Both Humoral Mesenchymal Factors and the Close Association between the Hepatic Endoderm and Mesenchyme can be Involved in Liver Formation of Mouse Embryos

Previous studies with tissue recombination experiments demonstrated that the splanchnic mesenchymes, including hepatic, pulmonary and stomach mesenchymes can support hepatocyte differentiation from the hepatic endoderm in 9.5-day mouse embryos. This phenomenon corresponds to the second hepatic induction. The present study was undertaken to determine whether direct cell-cell contacts between the hepatic endoderm and mesenchyme are required for hepatocyte differentiation, using transfilter experiments in which membrane filters with various pore sizes were inserted between the endoderm and the hepatocyte-inducing mesenchyme (the chick lung mesenchyme). Hepatocyte differentiation occurred even when the direct cell-cell contacts between the hepatic endoderm and the mesenchyme were absent, suggesting that humoral factors may work in this interaction. However, growth of hepatocytes was most prominent in the transfilter experiments with filters having pore sizes of 0.2 and 0.8 mum, which permitted mesenchymal cells or their cell processes to penetrate to the side of the endoderm. These results suggest that two types of tissue interactions, including humoral mesenchymal factors and very local tissue interactions such as direct cell-cell contacts, may be involved in the second step of hepatic induction.     

A simple and economical route to generate functional hepatocyte-like cells from hESCs and their application in evaluating alcohol induced liver damage

The in vitro derived hepatocytes from human embryonic stem cells (hESC) is a promising tool to acquire improved knowledge of the cellular and molecular events underlying early human liver development under physiological and pathological conditions. Here we report a simple two-step protocol employing conditioned medium (CM) from human hepatocellular carcinoma cell line, HepG2 to generate functional hepatocyte-like cells from hESC. Immunocytochemistry, flow cytometry, quantitative RT-PCR, and biochemical analyses revealed that the endodermal progenitors appeared as pockets in culture, and the cascade of genes associated with the formation of definitive endoderm (HNF-3β, SOX-17, DLX-5, CXCR4) was consistent and in concurrence with the up-regulation of the markers for hepatic progenitors [alpha-feto protein (AFP), HNF-4α, CK-19, albumin, alpha-1-antitrypsin (AAT)], followed by maturation into functional hepatocytes [tyrosine transferase (TAT), tryptophan-2, 3-dioxygenase (TDO), glucose 6-phosphate (G6P), CYP3A4, CYP7A1]. We witnessed that the gene expression profile during this differentiation process recapitulated in vivo liver development demonstrating a gradual down-regulation of extra embryonic endodermal markers (SOX-7, HNF-1β, SNAIL-1, LAMININ-1, CDX2), and the generated hepatic cells performed multiple liver functions. Since prenatal alcohol exposure is known to provoke irreversible abnormalities in the fetal cells and developing tissues, we exposed in vitro generated hepatocytes to ethanol (EtOH) and found that EtOH treatment not only impairs the survival and proliferation, but also induces apoptosis and perturbs differentiation of progenitor cells into hepatocytes. This disruption was accompanied by alterations in the expression of genes and proteins involved in hepatogenesis. Our results provide new insights into the wider range of destruction caused by alcohol on the dynamic process of liver organogenesis.     

Embryonic stem cells: hepatic differentiation and regenerative medicine for the treatment of liver disease

Hepatocyte transplantation is considered a potential treatment for liver diseases and a bridge for patients awaiting liver transplantation, but its application has been hampered by a limited supply of hepatocytes. Embryonic stem (ES) cells established from early mouse and human embryos are pluripotent, and proliferate indefinitely in an undifferentiated state in vitro. Since differentiation from ES cells seems to recapitulate early embryonic development, if hepatocytes could be efficiently generated in vitro, ES cells might become a source of transplantable hepatocytes for cell replacement therapy. Hepatocytes have been generated from ES cells in vitro, and the hepatocytes differentiated from ES cells have been found to express many hepatocyte-related genes and perform hepatic functions. However, it remains unclear whether the hepatocytes differentiated from ES cells are derived from definitive endoderm or primitive endoderm. Because visceral endoderm, which expresses many hepatocyte-related genes, is derived from primitive endoderm and is fated to form extraembryonic yolk sac tissues, not to form hepatocytes, ES cells must be directed to a definitive endoderm lineage in vitro. This article discusses the differentiation of ES cells into hepatocytes in vitro in comparison with early embryogenesis, and describes the efficacy of ES cell-derived hepatocyte transplantation.     

Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.     

Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines

This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by 16 d of culture. EBs are formed, and cells can be induced to differentiate into definitive endoderm by culture in Activin A and fibroblast growth factor 2 (FGF-2). Hepatic differentiation and maturation of cells is accomplished by withdrawal of Activin A and FGF-2 and by exposure to liver nonparenchymal cell-derived growth factors, a deleted variant of hepatocyte growth factor (dHGF) and dexamethasone. Approximately 70% of differentiated embryonic stem (ES) cells express albumin and can be recovered by albumin promoter-based cell sorting. The sorted cells produce albumin in culture and metabolize ammonia, lidocaine and diazepam at approximately two-thirds the rate of primary mouse hepatocytes.     

In vitro differentiation of embryonic stem cells into hepatocytes induced by fibroblast growth factors and bone morphological protein-4

The feasibility of transforming embryonic endoderm into different cell types is tightly controlled by mesodermal and septum transversumal signalings during early embryonic development. Here, an induction protocol tracing embryonic liver development was designed, in which, three growth factors, acid fibroblast growth factor, basic fibroblast growth factor and bone morphological protein-4 that secreted from pre-cardiac mesoderm and septum transversum mesenchyme, respectively, were employed to investigate their specific potency of modulating the mature hepatocyte proportion during the differentiation process. Results showed that hepatic differentiation took place spontaneously at a low level, however, supplements of the three growth factors gave rise to a significant up-regulation of mature hepatocytes. Bone morphological protein-4 highlighted the differentiation ratio to 40-55%, showing the most effective promotion, and also exhibited a synergistic effect with the other two fibroblast factors, whereas no similar phenomenon was observed between the other two factors, which was reported for the first time. Our study not only provides a high-performance system of embryonic stem cells differentiating into hepatocytes, which would supply a sufficient hepatic population for related studies, but also make it clear of the inductive effects of three important growth factors, which could support for further investigation on the mechanisms of mesodermal and septumal derived signalings that regulate hepatic differentiation.     

Lhx2 is expressed in the septum transversum mesenchyme that becomes an integral part of the liver and the formation of these cells is independent of functional Lhx2

Liver development is based on reciprocal interactions between ventral foregut endoderm and adjacent mesenchymal tissues. Targeted disruption of the LIM-homeobox gene Lhx2 has revealed that it is important for the expansion of the liver during embryonic development, whereas it appears not to be involved in the induction of hepatic fate. It is not known whether Lhx2 is expressed in the endodermal or mesenchymal portion of the liver, or if the cells normally expressing Lhx2 are absent or present in the liver of Lhx2(-/-) embryos. To address this we have analyzed gene expression from the Lhx2 locus during hepatic development in wild type and Lhx2(-/-) mice. Lhx2 is expressed in cells of the septum transversum mesenchyme adjacent to the liver bud from embryonic day 9. The hepatic cords subsequently migrate into and intermingle with the Lhx2+ cells of the septum transversum mesenchyme. Lhx2 expression is thereafter maintained in a subpopulation of mesenchymal cells in the liver until adult life. In adult liver the Lhx2+ mesenchymal cells co-express desmin, a marker associated with stellate cells. At embryonic day 10.5, cells expressing the mutant Lhx2 allel are present in Lhx2(-/-) livers, and expression of Hlx, hepatocyte growth factor, Hex and Prox1, genes known to be important in liver development, is independent of functional Lhx2 expression. Thus, Lhx2 is specifically expressed in the liver-associated septum transversum mesenchyme that subsequently becomes an integral part of the liver and the formation of these mesenchymal cells does not require functional Lhx2.     

