The clinical application of human umbilical cord mesenchymal stem cells (hUCMSCs) in the treatment of liver failure faces challenges due to cell quality, short engraftment time, and limited efficacy. Here, gelatin microcryogel (GM) microcarriers with pore sizes ranging from 15 to 36 μm were tuned from mixed gelatin and glutaraldehyde to develop a 3D culture system of hUCMSCs with improved therapeutic effects. Bulk RNA sequencing and in vitro assays showed that 3D-hUCMSCs exhibited significant improvement in signaling pathways related to paracrine secretion and anti-inflammation. These 3D-hUCMSCs superior compared to 2D-hUCMSCs not only in terms of paracrine secretion, protection from oxidation, and resistance to mechanical force damage, but also had better liver function improvement effect than 2D-hUCMSCs when they were transplanted as single cells into liver injury mice. Furthermore, a gelatin sponge patch grafting (GSPG) strategy was developed to enable the direct engraftment of 3D-hUCMSCs within the GM microcarriers. The results showed that overall engraftment in the host liver was significantly improved, and the life span of transplanted hosts was extended. Our study provided a practical strategy to achieve high engraftment and long retraining time of 3D-hUCMSCs in rescuing acute liver failure with gelatin matrixes.

Cancer cells frequently undergo energy metabolic stress induced by the increased dynamics of nutrient supply. Hepatocyte nuclear factor 4A (HNF4A) is a master transcription factor (TF) in hepatocytes that regulates metabolism and differentiation. However, the mechanism underlying how HNF4A functions in cancer progression remains unclear due to conflicting results observed in numerous studies. To address the roles of HNF4A in hepatocellular carcinoma (HCC), we investigated the regulatory functions of HNF4A in HCC cells under different glucose supply conditions. We found that HNF4A exhibited tumor-suppressive effects on the proliferation and migration of HCC cells in glucose-sufficient conditions and tumor-promotive effects on HCC cells in glucose-insufficient conditions. Further investigation revealed that this diverse function of HNF4A was dependent upon the AMPK pathway activity. Similarly, the prognosis predicted by HNF4A was also correlated with whether the AMPKa expression levels were low or high in clinical HCC patients. Multiomics approaches consisting of proteomics and ChIP-seq revealed that key HNF4A target genes, including NEDD4 and RPS6KA2, are involved in the diverse function of HNF4A in HCC in response to the AMPK activity status. Specifically, HNF4A could bind to the promoter region of NEDD4 and RPS6KA2, and upregulating their expression. Our study has demonstrated the relationship between and synergism of AMPK and HNF4A in the progression of HCC under diverse nutrient conditions.

Cell plasticity enables the dynamic changes in cell identities necessary for normal development and tissue repair. Induced cell reprogramming, which leverages this plasticity, holds great promise for regenerative medicine and personalized therapies. However, the success of cell reprogramming is often impeded by various molecular barriers, such as epigenetic marks, cell senescence, and the activation of alternative or refractory routes. In this review, we examine the cell reprogramming events that occur within or between germ layers and adult stem cell lineages and propose that the overall similarity in the pre-existing chromatin accessibility landscape is a major determinant of reprogramming efficiency from one cell type to another. A better understanding of the regulation and control of chromatin accessibility should facilitate the development of new methods and strategies to improve cell reprogramming efficiency and advance translational research.

The creation of induced pluripotent stem cells (iPSCs) has enabled scientists to explore the function, mechanisms, and differentiation processes of many types of cells. One of the fastest and most efficient approaches is transcription factor (TF) over-expression. However, finding the right combination of TFs to over-express to differentiate iPSCs directly into other cell types is a difficult task. Here, we describe a machine-learning (ML) pipeline, called CellCartographer, that uses chromatin accessibility and transcriptomics data to design multiplex TF pooled-screening experiments for cell-type conversions that then may be iteratively refined. We validate this method by differentiating iPSCs into twelve cell types at low efficiency in preliminary screens and iteratively refine our TF combinations to achieve high-efficiency differentiation for six of these cell types in <6 days. Finally, we functionally characterize iPSC-derived cytotoxic T cells (iCytoTs), regulatory T cells (iTregs), type II astrocytes (iAstIIs), and hepatocytes (iHeps) to validate functionally accurate differentiation.

All biological systems are subject to perturbations arising from thermal fluctuations, external environments, or mutations. Yet, while biological systems consist of thousands of interacting components, recent high-throughput experiments have shown that their response to perturbations is surprisingly low dimensional: confined to only a few stereotyped changes out of the many possible. In this review, we explore a unifying dynamical systems framework-soft modes-to explain and analyze low dimensionality in biology, from molecules to ecosystems. We argue that this soft mode framework makes nontrivial predictions that generalize classic ideas from developmental biology to disparate systems, namely phenocopying, dual buffering, and global epistasis. While some of these predictions have been borne out in experiments, we discuss how soft modes allow for a surprisingly far-reaching and unifying framework in which to analyze data from protein biophysics to microbial ecology.

