Meghnad Joshi

Fetal liver-derived mesenchymal stromal cells augment engraftment of transplanted hepatocytes

Background aims One important problem commonly encountered after hepatocyte transplantation is the low numbers of transplanted cells found in the graft. If hepatocyte transplantation is to be a viable therapeutic approach, significant liver parenchyma repopulation is required. Mesenchymal stromal cells (MSC) produce high levels of various growth factors, cytokines and metalloproteinases, and have immunomodulatory effects. We therefore hypothesized that co-transplantation of MSC with human fetal hepatocytes (hFH) could augment in vivo expansion after transplantation. We investigated the ability of human fetal liver MSC (hFLMSC) to augment expansion of phenotypically and functionally well-characterized hFH. Methods Two million hFH (passage 6) were either transplanted alone or together (1:1 ratio) with green fluorescence protein-expressing hFLMSC into the spleen of C57BL/6 nude mice with retrorsine-induced liver injury. Results After 4 weeks, engraftment of cells was detected by fluorescence in situ hybridization using a human-specific DNA probe. Significantly higher numbers of cells expressing human cytokeratin (CK)8, CK18, CK19, Cysteine-rich MNNG HOS Transforming gene (c-Met), alpha-fetoprotein (AFP), human nuclear antigen, mitochondrial antigen, hepatocyte-specific antigen and albumin (ALB) were present in the livers of recipient animals co-transplanted with hFLMSC compared with those without. Furthermore, expression of human hepatocyte nuclear factor (HNF)-4α and HNF-1β, and cytochrome P450 (CYP) 3A7 mRNA was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) in these animals. In addition, significantly increased amounts of human ALB were detected. Importantly, hFLMSC did not transdifferentiate into hepatocytes. Conclusions Our study reports the use of a novel strategy for enhanced liver repopulation and thereby advances this experimental procedure closer to clinical liver cell therapy.

Improved intestinal preservation using an intraluminal macromolecular solution: evidence from a rat model.

Background. Intestinal preservation injury consists of progressive submucosal edema, with fluid originating both from the lumen and the interstitium. Although vascular flushing aims to control electrolyte shifts in the tissue, the lumen is not addressed, and luminal water and electrolytes enter the tissue during ischemia. Because macromolecular solutions may retain water and electrolytes intraluminally, we investigated whether these solutions administered intraluminally may alleviate preservation injury. Methods. Sprague-Dawley rat intestines were perfused with University of Wisconsin solution. After excision of the intestines, we intraluminally introduced solutions containing polyethylene glycol 3350 with high (125 mEq) or low (65 mEq) sodium before cold preservation. Controls underwent only vascular flush. After 8, 14, or 20 hr of cold storage, the intestines were analyzed for extent of tissue injury, water retention, brush-border maltase, and tight junction proteins zonula occludens-1 and claudin-3. Results. Intraluminal composition changed over time, indicating sodium absorption and potassium secretion. After 8 and 14 hr of cold storage, intestines from the low-sodium group had the best morphology and least edema, followed by the controls. Maltase activity slightly decreased in all groups over time and was not affected by the intraluminal polyethylene glycol solutions. Various degrees of delocalization and degradation of zonula occludens-1 and claudin-3 were recorded within the tight junctions, with the most significant effects in intestines from the high-sodium group. Conclusions. Intraluminal macromolecular solutions may modulate the preservation injury in University of Wisconsin- perfused intestines. Low-sodium solutions administered immediately before preservation may improve preservation injury, but high-sodium solutions may be detrimental.

Intraluminal Polyethylene Glycol Stabilizes Tight Junctions and Improves Intestinal Preservation in the Rat

Rapidly progressing mucosal breakdown limits the intestinal preservation time below 10 h. Recent studies indicate that intraluminal solutions containing polyethylene glycol (PEG) alleviate preservation injury of intestines stored in UW‐Viaspan. We investigated whether a low‐sodium PEG solution is beneficial for intestines stored in histidine‐tryptophane‐ketoglutarate (HTK) preservation solution. Rat intestines used as control tissue (group 1) were perfused with HTK, groups 2 and 3 received either a customized PEG‐3350 (group 2) or an electrolyte solution (group 3) intraluminally before cold storage. Tissue injury, brush‐border maltase activity, zonula occludens‐1 (ZO‐1) and claudin‐3 expression in the tight junctions (TJ) were analyzed after 8, 14 and 20 h. We measured epithelial resistance and permeability (Ussing chamber) after 8 and 14 h. Group 2 had superior morphology while maltase activity was similar in all groups. TJ proteins rapidly decreased and decolocalized in groups 1 3; these negative events were delayed in group 2, where colocalization persisted for about 14 h. Intestines in group 2 had higher epithelial resistance and lower permeability than the other groups. These results suggest that a customized PEG solution intraluminally reduces the intestinal preservation injury by improving several major epithelial characteristics without negatively affecting the brush‐border enzymes or promoting edema.

