Fabio Marongiu

Concise review: Isolation and characterization of cells from human term placenta: Outcome of the First International Workshop on Placenta Derived Stem Cells

Placental tissue draws great interest as a source of cells for regenerative medicine because of the phenotypic plasticity of many of the cell types isolated from this tissue. Furthermore, placenta, which is involved in maintaining fetal tolerance, contains cells that display immunomodulatory properties. These two features could prove useful for future cell therapy‐based clinical applications. Placental tissue is readily available and easily procured without invasive procedures, and its use does not elicit ethical debate. Numerous reports describing stem cells from different parts of the placenta, using nearly as numerous isolation and characterization procedures, have been published. Considering the complexity of the placenta, an urgent need exists to define, as clearly as possible, the region of origin and methods of isolation of cells derived from this tissue. On March 23–24, 2007, the first international Workshop on Placenta Derived Stem Cells was held in Brescia, Italy. Most of the research published in this area focuses on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. The aim of this review is to summarize and provide the state of the art of research in this field, addressing aspects such as cell isolation protocols and characteristics of these cells, as well as providing preliminary indications of the possibilities for use of these cells in future clinical applications.

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.

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;)

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.

Antiviral Activity of Benzimidazole Derivatives. I. Antiviral Activity of 1-Substituted-2-[(Benzotriazol-1/2-yl)methyl]benzimidazoles†

Forty‐three 2‐[(benzotriazol‐1/2‐yl)methyl]benzimidazoles, bearing either linear (dialkylamino)alkyl‐ or bulkier (quinolizidin‐1‐yl)alkyl moieties at position 1, were evaluated in cell‐based assays for cytotoxicity and antiviral activity against viruses representative of two of the three genera of the Flaviviridae family, i.e. Flaviviruses (Yellow Fever Virus (YFV)) and Pestiviruses (Bovine Viral Diarrhoea Virus (BVDV)), as Hepaciviruses can hardly be used in routine cell‐based assays. Compounds were also tested against representatives of other virus families. Among ssRNA+ viruses were a retrovirus (Human Immunodeficiency Virus type 1 (HIV‐1)), two picornaviruses (Coxsackie Virus type B2 (CVB2), and Poliovirus type‐1, Sabin strain (Sb‐1)); among ssRNA− viruses were a Paramyxoviridae (Respiratory Syncytial Virus (RSV)) and a Rhabdoviridae (Vesicular Stomatitis Virus (VSV)) representative. Among double‐stranded RNA (dsRNA) viruses was a Reoviridae representative (Reo‐1). Two representatives of DNA virus families were also included: Herpes Simplex type 1, (HSV‐1; Herpesviridae) and Vaccinia Virus (VV; Poxviridae). Most compounds exhibited potent activity against RSV, with EC50 values as low as 20 nM. Moreover, some compounds, in particular when bearing a (quinolizidin‐1‐yl)alkyl residue, were also moderately active against BVDV, YFV, and CVB2.

Liver Repopulation and Carcinogenesis : Two Sides of the Same Coin?

Liver repopulation by transplanted normal hepatocytes has been described in a number of experimental settings. Extensive repopulation can also occur from the selective proliferation of endogenous normal hepatocytes, both in experimental animals and in the human liver. This review highlights the intriguing association between clinical and experimental conditions related to liver repopulation and an increased risk for development of hepatocellular carcinoma. It is suggested that any microenvironment that is able to sustain the clonal growth of normal transplanted (or endogenous) hepatocytes is also geared to select for the emergence of rare resistant cells with an altered phenotype. Whereas the first pathway leads to liver repopulation with normal histology, the latter results in the growth of focal proliferative lesions and carries an increased risk of neoplastic disease. The implications of this association are discussed, both in terms of pathogenetic significance and possible therapeutic exploitation.

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 effect of environmental chemicals on the tumor microenvironment

Potentially carcinogenic compounds may cause cancer through direct DNA damage or through indirect cellular or physiological effects. To study possible carcinogens, the fields of endocrinology, genetics, epigenetics, medicine, environmental health, toxicology, pharmacology and oncology must be considered. Disruptive chemicals may also contribute to multiple stages of tumor development through effects on the tumor microenvironment. In turn, the tumor microenvironment consists of a complex interaction among blood vessels that feed the tumor, the extracellular matrix that provides structural and biochemical support, signaling molecules that send messages and soluble factors such as cytokines. The tumor microenvironment also consists of many host cellular effectors including multipotent stromal cells/mesenchymal stem cells, fibroblasts, endothelial cell precursors, antigen-presenting cells, lymphocytes and innate immune cells. Carcinogens can influence the tumor microenvironment through effects on epithelial cells, the most common origin of cancer, as well as on stromal cells, extracellular matrix components and immune cells. Here, we review how environmental exposures can perturb the tumor microenvironment. We suggest a role for disrupting chemicals such as nickel chloride, Bisphenol A, butyltins, methylmercury and paraquat as well as more traditional carcinogens, such as radiation, and pharmaceuticals, such as diabetes medications, in the disruption of the tumor microenvironment. Further studies interrogating the role of chemicals and their mixtures in dose-dependent effects on the tumor microenvironment could have important general mechanistic implications for the etiology and prevention of tumorigenesis.

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.