Proliferating hepatocytes often undergo ductal metaplasia to balance the energy trade-off between cellular functions and replication, hindering the expansion of human adult hepatocytes with functional competency1. Here we demonstrate that the combined activation of Wnt and STAT3 signalling enables long-term self-renewal of human adult hepatocyte organoids. YAP activation facilitates hepatocyte proliferation but commits it towards the biliary duct lineage. By contrast, STAT3 activation by oncostatin M induces hepatocyte proliferation while counteracting ductal metaplasia and maintaining the hepatic identity. Xenotransplanted hepatocyte organoids repopulate the recipient mouse liver and reconstitute the metabolic zonation structure. Upon niche factor removal and hormone supplementation, hepatocyte organoids form cord-like structures with bile canalicular networks and exhibit major liver metabolic functions comparable to those of in vivo hepatocytes. Hepatocyte organoids are amenable to gene editing, prompting functional modelling of inherent metabolic liver diseases. The new culture system offers a promising avenue for developing therapeutic strategies against human liver diseases.

Hepatocytes are crucial for drug screening, disease modeling, and clinical transplantation, yet generating functional hepatocytes in vitro is challenging due to the difficulty of establishing their authentic gene regulatory networks (GRNs). We have previously developed a two-step lineage reprogramming strategy to generate functionally competent human induced hepatocytes (hiHeps), providing an effective model for studying the establishment of hepatocyte-specific GRNs. In this study, we utilized high-throughput single-cell RNA sequencing (scRNA-seq) to explore the cell-fate transition and the establishment of hepatocyte-specific GRNs involved in the two-step reprogramming process. Our findings revealed that the late stage of the reprogramming process mimics the natural trajectory of liver development, exhibiting similar transcriptional waves of developmental genes. CD24 and DLK1 were identified as surface markers enriching two distinct hepatic progenitor populations respectively. Lipid metabolism emerged as a key enhancer of hiHeps maturation. Furthermore, transcription factors HNF4A and HHEX were identified as pivotal gatekeepers directing cell fate decisions between hepatocytes and intestinal cells. Collectively, this study provides valuable insights into the establishment of hepatocyte-specific GRNs during hiHeps induction at single-cell resolution, facilitating more efficient production of functional hepatocytes for therapeutic applications.

End-stage liver disease represents a critical hepatic condition with high mortality, for which liver transplantation remains the only effective treatment. However, the scarcity of suitable donors results in numerous patients dying while awaiting transplantation. Novel strategies, including cell therapies and technologies mimicking liver organogenesis, offer promising alternatives for treating end-stage liver disease by potentially providing new sources of liver grafts. Recently, significant progress has been made in this field, including stem cell transplantation, hepatocyte transplantation, in vitro liver tissue generation, and liver replacement technologies. Several clinical studies have demonstrated that stem cell transplantation and hepatocyte transplantation can prolong patient survival and serve as a bridge to liver transplantation. Furthermore, in vitro liver tissue generation technologies, such as liver organoids and three-dimensional bioprinting, can generate hepatic tissues with sophisticated structures and functions, making them promising transplantation materials. Notably, liver replacement technologies hold considerable potential for producing biologically functional and transplantable liver grafts. In this review, we discuss the fundamental principles and recent advancements in cell therapies and liver organogenesis technologies while also addressing the challenges and future prospects in this rapidly evolving field.

Cell reprogramming represents a powerful approach to achieve the conversion cells of one type into cells of another type of interest, which has substantially changed the landscape in the field of developmental biology, regenerative medicine, disease modeling, drug discovery and cancer immunotherapy. Cell reprogramming is a complex and ordered process that involves the coordination of transcriptional, epigenetic, translational and metabolic changes. Over the past two decades, a range of questions regarding the facilitators/barriers, the trajectories, and the mechanisms of cell reprogramming have been extensively investigated. This review summarizes the recent advances in cell reprogramming mediated by transcription factors or chemical molecules, followed by elaborating on the important roles of biophysical cues in cell reprogramming. Additionally, this review will detail our current understanding of the mechanisms that govern cell reprogramming, including the involvement of the recently discovered biomolecular condensates. Finally, the review discusses the broad applications and future directions of cell reprogramming in developmental biology, disease modeling, drug development, regenerative/rejuvenation therapy, and cancer immunotherapy.

