8-bromo-7-methoxychrysin inhibits properties of liver cancer stem cells via downregulation of β-catenin

AIM:To evaluate whether 8-bromo-7-methoxychrysin(BrMC),a synthetic analogue of chrysin,inhibits the properties of cancer stem cells derived from the human liver cancer MHCC97 cell line and to determine the potential mechanisms.METHODS:CD133+cells were sorted from the MHCC97 cell line by magnetic activated cell sorting,and amplified in stem cell-conditioned medium to obtain the enriched CD133+sphere forming cells(SFCs).The stem cell properties of CD133+SFCs were validated by the tumorsphere formation assay in vitro and the xenograft nude mouse model in vivo,and termed liver cancer stem cells(LCSCs).The effects of BrMC on LCSCs in vitro were evaluated by MTT assay,tumorsphere formation assay and transwell chamber assay.The effects of BrMC on LCSCs in vivo were determined using a primary and secondary xenograft model in Balb/c-nu mice.Expressions of the stem cell markers,epithelialmesenchymal transition(EMT)markers andβ-catenin protein were analyzed by western blotting or immunohistochemical analysis.RESULTS:CD133+SFCs exhibited stem-like cell properties of tumorsphere formation and tumorigenesis capacity in contrast to the parental MHCC97 cells.We found that BrMC preferentially inhibited proliferation and self-renewal of LCSCs(P<0.05).Furthermore,BrMC significantly suppressed EMT and invasion of LCSCs.Moreover,BrMC could efficaciously eliminate LCSCs in vivo.Interestingly,we showed that BrMC decreased the expression ofβ-catenin in LCSCs.Silencing ofβ-catenin by small interfering RNA could synergize the inhibition of self-renewal of LCSCs induced by BrMC,while Wnt3a treatment antagonized the inhibitory effects of BrMC.CONCLUSION:BrMC can inhibit the functions and characteristics of LCSCs derived from the liver cancer MHCC97 cell line through downregulation ofβ-catenin expression.     

The evolution of leukaemia from pre-leukaemic and leukaemic stem cells

Haematopoietic stem and progenitor cells (HSPCs) are defined as unspecialized cells that give rise to more differentiated cells. In a similar way, leukaemic stem and progenitor cells (LSPCs) are defined as unspecialized leukaemic cells, which can give rise to more differentiated cells. Leukaemic cells carry leukaemic mutations/variants and have clear differentiation abnormalities. Pre-leukaemic HSPCs (PreL-HSPCs) carry pre-leukaemic mutations/variants (pLMs) and are capable of producing mature functional cells, which will carry the same variants. Under the roof of LSPCs, one can find a broad range of cell types genetic and disease phenotypes. Present-day knowledge suggests that this phenotypic heterogeneity is the result of interactions between the cell of origin, the genetic background and the microenvironment background. The combination of these attributes will define the LSPC phenotype, frequency, differentiation capacity and evolutionary trajectory. Importantly, as LSPCs are leukaemia-initiating cells that sustain clinical remission and are the source of relapse, an improved understanding of LSPCs phenotype would offer better clinical opportunities for the treatment and hopefully prevention of human leukaemia. The current review will focus on LSPCs attributes in the context of human haematologic malignancies.     

Overview of recent experimental studies on liver stem cells

Since our review of the experimental evidence for liver epithelial stem cells in 1997, a large body of new data has been added. The new studies have focused on the analysis of differentiation of candidate liver stem cells when they are transplanted into the liver in vivo and when cultured in vitro under defined conditions. The new studies that we review substantiate the existence of stem cells that can generate new lineages of hepatocytes and cholangiocytes, especially in severely damaged livers. However, the recent studies have introduced new areas of controversy about the essential nature of liver stem cells and about appropriate methods to analyze their properties and functions. Whether the major stem cell for hepatocytes and cholangiocytes is an endodermal epithelial cell that resides in the liver or a mesodermal cell from bone marrow is a current area of controversy. Another controversial topic concerns the appropriateness of cell culture to analyze liver epithelial cells. Both of these situations are addressed, if not yet conclusively solved, by the new data. From our review of recent data, we conclude that the major liver stem cell is an epithelial cell that is a liver resident, and that cell culture is a valuable, even necessary, adjunct for analyzing the properties of liver stem cells and for eventually identifying the molecular mechanisms that regulate their activation, proliferation, and differentiation.     

