Refinement of in vivo surgical procedures for cardiac gene and cell transfer in rats.

In studies of gene and cell transfer for the treatment of heart disease, direct intramyocardial injection and antegrade intracoronary injection are common methods of delivering biomaterials to the heart. The authors, who carried out these surgical procedures in 377 rats, describe their methodology in detail and discuss surgical refinements that substantially reduced rat mortality. These refinements include a rigorous fluid replacement regimen, use of inhalational anesthesia instead of injectable agents, exposure of the heart without direct contact and use of a chest drainage cannula to remove air from the pleural cavity and prevent lung collapse.

Effect of intrasplenic transplantation of immortalized human hepatocytes in the treatment of acetaminophen-induced acute liver failure SCID mice.

OBJECTIVE: We have established immortalized human hepatocytes by transduction of HPV16 E6/E7 and hTERT (HHE6E7T-1). The cells retained the characteristics of differentiated hepatocytes, but the functional characteristics such as albumin secretion, ureogenesis, and glyconeogenesis decreased gradually as the passages progressed beyond 200 population doublings (PDs). In this report, we transplanted minimally differentiated HHE6E7T-1 cells into the spleens of acute liver failure severe combined immunodeficiency (SCID) mice to examine the potential of the cells to redifferentiate in vivo. MATERIALS AND METHODS: Acute liver failure was induced in SCID mice by intraperitoneal injection of 400 mg/kg acetaminophen. Two hours later, HHE6E7T-1 cells at 200 PDs were transplanted: group 1 (n = 10) 50 microL phosphate-buffered saline (PBS); group 2 (n = 9), lysate of 1 x 10(6) HHE6E7T-1 cells at 200 PDs resuspended in 50 microL PBS; and group 3 (n = 8), 1 x 10(6) HHE6E7T-1 cells. Survival rates at 7 days after transplantation were compared. Blood glucose levels, plasma ammonia levels, and spleen histology were examined at 24 hours after transplantation. RESULTS: Survivals in each group were: 30% for group 1, 33% for group 2, and 100% for group 3. The survival of group 3 was significantly higher than groups 1 or 2 (P < .01). Plasma ammonia levels in group 3 (200 +/- 34 microg/dL) were significantly lower than those in group 1 (325 +/- 92 microg/dL; P < .05). Blood glucose levels in group 3 (110 +/- 20 mg/dL) were significantly higher than those in group 1 (83 +/- 14 mg/dL; P < .05). Upon histologic examination of spleen, the clusters of HHE6E7T-1 cells were clearly identified. CONCLUSIONS: The immortalized human hepatocytes, HHE6E7T-1 at 200 PDs, improved the survival of acute liver failure mice through possible redifferentiation in vivo.

Preservation of left ventricular function and attenuation of remodeling after transplantation of human epicardium-derived cells into the infarcted mouse heart.

BACKGROUND: Proper development of compact myocardium, coronary vessels, and Purkinje fibers depends on the presence of epicardium-derived cells (EPDCs) in embryonic myocardium. We hypothesized that adult human EPDCs might partly reactivate their embryonic program when transplanted into ischemic myocardium and improve cardiac performance after myocardial infarction. METHODS AND RESULTS: EPDCs were isolated from human adult atrial tissue. Myocardial infarction was created in immunodeficient mice, followed by intramyocardial injection of 4x10(5) enhanced green fluorescent protein-labeled EPDCs (2-week survival, n=22; 6-week survival, n=15) or culture medium (n=24 and n=18, respectively). Left ventricular function was assessed with a 9.4T animal MRI unit. Ejection fraction was similar between groups on day 2 but was significantly higher in the EPDC-injected group at 2 weeks (short term), as well as after long-term survival at 6 weeks. End-systolic and end-diastolic volumes were significantly smaller in the EPDC-injected group than in the medium-injected group at all ages evaluated. At 2 weeks, vascularization was significantly increased in the EPDC-treated group, as was wall thickness, a development that might be explained by augmented DNA-damage repair activity in the infarcted area. Immunohistochemical analysis showed massive engraftment of injected EPDCs at 2 weeks, with expression of alpha-smooth muscle actin, von Willebrand factor, sarcoplasmic reticulum Ca2+-ATPase, and voltage-gated sodium channel (alpha-subunit; SCN5a). EPDCs were negative for cardiomyocyte markers. At 6-weeks survival, wall thickness was still increased, but only a few EPDCs could be detected. CONCLUSIONS: After transplantation into ischemic myocardium, adult human EPDCs preserve cardiac function and attenuate ventricular remodeling. Autologous human EPDCs are promising candidates for clinical application in infarcted hearts.

