Evidence for mesenchymal-epithelial transition associated with mouse hepatic stem cell differentiation

Mesenchymal-epithelial transition events are related to embryonic development, tissue construction, and wound healing. Stem cells are involved in all of these processes, at least in part. However, the direct evidence of mesenchymal-epithelial transition associated with stem cells is unclear. To determine whether mesenchymal-epithelial transition occurs in liver development and/or the differentiation process of hepatic stem cells in vitro, we analyzed a variety of murine liver tissues from embryonic day 11.5 to adults and the colonies derived from hepatic stem/progenitor cells isolated with flow cytometry. The results of gene expression, immunohistochemistry and Western blot showed that as liver develops, the expression of epithelial markers such as Cytokeratin18 and E-cadherin increase, while expression of mesenchymal markers such as vimentin and N-cadherin decreased. On the other hand, in freshly isolated hepatic stem cells, the majority of cells (65.0%) co-express epithelial and mesenchymal markers; this proportion is significantly higher than observed in hematopoietic cells, non-hematopoietic cells and non-stem cell fractions. Likewise, in stem cell-derived colonies cultured over time, upregulation of epithelial genes (Cytokeratin-18 and E-cadherin) occurred simultaneously with downregulation of mesenchymal genes (vimentin and Snail1). Furthermore, in the fetal liver, vimentin-positive cells in the non-hematopoietic fraction had distinct proliferative activity and expressed early the hepatic lineage marker alpha-fetoprotein. CONCLUSION: Hepatic stem cells co-express mesenchymal and epithelial markers; the mesenchymal-epithelial transition occurred in both liver development and differentiation of hepatic stem/progenitor cells in vitro. Besides as a mesenchymal marker, vimentin is a novel indicator for cell proliferative activity and undifferentiated status in liver cells.     

Bone marrow-derived fibrocytes contribute to liver fibrosis

Chronic liver injury often leads to hepatic fibrosis, a condition associated with increased levels of circulating TGF-β1 and lipopolysaccharide, activation of myofibroblasts, and extensive deposition of extracellular matrix, mostly collagen Type I. Hepatic stellate cells are considered to be the major¹ but not the only source of myofibroblasts in the injured liver.² Hepatic myofibroblasts may also originate from portal fibroblasts, mesenchymal cells, and fibrocytes.³ Since the discovery of fibrocytes in 1994 by Dr. Bucala and colleagues, this bone marrow (BM)-derived collagen Type I-producing CD45⁺ cells remain the most fascinating cells of the hematopoietic system. Due to the ability to differentiate into collagen Type I producing cells/myofibroblasts, fibrocytes were implicated in the pathogenesis of liver, skin, lung, and kidney fibrosis. However, studies of different organs often contain controversial results on the number of fibrocytes recruited to the site of injury and their biological function. Furthermore, fibrocytes were implicated in the pathogenesis of sepsis and were shown to possess antimicrobial activity. Finally, in response to specific stimuli, fibrocytes can give rise to fully differentiated macrophages, suggesting that in concurrence with the high plasticity of hematopoietic cells, fibrocytes exhibit progenitor properties. Here, we summarize our current understanding of the role of CD45⁺Collagen Type I⁺ BM-derived cells in response to fibrogenic liver injury and septicemia and discuss the most recent evidence supporting the critical role of fibrocytes in the mediation of pro-fibrogenic and/or pro-inflammatory responses.     

Effect of human amniotic epithelial cells on pro‐fibrogenic resident hepatic cells in a rat model of liver fibrosis

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial–mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF‐β, the main fibrogenic growth factor, through αvβ6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvβ6 integrin expression, with a consequent decrease in TGF‐β activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury‐induced and fibrosis‐promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.     

