Differentiation of human and mouse embryonic stem cells along a hepatocyte lineage

Embryonic stem (ES) cells may differentiate along a hepatocyte lineage; however, currently there are no reports of culture conditions yielding high levels of hepatocyte-specific gene expression in these cells. We investigated culture conditions for differentiating ES cells into hepatocyte-like cells in vitro. Various combinations of culture media, growth and differentiation factors, and substratum precoatings were evaluated, and it was determined that a combination of Iscove's modified Dulbecco's medium with 20% fetal bovine serum, human insulin, dexamethasone. and collagen type I precoating was optimal for directing mouse ES cells along a hepatocyte lineage. Treatment of mouse ES cell with the optimal condition led to prealbumin gene expression 20% as high, and albumin synthesis 7% as high, as in mouse liver. The optimal culture condition also induced albumin gene expression in differentiated human ES cells 1% as high as in normal human hepatocytes as shown by Western blot analysis, and cells were positive for human albumin by immunocytochemistry. In addition, our optimal condition led to high levels of albumin gene expression in primary mouse hepatocytes after 35 days of culture, levels 10-fold higher than with other hepatocyte differentiation media. In conclusion, our optimal condition directed both mouse and human ES cells along a hepatocyte lineage. This represents the initial step in establishing cell lines that can be employed in cell-based therapeutics in humans and for toxicology and pharmacology studies.     

Murine embryonic stem cell-derived hepatic progenitor cells engraft in recipient livers with limited capacity of liver tissue formation

Directed endodermal differentiation of murine embryonic stem (ES) cells gives rise to a subset of cells with a hepatic phenotype. Such ES cell-derived hepatic progenitor cells (ES-HPC) can acquire features of hepatocytes in vitro, but fail to form substantial hepatocyte clusters in vivo. In this study, we investigated whether this is due to inefficient engraftment or an immature phenotype of ES-HPC. ES cells engrafted into recipient livers of NOD/SCID mice with a similar efficacy as adult hepatocytes after 28 days. Because transplanted unpurified ES-HPC formed teratomas in the spleen and liver, we applied an albumin promoter/enhancer-driven reporter system to purify ES-HPC by cell sorting. RT-PCR analyses for hepatocyte-specific genes showed that the cells exhibited a hepatic phenotype, lacking the expression of the pluripotency marker Oct4, comparable to cells of day 11.5 embryos. Sorted ES-HPC derived from beta-galactosidase transgenic ES cells were injected into fumaryl-acetoacetate-deficient (FAH(-/-)) SCID mice and analyzed after 8 to 12 weeks. Staining with X-gal solution revealed the presence of engrafted cells throughout the liver. However, immunostaining for the FAH protein indicated hepatocyte formation at a very low frequency, without evidence for large hepatocyte cluster formation. In conclusion, the limited repopulation capacity of ES-HPC is not caused by a failure of primary engraftment, but may be due to an immature hepatic phenotype of the transplanted ES-HPC.     

Hepatocyte differentiation from embryonic stem cells and umbilical cord blood cells

With the development of regeneration medicine, many researchers have attempted hepatic differentiation from nonhepatic-origin cell sources. The differentiation of embryonic stem (ES) cells into hepatocyte-like cells has been reported in several papers. Mouse ES cells have shown a potential to develop into hepatocyte-like cells in vitro on the basis of hepatic gene expression after adding several growth factors. We transplanted cultured embryoid body (EB) cells (male) into female mice. A liver specimen of the recipient was examined by immunohistochemical staining for albumin and fluorescence in situ hybridization for the Y chromosome after transplantation. Both Y chromosome- and albumin-positive cells were recognized in the recipient female liver, and were considered to be hepatocyte-like cells derived from ES cells containing the Y chromosome. Many groups, including ourselves, have studied hepatocyte-like cell differentiation from umbilical cord blood cells (UBCs). We cultured nucleated cells isolated from UBCs. Using immunostaining, ALB-positive and CK-19-positive cells were recognized in the culture. Dual staining of ALB and CK-19 demonstrated that ALB was coexpresed with CK-19, suggesting the existence of hepatic progenitors. In this review, we consider recent studies of the differentiation of hepatocytes from nonhepatic origins, especially ES cells and umbilical cord blood.     