Reprogramming into pancreatic endocrine cells based on developmental cues

Due to the increasing prevalence of type 1 diabetes and the complications arising from actual therapies, alternative treatments need to be established. In order to compensate the beta-cell deficiency associated with type 1 diabetes, current researches focus on new strategies to generate insulin-producing beta cells for transplantation purpose, including the differentiation of stem or progenitor cells, as well as the transdifferentiation of dispensable mature cell types. However, to successfully force any cell to adopt a functional beta-cell fate or phenotype, a better understanding of the molecular mechanisms underlying the genesis of these in vivo is required. The present short review summarizes the hitherto known functions and interplays of several key factors involved in the differentiation of the endocrine cell lineages during pancreas morphogenesis, as well as there potential in generating beta cells. Furthermore, an emphasize is made on beta-cell regeneration and the determinants implicated.     

Research progress and prospects of markers for liver cancer stem cells

Liver cancer is a common malignancy and surgery is the main treatment strategy. However, the prognosis is still poor because of high frequencies of postoperative recurrence and metastasis. In recent years, cancer stem cell(CSC) theory has evolved with the concept of stem cells, and has been applied to oncological research. According to cancer stem cell theory, liver cancer can be radically cured only by eradication of liver cancer stem cells(LCSCs). This notion has lead to the isolation and identification of LCSCs, which has become a highly researched area. Analysis of LCSC markers is considered to be the primary method for identification of LCSCs. Here, we provide an overview of the current research progress and prospects of surface markers for LCSCs.     

Stem and progenitor cell systems in liver development and regeneration

The liver comprises two stem/progenitor cell systems: fetal and adult liver stem/progenitor cells. Fetal hepatic progenitor cells, derived from foregut endoderm, differentiate into mature hepatocytes and cholangiocytes during liver development. Adult hepatic progenitor cells contribute to regeneration after severe and chronic liver injuries. However, the characteristics of these somatic hepatic stem/progenitor cells remain unknown. Culture systems that can be used to analyze these cells were recently established and hepatic stem/progenitor cell‐specific surface markers including delta‐like 1 homolog (DLK), cluster of differentiation (CD) 13, CD133, and LIV2 were identified. Cells purified using antibodies against these markers proliferate for an extended period and differentiate into mature cells both in vitro and in vivo. Methods to force the differentiation of human embryonic stem and induced pluripotent stem (iPS) cells into hepatic progenitor cells have been recently established. We demonstrated that the CD13⁺CD133⁺ fraction of human iPS‐derived cells contained numerous hepatic progenitor‐like cells. These analyses of hepatic stem/progenitor cells derived from somatic tissues and pluripotent stem cells will contribute to the development of new therapies for severe liver diseases.     

Differentiation of iPSCs into insulin-producing cells via adenoviral transfection of PDX-1, NeuroD1 and MafA

Aims

The aim of this study was to evaluate the effect of PDX-1 (pancreatic and duodenal homeobox-1), NeuroD1 (neurogenic differentiation-1) and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homolog A) in the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells and to explore this new approach of cell transplantation therapy for type 1 diabetes in mice.

Methods

iPSCs were infected with adenovirus (Ad-Mouse PDX-1-IRES-GFP, Ad-Mouse NeuroD1-IRES-GFP and Ad-Mouse Mafa-IRES-GFP) and then differentiated into insulin-producing cells in vitro. RT-PCR was applied to detect insulin gene expression, immunofluorescence to identify insulin protein, and mouse insulin enzyme-linked immunosorbent assay (ELISA) was used to evaluate the amount of insulin at different concentration of glucose. Insulin-producing cells were transplanted into the liver parenchyma of diabetic mice. Immunohistochemistry, intraperitoneal glucose tolerance test (IPGTT) and fasting blood glucose (FBG) were performed to assess the function of insulin-producing cells.

Results

Insulin biosynthesis and secretion were induced in iPSCs and insulin-producing cells were responsive to glucose in a dose-dependent manner. Gene expression of the three-gene-modified embryoid bodies (EBs) was similar to the mouse pancreatic β cell line MIN6. Transplantation of insulin-producing cells into type I diabetic mice resulted in hyperglycemia reversal.

Conclusions

The insulin-producing cells we obtained from three-gene-modified EBs may be used as seed cells for tissue engineering and may represent a cell replacement strategy for the production of β cells for the treatment of type 1 diabetes.     

