Engraftment of donor hepatocytes is a critical step that determines the success of hepatocyte transplantation. Rapid and efficient integration of donor cells would enable prompt liver repopulation of ...these cells in response to selective proliferative stimuli offered by a preparative regimen. We have earlier demonstrated that hepatic irradiation (HIR) in combination with a variety of hepatotrophic growth signals, such as partial hepatectomy and hepatocyte growth factor, can be used as a preparative regimen for liver repopulation of transplanted hepatocytes. In this study, we investigated the effects of HIR on engraftment of transplanted dipeptidyl peptidase IV (DPPIV)–positive hepatocytes in congeneic DPPIV‐deficient rats. HIR‐induced apoptosis of hepatic sinusoidal endothelial cells (SEC) within 6 hours of HIR resulted in dehiscence of the SEC lining in 24 hours. Although there was no change of the number of Kupffer cells after HIR, colloidal carbon clearance decreased 24 hours post HIR, indicating a suppression of phagocytic function. DPPIV+ donor cells were transplanted 24 hours after HIR (0–50 Gy). There was an HIR dose‐dependent increase in the donor hepatocyte mass engrafted in the liver parenchyma. The number of viable transplanted hepatocytes present in hepatic sinusoids or integrated in the parenchyma was greater in the HIR‐treated group at 3 and 7 days after transplantation compared with the sham controls. Finally, we validated these rodent studies in cynomolgus monkeys, demonstrating that a single 10‐Gy dose of HIR was sufficient to enhance engraftment of donor porcine hepatocytes. These data indicate that transient disruption of the SEC barrier and inhibition of the phagocytic function of Kupffer cells by HIR enhances hepatocyte engraftment and the integrated donor cell mass. Thus, preparative HIR could be potentially useful to augment hepatocyte transplantation. (HEPATOLOGY 2009;49:258‐267.)
This article is an updated report of a symposium held at the June 2000 annual meeting of the American Society for Pharmacology and Experimental Therapeutics in Boston. The symposium was sponsored by ...the ASPET Divisions for Drug Metabolism and Molecular Pharmacology. The report covers research from the authors' laboratories on the structure and regulation of UDP-glucuronosyltransferase (UGT) genes, glucuronidation of xenobiotics and endobiotics, the toxicological relevance of UGTs, the role of UGT polymorphisms in cancer susceptibility, and gene therapy for UGT deficiencies.
Crigler–Najjar syndrome type I is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia beginning at birth. The syndrome results from an absence of hepatic uridine ...diphosphoglucuronate (UDP) glucuronosyltransferase activity, which is essential for the conjugation and excretion of bilirubin. Because of the accumulation of unconjugated bilirubin in plasma, patients are at risk for kernicterus.
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Although phototherapy successfully reduces serum bilirubin levels, patients are again at risk for kernicterus around the time of puberty, when phototherapy becomes less effective.
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The necessary daily duration of phototherapy often approaches 14 to 16 hours. At present, liver transplantation is the only definitive treatment. . . .
Radiation-induced gastrointestinal syndrome (RIGS) results from a combination of direct cytocidal effects on intestinal crypt and endothelial cells and subsequent loss of the mucosal barrier, ...resulting in electrolyte imbalance, diarrhea, weight loss, infection and mortality. Because R-spondin1 (Rspo1) acts as a mitogenic factor for intestinal stem cells, we hypothesized that systemic administration of Rspo1 would amplify the intestinal crypt cells and accelerate the regeneration of the irradiated intestine, thereby, ameliorating RIGS.
Male C57Bl/6 mice received recombinant adenovirus expressing human R-spondin1 (AdRspo1) or E.coli Lacz (AdLacz), 1-3 days before whole body irradiation (WBI) or abdominal irradiation (AIR). Post-irradiation survival was assessed by Kaplan Meier analysis. RIGS was assessed by histological examination of intestine after hematoxilin and eosin staining, immunohistochemical staining of BrdU incorporation, Lgr5 and beta-catenin expression and TUNEL staining. The xylose absorption test (XAT) was performed to evaluate the functional integrity of the intestinal mucosal barrier. In order to examine the effect of R-spondin1 on tumor growth, AdRspo1 and AdLacZ was administered in the animals having palpable tumor and then exposed to AIR. There was a significant increase in survival in AdRspo1 cohorts compared to AdLacZ (p<0.003) controls, following WBI (10.4 Gy). Significant delay in tumor growth was observed after AIR in both cohorts AdRspo1 and AdLacZ but AdRspo1 treated animals showed improved survival compared to AdLacZ. Histological analysis and XAT demonstrated significant structural and functional regeneration of the intestine in irradiated animals following AdRspo1 treatment. Immunohistochemical analysis demonstrated an increase in Lgr5+ve crypt cells and the translocation of beta-catenin from the cytosol to nucleus and upregulation of beta-catenin target genes in AdRspo1-treated mice, as compared to AdLacz-treated mice.