Wilson's disease (WD) is a genetic disorder that impairs the excretion of copper in hepatocytes and results in excessive copper deposition in multiple organs. The replacement of disordered hepatocytes with functional hepatocytes can serve as a lifelong therapeutic strategy for the treatment of WD. The aim of this study was to determine the hepatocyte repopulation effects of fibroblast-derived hepatic progenitor cells in the treatment of WD.

Induced hepatic progenitor cells (iHPCs) were generated through direct reprogramming of adult mouse fibroblasts infected with lentivirus carrying both the Foxa3 and Hnf4α genes. These iHPCs were subsequently identified and transplanted into copper-overload WD mice with the Atp7b (H1071Q) mutation via caudal vein injection.

After lentivirus infection, the fibroblasts transformed into Foxa3- and Hnf4α-overexpressing cobblestone-like cells with reduced expression of fibroblast markers and increased expression of epithelial cell and hepatic progenitor cell markers, i.e., iHPCs. Sixteen weeks after transplantation into WD mice, approximately 2% of hepatocytes were derived from iHPCs, and these iHPC-derived hepatocytes expressed a tight junction-associated protein of the bile canal, tight junction protein 1 (Zo1). There was a decrease in the serum copper concentration and relative activity of serum ceruloplasmin at weeks 4 and 8 after iHPCs transplantation compared with those of WD fed mice administered saline or fibroblasts. Furthermore, iHPC transplantation markedly reduced the proportion of CD8+ T lymphocytes and natural killer cells compared with those in fibroblast-transplanted WD mice and downregulated the transcription of the inflammatory cytokines, including tumor necrosis factor α (Tnfα), interleukin 1β (IL-1β), and IL-6, compared with those in WD mice and in fibroblast-transplanted WD mice.

iHPCs reprogrammed from adult fibroblasts can repopulate hepatocytes and exert therapeutic effects in WD mice, representing a potential replacement therapy for clinical application.

CD146 plays a key role in cancer progression and metastasis. Cancer stem cells (CSCs) are responsible for tumor initiation, drug resistance, metastasis, and recurrence. In this study, we explored the role of CD146 in the regulation of liver CSCs. Here, we demonstrated that CD146 was highly expressed in liver CSCs. CD146 overexpression promoted the self-renewal ability and chemoresistance of Hepatocellular Carcinoma (HCC) cells in vitro and tumorigenicity in vivo. Inversely, knockdown of CD146 restrained these abilities. Mechanistically, CD146 activated the NF-κB signaling to up-regulate JAG2 expression and activated the Notch signaling, which resulted in increased stemness of HCC. Furthermore, JAG2 overexpression restored the Notch signaling activity, the stemness, and chemotherapeutic resistance caused by CD146 knockdown. These results demonstrated that CD146 positively regulates HCC stemness by activating the JAG2-NOTCH signaling. Combined targeting of CD146 and JAG2 may represent a novel therapeutic strategy for HCC treatment.

The generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells by Yamanaka factors, including pioneer transcription factors (TFs), has greatly reshaped our traditional understanding of cell plasticity and demonstrated the remarkable potential of pioneer TFs. In addition to iPSC reprogramming, pioneer TFs are pivotal in direct reprogramming or transdifferentiation where somatic cells are converted into different cell types without passing through a pluripotent state. Pioneer TFs initiate a reprogramming process through chromatin opening, thereby establishing competence for new gene regulatory programs. The action of pioneer TFs is both influenced by and exerts influence on epigenetic regulation. Despite significant advances, many direct reprogramming processes remain inefficient, which limits their reliability for clinical applications. In this review, we discuss the molecular mechanisms underlying pioneer TF-driven reprogramming, with a focus on their interactions with epigenetic modifiers, including Polycomb repressive complexes (PRCs), nucleosome remodeling and deacetylase (NuRD) complexes, and the DNA methylation machinery. A deeper understanding of the dynamic interplay between pioneer TFs and epigenetic modifiers will be essential for advancing reprogramming technologies and unlocking their full clinical potential.

Cellular identity requires the concerted action of multiple transcription factors (TFs) bound together to enhancers of cell-type-specific genes. Despite TFs recognizing specific DNA motifs within accessible chromatin, this information is insufficient to explain how TFs select enhancers1. Here we compared four different TF combinations that induce different cell states, analysing TF genome occupancy, chromatin accessibility, nucleosome positioning and 3D genome organization at the nucleosome resolution. We show that motif recognition on mononucleosomes can decipher only the individual binding of TFs. When bound together, TFs act cooperatively or competitively to target nucleosome arrays with defined 3D organization, displaying motifs in particular patterns. In one combination, motif directionality funnels TF combinatorial binding along chromatin loops, before infiltrating laterally to adjacent enhancers. In other combinations, TFs assemble on motif-dense and highly interconnected loop junctions, and subsequently translocate to nearby lineage-specific sites. We propose a guided-search model in which motif grammar on nucleosome fibres acts as signpost elements, directing TF combinatorial binding to enhancers.