Flow cytometric isolation of endodermal progenitors from mouse salivary gland differentiate into hepatic and pancreatic lineages.

Experimental injury is useful to induce tissue stem cells, which may exist in small numbers under normal conditions. The salivary glands originate from the endoderm and consist of acinar and ductal epithelial cells, which have exocrine function. After salivary gland duct ligation, acinar cells disappear as a result of apoptosis, and duct epithelium subsequently proliferates. In this study, we analyzed the tissue stem cells induced by salivary gland duct ligation in mice using immunohistochemistry and flow cytometry. We sorted the Sca-1(+)/c-Kit(+) fraction from adult mice salivary glands by way of fluorescence-activated cell sorting. The sorted cells were apparently homogeneous and were designated mouse salivary gland-derived progenitors (mSGPs). mSGP cells differentiated into a hepatic lineage when cultured in matrigel. In spherical culture in the presence of glucagon-like peptide-1 (GLP-1), these cells differentiated into a pancreatic endocrine lineage. When spheroidal bodies of mSGP, 20 to 30 microm in diameter, were transplanted into liver via the portal vein, the cells integrated into hepatic cords and expressed albumin and alpha1-antitrypsin, suggesting that they had differentiated into hepatic-type cells. Moreover, ductlike structures formed by mSGP cells also appeared, epithelial cells of which were positive for cytokeratin 19. In conclusion, fluorescence-activated cell sorting (FACS) based on histologic evidence is efficient in isolating adult tissue stem cells of the salivary gland. Tissue stem cells of endodermal origin (e.g., hepatic oval cells, pancreatic epithelial progenitor cells, and salivary gland progenitor cells) have similarities in their molecular markers and tissue location. Our findings suggest the existence of common tissue stem cells in endoderm-derived organs.

Myoblast transplantation for cardiac repair: a clinical perspective.

The incidence of heart failure is achieving epidemic proportions. Adult human myocytes cannot regenerate because these cells do not re-enter the cell cycle. In patients with heart failure, myoblast transplantation is emerging as a potential therapeutic option to augment the function of remaining myocytes. Both skeletal myoblasts and autologous bone marrow cell transplantation, after intensive preclinical experimental animal studies, have entered phase I safety studies in humans. Most of these clinical trials have involved small groups of patients and cell transplantation was carried out as an adjunct to coronary revascularization. Preliminary results show that the procedure is safe and leads to improved myocardial function. This paper reviews and summarizes the outcome of these phase I trials involving skeletal myoblast transplantation.

Influence of liver nonparenchymal cell infusion combined with cyclosporin A on rejection of rat small bowel transplantation.

AIM: To investigate the effect of liver nonparenchymal cell infusion combined with cyclosporin A (CsA) on rejection of heterostrain rat small bowel transplantation. METHODS: The liver nonparenchymal cell suspension was prepared by density gradient centrifugation method with Percoll centrifugal solution. Heterotopic small bowel transplantation was performed. Then the rats were divided into four groups. Group one: homogenic transplantation (F344/N-F344/N), group two: allotransplantation (F344/N-Wistar), group three: allotransplantation (F344/N-Wistar) + CsA, with CsA 10 mg/kg(-1)/d(-1) after transplantation, group four: allotransplantation + CsA (F344/N-Wistar) + liver nonparenchymal cell infusion + CsA (F344/N-Wistar), in which recipient Wistar rats had been injected with 2x10(8) F344/N liver nonparenchymal cells 20 days before transplantation, and treated with CsA after transplantation. Finally, the survival time after small bowel transplantation, gross and histopathological examination, and IL-2 levels in serum were observed. RESULTS: The survival time after small bowel transplantation was 7.14 +/- 0.33 d, 16.32 +/- 0.41 d and 31.41 +/- 0.74 d in group 2, 3, and 4, respectively. The survival time was significant longer (P<0.01) in group 4. The gross and histopathological examination showed that the rejection degree in group 4 was lower than those in groups 2 and 3. Serum IL-2 level in group 4 was also lower than those in groups 2 and 3 (P<0.01). CONCLUSION: Liver nonparenchymal cell infusion combined with CsA can prolong the survival time of rat small bowel transplantation, and the anti-rejection effect is good.