Evaluation of a hybrid artificial liver module based on a spheroid culture system of embryonic stem cell-derived hepatic cells

Hybrid artificial liver (HAL) is an extracorporeal circulation system comprised of a bioreactor containing immobilized functional liver cells. It is expected to not only serve as a temporary liver function support system, but also to accelerate liver regeneration in recovery from hepatic failure. One of the most difficult problems in developing a hybrid artificial liver is obtaining an adequate cell source. In this study, we attempt to differentiate embryonic stem (ES) cells by hepatic lineage using a polyurethane foam (PUF)/spheroid culture in which the cultured cells spontaneously form spherical multicellular aggregates (spheroids) in the pores of the PUF. We also demonstrate the feasibility of the PUF-HAL system by comparing ES cells to primary hepatocytes in in vitro and ex vivo experiments. Mouse ES cells formed multicellular spheroids in the pores of PUF. ES cells expressed liver-specific functions (ammonia removal and albumin secretion) after treatment with the differentiation-promoting agent, sodium butyrate (SB). We designed a PUF-HAL module comprised of a cylindrical PUF block with many medium-flow capillaries for hepatic differentiation of ES cells. The PUF-HAL module cells expressed ammonia removal and albumin secretion functions after 2 weeks of SB culture. Because of high proliferative activity of ES cells and high cell density, the maximum expression level of albumin secretion function per unit volume of module was comparable to that seen in primary mouse hepatocyte culture. In the animal experiments with rats, the PUF-HAL differentiating ES cells appeared to partially contribute to recovery from liver failure. This outcome indicates that the PUF module containing differentiating ES cells may be a useful biocomponent of a hybrid artificial liver support system.     

Enrichment of hepatocytes differentiated from mouse embryonic stem cells as a transplantable source

BACKGROUND: We previously reported that hepatocytes can be differentiated from embryonic stem (ES) cells by way of embryoid body (EB) formation and are transplantable into the mouse liver. However, the transplantation of EB-derived cells frequently resulted in teratoma formation in the recipient liver. In the present study, we eliminated the tumorigenic cells from EB outgrowths and examined the effects of enriched ES-cell-derived hepatocyte transplantation into an injured liver. METHODS: On day 15 in culture, the EBs were partially disaggregated and subcultured. Hepatocytes in the subcultured cells were examined by the expression of hepatocyte markers. Undifferentiated cells contaminating in the EB-derived cells were eliminated by Percoll discontinuous gradient centrifugation. Furthermore, undifferentiated cells, endothelial cells, and macrophages were eliminated by magnetic cell sorting using platelet/endothelial cell adhesion molecule (PECAM)-1 and Mac-1 antibodies. These enriched ES-cell-derived hepatocytes were then transplanted into the injured mouse liver. RESULTS: Percoll centrifugation and PECAM-1 antibodies eliminated the undifferentiated cells expressing Oct-3/4 from the EB-derived cells. ES-cell-derived hepatocytes showed expression of liver-related genes, synthesis of urea and glycogen, and structural characteristics during subculture. A transplantation study showed that the enriched ES-cell-derived hepatocytes integrated into the injured mouse liver and produced no teratomas. When the ES-cell-derived hepatocytes were transplanted into a CCl4-injured liver, the liver function was subsequently improved. CONCLUSIONS: Functional hepatocytes can be differentiated from mouse ES cells by way of EB formation. The elimination of undifferentiated cells from the EBs provides transplantable cells for liver failure without tumorigenicity.     

Establishment of an immortalized porcine liver cell line JSNK-1 with retroviral transduction of SV40T

Maintenance of freshly isolated porcine liver cells in vitro is limited for a short period of time. Therefore, establishment of easy handling cell lines is extremely important for in vitro study for liver cells and their possible utilization for cell differentiation and growth of stem cells. Porcine liver cells were transduced with a retroviral vector SSR#69 expressing SV40T, one of SSR#69-immortalized porcine liver cell lines, JSNK-1, was established and characterized. Morphology of JSNK-1 cells was spindle shaped. When the cells became confluent, JSNK-1 cells revealed hills-and-valleys pattern. In the presence of vitamin A, JSNK-1 cells showed big droplets inside the cytoplasm, which were positive with PAS staining. JSNK-1 cells showed the gene expression of collagen type 1α1, collagen type 1α2, FLT-1, β-actin, and SV40T. Immunostaining study revealed that JSNK-1 cells produced collagen, vimentin, and α-smooth muscle actin. JSNK-1 cells possessed the characteristics of the liver stellate cells. JSNK-1 cells produced hepatocyte growth factor (HGF) in a time-dependent manner. When cocultured with iPS cells towards the hepatic differentiation, JSNK-1 cells facilitated their hepatic differentiation in terms of albumin production. In conclusion, JSNK-1 cells would be valuable in the study of liver stellate cell pathophysiology and contribute to the optimization of hepatic differentiation of iPS cells.     

