Kuldeep K. Bhargava

Efficacy and safety of repeated hepatocyte transplantation for significant liver repopulation in rodents

BACKGROUND & AIMS Significant liver repopulation by hepatocyte transplantation will advance clinical applications. The aim of this study was to test the hypothesis that translocation of transplanted cells into liver plates will allow repeated cell transplantation for increasing the transplanted hepatocyte mass. METHODS Hepatocytes were transplanted via spleen from either F344 rats into syngeneic recipients deficient in dipeptidyl peptidase IV or from transgenic hepatitis B surface antigen-producing G26 mice with hepatitis B virus into nontransgenic congeneic recipients. Portosystemic shunting was shown by radiological methods. RESULTS Repeated hepatocyte transplantation led to progressively increased liver repopulation. Transplantation of 1.75 x 10(8) hepatocytes in three divided doses repopulated more than an estimated 5% of the host rat liver, with 3.8 x 10(6) +/- 0.1 x 10(6) transplanted cells/cm3 liver. This was a tenfold or threefold mean increase in transplanted cell number compared with recipients of 2.0 x 10(7) or 7.5 x 10(7) cells transplanted in single sessions, respectively (P < 0.001). Repeated hepatocyte transplantation interfered with neither cell integrations in liver parenchyma nor secretory function of transplanted cells. Portal hypertension, portasystemic collaterals, or intrahepatic shunting were not observed in cell recipients. CONCLUSIONS Repeated transplantation of hepatocytes in large numbers is safe and effective and should advance strategies for liver repopulation.

Transplanted hepatocytes engraft, survive, and proliferate in the liver of rats with carbon tetrachloride‐induced cirrhosis

Repopulation of the cirrhotic liver with disease‐resistant hepatocytes could offer novel therapies, as well as systems for biological studies. Establishing whether transplanted hepatocytes can engraft, survive, and proliferate in the cirrhotic liver is a critical demonstration. Dipeptidyl peptidase IV‐deficient F344 rats were used to localize transplanted hepatocytes isolated from the liver of syngeneic normal F344 rats. Cirrhosis was induced by administration of carbon tetrachloride with phenobarbitone and these drugs were withdrawn prior to cell transplantation. Cirrhotic rats showed characteristic hepatic histology, as well as significant portosystemic shunting. When hepatocytes were transplanted via the spleen, cells were distributed immediately in periportal areas, fibrous septa, and regenerative nodules of the cirrhotic liver. Although some transplanted cells translocated into pulmonary capillaries, this was not deleterious. At 1 week, transplanted cells were fully integrated in the liver parenchyma, along with expression of glucose‐6‐phosphatase and glycogen as reporters of hepatic function. Transplanted cells proliferated in the liver of cirrhotic animals and survived indefinitely. At 1 year, transplanted hepatocytes formed large clusters containing several‐fold more cells than normal control animals, which was in agreement with increased cell turnover in the cirrhotic rat liver. The findings indicate that the cirrhotic liver can be repopulated with functionally intact hepatocytes that are capable of proliferating. Liver repopulation using disease‐resistant hepatocytes will be applicable in chronic conditions, such as viral hepatitis or Wilsons disease. Copyright

Transplantation of bone marrow-derived MSCs improves cisplatinum-induced renal injury through paracrine mechanisms

Mesenchymal stem cells (MSCs) have been reported to preserve renal function in various models of acute kidney injury (AKI). Different routes were used to transplant MSCs but the role of cell transplantation routes in directing outcomes has been unknown. In the present study, we evaluated organ bio-distributions of transplanted MSCs, and correlated survival of transplanted cells with outcomes in mice with cisplatinum-induced AKI. We found that after intravenous administration, MSCs were largely localized in pulmonary capillaries and only a minute fraction of MSCs entered kidneys and the cells survived only transiently. Therefore, we also transplanted MSCs via intraperitoneal and renal subcapsular routes. Transplanted MSCs survived longer in peritoneal cavity and renal subcapsular space. Interestingly, when MSC transplantation was followed by cisplatinum-induced AKI, renal morphology and renal functions were better preserved, irrespective of the cell transplantation route. As transplanted MSCs did not migrate to kidneys from either peritoneal cavity or renal subcapsular space, this finding suggested that migration of cells was not required for the beneficial response. The possibility of indirect mechanisms was confirmed when administration of the conditioned medium from MSCs also protected renal tubular cells from cisplatinum-induced cytotoxicity. We identified presence of over forty regulatory cytokines in the conditioned medium obtained from MSCs. Since paracrine factors released by transplanted cells accounted for improvements, it appears that the route of cell transplantation is not critical for realizing benefits of cell therapy with MSCs in AKI. Studies of specific cytokines secreted by MSCs will help to obtain new therapeutic mechanisms for renal protection.