Rspo1 promoted radioprotection against RIGS and improved survival of mice exposed to WBI. The mechanism was likely related to induction of the Wnt-beta-catenin pathway and promotion of intestinal stem cell regeneration. Rspo1 has protective effect only on normal intestinal tissue but not in tumors after AIR and thereby may increase the therapeutic ratio of chemoradiation therapy in patients undergoing abdominal irradiation for GI malignancies.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Although several types of somatic cells have been reprogrammed into induced pluripotent stem cells (iPSCs) and then differentiated to hepatocyte-like cells (iHeps), the method for generating such ...cells from renal tubular epithelial cells shed in human urine and transplanting them into animal livers has not been described systematically. We report reprogramming of human urinary epithelial cells into iPSCs and subsequent hepatic differentiation, followed by a detailed characterization of the newly generated iHeps. The epithelial cells were reprogrammed into iPSCs by delivering the pluripotency factors OCT3/4, SOX2, KLF4, and MYC using methods that do not involve transgene integration, such as nucleofection of episomal (oriP/EBNA-1) plasmids or infection with recombinant Sendai viruses. After characterization of stable iPSC lines, a three-step differentiation toward hepatocytes was performed. The iHeps expressed a large number of hepatocyte-preferred genes, including nuclear receptors that regulate genes involved in cholesterol homeostasis, bile acid transport, and detoxification. MicroRNA profile of the iHeps largely paralleled that of primary human hepatocytes. The iHeps engrafted into the livers of Scid mice transgenic for mutant human SERPINA1 after intrasplenic injection. Thus, urine is a readily available source for generating human iHeps that could be potentially useful for disease modeling, pharmacological development, and regenerative medicine.
Hepatocyte transplantation has the potential to cure inherited liver diseases, but its application is impeded by a scarcity of donor livers. Therefore, we explored whether transplantation of ...hepatocyte-like cells (iHeps) differentiated from human induced pluripotent stem cells (iPSCs) could ameliorate inherited liver diseases. iPSCs reprogrammed from human skin fibroblasts were differentiated to iHeps, which were transplanted into livers of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-deficient Gunn rats, a model of Crigler-Najjar syndrome 1 (CN1), where elevated unconjugated bilirubin causes brain injury and death. To promote iHep proliferation, 30% of the recipient liver was X-irradiated before transplantation, and hepatocyte growth factor was expressed. After transplantation, UGT1A1+ iHep clusters constituted 2.5%–7.5% of the preconditioned liver lobe. A decline of serum bilirubin by 30%–60% and biliary excretion of bilirubin glucuronides indicated that transplanted iHeps expressed UGT1A1 activity, a postnatal function of hepatocytes. Therefore, iHeps warrant further exploration as a renewable source of hepatocytes for treating inherited liver diseases.
•Human skin fibroblast-derived iPSCs were differentiated to hepatocyte-like iHeps•iHeps were transplanted into Gunn rats, a model of Crigler-Najjar syndrome 1•Engraftment of the iHeps in Gunn rat livers reduced serum bilirubin levels•iHeps may be potentially useful in treating liver-based metabolic disorders
Roy-Chowdhury and colleagues show that human iPSCs differentiated to hepatocyte-like iHep cells engraft into the liver of UGT1A1-deficient Gunn rats, a model of Crigler-Najjar syndrome 1, where hyperbilirubinemia causes brain injury and death. After iHep transplantation, serum bilirubin declined by 30%–60% and bilirubin glucuronides appeared in bile, indicating that the engrafted iHeps expressed UGT1A1, a postnatal hepatocyte function.