The liver's role in metabolism, detoxification, and immune regulation underscores the urgency of addressing liver diseases, which claim millions of lives annually. Due to donor shortages in liver transplantation, liver tissue engineering (LTE) offers a promising alternative. Hydrogels, with their biocompatibility and ability to mimic the liver's extracellular matrix (ECM), support cell survival and function in LTE. This review analyzes recent advances in hydrogel-based strategies for LTE, including decellularized liver tissue hydrogels, natural polymer-based hydrogels, and synthetic polymer-based hydrogels. These materials are ideal for in vitro cell culture and obtaining functional hepatocytes. Hydrogels' tunable properties facilitate creating artificial liver models, such as organoids, 3D bioprinting, and liver-on-a-chip technologies. These developments demonstrate hydrogels' versatility in advancing LTE's applications, including hepatotoxicity testing, liver tissue regeneration, and treating acute liver failure. This review highlights the transformative potential of hydrogels in LTE and their implications for future research and clinical practice.

The differentiation of mesenchymal stem cells (MSCs) into hepatocyte-like cells (HLCs) is considered one of the most promising strategies for alternative hepatocyte transplantation to treat end-stage liver disease. To advance this method, it is crucial to gain a deeper understanding of the mechanisms governing hepatogenic differentiation. The study demonstrated that suppression of the intracellular domain release of the Notch pathway receptor via the γ-secretase inhibitor N-[(3, 5-difluorophenyl)acetyl]-L-alanyl-2-phenylglycine-1, 1-dimethylethyl ester (DAPT) significantly promotes the expression of hepatocyte-related genes and proteins in HLCs. Increased expression of intracellular inducible NO synthase (iNOS) during differentiation led to elevated endogenous NO production. Biotin switch assays revealed a gradual increase in S-nitrosylation (SNO)-NOTCH1 and a decrease in overall NOTCH1 expression during hepatogenic differentiation. The addition of the exogenous NO donor S-nitrosoglutathione (GSNO) and the SNO inhibitor dithiothreitol (DTT) further demonstrated that the elevated expression of SNO-NOTCH1 promotes the differentiation of MSCs into mature hepatocytes. Briefly, our results fully demonstrated that the modification of the extracellular domain of NOTCH1 by NO, leading to the formation of SNO-NOTCH1, significantly promotes hepatogenic differentiation by inhibiting the Notch signalling pathway. Our study highlights the critical role of SNO-NOTCH1 in regulating the Notch signalling pathway and offers new insights into the mechanisms driving this differentiation process.

Hepatocyte transplantation (HTx) has been a novel cell-based therapy for severe liver diseases, as the donor livers for orthotopic liver transplantation are of great shortage. However, HTx has been confronted with two main hurdles: limited high-quality hepatocyte sources and low cell engraftment and repopulation rate. To cope with, researchers have investigated on various strategies, including small molecule drugs with unique advantages. Small molecules are promising chemical tools to modulate cell fate and function for generating high quality hepatocyte sources. In addition, endothelial barrier, immune responses, and low proliferative efficiency of donor hepatocytes mainly contributes to low cell engraftment and repopulation rate. Interfering these biological processes with small molecules is beneficial for improving cell engraftment and repopulation. In this review, we will discuss the applications and advances of small molecules in modulating cell differentiation and reprogramming for hepatocyte resources and in improving cell engraftment and repopulation as well as its underlying mechanisms.

Utilizing induced pluripotent stem cells (iPSCs) in drug screening and cell replacement therapy has emerged as a method with revolutionary applications. With the advent of patient-specific iPSCs and the subsequent development of cells that exhibit disease phenotypes, the focus of medication research will now shift toward the pathology of human diseases. Regular iPSCs can also be utilized to generate cells that assess the negative impacts of medications. These cells provide a much more precise and cost-efficient approach compared to many animal models. In this review, we explore the utilization of small-molecule drugs to enhance the growth of iPSCs and gain insights into the process of reprogramming. We mainly focus on the functions of small molecules in modulating different signaling pathways, thereby modulating cell fate. Understanding the way small molecule drugs interact with iPSC technology has the potential to significantly enhance the understanding of physiological pathways in stem cells and practical applications of iPSC-based therapy and screening systems, revolutionizing the treatment of diseases.