Monocrotaline promotes transplanted cell engraftment and advances liver repopulation in rats via liver conditioning

Disruption of the hepatic endothelial barrier or Kupffer cell function facilitates transplanted cell engraftment in the liver. To determine whether these mechanisms could be activated simultaneously, we studied the effects of monocrotaline, a pyrollizidine alkaloid, with reported toxicity in liver sinusoidal endothelial cells and Kupffer cells. The effects of monocrotaline in Fischer 344 rats were examined by tissue morphology, serum hyaluronic acid levels, and liver tests (endothelial and hepatocyte injury) or incorporation of carbon and 99mTc‐sulfur colloid (Kupffer cell damage). To study changes in cell engraftment and liver repopulation, Fischer 344 rat hepatocytes were transplanted into syngeneic dipeptidyl peptidase IV–deficient rats followed by histological assays. We observed extensive endothelial injury without Kupffer cell or hepatocyte damage in monocrotaline‐treated rats. Monocrotaline enhanced transplanted cell engraftment without changes in transplanted cell numbers or induction of proliferation in native hepatocytes over 3 months. In monocrotaline‐treated rats, transplanted cells integrated into the liver parenchyma and survived in vascular spaces. To determine whether native hepatocytes suffered inapparent damage after monocrotaline, we introduced further liver injury with carbon tetrachloride subsequent to cell transplantation. Monocrotaline sensitized the liver to carbon tetrachloride–induced necrosis, which advanced transplanted cell proliferation, leading to significant liver repopulation. During this process, we observed proliferation of bile duct cells and small epithelial cells, although transplanted hepatocytes did not appear to reconstitute bile ducts. The studies showed that perturbation of multiple liver cell compartments by monocrotaline promoted transplanted cell engraftment and proliferation. In conclusion, development of drugs with monocrotaline‐like effects will help advance liver cell therapy. (HEPATOLOGY 2006;44:1411–1420.)

Integration and proliferation of transplanted cells in hepatic parenchyma following D-galactosamine-induced acute injury in F344 rats

To determine whether liver repopulation with cell transplantation could be of therapeutic value in acute hepatic failure, it is necessary to establish the fate of transplanted hepatocytes. This study used dipeptidyl peptidase IV‐deficient F344 rats as recipients to analyse the engraftment and proliferation of transplanted hepatocytes. Syngeneic hepatocytes were transplanted intrasplenically 24–30 h after induction of liver injury by D‐galactosamine (GalN). Portosystemic shunting was analysed with 99m‐Tc‐labelled albumin microspheres. GalN‐treated rats showed characteristic hepatic necrosis, inflammation, gamma‐glutamyl transpeptidase activation, and regenerative activity, without increased portosystemic shunting (>99% 99m‐Tc activity was in the liver in normal and GalN‐treated rats). Transplanted cells entered hepatic sinusoids promptly and were observed in liver plates at 48 h. The number of transplanted cells increased in GalN‐treated rats by approximately seven‐fold (range two‐ to 12‐fold), along with evidence for DNA synthesis between 3 and 14 days after cell transplantation and greater prevalence of larger transplanted cell clusters. These findings indicate that the liver can be safely repopulated in animals with acute liver failure, although the time required for regenesis of plasma membrane structures and proliferation in transplanted hepatocytes will need to be considered in developing therapeutic strategies. Copyright

In vitro human leukocyte labeling with 64Cu: an intraindividual comparison with 111In-oxine and 18F-FDG☆