Teratoma formation and hepatocyte differentiation in mouse liver transplanted with mouse embryonic stem cell-derived embryoid bodies

We previously reported that mouse embryonic stem (ES) cells are capable of differentiating into hepatocytes in cultured embryoid bodies (EBs) and that hepatocytes generate in the recipient liver injected with cultured day-9 EB cells via spleen without the formation of a teratoma. Because ES cells frequently form teratomas in recipient mice, we investigated incidence of teratoma formation when day-9 EBs derived from ES cells were transplanted directly into the subcapsule of mouse liver. In contrast to injection of day-9 EB cells through the portal vein via the spleen, direct subcapsular injection of cultured day-9 EB cells into liver, and even of cultured day-15 EBs, resulted in an high incidence of teratoma in the liver. In teratomas of livers injected directly with day-15 EBs, hepatocytes were detected singly and in clusters. These results imply that undifferentiated cells capable of developing into teratomas exist in cultured EBs, and even in cultured day-15 EBs containing differentiated hepatocytes.     

Hepatic differentiation of mouse embryonic stem cells in a bioreactor using polyurethane/spheroid culture

BACKGROUND: We have previously developed a hybrid artificial liver (HAL) using polyurethane foam (PUF)/hepatocyte spheroid culture. The PUF-HAL has been successfully scaled up to a clinical level. However, one of the most difficult problems for clinical application of HALs is obtaining a cell source. We now focused our attention on embryonic stem (ES) cells as a potential source for HAL. In this study, we investigated the differentiation of mouse ES (mES) cells into functional hepatocytes in the PUF-HAL module. METHODS: The PUF-HAL module included a cylindrical PUF block having many capillaries for medium flow. mES cells were immobilized in the module. To induce hepatic differentiation, growth factors were added to the culture medium. We evaluated cell density, gene expression analysis, and liver-specific functions. RESULTS: mES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of PUF. mES cells proliferated by 20 days, achieving a high cell density (about 1 x 10(8) cells/cm3 PUF). Differentiating ES cells expressed endodermal-specific genes such as alpha-fetoprotein, albumin, and tryptophan 2, 3-deoxygenase. The activity of ammonia removal of mES cells per unit volume of the module was detectable by 15 days and increased with culture time. Maximal expression levels were comparable to those of primary (porcine and human) hepatocytes. SUMMARY: mES cells immobilized in the PUF module expressed liver-specific functions at high level, because of high cell density in culture and hepatic differentiation. These results indicated that PUF module-immobilized mES cells may be useful as a biocomponent of HALs.     

Partial hepatectomy and subsequent radiation facilitates engraftment of mouse embryonic stem cells in the liver

For liver-targeted regenerative medicine, embryonic stem (ES) cell-derived hepatocyte-like cells proffer great expectation. In vitro exposure to a combination of various growth factors, such as hepatocyte growth factor and fibroblast growth factor-4, as well as cytokines, leads to differentiation of ES cells into hepatocyte-like cells. We sought to determine the in vivo environment that allowed engraftment of ES cells transplanted to the liver. Thus, we examined the effect of partial hepatectomy (50%) (PHT) and subsequent radiation (RT) of the male Balb/c mouse host liver on ES cell engraftment. ES cells (5 x 10(6)) derived from 129Sv mice were transplanted into the residual liver. The controls were ES cells transplanted into a normal liver. Bromo-deoxy-residine (BrdU)-uptake was performed to evaluate the effect of hepatectomy and RT on hepatocyte regeneration. Mouse ES cells engrafted, forming teratomas in the normal liver without showing any mononuclear infiltration. A liver modified by PHT and RT facilitated engraftment of mouse ES cells compared with a normal liver. Hepatic RT significantly suppressed hepatocytic uptake of BrdU.     