Cellular senescence associates with pathological aging and tissue dysfunctions. Studies utilizing mouse models for cell lineage tracings have emphasized the importance of senescence heterogeneity in different organs and cell types. Here, we constructed a p21- (Akaluc - tdTomato - Diphtheria Toxin Receptor [DTR]) (ATD) mouse model to specifically study the undefined mechanism for p21-expressing senescent cells in the aged and liver injury animals. The successful expressions of these genes enabled in vitro flow cytometric sorting, in vivo tracing, and elimination of p21-expressing senescent cells. During the natural aging process, p21-expressing cells were found in various tissues of p21-ATD mice. Eliminating p21-expressing cells in the aged p21-ATD mice recovered their multiple biological functions. p21-ATD/Fah-/- mice, bred from p21-ATD mice and fumarylacetoacetate hydrolase (Fah)-/- mice of liver injury, showed that the majority of their senescent hepatocytes were the phenotype of p21+ rather than p16+. Furthermore, eliminating the p21-expressing hepatocytes significantly promoted the engraftment of grafted hepatocytes and facilitated liver repopulation, resulting in significant recovery from liver injury. Our p21-ATD mouse model serves as an optimal model for studying the pattern and function of p21-expressing senescent cells under the physical and pathological conditions during aging.

Organoids refer to 3D cultures established to recapitulate histology, pathology, architecture, and genetic traits of various organs and tissues in the body, thereby replacing 2D cell cultures, xenograft, and animal models. Organoids form a 3D in vitro mimic of original tissues like the liver and are derived from embryonic or adult tissue stem cells. Liver and bile duct tumor organoids, also called, tumoroids capture genetic diversity, cellular, and pathophysiological properties of original tumors. Moreover, co-culture techniques along with genetic modulation of organoids allow for using tumoroids in liver and bile duct cancer research and drug screening/testing. Therefore, tumoroids are promising platforms for studying liver and bile duct cancer, which paves the way for the new era of personalized therapies. In the current review, we aimed to discuss liver and bile duct organoids with special emphasis on tumoroids and their applications, advantages, and shortcomings.

The liver exhibits remarkable regenerative capacity. However, the limited ability of primary human hepatocytes to proliferate in vitro, combined with a compromised regenerative capacity induced by pathological conditions in vivo, presents significant obstacles to effective liver regeneration following liver injuries and diseases. Developing strategies to compensate for the loss of endogenous hepatocytes is crucial for overcoming these challenges, and this remains an active area of investigation. Lineage reprogramming, the process of directly converting one cell type into another bypassing the intermediate pluripotent state, has emerged as a promising method for generating specific cell types for therapeutic purposes in regenerative medicine. Here, we discuss the recent progress and emergent technologies in lineage reprogramming into hepatic cells, and their potential applications in enhancing liver regeneration or treating liver disease models. We also address controversies and challenges that confront this field.

Development is regulated by coordinated changes in gene expression. Control of these changes in expression is largely governed by the binding of transcription factors to specific regulatory elements. However, the packaging of DNA into chromatin prevents the binding of many transcription factors. Pioneer factors overcome this barrier owing to unique properties that enable them to bind closed chromatin, promote accessibility and, in so doing, mediate binding of additional factors that activate gene expression. Because of these properties, pioneer factors act at the top of gene-regulatory networks and drive developmental transitions. Despite the ability to bind target motifs in closed chromatin, pioneer factors have cell type-specific chromatin occupancy and activity. Thus, developmental context clearly shapes pioneer-factor function. Here, we discuss this reciprocal interplay between pioneer factors and development: how pioneer factors control changes in cell fate and how cellular environment influences pioneer-factor binding and activity.

The utilization of three-dimensional (3D) bioprinting technology to create a transplantable bioartificial liver emerges as a promising remedy for the scarcity of liver donors. This study outlines our strategy for constructing a 3D-bioprinted liver, using in vitro-expanded primary hepatocytes recognized for their safety and enhanced functional robustness as hepatic cell sources for bioartificial liver construction. In addition, we have developed bioink biomaterials with mechanical and rheological properties, as well as printing capabilities, tailored for 3D bioprinting. Upon heterotopic transplantation into the mesentery of tyrosinemia or 90% hepatectomy mice, our 3D-bioprinted liver effectively restored lost liver functions, consequently extending the life span of mice afflicted with liver injuries. Notably, the inclusion of an artificial blood vessel in our 3D-bioprinted liver allowed for biomolecule exchange with host blood vessels, demonstrating, in principle, the rapid integration of the bioartificial liver into the host vascular system. This model underscores the therapeutic potential of transplantation for the treatment of liver failure diseases.