Smad2 and Smad3 play different roles in rat hepatic stellate cell function and alpha-smooth muscle actin organization

Hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis, transdifferentiating in chronic liver disease from "quiescent" HSC to fibrogenic myofibroblasts. Transforming growth factor-beta (TGF-beta), acting both directly and indirectly, is a critical mediator of this process. To characterize the function of the TGF-beta signaling intermediates Smad2 and Smad3 in HSC, we infected primary rat HSC in culture with adenoviruses expressing wild-type and dominant negative Smads 2 and 3. Smad3-overexpressing cells exhibited increased deposition of fibronectin and type 1 collagen, increased chemotaxis, and decreased proliferation compared with uninfected cells and those infected with Smad2 or either dominant negative, demonstrating different biological functions for the two Smads. Additionally, coinfection experiments suggested that Smad2 and Smad3 signal via independent pathways. Smad3-overexpressing cells as well as TGF-beta-treated cells demonstrated more focal adhesions and increased alpha-smooth muscle actin (alpha-SMA) organization in stress fibers, although all cells reached the same level of alpha-SMA expression, indicating that Smad3 also regulates cytoskeletal organization in HSC. We suggest that TGF-beta, signaling via Smad3, plays an important role in the morphological and functional maturation of hepatic myofibroblasts.     

Cellular composition and regulatory function of fetal liver stroma

Hematopoietic differentiation and formation of hepatic tissue both take place in mammalian liver during its prenatal development. Hematopoietic and hepatic stem cells self-renew, proliferate and differentiate within specific microenvironment that is organized by stromal elements. Stroma of developing liver consists of different cell populations such as mesenchymal stromal cells, Ito cells, portal fibroblasts and myofibroblasts, vascular endothelial and smooth muscle cells, cells undergoing epithelial-to-mesenchymal transition. In this review, their phenotypical and functional properties, possible derivation and role in the regulation of hematopoiesis and hepatogenesis are discussed.     

Progenitor cells of the biliary epithelial cell lineage

Stem-like cells have been identified in liver that are able to differentiate in vivo and in culture to biliary epithelial cells (BEC), hepatocytes and oval cells. The growth factors/cytokines and signal pathways required for the differentiation processes are beginning to be evaluated. There is increasing evidence to suggest that these stem-like cells may originate from both the bone marrow population or from a precursor remnant from liver embryogenesis, as they share many of the same markers (CD34, c-kit, CD45). Most recently, it has been shown that a population of progenitor cells can copurify with mesenchymal bone marrow cells and differentiate under specific culture conditions to form both hepatic epithelial and also endothelial cells. The interaction of haemopoietic and mesenchymal stem cells needs further evaluation. The close association of ductular reactive cells and neovessels in end-stage cholestatic liver diseases and the relation to Jagged/Notch signalling pathway may be important in the regulation of stem cells to form both biliary epithelial and endothelial cells.     

Hepatic stellate cells: unique characteristics in cell biology and phenotype

Hepatic stellate cells (HSCs), a mesenchymal cell type in hepatic parenchyma, have unique features with respect to their cellular origin, morphology, and function. Normal, quiescent HSCs function as major vitamin A-storing cells containing over 80% of total vitamin A in the body to maintain vitamin A homeostasis. HSCs are located between parenchymal cell plates and sinusoidal endothelial cells, and extend well-developed, long processes surrounding sinusoids in vivo as pericytes. However, HSCs are known to be 'activated' or 'transdifferentiated' to myofibroblast-like phenotype lacking cytoplasmic lipid droplets and long processes in pathological conditions such as liver fibrosis and cirrhosis, as well as merely during cell culture after isolation. HSCs are the predominant cell type producing extracellular matrix (ECM) components as well as ECM degrading metalloproteases in hepatic parenchyma, indicating that they play a pivotal role in ECM remodeling in both normal and pathological conditions. Recent findings have suggested that HSCs have a neural crest origin from their gene expression pattern similar to neural cell type and/or smooth muscle cells and myofibroblasts. The morphology and function of HSCs are regulated by ECM components as well as by cytokines and growth factors in vivo and in vitro. Liver regeneration after partial hepatectomy might be an invaluable model to clarify the HSC function in elaborate organization of liver tissue by cell-cell and cell-ECM interaction and by growth factor and cytokine regulation.     

