A biliary immune landscape map of primary sclerosing cholangitis reveals a dominant network of neutrophils and tissue-resident T cells.

The human biliary system, a mucosal barrier tissue connecting the liver and intestine, is an organ often affected by serious inflammatory and malignant diseases. Although these diseases are linked to immunological processes, the biliary system represents an unexplored immunological niche. By combining endoscopy-guided sampling of the biliary tree with a high-dimensional analysis approach, comprehensive mapping of the human biliary immunological landscape in patients with primary sclerosing cholangitis (PSC), a severe biliary inflammatory disease, was conducted. Major differences in immune cell composition in bile ducts compared to blood were revealed. Furthermore, biliary inflammation in patients with PSC was characterized by high presence of neutrophils and T cells as compared to control individuals without PSC. The biliary T cells displayed a CD103+CD69+ effector memory phenotype, a combined gut and liver homing profile, and produced interleukin-17 (IL-17) and IL-22. Biliary neutrophil infiltration in PSC associated with CXCL8, possibly produced by resident T cells, and CXCL16 was linked to the enrichment of T cells. This study uncovers the immunological niche of human bile ducts, defines a local immune network between neutrophils and biliary-resident T cells in PSC, and provides a resource for future studies of the immune responses in biliary disorders.

Chenodeoxycholic Acid Modulates Bile Acid Synthesis Independent of Fibroblast Growth Factor 19 in Primary Human Hepatocytes.

Bile acids (BAs) are detergents essential for intestinal absorption of lipids. Disruption of BA homeostasis can lead to severe liver damage. BA metabolism is therefore under strict regulation by sophisticated feedback mechanisms. The hormone-like protein Fibroblast growth factor 19 (FGF19) is essential for maintaining BA homeostasis by down regulating BA synthesis. Here, the impact of both FGF19 and chenodeoxycholic acid (CDCA) on primary human hepatocytes was investigated and a possible autocrine/paracrine function of FGF19 in regulation of BA synthesis evaluated. Primary human hepatocytes were treated with CDCA, recombinant FGF19 or conditioned medium containing endogenously produced FGF19. RNA sequencing revealed that treatment with CDCA causes deregulation of transcripts involved in BA metabolism, whereas treatment with FGF19 had minor effects. CDCA increased FGF19 mRNA expression within 1 h. We detected secretion of the resulting FGF19 protein into medium, mimicking in vivo observations. Furthermore, medium enriched with endogenously produced FGF19 reduced BA synthesis by down regulating CYP7A1 gene expression. However, following knockdown of FGF19, CDCA still independently decreased BA synthesis, presumably through the regulatory protein small heterodimer partner (SHP). In summary, we show that in primary human hepatocytes CDCA regulates BA synthesis in an FGF19-independent manner.

Diiodothyronines regulate metabolic homeostasis in primary human hepatocytes by modulating mTORC1 and mTORC2 activity.

Until three decades, ago 3,5-diiodothyronine (3,5-T2) and 3,3'-diiodothyronine (3,3'-T2) were considered products of thyroid hormone catabolism without biological activity. Some metabolic effects have been described in rodents, but the physiological relevance in humans and the mechanisms of action are unknown. Aim of this work was to investigate the role and the mechanisms of action of 3,5-T2 and 3,3'-T2 in the regulation of metabolic homeostasis in human liver. We used primary human hepatocytes freshly isolated from donors and grown on Matrigel as the golden standard in vitro model to study human hepatic metabolism. Results show that diiodothyronines in the range of plasma physiological concentrations reduced hepatic lipid accumulation, by modulating the activity of the mTORC1/Raptor complex through an AMPK-mediated mechanism, and stimulated the mTORC2/Rictor complex-activated pathway, leading to the down regulation of the expression of key gluconeogenic genes. Hence, we propose that diiodothyronines act as key regulators of hepatic metabolic homeostasis in humans.

Insights From Liver-Humanized Mice on Cholesterol Lipoprotein Metabolism and LXR-Agonist Pharmacodynamics in Humans.

