Lola M. Reid

Human hepatic stem cells from fetal and postnatal donors

Human hepatic stem cells (hHpSCs), which are pluripotent precursors of hepatoblasts and thence of hepatocytic and biliary epithelia, are located in ductal plates in fetal livers and in Canals of Hering in adult livers. They can be isolated by immunoselection for epithelial cell adhesion molecule–positive (EpCAM+) cells, and they constitute ∼0.5–2.5% of liver parenchyma of all donor ages. The self-renewal capacity of hHpSCs is indicated by phenotypic stability after expansion for >150 population doublings in a serum-free, defined medium and with a doubling time of ∼36 h. Survival and proliferation of hHpSCs require paracrine signaling by hepatic stellate cells and/or angioblasts that coisolate with them. The hHpSCs are ∼9 μm in diameter, express cytokeratins 8, 18, and 19, CD133/1, telomerase, CD44H, claudin 3, and albumin (weakly). They are negative for α-fetoprotein (AFP), intercellular adhesion molecule (ICAM) 1, and for markers of adult liver cells (cytochrome P450s), hemopoietic cells (CD45), and mesenchymal cells (vascular endothelial growth factor receptor and desmin). If transferred to STO feeders, hHpSCs give rise to hepatoblasts, which are recognizable by cordlike colony morphology and up-regulation of AFP, P4503A7, and ICAM1. Transplantation of freshly isolated EpCAM+ cells or of hHpSCs expanded in culture into NOD/SCID mice results in mature liver tissue expressing human-specific proteins. The hHpSCs are candidates for liver cell therapies.

EpCAM expression in normal, non-pathological tissues.

Epithelial Cell Adhesion Molecule (EpCAM) is a transmembrane glycoprotein that is associated with various cancers. Most normal, non-pathological epithelial tissue is EpCAM positive with the exception of epidermal keratinocytes, gastric parietal cells, myoepithelial cells, thymic cortical epithelial, and hepatocytes. However, during early liver development EpCAM expression is also observed. In our studies, we have demonstrated that EpCAM is expressed in non-pathological human livers on hepatic progenitors in livers of all donor ages, from 16 weeks gestation fetal livers to adult. Hepatic progenitors of the liver consist of the stem cells and their descendants, the hepatoblasts, that give rise to the hepatocytic and biliary lineages. Both hepatic stem cells and most hepatoblasts express EpCAM, but only hepatoblasts are alpha-fetoprotein positive. The percentage of EpCAM positive progenitors in human livers varies with donor age and is about 2.5% in the adult and 12.1% in fetuses. In vivo, hepatic stem cells have been found associated with the canals of Hering. Xeno-transplantation experiments with EpCAM positive human liver cells have revealed their potential for proliferation and differentiation to mature liver parenchymal cells.

Biliary tree stem cells, precursors to pancreatic committed progenitors: evidence for possible life-long pancreatic organogenesis.

Peribiliary glands (PBGs) in bile duct walls, and pancreatic duct glands (PDGs) associated with pancreatic ducts, in humans of all ages, contain a continuous, ramifying network of cells in overlapping maturational lineages. We show that proximal (PBGs)‐to‐distal (PDGs) maturational lineages start near the duodenum with cells expressing markers of pluripotency (NANOG, OCT4, and SOX2), proliferation (Ki67), self‐replication (SALL4), and early hepato‐pancreatic commitment (SOX9, SOX17, PDX1, and LGR5), transitioning to PDG cells with no expression of pluripotency or self‐replication markers, maintenance of pancreatic genes (PDX1), and expression of markers of pancreatic endocrine maturation (NGN3, MUC6, and insulin). Radial‐axis lineages start in PBGs near the ducts fibromuscular layers with stem cells and end at the ducts lumens with cells devoid of stem cell traits and positive for pancreatic endocrine genes. Biliary tree‐derived cells behaved as stem cells in culture under expansion conditions, culture plastic and serum‐free Kubotas Medium, proliferating for months as undifferentiated cells, whereas pancreas‐derived cells underwent only approximately 8–10 divisions, then partially differentiated towards an islet fate. Biliary tree‐derived cells proved precursors of pancreas committed progenitors. Both could be driven by three‐dimensional conditions, islet‐derived matrix components and a serum‐free, hormonally defined medium for an islet fate (HDM‐P), to form spheroids with ultrastructural, electrophysiological and functional characteristics of neoislets, including glucose regulatability. Implantation of these neoislets into epididymal fat pads of immunocompromised mice, chemically rendered diabetic, resulted in secretion of human C‐peptide, regulatable by glucose, and able to alleviate hyperglycemia in hosts. The biliary tree‐derived stem cells and their connections to pancreatic committed progenitors constitute a biological framework for life‐long pancreatic organogenesis. Stem Cells 2013;31:1966‐1979

