Hepatic differentiation of murine embryonic stem cells.

Murine embryonic stem (ES) cells can replicate indefinitely in culture and can give rise to all tissues, including the germline, when reimplanted into a murine blastocyst. ES cells can also be differentiated in vitro into a wide range of cell types. We have utilized a liver-specific marker to demonstrate that murine ES cells can differentiate into hepatocytes in vitro. We have used ES cells carrying a gene trap vector insertion (I.114) into an ankyrin repeat-containing gene (Gtar) that we have previously shown provides an exclusive beta-galactosidase marker for the early differentiation of hepatocytes in vivo. beta-Galactosidase-positive cells were differentiated from I.114 ES cells in vitro. The identity of these cells was confirmed by the expression of the proteins alpha-fetoprotein, albumin, and transferrin and by the fact that they have an ultrastructural appearance consistent with that of embryonic hepatocytes. We propose that this model system of hepatic differentiation in vitro could be used to define factors that are involved in specification of the hepatocyte lineage. In addition, human ES cells have recently been derived and it has been proposed that they may provide a source of differentiated cell types for cell replacement therapies in the treatment of a variety of diseases.     

Sequential specification of neurons and glia by developmentally regulated extracellular factors

Cortical progenitor cells give rise to neurons during embryonic development and to glia after birth. While lineage studies indicate that multipotent progenitor cells are capable of generating both neurons and glia, the role of extracellular signals in regulating the sequential differentiation of these cells is poorly understood. To investigate how factors in the developing cortex might influence cell fate, we developed a cortical slice overlay assay in which cortical progenitor cells are cultured over cortical slices from different developmental stages. We find that embryonic cortical progenitors cultured over embryonic cortical slices differentiate into neurons and those cultured over postnatal cortical slices differentiate into glia, suggesting that the fate of embryonic progenitors can be influenced by developmentally regulated signals. In contrast, postnatal progenitor cells differentiate into glial cells when cultured over either embryonic or postnatal cortical slices. Clonal analysis indicates that the postnatal cortex produces a diffusible factor that induces progenitor cells to adopt glial fates at the expense of neuronal fates. The effects of the postnatal cortical signals on glial cell differentiation are mimicked by FGF2 and CNTF, which induce glial fate specification and terminal glial differentiation respectively. These observations indicate that cell fate specification and terminal differentiation can be independently regulated and suggest that the sequential generation of neurons and glia in the cortex is regulated by a developmental increase in gliogenic signals.     

Notch is the key factor in the process of fetal liver stem/progenitor cells differentiation into hepatocytes

Cell transplantation is efficient method to therapy end-stage liver disease (ESLD). How to punctually induce stem cell differentiation into hepatocyte is still a challenge. Notch plays important roles in embryonic development and cell differentiation. However, during the differentiation process from fetal liver stem/progenitor cells (FLSPCs) to mature hepatocytes, the contribution of Notch, especially which Notch receptor is primarily responsible, is unknown. First, specific Notch receptor responsible for FLSPCs differentiation was identified. On both tissue level and cell level, we found that Notch3 was the only receptor greater expressed in liver tissue at embryonic day (ED) 14 and FLSPCs, compared with the adult liver and BRL cells, respectively. Second, morphological phenotypic and functional aspects were analyzed to evaluate whether Notch inhibition by GSIs (γ-secretase inhibitors, inhibitor of Notch) promotes the differentiation of FLSPCs into hepatocytes. Results showed that N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) as GSIs was able to induce FLSPCs differentiation into hepatocytes. The differentiated FLSPCs showed similar morphology to mature hepatocytes, expressed hepatic markers indicative of a mature developmental stage, and displayed similar functionality to mature hepatocytes. The differentiation efficiency by GSIs was similar to that by hepatocyte growth factor (HGF) induction. More specifically, as the differentiation of FLSPCs progressed towards hepatocytes, the expression of Notch3 was gradually down-regulated, consistent with the down-regulation of other stem cell markers. These findings imply that Notch3 may not only be a regulator of FLSPCs differentiation into hepatocytes, but also be a potential marker of FLSPCs.     

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.     

Human Embryonic and Rat Adult Stem Cells with Primitive Endoderm-Like Phenotype Can Be Fated to Definitive Endoderm,and Finally Hepatocyte-Like Cells

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10–20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development.     