UNLABELLED We investigated labeling human leukocytes [white blood cells (WBCs)] in vitro with copper-64 (Cu) comparing labeling efficiency, viability and stability of Cu-WBCs with (111)In-oxine (In) WBCs and (18)F-FDG (FDG) WBCs. METHODS Leukocytes from 10 volunteers were labeled with Cu, In and FDG. Forty milliliters of venous blood was collected and leukocyte separation was performed according to standard methods. In-WBCs and FDG-WBCs were labeled according to published methods. For Cu-WBCs, tropolone initially was used as a single chelating agent. Because of poor intracellular Cu retention (54+/-4% at 3 h and 24+/-5% at 24 h), the fluorinated, membrane-permeable divalent cation chelator quin-MF was added. WBCs were incubated in 5 ml saline containing 100 microl of 1mM quin-MF/AM in 2% dimethyl sulfoxide and 74-185 MBq Cu-tropolone for 45 min at 37 degrees C. Labeling efficiencies; in vitro cellular viabilities at 1, 3 and 24 h; and in vitro stabilities at 1, 2, 3, 4 and 24 h (except FDG-WBCs) were determined. RESULTS Mean Cu-WBCs (87+/-4%) and In-WBCs (86+/-4%) labeling efficiencies were comparable and were significantly higher than FDG-WBCs (60+/-19%, P<.001). Cell viabilities, similar at 1 h, were significantly higher for (64)Cu-WBCs at 3 and 24 h. Intracellular retention of activity was always significantly higher for In-WBCs than for Cu-WBCs and FDG-WBCs. At 24 h, intracellular retention was 88+/-4% for In-WBCs and 79+/-6% for Cu-WBCs. CONCLUSION Cu-WBC labeling efficiency and viability were comparable or superior to In-WBCs and significantly higher than FDG-WBCs. Although significantly more activity eluted from Cu-WBCs than from In-WBCs, Cu-WBC probably is adequate for imaging. These data suggest that further investigation of in vitro copper-64-labeled leukocytes for PET imaging of infection is warranted.

Hepatic targeting of transplanted liver sinusoidal endothelial cells in intact mice

Targeting of cells to specific tissues is critical for cell therapy. To study endothelial cell targeting, we isolated mouse liver sinusoidal endothelial cells (LSEC) and examined cell biodistributions in animals. To identify transplanted LSEC in tissues, we labeled cells metabolically with DiI‐conjugated acetylated low density lipoprotein particles (DiI‐Ac‐LDL) or 111Indium‐oxine, used LSEC from Rosa26 donors expressing β‐galactosidase or Tie‐2‐GFP donors with green fluorescent protein (GFP) expression, and tranduced LSEC with a GFP‐lentiviral vector. LSEC efficiently incorporated 111Indium and DiI‐Ac‐LDL and expressed GFP introduced by the lentiviral vector. Use of radiolabeled LSEC showed differences in cell biodistributions in relation to the cell transplantation route. After intraportal injection, LSEC were largely in the liver (60 ± 13%) and, after systemic intravenous injection, in lungs (67 ± 9%); however, after intrasplenic injection, only some LSEC remained in the spleen (29 ± 10%; P < .01), whereas most LSEC migrated to the liver or lungs. Transplanted LSEC were found in the liver, lungs, and spleen shortly after transplantation, whereas longer‐term cell survival was observed only in the liver. Transplanted LSEC were distinct from Kupffer cells with expression of Tie‐2 promoter‐driven GFP and of CD31, without F4/80 reactivity. In further studies using radiolabeled LSEC, we established that the manipulation of receptor‐mediated cell adhesion in liver sinusoids or the manipulation of blood flow–dependent cell exit from sinusoids improved intrahepatic retention of LSEC to 89 ± 7% and 89 ± 5%, respectively (P < .01). In conclusion, the targeting of LSEC to the liver and other organs is directed by vascular bed–specific mechanisms, including blood flow–related processes, and cell‐specific factors. These findings may facilitate analysis of LSEC for cell and gene therapy applications. (HEPATOLOGY 2005.)

