Neonatal Liver Cells Research Articles

(176) - Neonatal Tolerance to Heart Transplants Induced with Neonatal Allogeneic Liver Cells: Trafficking, Interactions and Fates of Donor Cell Types

(176) - Neonatal Tolerance to Heart Transplants Induced with Neonatal Allogeneic Liver Cells: Trafficking, Interactions and Fates of Donor Cell Types

Multisite Injection of Bioengineered Hepatic Units from Collagen Hydrogel and Neonatal Liver Cells in Parenchyma Improves Liver Cirrhosis.

Intrahepatic transplantation of hepatocytes/progenitor cells remodels or repopulates livers resulting in improved hepatic function, which represents a promising treatment option for liver diseases, but poor engraftment of transplanted liver cells in livers has been the major obstacle for exerting therapeutic efficacy on liver diseases. We herein demonstrated great potential of multisite injection of bioengineered hepatic units generating from collagen hydrogel and neonatal liver cells (MSI) in promoting engraftment, survival of liver cells, and the therapeutic potential of liver diseases in livers. The MSI allowed transplantation of large number of liver cells into parenchyma in one time. Superiorly, the MSI promoted the engraftment and survival of liver cells, and generated large, dispersively distributed tissue masses whose sizes were positively correlated with the number of injection sites in livers. Consistently, the MSI improved liver cirrhosis as evidenced by the reduction of collagen matrix deposit, improved liver architecture, and hepatic function in the MSI group compared to that of the nontreated control. These data indicate that the MSI represents a novel, improved bioengineered strategy that can exert therapeutic efficacy on liver diseases.

Liver Scaffolds Support Survival and Metabolic Function of Multilineage Neonatal Allogenic Cells.

Organ scaffold bioengineering is currently limited by the inability to effectively repopulate the scaffold with appropriately distributed functional cells. We examined the feasibility of a decellularized liver scaffold to support the growth and function of multilineage allogenic cells derived from either adult or neonatal liver cells. Cell slurries from neonatal and adult rat livers containing hepatocytes, cholangiocytes, and endothelial cells were introduced into decellularized adult rat liver scaffolds via the bile duct. Recellularized grafts were perfused with cell growth medium through the portal vein for 7 days. Concurrently, the same cell slurries were incubated on culture dishes. Albumin levels were measured from graft perfusates and cell culture media. Immunofluorescent assays were used to verify the colocalization of cholangiocytes, hepatocytes, endothelial cells, and Kupffer cells in the recellularized grafts by using anti-CK7, anti-hepatocyte antigen, anti-CD34, and anti-CD68, respectively. More robust albumin production was detected in the perfusate of scaffolds recellularized with a neonatal liver cell slurry compared with those with an adult liver cell slurry. The perfusates from all recellularized grafts showed increasing albumin concentration over 7 days; higher levels were detected in the constructs compared with the cell culture. Scaffolds seeded with a neonatal liver cell slurry showed the presence of hepatocytes, cholangiocytes, endothelial cells, and Kupffer cells. Results demonstrated the superiority of neonatal allogenic cells over adult cells of the same origin, possibly because of their pluripotent behavior. Liver bio-scaffolds supported the growth of four different liver cell lines. Recellularized grafts exhibited preserved functionality as demonstrated by albumin production, and constructs seeded with a neonatal cell slurry demonstrated proliferation on Ki-67 assay, thus representing a promising model for a transplantable construct.

