Raymond D. Hickey

The ploidy-conveyor of mature hepatocytes as a source of genetic variation

Mononucleated and binucleated polyploid hepatocytes (4n, 8n, 16n and higher) are found in all mammalian species, but the functional significance of this conserved phenomenon remains unknown. Polyploidization occurs through failed cytokinesis, begins at weaning in rodents and increases with age. Previously, we demonstrated that the opposite event, ploidy reversal, also occurs in polyploid hepatocytes generated by artificial cell fusion. This raised the possibility that somatic ‘reductive mitoses’ can also happen in normal hepatocytes. Here we show that multipolar mitotic spindles form frequently in mouse polyploid hepatocytes and can result in one-step ploidy reversal to generate offspring with halved chromosome content. Proliferating hepatocytes produce a highly diverse population of daughter cells with multiple numerical chromosome imbalances as well as uniparental origins. Our findings support a dynamic model of hepatocyte polyploidization, ploidy reversal and aneuploidy, a phenomenon that we term the ‘ploidy conveyor’. We propose that this mechanism evolved to generate genetic diversity and permits adaptation of hepatocytes to xenobiotic or nutritional injury.

Ploidy reductions in murine fusion-derived hepatocytes

We previously showed that fusion between hepatocytes lacking a crucial liver enzyme, fumarylacetoacetate hydrolase (FAH), and wild-type blood cells resulted in hepatocyte reprogramming. FAH expression was restored in hybrid hepatocytes and, upon in vivo expansion, ameliorated the effects of FAH deficiency. Here, we show that fusion-derived polyploid hepatocytes can undergo ploidy reductions to generate daughter cells with one-half chromosomal content. Fusion hybrids are, by definition, at least tetraploid. We demonstrate reduction to diploid chromosome content by multiple methods. First, cytogenetic analysis of fusion-derived hepatocytes reveals a population of diploid cells. Secondly, we demonstrate marker segregation using ß-galactosidase and the Y-chromosome. Approximately 2–5% of fusion-derived FAH-positive nodules were negative for one or more markers, as expected during ploidy reduction. Next, using a reporter system in which ß-galactosidase is expressed exclusively in fusion-derived hepatocytes, we identify a subpopulation of diploid cells expressing ß-galactosidase and FAH. Finally, we track marker segregation specifically in fusion-derived hepatocytes with diploid DNA content. Hemizygous markers were lost by ≥50% of Fah-positive cells. Since fusion-derived hepatocytes are minimally tetraploid, the existence of diploid hepatocytes demonstrates that fusion-derived cells can undergo ploidy reduction. Moreover, the high degree of marker loss in diploid daughter cells suggests that chromosomes/markers are lost in a non-random fashion. Thus, we propose that ploidy reductions lead to the generation of genetically diverse daughter cells with about 50% reduction in nuclear content. The generation of such daughter cells increases liver diversity, which may increase the likelihood of oncogenesis.

Efficient Production of Fah-null Heterozygote Pigs by Chimeric Adeno-Associated Virus-Mediated Gene Knockout and Somatic Cell Nuclear Transfer

Hereditary tyrosinemia type I (HT1) results in hepatic failure, cirrhosis, and hepatocellular carcinoma (HCC) early in childhood and is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase (FAH). In a novel approach we used the chimeric adeno‐associated virus DJ serotype (AAV‐DJ) and homologous recombination to target and disrupt the porcine Fah gene. AAV‐DJ is an artificial chimeric AAV vector containing hybrid capsid sequences from three naturally occurring serotypes (AAV2, 8, and 9). The AAV‐DJ vector was used to deliver the knockout construct to fetal pig fibroblasts with an average knockout targeting frequency of 5.4%. Targeted Fah‐null heterozygote fibroblasts were used as nuclear donors for somatic cell nuclear transfer (SCNT) to porcine oocytes and multiple viable Fah‐null heterozygote pigs were generated. Fah‐null heterozygotes were phenotypically normal, but had decreased Fah transcriptional and enzymatic activity compared to wildtype animals. Conclusion: This study is the first to use a recombinant chimeric AAV vector to knockout a gene in porcine fibroblasts for the purpose of SCNT. In using the AAV‐DJ vector we observed targeting frequencies that were higher than previously reported with other naturally occurring serotypes. We expect that the subsequent generation of FAH‐null homozygote pigs will serve as a significant advancement for translational research in the areas of metabolic liver disease, cirrhosis, and HCC. (HEPATOLOGY 2011;)