BACKGROUND AND AIMS: Genetically modified mice have been used extensively to study human disease. However, the data gained are not always translatable to humans because of major species differences. Liver-humanized mice (LHM) are considered a promising model to study human hepatic and systemic metabolism. Therefore, we aimed to further explore their lipoprotein metabolism and to characterize key hepatic species-related, physiological differences. APPROACH AND RESULTS: Fah-/- , Rag2-/- , and Il2rg-/- knockout mice on the nonobese diabetic (FRGN) background were repopulated with primary human hepatocytes from different donors. Cholesterol lipoprotein profiles of LHM showed a human-like pattern, characterized by a high ratio of low-density lipoprotein to high-density lipoprotein, and dependency on the human donor. This pattern was determined by a higher level of apolipoprotein B100 in circulation, as a result of lower hepatic mRNA editing and low-density lipoprotein receptor expression, and higher levels of circulating proprotein convertase subtilisin/kexin type 9. As a consequence, LHM lipoproteins bind to human aortic proteoglycans in a pattern similar to human lipoproteins. Unexpectedly, cholesteryl ester transfer protein was not required to determine the human-like cholesterol lipoprotein profile. Moreover, LHM treated with GW3965 mimicked the negative lipid outcomes of the first human trial of liver X receptor stimulation (i.e., a dramatic increase of cholesterol and triglycerides in circulation). Innovatively, LHM allowed the characterization of these effects at a molecular level. CONCLUSIONS: LHM represent an interesting translatable model of human hepatic and lipoprotein metabolism. Because several metabolic parameters displayed donor dependency, LHM may also be used in studies for personalized medicine.

Exogenous alpha 1-antitrypsin down-regulates SERPINA1 expression.

The main goal of the therapy with purified human plasma alpha1-antitrypsin (A1AT) is to increase A1AT levels and to prevent lungs from elastolytic activity in patients with PiZZ (Glu342Lys) A1AT deficiency-related emphysema. Potential hepatic gains of this therapy are unknown. Herein, we investigated the effect of A1AT therapy on SERPINA1 (gene encoding A1AT) expression. The expression of SERPINA1 was determined in A1AT or A1AT plus Oncostatin M (OSM) treated primary human hepatocytes isolated from liver tissues from A1AT deficient patients and control liver tissues. In addition, SERPINA1 mRNA was assessed in lung tissues from PiZZ emphysema patients with and without A1AT therapy, and in adherent human peripheral blood mononuclear cells (PBMC) isolated from healthy PiMM donors. In a dose-dependent manner purified A1AT lowered SERPINA1 expression in hepatocytes. This latter effect was more prominent in hepatocytes stimulated with OSM. Although it did not reach statistical significance (P = 0.0539)-analysis of lung tissues showed lower SERPINA1 expression in PiZZ emphysema patients receiving augmentation therapy relative to those without therapy. Finally, exogenously added purified A1AT (1mg/ml) reduced SERPINA1 expression in naïve as well as in lipopolysaccharide (LPS)-stimulated human adherent PBMCs. Exogenous A1AT protein reduces its own endogenous expression. Hence, augmentation with native M-A1AT protein and a parallel reduction in expression of dysfunctional mutant Z-A1AT may be beneficial for PiZZ liver, and this motivates further studies.

Addition of Dexamethasone Alters the Bile Acid Composition by Inducing CYP8B1 in Primary Cultures of Human Hepatocytes.

BACKGROUND: Primary human hepatocytes offer the best human in vitro model for studies on human liver cell metabolism. Investigators use a variety of different media supplements and matrix biocoatings and the type of culture system used may influence the outcome. OBJECTIVES: To optimize in vitro conditions for primary human hepatocytes with regard to bile acid synthesis. METHODS: Human hepatocytes were isolated and cultured on collagen type I or EHS matrigel in cell media with or without dexamethasone. The glucocorticoid receptor (GR) antagonist RU486 was used to elucidate the involvement of GR. RESULTS: Hepatocytes cultured on EHS matrigel produced more bile acids and expressed higher levels of cholesterol 7α-hydroxylase (CYP7A1) than cells cultured on rat tail collagen. Supplementation with dexamethasone increased the formation of cholic acid (CA) and decreased chenodeoxycholic acid formation. In line with these results, the mRNA expression of sterol 12α-hydroxylase (CYP8B1) increased following dexamethasone treatment. Surprisingly, the mRNA expression of CYP7A1 and CYP27A1 was not increased to the same extent. By using the GR antagonist RU486, we concluded that CYP8B1 induction is mediated via a GR-independent pathway. An altered expression of retinoid-related orphan receptor (ROR) α and ROR α target gene Glucose-6-phosphatase (G6Pase) suggests that ROR α signaling may regulate CYP8B1 expression. CONCLUSION: Primary human hepatocytes have an increased bile acid synthesis rate when cultured on matrigel as compared to collagen. Exposure to glucocorticoid hormones stimulates the expression of CYP8B1, leading to an increased formation of CA and alteration of the bile acid composition. The effect is most likely mediated through a GR-independent pathway, possibly through ROR α.