Hepatocyte transplantation is an attractive alternative to liver transplantation. Thus far, however, extensive liver repopulation by adult hepatocytes has required ongoing genetic, physical, or ...chemical injury to host liver. We hypothesized that providing a regulated proliferative and/or survival advantage to transplanted hepatocytes should enable repopulation in a normal liver microenvironment. Here, we repopulated livers of DPPIV− (dipeptidyl peptidase‐4) rats and Ugt1a1 (uridinediphosphoglucuronate glucuronosyltransferase 1a1)‐deficient Gunn rats (model of Crigler‐Najjar syndrome type 1), both models without underlying liver injury, for up to 1 year by transplanting lenti‐hYAP‐ERT2 (mutated estrogen receptor ligand‐binding domain 2)‐transduced hepatocytes (YAP‐Hc). Yap (yes‐associated protein) nuclear translocation/function in YAP‐Hc was regulated by tamoxifen. Repopulating YAP‐Hc and host hepatocytes were fluorescence‐activated cell sorting–purified and their transcriptomic profiles compared by RNAseq. After 1 year of liver repopulation, YAP‐Hc clusters exhibited normal morphology, integration into hepatic plates and hepatocyte‐specific gene expression, without dysplasia, dedifferentiation, or tumorigenesis. RNAseq analysis showed up‐regulation of 145 genes promoting cell proliferation and 305 genes suppressing apoptosis, including hepatocyte growth factor and connective tissue growth factor among the top 30 in each category and provided insight into the mechanism of cell competition that enabled replacement of host hepatocytes by YAP‐Hc. In Gunn rats transplanted with YAP‐Hc+tamoxifen, there was a 65%‐81% decline in serum bilirubin over 6 months versus 8%‐20% with control‐Hc, representing a 3‐4‐fold increase in therapeutic response. This correlated with liver repopulation as demonstrated by the presence of Ugt1a1‐positive hepatocyte clusters in livers and western blot analysis of tissue homogenates. Conclusion: Tamoxifen‐regulated nuclear translocation/function of hYAP‐ERT2 enabled long‐term repopulation of DPPIV−/− and Gunn rat livers by hYAP‐ERT2‐transduced hepatocytes without tumorigenesis. This cell transplantation strategy may offer a potential therapy for most of the inherited monogenic liver diseases that do not exhibit liver injury.
The liver of DPPIV‐ and UGT1A1‐deficient Gunn rats were repopulated for one year in a normal liver microenvironment by transplanting hERT2‐YAP transduced hepatocytes under tight tamoxifen control. Repopulating YAP+ clusters exhibited normal hepatic morphology, integration into hepatic parenchyma, normal membrane polarity and orientation within hepatic plates, no change in ploidy and no evidence for dysplasia, dedifferentiation, transdifferentiation, fibrosis or oncogenesis. Comparable repopulation was obtained in jaundiced Gunn rats with 75‐80% reduction in serum bilirubin and excretion of bilirubin glucuronides in bile, thus demonstrating a potential therapy for monogenic liver disorders that do not cause liver injury.
The treatment of inherited metabolic liver diseases by hepatocyte transplantation (HT) would be greatly facilitated if the transplanted normal hepatocytes could be induced to proliferate ...preferentially over the host liver cells. We hypothesized that preparative hepatic irradiation (HIR) should inhibit host hepatocyte proliferation in response to partial hepatectomy (PH). Normal nonirradiated hepatocytes transplanted in this setting should have a selective growth advantage over the host liver cells and should progressively repopulate the liver. To test this hypothesis, we transplanted 5 million hepatocytes from normal Wistar-Roman High Avoidance (RHA) rats into the livers of congeneic bilirubin-uridine 5'-diphosphoglucuronate glucuronosyltransferase (UGT1A1)-deficient jaundiced Gunn rats by intrasplenic injection after one of the following treatments: (1) 68% PH, (2) HIR (50 Gy), or (3) HIR + PH. In rats receiving either PH or HIR alone before HT, serum bilirubin concentrations declined by 25% to 30% in 28 weeks. In contrast, serum bilirubin levels were normalized completely in rats receiving HIR + PH before HT. Massive repopulation of the Gunn rat liver by the UGT1A1-positive Wistar-RHA hepatocytes was shown by UGT1A1 enzyme assay, immunoblot analysis, and immunohistochemical staining of the recipient liver. High-performance liquid chromatography analysis of the bile collected from Gunn rats 5 months after PH, HIR, and HT showed normalization of the pigment profile, with bilirubin diglucuronide and monoglucuronide as the predominant pigments. In conclusion, a preparative regimen of HIR + PH results in massive repopulation of the liver with functionally normal transplanted hepatocytes, resulting in complete correction of a metabolic deficiency. Noninvasive strategies to replace PH for providing proliferative stimuli to the transplanted cells should make this regimen valuable in augmenting the effects of HT for the treatment of liver diseases. (H
EPATOLOGY 2002;36:354-362.)
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