Transplanted cryopreserved encapsulated porcine hepatocytes are as effective as fresh hepatocytes in preventing death from acute liver failure in rats.

BACKGROUND: An implantable bioartificial liver (BAL) using xenogeneic isolated hepatocytes may be an alternative method to orthotopic liver transplantation for treatment of acute liver failure. The purpose of this study was to demonstrate that not only fresh but also cryopreserved porcine hepatocytes could be used in a BAL to prevent death after the onset of acute liver failure in rats. METHODS: Acute liver failure was induced by two-stage 95% hepatectomy. At the time of completion of liver resection, 100 rats were assigned to undergo or not undergo transplantation into the peritoneum of 4 meters of hollow fibers filled with 60 million either fresh or cryopreserved porcine hepatocytes, or syngeneic hepatocytes, or culture medium, or of 60 million nonencapsulated cryopreserved porcine hepatocytes without immunosuppressive therapy. Survival rates at 7 days were compared between the different groups. RESULTS: In the control groups of hepatectomized animals not receiving encapsulated hepatocytes, 69-79% of the rats died from acute liver failure. The mortality rate was reduced to 15% (2 of 13) in rats receiving fresh porcine hepatocytes (P<0.01), 25% (4 of 16) in rats transplanted with either cryopreserved or syngeneic hepatocytes (P<0.05). Survival rates were maintained when hollow fibers were explanted > or =4 days after hepatectomy. In surviving rats, the weight of the remnant native liver increased with time and returned to the initial weight after 1 month. CONCLUSIONS: The implantable BAL using xenogeneic porcine hepatocytes was able in preventing death from acute liver failure without immunosuppressive therapy. Encapsulated cryopreserved hepatocytes were as effective as fresh hepatocytes.

Treatment of surgically induced acute liver failure by transplantation of conditionally immortalized hepatocytes.

The shortage of human livers available for hepatocyte isolation limits its clinical application. The availability of cloned, conditionally immortalized hepatocytes that could be grown in culture but would lose their transformed phenotype and provide metabolic support upon transplantation would greatly facilitate the treatment of acute liver failure. Toward this goal, we transduced isolated Lewis rat hepatocytes using a replication-defective recombinant retrovirus capable of transferring a gene encoding a thermolabile mutant simian virus 40 T antigen (SV40ts). The cloned, immortalized hepatocytes proliferate at 33 degrees C. At the nonpermissive temperatures (37-39 degrees C), they stop growing and exhibit characteristics of differentiated hepatocytes. These cells did not produce tumors when transplanted in mice with severe combined immunodeficiency disease or in syngeneic rats. To induce acute liver failure, Lewis rats were subjected to 90% hepatectomy (Hpx) and given 5% oral dextrose. All rats that did not undergo hepatocyte transplantation died within 96 hr. Fifty percent of rats that received intrasplenic injection of 10 x 10(6) primary Lewis rat hepatocytes (G2, n=6) or 10 x 10(6) SV40ts-conditionally immortalized (SV40ts-ci) hepatocytes (G3, n=8) 1 day before 90% hepatectomy survived, whereas 80% of rats that received an intraperitoneal injection of 200 x 10(6) primary Lewis rat hepatocytes (G4, n=10) or 200 x 10(6) SV40ts-ci hepatocytes (G5, n=10) on the day of hepatectomy survived. Survival after intraperitoneal injection of a cellular homogenate of 200 x 10(6) primary Lewis rat (G7, n=9) or SV40ts-ci hepatocytes (G8, n=10) on the day of Hpx was 33% and 40%, respectively, whereas survival after intraperitoneal injection of 200 x 10(6) Lewis rat bone marrow cells (G6, n=7) was 29%. Thus, transplanted, conditionally immortalized hepatocytes can be as effective as primary hepatocytes in supporting life during acute liver insufficiency. This work represents the first step in developing an hepatocyte cell line that would partially alleviate the organ-donor shortage and could be of potential clinical value.

