Search

Background Research directed towards drug development, metabolism, and liver functions often utilize primary hepatocytes (PH) for preliminary in vitro studies. Variability in the in vitro functionality of PH and the unsuitability of hepatocarcinoma cells for these studies have driven researchers to look to ESC, iPS, and other stem cell types using differentiation protocols to provide more reliable and available cells. This study describes the development of hepatocyte-like cells through the in vitro differentiation of human TERT-immortalized cord blood-derived multi-lineage progenitor cells (MLPC). The E12 clonal cell line derived from polyclonal TERT-transfected cells was used throughout the study. Methods E12 MLPC were subjected to a three-step differentiation protocol using alternating combinations of growth factors, cytokines, and maturational factors. Cells at various stages of differentiation were analyzed for consistency with PH by morphology, immunohistochemistry, urea production, and gene expression. Results E12 MLPC were shown to significantly change morphology with each stage of differentiation. Coincidental with the morphological changes in the cells, immunohistochemistry data documented the differentiation to committed endoderm by the expression of SOX-17 and GATA-4; the progression to committed hepatocyte-like cells by the expression of a large number of markers including α-fetoprotein and albumin; and the final differentiation by the expression of nuclear and cytoplasmic HNF4. Fully differentiated cells demonstrated gene expression, urea production, and immunohistochemistry consistent with PH. A methodology and medium formulation to continuously expand the E12-derived hepatocyte-like cells is described. Conclusion The availability of immortalized hepatocyte-like cell lines could provide a consistent tool for the study of hepatic diseases, drug discovery, and the development of cellular therapies for liver disorders. Utilization of these techniques could provide a basis for the development of bridge therapies for liver failure patients awaiting transplant.

Background Primary human hepatocytes (PHHs) are the ideal candidates for studying critical liver functions such as drug metabolism and toxicity. However, as they are isolated from discarded livers that are unsuitable for transplantation, they possess limited expansion ability in vitro and their enzymatic functions deteriorate rapidly because they are often of poor quality. Therefore, there is a compelling reason to find reliable alternative sources of hepatocytes. Methods In this study, we report on efficient and robust differentiation of embryonic stem cells (ESC) from the common marmoset Callithrix jacchus into functional hepatocyte-like cells (HLC) using a simple, and reproducible three-step procedure. ESC-derived HLCs were examined by morphological analysis and tested for their expression of hepatocyte-specific markers using a combination of immunohistochemistry, RT-PCR, and biochemical assays. Primary human hepatocytes were used as controls. Results ESC-derived HLCs expressed each of the hepatocyte-specific markers tested, including albumin; α-fetoprotein; asialoglycoprotein receptor 1; α-1 antitrypsin; hepatocyte nuclear factors 1α and 4; cytokeratin 18; hepatocyte growth factor receptor; transferrin; tyrosine aminotransferase; alkaline phosphatase; c-reactive protein; cytochrome P450 enzymes CYP1A2, CYP2E1 and CYP3A4; and coagulation factors FVII and FIX. They were functionally competent as demonstrated by biochemical assays in addition to producing urea. Conclusion Our data strongly suggest that marmoset HLCs possess characteristics similar to those of PHHs. They could, therefore, be invaluable for studies on drug metabolism and cell transplantation therapy for a variety of liver disorders. Because of the similarities in the anatomical and physiological features of the common marmoset to that of humans, Callithrix jacchus is an appropriate animal model to study human disease conditions and cellular functions.

Blastocyst complementation combined with gene editing is an emerging approach in the field of regenerative medicine that could potentially solve the worldwide problem of organ shortages for transplantation. In theory, blastocyst complementation can generate fully functional human organs or tissues, grown within genetically engineered livestock animals. Targeted deletion of a specific gene(s) using gene editing to cause deficiencies in organ development can open a niche for human stem cells to occupy, thus generating human tissues. Within this review, we will focus on the pancreas, liver, heart, kidney, lung, and skeletal muscle, as well as cells of the immune and nervous systems. Within each of these organ systems, we identify and discuss (i) the common causes of organ failure; (ii) the current state of regenerative therapies; and (iii) the candidate genes to knockout and enable specific exogenous organ development via the use of blastocyst complementation. We also highlight some of the current barriers limiting the success of blastocyst complementation.

