Intrasplenic transplantation of encapsulated genetically engineered mouse insulinoma cells reverses streptozotocin-induced diabetes in rats.

Pancreatic islet transplantation is limited by shortage of donor organs. Although beta-cell lines could be used, their secretion of insulin is characteristically glucose independent and immunoisolation is required. Here we show that intrasplenic transplantation of encapsulated glucose-responsive mouse insulinoma cells reversed streptozotocin (STZ)-induced diabetes in rats. MIN-6 cells derived from a transgenic mouse expressing SV 40 large T antigen in pancreatic beta-cells were transfected with minigene encoding for human glucagon-like-peptide-1 under the control of rat insulin promoter. The cells were encapsulated in alginate/poly-L-lysine and used for cell transplantation in STZ-diabetic rats. Rats with nonfasting blood glucose (n-FBG) greater than 350 mg/dl were used. In group I rats (n=6) 20 million encapsulated cells were injected into the spleen. Group II rats (n=6) received empty capsules. n-FBG was measured biweekly. After 4 and 8 weeks, an intraperitoneal glucose tolerance test (IPGTT) was performed in group I; normal rats served as controls. Plasma insulin level was measured every other week (RIA). After 8 weeks, spleens were removed 1 day before sacrifice. In rats transplanted with cells the n-FBG was 100-150 mg/dl until the end of the study. After splenectomy, all cell recipients became diabetic (glucose 400 +/- 20 mg/dl). Transplanted rats showed increase in body weight and insulin production (3.3 +/- 1.0 ng/ml versus 0.92 +/- 0.3 ng/ml; p < 0.01) and had normal IPGTT. Spleens contained capsules with insulin-positive cells. Overall, data from this work indicate that intrasplenic transplantation of xenogeneic encapsulated insulin-producing cells without immunosuppression reversed diabetes in rats. Excellent survival and function of the transplanted cells was due to the fact that the cells were separated from the bloodstream by the immunoisolatory membrane only and insulin was delivered directly to the liver (i.e., in a physiological manner).

A novel molecular marker of pituitary tumor transforming gene involves in a rat liver regeneration.

BACKGROUND: Pituitary tumor transforming gene (PTTG), homologous to a mammalian securin, plays a pivotal role in cell transformation, however, its biological function(s) in normal tissues is not fully understood. Because the liver is a regenerative organ, the relevant biological function of PTTG in the liver would be more feasible to understand PTTG. Also, PTTG may be involved in the liver regeneration. MATERIALS AND METHODS: Expressions of rat hepatic PTTG messengerRNA (mRNA) and cellular immunoreactivities during the cell proliferative period of the liver regeneration both in vitro and in vivo were tested. RESULTS: PTTG expression of the rat primary hepatocyte was stimulated by HGF in a dose dependent manner, and was suppressed when hepatocyte proliferation was inhibited by transforming growth factor-beta1. A positive PTTG immunoreactive co-localizing with 5-bromo-2'-deoxyuridine (BrdU) in the hepatocyte nucleus was found and there was a concurrent sister chromatin itself by the immunofluorescent labeling of PTTG with cytokeratin 18 (CK18). DISCUSSION: Since the correlation of PTTG mRNA expression, cell proliferation and immunoreactivity were observed in primary rat cultured hepatocytes, PTTG may be a novel marker of cell proliferation both in vitro and in vivo liver regeneration.

Intrasplenic transplantation of encapsulated cells: a novel approach to cell therapy.

Cell therapy is likely to succeed clinically if cells survive at the transplantation site and are protected against immune rejection. We hypothesized that this could be achieved with intrasplenic transplantation of encapsulated cells because the cells would have instant access to oxygen and nutrients while being separated from the host immune system. In order to provide proof of the concept, primary rat hepatocytes and human hepatoblastoma-derived HepG2 cells were used as model cells. Rat hepatocytes were encapsulated in 100-kDa hollow fibers and cultured for up to 28 days. Rat spleens were implanted with hollow fibers that were either empty or contained I x 10(7) rat hepatocytes. Human HepG2 cells were encapsulated using alginate/ poly-L-lysine (ALP) and also transplanted into the spleen; control rats were transplanted with free HepG2 cells. Blood human albumin levels were measured using Western blotting and spleen sections were immunostained for albumin. Hepatocytes in monolayer cultures remained viable for only 6-10 days, whereas the cells cultured in hollow fibers remained viable and produced albumin throughout the study period. Allogeneic hepatocytes transplanted in hollow fibers remained viable for 4 weeks (end of study). Free HepG2 transplants lost viability and function after 7 days, whereas encapsulated HepG2 cells remained viable and secreted human albumin at all time points studied. ALP capsules, with or without xenogeneic HepG2 cells, produced no local fibrotic response. These data indicate that intrasplenic transplantation of encapsulated cells results in excellent survival and function of the transplanted cells and that the proposed technique has the potential to allow transplantation of allo- and xenogeneic cells (e.g., pancreatic islets) without immunosuppression.