Characterization and engraftment of long-term serum-free human fetal liver cell cultures

BACKGROUND AIMSnCultured human hepatocytes have extensive diagnostic and clinical applications. However, the setting-up of new in vitro culture techniques allowing the long-term survival and functional maintenance of adult human hepatocytes represents a formidable challenge. Fetal liver cells (FLC) are attractive candidate donor cells because of their high proliferative capacity.nnnMETHODSnUsing cell culture and molecular techniques, we studied the in vitro and in vivo characteristics of FLC grown long-term in serum-free conditions.nnnRESULTSnSerum-free FLC obtained from 6-10-week-old human fetal livers grew as multiple clusters in suspension and could be subcultured for at least six passages. These cells maintained stable hepatocyte phenotypes and gene expression patterns in culture for up to 6 months. When a cluster of these cells in various passages was placed on collagen-coated plates, they formed a monolayer and morphologically resembled hepatocytes. The cells expressed alpha -fetoprotein, cytokeratin (CK) 8, CK18 and CK19 and albumin (ALB). Hepatocyte nuclear factor 4alpha and 1beta and cytochrome P450 (CYP) 3A4 and CYP3A7 mRNA expression was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). Cells at different passages, when transplanted into nude mice with liver injury, engrafted successfully, as detected by in situ hybridization using a human-specific DNA probe. Colonies of human-specific CK8, CK18, c-Met nuclear antigen (Ag), mitochondrial Ag, hepatocyte-specific Ag and ALB-expressing cells were present in the livers of recipient animals.nnnCONCLUSIONSnPrimary human FLC can be kept in culture consistently over a long time period and are potential candidates for cell therapy and in vitro diagnostics.

Expression of major histocompatibility complex class I-related chain A/B (MICA/B) in pancreatic carcinoma

Major histocompatibility complex class I-related chain A and B (MICA/B) are two stress-inducible ligands that bind to the immunoreceptor NKG2D and play an important role in mediating cytotoxicity of NK and T cells. Release of MIC molecules from the cell surface is thought to constitute an immune escape mechanism of tumor cells and thus could be associated with more aggressive course of tumor growth. In this study, we investigated the expression of MICA/B in ductal pancreatic carcinoma and serum in relation to tumor stage, differentiation and survival. MICA/B expression in tumor tissues and sera from patients with pancreatic cancer were analyzed by immunohistochemical staining (IHC), western blotting and ELISA, respectively. MICA/B expression was present in 17 of 22 (77%) of the tumors but not in normal pancreatic ductal epithelial cells. Poorly differentiated tumors showed more pronounced MICA/B expression compared to differentiated tumors, but did not correlate significantly to other tumor characteristics. MICA/B-negative tumors displayed significantly lower incidence of lymph node metastases (p<0.01), and less mortality within 3 years following resection (p<0.02). In conclusion, tissue levels of MICA/B expression were elevated in pancreatic cancer cells without elevated levels in serum, despite well-recognized acute phase reactants in serum. Poorly differentiated tumors showed high MICA/B expression, which was related to extended tumor lymph node metastases and less frequent long-term survival.

TEMPORARY REMOVAL: CD271 identifies functional human hepatic stellate cells, which localize in peri-sinusoidal and portal areas in liver after partial hepatectomy

BACKGROUND AIMSnHepatic stellate cells (HSCs) are liver-resident mesenchymal cells involved in essential processes in the liver. However, knowledge concerning these cells in human livers is limited because of the lack of a simple isolation method.nnnMETHODSnWe isolated fetal and adult human liver cells by immunomagnetic beads coated with antibodies to a mesenchymal stromal cell marker (CD271) to enrich a population of HSCs. The cells were characterized by cell cultivation, immunocytochemistry, flow cytometry, reverse-transcription polymerase chain reaction and immunohistochemistry. Cells were injected into nude mice after partial hepatectomy to study in vivo localization of the cells.nnnRESULTSnIn vitro, CD271(+) cells were lipid-containing cells expressing several HSC markers: the glial fibrillary acidic protein, desmin, vimentin and α-smooth muscle actin but negative for CK8, albumin and hepatocyte antigen. The cells produced several inflammatory cytokines such as interleukin (IL)-6, IL-1A, IL-1B and IL-8 and matrix metalloproteinases MMP-1 and MMP-3 and inhibitors TIMP-1 and TIMP-2. In vivo, fetal CD271(+) cells were found in the peri-sinusoidal space and around portal vessels, whereas adult CD271(+) cells were found mainly in the portal connective tissue and in the walls of the portal vessels, which co-localized withxa0α-smooth muscle actin or desmin. CD271(-) cells did not show this pattern of distribution in the liver parenchyma.nnnCONCLUSIONSnThe described protocol establishes a method for isolation of mesenchymal cell precursors for hepatic stellate cells, portal fibroblasts and vascular smooth muscle cells. These cells provide a novel culture system to study human hepatic fibrogenesis, gene expression and transcription factors controlling HSC regulation.

