Application of stem cells in the precise diagnosis and treatment of liver diseases

Abstract

Liver diseases caused by diverse inflammation or cancer have serious damaged people’s physical and mental health and become a heavy social burden. Stem cells possess unique properties of self-renewal and multi-directional differentiation, which are widely used for disease remodeling and regenerative medicine. Of them, human induced pluripotent stem cell-based liver organoids with self-organization of sinusoidal vessels have been reported for diverse liver cancer remodeling and drug sensitivity test, which provide unique platforms to dissect functional consequences of diverse levels of heteroplasmy of target gene mutation in liver cancers. Meanwhile, mesenchymal stem/stromal cells have been adopted for clinical treatment research on various liver diseases, including acute liver injury, decompensated liver cirrhosis, hepatic fibrosis and acute-on-chronic liver failure. In this review article, we mainly focus on the state-of-the-art literatures upon stem cell-based disease modeling and cell therapy for multifarious liver diseases from the view of basic research and clinical progress, which will provide references for the development of stem cell-based regenerative medicine in the precise diagnosis and treatment of liver diseases.

Key Words: Stem cells; Liver diseases; Organoids; Cell therapy; Human induced pluripotent stem cells; Mesenchymal stem/stromal cells

Core Tip: Liver diseases of multitudinous types have caused great burden to public health and social economics. In this review article, we outline the fundamental and clinical progress of stem cell-based strategies for liver disease diagnosis and intervention, and in particular, the human induced pluripotent stem cells-based organoids for disease modeling and mesenchymal stem/stromal cells-based regimens for therapeutic purposes. Overall, our review will provide new references for facilitating the development of stem cell-based precision medicine for liver diseases.

INTRODUCTION

The liver is composed of primarily hepatocytes, hepatic parenchymal cells (e.g., hepatocytes) and non-parenchymal cells [e.g., hepatic stellate cells (HSCs), Kupffer cells, liver sinusoidal endothelial cells (LSECs)]. Proverbially, the liver plays a critical role in metabolism (e.g., protein synthesis, nutrient metabolism), detoxification and biochemical processes[1,2]. The dysregulation of homeostasis and abnormity in cellular interactions commonly results in metabolic zonation destruction and liver diseases[3], including liver injury[4], liver fibrosis[5], liver cirrhosis[6,7], metabolic associated fatty liver disease[8], metabolic dysfunction-associated steatotic liver disease (MASLD)[9,10], alcoholic steatohepatitis[11], acute-on-chronic liver failure (ACLF)[12], and even hepatocellular carcinoma (HCC)[13,14]. To date, diverse pathogenic factors have been identified as modulators of liver disease trajectory, including virus infection (e.g., hepatitis B virus[15], hepatitis C virus[16], hepatitis D virus[17]), gut microbiota[18], physical and chemical factors (ethanol[19], radiation[20]), oxidative stress (e.g., oxygen gradient within liver acinus[3]). Meanwhile, diverse liver disorders are also related with chronic inflammation and atherogenic dyslipidemia[21]. The liver disease-associated endothelial dysfunction and aging-associated cellular senescence[22] are orchestrated by a variety of proinflammatory signal transduction networks in the microenvironment, e.g., Wnt signaling[23], interleukin (IL) 6 trans-signaling[24], phosphoinositide 3-kinases (PI3K)-protein kinase B (AKT) signaling[25], nuclear factor-κB signaling[26,27], transforming growth factor-β (TGF-β) signaling[28]. Meanwhile, multifarious therapeutics and the relative interventions have been developed for liver disease administration, including surgical treatment (e.g., liver transplantation), pharmacotherapy, chemotherapy[20], and even chimeric antigen receptor-transduced T cell- and natural killer cells-based cell therapy (e.g., chimeric antigen receptor-modified T[29,30], chimeric antigen receptor natural killer[31-33]).

Stem cells are cell populations with self-renewal and multi-lineage differentiation properties, which play a prominent role in embryonic development, tissue repair and regeneration, disease modeling and drug screening[34-36]. Overall, stem cells can be classified in many ways. According to the developmental stages, stem cells can be divided into embryonic stem cells (ESCs)[37,38], adult stem cells[39] (e.g., dental pulp stem cells[40,41], bone marrow (BM)-derived mesenchymal stem/stromal cells (MSCs)[42], adipose tissue-derived stem cells[43,44]), and perinatal stem cells (e.g., umbilical cord-derived stem cells[45], placenta tissue-derived stem cells[46], amniotic stem cells[47]). According to the differentiation potential, stem cells include various subtypes such as totipotent stem cells, pluripotent stem cells [e.g., ESCs[35,48], induced pluripotent stem cells (iPSCs)[49]], multipotent stem cells (hematopoietic stem cells[50], MSCs[51]), and unipotent stem cells (e.g., neural stem cells[52]). Additionally, stem cells can be catalogued into natural stem cells (e.g., ESCs[53]) and artificial stem cells (e.g., haploid stem cells[54-56], iPSCs[57]), or classified into autologous stem cells[58] (e.g., autologous hematopoietic stem cells[59,60], autologous adipose tissue-derived stem cells[61], autologous iPSCs[62]) and allogeneic stem cells[63] (e.g., allogeneic germline stem cells[64], allogeneic iPSCs[65], allogeneic adipose-derived mesenchymal stem cells[66]) according to the origins.

Longitudinal studies have indicated the generation of diverse functional liver cell lineages from human pluripotent stem cells (hPSCs) for precision medicine[67], including hepatocytes[68], HSCs, LSECs, and cholangiocytes[1]. Meanwhile, a variety of organoids have been derived from hPSCs for faithful recapitulation of liver diseases and drug susceptibility test[69]. Of note, MSCs reveal robust therapeutic prospect upon certain liver diseases from bench to bedside. However, the differentiation efficiency and the concomitant high cost of hPSCs towards functionally mature cells should be further optimized. Collectively, in this review article, we outline the latest updates of stem cell-based applications in liver diseases, which would benefit the development of novel diagnosis and intervention in future.

DIFFERENTIATION OF STEM CELLS INTO LIVER LINEAGE CELLS

Differentiation into hepatocytes

Proverbially, hepatocytes are highly polarized epithelial cells and heterogeneous parenchymal cells within liver lobule that execute the main synthetic and metabolic functions of the liver[3,67]. Hepatocytes, together with cholangiocytes, are originally differentiated from ventral endoderm-derived hepatoblasts, which are recognized as the main cell type of liver as well as liver progenitor cells for liver development[2,70]. As to liver differentiation, Ang et al[71] detailed described the roadmap of hepatocyte induction from hPSCs via consecutive branching lineage choices orchestrated by spatiotemporally manipulation of extracellular signals (e.g., bone morphogenetic protein, TGF-β, basic fibroblast growth factor, Wnt)[67,71-73].

Currently, hepatocytes and fumarylacetoacetate hydrolase⁺ hepatocyte-like cells with biofunction have been generated from hPSCs [including human ESCs (hESCs) and human induced pluripotent stem cells (hiPSCs)] by utilizing cell programming and reprogramming technologies[4] (Table 1). For instance, Cai et al[74] reported the three-step induction of maturated hepatic cells with liver cell functions from hESCs within 18 days, while Song et al[75] took advantage of a four-step strategy for efficient differentiation of hiPSCs towards hepatocyte-like cells, including endoderm induction, hepatic specification, hepatoblast amplification, and hepatic maturation. Instead, Chen et al[76] reported a three-step protocol (endodermal induction, hepatic commitment and maturation) for the preparation of functional hepatocyte-like cells from hiPSCs (e.g., urea secretion, low-density lipoprotein uptake, glycogen store). For modeling hepatic tumorigenesis with distinct mutational patterns, single or combined oncogenic alterations were introduced into hepatocytes derived from hPSCs, thereby PI3KCA E542K mutant and c-MYC were demonstrated with liver neoplasia-promoting effects by facilitating cancer metabolic reprogramming and inhibiting hepatocyte functions[4]. Additionally, relative strategies have also applied for efficient hPSC-hepatocyte induction and preservation of the functionality, including the “5C” (short for a combination of five chemicals) culture method[77], the ProliHH (short for proliferation human hepatocytes condition) method[4], liver organoid models[78,79], and liver-on-a-chip models[80,81]. Meanwhile, various approaches have been introduced for retaining the function and viability of hepatocytes in vitro by mimicking the in vivo environment (e.g., sandwich culture, coculture with Kupffer cells, and aggregation into spheroids)[82].

Table 1 Differentiation of human pluripotent stem cells into hepatic parenchymal and non-parenchymal cells.