Concise Review: Clinical Programs of Stem Cell Therapies for Liver and Pancreas

Regenerative medicine is transitioning into clinical programs using stem/progenitor cell therapies for repair of damaged organs. We summarize those for liver and pancreas, organs that share endodermal stem cell populations, biliary tree stem cells (hBTSCs), located in peribiliary glands. They are precursors to hepatic stem/progenitors in canals of Hering and to committed progenitors in pancreatic duct glands. They give rise to maturational lineages along a radial axis within bile duct walls and a proximal‐to‐distal axis starting at the duodenum and ending with mature cells in the liver or pancreas. Clinical trials have been ongoing for years assessing effects of determined stem cells (fetal‐liver‐derived hepatic stem/progenitors) transplanted into the hepatic artery of patients with various liver diseases. Immunosuppression was not required. Control subjects, those given standard of care for a given condition, all died within a year or deteriorated in their liver functions. Subjects transplanted with 100‐150 million hepatic stem/progenitor cells had improved liver functions and survival extending for several years. Full evaluations of safety and efficacy of transplants are still in progress. Determined stem cell therapies for diabetes using hBTSCs remain to be explored but are likely to occur following ongoing preclinical studies. In addition, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are being used for patients with chronic liver conditions or with diabetes. MSCs have demonstrated significant effects through paracrine signaling of trophic and immunomodulatory factors, and there is limited evidence for inefficient lineage restriction into mature parenchymal or islet cells. HSCs effects are primarily via modulation of immune mechanisms. Stem Cells 2013;31:2047–2060

CLINICAL PROGRAMS OF STEM CELL THERAPIES FOR LIVER AND PANCREAS

Regenerative medicine is transitioning into clinical programs using stem/progenitor cell therapies for repair of damaged organs. We summarize those for liver and pancreas, organs that share endodermal stem cell populations, biliary tree stem cells (hBTSCs), located in peribiliary glands. They are precursors to hepatic stem/progenitors in canals of Hering and to committed progenitors in pancreatic duct glands. They give rise to maturational lineages along a radial axis within bile duct walls and a proximal‐to‐distal axis starting at the duodenum and ending with mature cells in the liver or pancreas. Clinical trials have been ongoing for years assessing effects of determined stem cells (fetal‐liver‐derived hepatic stem/progenitors) transplanted into the hepatic artery of patients with various liver diseases. Immunosuppression was not required. Control subjects, those given standard of care for a given condition, all died within a year or deteriorated in their liver functions. Subjects transplanted with 100‐150 million hepatic stem/progenitor cells had improved liver functions and survival extending for several years. Full evaluations of safety and efficacy of transplants are still in progress. Determined stem cell therapies for diabetes using hBTSCs remain to be explored but are likely to occur following ongoing preclinical studies. In addition, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are being used for patients with chronic liver conditions or with diabetes. MSCs have demonstrated significant effects through paracrine signaling of trophic and immunomodulatory factors, and there is limited evidence for inefficient lineage restriction into mature parenchymal or islet cells. HSCs effects are primarily via modulation of immune mechanisms. Stem Cells 2013;31:2047–2060