A requirement for Notch1 distinguishes 2 phases of definitive hematopoiesis during development

Notch1 is known to play a critical role in regulating fates in numerous cell types, including those of the hematopoietic lineage. Multiple defects exhibited by Notch1-deficient embryos confound the determination of Notch1 function in early hematopoietic development in vivo. To overcome this limitation, we examined the developmental potential of Notch1(-/-) embryonic stem (ES) cells by in vitro differentiation and by in vivo chimera analysis. Notch1 was found to affect primitive erythropoiesis differentially during ES cell differentiation and in vivo, and this result reflected an important difference in the regulation of Notch1 expression during ES cell differentiation relative to the developing mouse embryo. Notch1 was dispensable for the onset of definitive hematopoiesis both in vitro and in vivo in that Notch1(-/-) definitive progenitors could be detected in differentiating ES cells as well as in the yolk sac and early fetal liver of chimeric mice. Despite the fact that Notch1(-/-) cells can give rise to multiple types of definitive progenitors in early development, Notch1(-/-) cells failed to contribute to long-term definitive hematopoiesis past the early fetal liver stage in the context of a wild-type environment in chimeric mice. Thus, Notch1 is required, in a cell-autonomous manner, for the establishment of long-term, definitive hematopoietic stem cells (HSCs).     

Model systems and clinical applications of hepatic stem cells for liver regeneration

Background  

Hepatic stem cells are capable to self-renew and to differentiate into all types of cells in the liver, including hepatic progenitors, mature hepatocytes and cholangiocytes.     

Lack of evidence that bone marrow cells contribute to cholangiocyte repopulation during experimental cholestatic ductal hyperplasia.

BACKGROUND: Ductopenia is observed in end-stage human cholestatic diseases. The limited capability of cholangiocytes for proliferation is suggested to be the principal reason. Recently, bone marrow cells (BMCs) have been reported to behave as hepatic stem cells; however, their capability to differentiate into cholangiocytes in cholestasis remains unclear. METHODS: Normal mice were lethally irradiated to suppress the proliferation of self-BMCs; thereafter, the BMCs from enhanced green fluorescent protein (EGFP)-transgenic mice were transferred to recipients. Chronic cholestasis was induced by 0.1%alpha-naphtylisothiocyanate (ANIT) feeding. The proliferation of cholangiocytes and oval cells was assessed morphologically and immunohistchemically (cytokeratin-7 (CK-7), A6). Proliferative activity (proliferating cell nuclear antigen (PCNA) protein expression), hepatic growth factor (HGF) receptor (c-Met), stem cell factor receptor (c-kit), Notch2 and Hes1 expression were also evaluated. RESULTS: Marked cholangiocyte proliferation was observed in ANIT-fed mice. However, no EGFP/CK-7 double positive cells were identified in any of the liver specimens after BMCs transfer (Tx). In hepatic parenchyma, there were scattered EGFP-positive cells, although none of them were positive for CK-7. CONCLUSIONS: In spite of the significant ductular proliferations after ANIT feeding, no EGFP-positive cholangiocytes were confirmed by any other means in this chronic cholestasis model. Thus, different from hepatocytes, BMCs Tx seems not to contribute to the differentiation of cholangiocytes. Future studies are feasible to clarify the origin of proliferative cholangiocytes observed in this chronic cholestatic ductular hyperplasia model.     

Cell therapy for diabetes: Stem cells,progenitors or beta-cell replication?

The cure for type 1 diabetes (T1D) will require either the replacement or regeneration of insulin-producing cells, together with measures that prevent their immune-mediated destruction. Experiments in rodent models have found that pancreatic stem cells, committed progenitors and replicating beta-cells can all contribute to insulin-producing cell regeneration. The cellular and molecular mechanisms of these cells, both in vitro and in vivo, have been investigated by us and by others. Furthermore, our surgical research laboratory has developed a unique in vivo chamber model of T1D, allowing the assessment of the behaviour of different sources of insulin-producing cells with a view to their potential use in cell-based therapies.     

Morphogenesis of liver epithelial cells

The mammalian liver is a physiologically important organ performing various types of metabolism, producing serum proteins, detoxifying bilirubin and ammonia, and protecting the body from infection. Those physiological functions are achieved with the 3D tissue architecture of liver epithelial cells. The liver contains two types of epithelial cells, namely, hepatocytes and cholangiocytes. They split from hepatoblasts (embryonic liver stem cells) in mid‐gestation and differentiate into structurally and functionally mature cells. Analyses of mutant mice showing abnormal liver organogenesis have identified genes involved in liver development. In vitro culture systems have been used to examine the mechanism in which each molecule or signaling pathway regulates the morphogenesis and functional differentiation of hepatocytes and cholangiocytes. In addition, liver epithelial cells as well as mesenchymal, sinusoidal endothelial and hematopoietic cells can be purified from developing livers, which enables us to perform genome‐wide screening to identify novel genes regulating epithelial morphogenesis in the liver. By combining these in vivo and in vitro systems, the liver could be a unique and suitable model for revealing a principle, governing epithelial morphogenesis. In this review, we summarize recent progress in the understanding of the development of liver epithelial tissue structures.     