Norepinephrine and neuropeptide Y promote proliferation and collagen gene expression of hepatic myofibroblastic stellate cells

The mechanisms initiating and perpetuating the fibrogenic response in the injured liver are not well understood. Hepatic stellate cells are activated by liver injury to become proliferative and fibrogenic myofibroblasts. Emerging evidence suggests that the sympathetic nervous system may play a role in the development of cirrhosis. It is not known, however, whether this requires a direct interaction between sympathetic neurotransmitters and stellate cell receptors, or results indirectly, from sympathetic effects on the vasculature. Using cultured hepatic stellate cells, we show that the sympathetic neurotransmitters, norepinephrine and neuropeptide Y, markedly stimulate the proliferation of activated, myofibroblastic, hepatic stellate cells. Norepinephrine, but not neuropeptide Y, also induces collagen gene expression. In conclusion, physiologically relevant concentrations of sympathetic neurotransmitters directly modulate the phenotype of hepatic stellate cells. This suggests that targeted interruption of sympathetic nervous system signaling in hepatic stellate cells may be useful in constraining the fibrogenic response to liver injury.     

Stem cells in liver regeneration and therapy

The liver has adapted to the inflow of ingested toxins by the evolutionary development of unique regenerative properties and responds to injury or tissue loss by the rapid division of mature cells. Proliferation of the parenchymal cells, i.e. hepatocytes and epithelial cells of the bile duct, is regulated by numerous cytokine/growth-factor-mediated pathways and is synchronised with extracellular matrix degradation and restoration of the vasculature. Resident hepatic stem/progenitor cells have also been identified in small numbers in normal liver and implicated in liver tissue repair. Their putative role in the physiology, pathophysiology and therapy of the liver, however, is not yet precisely known. Hepatic stem/progenitor cells also known as “oval cells” in rodents have been implicated in liver tissue repair, at a time when the capacity for hepatocyte and bile duct replication is exhausted or experimentally inhibited (facultative stem/progenitor cell pool). Although much more has to be learned about the role of stem/progenitor cells in the physiology and pathophysiology of the liver, experimental analysis of the therapeutic value of these cells has been initiated. Transplantation of hepatic stem/progenitor cells or in vivo pharmacological activation of the pool of hepatic stem cells may provide novel modalities for the therapy of liver diseases. In addition, extrahepatic stem cells (e.g. bone marrow cells) are being investigated for their contribution to liver regeneration. Hepatic progenitor cells derived from embryonic stem cells are included in this review, which also discusses future perspectives of stem cell-based therapies for liver diseases.     

Intestinal myofibroblasts: targets for stem cell therapy

The subepithelial intestinal myofibroblast is an important cell orchestrating many diverse functions in the intestine and is involved in growth and repair, tumorigenesis, inflammation, and fibrosis. The myofibroblast is but one of several α-smooth muscle actin-positive (α-SMA(+)) mesenchymal cells present within the intestinal lamina propria, including vascular pericytes, bone marrow-derived stem cells (mesenchymal stem cells or hematopoietic stem cells), muscularis mucosae, and the lymphatic pericytes (colon) and organized smooth muscle (small intestine) associated with the lymphatic lacteals. These other mesenchymal cells perform many of the functions previously attributed to subepithelial myofibroblasts. This review discusses the definition of a myofibroblast and reconsiders whether the α-SMA(+) subepithelial cells in the intestine are myofibroblasts or other types of mesenchymal cells, i.e., pericytes. Current information about specific, or not so specific, molecular markers of lamina propria mesenchymal cells is reviewed, as well as the origins of intestinal myofibroblasts and pericytes in the intestinal lamina propria and their replenishment after injury. Current concepts and research on stem cell therapy for intestinal inflammation are summarized. Information about the stem cell origin of intestinal stromal cells may inform future stem cell therapies to treat human inflammatory bowel disease (IBD).     

