Optimierung der Kultur und Charakterisierung primärer embryonaler Hepatozyten der Ratte

In dieser Arbeit wurde in vitro die Proliferation und Differenzierung embryonaler Hepatozyten untersucht. Im Rattenmodell wurden daher die Kulturbedingungen der Hepatozyten variiert und optimiert. Mit Hilfe von Analysen sowohl auf genomischer als auch funktioneller Ebene wurde eine umfassende Charakterisierung der Hepatozyten in Kultur durchgeführt. Untersucht wurde der Einfluß verschiedener Matrizes (Collagen I, IV, Matrigel, Laminin), feeder-Zellen (3T3, MRC-5, primäre Fibroblasten) und Wachstumsfaktoren einzeln oder in Kombination (Thrombopoietin [TPO], Hepatocyte-, Epidermal- und Transforming Growth Factor) auf die Proliferation und Differenzierung der embryonalen Hepatozyten. Die Proliferation und Differenzierung embryonaler Hepatozyten in Kultur werden eindeutig von der Art der verwendeten Beschichtung bzw. feeder-Zellen beeinflußt. Maximale, über 30fache Zellvermehrung, wurde nach 12 Tagen in Kultur auf 3T3 feeder-Zellen mit Zusatz von TPO erreicht. Es konnte erstmalig ein proliferativer Effekt von TPO auf Hepatozyten in Kultur gezeigt werden. Die auf 3T3 feeder-Zellen mit TPO kultivierten Zellen wiesen ein RNA Expressionsprofil ähnlich dem adulter Hepatozyten auf. Zusätzlich wurden Differenzierungsmarker nachgewiesen: Es konnte Albuminsekretion über einen Untersuchungszeitraum von 30 Tagen gemessen werden; durch Immuncytochemie wurden Albumin, GST und verschiedene Cytochrome detektiert; es konnte Cytochrom-P450 Aktivität nachgewiesen werden.The aim of the present dissertation was to investigate in vitro proliferation and differentiation of embryonic rat hepatocytes. Therefore, culture conditions of hepatocytes were varied and optimised. A widespread characterisation of embryonic hepatocytes in culture was realised by using genomic as well as functional methods of analysis. The influence of various matrices (collagen I, IV, matrigel, laminin), feeder cells (3T3, MRC-5, primary fibroblasts) and growth factors (thrombopoietin [TPO], hepatocyte growth factor, epidermal growth factor and transforming growth factor alpha) alone or in combination on proliferation and differentiation of these embryonic hepatocytes was studied. Proliferation and differentiation of embryonic hepatocytes in culture is clearly affected by type of coating and feeder cell, respectively. After 12 days in culture maximal increase in cell number (more than 30 fold) was observed using 3T3 as feeder cells in combination with TPO. For the first time a proliferative effect of TPO on hepatocytes could be demonstrated. The RNA profile of expression of hepatocytes cultured on 3T3 feeder cells supplemented with TPO bear resemblance to that of adult hepatocytes. In addition, markers of differentiation could be verified. Secretion of albumin could be measured during an observation period of 30 days. Immuncytochemistry revealed expression of albumin, GST, and various cytochromes. Cytochrome P450 activities could be detected for several days in culture

Human telomerase activity, telomerase and telomeric template expression in hepatic stem cells and in livers from fetal and postnatal donors

Even though telomerase activity has been analyzed in various normal and malignant tissues, including liver, it is still unknown to what extent telomerase can be associated with specific maturational lineage stages

Ex Vivo Conditions for Self-Replication of Human Hepatic Stem Cells

Human hepatic stem cells (hHpSCs), identifiable by a unique antigenic profile, have been isolated from human livers and established ex vivo under expansion conditions permissive for self-replication. The conditions consist of a substratum of type III collagen, ideally on Transwell inserts, and Kubota's medium, a serum-free medium developed for hepatic progenitors. Under these conditions the cells demonstrated a doubling time of ∼24 h, generating at least a 16-fold increase in cell number within 7–10 days; were stable at confluence for up to 2 weeks; could be passaged, if on type III collagen, to initiate colonies that went through log-phase growth and saturation density kinetics; and expressed telomerase, indicative of regenerative capacity. The hHpSC colonies remained morphologically and phenotypically stable throughout expressing epithelial cell adhesion molecule, neural cell adhesion molecule, albumin, cytokeratins 8, 18, and 19, but not α-fetoprotein, or intercellular adhesion molecule-1 (ICAM-1). Those maintained under self-replication conditions for more than a month were transplanted and found to engraft in the livers of SCID/nod mice yielding human liver tissue expressing adult liver–specific proteins. The conditions for self-replication should offer ideal culture conditions for generating large numbers of hHpSCs for use in commercial and clinical programs

Phases I-II Matched Case-Control Study of Human Fetal Liver Cell Transplantation for Treatment of Chronic Liver Disease.