Biological Liver Scaffolds Promote Engraftment and Functionality of Multilineage Allogenic Neonatal Liver Cells

Biological Liver Scaffolds Promote Engraftment and Functionality of Multilineage Allogenic Neonatal Liver Cells

Recellularization via the bile duct supports functional allogenic and xenogenic cell growth on a decellularized rat liver scaffold

ABSTRACTRecent years have seen a proliferation of methods leading to successful organ decellularization. In this experiment we examine the feasibility of a decellularized liver construct to support growth of functional multilineage cells. Bio-chamber systems were used to perfuse adult rat livers with 0.1% SDS for 24 hours yielding decellularized liver scaffolds. Initially, we recellularized liver scaffolds using a human tumor cell line (HepG2, introduced via the bile duct). Subsequent studies were performed using either human tumor cells co-cultured with human umbilical vein endothelial cells (HUVECs, introduced via the portal vein) or rat neonatal cell slurry (introduced via the bile duct). Bio-chambers were used to circulate oxygenated growth medium via the portal vein at 37C for 5-7 days. Human HepG2 cells grew readily on the scaffold (n = 20). HepG2 cells co-cultured with HUVECs demonstrated viable human endothelial lining with concurrent hepatocyte growth (n = 10). In the series of neonatal cell slurry infusion (n = 10), distinct foci of neonatal hepatocytes were observed to repopulate the parenchyma of the scaffold. The presence of cholangiocytes was verified by CK-7 positivity. Quantitative albumin measurement from the grafts showed increasing albumin levels after seven days of perfusion. Graft albumin production was higher than that observed in traditional cell culture. This data shows that rat liver scaffolds support human cell ingrowth. The scaffold likewise supported the engraftment and survival of neonatal rat liver cell slurry. Recellularization of liver scaffolds thus presents a promising model for functional liver engineering.

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PC-FACS

Successful xenotransplantation with re-aggregated and encapsulated neonatal pig liver cells for treatment of mice with acute liver failure.

Hepatocyte transplantation is a promising therapy for acute liver failure. Cell therapy using xenogeneic sources has emerged as an alternative treatment for patients with organ failure due to the shortage of transplantable human organs. The purpose of this study was to improve the survival of mice with acute liver failure by transplanting encapsulated neonatal pig re-aggregated liver cells (NPRLC). Liver injury was induced in C57/BL6 male mice by the injection of 600 mg/kg of acetaminophen. Xenogeneic liver cells were isolated from a neonatal pig and processed via re-aggregation and encapsulation to improve the efficiency of the xenogeneic liver cell transplantation. The neonatal pig liver showed abnormal lobule structure. Isolated cells were re-aggregated and intraperitoneally transplanted into acute liver failure mice models. Re-aggregated cells showed significantly enhanced viability and significantly greater synthesis of albumin and urea than cells cultured in monolayers. Further, we observed improved serum levels of ALT/AST, and the survival rate of mice with acute liver failure was improved by the intraperitoneal transplantation of encapsulated hepatocytes (48,000 equivalent (Eq) per mouse). This study shows that using encapsulated NPRLCs improves the efficacy of xenogeneic liver cell transplantation for the treatment of mice with acute liver failure. Therefore, this may be a good strategy for bridge therapy for the treatment of acute liver failure in humans.

Three-dimensional culture in a microgravity bioreactor improves the engraftment efficiency of hepatic tissue constructs in mice.

Tissue-engineered liver using primary hepatocytes has been considered a valuable new therapeutic modality as an alternative to whole organ liver transplantation for different liver diseases. The development of clinically feasible liver tissue engineering approaches, however, has been hampered by the poor engraftment efficiency of hepatocytes. We developed a three-dimensional (3D) culture system using a microgravity bioreactor (MB), biodegradable scaffolds and growth-factor-reduced Matrigel to construct a tissue-engineered liver for transplantation into the peritoneal cavity of non-obese diabetic severe combined immunodeficient mice. The number of viable cells in the hepatic tissue constructs was stably maintained in the 3D MB culture system. Hematoxylin-eosin staining and zonula occludens-1 expression revealed that neonatal mouse liver cells were reorganized to form tissue-like structures during MB culture. Significantly upregulated hepatic functions (albumin secretion, urea production and cytochrome P450 activity) were observed in the MB culture group. Post-transplantation analysis indicated that the engraftment efficiency of the hepatic tissue constructs prepared in MB cultures was higher than that of those prepared in the static cultures. Higher level of hepatic function in the implants was confirmed by the expression of albumin. These findings suggest that 3D MB culture systems may offer an improved method for creating tissue-engineered liver because of the higher engraftment efficiency and the reduction of the initial cell function loss.