Liver lineages	Stem cell types	Culture medium	Supplements	Days of induction	Biomarkers	Ref.
Hepatocyte-like cells	hiPSCs	RPMI/B27, KO-DMEM with KSR, IMDM	Activin A, Wnt3a, HGF, OSM, Dex, ITS premix	12 days	AFP, albumin, CK18, HNF4, CYP3A4, CYP7A1	Chen et al[76]
Hepatocyte-like cells	hiPSCs	Hepatocyte culture medium, IMDM, N2/B27	Acitivin A, FGF4, BMP2, HGF, KGF, OSM, Dex	21 days	AFP, albumin, CK8, CK18, CK19, PEPCK, HNF4α, HNF6, CEBPα, GATA4, HEX	Song et al[75]
Hepatocyte cells	GATA6- hiPSCs	StemFit04, IMDM/Ham’s F-12K, N2/B27	L-ascorbic acid, Dex, 2-phosphate, basic fibroblast growth factor, 1-thioglycerol, HGF, nicotinamide, OSM	18 days	Albumin, AAT, AFP, HNF4A, CYP3A4, CYP3A5	Luo et al[126]
Hepatic cells	hESCs	DMEM/F12, KSR, 1640 medium	Activin A, aFGF, basic fibroblast growth factor, FGF4, HGF, BMP2, BMP4, Noggin, OSM, Dex, Su5402	18 days	PEPCK, CYP7A1, CYP3A4, CYP2B6	Cai et al[74]
Hepatocytes	hPSCs	DEI medium A/B, CDM3 medium, CDM5 medium	A8301, FGF2, Acitivin A, BMP4, ATRA, AAP, TTNPB, OSM, insulin, forskolin, SB505124, 8-bromo-cAMP, DAPT, CHIR99021, AA2P, RO4929097, Dex	18 days	Fumarylacetoacetate hydrolase, albumin, HGD	Ang et al[71]
Hepatic stellate cells	hiPSCs	Liver differentiation medium	BMP4, FGF1, FGF3, retinol, palmitic acid	12 days	PDGFRβ, PCHD7, COL1α1	Vallverdú et al[2]
Hepatic stellate cells	hPSCs	DMEM low glucose	BMP4, FGF1, FGF3, retinol, palmitic acid, ITS, Dex	12 days	DES, P75NTR, ALCAM, ACTA2, COL1α1, LRAT, RELN, PCDH7, PDGFRβ	Coll et al[91]
Hepatic stellate cells	hESCs	DMEM/F-12, B27, FBS	Activin A, CHIR99021, BMP4, FGF1, FGF3, Y-27632, palmitic acid, retinol	12 days	PDGFRβ, NCAM1, ALCAM, LRAT, ACTA2, vimentin	Wilhelmsen et al[92]
LSECs	hPSCs	EBM-2 medium, IMDM	BMP4, basic fibroblast growth factor, VEGF-A, CHIR99021, cAMP, GSI, SB431542, ITS-X, ascorbic acid, transferrin	22 days	CD34, PECAM1, VWF, LYVE1, CD14, FCGR2B, CD36, CD54, STAB1, FVIII, STAB2, CLEC1B	Gage et al[97]
LSECs	hPSCs	IMDM, DMEM-F12, StemPro34, FCS	BMP4, Activin A, ascorbic acid, basic fibroblast growth factor, L-685-458, ITS-X, VEGF-A, CHIR99021, transferrin	21 days	FCGR2B, LYVE1, STAB2, F8, CD14, MRC1, CD36, RAMP3, CD32B, FVIII	Gage et al[96]
LSECs	Mouse ESCs	IMEM, EGM2-MV, FBS	Adrenomedullin, SB431542, β-ME, VEGF-A, ITS	20 days	F8, Fcgr2b, Mrc1	Arai et al[100]
Cholangiocytes	hPSCs	RPMI 1640 medium, low glucose DMEM, B27, DMEM/F12, a-MEM	MTG, Acitivin A, CHIR99021, ascorbic acid, basic fibroblast growth factor, BMP4, HGF, EGF, OSM, Dex, RA, Noggin, Y-27632, forskolin	49 days	Albumin, AFP, CFTR, TRPV4, PKD1, PKD2	Ogawa et al[101]
Cholangiocytes	hiPSCs	RPMI, B27, William’s E medium	Acitivin A, FGF2, FGF10, BMP4, Ly294002, SB431542, RA, EGF	26 days	SSTR2, ALP, KRT7, SOX9	Sampaziotis et al[102]
Kupffer cells	hiPSCs	X-VIVO 15 media, PHCM, advanced DMEM	M-CSF, IL-3, streptomycin	4-5 weeks	CD163, CD200R, CD86, IL-6, CSF1, HLA-DRB1, IL-1β	Tasnim et al[106]
Kupffer cells	hESCs; cord blood	DMEM/F12, KSR, IMDM, RPMI 1640	BMP4, basic fibroblast growth factor, Activin A, IL-3, IL-7, M-CSF, TPO, SCF	9-17 days (cord blood); > 90 days (hESCs)	MARCO, CD5 L, TIMD4, VCAM1, CPVL, VSIG4, FOLR2, CD163	Kent et al[99]

LSECs: Liver sinusoidal endothelial cells; hiPSCs: Human induced pluripotent stem cells; hESCs: Human embryonic stem cells; hPSCs: Human pluripotent stem cells; ESCs: Embryonic stem cells; KO-DMEM: Knockout Dulbecco’s modified eagle medium; KSR: Knockout serum replacement; IMDM: Iscove’s modified Dulbecco’s medium; CDM3: Chemically defined medium 3; CDM5: Chemically defined medium 5; FBS: Fetal bovine serum; EBM-2: Endothelial basal medium-2; FCS: Fetal calf serum; IMEM: Iscove’s modified eagle medium; EGM2-MV: Endothelial growth medium-2 microvascular; a-MEM: Minimum essential medium alpha modification; PHCM: Primary hepatocyte culture medium; HGF: Hepatocyte growth factor; OSM: Oncostatin M; Dex: Dexamethasone; ITS: Insulin-transferrin-selenium; FGF4: Fibroblast growth factor 4; BMP2: Bone morphogenetic protein 2; KGF: Keratinocyte growth factor; aFGF: Acidic fibroblast growth factor; BMP4: Bone morphogenetic protein 4; FGF2: Fibroblast growth factor 2; ATRA: All-trans retinoic acid; AAP: Ascorbic acid 2-phosphate; AA2P: Ascorbic acid 2-phosphate; FGF1: Fibroblast growth factor 1; FGF3: Fibroblast growth factor 3; VEGF-A: Vascular endothelial growth factor A; cAMP: Cyclic adenosine monophosphate; GSI: Gamma-secretase inhibitor; ITS-X: Insulin-transferrin-selenium-X; β-ME: Β-mercaptoethanol; EGF: Epidermal growth factor; RA: Retinoic acid; M-CSF: Macrophage colony-stimulating factor; IL-3: Interleukin-3; IL-7: Interleukin-7; TPO: Thrombopoietin; SCF: Stem cell factor; AFP: Alpha-fetoprotein; CK18: Cytokeratin 18; HNF4: Hepatocyte nuclear factor 4; CYP3A4: Cytochrome P450 3A4; CYP7A1: Cytochrome P450 7A1; CK8: Cytokeratin 8; CK19: Cytokeratin 19; PEPCK: Phosphoenolpyruvate carboxykinase; HNF4α: Hepatocyte nuclear factor 4 alpha; HNF6: Hepatocyte nuclear factor 6; CEBPα: CCAAT/enhancer binding protein alpha; AAT: Alpha-1-antitrypsin; HNF4A: Hepatocyte nuclear factor 4 alpha; CYP3A5: Cytochrome P450 3A5; CYP2B6: Cytochrome P450 2B6; HGD: Homogentisate 1,2-dioxygenase; PDGFRβ: Platelet-derived growth factor receptor beta; PCDH7: Protocadherin 7; COL1α1: Collagen type I alpha 1 chain; DES: Desmin; P75NTR: P75 neurotrophin receptor; ALCAM: Activated leukocyte cell adhesion molecule; ACTA2: Actin alpha 2, smooth muscle; LRAT: Lecithin retinol acyltransferase; RELN: Reelin; NCAM1: Neural cell adhesion molecule 1; PECAM1: Platelet endothelial cell adhesion molecule 1; VWF: Von willebrand factor; LYVE1: Lymphatic vessel endothelial hyaluronan receptor 1; FCGR2B: Fc gamma receptor IIb; STAB1: Stabilin 1; FVIII: Factor VIII; STAB2: Stabilin 2; CLEC1B: C-type lectin domain family 1 member B; RAMP3: Receptor activity-modifying protein 3; CFTR: Cystic fibrosis transmembrane conductance regulator; TRPV4: Transient receptor potential vanilloid 4; PKD1: Polycystin 1; PKD2: Polycystin 2; SSTR2: Somatostatin receptor; ALP: Alkaline phosphatase; KRT7: Keratin 7; SOX9: SRY-box transcription factor 9; IL-6: Interleukin-6; CSF1: Colony-stimulating factor 1; HLA-DRB1: Human leukocyte antigen-DRB1; IL-1β: Interleukin-1 beta; MARCO: Macrophage receptor with collagenous structure; TIMD4: T-cell immunoglobulin and mucin domain-containing protein 4; VCAM1: Vascular cell adhesion molecule 1; CPVL: Carboxypeptidase, vitellogenic-like; VSIG4: V-set and immunoglobulin domain-containing protein 4; FOLR2: Folate receptor 2.

Adult tissue-derived stem cells are also advantaged alternative sources for generating hepatocytes. For example, Park[83] reported the hepatic differentiation of human adipose-derived stem cells (hASCs) under the effective bio-stimulators (hypoxia, photobiomodulation therapy), and demonstrated the hepatic gene expression profiling (e.g., albumin, alpha-fetoprotein, cytokeratin 8/18) and growth factor secretion of generated hepatocytes. Taken together, both hPSCs and adult stem cells have served as alternative seeding cells for preparation of human hepatocyte source.

