodeling, nuclear translocation, and epigenetic transition. This study provides insights into the mechanical cue-enhanced direct reprogramming and cell therapy.

In prolonged liver injury, hepatocytes undergo partial identity loss with decreased regenerative capacity, resulting in liver failure. Here, we identified a five compound (5C) combination that could restore hepatocyte identity and reverse the damage-associated phenotype (e.g., dysfunction, senescence, epithelial to mesenchymal transition, growth arrest, and pro-inflammatory gene expression) in damaged hepatocytes (dHeps) from CCl4-induced mice with chronic liver injury, resembling a direct chemical reprogramming approach. Systemic administration of 5C in mice with chronic liver injury promoted hepatocyte regeneration, improved liver function, and ameliorated liver fibrosis. The hepatocyte-associated transcriptional networks were reestablished with chemical treatment as revealed by motif analysis of ATAC-seq, and a hepatocyte-enriched transcription factor, Foxa2, was found to be essential for hepatocyte revitalization. Overall, our findings indicate that the phenotype and transcriptional program of dHeps can be reprogrammed to generate functional and regenerative hepatocytes by using only small molecules, as an alternative approach to liver repair and regeneration.

Cell plasticity theoretically extends to all possible cell types, but naturally decreases as cells differentiate, whereas injury-repair re-engages the developmental plasticity. Here we show that the lung alveolar type 2 (AT2)-specific transcription factor (TF), CEBPA, restricts AT2 cell plasticity in the mouse lung. AT2 cells undergo transcriptional and epigenetic maturation postnatally. Without CEBPA, both neonatal and mature AT2 cells reduce the AT2 program, but only the former reactivate the SOX9 progenitor program. Sendai virus infection bestows mature AT2 cells with neonatal plasticity where Cebpa mutant, but not wild type, AT2 cells express SOX9, as well as more readily proliferate and form KRT8/CLDN4+ transitional cells. CEBPA promotes the AT2 program by recruiting the lung lineage TF NKX2-1. The temporal change in CEBPA-dependent plasticity reflects AT2 cell developmental history. The ontogeny of AT2 cell plasticity and its transcriptional and epigenetic mechanisms have implications in lung regeneration and cancer.

Regenerative medicine is a highly advanced medical field that aims to restore tissues and organs lost due to diseases and injury using a person's own cells or those of others. Direct cellular reprogramming is a promising technology that can directly induce cell-fate conversion from terminally differentiated cells to other cell types and is expected to play a pivotal role in applications in regenerative medicine. The induction of direct cellular reprogramming requires one or more master transcription factors with the potential to reconstitute cell type-specific transcription factor networks. The set of master transcription factors may contain unique transcription factors called pioneer factors that can open compacted chromatin structures and drive the transcriptional activation of target genes. Therefore, pioneer factors may play a central role in direct cellular reprogramming. However, our understanding of the molecular mechanisms by which pioneer factors induce cell-fate conversion is still limited. This review briefly summarizes the outcomes of recent findings and discusses future perspectives, focusing on the role of pioneer factors in direct cellular reprogramming.

Cell plasticity theoretically extends to all possible cell types, but naturally decreases as cells differentiate, whereas injury-repair re-engages the developmental plasticity. Here we show that the lung alveolar type 2 (AT2)-specific transcription factor (TF), CEBPA, restricts AT2 cell plasticity in the mouse lung. AT2 cells undergo transcriptional and epigenetic maturation postnatally. Without CEBPA, both neonatal and mature AT2 cells reduce the AT2 program, but only the former reactivate the SOX9 progenitor program. Sendai virus infection bestows mature AT2 cells with neonatal plasticity where Cebpa mutant, but not wild type, AT2 cells express SOX9, as well as more readily proliferate and form KRT8/CLDN4+ transitional cells. CEBPA promotes the AT2 program by recruiting the lung lineage TF NKX2-1. The temporal change in CEBPA-dependent plasticity reflects AT2 cell developmental history. The ontogeny of AT2 cell plasticity and its transcriptional and epigenetic mechanisms have implications in lung regeneration and cancer.

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.