The construction of bioartificial livers, such as liver organoids, offers significant promise for disease modeling, drug development, and regenerative medicine. However, existing methods for generating liver organoids have limitations, including lengthy and complex processes (taking 6-8 weeks or longer), safety concerns associated with pluripotency, limited functionality of pluripotent stem cell-derived hepatocytes, and small, highly variable sizes (typically ≈50-500 µm in diameter). Prolonged culture also leads to the formation of necrotic cores, further restricting size and function. In this study, a straightforward and time-efficient approach is developed for creating rapid self-assembly mini-livers (RSALs) within 12 h. Additionally, primary hepatocytes are significantly expanded in vitro for use as seeding cells. RSALs exhibit consistent larger sizes (5.5 mm in diameter), improved cell viability (99%), and enhanced liver functionality. Notably, RSALs are functionally vascularized within 2 weeks post-transplantation into the mesentery of mice. These authentic hepatocyte-based RSALs effectively protect mice from 90%-hepatectomy-induced liver failure, demonstrating the potential of bioartificial liver-based therapy.

Hepatic stellate cells (HSCs) play a crucial role in the development of fibrosis in non-alcoholic fatty liver disease (NAFLD). Small extracellular vesicles (sEV) act as mediators for intercellular information transfer, delivering various fibrotic factors that impact the function of HSCs in liver fibrosis. In this study, we investigated the role of lipotoxic hepatocyte derived sEV (LTH-sEV) in HSCs activation and its intrinsic mechanisms.

High-fat diet (HFD) mice model was constructed to confirm the expression of LIMA1. The relationship between LIMA1-enriched LTH-sEV and LX2 activation was evaluated by measurement of fibrotic markers and related genes. Levels of mitophagy were detected using mt-keima lentivirus. The interaction between LIMA1 and PINK1 was discovered through database prediction and molecular docking. Finally, sEV was injected to investigate whether LIMA1 can accelerate HFD induced liver fibrosis in mice.

LIMA1 expression was upregulated in lipotoxic hepatocytes and was found to be positively associated with the expression of the HSCs activation marker α-SMA. Lipotoxicity induced by OPA led to an increase in both the level of LIMA1 protein in LTH-sEV and the release of LTH-sEV. When HSCs were treated with LTH-sEV, LIMA1 was observed to hinder LX2 mitophagy while facilitating LX2 activation. Further investigation revealed that LIMA1 derived from LTH-sEV may inhibit PINK1-Parkin-mediated mitophagy, consequently promoting HSCs activation. Knocking down LIMA1 significantly attenuates the inhibitory effects of LTH-sEV on mitophagy and the promotion of HSCs activation.

Lipotoxic hepatocyte-derived LIMA1-enriched sEVs play a crucial role in promoting HSCs activation in NAFLD-related liver fibrosis by negatively regulating PINK1 mediated mitophagy. These findings provide new insights into the pathological mechanisms involved in the development of fibrosis in NAFLD.

Long non-coding RNAs (lncRNAs) outnumber protein-coding transcripts, but their functions remain largely unknown. In this Review, we discuss the emerging roles of lncRNAs in the control of gene transcription. Some of the best characterized lncRNAs have essential transcription cis-regulatory functions that cannot be easily accomplished by DNA-interacting transcription factors, such as XIST, which controls X-chromosome inactivation, or imprinted lncRNAs that direct allele-specific repression. A growing number of lncRNA transcription units, including CHASERR, PVT1 and HASTER (also known as HNF1A-AS1) act as transcription-stabilizing elements that fine-tune the activity of dosage-sensitive genes that encode transcription factors. Genetic experiments have shown that defects in such transcription stabilizers often cause severe phenotypes. Other lncRNAs, such as lincRNA-p21 (also known as Trp53cor1) and Maenli (Gm29348) contribute to local activation of gene transcription, whereas distinct lncRNAs influence gene transcription in trans. We discuss findings of lncRNAs that elicit a function through either activation of their transcription, transcript elongation and processing or the lncRNA molecule itself. We also discuss emerging evidence of lncRNA involvement in human diseases, and their potential as therapeutic targets.

Ectopic liver regeneration in the spleen is a promising alternative to organ transplantation for treating liver failure. To accommodate transplanted liver cells, the splenic tissue must undergo structural changes to increase extracellular matrix content, demanding a safe and efficient approach for tissue remodelling.

We synthesised sulphated hyaluronic acid (sHA) with an affinity for the latent complex of transforming growth factor-β (TGF-β) and cross-linked it into a gel network (sHA-X) via click chemistry. We injected this glycan into the spleens of mice to induce splenic tissue remodelling via supraphysiological activation of endogenous TGF-β.

sHA-X efficiently bound to the abundant latent TGF-β in the spleen. It provided the molecular force to liberate the active TGF-β dimers from their latent complex, mimicking the 'bind-and-pull' mechanism required for physiological activation of TGF-β and reshaping the splenic tissue to support liver cell growth. Hepatocytes transplanted into the remodelled spleen developed into liver tissue with sufficient volume to rescue animals with a metabolic liver disorder (Fah-/- transgenic model) or following 90% hepatectomy, with no adverse effects observed and no additional drugs required.