Differentiation into HSCs

HSCs, including the quiescent HSCs and the activated counterparts, are nonparenchymal cells and specialized liver pericytes that mainly involve in extracellular matrix (ECM) homeostasis, vitamin A storage and the process of liver fibrosis after injury via communications with Kupffer cells, endothelial cells, and hepatocytes[2,84]. For example, HSCs act as the main contributors to the deposition of ECM and fibrosis progression during chronic liver injury in a fibrosis-independent manner, which thus renders HSCs as a primary candidate target for antifibrotic therapies[85,86]. Differ from hepatocytes, both HSCs and LSECs that originated from mesoderm for hepatocyte nuclear factor-4 alpha+ liver bud progenitor formation, which are essential for the maintenance of liver structure, homeostasis, and adequate response to inflammation and liver fibrosis[87]. Nowadays, HSCs have also been reported with fibropathogenic functions in the development of diverse liver disorders, including cirrhosis, liver fibrosis, portal hypertension, steatotic liver diseases (e.g., MASLD, and alcohol-associated liver disease), and HCC via modulating R-spondin-leucine-rich repeat containing G protein-coupled receptor 4/5-β-catenin signal cascades[86,88-90].

In recent years, investigators have benchmarked multiple culture techniques for functional HSC induction and long-term maintenance of mature HSC characteristics from hPSCs (Table 1). For instance, Vallverdú et al[2] reported a stepwise protocol with cytokine cocktail stimulation at different timepoints for the direct differentiation of hiPSCs into HSCs within 12 days. Coll et al[91] induced HSCs from both H9 hESCs and hiPSCs, which could further form liver spheroids with the in vitro HepaRG hepatocytes and thus competent for liver fibrosis and drug toxicity. Similarly, Wilhelmsen et al[92] detailed described the induction of hESC-HSCs benchmarked to human primary counterparts by integrating TGF-β-induced activation and vitamin A and palmitic acid starvation within 12 days, which highlighted the pivotal effect of energy metabolism in HSC activation. Hence, hPSC differentiation towards HSCs supplies an extensive framework for illuminating the physiological and pathological effects of HSCs during liver homeostasis and diseases[93].

Differentiation into LSECs

LSECs are mesoderm derivatives and function essentially for preserving liver homeostasis, together with collagenization, immunologic responses, pathological adaptation, and pharmacological responses[94,95]. In chronic liver diseases such as MASLD and metabolic dysfunction-associated steatohepatitis (MASH), LSECs revealed cellular dysfunction and capillarization due to the hyperactivation of diverse pro-fibrotic signaling pathways (e.g., TGF-β). These data collectively propose the possibility of LSECs as alternative therapeutic targets in hepatic disorders[94,96].

Of late years, pioneering investigators in the field turned to hPSCs for LSEC generation and the concomitant research on pathogenesis (Figure 1, Table 1). For instance, Gage et al[97] developed a novel induction system for the generation of functional hPSC-LSECs by modulating hypoxia, activating cyclic adenosine monophosphate and TGF-β signaling. The hPSC-LSECs revealed scavenger functions and gene expression profiles of primary human LSECs. By integrating the organoid technology and Wnt 2 stimulation, liver sinusoidal endothelial progenitors and perfused vessels were induced from hiPSCs with functional sinusoid-like features[98]. Interestingly, Ken et al[99] verified that hPSC-LSECs facilitated the development of functional hPSC-derived kupffer cells by practicing the macrophage engraftment test. Additionally, Arai et al[100] reported the enhanced induction of functional LSECs (e.g., endocytosis of acetylated low-density lipoprotein, LSECs-specific biomarkers, and fenestrae-like structure) from mouse ESC-derived lymphatic vessel endothelial hyaluronan receptor 1 + stabilin-2 + endothelial cells by adrenomedullin stimulation and TGF-β signaling abolishment. Therefore, hPSCs supply as advantageous ex vivo models for dissecting LSEC generation and the concomitant pathogenesis of liver diseases.

Figure 1 Illustration of the constituents for human pluripotent stem cells differentiation towards liver lineages. The image shows the constituents for major liver lineage generation from stem cells, including medium, the supplements and cytokine cocktails, and lineage-specific biomarkers. HSCs: Hepatic stellate cells; LSECs: Liver sinusoidal endothelial cells; DMEM: Dulbecco’s modified eagle medium; MARCO: Macrophage receptor with collagenous structure; VCAM1: Vascular cell adhesion molecule 1; bFGF: Basic fibroblast growth factor; TPO: Thrombopoietin; BMP4: Bone morphogenetic protein 4; ALB: Albumin; AFP: Alpha-fetoprotein; CFTR: Cystic fibrosis transmembrane conductance regulator; SSTR2: Somatostatin receptor 2; KRT7: Keratin 7; SOX9: SRY-box transcription factor 9; HGF: Hepatocyte growth factor; MTG: Methyl-β-cyclodextrin; EGF: Epidermal growth factor; LYVE1: Lymphatic vessel endothelial hyaluronan receptor 1; VEGF-A: Vascular endothelial growth factor A; ITS: Insulin-transferrin-selenium; PDGFRβ: Platelet-derived growth factor receptor β; FGF1: Fibroblast growth factor 1; DEX: Dexamethasone; KGF: Keratinocyte growth factor; ATRA: All-trans retinoic acid; FGF4: Fibroblast growth factor 4; IL: Interleukin; M-CSF: Macrophage colony-stimulating factor.

Differentiation into cholangiocytes

Cholangiocytes, also known as bile duct epithelial cells, are cell populations for the formation of the biliary system, which thus play an essential in the production of bile and the lipid digestion[101]. As described by Ogawa et al[101] and Sampaziotis et al[102], cholangiocytes in response to diverse sensory signaling (e.g., IL-6, Notch, epidermal growth factor, TGF-β, retinoic acid) or ion channel alterations in bile flow via primary cilia, and thus modulate liver homeostasis as well as cholangiocyte dysfunction[101,102].

The derivation of cholangiocytes from hPSCs extensively facilitates the study of the physiological cholangio-genesis and pathogenesis of cholangiopathies (e.g., Alagille syndrome, biliary atresia, cystic fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis), together with novel cellular therapies[103] (Table 1). For example, Ogawa et al[104] reported the cystic fibrosis transmembrane conductance regulator+ cholangiocyte lineage specification from hPSC-derived hepatoblasts via staged Notch signal activation and the following cocktail stimulation for DHIC5-4D9+ mature cholangiocyte cysts. Similarly, Sampaziotis et al[102] introduced a five-step protocol for cholangiocyte-like cell (CLC) differentiation from hiPSCs, including definitive endoderm, foregut progenitors, hepatoblasts, cholangiocyte progenitors, and CLCs. To date, both the spontaneous and guided cholangiocyte differentiation of hPSCs has been achieved, yet the poor differentiation efficiency and the considerable differences from primary biliary tissue should be further optimized[1,105].

Differentiation into Kupffer cells

Kupffer cells, also known as resident liver macrophages, are structurally located at the luminal side of sinusoids and play a pivotal role in drug-induced liver injury and diverse liver diseases (e.g., drug-induced liver injury, cholestasis)[67,106]. In mice, Kupffer cells are also recognized as the first liver-resident macrophages derived from yolk sac hematopoietic progenitors prior to hematopoietic stem cell emergence in embryonic life[99,107-109].

For the recapitulation of yolk sac-like hematopoiesis, hPSC-derived kupffer cells were transplanted into NSG mice, and the homogeneous MARCO (macrophage receptor with collagenous structure)- expressing genetic signature and functional maturation (e.g., phagocytosis, erythrophagocytosis) were observed in the engrafted hPSC-derived kupffer cells according to single-cell RNA sequencing (scRNA-seq) analysis[99]. Instead, Tasnim et al[106] recapitulated Kupffer cell ontogeny in vitro from MYB-independent hiPSC-derived macrophage- precursors, which phagocytosed and secreted cytokines (e.g., IL-6, TNF-α) upon inflammation-associated drug stimulation (e.g., acetaminophen, chlorpromazine, and trovafloxacin) comparable to donor-mismatched counterpart. Of note, despite the signatures of Kupffer cells have been extensively explored and well established, yet the underlying molecular mechanisms modulating the derivation and maturation from hPSCs and the progenitor cells are largely obscure[110] (Figure 1, Table 1).

STEM CELL-BASED ORGANOIDS FOR LIVER DISEASE DIAGNOSIS

Liver organoids

Organoids are three-dimensional (3D) “in a dish” models with remarkable self-organizing structures for exploring the physiological and pathological processes, which commonly generate from stem cells (e.g., adult stem cells, hPSCs) and recapitulate the key structural and functional signatures of the native organs[69]. For decades, liver organoids have been extensively reported for recapturing spatial liver architecture, cellular heterogeneity, and stimulations of liver microenvironment[69,111] (Figure 2, Table 2).