Neonatal liver cell donation: a case report.

Traditional organ transplant options for newborns have been rare. There continues to be an increasing need for organs for transplant and a limited number of available organs, especially for small children. Liver cell transplantation is a promising alternative to orthotopic liver transplantation to treat liver-based inborn errors of metabolism.1 The procedure is minimally invasive and can be performed repeatedly. The safety of the procedure has been well established, and the clinical results are encouraging.1 The liver cell donation process is an option for families who experience the loss of a newborn and offers them a legacy for their child by providing life for others. The purpose of this article is to discuss the neonatal liver cell donation process and present a case report of an anencephalic infant whose parents chose to participate in this unique program.

Generation of Functional Insulin-Producing Cells from Neonatal Porcine Liver-Derived Cells by PDX1/VP16, BETA2/NeuroD and MafA

Surrogate β-cells derived from stem cells are needed to cure type 1 diabetes, and neonatal liver cells may be an attractive alternative to stem cells for the generation of β-cells. In this study, we attempted to generate insulin-producing cells from neonatal porcine liver-derived cells using adenoviruses carrying three genes: pancreatic and duodenal homeobox factor1 (PDX1)/VP16, BETA2/NeuroD and v-maf musculo aponeurotic fibrosarcoma oncogene homolog A (MafA), which are all known to play critical roles in pancreatic development. Isolated neonatal porcine liver-derived cells were sequentially transduced with triple adenoviruses and grown in induction medium containing a high concentration of glucose, epidermal growth factors, nicotinamide and a low concentration of serum following the induction of aggregation for further maturation. We noted that the cells displayed a number of molecular characteristics of pancreatic β-cells, including expressing several transcription factors necessary for β-cell development and function. In addition, these cells synthesized and physiologically secreted insulin. Transplanting these differentiated cells into streptozotocin-induced immunodeficient diabetic mice led to the reversal of hyperglycemia, and more than 18% of the cells in the grafts expressed insulin at 6 weeks after transplantation. These data suggested that neonatal porcine liver-derived cells can be differentiated into functional insulin-producing cells under the culture conditions presented in this report and indicated that neonatal porcine liver-derived cells (NPLCs) might be useful as a potential source of cells for β-cell replacement therapy in efforts to cure type I diabetes.

Protective Effect of Transplantation of Neonatal Liver Cell Nuclei on the Model of Acute Toxic Hepatitis

We present the results of comparative analysis of functional and morphological changes in the liver of animals with experimental CCl(4)-induced hepatitis under conditions of transplantation of neonatal liver cells and nuclei. It was found that transplantation of neonatal liver cell nuclei in acute toxic hepatitis provides better functional and structural state of the target organ.

Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor

Reconstitution of tissue architecture in vitro is important because it enables researchers to investigate the interactions and mutual relationships between cells and cellular signals involved in the three-dimensional (3D) construction of tissues. To date, in vitro methods for producing tissues with highly ordered structure and high levels of function have met with limited success although a variety of 3D culture systems have been investigated. In this study, we reconstituted functional hepatic tissue including mature hepatocyte and blood vessel-like structures accompanied with bile duct-like structures from E15.5 fetal liver cells, which contained more hepatic stem/progenitor cells comparing with neonatal liver cells. The culture was performed in a simulated microgravity environment produced by a rotating wall vessel (RWV) bioreactor. The hepatocytes in the reconstituted 3D tissue were found to be capable of producing albumin and storing glycogen. Additionally, bile canaliculi between hepatocytes, characteristics of adult hepatocyte in vivo were also formed. Apart from this, bile duct structure secreting mucin was shown to form complicated tubular branches. Furthermore, gene expression analysis by semi-quantitative RT-PCR revealed the elevated levels of mature hepatocyte markers as well as genes with the hepatic function. With RWV culture system, we could produce functionally reconstituted liver tissue and this might be useful in pharmaceutical industry including drug screening and testing and other applications such as an alternative approach to experimental animals.