Neuron navigator 3a regulates liver organogenesis during zebrafish embryogenesis

Endodermal organogenesis requires a precise orchestration of cell fate specification and cell movements, collectively coordinating organ size and shape. In Caenorhabditis elegans, uncoordinated-53 (unc-53) encodes a neural guidance molecule that directs axonal growth. One of the vertebrate homologs of unc-53 is neuron navigator 3 (Nav3). Here, we identified a novel vertebrate neuron navigator 3 isoform in zebrafish, nav3a, and we provide genetic evidence in loss- and gain-of-function experiments showing its functional role in endodermal organogenesis during zebrafish embryogenesis. In zebrafish embryos, nav3a expression was initiated at 22 hpf in the gut endoderm and at 40 hpf expanded to the newly formed liver bud. Endodermal nav3a expression was controlled by Wnt2bb signaling and was independent of FGF and BMP signaling. Morpholino-mediated knockdown of nav3a resulted in a significantly reduced liver size, and impaired development of pancreas and swim bladder. In vivo time-lapse imaging of liver development in nav3a morphants revealed a failure of hepatoblast movement out from the gut endoderm during the liver budding stage, with hepatoblasts being retained in the intestinal endoderm. In hepatocytes in vitro, nav3a acts as a positive modulator of actin assembly in lamellipodia and filipodia extensions, allowing cellular movement. Knockdown of nav3a in vitro impeded hepatocyte movement. Endodermal-specific overexpression of nav3a in vivo resulted in additional ectopic endodermal budding beyond the normal liver and pancreatic budding sites. We conclude that nav3a is required for directing endodermal organogenesis involving coordination of endodermal cell behavior.     

The inhibitory role of stromal cell mesenchyme on human embryonic stem cell hepatocyte differentiation is overcome by Wnt3a treatment

Pluripotent stem cells are derived from the inner cell mass of preimplantation embryos, and display the ability of the embryonic founder cells by forming all three germ lineages in vitro. It is well established that the cellular niche plays an important role in stem cell maintenance and differentiation. Stem cells generally have limited function without the specialized microenvironment of the niche that provides key cell-cell contact, soluble mediators, and extracellular matrices. We were interested in the role that Wnt signaling, in particular Wnt3a, played in human embryonic stem cell (hESC) differentiation to hepatic endoderm in vitro. hESC differentiation to hepatic endoderm was efficient in pure stem cell populations. However, in younger hESC lines, generating stromal cell mesenchyme, our model was very inefficient. The negative effect of stroma could be reversed by pretreating hESCs with Wnt3a prior to the onset of hepatocyte differentiation. Wnt3a pretreatment reinstated efficient hESC differentiation to hepatic endoderm. These studies represent an important step in understanding hepatocyte differentiation from hESCs and the role played by the cellular niche in vitro.     

Endoderm- and mesenchyme-dependent commitment of the differentiated epithelial cell types in the developing intestine of rat

During organogenesis, the intestinal tract progressively acquires a functional regionalization along the antero-posterior axis. Positional information needed for enterocytes has been studied, but the mechanisms that control Paneth and endocrine cell differentiation are poorly understood. We have used a model of endoderm/mesenchyme cross-associations to evaluate the respective roles of endoderm and mesenchyme in the cytodifferentiation of these epithelial cells. Heterotopic cross-associations comprising endoderm and mesenchyme from the presumptive proximal jejunum and colon were developed as xenografts in nude mice. Our results show that endoderm from the presumptive proximal jejunum when associated with colonic mesenchyme generate small intestinal enterocytes. Interestingly, no lysozyme-producing cells were generated. On the other hand, associations comprising colon endoderm and jejunal mesenchyme showed heterodifferentiation with typical small intestinal morphology with sucrase-isomaltase expression and Paneth cell differentiation. Heterotopic associations developed enteroendocrine cell patterns according to the normal fate of the endodermal moiety. As enteroendocrine cell commitment seems to occur before the other intestinal cell types, we cannot exclude a role of instructive signals from the mesenchyme on endocrine cell differentiation earlier in the development. These results identified a complex pattern of cell commitment, dependent of the differentiation type of the epithelial cell, on the regional origin of the endoderm and the associated mesenchyme.     

Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice

During embryogenesis, the pancreas arises from dorsal and ventral pancreatic protrusions from the primitive gut endoderm upon induction by different stimuli from neighboring mesodermal tissues. Recent studies have shown that Retinoic Acid (RA) signaling is essential for the development of the pancreas in non-mammalian vertebrates. To investigate whether RA regulates mouse pancreas development, we have studied the phenotype of mice with a targeted deletion in the retinaldehyde dehydrogenase 2 (Raldh2) gene, encoding the enzyme required to synthesize RA in the embryo. We show that Raldh2 is expressed in the dorsal pancreatic mesenchyme at the early stage of pancreas specification. RA-responding cells have been detected in pancreatic endodermal and mesenchymal cells. Raldh2-deficient mice do not develop a dorsal pancreatic bud. Mutant embryos lack Pdx 1 expression, an essential regulator of early pancreas development, in the dorsal but not the ventral endoderm. In contrast to Pdx 1-deficient mice, the early glucagon-expressing cells do not develop in Raldh2 knockout embryos. Shh expression is, as in the wild-type embryo, excluded from the dorsal endodermal region at the site where the dorsal bud is expected to form, indicating that the dorsal bud defect is not related to a mis-expression of Shh. Mesenchymal expression of the LIM homeodomain protein Isl 1, required for the formation of the dorsal mesenchyme, is altered in Raldh2--/-- embryos. The homeobox gene Hlxb9, which is essential for the initiation of the pancreatic program in the dorsal foregut endoderm, is still expressed in Raldh2--/-- dorsal epithelium but the number of HB9-expressing cells is severely reduced. Maternal supplementation of RA rescues early dorsal pancreas development and restores endodermal Pdx 1 and mesenchymal Isl 1 expression as well as endocrine cell differentiation. These findings suggest that RA signaling is important for the proper differentiation of the dorsal mesenchyme and development of the dorsal endoderm. We conclude that RA synthesized in the mesenchyme is specifically required for the normal development of the dorsal pancreatic endoderm at a stage preceding Pdx 1 function.     

Early embryonic expression of a LIM-homeobox gene Cs-lhx3 is downstream of beta-catenin and responsible for the endoderm differentiation in Ciona savignyi embryos

In early Ciona embryos, nuclear accumulation of beta-catenin is most probably the first step of endodermal cell specification. If beta-catenin is mis- and/or overexpressed, presumptive notochord cells and epidermal cells change their fates into endodermal cells, whereas if beta-catenin nuclear localization is downregulated by the overexpression of cadherin, the endoderm differentiation is suppressed, accompanied with the differentiation of extra epidermal cells ( Imai, K., Takada, N., Satoh, N. and Satou, Y. (2000) Development 127, 3009-3020). Subtractive hybridization screens of mRNAs between beta-catenin overexpressed embryos and cadherin overexpressed embryos were conducted to identify potential beta-catenin target genes that are responsible for endoderm differentiation in Ciona savignyi embryos. We found that a LIM-homeobox gene (Cs-lhx3), an otx homolog (Cs-otx) and an NK-2 class gene (Cs-ttf1) were among beta-catenin downstream genes. In situ hybridization signals for early zygotic expression of Cs-lhx3 were evident only in the presumptive endodermal cells as early as the 32-cell stage, those of Cs-otx in the mesoendodermal cells at the 32-cell stage and those of Cs-ttf1 in the endodermal cells at the 64-cell stage. Later, Cs-lhx3 was expressed again in a set of neuronal cells in the tailbud embryo, while Cs-otx was expressed in the anterior nervous system of the embryo. Expression of all three genes was upregulated in beta-catenin overexpressed embryos and downregulated in cadherin overexpressed embryos. Injection of morpholino oligonucleotides against Cs-otx did not affect the embryonic endoderm differentiation, although the formation of the central nervous system was suppressed. Injection of Cs-ttf1 morpholino oligonucleotides also failed to suppress the endoderm differentiation, although injection of its synthetic mRNAs resulted in ectopic development of endoderm differentiation marker alkaline phosphatase. By contrast, injection of Cs-lhx3 morpholino oligo suppressed the endodermal cell differentiation and this suppression was rescued by injection of Cs-lhx3 mRNA into eggs. In addition, although injection of delE-Ci-cadherin mRNA into eggs resulted in the suppression of alkaline phosphatase development, injection of delE-Ci-cadherin mRNA with Cs-lhx3 mRNA rescued the alkaline phosphatase development. These results strongly suggest that a LIM-homeobox gene Cs-lhx3 is one of the beta-catenin downstream genes and that its early expression in embryonic endodermal cells is responsible for their differentiation.     