Figure 2 Stem cell-based liver organoids. Illustration image shows the major subtypes of stem cell-based liver organoids and the corresponding biomarkers. ALB: Albumin; HNF4A: Hepatocyte nuclear factor 4 alpha; HNF1B: Hepatocyte nuclear factor 1 beta; KRT7: Keratin 7; KRT19: Keratin 19; AFP: Alpha-fetoprotein; A1AT: Alpha-1-antitrypsin; CEBPA: CCAAT/enhancer binding protein alpha; CYP: Cytochrome P450; CK7: Cytokeratin 7; STAB2: Stabilin 2; LYVE1: Lymphatic vessel endothelial hyaluronan receptor 1; CK18: Cytokeratin 18; FCGR2B: Fc gamma receptor IIb; ADRA1B: Alpha-1B adrenergic receptor; TBX3: T-box transcription factor 3; WT1: Wilms tumor 1; ASGR1: Asialoglycoprotein receptor 1; ASGR2: Asialoglycoprotein receptor 2; SERPINA1: Serpin family a member 1; TTR: Transthyretin; TDO2: Tryptophan 2,3-dioxygenase; COL1A1: Collagen type I alpha 1 chain; TIMP1: Tissue inhibitor of metalloproteinases 1; KRT17: Keratin 17; TACSTD2: Tumor-associated calcium signal transducer 2.

Table 2 Liver organoids generated from human pluripotent stem cells.

Organoid types	Stem cells	Biomarkers	Characteristics	Ref.
Vascularized mLOs	hPSCs	HNF1B, HNF4A, albumin, CEBPA, CYP	The mLOs was superior to 2D Hep for HPC maturation; mLOs comprise hepatic- and non-parenchymal cells; mLOs with enhanced structural complexity and functionality	Chi et al[112]
Liver organoids	hPSCs	AFP, albumin, HNF4α, A1AT, KRT19, KRT7, albumin	The hPSC-LOs with various liver cell types highly simulated DENV infection and screened for antivirals	Li et al[69]
Liver organoids	hiPSCs	Albumin, HNF4α, KRT7, KRT19, CK7	The in situ formation of hiPSC-LOs were achieved in microporous array chips; the hiPSC-LOs were competent for evaluating the efficacy of semaglutide for NAFLD	You et al[221]
Liver bud organoids (hLBOs)	hPSCs	STAB2, LYVE1, CD32, CD36, FCGR2B	The vascularized hLBOs contain hepatic and endothelial subpopulations that show key organ-specific functional features, and can restore coagulation factor deficiencies	Saiki et al[98]
Liver bud organoids (hLBOs)	hiPSCs	Albumin, CK18, HNF4α, AFP	The hiPSC-derived hLBOs were competent for whole body in vivo cell tracking and the resultant cell therapy development	Ashmore-Harris et al[119]
Liver bud organoids (hLBOs)	hPSCs	TBX3, ADRA1B, HNF4α, WT1, ADRA1B, LHX2, CD144, CD31	The strategy fulfilled the mass production and batch validation of self-organizing hLBOs with in vitro functions and in vivo therapeutic potential from hPSCs	Takebe et al[118]
Hepatocyte organoids	hiPSCs	Albumin, ASGR1, ASGR2, A1AT, AFP, SERPINA1	HiPSCs were transduced with adeno-associated virus vectors and the hepatocyte organoids enhanced the translation of gene therapies	Berreur et al[125]
Hepatostellate organoids	hiPSCs	Albumin, TTR, TDO2, COL1A1, TIMP1, KRT17, TACSTD2	The hepatostellate organoids were derived from hiPSCs with dyskerin 1 mutation, and would benefit the studies upon telomere dysfunction–induced liver disease	Choi et al[128]

mLO: Multi-lineage liver organoids; hLBOs: Human liver bud organoids; hPSCs: Human pluripotent stem cells; hiPSCs: Human induced pluripotent stem cells; HNF1B: Hepatocyte nuclear factor 1 beta; HNF4A: Hepatocyte nuclear factor 4 alpha; CEBPA: CCAAT/enhancer binding protein alpha; CYP: Cytochrome P450; AFP: Alpha-fetoprotein; A1AT: Alpha-1-antitrypsin; KRT19: Keratin 19; KRT7: Keratin 7; CK7: Cytokeratin 7; STAB2: Stabilin 2; LYVE1: Lymphatic vessel endothelial hyaluronan receptor 1; FCGR2B: Fc gamma receptor IIb; CK18: Cytokeratin 18; TBX3: T-box transcription factor 3; ADRA1B: Alpha-1B adrenergic receptor; WT1: Wilms tumor 1; LHX2: LIM homeobox 2; ASGR1: Asialoglycoprotein receptor 1; ASGR2: Asialoglycoprotein receptor 2; SERPINA1: Serpin family a member 1; TTR: Transthyretin; TDO2: Tryptophan 2,3-dioxygenase; COL1A1: Collagen type I alpha 1 chain; TIMP1: Tissue inhibitor of metalloproteinases 1; KRT17: Keratin 17; TACSTD2: Tumor-associated calcium signal transducer 2; 2D Hep: 2D Hepatocytes; HPC: Hepatic progenitor cells; hPSC-LOs: Human pluripotent stem cell-derived liver organoids; DENV: Dengue virus; hiPSC-LOs: Human induced pluripotent stem cell-derived liver organoids; NAFLD: Non-alcoholic fatty liver disease.

Recently, Li et al[69] integrated the hPSC-derived liver organoid platform and scRNA-seq technology for recapitulating dengue virus infection and antiviral screening. The functional and expandable hPSC-derived liver organoids are consisted of multitudinous liver cell types (e.g., hepatocyte-like cells, intrahepatic CLCs, hepatocyte-like cells, and hepatic stellate-like cells), and thus competent for mimicking liver functional disability and validating drug response during infection. Multi-lineage liver organoids (mLOs) composed of hepatobiliary cell lineages are promising for simulating the functional maturity and structural complexity of the organ physiologically and pathologically[112]. By modulating the cyclic adenosine monophosphate/Wnt/Hippo signaling pathways, Chi et al[112] generated vascularized mLOs containing hepatic parenchymal and non-parenchymal cells (e.g., hepatocytes, endothelial cells cholangiocytes, and HSCs) from hPSCs within 16 days. With the aid of liver organoids, Kim et al[113] proposed hepcidin antimicrobial peptide 1 as predicting biomarkers for monitoring and optimizing saponin response (e.g., fat accumulation, hepatic inflammation) in MASLD, while it was developed that non-alcoholic fatty liver disease (NAFLD)-specific hiPSC-derived liver organoids recapitulating disease hallmarks for semaglutide efficacy evaluation. Of note, region-specific liver organoids were fabricated by 3D bioprinting, which exhibited characteristics of hierarchical functional liver lobule and manifested considerable performance in liver disease modeling and hepatotoxicity prediction[95]. Additionally, liver organoids derived from hPSCs or MSCs have been involved in pharmaceutical testing and relative liver disease modeling such as liver cirrhosis[114], periductal fibrosis[115], and HCC[116,117]. Taken together, stem cell-derived liver organoids display an uncanny self-organizing capacity for reflecting crucial aspects of the organ.

Liver bud organoids

In recent years, talented investigators have reported the massive and reproducible induction of human liver bud organoids from hESCs and hiPSCs under the two-dimensional (2D) and 3D culture conditions[118] (Figure 2, Table 2). Very recently, Saiki et al[98] reported the direct differentiation of hPSCs into CD32b+ putative liver sinusoidal progenitors by utilizing a multilayered air-liquid interface culture for liver bud organoid formation. In detail, a stepwise differentiation system was established for the induction of vascularized 3D liver bud organoids and the derivatives from hPSCs, including LSEC progenitors, hepatic and endothelial subpopulations. Another study by Ashmore-Harris et al[119] turned to hiPSCs with a radionuclide reporter for the construction of in vivo trackable liver bud organoids in liver-injured mice, and demonstrated the differentiated function towards hepatocyte-like cells. Interestingly, with the aid of 2D culture and the organ-on-a-chip platform, Yoshimoto and the colleagues observed the promoting effect of cyclic mechanical stretching upon angiocrine signals during hPSC differentiation towards vascularized liver organoids during the liver bud stage[120]. These findings would help improve the engineered liver tissue for patients with hepatopathy.

Hepatocyte organoids

The ex vivo maintenance and expansion of primary hepatocytes with functional characteristics are long-standing challenges for investigators in the field[121]. Over the years, a variety of 2D and 3D methodologies have been developed for the construction and long-term expansion of primary hepatocyte organoids from stem cells and liver counterparts (e.g., hepatocytes, bile-duct epithelial cells)[122,123]. Longitudinal studies have demonstrated the feasibility of hPSCs for ex vivo hepatocyte organoid induction[124] (Figure 2, Table 2). For instance, Berreur et al[125] transduced eight serotypes of recombinant adeno-associated virus vectors into hiPSCs, and tested the efficiency and hepatotoxicity in iPSC-derived hepatocyte organoids. Differ from the conventional strategies with diverse limitations, Luo et al[126] recently turned to hiPSCs with a doxycycline-inducible GATA6 expression for the synchronous differentiation into homogeneous hepatocytes and multi-lineage non-parenchymal liver cells in hepatocyte organoids within 18 days, which offered new opportunities for liver disorder modeling, liver development investigation, and regenerative medicine. Besides stem cells, liver organoids can also be derived from fetal or adult liver cells (e.g., adult hepatocytes) encapsulated in ECM via self-organization, which retain the tissue-of-origin commitment of liver cells and are qualified for genetic manipulation in vitro[23,127]. Therefore, stem cells of diverse subtypes hold promising prospect for the generation of hepatocyte organoids and homogeneous hepatocytes.