Bone marrow-derived liver stem cell and mature hepatocyte engraftment in livers undergoing rejection.

BACKGROUND: The definitive therapy for end-stage liver disease is orthotopic liver transplantation (OLT). However, rejection is still a major cause of mortality and morbidity following OLT. Hepatocyte transplantation has been used experimentally to treat liver diseases. The aim of this study was to investigate whether bone marrow-derived liver stem cells (BDLSC) and mature hepatocytes could repopulate transplanted livers undergoing rejection. METHODS: OLT was carried out from D'Agouti (C3-positive female) into Lewis (C3-negative female) rats. BDLSC were transplanted from Lewis (male) into livers of D'Agouti (female) rats. Group A (n = 9) received intraportal normal saline. Groups B (n = 9) and C (n = 9) underwent intraportal transplantation of mature hepatocytes (Lewis female, 0.75 x 10(7)) and DBLSC (Lewis male, 5 x 10(4)) respectively. All groups received subtherapeutic immunosuppression (Cyclosporin 0.25 mg/kg/d) for 13 days. Liver repopulation was assessed using immunohistochemistry (C3 antigen-negative cells), in-situ hybridization, (Y-chromosome-positive BDLSC) and histologic assessment (hematoxylin and eosin) for rejection. RESULTS: BDLSC and mature hepatocytes repopulated 62 +/- 12.3% and 2.5 +/- 1.7% of rejecting livers, respectively. BDLSC demonstrated formation of hepatic lobules and portal triads with little evidence of rejection 36 days after discontinuation of immunosuppression. CONCLUSIONS: BDLSC can repopulate livers undergoing severe rejection. Moreover, BDLSC can differentiate into hepatocytes and cholangiocytes. This finding may have important clinical implications.

Glycerol suppresses proliferation of rat hepatocytes and human HepG2 cells.

BACKGROUND: In fulminant hepatic failure (FHF), the ability of surviving hepatocytes to proliferate is diminished. Therefore, it is important that medical therapy cause no further impairment of liver regeneration. In FHF, intracranial hypertension secondary to brain edema is the most common cause of brain injury and death and glycerol is used in some countries to treat this complication. Glycerol has been long known to suppress the growth of various cell types. We therefore decided to examine the effect of glycerol on hepatocyte proliferation in vitro and in vivo in rats subjected to partial (2/3) hepatectomy. Additionally, we investigated the effect of glycerol on the proliferation of HepG2 cells. MATERIALS AND METHODS: Mitogen-induced primary rat hepatocytes were cultured in a hormonally defined Dulbecco's modified Eagle's medium containing increasing amounts of glycerol (0.5, 1.0, 2.0, 4.0%). HepG2 cells were cultured in minimal essential medium/10% FBS. After 2 days, HepG2 cells were exposed to glycerol (1.0-2.0-4.0%) and harvested after 48 h. Control dishes contained no glycerol. Cell proliferation was measured by the incorporation of [(3)H]thymidine and/or bromodeoxyuridine (BrdU). In vivo, Sprague-Dawley rats were subjected to standard partial 2/3 hepatectomy and assigned to intraportal administration of either 400 microl of glycerol or saline. Rats were killed after 1, 2, 3, 5, and 7 days. Liver weight/body weight ratio and BrdU uptake were measured. RESULTS: In all cultures tested, glycerol suppressed the growth of cells in a dose-dependent manner. In vivo, a single intraportal dose of glycerol slowed the liver regenerative response. CONCLUSIONS: This study demonstrated that glycerol has a potent growth-inhibitory effect on hepatocyte proliferation in vivo and in vitro. Remarkably, glycerol inhibited the proliferation of liver cancer cells as well. The results of this study have important clinical implications.

Immediate early genes and p21 regulation in liver of rats with acute hepatic failure.

It has been observed that liver regeneration in acute hepatic failure (AHF) is suppressed [Eguchi et al. Hepatology 1996;24(6):1452-9]. The molecular mechanism regulating this inhibition is not known. We previously reported that in AHF rats, hepatocyte proliferation was significantly impaired with elevation in serum IL-6, TGF-beta1, and HGF [Kamohara et al. Biochem Biophys Res Commun 2000;273(1):129-35]. Following either 70% partial hepatectomy (PH) or liver injury, quiescent mature hepatocytes are "primed" to re-enter the cell cycle. The process of "priming" appears to be triggered by extracellular cytokines (IL-6 and TNF-alpha) and is characterized by expression of immediate early genes. Under the stimulation of growth factors such as HGF, "primed" hepatocytes exit the G1 phase of the cell cycle. G1-associated cyclins and their inhibitors play a pivotal role in G1/S cell cycle transition. Here, we demonstrate that immediate early gene (i.e. c-myc, c-fos) expression and AP-1 activity are preserved in AHF rat livers despite absence of hepatocyte proliferation. In contrast, p21 mRNA and protein are both over-expressed in AHF livers compared to livers from rats undergoing PH; this elevation leads to inhibition in Cdk2 activity, resulting in G1 cell cycle arrest and inhibition of regeneration.