Differentiation of embryonic stem cells after transplantation into peritoneal cavity of irradiated mice and expression of specific germ cell genes in pluripotent cells

Permanent embryonic stem cell lines (ES cells) are considered as one of the most promising cellular sources for regenerative medicine. ES cells have a high proliferative potency and ability to differentiate into all kinds of somatic and germ cells. However, transplantation of undifferentiated ES cells into adult recipient tissue results in the formation of teratomas. To understand the mechanisms underlying self-renewal and determination of pluripotent cells, we investigated differentiation potencies of undifferentiated ES cells and differentiating embryoid bodies (EB). ES cells and EBs growing on acetate-cellulose membranes were transplanted into the peritoneal cavity of irradiated mice. Behavior and differentiation of transplanted cells were studied within 1, 2, 3, and 6 weeks after transplantation. No differences in the cell composition were found in the teratomas formed by ES cells and differentiating EBs. The pattern of expression of the genes specific for pluripotent and germ cells was studied in all types of experimental teratomas. The expression of oct4, stella, fragilis was detected in the teratomas, but nanog was not expressed. We conclude that pluripotent cells are retained in the experimental teratomas formed after transplantation of ES cells and EBs but the pattern of expression of the studied genes underwent changes.     

Cholestasis without cirrhosis alters regulatory liver gene expression and inhibits hepatic regeneration.

Partial hepatectomy (PH) initiates cellular signals for regeneration. Sequential expression of nuclear and cytosolic protooncogenes accompanies the restoration of normal liver function and architecture. Although cirrhosis is known to inhibit liver regeneration, the effects of noncirrhotic cholestasis on hepatocellular proliferation, differentiation, and regulatory gene expression are unknown. To examine this, 25 male Fisher rats underwent common bile duct ligation and division. A 47% +/- 5% PH was performed 10 days after common bile duct ligation and division when histologic analysis revealed cholestasis without cirrhosis. Despite early elevations of total hepatic DNA and RNA values, cholestatic livers demonstrated a significant threefold suppression of expected hepatocyte mitotic indexes 48 and 72 hours after PH, compared with livers after PH alone. Weight restoration in cholestatic livers was 11% +/- 5.2% compared with 40% +/- 4.3% in control livers (+/- SEM; p less than 0.001) 5 days after PH. Analysis of regenerating liver messenger RNA with complementary DNA probes revealed an abnormal, sustained elevation of K-ras expression in cholestatic livers through all time points. Cholestasis blunted but did not obliterate normal sequential elevations in H-ras found in control livers. The expression of c-myc was inhibited threefold with cholestasis 72 hours after PH. These results are the first indication that cholestasis alone inhibits hepatocyte proliferation and the expression of c-myc that normally precedes the first wave of mitosis. This implies that cholestasis without cirrhosis may alter programmed liver gene expression, inhibiting normal hepatic regeneration.     

肝硬变门静脉高压症患者肝细胞凋亡与原癌基因表达的研究

目的探讨肝细胞凋亡与肝硬变及原癌基因的关系。方法取正常人及肝硬变门静脉高压症患者的肝组织。行透射电镜检查，并检测其凋亡细胞及原癌基因表达。结果肝硬变患者肝组织内的凋亡细胞及凋亡指数高于正常人；肝组织原癌基因c-myc，c-fos和bcl-2均呈过度表达，而正常人肝组织则无表达。结论肝细胞凋亡在肝硬变的演变过程中发挥一定作用，原癌基因c-myc，c-fos和bcl-2参与肝细胞凋亡的调控。     

Liver-targeted regenerative medicine

Recently, much attention has been drawn to liver-targeted regenerative medicine for the treatment of liver failure. Researchers in various fields consider that the following cells can be used for such therapy: human embryonic stem (ES) cells, somatic stem cells, hepatic stem cells, small hepatocytes, bone marrow- and cord blood-derived hepatic progenitor cells, and human hepatocyte cell lines. The Cre/loxP-based reversible immortalization of human hepatocytes is also introduced in this review article. Here we describe the candidates that can contribute to hepatic regeneration. This reversible immortalization system allows us to establish a highly safe human hepatocyte line that is capable of differentiation at a lower cost and on a larger scale.     

