Roberto Gramignoli

A Whole-Organ Regenerative Medicine Approach for Liver Replacement

BACKGROUND & AIMS The therapy of choice for end-stage liver disease is whole-organ liver transplantation, but this option is limited by a shortage of donor organs. Cell-based therapies and hepatic tissue engineering have been considered as alternatives to liver transplantation, but neither has proven effective to date. A regenerative medicine approach for liver replacement has recently been described that includes the use of a three-dimensional organ scaffold prepared by decellularization of xenogeneic liver. The present study investigates a new, minimally disruptive method for whole-organ liver decellularization and three different cell reseeding strategies to engineer functional liver tissue. METHODS A combination of enzymatic, detergent, and mechanical methods are used to remove all cells from isolated rat livers. Whole-organ perfusion is used in a customized organ chamber and the decellularized livers are examined by morphologic, biochemical, and immunolabeling techniques for preservation of the native matrix architecture and composition. Three different methods for hepatocyte seeding of the resultant three-dimensional liver scaffolds are evaluated to maximize cell survival and function: (1) direct parenchymal injection, (2) multistep infusion, or (3) continuous perfusion. RESULTS The decellularization process preserves the three-dimensional macrostructure, the ultrastructure, the composition of the extracellular matrix components, the native microvascular network of the liver, and the bile drainage system, and up to 50% of growth factor content. The three-dimensional liver matrix reseeded with the multistep infusion of hepatocytes generated ∼90% of cell engraftment and supported liver-specific functional capacities of the engrafted cells, including albumin production, urea metabolism, and cytochrome P450 induction. CONCLUSIONS Whole-organ liver decellularization is possible with maintenance of structure and composition suitable to support functional hepatocytes.

Hepatic differentiation of amniotic epithelial cells.

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. (HEPATOLOGY 2011;)

FoxO6 Integrates Insulin Signaling With Gluconeogenesis in the Liver

OBJECTIVE Excessive endogenous glucose production contributes to fasting hyperglycemia in diabetes. This effect stems from inept insulin suppression of hepatic gluconeogenesis. To understand the underlying mechanisms, we studied the ability of forkhead box O6 (FoxO6) to mediate insulin action on hepatic gluconeogenesis and its contribution to glucose metabolism. RESEARCH DESIGN AND METHODS We characterized FoxO6 in glucose metabolism in cultured hepatocytes and in rodent models of dietary obesity, insulin resistance, or insulin-deficient diabetes. We determined the effect of FoxO6 on hepatic gluconeogenesis in genetically modified mice with FoxO6 gain- versus loss-of-function and in diabetic db/db mice with selective FoxO6 ablation in the liver. RESULTS FoxO6 integrates insulin signaling to hepatic gluconeogenesis. In mice, elevated FoxO6 activity in the liver augments gluconeogenesis, raising fasting blood glucose levels, and hepatic FoxO6 depletion suppresses gluconeogenesis, resulting in fasting hypoglycemia. FoxO6 stimulates gluconeogenesis, which is counteracted by insulin. Insulin inhibits FoxO6 activity via a distinct mechanism by inducing its phosphorylation and disabling its transcriptional activity, without altering its subcellular distribution in hepatocytes. FoxO6 becomes deregulated in the insulin-resistant liver, accounting for its unbridled activity in promoting gluconeogenesis and correlating with the pathogenesis of fasting hyperglycemia in diabetes. These metabolic abnormalities, along with fasting hyperglycemia, are reversible by selective inhibition of hepatic FoxO6 activity in diabetic mice. CONCLUSIONS Our data uncover a FoxO6-dependent pathway by which the liver orchestrates insulin regulation of gluconeogenesis, providing the proof-of-concept that selective FoxO6 inhibition is beneficial for curbing excessive hepatic glucose production and improving glycemic control in diabetes.

ChREBP Mediates Glucose-Stimulated Pancreatic β-Cell Proliferation

Glucose stimulates rodent and human β-cell replication, but the intracellular signaling mechanisms are poorly understood. Carbohydrate response element-binding protein (ChREBP) is a lipogenic glucose-sensing transcription factor with unknown functions in pancreatic β-cells. We tested the hypothesis that ChREBP is required for glucose-stimulated β-cell proliferation. The relative expression of ChREBP was determined in liver and β-cells using quantitative RT-PCR (qRT-PCR), immunoblotting, and immunohistochemistry. Loss- and gain-of-function studies were performed using small interfering RNA and genetic deletion of ChREBP and adenoviral overexpression of ChREBP in rodent and human β-cells. Proliferation was measured by 5-bromo-2′-deoxyuridine incorporation, [3H]thymidine incorporation, and fluorescence-activated cell sorter analysis. In addition, the expression of cell cycle regulatory genes was measured by qRT-PCR and immunoblotting. ChREBP expression was comparable with liver in mouse pancreata and in rat and human islets. Depletion of ChREBP decreased glucose-stimulated proliferation in β-cells isolated from ChREBP−/− mice, in INS-1–derived 832/13 cells, and in primary rat and human β-cells. Furthermore, depletion of ChREBP decreased the glucose-stimulated expression of cell cycle accelerators. Overexpression of ChREBP amplified glucose-stimulated proliferation in rat and human β-cells, with concomitant increases in cyclin gene expression. In conclusion, ChREBP mediates glucose-stimulated proliferation in pancreatic β-cells.