Abstract 4246: Granulin-epithelin precursor (GEP) is an oncofetal protein involved in fetal liver development and liver cancer

Abstract Background and objectives: Granulin-epithelin precursor (GEP) is a novel pluripotent growth factor regulating embryo development, wound repair and tumorigenesis. Our group previously has showed that GEP was over-expressed in over 70% of liver cancer. Besides, functional studies demonstrated that GEP controlled liver cancer proliferation, invasion and tumorigenicity. In this study, we aim to examine the time-frame of GEP expression during fetal liver development, and to characterize the properties of GEP-expressing liver cells. Methods: GEP expression was examined by immunofluorescence using flow cytometry and immunohistochemistry. The properties of GEP-expressing cells were characterized by measuring the co-expression of GEP and various stem/progenitor cell markers using immunofluorescence and flow cytometry. Correlation of GEP level with the expression of stem/progenitor markers was studied by transfection experiments. Results: In murine model, GEP expression was observed to increase gradually from embryonic day (E) 11 to E17, with up to 49% fetal liver cells were GEP positive. Immediately after birth, GEP expression decreased abruptly from day 1, and by day 7, less than 1% neonatal liver cells were GEP positive, and GEP was not detectable in adult liver. In human, fetal liver at 10 weeks old were demonstrated to express GEP protein, while normal adult livers were devoid of GEP-expressing cells. GEP expression was up-regulated in liver cancer cells compared with the adjacent non-tumor liver cells. Moreover, we showed that GEP co-expressed with hematopoietic stem/progenitor cell marker CD34, liver cancer stem cell marker CD326, and stem cell-related signaling molecules β-catenin and Oct4, suggesting a stem/progenitor cell feature of the GEP-expressing cells. Furthermore, up-regulation of GEP level enhanced the expression of β-catenin and Oct4, and suppression of GEP level resulted in decreased expression of these molecules. Conclusion: The present data suggests that GEP is an oncofetal protein, and GEP regulates the expression of stem cell-related signaling molecules. This information advances our knowledge on the fundamental role of GEP in fetal liver development and liver carcinogenesis, and provides valuable insights on the use of GEP as a therapeutic target in the treatment of liver cancer. Category: Tumor Biology Sub-classification: TB2: stem cell biology/cancer stem cells/developmental oncobiology Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4246.

In-Vitro Reconstitution of Hepatic Tissue Architectures with Neonatal Mouse Liver Cells Using Three-Dimensional Culture

Stem cells have the potential to differentiate into multiple lineages, and the capabilities to self-renew, and reconstitute tissues following transplantation. Thus, stem cells are expected to be useful for regenerative medicine; however, the mechanisms that regulate the reconstitution of three-dimensional (3-D) tissues remain to be elucidated. To study such mechanisms, we have established a novel procedure of 3-D culture that supports the formation of tissues from isolated cells, including hepatic stem/progenitor cells in vitro. We cultured neonatal mouse liver cell populations, including hepatic stem/progenitor cells, in a simulated microgravity environment produced by a Rotating Wall Vessel bioreactor. After 8 days in culture, we obtained a 3-D tissue architecture. Histological analysis showed that bile duct structures secreting mucin formed complicated tubular branches in the peripheral region. In the non-bile duct structure region, we observed mature hepatocytes that were capable of producing albumin and storing glycogen. Thus, we were able to establish a novel 3-D culture system that is able to reconstitute functional hepatic tissue architecture from isolated neonatal mouse liver cells.