Hepatostellate organoids

Compared to the abovementioned types of hPSC-derived liver organoids, there are minimal pieces of literatures upon hepatostellate organoid construction (Figure 2, Table 2). For example, Choi et al[128] reported the generation of hepatostellate organoids from patient hiPSCs with a causal dyskeratosis congenital mutation in dyskerin 1, which supplied novel platforms for dissecting telomere dysfunction-induced liver diseases. In details, the hiPSCs were incipiently programmed into hepatocytes or HSCs, and followed by the formation of genotype-admixed hepatostellate organoids[128]. Overall, the hepatostellate organoids provide new avenues for illuminating genotype-phenotype relationships and liver pathologies in telomeropathies.

Organoid-based clinical trials

Due to the robust attributes of stem cell-derived organoids, clinicians are devoted to organoid-based precision medicine for liver disease prediction (e.g., liver resection, drug screen, the response to drugs, hepatic disease development). According to ClinicalTrials.gov website of National Institutes of Health (https://clinicaltrials.gov/), a total number of 9 trials (4 interventional trials and 5 observational ones) with 662 participants have been registered (up to September 10, 2025) for liver diseases (Figure 3A and B, Table 3). The registered clinical trials are distributed in China (4 trials), Italy (4 trials) and United Kingdom (1 trial) (Figure 3C). Of them, except one for non-alcoholic steatohepatitis, all the other trials were registered upon liver cancers, including HCC (n = 6), cholangiocarcinoma (n = 1) and liver metastases (n = 1) during colorectal cancer (n = 1). Furthermore, the enrolled participants (shown as log₁₀N12) in the corresponding types of trials [shown as condition (N11)] were shown according to the line (Figure 3D, Table 3). Considering the recruiting and active (not recruiting) status of the abovementioned studies, there’s still far from adequate for large-scale application for patients with intractable liver diseases.

Figure 3 Organoid-based clinical trials for liver diseases. A: The categories of the registered liver organoid-based clinical trials according to ClinicalTrial.gov; B and C: The status and location of the abovementioned liver organoid-based clinical trials; D: The distribution of the organoid-based clinical trials for liver diseases according to clinical trial number [condition (N11)] and the corresponding enrolled participants [enrollment (Log₁₀N12)]. HCC: Hepatocellular carcinoma; NASH: Non-alcoholic steatohepatitis.

Table 3 Clinical trials of liver organoids for liver diseases.

NCT No.	Study title	Study status	Condition	Enrollment	Study type	Location
NCT06929845	“Organoid Models of Hepatocellular Carcinoma”	Recruiting	HCC	150	Interventional	Italy
NCT02436564	“In Vitro Models of Liver and Pancreatic Cancer”	Unknown	Cholangiocarcinoma	75	Observational	United Kingdom
NCT05913141	“PDO/PDO-TIL/PDOTS for Drug Screen”	Recruiting	HCC	30	Observational	China
NCT06077591	“Prospective Clinical Validation of Next Generation Sequencing and Patient-Derived Tumor Organoids (PDO) Guided Therapy in Patients with Advanced/ Inoperable Solid Tumors”	Recruiting	HCC	40	Interventional	China
NCT06355700	“Hepatocellular Carcinoma Liver Organoids”	Recruiting	HCC	10	Interventional	Italy
NCT06699524	“Construction of a Recurrence Risk Prediction Model for Liver Resection Based on Drug Sensitivity of Patient-derived Hepatocellular Carcinoma Organoid”	Active not recruiting	HCC	122	Observational	China
NCT06856252	“Human Liver Organoids as a Model to Study the Development of NASH”	Recruiting	NASH	60	Interventional	Italy
NCT06787625	“Development of Organoids From Primary Colorectal Tumors and Synchronous Liver Metastases”	Recruiting	Colorectal cancer	10	Observational	Italy
NCT05932836	“An Organoid-on-chips Technique Based on Biopsy Samples and Its Efficacy in Predicting the Response to HAI in HCC”	Active not recruiting	HCC	165	Observational	China

HCC: Hepatocellular carcinoma; NASH: Non-alcoholic steatohepatitis.

STEM CELLS FOR LIVER DISEASE INTERVENTION

Acute liver injury

Acute liver injury (ALI) serves as a main inducement of liver failure and the concomitant microcirculatory dysfunction, which is commonly caused by drug-induced hepatotoxicity and proinflammatory factors[5,129]. For example, numerous literatures have indicated the drug overdose resultant ALI and the molecular mechanism by acetaminophen[130], together with the hepatoprotective effects of regimens (e.g., CpG-oligodeoxynucleotides[131], crizotinib[132], pristimerin[133]). Among them, formoterol acetaminophen reveals alleviative effect against acetaminophen-induced ALI via suppressing the lipid peroxidation-mediated ferroptosis, while liraglutide ameliorates radiation-induced hepatotoxicity in ALI via modulating the liver kinase B1/AMP-activated protein kinase/mammalian target of rapamycin axis[130,134].

State-of-the-art literatures have highlighted the feasibility of stem cells (e.g., MSCs) and the derivatives (e.g., microvesicles, MSC-exosomes, hPSC-derived hepatocytes) for ALI intervention[135-137]. For example, Chen et al[138] invigorated hASCs with anti-inflammation and antioxidant defenses for elevated efficacy upon ACLF via orchestrating the shift from the anti-regenerative interferon-γ/signal transducer and activator of transcription 1 axis to the pro-regenerative IL-6/signal transducer and activator of transcription 3 axis, whereas Lin et al[137] put forward the protective response of MSCs against ferroptosis in ALI. Instead, Zhang et al[5] turned to cell programming strategy for the efficient induction of MSCs from hESCs, and confirmed the ameliorative efficacy upon ALI mice comparable to BM-MSCs.

Meanwhile, current literatures have also indicated the ameliorative effect of MSC-extracellular vesicles (EVs) upon ALI[135,139,140]. For example, exosomes enriched from human exfoliated deciduous teeth-derived stem cells revealed notable regenerative effects in the context of ALI model, including mitigated damage, enhanced hepatocyte proliferation, macrophage polarization, and reduced inflammation[141]. With the aid of MSC-exosomes, Cai et al[140] verified the attenuated effect and potential mechanisms of MALAT1/miR-26a-5p axis-mediated ALI. Notably, the inherent interindividual and batch-to-batch variability in biological functions (e.g., differentiation capacity, immunomodulatory property) and genetic phenotypes (e.g., gene expression profiles) of MSCs and the derivatives including EVs for ALI administration should not be neglected in clinical translation.

Acute liver failure

ALF is recognized as a life-threatening disorder and critical clinical syndrome characterized by substantial hepatocyte loss, massive tissue necrosis, severe functional deterioration, hepatic encephalopathy, excessive oxidative stress, coagulopathy, metabolic dysregulation and massive hepatocyte death[142,143]. ALF is commonly caused by a series of inducements such as drug hepatotoxicity, herbal and dietary supplements, hepatotropic and non-hepatotropic viruses, and autoimmune hepatitis[144]. Recently, He et al[142] verified ferroptosis and ferroptotic cell death as predominant pathogenesis in hepcidin-ferroportin-related ALF.

Considering the bidirectional immunomodulatory capacity of MSCs, it is imperative to develop innovative cell therapy for triggering the progression of ALF[142,145]. For instance, MSCs manifested superior therapeutic efficacy upon ALF via modulating hepcidin-ferroportin axis and suppressing PI3K/AKT/nuclear factor erythroid-2-related factor 2 pathway or Notch1-yes-associated protein 1 circuit, which put forward the significance of liver management in ALF via iron homeostasis and ferroptosis[142,146]. Instead, Tao et al[147] demonstrated the alleviative effect of human umbilical cord-derived MSCs (hUC-MSCs) upon ALF and liver inflammation through synchronously inhibiting c-Jun N-terminal kinase/nuclear factor-кB activation-induced hepatocyte apoptosis and proinflammatory macrophage M1 polarization[147]. Interestingly, Zhang et al[143] integrated MSC spheroids and immunoregulatory decellularized EVs for the amelioration of ALF, which revealed enhanced anti-oxidative and pro-angiogenic effects, together with superior hepatic cell differentiation. Of note, MSC-derived EVs (MSC-EVs) alone or engineered with biomacromolecules like chimeric antigen receptor also reveal considerable efficacy in ALF[148-150]. In all, MSC-based regimens propose a novel multifaceted strategy dispense with liver transplantation for conquering refractory liver failure like ALF.

MASLD

MASLD, previously known as NAFLD, is characterized by hepatocyte ballooning and liver damage in analogy with alcoholic fatty liver disease[113,151]. MASLD has been recognized as a metabolic and systemic disorder encompasses the metabolic dysfunction-related fatty liver and MASH subtypes based on hepatic steatosis[152,153]. The latest updates emphasize the superior attributes of MSCs and MSC-EVs in culture supernatant for MASLD management[154-157]. Very recently, Tunstead et al[158] combined MSCs with exogenous mitochondrial pretreatment or palmitate for enhancing the therapeutic efficacy or immunomodulation capacity of macrophages upon NAFLD, respectively. These findings suggest that stem cells like MSCs serve as novel avenues for chronic liver disease treatment including MASLD.