Nuclear Magnetic Resonance Metabolomic Footprinting of Human Hepatic Stem Cells and Hepatoblasts Cultured in Hyaluronan‐Matrix Hydrogels

Human hepatoblasts (hHBs) and human hepatic stem cells (hHpSCs) were maintained in serum‐free Kubotas medium, a defined medium tailored for hepatic progenitors, and on culture plastic versus hyaluronan hydrogels mixed with specific combinations of extracellular matrix components (e.g., type I collagen and laminin). Nuclear magnetic resonance spectroscopy was used to define metabolomic profiles for each substratum tested. The hHpSCs on culture plastic survived throughout the culture study, whereas hHBs on plastic died within 7–10 days. Both survived and expanded in all hydrogel‐matrix combinations tested for more than 4 weeks. Profiles of hundreds of metabolites were narrowed to a detailed analysis of eight, such as glucose, lactate, and glutamine, shown to be significant components of cellular pathways, including the Krebs and urea cycles. The metabolomic profiles indicated that hHpSCs on plastic remained as stem cells expressing low levels of albumin but no α‐fetoprotein (AFP); those in hydrogels were primarily hHBs, expressing AFP, albumin, and urea. Both hHpSCs and hHBs used energy provided by anaerobic metabolism. Variations in hyaluronan‐matrix chemistry resulted in distinct profiles correlating with growth or with differentiative responses. Metabolomic footprinting offers noninvasive and nondestructive assessment of physiological states of stem/progenitor cells ex vivo.

Virus Carrier State Suppresses Tumorigenicity of Tumor Cells in Athymic (Nude) Mice

Nude mice injected subcutaneously with normal uninfected BHK 21 cells or HeLa cells regularly develop large, rapidly-growing tumours at the subcutaneous site of inoculation. However, these same tumour cell lines when persistently infected with VSV or other enveloped RNA viruses are either rejected or form small nodules in nude mice. This rejection phenomenon probably involves some type of immunocyte since heavily-irradiated nude mice (500 rads) cannot reject persistently infected cells but develop large, rapidly-growing tumours which shed virus and defective interfering virus (DI) and which do not exhibit the lymphocytic infiltration observed in the nodules of unirradiated mice given persistently infected cells. Finally, it was possible to select a subline of BHK 21-VSV carrier cells which regularly produces large rapidly-growing tumours in normal unirradiated nude mice, although all these carrier cells express virus antigen and shed large amounts of mature infectious virus and DI both in vivo and in vitro.

Identification of microRNAs specific for epithelial cell adhesion molecule–positive tumor cells in hepatocellular carcinoma

Therapies that target cancer stem cells (CSCs) hold promise in eliminating cancer burden. However, normal stem cells are likely to be targeted owing to their similarities to CSCs. It is established that epithelial cell adhesion molecule (EpCAM) is a biomarker for normal hepatic stem cells (HpSCs), and EpCAM+AFP+ hepatocellular carcinoma (HCC) cells have enriched hepatic CSCs. We sought to determine whether specific microRNAs (miRNAs) exist in hepatic CSCs that are not expressed in normal HpSCs. We performed a pair‐wise comparison of the miRNA transcriptome of EpCAM+ and corresponding EpCAM− cells isolated from two primary HCC specimens, as well as from two fetal livers and three healthy adult liver donors by small RNA deep sequencing. We found that miR‐150, miR‐155, and miR‐223 were preferentially highly expressed in EpCAM+ HCC cells, which was further validated. Their gene surrogates, identified using miRNA and messenger RNA profiling in a cohort of 292 HCC patients, were associated with patient prognosis. We further demonstrated that miR‐155 was highly expressed in EpCAM+ HCC cells, compared to corresponding EpCAM− HCC cells, fetal livers with enriched normal hepatic progenitors, and normal adult livers with enriched mature hepatocytes. Suppressing miR‐155 resulted in a decreased EpCAM+ fraction in HCC cells and reduced HCC cell colony formation, migration, and invasion in vitro. The reduced levels of identified miR‐155 targets predicted the shortened overall survival and time to recurrence of HCC patients. Conclusion: miR‐155 is highly elevated in EpCAM+ HCC cells and might serve as a molecular target to eradicate the EpCAM+ CSC population in human HCCs. (Hepatology 2015;62:829–840)