Assay of Bile Acid Conjugation and Excretion in Human Hepatocytes.

Primary hepatocytes isolated from human, mouse and rat liver as well as cell lines, such as HepG2 cells, are frequently used in vitro systems in liver research. In regenerative medicine, stem cells are used for differentiation towards hepatocyte-like cells with the goal of creating differentiated functional hepatocytes. It is therefore important to measure the quality and function of highly specialized hepatocyte-specific functions using appropriate methods. In this chapter, we describe an assay to assess conjugation and excretion of labeled bile acids in cultured hepatocytes.

Rapid and sensitive assessment of human hepatocyte functions.

Transplantation of human hepatocytes (HTx) has gained recognition as a bridge to, or an alternative to, orthotopic liver transplantation for patients with acute liver failure or genetic defects in liver function. Although the quality of the hepatocytes used for cell transplantation is critical, no consensus exists on protocols to assess the function of hepatocytes prior to HTx. Application of this cell therapy in clinical practice could be aided by fast and reliable assays to evaluate the functional competence of isolated hepatocytes prior to clinical transplantation. Traditional assays for measuring metabolic functions in primary hepatocytes frequently involve highly technical equipment, time-consuming methods, and large numbers of cells. We describe a novel approach for the rapid assessment of the metabolic capabilities of human hepatocytes. This report details simple procedures to evaluate 11 endpoints from cells isolated from human liver that can be performed by a single operator within approximately 2 h of isolation. Longer term cultured hepatocytes were also analyzed to determine if the results from the 2-h tests were predictive of long-term hepatic function. The assays simultaneously measure five cytochrome P450 activities, one phase II activity, plating efficiency, and ammonia metabolism in addition to viability and cell yield. The assays require fewer than 20 million cells and can be completed using commonly available and inexpensive laboratory equipment. The protocol details methods that can be used in a time frame that would allow analysis of hepatic functions in freshly isolated hepatocytes prior to their use for clinical transplantation.

Hepatocyte transplantation ameliorates the metabolic abnormality in a mouse model of acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an autosomal dominant disorder characterized by insufficient porphobilinogen deaminase (PBGD) activity. When hepatic heme synthesis is induced, porphobilinogen (PBG) and 5-aminolevulinic acid (ALA) accumulate, which causes clinical symptoms such as abdominal pain, neuropathy, and psychiatric disturbances. Our aim was to investigate if hepatocyte transplantation can prevent or minimize the metabolic alterations in an AIP mouse model. We transplanted wild-type hepatocytes into PBGD-deficient mice and induced heme synthesis with phenobarbital. ALA and PBG concentrations in plasma were monitored, and the gene transcriptions of hepatic enzymes ALAS1, PBGD, and CYP2A5 were analyzed. Results were compared with controls and correlated to the percentage of engrafted hepatocytes. The accumulation of ALA and PBG was reduced by approximately 50% after the second hepatocyte transplantation. We detected no difference in mRNA levels of PBGD, ALAS1, or CYP2A5. Engraftment corresponding to 2.7% of the total hepatocyte mass was achieved following two hepatocyte transplantations. A lack of precursor production in less than 3% of the hepatocytes resulted in a 50% reduction in plasma precursor concentrations. This disproportional finding suggests that ALA and PBG produced in PBGD-deficient hepatocytes crossed cellular membranes and was metabolized by transplanted cells. The lack of effect on enzyme mRNA levels suggests that no significant efflux of heme from normal to PBGD-deficient hepatocytes takes place. Further studies are needed to establish the minimal number of engrafted hepatocytes needed to completely correct the metabolic abnormality in AIP and whether amelioration of the metabolic defect by partial restoration of PBGD enzyme activity translates into a clinical effect in human AIP.

Mice with chimeric livers are an improved model for human lipoprotein metabolism.