Summary A reliable source of human hepatocytes and transplantable livers is needed. Interspecies embryo complementation, which involves implanting donor human stem cells into early morula/blastocyst stage animal embryos, is an emerging solution to the shortage of transplantable livers. We review proposed mutations in the recipient embryo to disable hepatogenesis, and discuss the advantages of using fumarylacetoacetate hydrolase knockouts and other genetic modifications to disable hepatogenesis. Interspecies blastocyst complementation using porcine recipients for primate donors has been achieved, although percentages of chimerism remain persistently low. Recent investigation into the dynamic transcriptomes of pigs and primates have created new opportunities to intimately match the stage of developing animal embryos with one of the many varieties of human induced pluripotent stem cell. We discuss techniques for decreasing donor cell apoptosis, targeting donor tissue to endodermal structures to avoid neural or germline chimerism, and decreasing the immunogenicity of chimeric organs by generating donor endothelium., In this article, Nyberg and colleagues discuss the possibility of generating human livers through interspecies blastocyst complementation, review candidate hepatogenesis-disabling mutations in recipient blastocysts with a focus on fumarylacetoacetate hydrolase knockouts as an advantageous model, and present recent advances in blastocyst complementation to improve the effectiveness and ethics of interspecies hepatogenesis.

The development of innovative genome editing techniques in recent years has revolutionized the field of biomedicine. Among the novel approaches, the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas9) technology has become the most popular, in part due to its matchless ability to carry out gene editing at the target site with great precision. With considerable successes in animal and preclinical studies, CRISPR/Cas9-mediated gene editing has paved the way for its use in human trials, including patients with a variety of liver diseases. Gene editing is a logical therapeutic approach for liver diseases because many metabolic and acquired disorders are caused by mutations within a single gene. In this review, we provide an overview on current and emerging therapeutic strategies for the treatment of liver diseases using the CRISPR/Cas9 technology.

The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.

Human primary hepatocytes (PHs) are critical to studying liver functions, drug metabolism and toxicity. PHs isolated from livers that are unacceptable for transplantation have limited expansion and culture viability in vitro, in addition to rapidly deteriorating enzymatic functions. The unsuitability of immortalized hepato-carcinoma cell lines for this function has prompted studies to develop hepatocyte-like cells from alternative sources like ESC, iPS, and other stem cell types using differentiation protocols. This study describes a novel technique to produce expandable and functional hepatocyte-like cells from the fusion of an immortalized human umbilical cord blood derived cell line (E12 MLPC) to normal human primary hepatocytes. Multi-lineage progenitor cells (MLPC) comprise a small subset of mesenchymal-like cells isolated from human umbilical cord blood. MLPC are distinguishable from other mesenchymal-like cells by their extended expansion capacity (up to 80 cell doublings before senescence) and the ability to be differentiated into cells representative of endo-, meso- and ectodermal origins. Transfection of MLPC with the gene for telomerase reverse transcriptase (TERT) resulted in clonal cell lines that were capable of differentiation to different cellular outcomes while maintaining their functional immortality. A methodology for the development of immortalized hepatocyte-like hybrid cells by the in vitro fusion of human MLPC with normal human primary hepatocytes is reported. The resultant hybrid cells exhibited homology with hepatocytes by morphology, immunohistochemistry, urea and albumin production and gene expression. A medium that allows stable long-term expansion of hepatocyte-like fusion cells is described.

Rajagopal N AravalliDepartment of Radiology, University of Minnesota Medical School, Minneapolis, MN, USAAbstract: Primary hepatocellular carcinoma (HCC) is a common malignancy that has a poor prognosis because it is often diagnosed at an advanced stage. HCC normally develops as a consequence of underlying liver disease and is most often associated with cirrhosis. Surgical resection and liver transplantation are the current best options to treat liver cancer. However, problems associated with liver transplantation, such as shortage of donors, risk of immune rejection, and tissue damage following surgery provided the impetus for development of alternative therapies. The emerging field of stem cell therapy has raised hopes for finding curative options for liver cancer. Stem cells have the ability not only to proliferate after transplantation but also to differentiate into most mammalian cell types in vivo. In this review, progress on stem cell-derived technologies for the treatment of liver cancer is discussed.Keywords: liver, cancer, stem cell, therapy, hepatocellular carcinoma

Porcine hepatocytes are potentially important in liver regeneration and in the treatment of humans with acute and chronic liver diseases. Induced pluripotent stem (iPS) cells are a valuable source of hepatocytes for these applications as they have unlimited potential to propagate in vitro. An efficient and robust differentiation of iPS cells generated from porcine fetal fibroblasts into functional hepatocyte-like cells in vitro is reported. The methodology followed a three-step differentiation protocol using several growth factors, namely, activin A, basic fibroblast growth factor, bone morphogenetic protein-4, and oncostatin M. Porcine iPS cell-derived hepatocyte-like (piPS-Hep) cells were characterized by morphological analysis and were tested for the expression of hepatocyte-specific genes using RT-PCR. Functional analyses for albumin production and glycogen storage were also carried out. These differentiated hepatocyte-like cells could represent a valuable source for studies of drug metabolism and for cell transplantation therapy for a variety of liver disorders.