Our findings highlight the efficacy and translational potential of using sHA-X to remodel a specific organ by mechanically activating one single cytokine, representing a novel strategy for the design of biomaterials-based therapies for organ regeneration.

Cell transplantation may provide a lifeline to millions of patients with end-stage liver diseases, but their severely damaged livers being unable to accommodate the transplanted cells is a crucial hurdle. Herein, we report an approach to restore liver functions in another organ - the spleen - by activating one single growth factor in situ. This approach, based on a chemically designed polysaccharide that can mechanically liberate the active transforming growth factor-β to an unusually high level, promotes the function of abundant allogenic liver cells in the spleen, rescuing animals from lethal models of liver diseases and showing a high potential for clinical translation.

Although lineage reprogramming from one cell type to another is becoming a breakthrough technology for cell-based therapy, several limitations remain to be overcome, including the low conversion efficiency and subtype specificity. To address these, many studies have been conducted using genetics, chemistry, physics, and cell biology to control transcriptional networks, signaling cascades, and epigenetic modifications during reprogramming. Here, we summarize recent advances in cellular reprogramming and discuss future directions.

Previous studies have shown that hepatocyte-like cells can be generated from fibroblasts using either lineage-specific transcription factors or chemical induction methods. However, these methods have their own deficiencies that restrict the therapeutic applications of such induced hepatocytes. In this study, we present a transgene-free, highly efficient chemical-induced direct reprogramming approach to generate hepatocyte-like cells from mouse embryonic fibroblasts (MEFs). Using a small molecule cocktail (SMC) as an inducer, MEFs can be directly reprogrammed into hepatocyte-like cells, bypassing the intermediate stages of pluripotent and immature hepatoblasts. These chemical-induced hepatocyte-like cells (ciHeps) closely resemble mature primary hepatocytes in terms of morphology, biological behavior, gene expression patterns, marker expression levels, and hepatic functions. Furthermore, transplanted ciHeps can integrate into the liver, promote liver regeneration, and improve survival rates in mice with acute liver damage. ciHeps can also ameliorate liver fibrosis caused by chronic injuries and enhance liver function. Notably, ciHeps exhibit no tumorigenic potential either in vitro or in vivo. Mechanistically, SMC-induced mesenchymal-to-epithelial transition and suppression of SNAI1 contribute to the fate conversion of fibroblasts into ciHeps. These results indicate that this transgene-free, chemical-induced direct reprogramming technique has the potential to serve as a valuable means of producing alternative hepatocytes for both research and therapeutic purposes. Additionally, this method also sheds light on the direct reprogramming of other cell types under chemical induction.

3D bioprinting has unlocked new possibilities for generating complex and functional tissues and organs. However, one of the greatest challenges lies in selecting the appropriate seed cells for constructing fully functional 3D artificial organs. Currently, there are no cell sources available that can fulfill all requirements of 3D bioprinting technologies, and each cell source possesses unique characteristics suitable for specific applications. In this review, we explore the impact of different 3D bioprinting technologies and bioink materials on seed cells, providing a comprehensive overview of the current landscape of cell sources that have been used or hold potential in 3D bioprinting. We also summarized key points to guide the selection of seed cells for 3D bioprinting. Moreover, we offer insights into the prospects of seed cell sources in 3D bioprinted organs, highlighting their potential to revolutionize the fields of tissue engineering and regenerative medicine.

Significant advances have been made in reprogramming various somatic cells into induced pluripotent stem cells (iPSCs) and in multi-lineage differentiation (transdifferentiation) into different tissues. These manipulable transdifferentiating techniques may be applied in cancer therapy. Limited works have been reported that cancer cell malignancy can be switched to benign phenotypes through reprogramming techniques. Here, we reported that two colorectal cancer (CRC) cell lines (DLD1, HT29) could be reprogrammed into iPSCs (D-iPSCs, H-iPSCs). D- and H-iPSCs showed reduced tumorigenesis. Furthermore, we successfully induced D- and H-iPSCs differentiation into terminally differentiated cell types such as cardiomyocyte, neuron, and adipocyte-like cells. Impressively, the differentiated cells exhibited further attenuated tumorigenesis in vitro and in vivo. RNA-Seq further indicated that epigenetic changes occurred after reprogramming and transdifferentiation that caused reduced tumorigenicity. Overall, our study indicated that CRC cells can be reprogrammed and further differentiated into terminally differentiated lineages with attenuation of their malignancy in vitro and in vivo. The current work sheds light on a potential multi-lineage differentiation therapeutic strategy for colorectal cancer.