As to EVs of different origins including stem cells, investigators have also detailed described the applications for MASLD administration[159]. For instance, MSC-EVs were adequate to suppress inhibitor of κB alpha/nuclear factor-κB/angiopoietin-2 pathway hyperactivation-mediated LSEC angiogenesis in MASH mice, and the promoting effect could be attenuated by USP9X knockdown[160]. By conducting high-fat diet-induced MASLD model, researchers observed the inhibitory effect of MSC-EVs upon mitochondrial fission and lipid deposition in hepatocytes, which indicated the potential mechanism in hepatocyte steatosis and MASLD progression[161]. Meanwhile, MSC-EVs play an important role in counteracting oxidative stress in both in vivo and in vitro models, and represent a novel therapeutic avenue for halting or reversing liver disease progression including MASLD and MASH[154]. Instead, Kasahara et al[162] reported the controlled release of MSC conditioned medium encapsulated in hydrogel for MASLD rat treatment, which increased immunoregulatory cytokines and hepatocyte size and functionality (e.g., hepatic ATP, β-hydroxybutyrate). Even though the remarkable progress in preclinical investigations, the potential variations in clinical practice owing to the differences in species and infusion doses should be further dissected during MSCs-based regimens for MASLD administration.

Liver fibrosis

As a complex progressive condition and critical health issue, liver fibrosis is characterized by excessive accumulation of ECM and scar tissue buildup, and commonly results in functional impairment and liver cirrhosis[163,164]. Generally, liver fibrosis initially encapsulates the injury and thus acts as a protective and reversible response, yet the prolonged damage usually leads to liver cirrhosis and even the life-threatening HCC[165]. There are two major approaches for liver fibrosis reversion in clinical practice, including the fibrosis process abolishment and the underlying insult elimination[166]. Despite diverse causes and underlying mechanisms have been identified for pathogenesis, yet the limitations in liver fibrosis treatment options largely hinder the reversibility and recovery of liver fibrosis[166].

Nowadays, stem cells like MSCs of different origins have showed robust prospects in the administration of liver fibrosis attribute to the intercellular communications and the suppression of key fibrotic pathways. For instance, BM-MSCs infusion is adequate to ameliorate liver fibrosis in tetrachloromethane-induced liver fibrosis model via anti-inflammation and immunoregulation[167,168]. Instead, Gunardi et al[169] compared the intrahepatic and intrasplenic infusion of MSCs for liver fibrosis treatment in a bile duct ligation rabbit model, and confirmed the facilitating effect upon hepatocyte proliferation whereas minimal differences were observed in liver function and liver fibrosis severity between the two routes. Of note, hepatocyte-like cells derived from hUC-MSCs revealed minimal effect upon the acceleration of the venularization and capillarization of hepatic sinusoids in a chronic liver fibrosis model[170]. Simultaneously, EVs derived from MSCs (MSC-exosomes) and liver-resident cells integrated with biomaterials stand out for their pharmacological and therapeutic potential against liver fibrosis. For example, Sani et al[171] put forward the synergistic effect of MSC-exosomes and anti-miR17-5p for the enhanced efficacy upon preclinical liver fibrosis model, and thus demonstrated the feasibility of MSC-exosome as promising therapeutic options against liver fibrosis. Despite the numerous progress in stem cell-based tactics for liver fibrosis intervention, the concomitant practical challenges should not be neglected such as efficacy heterogeneity, variations in cell sources, and post-transplantation survival rates. For example, we and Zhang et al[172] reported the variations of MSCs at diverse passages in long-term effectiveness and homing ability, which collectively indicated the urgency for the establishment of standardization and guidelines for therapeutic purposes[45,172,173].

Liver cirrhosis

Liver cirrhosis is the final phase of the reversible liver fibrosis, characterized by extensive liver scarring and the resultant hepatic dysfunction and fatal complications[174,175]. Patients with the inflammatory and fibrosing biliary atresia commonly lead to a rapid deterioration towards severe liver cirrhosis[176]. To overcome the issue in the chronic liver disease, Nguyen et al[177] conducted allogeneic UC-MSC administration, and confirmed the safety and improved liver function (e.g., improved alkaline phosphatase, better biochemical profiles) in patients with liver cirrhosis.

Currently, stem cell-based regimens have been recognized as encouraging innovative therapy for end-stage liver diseases including liver cirrhosis[178,179]. Of them, MSCs are splendid cell sources for liver cirrhosis management ascribe to the unique properties, including cytokine paracrine (e.g., vascular endothelial growth factor, hepatocyte growth factor) and multi-lineage lineage differentiation capacity towards hepatocyte-like cells[174,180]. The trophic signaling pathways of MSCs are vastly involved in angiogenesis, hepatocyte proliferation, liver recovery and regeneration such as PI3K/AKT pathway and TGF-β/Smad pathway[174,181]. In a randomized controlled clinical trial upon hepatitis B virus-related decompensated liver cirrhosis, Shi et al[178] observed the improved long-term survival rate and liver function in decompensated liver cirrhosis patients after infusion of UC-MSCs. In another phase Ia/Ib trials, Shi et al[178] recently verified the outcomes and dose-effect relationship of liver cirrhosis patients with three doses of MSC infusion, and observed the stronger immunomodulatory effects induced by higher MSC doses[182]. Therefore, stem cells like MSCs provide novel paradigms for irreversible liver cirrhosis and the severe complications. Meanwhile, it’s noteworthy that the safety and effectiveness of stem cell infusion for patients with liver cirrhosis should be further verified due to the limitations of small-scale enrollments.

ACLF

ACLF, a severe form of cirrhosis, is a syndrome characterized by liver function decline, acute liver deterioration, multiorgan failure and high short-term mortality, which has become a major challenge in hepatology and global health burden[183-185]. Of the therapeutic regimens, liver transplantation serves as the most effective option for ACLF patients, yet multifaceted undeniable limitations should be further resolved such as high costs, surgical complications, liver donor shortage and immunosuppressive therapy[186].

Encouragingly, more and more investigators are devoted to developing the stem cell-based iatreusis for liver diseases including ACLF in preclinical disease models and clinical practice[187,188]. Of them, ACLF patients with MSC infusion have been reported with safety (e.g., adverse events, serious adverse events) and liver function improvement (elevated albumin level, decreased MELD score)[189,190]. For instance, Heo et al[186] reported the enhanced differentiation of hBM-MSCs into functional hepatocyte-like cells, while Liu et al[190] performed systematic meta-analysis and concluded the safety and efficacy of both hUC-MSCs- and BM-MSCs- based novel therapeutics upon ACLF and the concomitant clinical symptoms (e.g., encephalopathy, gastrointestinal hemorrhage events). As to the cell-free MSC-exosomes, the beneficial pleiotropic properties (e.g., low immunogenicity, good safety profile, anti-fibrotic cytokines) and hepatoprotetive effects (e.g., suppress HSC activation, improve metabolism, anti-apoptosis, anti-inflammatory, anti-ferroptosis) upon ACLF have also been repeatedly highlighted[185]. Additionally, the let-7a-5p/MAP4K3 (mitogen-activated protein kinase kinase kinase kinase 3) axis in MSC-exosomes were reported in mediating autophagy repairmen in ACLF, which would benefit the illumination of the intrinsic mechanisms and targeted cell therapy in future[191].

Clinical trials of stem cell-based therapeutics

To present the systematic and detailed information of stem cell-based therapeutics, we turned to the registered clinical trials according to the ClinicalTrial.gov database (up to September, 2025). In total, 169 registered stem cell-related trials were gained according to the keywords (condition/disease: Liver diseases; intervention/treatment: Stem cells), and finally 45 trials with the “completed” (n = 33), “recruiting” (n = 9) or “terminated” (n = 3) status were further enriched for liver disease diagnosis and treatment including 36 interventional trials and 9 observational ones (Figure 4A and B, Table 4). Of them, most of the trials were under the early stage for verifying the safety and efficacy of stem cell transplantation upon liver diseases, including 7 in phase 1, 15 in phase 1/2, 5 in phase 2, 2 in phase 3, 1 in phase 4, and 15 with the unknown phase (N/A) (Figure 4C, Table 4).

Figure 4 Stem cell-based clinical trials for liver diseases. A: The categories of the registered stem cell-based clinical trials for liver diseases according to ClinicalTrial.gov; B-D: The status, clinical stage and location of the abovementioned stem cell-based clinical trials; E and F: The distribution of stem cell-based clinical trials for liver diseases according to the enrolled participants [enrollment (Log₂N21)] and the corresponding cell types (N22) or the clinical trial number [condition (N23)]. BM-EPCs: Bone marrow-derived endothelial progenitor cells; LC: Liver cells; MSCs: Mesenchymal stem/stromal cells; hiPSCs: Human induced pluripotent stem cells; BM-MSCs: Bone marrow-derived mesenchymal stem/stromal cells; UC-MSCs: Umbilical cord-derived mesenchymal stem/stromal cells; ACLF: Acute-on-chronic liver failure; NASH: Non-alcoholic steatohepatitis.

Table 4 Clinical trials of stem cell-based observational and interventional studies for liver diseases.