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 μm in diameter. RA1 cells proliferated in vitro with a doubling time of 5–7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers.

Microglial cells respond to the herpes simplex virus (HSV)-1 by producing proinflammatory cytokines and chemokines. After this inflammatory burst, these cells undergo apoptotic cell death. We have recently demonstrated that both virus-induced immune mediator production and apoptosis were induced through Toll-like receptor 2 (TLR2) signaling. Based upon these findings, we hypothesized that the inhibition of TLR2 signaling may serve as a means to alleviate excessive neuroinflammation. In the present study, we cloned four vaccinia virus (VV) proteins, which have been reported to disrupt either TLR signaling or NF-kappaB activation, and overexpressed them in HEK293T cells stably expressing murine TLR2 and in primary murine microglia. Using an NF-kappaB-driven luciferase reporter gene assay, we show that upon stimulation with HSV and Listeria monocytogenes, all four vaccinia proteins inhibited TLR2 signaling with different levels of inhibition in the TLR2-expressing cell line and primary microglia. We found similar results when microglial cells were stimulated with the TLR4 ligand LPS and the TLR9 ligand CpG ODN. Taken together, these data provide evidence that these VV proteins can function as inhibitors of TLR signaling in primary microglial cells.

Background Information regarding the response of brain cells to infection with herpes simplex virus (HSV)-1 is needed for a complete understanding of viral neuropathogenesis. We have recently demonstrated that microglial cells respond to HSV infection by producing a number of proinflammatory cytokines and chemokines through a mechanism involving Toll-like receptor 2 (TLR2). Following this cytokine burst, microglial cells rapidly undergo cell death by apoptosis. We hypothesized that TLR2 signaling might mediate the cell death process as well. Methods To test this hypothesis, we investigated HSV-induced cell death of microglia obtained from both wild-type and TLR2-/- mice. Cell death was studied by oligonucleosomal ELISA and TUNEL staining, and the mechanisms of apoptosis were further analyzed using murine apoptosis-specific microarrays. The data obtained from microarray analysis were then validated using quantitative real-time PCR assays. Results HSV infection induced apoptotic cell death in microglial cells from wild-type as well as TLR2 cells. However, the cell death at 24 h p.i. was markedly lower in knockout cells. Furthermore, microarray analyses clearly showed that the expression of pro-apoptotic genes was down-regulated at the time when wild-type cells were actively undergoing apoptosis, indicating a differential response to HSV in cells with or without TLR2. Conclusion We demonstrate here that HSV induces an apoptotic response in microglial cells which is mediated through TLR2 signaling.

Microglial cells and astrocytes are glial cell types that perform distinct functions and generate innate immune responses to counter invading pathogens. Herpes simplex virus 1 (HSV-1) is a neurotropic virus that is capable of causing severe, necrotizing encephalitis. HSV-1 infects both of these glial cell types. Microglial cells undergo an abortive infection, yet respond to viral infection by inducing a burst of proinflammatory cytokine and chemokine production. Following this cytokine burst, they rapidly succumb to cell death. In contrast, astrocytes do permit productive viral replication, but do not generate these same innate immune mediators. Although apoptosis has been implicated in a number of acute and chronic neurological disorders, little is known about apoptosis during viral encephalitis. In the present study, the authors investigated the effect of HSV-1 infection on cell survival and studied the mechanisms of cell-death in virus-infected, primary murine glial cells. The authors report that although apoptosis occurred rapidly in microglia, it was delayed during productive infection of astrocytes. Furthermore, microarray studies revealed significant variations in the expression of apoptotic genes between these two types of glial cells, indicating crucial differences in signaling pathways. Intrinsic as well as extrinsic pathways of apoptosis were found to be activated in both glial cell types. Specifically, genes involved in the tumor necrosis factor (TNF) signaling pathway were predominantly up-regulated in microglia, whereas genes of the Fas pathway were induced during HSV infection of astrocytes.