胚胎干细胞源性肝细胞的获得及体内移植研究

目的 探讨体外定向诱导小鼠胚胎干细胞(ES细胞)分化为肝细胞的方法及肝损伤模型肝内移植的可行性.方法 常规培养ES细胞后,继续悬浮培养4 d以形成拟胚体(EBs),转移EBs到铺有明胶的6孔板中贴壁培养,并添加3 mmol/L丁酸钠开始诱导分化,7 d后加入淤胆血清筛选、纯化ES源性肝细胞,分化过程中用光学显微镜和电子显微镜观察细胞形态及超微结构的改变;用逆转录-聚合酶链反应(RT-PCR)方法检测肝细胞特异性标志基因:白蛋白(ALB)、甲胎蛋白(AFP)、甲状腺素运载蛋白(TTR)、α1抗胰蛋白酶(AAT)、葡萄糖6磷酸酶(G6P)、酪氨酸转氨酶(TAT)mRNA水平的表达;以荧光示踪剂CFDA-SE标记诱导获得的肝细胞,并移植到肝损伤小鼠肝内,观察移植细胞在肝内的定居、增殖情况.结果 诱导分化过程中,ES细胞形态逐渐出现肝细胞样改变,其超微结构与小鼠肝细胞超微结构十分相似;RT-PCR结果显示,随着诱导时间的推进,标志肝细胞发育过程的ALB、AFP、TTR、AAT、G6P、TAT mRNA顺序表达;肝内移植实验结果显示:ES源性肝细胞可在肝损伤小鼠肝内定居并增殖.结论 丁酸钠联合淤胆血清可以诱导ES细胞分化为肝细胞,ES源性肝细胞肝损伤模型体内移植是可行的,这有可能为细胞移植治疗难治性肝病提供一种新的细胞来源.     

小鼠骨髓干细胞体外定向诱导为肝细胞样细胞的实验研究

目的来源于骨髓干细胞的有功能的干细胞,可能成为生物人工肝和肝细胞移植的细胞来源。我们建立恰当的体外诱导培养体系,促使骨髓干细胞转化为肝细胞。方法获取骨髓干细胞,建立以FGF、HGF、OSM、EGF为主的细胞诱导培养体系。在细胞分化过程中,观察细胞形态和数量的变化,RT-PCR检测基因表达的变化,免疫荧光染色技术在蛋白水平检测ALB和CK18表达情况。PAS染色检测其糖代谢功能。结果在诱导过程中,多极性的肝细胞样细胞在12 d内可以观察到,且其随着细胞分化过程逐渐增多、集落增大。诱导细胞在7 d内表达AFPmRNA并维持到第21天,但后期表达减弱;在7天内表达ALBmRNA和CK18mRNA,随着分化时间的延长,其表达不断增强;在14 d内表达TTRmRNA,随着分化时间的延长,其表达不断增强。免疫荧光染色进行定位:在诱导组,诱导21 d的细胞表达ALB和CK18,其中ALB表达于胞浆和胞膜,而CK18表达于胞浆。糖原染色发现诱导组中,细胞胞浆内出现大小不等的红染的糖原颗粒。结论我们建立的以细胞因子FGF、HGF、OSM、EGF为主的细胞诱导培养体系是有效的,通过诱导骨髓干细胞,我们获得了有部分肝细胞形态结构和功能的肝细胞样细胞。     

Isolation of hepatocyte-like cells from mouse embryoid body cells

We previously reported that embryoid body (EB) cells derived from embryonic stem (ES) cells are capable of differentiating into functional hepatocyte-like cells both in vitro and in vivo. Because transplantation of EB-derived cells into the liver via the spleen resulted in a low incidence of teratoma formation, purification of hepatocyte-like cells is required to prevent teratoma formation. The aim of this study was to purify hepatocyte-like cells from cultured EBs. For the isolation of hepatocyte-like cells, EBs cultured for 15 days were treated with trypsin-EDTA. The disaggregated cells were plated on a gelatin-coated dish as a monolayer. These cells were separated by Percoll gradient centrifugation, enriched by magnetic cell sorting, and purified by FACS. The purified hepatocyte-like cells in monolayer cultures were positive for immunostaining for albumin and expressed albumin mRNA, but not Oct3/4 mRNA. Transplantation of the purified hepatocyte-like cells derived from mouse ES cells might be an effective treatment for liver failure.