Humberto E. Soriano

Induction of three-dimensional assembly of human liver cells by simulated microgravity

SummaryThe establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.

Cultures of human liver cells in simulated microgravity environment

We used microgravity-simulated bioreactors that create the unique environment of low shear force and high-mass transfer to establish long-term cultures of primary human liver cells (HLC). To assess the feasibility of establishing HLC cultures, human liver cells obtained either from cells dissociated by collagenase perfusion or minced tissues were cultured in rotating vessels. Formation of multidimensional tissue-like spheroids (up to 1.0 cm) comprised of hepatocytes and biliary epithelial cells that arranged as bile duct-like structures along newly formed vascular sprouts were observed. Electron microscopy revealed clusters of round hepatocytes and bile canaliculi with multiple microvilli and tight junctions. Scanning EM revealed rounded hepatocytes that were organized in tight clusters surrounded by a complex mesh of extracellular matrix. Also, we observed that co-culture of hepatocytes with endothelial cells stimulate albumin mRNA expression. In summary, a simulated microgravity environment is conducive for the establishment of long-term HLC cultures and allows the dissection of the mechanism of liver regeneration and cell-to-cell interactions that resembles in vivo conditions.

Development of a clinical protocol for hepatic gene transfer: Lessons learned in preclinical studies

ABSTRACT: Strategies for hepatic gene therapy have been proposed that involve isolation of primary hepatocytes and introduction of recombinant genes into these cells in culture, followed by autologous hepatocellular transplantation (HCT). Consideration of clinical applications requires data suggesting that HCT can be performed safely in human subjects in addition to data indicating that recombinant gene expression can reverse a disease process. This report describes preclinical studies that underlie a clinical trial of HCT in which hepatocytes would be labeled with a marker gene to facilitate assessment of engraftment in the recipient. Human hepatocytes were harvested from liver segments preserved in Belzars solution and transduced with an amphotropic retroviral vector carrying a recombinant marker gene (neomycin phosphotransferase II). Human hepatocytes were recovered from monolayer culture, stained with the fluorescent dye 1,1′-dioctadecyl-3,3,3,3′-tetra-methylindo-carbocyanine perchlorate (DiI) and transplanted into severe combined immunodeficient mice by splenic injection. Engrafted hepatocytes were identified in the liver and spleen of severe combined immunodeficient mice but not immunocompetent controls. Two large animal models of HCT are described. In a dog model, neomycin phosphotransferase II-containing hepatocytes were identified in the liver 7 wk after transplantation. In a baboon model, autologous HCT with DiI-stained cells demonstrated that transplanted cells assume a normal morphology and constitute up to 5% of hepatocytes. These data demonstrate transduction and transplantation of human hepatocytes and the feasibility of HCT in large animals. On the basis of these studies, the proposed clinical trial for gene transfer and transplantation in human subjects has been approved by the National Institutes of Health and the Food and Drug Administration. These studies illustrate the nature and extent of preclinical data required to gain approval for clinical trials involving gene transfer into human subjects. These data also illustrate the limitations of existing methods that may be used for hepatic gene therapy in the future.

DNA binding by C/EBP proteins correlates with hepatocyte proliferation

SummaryThe leucine zipper transcription factors C/EBPα and C/EBPβ exhibit growth-related variations of expression and DNA binding during liver regeneration. We examined the expression of C/EBP proteins in relation to hepatocyte proliferation by studying their DNA-binding activity in primary mouse hepatocytesin vitro. Mouse hepatocytes were dissociated by collagenase perfusion and cultured in a serum-free, defined medium containing a variety of growth factors and hormones. Cell protein extracts were collected every 24 h for up to 10 d and examined for DNA-binding activity by gel retardation analysis using a C/EBP consensus sequence oligomer (bZIP). C/EBPα is the major bZIP-binding protein present in the dissociated cells prior to plating. With the culture conditions we employed, little or no binding of C/EBP proteins was observed in the first 24 to 48 h of cultivation. After 48 h, C/EBPβ binding activity was elevated relative to the level seen in freshly dissociated cells. In contrast, C/EBPα binding continued to be greatly reduced and no C/EBPδ binding was observed. C/EBPβ binding remained elevated for the duration of the experiment. Additional growth factor treatment (EGF, FGF, TGFα, and HGF) of the hepatocytes did not appreciably alter the pattern of C/EBP binding. However, TGFβ treatment, known to decrease hepatocyte proliferation, increased C/EBPβ binding activity earlier and more actively than in control cells. This study confirms a negative correlation between DNA binding by the C/EBP transactivator proteins and the proliferation of primary mouse hepatocytesin vitro.