胚胎期及成年大白鼠肝脏的表皮角蛋白免疫细胞化学定位

应用自制的表皮角蛋白(EK)家兔抗体,对胚胎期及成年大白鼠的肝脏进行免疫细胞化学定位。结果表明,大白鼠肝脏内,肝细胞EK阴性,胆管上皮细胞EK明确阳性;大白鼠胚胎肝脏也显示出这种EK染色差别。随着肝内胆管上皮的形成,EK阳性物质在其胞浆内出现。作者认为,EK阳性物质的出现是肝内胆管上皮结构分化的结果。     

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.     

Sdmg1 is a conserved transmembrane protein associated with germ cell sex determination and germline-soma interactions in mice

In mammals, the supporting cell lineage in an embryonic gonad communicates the sex-determining decision to various sexually dimorphic cell types in the developing embryo, including the germ cells. However, the molecular nature of the sex-determining signals that pass from the supporting cells to the germ cells is not well understood. We have identified a conserved transmembrane protein, Sdmg1, owing to its male-specific expression in mouse embryonic gonads. Sdmg1 is expressed in the Sertoli cells of embryonic testes from 12.5 dpc, and in granulosa cells of growing follicles in adult ovaries. In Sertoli cells, Sdmg1 is localised to endosomes, and knock-down of Sdmg1 in Sertoli cell lines causes mis-localisation of the secretory SNARE Stx2 and defects in membrane trafficking. Upregulation of Sdmg1 appears to be part of a larger programme of changes to membrane trafficking pathways in embryonic Sertoli cells, and perturbing secretion in male embryonic gonads in organ culture causes male-to-female germ cell sex reversal. These data suggest that changes that occur in the cell biology of embryonic Sertoli cells may facilitate the communication of male sex-determining decisions to the germ cells during embryonic development.     

Elevated expression of hormone-regulated rat hepatocyte functions in a new serum-free hepatocyte-stromal cell coculture model

Summary The specific performance of the adult hepatic parenchymal cell is maintained and controlled by factors deriving from the stromal bed; the chemical nature of these factors is unknown. This study aimed to develop a serum-free hierarchical hepatocyte-nonparenchymal (stromal) cell coculture system. Hepatic stromal cells proliferated on crosslinked collagen in serum-free medium with epidermal growth factor, basic fibroblast growth factor, and hepatocyte-conditioned medium; cell type composition changed during the 2-wk culture period. During the first wk, the culture consisted of proliferating sinusoidal endothelial cells with well-preserved sieve plates, proliferating hepatic stellate cells, and partially activated Kupffer cells. The number of endothelial cells declined thereafter; stellate cells and Kupffer cells became the prominent cell types after 8 d. Hepatocytes were seeded onto stromal cells precultured for 4–14 d; they adhered to stellate and Kupffer cells, but spared the islands of endothelial cells. Stellate cells spread out on top of the hepatocytes; Kupffer cell extensions established multiple contacts to hepatocytes and stellate cells. Hepatocyte viability was maintained by coculture; the positive influence of stromal cell signals on hepatocyte differentiation became evident after 48 h; a strong improvement of cell responsiveness toward hormones could be observed in cocultured hepatocytes. Hierarchial hepatocyte coculture enhanced the glucagon-dependent increases in phosphoenolpyruvate carboxykinase activity and messenger ribonucleic acid (mRNA) content three- and twofold, respectively; glucagon-activated urea production was elevated twofold. Coculturing also stimulated glycogen deposition; basal synthesis was increased by 30% and the responsiveness toward insulin and glucose was elevated by 100 and 55%, respectively. The insulin-dependent rise in the glucokinase mRNA content was increased twofold in cocultured hepatocytes. It can be concluded that long-term signals from stromal cells maintain hepatocyte differentiation. This coculture model should, therefore, provide the technical basis for the investigation of stroma-derived differentiation factors.     