Combined foscarnet-ganciclovir treatment for cytomegalovirus infections after allogeneic hemopoietic stem cell transplantation.

In a previous study, we showed that patients undergoing allogeneic hemopoietic stem cell transplantation (HSCT) who had cytomegalovirus (CMV) antigenemia with more than 4 CMV antigen-positive cells/200,000 have a high transplant-related mortality (TRM) rate, despite treatment with ganciclovir or foscarnet. In an attempt to reduce TRM, 32 allogeneic HSCT recipients, between the ages of 16 and 55 years (median, 35 years), with CMV antigenemia (> or = 5 positive cells) developing at a median interval from HSCT of 49 days, were given combination treatment with foscarnet and ganciclovir for 15 days. The prescribed dose was 180 mg/kg/day of foscarnet and 10 mg/kg/day of ganciclovir: the median administered dose in the first 15 days, after adjusting for creatinine levels and peripheral blood counts, was 64% for foscarnet and 53% for ganciclovir. Maintenance was given with foscarnet and ganciclovir on alternate days for an additional 2 weeks. Thirty-one of 32 patients were on cyclosporine, 30 were on systemic antibiotics, and 9 were on intravenous amphotericin. Median laboratory values on days 1 and 15 of treatment were 1.0 and 1.1 mg/100 ml creatinine, 5.7 x 10(9)/L, and 4.1 x 10(9)/L white blood cells, and 78 x lO(9)/L and 72 x 10(9)/L platelets. All patients cleared CMV antigenemia by day +15, although CMV antigenemia recurred in 5 patients on maintenance therapy and in 14 patients off maintenance therapy: the dose of foscarnet (but not ganciclovir) received in the first 15 days was significantly lower in patients in whom antigenemia recurred within 30 days (P=0.0002). Six patients died, one with interstitial pneumonia, one with multiorgan failure, and four with infections. Twenty-six patients survived 119-1051 days after transplant. The actuarial TRM rate at 1 year is 23%. Eighteen patients who had received unmanipulated bone marrow transplants from HLA-identical siblings were compared with 15 matched controls who had been treated with a single drug (either foscarnet or ganciclovir) for CMV antigenemia (> or = 5 cells): the actuarial 1 year TRM rate was 13% for patients receiving combined treatment, compared with 47% for controls receiving a single drug (P=0.02). This study shows that combined foscarnet-ganciclovir is one therapeutic option for allogeneic HSCT recipients who develop CMV antigenemia with a high number of CMV antigen-positive cells. Treatment can be given together with cyclosporine and antibiotics with appropriate dose reductions. It produces prompt clearing of CMV infection, and may reduce TRM rates in comparison to single-agent therapy.

Age-related changes in graft-versus-host reactivity of spleen cells from male, virgin, or multiparous female Long-Evans rats.

Spleen cell graft-versus-host (GVH) reactivity was determined in male and female, either virgin or breeder, Long-Evans (LE) rats from 3 to 24 months of age. The tests of a regional (popliteal lymph node enlargement index) and a systemic (splenomegaly index, mortality assay) GVH reaction was used. Although the GVH reactivity declined with age in both sexes, the onset of this decline was significantly delayed in 18-month-old virgin females in comparison with 12-month-old males. Moreover, 6 or 7 consecutive pregnancies resulted in significantly enhanced GVH reactivity of 18-24-month-old females. This long-lasting effect of multiparity was observed in females mated either syngeneically or allogeneically. The possible role of neuroendocrine factors in delaying the age-related process of thymic involution in multiparous females is suggested.