肝癌干细胞与肝癌复发转移的研究进展

影响肝癌预后的因素很多,其中包括确诊时间过晚、合并肝硬化、肿瘤细胞对放化疗不敏感以及术后的转移与复发等.以往对肝癌复发转移的研究重点在:(1)肝癌细胞的脱落、迁移;(2)肝癌细胞在局部组织的黏附;(3)肝癌细胞周围血管的生成.随着对肝癌干细胞的研究报道,使我们对肝癌的转移和复发有了更进一步的认识.本文通过基因层面对肝癌...     

Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro

Human pluripotent stem cells (hPSCs) have been derived from the inner cell mass cells of blastocysts (embryonic stem cells) and primordial germ cells of the developing gonadal ridge (embryonic germ cells). Like their mouse counterparts, hPSCs can be maintained in culture in an undifferentiated state and, upon differentiation, generate a wide variety of cell types. Embryoid body (EB) formation is a requisite step in the process of in vitro differentiation of these stem cells and has been used to derive neurons and glia, vascular endothelium, hematopoietic cells, cardiomyocytes, and glucose-responsive insulin-producing cells from mouse PSCs. EBs generated from human embryonic germ cell cultures have also been found to contain a wide variety of cell types, including neural cells, vascular endothelium, muscle cells, and endodermal derivatives. Here, we report the isolation and culture of cells from human EBs as well as a characterization of their gene expression during growth in several different culture environments. These heterogeneous cell cultures are capable of robust and long-term [>70 population doublings (PD)] proliferation in culture, have normal karyotypes, and can be cryopreserved, clonally isolated, and stably transfected. Cell cultures and clonal lines retain a broad pattern of gene expression including simultaneous expression of markers normally associated with cells of neural, vascular/hematopoietic, muscle, and endoderm lineages. The growth and expression characteristics of these EB-derived cells suggest that they are relatively uncommitted precursor or progenitor cells. EB-derived cells may be suited to studies of human cell differentiation and may play a role in future transplantation therapies.     

Generation of human/rat xenograft animal model for the study of human donor stem cell behaviors in vivo

AIM: To accurately and realistically elucidate human stem cell behaviors in vivo and the fundamental mechanisms controlling human stem cell fates in vivo, which is urgently required in regenerative medicine and treatments for some human diseases, a surrogate human-rat chimera model was developed.METHODS: Human-rat chimeras were achieved by in utero transplanting low-density mononuclear cells from human umbilical cord blood into the fetal rats at 9-11 d of gestation, and subsequently, a variety of methods, including flow cytometry, PCR as well as immunohistochemical assay, were used to test the human donor contribution in the recipients.RESULTS: Of 29 live-born recipients, 19 had the presence of human CD45+ cells in peripheral blood (PB) detected by ? ow cytometry, while PCR analysis on genomic DNA from 11 different adult tissues showed that 14 selected from ? ow cytometry-positive 19 animals possessed of donor-derived human cell engraftment in multiple tissues (i.e. liver, spleen, thymus, heart, kidney, blood, lung, muscle, gut and skin) examined at the time of tissue collection, as conf irmed by detecting human β2-microglobulin expression using immunohistochemistry.In this xenogeneic system, the engrafted donor-derived human cells persisted in multiple tissues for at least 6 mo after birth. Moreover, transplanted human donor cells underwent site-specifi c differentiation into CK18-positive human cells in chimeric liver and CD45-positive human cells in chimeric spleen and thymus of recipients.CONCLUSION: Taken together, these fi ndings suggest that we successfully developed human-rat chimeras, in which xenogeneic human cells exist up to 6 mo later. This humanized small animal model, which offers an in vivo environment more closely resembling to the situations in human, provides an invaluable and effective approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future. The potential for new advances in our better understanding the living biological systems in human provided by investigators in humanized animals will remain promising.     

The diversity and plasticity of adult hepatic progenitor cells and their niche

The liver is a unique organ for homoeostasis with regenerative capacities. Hepatocytes possess a remarkable capacity to proliferate upon injury; however, in more severe scenarios liver regeneration is believed to arise from at least one, if not several facultative hepatic progenitor cell compartments. Newly identified pericentral stem/progenitor cells residing around the central vein is responsible for maintaining hepatocyte homoeostasis in the uninjured liver. In addition, hepatic progenitor cells have been reported to contribute to liver fibrosis and cancers. What drives liver homoeostasis, regeneration and diseases is determined by the physiological and pathological conditions, and especially the hepatic progenitor cell niches which influence the fate of hepatic progenitor cells. The hepatic progenitor cell niches are special microenvironments consisting of different cell types, releasing growth factors and cytokines and receiving signals, as well as the extracellular matrix (ECM) scaffold. The hepatic progenitor cell niches maintain and regulate stem cells to ensure organ homoeostasis and regeneration. In recent studies, more evidence has been shown that hepatic cells such as hepatocytes, cholangiocytes or myofibroblasts can be induced to be oval cell-like state through transitions under some circumstance, those transitional cell types as potential liver-resident progenitor cells play important roles in liver pathophysiology. In this review, we describe and update recent advances in the diversity and plasticity of hepatic progenitor cell and their niches and discuss evidence supporting their roles in liver homoeostasis, regeneration, fibrosis and cancers.     