OBJECTIVE: Rodents are poor model for human hyperlipidemias because total cholesterol and low density lipoprotein levels are very low on a normal diet. Lipoprotein metabolism is primarily regulated by hepatocytes and we therefore assessed whether chimeric mice extensively repopulated with human cells can model human lipid and bile acid metabolism. DESIGN: FRG [ F ah(-/-) R ag2(-/-)Il2r g (-/-)]) mice were repopulated with primary human hepatocytes. Serum lipoprotein lipid composition and distribution (VLDL, LDL, and HDL) was analyzed by size exclusion chromatography. Bile was analyzed by LC-MS or by GC-MS. RNA expression levels were measured by quantitative RT-PCR. RESULTS: Chimeric mice displayed increased LDL and VLDL fractions and a lower HDL fraction compared to wild type, thus significantly shifting the ratio of LDL/HDL towards a human profile. Bile acid analysis revealed a human-like pattern with high amounts of cholic acid and deoxycholic acid (DCA). Control mice had only taurine-conjugated bile acids as expcted, but highly repopulated mice had glycine-conjugated cholic acid as found in human bile. RNA levels of human genes involved in bile acid synthesis including CYP7A1, and CYP27A1 were significantly upregulated as compared to human control liver. However, administration of recombinant hFGF19 restored human CYP7A1 levels to normal. CONCLUSION: Humanized-liver mice showed a typical human lipoprotein profile with LDL as the predominant lipoprotein fraction even on a normal diet. The bile acid profile confirmed presence of an intact enterohepatic circulation. Although bile acid synthesis was deregulated in this model, this could be fully normalized by FGF19 administration. Taken together these data indicate that chimeric FRG-mice are a useful new model for human lipoprotein and bile-acid metabolism.

New potential cell source for hepatocyte transplantation: discarded livers from metabolic disease liver transplants.

UNLABELLED: Domino liver transplantation is a method used to increase the number of liver grafts available for orthotopic liver transplantation (OLT). Reports indicate that livers from patients with metabolic liver disease can be safely transplanted into select recipients if the donor's defect and the recipient's metabolic needs are carefully considered. The liver of patients with many types of metabolic liver disease is morphologically and biochemically normal, except for the mutation that characterizes that disease. Other biochemical functions normally performed by the liver are present and presumably "normal" in these hepatocytes. Hepatocytes were isolated from the liver of 35 organ donors and 35 liver tissues taken at OLT from patients with liver disease were analyzed for 9 different measures of viability and function. The data indicate that cells isolated from some diseased livers performed as well or better than those isolated from organ donors with respect to viability, cell yield, plating efficiency and in assays of liver function, including drug metabolism, conjugation reactions and ammonia metabolism. Cells from metabolic diseased livers rapidly and efficiently repopulated a mouse liver upon transplantation. CONCLUSIONS: As with domino liver transplantation, domino cell transplantation deserves consideration as method to extend the pool of available organs and cells for transplantation.

Hepatobiliary disposition of 17-OHPC and taurocholate in fetal human hepatocytes: a comparison with adult human hepatocytes.

Little information is available in the literature regarding the expression and activity of transporters in fetal human liver or cultured cells. A synthetic progesterone structural analog, 17α-hydroxyprogesterone caproate (17-OHPC), is used in the prevention of spontaneous abortion in women with a history of recurrent miscarriage (habitual abortion). 17-OHPC has been reported to traverse the placental barrier and gain access to fetal circulation. In this study, the role of transporters in the disposition of 17-OHPC in fetal and adult human hepatocytes was examined. Progesterone metabolites have been reported to induce trans-inhibition of bile acid transporter, ABCB11. Thus, we investigated the effect of 17-OHPC or its metabolites on [(3)H]taurocholic acid transport in sandwich-cultured human fetal and adult hepatocytes. 17-OHPC was taken up rapidly into the cells and transported out partially by an active efflux process that was significantly inhibited by cold temperature, cyclosporine, verapamil, and rifampin. The active efflux mechanism was observed in both adult and fetal hepatocyte cultures. 17-OHPC produced a concentration-dependent inhibition of taurocholate efflux into canaliculi in sandwich-cultured adult and fetal human hepatocytes. However, given the high concentrations required to cause inhibition of these transport processes, no adverse effects would be anticipated from therapeutic levels of 17-OHPC. We also evaluated the expression of various hepatic transporters (ABCB1, ABCB4, SLCO1B1, SLCO1B3, SLCO2B1, ABCB11, SLC10A1, ABCC2, ABCC3, ABCC4, and ABCG2) in fetal and adult hepatocytes. With the exception of ABCB4, all transporters examined were expressed, albeit at lower mRNA levels in fetal hepatocytes compared with adults.

Improving the techniques for human hepatocyte transplantation: report from a consensus meeting in London.