Hepatic targeting and biodistribution of human fetal liver stem/progenitor cells and adult hepatocytes in mice

Tracking stem/progenitor cells through noninvasive imaging is a helpful means of assessing the targeting of transplanted cells to specific organs. We performed in vitro and in vivo studies wherein adult human hepatocytes and human fetal liver stem/progenitor cells were labeled with indium‐111 (111In)‐oxine and technetium‐99m (99mTc)‐Ultratag or 99mTc‐Ceretec. The labeling efficiency and viability of cells was analyzed in vitro, and organ biodistribution of cells was analyzed in vivo after transplantation in xenotolerant nonobese diabetic/severe combined immunodeficiency mice through intrasplenic or intraportal routes. We found that adult hepatocytes and fetal liver stem/progenitor cells incorporated 111In but not 99mTc labels. After radiolabeling, cell viability was unchanged. Transplanted adult hepatocytes or fetal liver stem/progenitor cells were targeted to the liver more effectively by the intraportal rather than the intrasplenic route. Transplanted cells were retained in the liver after intraportal injection and in the liver and spleen after intrasplenic injection, without translocations into pulmonary or systemic circulations. Compared with fetal liver stem/progenitor cells, fewer adult hepatocytes were retained in the spleen after intrasplenic transplantation. The distribution of transplanted cells in organs was substantiated by genetic assays, including polymerase chain reaction amplification of DNA sequences from a primate‐specific Charcot‐Marie‐Tooth element, and in situ hybridization for primate alphoid satellite sequences ubiquitous in all centromeres. Conclusion: 111In labeling of human fetal liver stem/progenitor cells and adult hepatocytes was effective for noninvasive localization of transplanted cells. This should facilitate continued development of cell therapies through further animal and clinical studies. (HEPATOLOGY 2009.)

Rapid clearance of transplanted hepatocytes from pulmonary capillaries in rats indicates a wide safety margin of liver repopulation and the potential of using surrogate albumin particles for safety analysis

BACKGROUND/AIMS Applications of liver repopulation by hepatocyte transplantation require analysis of cell biodistributions, particularly when portasystemic shunting coexists. The aims of this study were to determine the fate of hepatocytes transplanted into the pulmonary vascular bed and to examine whether cell biodistributions could be approximated by convenient surrogates. METHODS Rat hepatocytes and macroaggregated serum albumin particles of similar sizes were injected into the portal and pulmonary vascular beds of rats, followed by biodistribution, survival and function analyses. RESULTS Although functionally intact, virtually all hepatocytes were cleared from the pulmonary capillaries within 24 h. Serum albumin levels increased minimally in Nagase analbuminemic rats with or without portacaval shunting to enhance delivery of portal factors to transplanted cells in lungs. Despite intravenous injection of hepatocytes approaching >1x10(9) cells in humans, the hemodynamic changes were limited to transient increases in right atrial pressures. The hepatocyte distributions in specific vascular beds were largely reproduced by macroaggregated human serum albumin particles. CONCLUSIONS Incidental intrapulmonary cell translocations during liver repopulation will have a wide safety margin. Use of macroaggregated serum albumin particles as surrogates for initial short-term biodistribution and safety analysis will advance hepatocyte transplantation, as the cost of GLP-certified laboratories and consumption of scarce donor livers will be avoided.

Analysis of hepatocyte distribution and survival in vascular beds with cells marked by 99mTC or endogenous dipeptidyl peptidase IV activity

Knowledge of the kinetics of cell distribution in vascular beds will help optimize engraftment of transplanted hepatocytes. To noninvasively localize transplanted cells in vivo, we developed conditions for labeling rat hepatocytes with 99mTc-pertechnetate. The incorporated o9mTc was bound to intracellular proteins and did not impair cell viability. When 99mTc hepatocytes were intrasplenically injected into normal rats, cells entered liver sinusoids with time-activity curves demonstrating instantaneous cell translocations. 99mTc activity in removed organs was in liver or spleen, and lungs showed little activity. However, when cells were intrasplenically transplanted into rats with portasystemic collaterals, 99mTc appeared in both liver sinusoids and pulmonary alveolar capillaries. To further localize cells, we transplanted DPPIV+ F344 rat hepatocytes into syngeneic DPPIV-recipients. Histochemical staining for DPPIV activity demonstrated engraftment of intrasplenically transplanted cells in liver parenchyma. In contrast, when 99mTc hepatocytes were injected into a peripheral vein, cells were entrapped in pulmonary capillaries but were subsequently broken down with redistribution of 99mTc activity elsewhere. Intact DPPIV+ hepatocytes were identified in lungs, whereas only cell fragments were present in liver, spleen, or kidneys. These findings indicate that although the pulmonary vascular bed offers advantages of easy accessibility and a relatively large capacity, significant early cell destruction is an important limitation.