Xenotransplantation of neonatal porcine liver cells

Xenotransplantation of porcine liver cell types may provide a means of overcoming the shortage of suitable donor tissues to treat hepatic diseases characterized by inherited inborn errors of metabolism or protein production. Here we report the successful isolation, culture, and xenotransplantation of liver cells harvested from 7- to 10-day-old piglets. Liver cells were isolated and cultured immediately after harvesting. Cell viability was excellent (>90%) over the duration of the in vitro studies (3 weeks) and the cultured cells continued to significantly proliferate. These cells also retained their normal secretory and metabolic capabilities as determined by continued release of albumin, factor 8, and indocyanin green (ICG) uptake. After 3 weeks in culture, porcine liver cells were loaded into immunoisolatory macro devices (Theracyte devices) and placed into the intraperitoneal cavity of immunocompetant CD1 mice. Eight weeks later, the devices were retrieved and the cells analyzed for posttransplant determinations of survival and function. Post mortem analysis confirmed that the cell-loaded devices were biocompatible, and were well-tolerated without inducing any notable inflammatory reaction in the tissues immediately surrounding the encapsulated cells. Finally, the encapsulated liver cells remained viable and functional as determined by histologic analyses and ICG uptake/release. The successful harvesting, culturing, and xenotransplantation of functional neonatal pig liver cells support the continued development of this approach for treating a range of currently undertreated or intractable hepatic diseases.

Epithelial-mesenchymal transition of cultured rat neonatal hepatocytes is differentially regulated in response to epidermal growth factor and dimethyl sulfoxide.

Neonatal rat hepatocytes cultured in the absence of added growth factors dedifferentiate by epithelial-mesenchymal transition (EMT). This involves the loss of their typical differentiation markers, the acquisition of a migrating morphology, and a change in the expression of the intermediate filament (IF) proteins. We attempted to determine whether the EMT of cultured neonatal rat hepatocytes could be modulated by factors that maintain and promote the differentiation state of adult and fetal hepatocytes such as epidermal growth factor (EGF) and dimethyl sulfoxide (DMSO). By (3H)-thymidine incorporation, Western blotting analysis, flow cytometry, and double-immunofluorescence studies, we found that both factors have marked but opposite effects on the EMT and on proliferation of neonatal liver cells. In DMSO treatment, albumin levels were higher than in the nontreated cells at all days studied. Moreover, DMSO reduced cytokeratin levels and inhibited cell proliferation, acquisition of the fibroblast-like morphology, and vimentin expression typical of the EMT. The increase in vimentin-positive cells in serum-free medium was not observed in DMSO cultures. EGF also increased albumin levels at all days studied. In contrast, EGF treatment induced hepatocyte proliferation and enhanced vimentin and cytokeratin expression. However, the increase in vimentin levels did not correlate with a significant increase in the number of vimentin-positive cells. Moreover, vimentin-positive cells in EGF treatment were also cytokeratin-positive and albumin-positive, and they maintained epithelioid morphology in spite of the vimentin network. These results indicate that EMT of cultured rat neonatal hepatocytes is differentially regulated in response to EGF and DMSO.

Vimentin Filaments Follow the Preexisting Cytokeratin Network during Epithelial–Mesenchymal Transition of Cultured Neonatal Rat Hepatocytes