Human telomerase activity, telomerase and telomeric template expression in hepatic stem cells and in livers from fetal and postnatal donors

Background Although telomerase activity has been analyzed in various normal and malignant tissues, including liver, it is still unknown to what extent telomerase can be associated with specific maturational lineage stages. Methods We assessed human telomerase activity, protein and gene expression for the telomerase reverse transcriptase, as well as expression of the telomeric template RNA hTER in hepatic stem cells and in various developmental stages of the liver from fetal to adult. In addition, the effect of growth factors on telomerase activity was analyzed in hepatic stem cells in vitro. Results Telomerase was found to be highly active in fetal liver cells and was significantly higher than in hepatic stem cells, correlating with gene and protein expression levels. Activity in postnatal livers from all donor ages varied considerably and did not correlate with age or gene expression levels. The hter expression could be detected throughout the development. A short stimulation by growth factors of cultured hepatic stem cells did not increase telomerase activity. Conclusion Telomerase is considerably active in fetal liver and variably in postnatal livers. Although telomerase protein is present at varying levels in liver cells of all donor ages, gene expression is solely associated with fetal liver cells.

VIRAL MUTATION IN PERSISTENT INFECTION

ABSTRACT Oligonucleotide mapping of viral RNA, peptide mapping of virus proteins, and virion RNA termini sequencing all show that long term persistent infection by Vesicular Stomatitis Virus (VSV) leads to extensive and continuous genome mutation. The Dl particles present in persistently infected cells apparently help drive this rapid mutation of infectious virus because serial high multiplicity passages of VSV in acute lytic infections generates more oligonucleotide map changes in virus RNA than does alternating high and low m.o.i. passage, or low m.o.i. passage. Furthermore, virus populations in persistently - infected cultures are selected for resistance to the Dl particles present initially in the carrier cultures. The mutant virus clones recovered after many years of persistence contain more than 2% of mutated bases at the 3′ end of virion RNA and about 15% at the 5′ end. The mutant virus recovered after years of persistence does not rapidly revert, and is as stable as the original input virus clone. A number of biologically interesting mutant phenotypes have been recovered from these persistently infected cells. These include: (I) ts smaII plaque, avirulent mutants; (2) mutants resistant to Dl particles originally present in the persistently infected cells; (3) mutants which give extremely low virus yields on initial isolation and which are able to establish persistent infection without added Dl particles; (4) mutants selected in vivo which allow persistently infected cells to form tumors and metastases in nude mice (these strongly induce interferon and natural killer cells, but escape the natural killer cell surveillance due to virus mutation); (5) mutants arising spontaneously in the carrier cells in culture which are poor inducers of interferon and of natural killer cells, and which also allow carrier cells to form tumors and metastases due to viral mutations; (6) mutants which show reduced neutralization kinetics with heterologous and homologous antisera; (7) mutants exhibiting apparent smaller size M protein, or a major new virion protein band near the G protein; (8) mutants which have lost all mouse virulence (VSV and rabies virus), and mutants which acquired mouse virulence (rabies HEP Flury vaccine strain). Many of the phenotypes must involve multiple mutations and a number of them may be due to the same mutation(s). Considerable further work will be required to map and understand these phenotypes, but they should provide considerable insight into virus functions. It is clear from this work that RNA virus genomes have an unexpected degree of mutational plasticity, and that viral persistence is a highly competitive, metastable situation which shows no signs of stabilizing as viral genomes and Dl particle genomes evolve over many years.