Generation of islet-like cells from mouse gall bladder by direct ex vivo reprogramming

Cell replacement is an emerging therapy for type 1 diabetes. Pluripotent stem cells have received a lot of attention as a potential source of transplantable β-cells, but their ability to form teratomas poses significant risks. Here, we evaluated the potential of primary mouse gall bladder epithelial cells (GBCs) as targets for ex vivo genetic reprogramming to the β-cell fate. Conditions for robust expansion and genetic transduction of primary GBCs by adenoviral vectors were developed. Using a GFP reporter for insulin, conditions for reprogramming were then optimized. Global expression analysis by RNA-sequencing was used to quantitatively compare reprogrammed GBCs (rGBCs) to true β-cells, revealing both similarities and differences. Adenoviral-mediated expression of NEUROG3, Pdx1, and MafA in GBCs resulted in robust induction of pancreatic endocrine genes, including Ins1, Ins2, Neurod1, Nkx2-2 and Isl1. Furthermore, expression of GBC-specific genes was repressed, including Sox17 and Hes1. Reprogramming was also enhanced by addition of retinoic acid and inhibition of Notch signaling. Importantly, rGBCs were able to engraft long term in vivo and remained insulin-positive for 15weeks. We conclude that GBCs are a viable source for autologous cell replacement in diabetes, but that complete reprogramming will require further manipulations.

Concise Review: Liver Regenerative Medicine: From Hepatocyte Transplantation to Bioartificial Livers and Bioengineered Grafts

Donor organ shortage is the main limitation to liver transplantation as a treatment for end‐stage liver disease and acute liver failure. Liver regenerative medicine may in the future offer an alternative form of therapy for these diseases, be it through cell transplantation, bioartificial liver (BAL) devices, or bioengineered whole organ liver transplantation. All three strategies have shown promising results in the past decade. However, before they are incorporated into widespread clinical practice, the ideal cell type for each treatment modality must be found, and an adequate amount of metabolically active, functional cells must be able to be produced. Research is ongoing in hepatocyte expansion techniques, use of xenogeneic cells, and differentiation of stem cell‐derived hepatocyte‐like cells (HLCs). HLCs are a few steps away from clinical application, but may be very useful in individualized drug development and toxicity testing, as well as disease modeling. Finally, safety concerns including tumorigenicity and xenozoonosis must also be addressed before cell transplantation, BAL devices, and bioengineered livers occupy their clinical niche. This review aims to highlight the most recent advances and provide an updated view of the current state of affairs in the field of liver regenerative medicine. Stem Cells 2017;35:42–50

Noninvasive 3-dimensional imaging of liver regeneration in a mouse model of hereditary tyrosinemia type 1 using the sodium iodide symporter gene

Cell transplantation is a potential treatment for the many liver disorders that are currently only curable by organ transplantation. However, one of the major limitations of hepatocyte (HC) transplantation is an inability to monitor cells longitudinally after injection. We hypothesized that the thyroidal sodium iodide symporter (NIS) gene could be used to visualize transplanted HCs in a rodent model of inherited liver disease: hereditary tyrosinemia type 1. Wild‐type C57Bl/6J mouse HCs were transduced ex vivo with a lentiviral vector containing the mouse Slc5a5 (NIS) gene controlled by the thyroxine‐binding globulin promoter. NIS‐transduced cells could robustly concentrate radiolabeled iodine in vitro, with lentiviral transduction efficiencies greater than 80% achieved in the presence of dexamethasone. Next, NIS‐transduced HCs were transplanted into congenic fumarylacetoacetate hydrolase knockout mice, and this resulted in the prevention of liver failure. NIS‐transduced HCs were readily imaged in vivo by single‐photon emission computed tomography, and this demonstrated for the first time noninvasive 3‐dimensional imaging of regenerating tissue in individual animals over time. We also tested the efficacy of primary HC spheroids engrafted in the liver. With the NIS reporter, robust spheroid engraftment and survival could be detected longitudinally after direct parenchymal injection, and this thereby demonstrated a novel strategy for HC transplantation. This work is the first to demonstrate the efficacy of NIS imaging in the field of HC transplantation. We anticipate that NIS labeling will allow noninvasive and longitudinal identification of HCs and stem cells in future studies related to liver regeneration in small and large preclinical animal models. Liver Transpl 21:442–453, 2015.