NCT No.	Study title	Cell types	Study status	Condition	Phase	Enrollment	Study type	Location
NCT00953693	“Patient Specific Induced Pluripotency Stem Cells (PSiPS)”	hiPSCs	Completed	Liver insufficiency	N/A	15	Observational	Iran
NCT01591200	“Dose Finding Study to Assess Safety and Efficacy of Stem Cells in Liver Cirrhosis”	MSCs	Completed	Alcoholic liver cirrhosis	Phase 2	40	Interventional	India
NCT01429038	“MSCs After Renal or Liver Transplantation”	MSCs	Completed	Liver failure	Phase 1/2	40	Interventional	Belgium
NCT00655707	“A Phase I/II Safety and Tolerability Dose Escalation Study of Autologous Stem Cells to Patients With Liver Insufficiency”	Adult stem cells	Completed	Liver insufficiency	Phase 1/2	5	Interventional	United Kingdom
NCT02089919	“Cancer Stem Cells Vaccine Therapy in Treating Hepatocellular Cancer Patients”	Cancer stem cells	Completed	HCC	Phase 1/2	40	Interventional	China
NCT00147043	“Adult Stem Cell Therapy in Liver Insufficiency”	Adult stem cells	Completed	Liver cirrhosis	N/A	5	Interventional	United Kingdom
NCT04243681	“Combination of Autologous MSC and HSC Infusion in Patients With Decompensated Cirrhosis”	MSCs + hematopoetic stem cells	Completed	Decompensated liver cirrhosis	Phase 4	5	Interventional	India
NCT01333228	“Evaluate Safety and Efficacy of Autologous Bone Marrow-derived Endothelial Progenitor Cells in Advanced Liver Cirrhosis”	BM-EPCs	Completed	Liver cirrhosis	Phase 1/2	14	Interventional	Spain
NCT05331872	“Umbilical Cord-derived MSC Infusion in the Management of Adult Liver Cirrhosis”	UC-MSCs	Completed	Liver cirrhosis	Phase 1	20	Interventional	Vietnam
NCT00420134	“Improvement of Liver Function in Liver Cirrhosis Patients After Autologous MSC Injection: A Phase I-II Clinical Trial”	Autologous MSCs	Completed	Liver cirrhosis	Phase 1/2	30	Interventional	Iran
NCT01454336	“Transplantation of Autologous MSC in Decompensated Cirrhotic Patients With Pioglitazone”	Autologous MSCs	Completed	Decompensated liver cirrhosis	Phase 1	3	Interventional	Iran
NCT02557724	“Mobilization of MSCs During Liver Transplantation”	MSCs	Completed	Liver failure, HCC	N/A	35	Observational	United States
NCT01875081	“REVIVE (Randomized Exploratory Clinical Trial to Evaluate the Safety and Effectiveness of Stem Cell Product in Alcoholic Liver Cirrhosis Patient)”	Stem cells	Completed	Alcoholic liver cirrhosis	Phase 2	72	Interventional	South Korea
NCT01013194	“Human Fetal Liver Cell Transplantation in Chronic Liver Failure”	Fetal liver cells	Completed	Liver cirrhosis	Phase 1/2	25	Interventional	Italy
NCT02297867	“Clinical Trial Study About Human Adipose-Derived Stem Cells in the Liver Cirrhosis”	ASCs	Completed	Liver cirrhosis	Phase 1	6	Interventional	China
NCT01378182	“Efficacy of In vitro Expanded Bone Marrow Derived Allogeneic MSC Transplantation Via Portal Vein or Hepatic Artery or Peripheral Vein in Patients With Wilson Cirrhosis”	BM-MSCs	Completed	Wilson’s disease	NA	10	Interventional	Turkey
NCT01120925	“Autologous Bone Marrow Derived Stem Cells in Decompensated Cirrhotic Patients”	BM-MSCs	Completed	Decompensated liver cirrhosis	Phase 1/2	30	Interventional	Iran
NCT01220492	“Umbilical Cord MSCs for Patients With Liver Cirrhosis”	UC-MSCs	Completed	Liver cirrhosis	Phase 1/2	266	Interventional	China
NCT01342250	“Human Umbilical Cord MSCs Transplantation for Patients With Decompensated Liver Cirrhosis”	UC-MSCs	Completed	Liver cirrhosis	Phase 1/2	20	Interventional	China
NCT03468699	“Autologous Bone Marrow Mononuclear Stem Cell for Children Suffering From Liver Cirrhosis Due to Biliary Atresia”	BM-MSCs	Completed	Liver fibrosis (biliary atresia)	Phase 2	17	Interventional	Vietnam
NCT04522869	“Umbilical Cord Derived MSC (UC -MSC) Transplantation for Children Suffering From Biliary Atresia”	UC-MSCs	Completed	Primary biliary cirrhosis	Phase 1/2	20	Interventional	Vietnam
NCT01625351	“A Study of CD45RA+ Depleted Haploidentical Stem Cell Transplantation in Children With Relapsed or Refractory Solid Tumors and Lymphomas”	Hematopoetic stem cells	Completed	Hepatic tumor	Phase 1	23	Interventional	United States
NCT00003966	“Defibrotide in Treating Patients With Liver Damage Following Peripheral Stem Cell Transplantation”	Peripheral stem cells	Completed	Veno-occlusive disease	Phase 2	151	Interventional	United States
NCT03132337	“Sinusoidal Obstruction Syndrome for Stem Cell Transplant Patients Biomarker Study”	Stem cells	Completed	Sinusoidal obstruction syndrome	N/A	80	Observational	United States
NCT03963921	“Safety and Tolerability of HepaStem in Patients With Cirrhotic and Pre-cirrhotic NASH Patients”	HepaStem	Completed	NASH	Phase 1/2	23	Interventional	Belgium, Spain
NCT00956891	“Therapeutic Effects of Liver Failure Patients Caused by Chronic Hepatitis B After Autologous MSCs Transplantation”	Autologous MSCs	Completed	Liver failure	N/A	158	Observational	China
NCT02727673	“Relationship Between Circulating Tumor Stem Cells and the Clinical Pathology”	Tumor stem cells	Completed	HCC	N/A	1000	Observational	China
NCT00358501	“Defibrotide for the Treatment of Severe Hepatic Veno-Occlusive Disease in Hematopoetic Stem Cell Transplant Patients”	Hematopoetic stem cells	Completed	Severe hepatic veno-occlusive disease	Phase 3	134	Interventional	United States, Canada
NCT00007813	“Peripheral Stem Cell Transplantation Plus Chemotherapy in Treating Patients With Malignant Solid Tumors”	Peripheral stem cells	Completed	Liver cancer	Phase 1	21	Interventional	United States
NCT04423237	“Risk Factors and Measures to Prevent Liver and Pancreas Complications in Pediatric Patients After HSCT”	Hematopoetic stem cells	Completed	Liver complication	N/A	39	Observational	Italy
NCT03511794	“Effectiveness of the Hepatitis B Vaccine Post-Hematopoietic Stem Cell Transplant”	Hematopoetic stem cells	Completed	Hepatitis B	N/A	52	Observational	United States
NCT01481649	“Risk of Hepatitis B Reactivation After Bone Marrow Transplantation With Prior Hepatitis B Virus (HBV) Exposure”	Stem cells	Completed	Exposure to hepatitis B virus	N/A	69	Observational	China
NCT00002515	“Combination Chemotherapy Followed by Bone Marrow Transplantation in Treating Patients With Rare Cancer”	Stem cells	Completed	HCC	Phase 2	N/A	Interventional	United States
NCT06892236	“Preparation of IPSC for Cell Gene Editing for the Treatment of AATD”	hiPSCs	Recruiting	Alpha1-antitrypsin deficiency	NA	3	Interventional	Italy
NCT06242405	“Effect of Different Frequencies of Umbilical Cord-MSCs Through Peripheral Vein in Patients with ESLD”	UC-MSCs	Recruiting	End-stage liver disease	NA	92	Interventional	China
NCT06564740	“Stem Cell Applications in Biliary Atresia Patients”	Stem cells	Recruiting	Liver fibrosis (biliary atresia)	NA	64	Interventional	Turkey
NCT06740149	“Efficacy and Safety of BMSCs (CG-BM1) for ACLF Patients”	BM-MSCs	Recruiting	ACLF	Phase 1/2	90	Interventional	China
NCT06904755	“Safety and Effectiveness of MSCs in Blood Purification for the Treatment of Liver Failure”	MSCs	Recruiting	Liver failure	Phase 1	10	Interventional	China
NCT05106972	“Umbilical Cord MSC Transplantation for Decompensated Hepatitis B Cirrhosis”	UC-MSCs	Recruiting	Liver cirrhosis	NA	30	Interventional	China
NCT05985863	“Human Umbilical Cord MSC Transplantation for The Treatment of Acute-on-Chronic Liver Failure”	UC-MSCs	Recruiting	ACLF	Phase 1/2	150	Interventional	China
NCT04689152	“Clinical Trial to Evaluate the Efficacy and Safety of Cellgram-LC Administration in Patients With Alcoholic Cirrhosis”	Cellgram-LC	Recruiting	Alcoholic cirrhosis	Phase 3	200	Interventional	South Korea
NCT03826433	“hUC-MSCs (19#iSCLife®-LC) in the Treatment of Decompensated Hepatitis b Cirrhosis hepatitis b Cirrhosis”	UC-MSCs	Recruiting	Hepatitis B	Phase 1	20	Interventional	China
NCT00382278	“Safety Study of Autologous Stem Cell in Liver Cirrhosis”	Autologous stem cells	Terminated	Liver cirrhosis	Phase 1/2	15	Interventional	Brazil
NCT03860155	“Allogeneic ABCB5-positive Stem Cells for Treatment of Acute-on-Chronic Liver Failure”	Allogeneic stem cells	Terminated	ACLF	Phase 1/2	5	Interventional	Germany
NCT00923052	“The Natural History of Solid Organ Cancer Stem Cells (SOCSC)”	Cancer stem cells	Terminated	Hepatic cancer	N/A	190	Observational	United States

hiPSCs: Human induced pluripotent stem cells; MSCs: Mesenchymal stem/stromal cells; BM-EPCs: Bone marrow-derived endothelial progenitor cells; UC-MSCs: Umbilical cord-derived mesenchymal stem/stromal cells; BM-MSCs: Bone marrow-derived mesenchymal stem/stromal cells; HCC: Hepatocellular carcinoma; NASH: Non-alcoholic steatohepatitis; ACLF: Acute-on-chronic liver failure; N/A: Not Applicable; NA: Not Available.