On September 6 and 7, 2009 a meeting was held in London to identify and discuss what are perceived to be current roadblocks to effective hepatocyte transplantation as it is currently practiced in the clinics and, where possible, to offer suggestions to overcome the blocks and improve the outcomes for this cellular therapy. Present were representatives of most of the active clinical hepatocyte transplant programs along with other scientists who have contributed substantial basic research to this field. Over the 2-day sessions based on the experience of the participants, numerous roadblocks or challenges were identified, including the source of cells for the transplants and problems with tracking cells following transplantation. Much of the discussion was focused on methods to improve engraftment and proliferation of donor cells posttransplantation. The group concluded that, for now, parenchymal hepatocytes isolated from donor livers remain the best cell source for transplantation. It was reported that investigations with other cell sources, including stem cells, were at the preclinical and early clinical stages. Numerous methods to modulate the immune reaction and vascular changes that accompany hepatocyte transplantation were proposed. It was agreed that, to obtain sufficient levels of repopulation of liver with donor cells in patients with metabolic liver disease, some form of liver preconditioning would likely be required to enhance the engraftment and/or proliferation of donor cells. It was reported that clinical protocols for preconditioning by hepatic irradiation, portal vein embolization, and surgical resection had been developed and that clinical studies using these protocols would be initiated in the near future. Participants concluded that sharing information between the groups, including standard information concerning the quality and function of the transplanted cells prior to transplantation, clinical information on outcomes, and standard preconditioning protocols, would help move the field forward and was encouraged.

Development and application of purified tissue dissociation enzyme mixtures for human hepatocyte isolation.

Human hepatocyte transplantation is gaining acceptance for the treatment of liver diseases. However, the reagents used to isolate hepatocytes from liver tissue are not standardized and show lot-to-lot variability in enzyme activity and endotoxin contamination. For clinical application, highly purified reagents are preferable to crude digest preparations. A purified tissue dissociating enzyme (TDE) preparation (CIzyme(TM) purified enzymes) was developed based on the enzyme compositions found in a superior lot of collagenase previously used by our group for human hepatocyte isolation. The performance of this enzyme preparation was compared to collagenase type XI on 110 liver cases by assessing hepatocyte yield, viability, and seven other functional assays that included plating efficiency, basal and induced CYP450 activities, phase II conjugation activity, and ammonia metabolism. No statistically significant difference was observed between these TDEs when they were used to isolate hepatocytes from liver resections or organ donor tissue on 54 hepatocyte isolations with type XI enzyme and 56 isolations using CIzyme(TM). These results show that a highly purified and defined TDE preparation can be formulated that provides excellent performance with respect to viability, yield, and functional activity of the isolated cells. In addition to reproducible formulation, these purified enzyme products have only 2-3% of the endotoxin of crude enzyme preparations. These results show that purified enzymes such as CIzyme(TM) will be a safe and effective for the isolation of human hepatocytes for clinical transplants.

Hepatic differentiation of amniotic epithelial cells.

UNLABELLED: Hepatocyte transplantation to treat liver disease is largely limited by the availability of useful cells. Human amniotic epithelial cells (hAECs) from term placenta express surface markers and gene characteristics of embryonic stem cells and have the ability to differentiate into all three germ layers, including tissues of endodermal origin (i.e., liver). Thus, hAECs could provide a source of stem cell-derived hepatocytes for transplantation. We investigated the differentiation of hAECs in vitro and after transplantation into the livers of severe combined immunodeficient (SCID)/beige mice. Moreover, we tested the ability of rat amniotic epithelial cells (rAECs) to replicate and differentiate upon transplantation into a syngenic model of liver repopulation. In vitro results indicate that the presence of extracellular matrix proteins together with a mixture of growth factors, cytokines, and hormones are required for differentiation of hAECs into hepatocyte-like cells. Differentiated hAECs expressed hepatocyte markers at levels comparable to those of fetal hepatocytes. They were able to metabolize ammonia, testosterone, and 17α-hydroxyprogesterone caproate, and expressed inducible fetal cytochromes. After transplantation into the liver of retrorsine (RS)-treated SCID/beige mice, naïve hAECs differentiated into hepatocyte-like cells that expressed mature liver genes such as cytochromes, plasma proteins, transporters, and other hepatic enzymes at levels equal to adult liver tissue. When transplanted in a syngenic animal pretreated with RS, rAECs were able to engraft and generate a progeny of cells with morphology and protein expression typical of mature hepatocytes. CONCLUSION: Amniotic epithelial cells possess the ability to differentiate into cells with characteristics of functional hepatocytes both in vitro and in vivo, thus representing a useful and noncontroversial source of cells for transplantation.