Induction of cardiogenesis in embryonic stem cells via downregulation of Notch1 signaling

Embryonic stem cells represent an attractive source of cardiomyocytes for cell-replacement therapies. However, before embryonic stem cells can be successfully used for the treatment of cardiac diseases, the precise molecular mechanisms that underlie their cardiogenic differentiation must be identified. A network of intrinsic and extrinsic factors regulates embryonic stem cell self-renewal and differentiation into a variety of different cell lineages. Here, we show that Notch signaling takes place in some but not all embryonic stem cells and that the Notch pathway is shut down during the course of differentiation concomitantly with downregulation of Notch receptor and ligand expression. Moreover, gain- and loss-of-function experiments for Notch signaling components show that this pathway is a crucial regulator of cardiomyocyte differentiation within ES cells. Differentiation of ES cells into cardiomyocytes is favored by inactivation of the Notch1 receptor, whereas endogenous Notch signaling promotes differentiation of ES cells into the neuronal lineage. We conclude that Notch signaling influences the cell fate decision between mesodermal and the neuroectodermal cell fates during embryonic stem cell differentiation. These findings should help to optimize the production of specific cell types via modulation of the Notch pathways and, in particular, to improve the production of embryonic stem cell-derived cardiomyocytes.     

Adenosine triphosphate release and purinergic regulation of cholangiocyte transport

The discovery of purinergic receptors on almost every cell type studied to date suggests that purinergic signaling is a fundamental process regulating cell and organ level functions. Purinergic receptors have been found on all principal liver cell types, including liver parenchymal cells, or hepatocytes, and biliary epithelial cells, or cholangiocytes. Both hepatocytes and cholangiocytes are capable of the regulated release of adenosine triphosphate (ATP), and both cell types express a range of purinergic receptors to mediate cellular processes. The role of extracellular nucleotides in liver function is presently being elucidated. Extracellular ATP, in addition to autocrine regulation of liver cell volume, has recently been shown to play an important role in paracrine signaling to coordinate specific hepatocyte and cholangiocyte cellular responses. The findings that (1) cholangiocytes are capable of the regulated release of ATP into bile, (2) ATP is present in bile in concentrations capable of stimulating purinergic receptors, and (3) P2 receptor stimulation results in brisk Cl(-) channel activation and fluid secretion suggest an important role of extracellular ATP in the regulation of bile formation. This article highlights important developments in our understanding of the role of purinergic signaling in cholangiocyte transport and bile formation.     

Proliferation of hepatic lineage cells of normal C57BL and interleukin-6 knockout mice after cocaine-induced periportal injury

The cellular response to periportal liver injury, induced by phenobarbital feeding and cocaine injection, is used to compare the restitutive proliferation of hepatocytes, cholangiocytes, and oval cells in the livers of normal control to those of interleukin-6 (IL-6) knockout mice. After this injury hepatocytes in noninjured middle and central zones start to proliferate first, followed by proliferation of cholangiocytes and intraportal oval cells. Proliferation of all cell types peaks at 2 days, but oval cells continue to proliferate and differentiate through days 4 and 6 as they reconstitute the necrotic zone. By day 10, the injured zone is completely repaired, and no dividing cells remain. During the first 3 to 4 days after injury, the number of proliferating hepatocytes, cholangiocytes, and sinusoidal cells is lower in IL-6 knockout mice than in normal mice, whereas the number of dividing oval cells is higher. However, overall repair of the injury is accomplished in the same time period in both groups. During repair of the periportal zone, oval cells acquire differentiation markers of hepatocytes as they cross the zone of injury. In conclusion, the phenobarbital/cocaine injury model is useful to study restitutive proliferation of mouse liver cell lineages. The proliferative response in IL-6 knockout mice shows that IL-6 is not required for proliferation of liver cells; timely repair of liver injury occurs in both normal and IL-6 knockout mice. Increased proliferation of oval cells in IL-6 knockout mice may compensate for the lower proliferation of other liver cell types.