As to the locations of the registered trials, China (n = 13), United States (n = 9) and Iran (n = 4) are the top three countries in the number of stem cell-based clinical studies for liver diseases (Figure 4D, Table 4). As to the subtypes of cell sources, both the autologous and allogeneic stem cells and the derivatives are applied, including adult stem cells, hBM-MSCs, hUC-MSCs, hASCs, patient-specific hiPSCs, circulating tumor stem cells, hematopoietic stem cells, solid organ cancer stem cells, peripheral stem cells, BM-derived endothelial progenitor cells, ABCB5-positive stem cells, bone barrow mononuclear stem cells, fetal liver cells, hematopoietic stem cells, cancer stem cells, and even the commercial stem cell products (e.g., HepaStem, Cellgram™, and 19#iSCLife^®-LC) (Figure 4E, Table 4). As shown in Figure 4F (also see Table 4), a total number of 3337 participants are distributed in a variety of liver disorders and liver metastasis of the relative tumors, including ACLF and liver failure, decompensated or alcoholic liver cirrhosis, HCC, non-alcoholic steatohepatitis, primary biliary cirrhosis, severe hepatic veno-occlusive disease, sinusoidal obstruction syndrome, end-stage liver disease, and Wilson’s Disease.

Furthermore, of the abovementioned registered trials with completed status, only 7 interventional ones were with results for the indicated liver disorders. For instance, in a phase I/II study (NCT00655707), investigators reported the safety and tolerability dose escalation in 5 patients with liver insufficiency by infusion of autologous CD34+ hematopoietic stem cells via either the hepatic artery or the portal vein, and one patient revealed serious adverse events (urinary tract infection). In another clinical trial upon liver insufficiency (NCT00147043), infusion of CD34+ hematopoietic stem cells were conducted via image guided scan into patients with chronic liver failure and abnormal liver function, and all the participants showed improvement in liver function including one with serious adverse events related to injection. Similarly, the safety and efficacy outcomes of patients after stem cell intervention were observed in the relative 5 clinical trials upon liver disorders, including nonalcoholic fatty liver diseases (NCT01875978), chronic liver failure (NCT01013194), hepatic veno-occlusive disease (NCT02851407, NCT00358501), and cancer pain (NCT00538850). Collectively, these findings indicated the robust prospects of stem cell-based cell therapy for liver disease, together with the inevitable adverse effects as well.

CONCLUSION

Stem cell, and particularly patient-specific hiPSCs with unlimited expansion and multipotent differentiation capacity, are splendid alternatives and renewable sources for pathogenic mechanism exploration and liver cell preparation[86]. At the meantime, hPSCs- and MSCs-guided cell therapy and regenerative medicine hold remarkable prospects to revolutionize paradigms of traditional treatments for liver disorders including end-stage liver diseases[174]. For the purpose, in this review article, we outline the conception and the latest renewal of stem cells and the derivatives in liver disease diagnosis and modeling, together with novel cell therapy for improving the outcomes of intractable liver disorders.

As to stem cell-based cell therapy, the preparation of cell sources with outstanding attributes (e.g., purity, functional mature, long-term survival and expansion) is of prime importance for liver diseases. As to MSCs, longitudinal studies have highlighted the heterogeneity in cellular viability and efficacy upon liver diseases largely attributes to the wide range of sources and the absence of unique biomarker[192,193]. For example, Zhao et al[45] and Zhang et al[172] reported the multifaceted variations of hUC-MSCs both at the cellular and genomic levels after continuous in vitro culture. Interestingly, we and other investigators reported the identification and high-efficient preparation of a vascular cellular adhesion molecule-1+ subset of MSCs (also known as CD106+ MSCs) with preferable immunomodulatory signatures and improved angiogenesis over the negative counterparts[194-196], which were further confirmed by preclinical investigation upon diverse disease intervention (e.g., acute lung injury[197], cerebral Infarction[198], experimental autoimmune encephalomyelitis[199]). These data indicated the importance of stem cell heterogeneity as well as the limitations of current technological limitations for disease intervention. Primary hepatocytes in 2D cultures promptly dedifferentiate and lose their function[200]. Meanwhile, the current in vitro three-step or four-step protocols for hepatic differentiation might not precisely mirror body liver development attribute to the variations with the in vivo liver development[71]. Compared to the abovementioned 2D or 3D monolayer cultures, stem cell-based 3D liver organoids (e.g., mLOs, liver assembloids) facilitate the long-term ex vivo expansion of primary human hepatocytes and serve as unlimited sources for large-scale preparation of mature hepatic parenchymal and non-parenchymal cells[112,201]. Nevertheless, there’s still an urgency for engineering multicellular complexity in liver organoids to more faithfully recapture native structures of individual organs, and in particular, the mimicking of liver organ-specific vasculature (e.g., liver-specific capillary plexus, blood vessels)[98].

The stem cell-based liver organoids with self-organizing properties have dramatically prompted the progression of liver development, liver disease pathogenesis, pharmacological screening, functional liver lineage generation, and innovative cell therapy and targeted therapy[157,177,202-204]. Simultaneously, it’s of prime importance for the induction of mature hepatic organoids with structural complexity and functional maturity (e.g., vascularization, immune cells, and sinusoidal endothelial cells) by 3D bioprinting or modulating signaling pathways with non-gene-editing strategies (e.g., small molecules, cytokine cocktails) for fulfilling precise liver disease diagnosis and regenerative medicine[205,206]. For instance, the limitations in the current technologies for the vast network of capillaries and vessels and the resultant insufficient vascularization of organoids (e.g., endothelial specialization, angiocrine factors) largely hinder the faithful simulation of the in vivo organ development, homeostasis and immune response[207-209]. Meanwhile, the vascularized organoids with organ-specific vascular phenotypes would also provide platforms for dissecting the physiological and angiocrine interactions dispense from substrate provision and perfusion in vivo[210,211]. As to immune cells, the involvement of Kupffer cells in hepatic organoids would help enhance the understanding of liver immunity, the contribution to liver diseases, and hepatotoxicity of drugs and relative xenobiotics[212]. Additionally, the integration of liver organoid technology and multidisciplinary new technologies (e.g., scRNA-seq, organ-on-a-chip technology, nano-biomaterials, lineage tracing technology) will further facilitate the dissection of liver diseases and optimization of treatment schedules[69,213,214]. For example, investigators recently gained high-throughput generation of pre-vascularized hepatobiliary organoids from hiPSCs on a chip via nonparenchymal cell grafting, which offers novel technical route for high-fidelity liver organoids formation[214].

Small EVs (sEVs) of diverse kinds are prestigious vehicles for intercellular communication and nutrient delivery, which play a critical role in physiological liver metabolism and pathological hepatopathy[80,215,216]. To date, a variety of sEVs have been applied to modulating hepatic signal transduction such as MSC-exosomes, MSC-sEVs[173], and bioengineered sEVs[217]. Of them, it was observed the differential alteration between intracellular and extracellular inhibition of cathepsin D upon liver lipidome in MASLD mice[157]. Meanwhile, MSC-EVs exercise therapeutic effects upon liver diseases by regulating the mitochondrial functions of immune cells (e.g., macrophages, lymphocytes), which provides new evidence and novel insights for stem cell-related immunotherapeutic remedies by targeting immune microenvironment (e.g., cell polarization, reactive oxygen, programmed apoptosis, and tumor microenvironment)[202,218-221]. Collectively, stem cells allow for interrogating core insights governing liver-specific organogenesis and pathogenesis, and pave the way for conquering coagulation liver disorders and the concomitant precision medicine.

ACKNOWLEDGEMENTS

The co-authors thank the members in The Fourth People’s Hospital of Jinan Affiliated to Shandong Second Medical University, Qingdao Medical College of Qingdao University, Shandong Provincial Hospital Affiliated to Shandong First Medical University, School of Basic Medicine of Gannan Medical University, Shandong Provincial Key Medical and Health Laboratory of Blood Ecology and Biointelligence, Jinan Key Laboratory of Medical Cell Bioengineering, and Shandong Health Youth Science and Technology Innovation Team for their suggestions and technical supports.