Hepatic oval 'stem' cell in liver regeneration

Hepatic oval cell activation, proliferation, and differentiation has been observed under certain physiological conditions, mainly when the proliferation of existing hepatocytes has been inhibited followed by severe hepatic injury. Hepatic oval cells display a distinct phenotype and have been shown to be a bipotential progenitor of two types of epithelial cells found in the liver, hepatocytes and bile ductular cells. Bone marrow stem cells have recently been shown to be a potential source of the hepatic oval cells and that reconstitution of an injured liver from a purified stem cell population is possible. The focus of this review is on the studies involving the activation, proliferation, and differentiation of these hepatic oval cells and the role that they play in regeneration of the damaged liver. In order to present the potentiality of the hepatic oval cell, an experimental model that involves the inhibition of normal hepatic growth and division as well as severe hepatic injury via chemical or surgical means has been employed. In this model, an as yet undetermined signal or perhaps the lack of regenerative capability in the hepatocytes activates the hepatic oval cell compartment. However, other than understanding a potential origin of these cells and some of the markers that characterize them, it still remains unclear as to how these cells migrate ('home') into the damaged areas and how they begin their differentiation into mature and functioning hepatic cells.     

Robust differentiation of fetal hepatocytes from human embryonic stem cells and iPS

Hepatocyte transplantation is considered as an alternative to organ transplantation in particular for the treatment of liver metabolic diseases. However, due to the difficulties to obtain a large number of hepatocytes, new sources of cells are needed. These cells could be either of hepatic origin (hepatic stem cells) or extrahepatic such as mesenchymal stem cells or pluripotent stem cells (human embryonic stem cells [hESC] or iPS). We developed a new method to differentiate hESCs into fetal hepatocytes. These conditions recapitulate the main liver developmental stages, using fully defined medium devoid of animal products or unknown factors. The differentiated cells express many fetal hepatocytes markers (cytochrome P450 3A7, albumin, alpha-1-antitrypsin, etc.). The cells display specific hepatic functions (ammonia metabolism, excretion of indocyanin green) and are capable to engraft and express hepatic proteins two months after transplantation into newborn uPAxrag2gc-/- mouse liver. We have also showed that this approach is transposable to human iPS, and further studies on animal models will allow us to compare the in vivo potential of these two sources of pluripotent cells. Finally, only studies on large animals such as nonhuman primates will validate an eventual clinical application.     

GITR/GITRL reverse signalling modulates the proliferation of hepatic progenitor cells by recruiting ANXA2 to phosphorylate ERK1/2 and Akt

Hepatic stem/progenitor cells are the major cell compartment for tissue repair when hepatocyte proliferation is compromised in chronic liver diseases, but the expansion of these cells increases the risk of carcinogenesis. Therefore, it is essential to explore the pathways restricting their expansion and abnormal transformation. The ligand of glucocorticoid-induced tumour necrosis factor receptor (GITRL) showed the most highly increased expression in hepatic progenitor cells treated with transforming growth factor (TGF)-β1. If overexpressed by hepatic progenitor cells, GITRL stimulated cell proliferation by activating the epithelial–mesenchymal transition pathway and enhancing ERK1/2 and Akt phosphorylation via GITRL binding to ANXA2. However, GITR, the specific GITRL receptor, suppressed the epithelial–mesenchymal transition pathway of GITRL-expressing cells and decreased their growth by dissociating ANXA2 from GITRL and reducing downstream ERK1/2 and Akt phosphorylation. This study identifies GITR/GITRL reverse signalling as a cross-interaction pathway between immune cells and hepatic stem/progenitor cells that restricts the expansion of hepatic stem/progenitor cells and reduces the possibility of carcinogenesis.Subject terms: Cell growth, Liver fibrosis     

The therapeutic potential of bone marrow-derived mesenchymal stem cells on hepatic cirrhosis

Hepatic cirrhosis is the end-stage of chronic liver diseases. The majority of patients with hepatic cirrhosis die from life-threatening complications occurring at their earlier ages. Liver transplantation has been the most effective treatment for these patients. Since liver transplantation is critically limited by the shortage of available donor livers, searching for an effective alternative therapy has attracted great interest in preclinical studies. The transplantation of autologous bone marrow-derived mesenchymal stem cells holds great potential for treating hepatic cirrhosis. Mesenchymal stem cells can differentiate to hepatocytes, stimulate the regeneration of endogenous parenchymal cells, and enhance fibrous matrix degradation. Experimental and clinical studies have shown promising beneficial effects. This review is intended to translate the bench study results to the patients' bedside. The potential interventions of mesenchymal stem cells on cirrhosis are illustrated in terms of the cellular and molecular mechanisms of hepatic fibrogenesis.     