人源诱导多能干细胞体外定向分化为功能性肝细胞的方法研究

摘要 目的：研究开发一种简易、快速在体外使多能诱导干细胞（induced pluripotent stem cells,iPSCs）定向分化为功能性肝样细胞的培养方法。方法：根据正常肝细胞在体内的发育规律,设计简化诱导方法使iPS细胞定向分化为内胚层细胞,应用qPCR和流式细胞术鉴定其纯度后进一步诱导分化为肝样细胞,并通过qPCR、ELISA、免疫荧光等技术鉴定肝细胞的性状和功能。结果：iPS细胞诱导7天后, OCT4和NANOG的表达水平显著下降,内胚层细胞相关基因CXCR4、FOXA2和HNF4A表达水平明显升高。内胚层细胞继续诱导培养15天后,肝细胞特异性标志基因ALB、TDO2、RBP4、G6PC和肝药酶基因CYPs等显著上调,同时产生高水平的白蛋白和尿素;PAS糖原染色为阳性,能主动摄取和释放吲哚菁绿,证实诱导成的肝样细胞具备正常肝细胞的部分功能。结论：该诱导方案能够在体外使iPS细胞遵循正常肝脏发育通路简易、高效地分化为功能性肝细胞。本研究为大量获得iPS来源的肝细胞及其在细胞疗法和药筛模型中的运用提供了可能性。     

Hepatocytic differentiation of rhesus monkey embryonic stem cells promoted by collagen gels and growth factors

rES (rhesus monkey embryonic stem) cells have similar characteristics to human ES (embryonic stem) cells, and might be useful as a substitute model for preclinical research. Before their clinical application, it is critical to understand the roles of factors that control the differentiation of ES cells into hepatocytes. Here, we analysed the effect of collagen gels on rES cells differentiation into hepatocytes by stepwise protocols. About 80% of DE (definitive endoderm) cells were generated from rES cells after being treated with activin A. The DE cells were then plated on to collagen gels or type I collagen-coated wells with growth factors to induce hepatocyte differentiation. In type I collagen systems, characteristics of immature hepatocytes were observed, including the expression of immature hepatic genes and the generation of 15±3% AFP (alpha fetoprotein)/CK (cytokeratin)18 double-positive cells. In collagen gel culture, differentiated cells exhibited typical hepatocyte morphology and expressed adult liver-specific genes. The mRNA expression of AFP (immature hepatic gene) was detected at day 11 but decreased at day 18. In contrast, mRNA expression of albumin (mature hepatic gene) was detected at day 11 and increased at day 18. Compared with type I collagen systems, significantly higher AFP/CK18 double-positive cells (68±7%) were produced in collagen gel culture. Furthermore, some differentiated cells acquired the hepatocytic function of glycogen storage. However, only immature hepatic genes were observed in collagen gel systems if growth factors were absent. Thus, collagen gels combined with hepatocyte-inducing growth factors efficiently promoted differentiation of hepatocytes from rES.