The use of human hepatocytes to investigate bile acid synthesis.

De novo synthesis of bile acids is a liver-specific function that is difficult to maintain in cultured cells. There are significant species differences in both types of bile acids formed and more importantly in the regulation of bile acid homeostasis. This highlights the need for a good human in vitro model. Isolated primary human hepatocytes have the capacity to synthesize normal conjugated bile acids at a rate similar to that in vivo. In this chapter we describe the importance of different culture conditions such as choice of substrate, media and supplements on the total bile acid production as wells as the bile acid composition.

Metabolism of 17alpha-hydroxyprogesterone caproate, an agent for preventing preterm birth, by fetal hepatocytes.

Preterm delivery (i.e., delivery before 37 completed weeks of gestation) is a major determinant of neonatal morbidity and mortality. Until recently, no effective therapies for prevention of preterm birth existed. In a recent multicentered trial, 17alpha-hydroxyprogesterone caproate (17-OHPC) was shown to reduce the rate of preterm birth by 33% in a group of high-risk women. Limited pharmacologic data exist for this drug. Previous studies have shown that CYP3A is involved in the metabolism of 17-OHPC. In this study, we evaluated the metabolism of 17-OHPC in adult and fetal human hepatocytes and in expressed cytochrome P450 enzymes. 17-OHPC was metabolized by expressed CYP3A7 and by fetal hepatocytes. The metabolite profile was qualitatively different between expressed CYP3A4 and CYP3A7. Expressed CYP3A4 demonstrated a significantly higher (>10 times) capacity to metabolize 17-OHPC than CYP3A7. Based on retention times, two unique metabolites were observed in the fetal and adult hepatocyte systems along with one common metabolite. The intrinsic clearance of 17-OHPC by fetal hepatocytes was observed to be one-half of that in adults. In summary, this study demonstrates that fetal hepatocytes and, in particular, the fetal form of CYP3A (i.e., CYP3A7) can metabolize 17-OHPC.

Isolation of amniotic epithelial stem cells.

Many of the cell types that can be isolated from placental tissues retain phenotypic plasticity that makes them an interesting source of cells for regenerative medicine. Several procedures for the isolation of stem cells from different parts of the placenta have been reported. This unit describes a detailed and simple protocol for the selective isolation of amniotic epithelial cells from human term placenta without disturbing the mesenchymal layer. We also introduce a simple density separation technique for the enrichment of the population for SSEA-4 positive cells.

Production of hepatocyte-like cells from human amnion.

Cells isolated from the placenta have been the subject of intense investigation because many of the cells express characteristics of multipotent or even pluripotent stem cells. Cells from the placental tissues such as amnion and chorion have been reported to display multilineage differentiation and surface marker and gene expression patterns consistent with embryonic stem (ES) and mesenchymal stem cells, respectively. We have reported that epithelial cells isolated from term placenta contain cells that express surface markers such as the stage-specific embryonic antigens (SSEA) and a gene expression profile that is similar to ES cells. When subjected to specific differentiation protocols, amniotic epithelial cells display markers of differentiation to cardiomyocytes, neurons, pancreatic cells and hepatocytes. If specific and efficient methods could be developed to induce differentiation of these cells to hepatocytes, the amnion may become a useful source of cells for hepatocyte transplants. Cells isolated from amnion also have some unique properties as compared to some other stem cell sources in that they are isolated from a tissue that is normally discarded following birth, they are quite plentiful and easily isolated and they do not produce tumors when transplanted. Cells isolated from the amnion may be a uniquely useful and noncontroversial stem cell source.

Isolation of mouse hepatocytes for transplantation: a comparison between antegrade and retrograde liver perfusion.

We compared antegrade with retrograde liver perfusion when isolating mouse hepatocytes for hepatocyte transplantation. Male mouse hepatocytes were isolated by different perfusion methods and transplanted into the spleen of congeneic female mice. Retrograde perfusion yielded a larger number of cells (4.90 x 10(7)) than antegrade (4.09 x 10(7), p < 0.05), but hepatocytes obtained by antegrade perfusion gave higher engraftment efficiency (p < 0.05). More of the transplanted hepatocytes could be recovered from recipient liver with antegrade perfusion than with retrograde perfusion (p < 0.05). Our results indicate that hepatocytes isolated by antegrade perfusion gave a higher engraftment efficiency.