Stroke is the leading cause of severe long-term disability. Cell therapy has recently emerged as an approach to facilitate functional recovery in stroke. Although administration of peripheral blood mononuclear cells preconditioned by oxygen-glucose deprivation (OGD-PBMCs) has been shown to be a therapeutic strategy for ischemic stroke, the recovery mechanisms remain largely unknown. We hypothesised that cell-cell communications within PBMCs and between PBMCs and resident cells are necessary for a polarising protective phenotype. Here, we investigated the therapeutic mechanisms underlying the effects of OGD-PBMCs through the secretome. We compared levels of transcriptomes, cytokines, and exosomal microRNA in human PBMCs by RNA sequences, Luminex assay, flow cytometric analysis, and western blotting under normoxic and OGD conditions. We also performed microscopic analyses to assess the identification of remodelling factor-positive cells and evaluate angiogenesis, axonal outgrowth, and functional recovery by blinded examination by administration of OGD-PBMCs after ischemic stroke in Sprague-Dawley rats. We found that the therapeutic potential of OGD-PBMCs was mediated by a polarised protective state through decreased levels of exosomal miR-155-5p, and upregulation of vascular endothelial growth factor and a pluripotent stem cell marker stage-specific embryonic antigen-3 through the hypoxia-inducible factor-1α axis. After administration of OGD-PBMCs, microenvironment changes in resident microglia by the secretome promoted angiogenesis and axonal outgrowth, resulting in functional recovery after cerebral ischemia. Our findings revealed the mechanisms underlying the refinement of the neurovascular unit by secretome-mediated cell-cell communications through reduction of miR-155-5p from OGD-PBMCs, highlighting the therapeutic potential carrier of this approach against ischemic stroke.

Forced expression of a specific set of transcription factors can reprogram terminally differentiated cells and convert them into induced pluripotent stem cells that correspond to cells in the inner cell mass of the developing embryo. It is now recognized that the scope of the reprogramming factors extends far beyond the stem cell biology. Studies using mouse models demonstrated that the induction of the reprogramming factors promotes cellular reprogramming in vivo. Closer inspection of these mice has revealed that expression of the reprogramming factors results in unique consequences that are not seen when cells are reprogrammed ex vivo, and can provide insights into development, tissue regeneration, cancer, and aging.

Stem cell-based regenerative therapies, which harness the self-renewal and differentiation properties of stem cells, have been in the spotlight due to their widespread applications in treating degenerative, aging, and other, generally intractable diseases. Therapeutically effective hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells have been used in numerous basic and translational studies with exciting results. However, pre-/post-transplantation issues of poor cell survival and retention, uncontrolled differentiation, and insufficient numbers of cells engrafted into host tissues are the major challenges in stem cell-based regenerative therapies. Engineered biomaterials have adjustable biochemical and biophysical properties that significantly affect cell behaviors, such as cell engraftment, survival, migration, and differentiation outcomes, thereby enhancing the engraftment of implanted stem cells and guiding tissue regeneration. Therefore, the combination of stem cell biology with bioengineered materials is a promising strategy to improve the therapeutic outcomes of stem cell-based regenerative therapy. In this review, we summarize the advances in the modulation of behaviors of stem cells via engineered biomaterials. We then present different approaches to harnessing bioengineered materials to enhance the transplantation of stem cells. Finally, we will provide future directions in regenerative therapy using stem cells.

The telomerase RNA component (TERC) gene plays an important role in telomerase-dependent extension and maintenance of the telomeres. In the event of TERC haploinsufficiency, telomere length is often affected; this, in turn, can result in the development of progeria-related diseases such as aplastic anemia (AA) and congenital keratosis. Cell reprogramming can reverse the differentiation process and can, therefore, transform cells into pluripotent stem cells with stronger differentiation and self-renewal abilities; further, cell reprograming can also extend the telomere length of these cells, which may be crucial in the diagnosis and treatment of telomere depletion diseases such as AA. In this study, we summarized the effects of TERC haploid cell reprogramming on telomere length and the correlation between this alteration and the pathogenesis of AA; by investigating the role of cell reprogramming in AA, we aimed to identify novel diagnostic indicators and therapeutic strategies for patients with AA.

Bridging knowledge gaps could enable regenerative therapy.

Mitochondrial dysfunction is a common denominator in both biological aging and cardiovascular disease (CVD) pathology. Understanding the protagonist role of mitochondria in the respective and independent progressions of CVD and biological aging will unravel the synergistic relationship between biological aging and CVD. Moreover, the successful development and implementation of therapies that can simultaneously benefit mitochondria of multiple cell types, will be transformational in curtailing pathologies and mortality in the elderly, including CVD. Several works have compared the status of mitochondria in vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in CVD dependent context. However, fewer studies have cataloged the aging-associated changes in vascular mitochondria, independent of CVD. This mini review will focus on the present evidence related to mitochondrial dysfunction in vascular aging independent of CVD. Additionally, we discuss the feasibility of restoring mitochondrial function in the aged cardiovascular system through mitochondrial transfer.