人羊膜间充质干细胞对四氯化碳诱导小鼠肝损伤定位修复的研究

目的：观察活体染料羧基荧光素乙酰乙酸（CFSE）标记的人羊膜间充质干细胞对四氯化碳诱导小鼠肝损伤模型的定位修复情况。方法：采用胰蛋白酶-胶原酶消化法从羊膜组织中分离间充质干细胞,通过流式细胞术和免疫荧光等方法进行鉴定。模型组按浓度为20μl/g剂量的四氯化碳和橄榄油混合液诱导小鼠肝损伤,治疗组经小鼠尾静脉注射羧基荧光素乙酰乙酸标记的人羊膜间充质干细胞约1×10⁶个/ml。分别取模型组和细胞移植的治疗组小鼠眼球血和肝组织进行相关检测。结果：分离得到纯度较高的羊膜间充质干细胞;冰冻切片免疫荧光显示移植1周后细胞向小鼠受损肝组织定植,CFSE标记的人羊膜间充质干细胞呈绿色荧光;细胞移植后4周,与模型组比较,细胞移植组小鼠血清中天冬氨酸转移酶、丙氨酸氨基转移酶显著降低,而白蛋白明显升高（P< 0.01）;肝组织病理切片模型组小鼠细胞水肿,坏死灶多见,脂肪变性,可见不同程度的炎性细胞浸润;治疗组小鼠肝组织病理学改变和损伤程度有较明显改善;小鼠肝组织冰冻切片的免疫荧光显示移植4周后人羊膜间充质干细胞周围分泌血清白蛋白。结论：羧基荧光素乙酰乙酸标记的人羊膜间充质干细胞可有效改善肝组织的生理功能,为细胞定位移植治疗肝脏疾病的修复情况提供实验数据。     

Origins and functions of liver myofibroblasts

Myofibroblasts combine the matrix-producing functions of fibroblasts and the contractile properties of smooth muscle cells. They are the main effectors of fibrosis in all tissues and make a major contribution to other aspects of the wound healing response, including regeneration and angiogenesis. They display the de novo expression of α-smooth muscle actin. Myofibroblasts, which are absent from the normal liver, are derived from two major sources: hepatic stellate cells (HSCs) and portal mesenchymal cells in the injured liver. Reliable markers for distinguishing between the two subpopulations at the myofibroblast stage are currently lacking, but there is evidence to suggest that both myofibroblast cell types, each exposed to a particular microenvironment (e.g. hypoxia for HSC-MFs, ductular reaction for portal mesenchymal cell-derived myofibroblasts (PMFs)), expand and exert specialist functions, in scarring and inflammation for PMFs, and in vasoregulation and hepatocellular healing for HSC-MFs. Angiogenesis is a major mechanism by which myofibroblasts contribute to the progression of fibrosis in liver disease. It has been clearly demonstrated that liver fibrosis can regress, and this process involves a deactivation of myofibroblasts, although probably not to a fully quiescent phenotype. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

Characterization and hepatogenic differentiation of mesenchymal stem cells from human amniotic fluid and human bone marrow: a comparative study

Since stem cells can differentiate into hepatocyte, stem cell-based therapy becomes a potential alternative treatment for terminal liver diseases. However, an appropriate source of human mesenchymal stem cells (hMSCs) for hepatocytes has not yet been clearly elucidated. The aim of the present study was to investigate the in vitro biological characterization and hepatic differentiation potential of human amniotic fluid-derived mesenchymal stem cells (AF-hMSCs) and human bone marrow-derived mesenchymal stem cells (BM-hMSCs). Our results show that AF-hMSCs possess higher proliferation and self-renewal capacity than BM-hMSCs. Cytogenetic studies indicate that AF-hMSCs are as genetically stabile as BM-hMSCs. Following incubation with specific hepatogenic agents, AF-hMSCs showed a higher hepatic differentiation potential than BM-hMSCs. Expression of several liver-specific markers was significantly greater in AF-hMSCs than in BM-hMSCs, as shown by real time RT-PCR and immunofluorescence (IF). In conclusion, AF-hMSCs possess superior potential for hepatic differentiation, making them more suitable for diverse terminal liver diseases.