Isolation of amniotic mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteocytes, chondrocytes, and adipocytes and possess immunomodulatory properties. Amniotic membrane from human term placenta is a potential source of multipotent MSCs that could be useful for regenerative medicine. This unit describes a detailed and simple protocol for the isolation of amniotic mesenchymal cells. We also introduce a simple density separation technique for the purification of this cell type from possible contamination with amniotic epithelial 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.

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™ 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™. 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™ will be a safe and effective for the isolation of human hepatocytes for clinical transplants.

The History and Use of Human Hepatocytes for the Treatment of Liver Diseases: The First 100 Patients

Orthotopic liver transplantation remains the only curative treatment for many end‐stage liver diseases, yet the number of patients receiving liver transplants remains limited by the number of organs available for transplant. There is a need for alternative therapies for liver diseases. The transplantation of isolated hepatocytes (liver cells) has been used as an experimental therapy for liver disease in a limited number of cases. Recently, the 100th case of hepatocyte transplantation was reported. This review discusses the history of the hepatocyte transplant field, the major discoveries that supported and enabled the first hepatocyte transplants, and reviews the cases and outcomes of the first 100 clinical transplants. Some of the problems that limit the application or efficacy of hepatocyte transplantation are discussed, as are possible solutions to these problems. In conclusion, hepatocyte transplants have proven effective particularly in cases of metabolic liver disease where reversal or amelioration of the characteristic symptoms of the disease is easily quantified. However, no patients have been completely corrected of a metabolic liver disease for a significant amount of time by hepatocyte transplantation alone. It is likely that future developments in new sources of cells for transplantation will be required before this cellular therapy can be fully implemented and available for large numbers of patients.

Clinical Hepatocyte Transplantation: Practical Limits and Possible Solutions

Since the first human hepatocyte transplants (HTx) in 1992, clinical studies have clearly established proof of principle for this therapy as a treatment for patients with acquired or inherited liver disease. Although major accomplishments have been made, there are still some specific limitations to this technology, which, if overcome, could greatly enhance the efficacy and implementation of this therapy. Here, we describe what in our view are the most significant obstacles to the clinical application of HTx and review the solutions currently proposed. The obstacles of significance include the limited number and quality of liver tissues as a cell source, the lack of clinical grade reagents, quality control evaluation of hepatocytes prior to transplantation, hypothermic storage of cells prior to transplantation, preconditioning treatments to enhance engraftment and proliferation of donor cells, tracking or monitoring cells after transplantation, and the optimal immunosuppression protocols for transplant recipients.

Placental stem cell correction of murine intermediate maple syrup urine disease

There is improved survival and partial metabolic correction of a mouse intermediate maple syrup urine disease (iMSUD) model after allogenic hepatocyte transplantation, confirming that a small number of enzyme‐proficient liver‐engrafted cells can improve phenotype. However, clinical shortages of suitable livers for hepatocyte isolation indicate a need for alternative cell sources. Human amnion epithelial cells (hAECs) share stem cell characteristics without the latters safety and ethical concerns and differentiate to hepatocyte‐like cells. Eight direct hepatic hAEC transplantations were performed in iMSUD mice over the first 35 days beginning at birth; animals were provided a normal protein diet and sacrificed at 35 and 100 days. Treatment at the neonatal stage is clinically relevant for MSUD and may offer a donor cell engraftment advantage. Survival was significantly extended and body weight was normalized in iMSUD mice receiving hAEC transplantations compared with untreated iMSUD mice, which were severely cachectic and died ≤28 days after birth. Branched chain α‐keto acid dehydrogenase enzyme activity was significantly increased in transplanted livers. The branched chain amino acids leucine, isoleucine, valine, and alloisoleucine were significantly improved in serum and brain, as were other large neutral amino acids. Conclusion: Placental‐derived stem cell transplantation lengthened survival and corrected many amino acid imbalances in a mouse model of iMSUD. This highlights the potential for their use as a viable alternative clinical therapy for MSUD and other liver‐based metabolic diseases. (HEPATOLOGY 2013)