Direct lineage reprogramming of one somatic cell into another without transitioning through a progenitor stage has emerged as a strategy to generate clinically relevant cell types. One cell type of interest is the pancreatic insulin-producing β cell whose loss and/or dysfunction leads to diabetes. To date it has been possible to create β-like cells from related endodermal cell types by forcing the expression of developmental transcription factors, but not from more distant cell lineages like fibroblasts. In light of the therapeutic benefits of choosing an accessible cell type as the cell of origin, in this study we set out to analyze the feasibility of transforming human skin fibroblasts into β-like cells. We describe how the timed-introduction of five developmental transcription factors (Neurog3, Pdx1, MafA, Pax4, and Nkx2-2) promotes conversion of fibroblasts toward a β-cell fate. Reprogrammed cells exhibit β-cell features including β-cell gene expression and glucose-responsive intracellular calcium mobilization. Moreover, reprogrammed cells display glucose-induced insulin secretion in vitro and in vivo. This work provides proof-of-concept of the capacity to make insulin-producing cells from human fibroblasts via transcription factor-mediated direct reprogramming.

Nervous system diseases present significant challenges to the neuroscience community due to ethical and practical constraints that limit access to appropriate research materials. Somatic cell reprogramming has been proposed as a novel way to obtain neurons. Various emerging techniques have been used to reprogram mature and differentiated cells into neurons. This review provides an overview of somatic cell reprogramming for neurological research and therapy, focusing on neural reprogramming and generating different neural cell types. We examine the mechanisms involved in reprogramming and the challenges that arise. We herein summarize cell reprogramming strategies to generate neurons, including transcription factors, small molecules, and microRNAs, with a focus on different types of cells.. While reprogramming somatic cells into neurons holds the potential for understanding neurological diseases and developing therapeutic applications, its limitations and risks must be carefully considered. Here, we highlight the potential benefits of somatic cell reprogramming for neurological disease research and therapy. This review contributes to the field by providing a comprehensive overview of the various techniques used to generate neurons by cellular reprogramming and discussing their potential applications.

The ageing process is a systemic decline from cellular dysfunction to organ degeneration, with more predisposition to deteriorated disorders. Rejuvenation refers to giving aged cells or organisms more youthful characteristics through various techniques, such as cellular reprogramming and epigenetic regulation. The great leaps in cellular rejuvenation prove that ageing is not a one-way street, and many rejuvenative interventions have emerged to delay and even reverse the ageing process. Defining the mechanism by which roadblocks and signaling inputs influence complex ageing programs is essential for understanding and developing rejuvenative strategies. Here, we discuss the intrinsic and extrinsic factors that counteract cell rejuvenation, and the targeted cells and core mechanisms involved in this process. Then, we critically summarize the latest advances in state-of-art strategies of cellular rejuvenation. Various rejuvenation methods also provide insights for treating specific ageing-related diseases, including cellular reprogramming, the removal of senescence cells (SCs) and suppression of senescence-associated secretory phenotype (SASP), metabolic manipulation, stem cells-associated therapy, dietary restriction, immune rejuvenation and heterochronic transplantation, etc. The potential applications of rejuvenation therapy also extend to cancer treatment. Finally, we analyze in detail the therapeutic opportunities and challenges of rejuvenation technology. Deciphering rejuvenation interventions will provide further insights into anti-ageing and ageing-related disease treatment in clinical settings.

Cellular senescence is a state of irreversible cell cycle arrest that often emerges after tissue damage and in age-related diseases. Through the production of a multicomponent secretory phenotype (SASP), senescent cells can impact the regeneration and function of tissues. However, the effects of senescent cells and their SASP are very heterogeneous and depend on the tissue environment and type as well as the duration of injury, the degree of persistence of senescent cells and the organism's age. While the transient presence of senescent cells is widely believed to be beneficial, recent data suggest that it is detrimental for tissue regeneration after acute damage. Furthermore, although senescent cell persistence is typically associated with the progression of age-related chronic degenerative diseases, it now appears to be also necessary for correct tissue function in the elderly. Here, we discuss what is currently known about the roles of senescent cells and their SASP in tissue regeneration in ageing and age-related diseases, highlighting their (negative and/or positive) contributions. We provide insight for future research, including the possibility of senolytic-based therapies and cellular reprogramming, with aims ranging from enhancing tissue repair to extending a healthy lifespan.

Organoids with region-specific architecture could facilitate the repair of injuries of the central nervous system. Here we show that human astrocytes can be directly reprogrammed into early neuroectodermal cells via the overexpression of OCT4, the suppression of p53 and the provision of the small molecules CHIR99021, SB431542, RepSox and Y27632. We also report that the activation of signalling mediated by fibroblast growth factor, sonic hedgehog and bone morphogenetic protein 4 in the reprogrammed cells induces them to form spinal-cord organoids with functional neurons specific to the dorsal and ventral domains. In mice with complete spinal-cord injury, organoids transplanted into the lesion differentiated into spinal-cord neurons, which migrated and formed synapses with host neurons. The direct reprogramming of human astrocytes into neurons may pave the way for in vivo neural organogenesis from endogenous astrocytes for the repair of injuries to the central nervous system.