Hepatocytes in heterokaryons do not retard the nuclei of stimulated fibroblasts entering the S period]

Serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with hepatocytes from intact, regenerating and embryonic mouse livers to elucidate mechanisms of liver cell proliferation, DNA synthesis being investigated in nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes in heterokaryons were found to have no inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period, but on the contrary they were involved in DNA synthesis. In addition, the nuclei in heterokaryons mutually stimulated each other to enter the S-period. In their turn, the resting fibroblasts did not prevent the proliferating hepatocytes from the regenerating and embryonic livers to enter the S-period. Possible reasons of the absence of inhibitory effect of differentiated cells in heterokaryons are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in resting immortalized cells differs from that in differentiated cells where proliferation seems to be stopped without affecting the endogenous inhibitor postulated for the resting and ageing fibroblasts.     

Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell-derived hepatocytes

Severe acute liver failure, even when transient, must be treated by transplantation and lifelong immune suppression. Treatment could be improved by bioartificial liver (BAL) support, but this approach is hindered by a shortage of human hepatocytes. To generate an alternative source of cells for BAL support, we differentiated mouse embryonic stem (ES) cells into hepatocytes by coculture with a combination of human liver nonparenchymal cell lines and fibroblast growth factor-2, human activin-A and hepatocyte growth factor. Functional hepatocytes were isolated using albumin promoter-based cell sorting. ES cell-derived hepatocytes expressed liver-specific genes, secreted albumin and metabolized ammonia, lidocaine and diazepam. Treatment of 90% hepatectomized mice with a subcutaneously implanted BAL seeded with ES cell-derived hepatocytes or primary hepatocytes improved liver function and prolonged survival, whereas treatment with a BAL seeded with control cells did not. After functioning in the BAL, ES cell-derived hepatocytes developed characteristics nearly identical to those of primary hepatocytes.     

Specific protein markers in monolayer hepatocyte cultures from adult rats

By indirect immunofluorescence it has been shown that syntheses of protein A, ligandin, cytochrome P-450 PhB, and serum albumin persist in hepatocytes of adult rats during the first 2-3 days in culture. A surface protein--fibronectin--was also synthesized in cultured cells to be localized on the lower side of the free cell edge. On the 4-5th day of cultivation large regions of the lammelar cytoplasm appeared in hepatocytes accompanied by cell polarization. As a result, cells acquired a "fibroblast-like" form. During this period of cultivation, cells were characterized by the loss of cytochrome P-450 PhB, by a drastic decrease in protein A, and ligandin synthesis. At the same time, gamma-glutamyl transpeptidase, the protein characteristic of the embryonic stages, was revealed histochemically. Therefore, the impairment of tissue organization accompanying the transfer of hepatocytes into the vitro conditions results in gradual changes of their morphology, in a reduction or complete loss of some specific "adult" synthesis and activation of the "embryonic" synthesis.     

Developmental Stage-Specific Hepatocytes Induce Maturation of HepG2 Cells by Rebuilding the Regulatory Circuit

On the basis of their characteristics, we presume that developmental stage-specific hepatocytes should have the ability to induce maturation of hepatoma cells. A regulatory circuit formed by hepatocyte nuclear factor (HNF)-4α, HNF-1α, HNF-6 and the upstream stimulatory factor (USF-1) play a key role in the maturation of embryonic hepatocytes; however, it is unclear whether the regulatory circuit mediates the embryonic induction of hepatoma cell maturation. In this study, 12.5-d to 15.5-d mouse embryonic hepatocytes or their medium were used to coculture or treat HepG2 cells, and the induced maturation was evaluated in vitro and in vivo. In the induced HepG2 cells, the components of the regulatory circuit were detected, their cross-regulation was evaluated and HNF-4α RNA interference was performed. We found that 13.5-d to 14.5-d embryonic hepatocytes could induce HepG2 cell maturation, demonstrated by morphological changes, increased maturation markers and decreased c-Myc and α-fetoprotein (AFP) in vitro. The majority of HepG2 tumors were eliminated by 13.5-d embryonic induction in vivo. All components of the regulatory circuit were upregulated and the binding of HNF-4α, HNF-1α, HNF-6 and USF-1 to their target sites was promoted to rebuild the regulatory circuit in the induced HepG2 cells. Moreover, RNA interference targeting HNF-4α, which is the core of the regulatory circuit, attenuated the induced maturation of HepG2 cells with downregulation of the regulatory circuit. These results revealed that developmental stage-specific hepatocytes could induce the maturation of HepG2 cells by rebuilding the regulatory circuit.