Changes in cell cytoskeleton are known to play an important role in differentiation and embryogenesis and also in carcinogenesis. Previous studies indicated that neonatal hepatocytes undergo an epithelial–mesenchymal transition when cultured in a serum-free medium for several days. Here we show by Western blotting of neonatal rat liver cells cultured for 3 days that vimentin and cytokeratin were expressed by these cells. Epidermal growth factor treatment induced high coexpression of vimentin and cytokeratin filaments in hepatocytes from neonatal livers, as detected by double immunofluorescence microscopy. Confocal scanning laser microscopy was used to determine the spatial and cell distribution of cytokeratin and vimentin intermediate filament networks. Vimentin-expressing hepatocytes were mainly located on the periphery of epithelial clusters and presented a migratory morphology, suggesting that vimentin expression was related to the loss of cell–cell contact. Short vimentin filaments were mainly located at the cytoplasmic sites behind the extending lamella. Horizontal and vertical dual imaging of double immunofluorescence with anti-vimentin and anti-cytokeratin antibodies indicated that both filaments colocalize strongly. Three-dimensional reconstruction of serial optical sections revealed that newly synthesized vimentin distributed following the preexisting cytokeratin network and, when present, both filament scaffolds codistributed inside cultured hepatocytes. Immunoelectron microscopy performed in whole-mount-extracted cultured cells revealed that both filaments are closely interrelated but independent. However, a high degree of immunogold colocalization was found in the knots of the filament network. Further experiments with colce- mide and cytochalasin treatment indicated that vimentin filament distribution, but not cytokeratin, was dependent on an intact microtubule network. These results are consistent with a mechanism of vimentin assembly, whereby growth of vimentin intermediate filaments is dependent on microtubules in topographically restricted cytoplasmic sites, in close relation to the cytokeratin cytoskeleton and to changes in cell–cell contact and cell shape.

?Tissue? transglutaminase is specifically expressed in neonatal rat liver cells undergoing apoptosis upon epidermal growth factor-stimulation

We recently reported that activation of "tissue" transglutaminase (EC 2.3.2.13; tTG) in liver cells undergoing apoptosis determines extensive cross-linking of cellular proteins resulting in the formation of SDS-insoluble shells in the so-called "apoptotic bodies". In attempt to obtain further insight into the role played by tTG in apoptosis of liver cells, we investigated its expression in primary cultures of neonatal rat liver cells stimulated with epidermal growth factor (EGF). EGF-treatment of neonatal rat liver cells induces first hyperplasia of hepatocytes, followed by involution characterized by a high incidence of apoptosis. The proliferative phase of hepatocytes is paralleled by a 10-fold increase in tTG mRNA level, which is followed, during the phase of involution, by sequential increases in enzyme activity and levels of SDS-insoluble apoptotic bodies. tTG immunostaining at both the light- and electron-microscopic levels shows that the most intensive reaction is present in globular structures showing the typical morphological appearance of mature apoptotic bodies. In early apoptotic stages, tTG protein is localized in the perinuclear region of the cell. Intense immunostaining is also found in the apoptotic bodies present inside phagosomes within the cytoplasm of neighboring cells. This evidence confirms and extends our previous findings, indicating that tTG induction and activation specifically takes place in cells undergoing apoptosis, suggesting a key role for the enzyme in the apoptotic program.

Acid lipase activity in neonatal rat liver cell types. Effect of starvation

The acid lipase activity in the liver of neonatal (1-day-old) rats was studied. It was found that (i) in whole liver, the activity was 50% lower than in adult rats; (ii) in neonatal livers, the activity was 7.7-fold higher in hepatocytes than in hemopoietic cells; (iii) neonatal hepatocytes contained about 25% of the activity detected in adult hepatocytes; (iv) all the differences disappeared when expressed per mg of protein; and (v) starvation did not affect the activity either in adult or in neonatal rat liver.

Reversal of ischemic liver failure by intrasplenic liver cell transplantation

A model of ischemic hepatic failure in Sprague-Dawley rats (SD) with 100% mortality has been developed by one-hour occlusion of the right portal vein and hepatic artery followed by left 70% hepatectomy. The intrasplenic injection of 40 x 10(6) syngeneic adult or three-day neonatal single liver cell suspensions decreased the mortality from 100% to 50% and 36%, respectively. Mortality decreased with increasing time from the intrasplenic injection of neonatal liver cells to the time of acute hepatic ischemia. Mortality also decreased with increasing interval between hepatic ischemia and removal of the transplanted liver cells by splenectomy. Intrasplenic injection of graded doses of neonatal liver cells decreased mortality from 75% at a dose of 10 x 10(6) cells, to 36% at 160 x 10(6) cells. Treatment of neonatal liver cells with metabolic inhibitors did not significantly affect their ability to reverse acute hepatic ischemia.