Phenotypic and in vivo functional characterization of immortalized human fetal liver cells

Abstract We report the establishment and characterization of immortalized human fetal liver progenitor cells by expression of the Simian virus 40 large T (SV40 LT) antigen. Well-characterized cells at various passages were transplanted into nude mice with acute liver injury and tested for functional capacity. The SV40LT antigen-immortalized fetal liver cells showed a morphology similar to primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers such as albumin, CK 8, CK18, transcription factors HNF-4α and HNF-1α and CYP3A/7. The cells did not stain for any of the tested cancer-associated markers. Albumin, HNF-4α and CYP3A7 expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry showed expression of some progenitor cell markers. In vivo study showed that the cells expressed both fetal and differentiated hepatocytes markers. Our study suggests new approaches to expand hepatic progenitor cells, analyze their fate in animal models aiming at cell therapy of hepatic diseases.

Chemokine-Mediated Robust Augmentation of Liver Engraftment: A Novel Approach

Effective repopulation of the liver is essential for successful clinical hepatocyte transplantation. The objective was to improve repopulation of the liver with human hepatocytes using chemokines. We used flow cytometry and immunohistochemistry assays to identify commonly expressed chemokine receptors on human fetal and adult hepatocytes. The migratory capacity of the cells to various chemokines was tested. For in vivo studies, we used a nude mouse model of partial hepatectomy followed by intraparenchymal injections of chemokine ligands at various concentrations. Human fetal liver cells transformed with human telomerase reverse transcriptase were used for intrasplenic cell transplantation. Repopulation and functionality were assessed 4 weeks after transplantation. The receptor CXCR3 was commonly expressed on both fetal and adult hepatocytes. Both cell types migrated efficiently toward corresponding CXC chemokine ligands 9, 10, and 11. In vivo, animals injected with recombinant chemokines showed the highest cell engraftment compared with controls (p < .05). The engrafted cells expressed several human hepatic markers such as cytokeratin 8 and 18 and albumin as well as transferrin, UGT1A1, hepatocyte nuclear factor (1α, 1β, and 4α), cytochrome CYP3A1, CCAAT/enhancer binding protein (α and β), and human albumin compared with controls. No inflammatory cells were detected in the livers at 4 weeks after transplantation. The improved repopulation of transplanted cells is likely a function of the chemokines to mediate cell homing and retention in the injured liver and might be an attractive strategy to augment repopulation of transplanted hepatocytes in vivo.

Natural killer group 2 member D cell recruitment driven by major histocompatibility complex class I chain-related antigens A and B: a possible mechanism during acute intestinal allograft rejection in the mouse.

Intestinal allograft rejection occurs frequently despite potent T-cell depletion protocols. We investigated the interaction of major histocompatibility complex class I chain-related antigens A and B (MICA/B; a ligand for natural killer [NK] cells) and NK group 2 member D (NKG2D) cells as an alternative mechanism for acute rejection (AR) of the intestinal graft. Heterotopic intestinal allotransplantation was performed from BalbC to C57Bl mice. Samples of grafted and native intestine were obtained at days 1, 3, 6, and 8 after transplantation (n = 4-6). We performed immunostaining for MICA/B and NKG2D. Moderate AR with increased crypt apoptosis was observed at day 6 and advanced AR with crypt destruction and mucosal sloughing was present by day 8. Low MICA/B levels were observed in grafted and native intestines on day 1. MICA/B expression gradually increased in the grafts during AR but not in the native intestines. The up-regulation was found mostly in the crypts. NKG2D+ cell counts that increased in the graft colocalized with MICA/B. The increase was most prominent in the crypt and villus. Together, these results suggest that MICA/B up-regulation and its subsequent interaction with the NK cells may represent an important link between innate and adaptive immune responses early after intestinal transplantation.

Fetal Liver Cell Transplantation

Fetal liver cell transplantation is a promising technique and is entering its clinical application phase. Temporary support of liver function is clearly obtained, and short-term benefit can be achieved for patients. The challenge is to obtain long-term efficacy and demonstrate engraftment and repopulation of the recipient liver. Numerous animal studies have been performed; however, there are many challenging issues that remain to be solved, including increasing engraftment and repopulation and in vitro cell expansion. This knowledge can be used to improve the function of hepatocyte-like cells for drug testing, bioartificial livers, and transplantation.