Hepatic organoids might provide a golden opportunity for realizing precision medicine in various hepatic diseases. Previously described hepatic organoid protocols from pluripotent stem cells rely on complicated multiple differentiation steps consisting of both 2D and 3D differentiation procedures. Therefore, the spontaneous formation of hepatic organoids from 2D monolayer culture is associated with a low-throughput production, which might hinder the standardization of hepatic organoid production and hamper the translation of this technology to the clinical or industrial setting. Here we describe the stepwise and fully 3D production of hepatic organoids from human pluripotent stem cells. We optimized every differentiation step by screening for optimal concentrations and timing of differentiation signals in each differentiation step. Hepatic organoids are stably expandable without losing their hepatic functionality. Moreover, upon treatment of drugs with known hepatotoxicity, we found hepatic organoids are more sensitive to drug-induced hepatotoxicity compared with 2D hepatocytes differentiated from PSCs, making them highly suitable for in vitro toxicity screening of drug candidates. The standardized fully 3D protocol described in the current study for producing functional hepatic organoids might serve as a novel platform for the industrial and clinical translation of hepatic organoid technology.

Direct reprogramming, inducing the conversion of one type of somatic cell into another by the forced expression of defined transcription factors, is a technology with anticipated medical applications. However, due to the many unresolved aspects of the induction mechanisms, it is essential to thoroughly analyze the epigenomic state of the generated cells. Here, we performed comparative genome-wide DNA methylation analyses of mouse embryonic fibroblasts (MEFs) and cells composing organoids formed by intestinal stem cells (ISCs) or induced ISCs (iISCs) that were directly induced from MEFs. We found that the CpG methylation state was similar between cells forming ISC organoids and iISC organoids, while they differed widely from those in MEFs. Moreover, genomic regions that were differentially methylated between ISC organoid- and iISC organoid-forming cells did not significantly affect gene expression. These results demonstrate the accuracy and safety of iISC induction, leading to the medical applications of this technology.

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.

The liver is one of the few organs that retain the capability to regenerate in adult mammals. This regeneration process is mainly facilitated by the dynamic behavior of hepatocytes, which are the major functional constituents in the liver. In response to liver injury, hepatocytes undergo remarkable alterations, such as reprogramming, wherein they lose their original identity and acquire properties from other cells. This phenomenon of hepatocyte reprogramming, coupled with hepatocyte expansion, plays a central role in liver regeneration, and its underlying mechanisms are complex and multifaceted. Understanding the fate of reprogrammed hepatocytes and the mechanisms of their conversion has significant implications for the development of innovative therapeutics for liver diseases. Herein, we review the plasticity of hepatocytes in response to various forms of liver injury, with a focus on injury-induced hepatocyte reprogramming. We provide a comprehensive summary of current knowledge on the molecular and cellular mechanisms governing hepatocyte reprogramming, specifically in the context of liver regeneration, providing insight into potential applications of this process in the treatment of liver disorders, including chronic liver diseases and liver 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.

Cardiovascular disease remains a leading cause of death worldwide despite important advances in modern medical and surgical therapies. As human adult cardiomyocytes have limited regenerative ability, cardiomyocytes lost after myocardial infarction are replaced by fibrotic scar tissue, leading to cardiac dysfunction and heart failure. To replace lost cardiomyocytes, a promising approach is direct cardiac reprogramming, in which cardiac fibroblasts are transdifferentiated into induced cardiomyocyte-like cells (iCMs). Here we review cardiac reprogramming cocktails (including transcription factors, microRNAs and small molecules) that mediate iCM generation. We also highlight mechanistic studies exploring the barriers to and facilitators of this process. We then review recent progress in iCM reprogramming, with a focus on single-cell '-omics' research. Finally, we discuss obstacles to clinical application.

Direct reprogramming of fibroblasts into induced cardiomyocytes holds great promise for heart regeneration. Although considerable progress has been made in understanding the transcriptional and epigenetic mechanisms of iCM reprogramming, its translational regulation remains largely unexplored. Here, we characterized the translational landscape of iCM reprogramming through integrative ribosome and transcriptomic profiling, and found extensive translatome repatterning during this process. Loss of function screening for translational regulators uncovered Ybx1 as a critical barrier to iCM induction. In a mouse model of myocardial infarction, removing Ybx1 enhanced in vivo reprogramming, resulting in improved heart function and reduced scar size. Mechanistically, Ybx1 depletion de-repressed the translation of its direct targets SRF and Baf60c, both of which mediated the effect of Ybx1 depletion on iCM generation. Furthermore, removal of Ybx1 allowed single factor Tbx5-mediated iCM conversion. In summary, this study revealed a new layer of regulatory mechanism that controls cardiac reprogramming at the translational level.