Ectopic expansion of engineered human liver tissue seeds using mature cell populations

The cure for many of the diverse pathological conditions affecting the liver, requires full organ replacement, including the most common reasons for liver transplantation, namely cirrhosis, acute liver failure, and hepatocellular carcinoma. Although perioperative mortality and posttransplant medical morbidities still pose some difficulties for patients receiving liver transplants, outcomes are in general excellent and demonstrate significantly prolonged survival in the majority of patients. The major concern is the growing number of patients in need of liver transplantation coupled with a stagnant donor supply. In a recent article in Science Translational Medicine, Stevens et al. have generated a multicellular human “liver seed,” which, upon implantation into a mouse model of liver injury, could perform many functions of the human liver, including the ability to grow and self-organize in response to regenerative cues from the host. Their work adds to the growing body of literature over the past three decades that has already demonstrated efficacy of a tissue engineering approach for treating liver disease. Attempts to address the scarcity of liver donors have met with limited success. Hepatocytes harvested from discarded human donor livers have been transplanted with some success into patients with metabolic liver disease, but use of this strategy for cirrhosis or acute liver failure has not been effective and likely is related to the altered microenvironments in these conditions. In addition, these efforts are also hampered by shortage of donor livers, and robust ex vivo expansion of hepatocytes has not been achieved to date. Novel cell sources, such as induced pluripotent stem cell (iPSC)or organoid-derived hepatocytes, have shown some promise, but these approaches cannot provide functional hepatocytes in the numbers needed for clinical use. In addition to the problem of simply producing enough hepatocytes, it has become increasingly clear that the three-dimensional structure of the liver contributes indispensably to its function, as does communication and support from the various nonparenchymal cells. Work in this area has focused on seeding of three-dimensional scaffolds, including previously decellularized livers, with the idea that incorporation of both hepatocytes and nonparenchymal cells into these scaffolds might recapitulate the necessary threedimensional structure for normal liver function. Perhaps the most notable was the recent use of iPSCderived cells combined with mesenchymal stromal cells and umbilical vein endothelial cells (“liver buds”), which, when transplanted into a mouse, resulted in vascularized liver tissue that could support a failing liver and provide a significant survival advantage. Generation of these liver buds in vitro occurred through self-organization of the three cell types into three-dimensional cell clusters. In the current work, Stevens et al. have taken liver tissue engineering using adult human cells a step further by demonstrating expansion of in vitro assembled liver tissue in vivo. The core concept of their work was controlled and guided construction of a liver “tissue seed” consisting of human hepatocytes, dermal fibroblasts, and umbilical vein endothelial cells patterned in a fibrin hydrogel. To begin, hepatocytes and fibroblasts were aggregated together in defined sizes using microwell technology. These aggregates were then added to patterned cord structures composed of endothelial cells produced using microtissue molding. Finally, hepatic aggregates and endothelial cells were coencapsulated in fibrin hydrogel. Although the fabricated structure did not resemble endogenous liver tissue, the goal of the investigators was achieved by generating a tissue in which the hepatocytes could respond to regenerative cues in the circulation. To test this functionally, an immunodeficient mouse model of liver failure was used that has previously been shown to robustly support expansion of human hepatocytes after engraftment in the liver: the Fah model of hereditary tyrosinemia type 1. Recipient animals were exposed to cycles of liver injury over the course of 80 days after transplantation of the tissue seeds into the mesenteric fat. Although the investigators could not demonstrate rescue of liver failure in this model, they did show significant functionality of the liver seed, more so when the native liver was injured to promote expansion of the transplanted seed. The investigators show production of human albumin, transferrin, and other synthesized hepatic proteins, and RNA-sequencing analysis showed up-regulation of key human hepatic metabolic genes related to drug metabolism. A potential limitation of this work, however, was the use of the Fah mouse model to demonstrate efficacy. This model for promoting expansion of transplanted cells or tissue seeds is reliant on massive hepatocyte death in the absence of fibrosis and inflammation, a model that has

549 Tyrosinemia Without Succinylacetone: Modeling a Novel Mutation in FAH in Family With Early Onset Cirrhosis and Hepatocellular Carcinoma

is high with rapid progression from adenoma to carcinoma. The penetrance of gastrointestinal neoplasms is extremely high. Among patients undergoing surveillance GI mortality has been zero. The presence of cafe-au-lait macules and consanguinity should raise clinical suspicion. Based on the early age of GI polyps we suggest that colorectal and small bowel surveillance commence at ages 5 and 8 years respectively.