Feasibility of hepatocellular transplantation via the umbilical vein in prenatal and perinatal lambs

Hepatocellular transplantation has previously been performed in experimental animals by infusion of hepatocyte suspensions into the spleen or portal venous system. Cells injected into these sites flow to the liver and engraft within the hepatic parenchyma. We designed this study to evaluate the feasibility of hepatocellular transplantation through the umbilical vein in the prenatal or perinatal periods. Allogeneic sheep hepatocytes were harvested, stained with the vital fluorescent dye DiI, and injected into the umbilical vein of fetal lambs at 85% gestation and term. Hemodynamic studies performed to assess the physiological impact of transplantation on the recipient animal demonstrated that the procedure was well tolerated. No significant short-term complications were encountered and no lesions were found by conventional histological examination at necropsy 1-17 days after transplantation. Engrafted cells were identified within the liver by fluorescent microscopy and flow cytometry in 4/7 animals constituting 1.2-5% of the hepatocyte population. Fluorescent cellular material with the morphology of hepatocytes, noncellular material, and fluorescent phagocytic cells were seen occasionally in other organs including lung, brain, adrenal, and placenta. These studies demonstrate the feasibility of performing hepatocellular transplantation in the fetus via the umbilical vein in experimental animals.

Risks of endoscopy in hospitalized pediatric patients with collagen vascular diseases

BACKGROUND The gastrointestinal manifestations of the collagen vascular diseases have been well described in the pediatric population. These patients frequently have symptoms that constitute indications for endoscopy. However, the risks and benefits of endoscopy in this population have not been examined. METHODS A retrospective review of all patients with collagen vascular diseases hospitalized during a 7-year period was undertaken, and those patients who underwent endoscopy were identified. RESULTS Nine patients (5%) underwent endoscopic procedures (eight upper and three lower endoscopy). Complications and outcomes were analyzed. Indications for endoscopy included abdominal pain, gastrointestinal (GI) bleeding, and/or vomiting and diarrhea. Two patients had complications that required surgery within 1 day of the endoscopic procedure. One of these patients subsequently died with GI bleeding. Five of the nine patients had changes in their management after endoscopy. Helicobacter pylori infection was identified and treated in two patients. Three patients had esophagitis or gastritis and acid suppression treatment was started or optimized. Vasculopathy was present in the patients who had complications. CONCLUSIONS This series suggests that endoscopy can provide useful information for the management of the pediatric patient with GI symptoms and collagen vascular diseases. However, because serious and potentially life-threatening complications can occur, great care is needed in evaluating the risk/benefit ratio of endoscopy in these patients.

Gene knockout animal models

Mouse lines carrying specific mutations in their genomes can be obtained using targeted recombination in murine embryonic stem (ES) cells. This is a powerful method for studying the significance of gene products both in vivo, in the whole animal, and in vitro, at the cellular level using cultures derived from the knockout mice. In addition, ES cells can be made that are homozygous for a particular mutation. Because ES cells can differentiate into various cell types in vitro, a variety of developmental and differentiated genes can be examined in mutant ES cell lines. Both loss-of-function (ablation of gene function) and gain-of-function (acquisition of a new activity conferred by mutation) are possible using this technique of gene targeting by homologous recombination in ES cells.

HEPATOCELLULAR TRANSPLANTATION (HCT) VIA PORTAL VEIN CATHETER IN A PATIENT WITH FULMINANT LIVER FAILURE. † 746

HEPATOCELLULAR TRANSPLANTATION (HCT) VIA PORTAL VEIN CATHETER IN A PATIENT WITH FULMINANT LIVER FAILURE. † 746

BENEFITS AND RISKS OF ENDOSCOPY IN PEDIATRIC PATIENTS WITH CHRONIC VASCULITIDES. 757

The gastrointestinal manifestations of collagen vascular diseases, such as systemic lupus erythematosus and polymyositis, have been well described in the pediatric population. These patients frequently have symptoms which are indications for endoscopic studies. However, the risks and benefits of gastrointestinal endoscopy in this population have not been examined. A complete review for all rheumatologic patients admitted to Texas Childrens Hospital during the past five years was performed and those patients undergoing endoscopy were identified. Ten patients (5%) underwent endoscopic procedures (nine upper and three lower endoscopies) which were analyzed with emphasis on resultant complications and therapeutic benefit. Indications for endoscopy included pain in six of ten patients, bleeding in three of ten patients and vomiting and diarrhea in one patient. Three patients had severe complications, two of which required surgery within one day of the endoscopic procedure. One of these patients subsequently died with GI bleeding. Five of the ten patients had changes in management following their endoscopy. Two patients had Helicobacter pylori and treatment was initiated. Three patients had esophagitis or gastritis and acid suppression treatment was started or optimized. Vasculopathy was present in the patients who had complications and may have contributed to their development. This study provides evidence that endoscopy can provide useful information for the management of the pediatric patient with GI symptoms and rheumatologic disease. However, since serious and potentially life-threatening complications occur, great care is needed in evaluating risk/benefit for endoscopic procedures in these high risk patients.