Fetal hepatocytes have a high regenerative capacity. The aim of the study was to assess treatment safety and clinical efficacy of human fetal liver cell transplantation through splenic artery infusion. Patients with endstage chronic liver disease on the waiting list for liver transplantation were enrolled. A retrospectively selected contemporary matched-pair group served as control. Nonsorted raw fetal liver cell preparations were isolated from therapeutically aborted fetuses. The end points of the study were safety and improvement of the Model for End-Stage Liver Disease (MELD) and Child-Pugh scores. Nine patients received a total of 13 intrasplenic infusions and were compared with 16 patients on standard therapy. There were no side effects related to the infusion procedure. At the end of follow-up, the MELD score (mean ± SD) in the treatment group remained stable from baseline (16.0 ± 2.9) to the last observation (15.7 ± 3.8), while it increased in the control group from 15.3 ± 2.5 to 19 ± 5.7 ( p = 0.0437). The Child-Pugh score (mean ± SD) dropped from 10.1 ± 1.5 to 9.1 ± 1.4 in the treatment group and increased from 10.0 ± 1.2 to 11.1 ± 1.6 in the control group ( p = 0.0076). All treated patients with history of recurrent portosystemic encephalopathy (PSE) had no further episodes during 1-year follow-up. No improvement was observed in the control group patients with PSE at study inclusion. Treatment was considered a failure in six of the nine patients (three deaths not liver related, one liver transplant, two MELD score increases) compared with 14 of the 16 patients in the control group (six deaths, five of which were caused by liver failure, four liver transplants, and four MELD score increases). Intrasplenic fetal liver cell infusion is a safe and well-tolerated procedure in patients with end-stage chronic liver disease. A positive effect on clinical scores and on encephalopathy emerged from this preliminary study

EpCAM and the biology of hepatic stem/progenitor cells

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein, which is frequently and highly expressed on carcinomas, tumor-initiating cells, selected tissue progenitors, and embryonic and adult stem cells. During liver development, EpCAM demonstrates a dynamic expression, since it can be detected in fetal liver, including cells of the parenchyma, whereas mature hepatocytes are devoid of EpCAM. Liver regeneration is associated with a population of EpCAM-positive cells within ductular reactions, which gradually lose the expression of EpCAM along with maturation into hepatocytes. EpCAM can be switched on and off through a wide panel of strategies to fine-tune EpCAM-dependent functional and differentiative traits. EpCAM-associated functions relate to cell–cell adhesion, proliferation, maintenance of a pluripotent state, regulation of differentiation, migration, and invasion. These functions can be conferred by the full-length protein and/or EpCAM-derived fragments, which are generated upon regulated intramembrane proteolysis. Control by EpCAM therefore not only depends on the presence of full-length EpCAM at cellular membranes but also on varying rates of the formation of EpCAM-derived fragments that have their own regulatory properties and on changes in the association of EpCAM with interaction partners. Thus spatiotemporal localization of EpCAM in immature liver progenitors, transit-amplifying cells, and mature liver cells will decisively impact the regulation of EpCAM functions and might be one of the triggers that contributes to the adaptive processes in stem/progenitor cell lineages. This review will summarize EpCAM-related molecular events and how they relate to hepatobiliary differentiation and regeneration

Human hepatic stem cells from fetal and postnatal donors

Human hepatic stem cells (hHpSCs), which are pluripotent precursors of hepatoblasts and thence of hepatocytic and biliary epithelia, are located in ductal plates in fetal livers and in Canals of Hering in adult livers. They can be isolated by immunoselection for epithelial cell adhesion molecule–positive (EpCAM+) cells, and they constitute ∼0.5–2.5% of liver parenchyma of all donor ages. The self-renewal capacity of hHpSCs is indicated by phenotypic stability after expansion for >150 population doublings in a serum-free, defined medium and with a doubling time of ∼36 h. Survival and proliferation of hHpSCs require paracrine signaling by hepatic stellate cells and/or angioblasts that coisolate with them. The hHpSCs are ∼9 μm in diameter, express cytokeratins 8, 18, and 19, CD133/1, telomerase, CD44H, claudin 3, and albumin (weakly). They are negative for α-fetoprotein (AFP), intercellular adhesion molecule (ICAM) 1, and for markers of adult liver cells (cytochrome P450s), hemopoietic cells (CD45), and mesenchymal cells (vascular endothelial growth factor receptor and desmin). If transferred to STO feeders, hHpSCs give rise to hepatoblasts, which are recognizable by cordlike colony morphology and up-regulation of AFP, P4503A7, and ICAM1. Transplantation of freshly isolated EpCAM+ cells or of hHpSCs expanded in culture into NOD/SCID mice results in mature liver tissue expressing human-specific proteins. The hHpSCs are candidates for liver cell therapies

Sensitive to critical incidents : the development and validation of the Swiss CSI-HC : a client satisfaction measure for homecare services in Switzerland

Background: Homecare services for the elderly and chronically ill are becoming increasingly important in many western countries, including Switzerland. Surveys of client satisfaction with services are conducted using various measurement instruments, some of which do not meet scientific quality criteria. In particular, the validity of existing measures is often questioned because less sensitive and favorable ratings lead to ceiling effects. Objectives: Development and validation of a satisfaction measure for homecare services in Switzerland which reflects (i) the service experience of clients, (ii) is sensitive to critical incidents, and (iii) offers a high explanatory power for overall satisfaction with homecare services. Method: After a step-by-step scale development process, the prototype was field-tested in a survey of Swiss homecare clients (sample n = 1,312). To assess validity, the ratings on the CSI-HC measure were compared with client-stated data on the occurrence of critical incidents, stated client experience, and the affective state of the client in relation to the homecare provider. Findings: The items of the measure explain a high proportion of overall satisfaction with homecare services. Furthermore, the measure appears to be sensitive to the occurrence of critical incidents and reflects the stated qualitative client experience very well. The measure correlates highly with affective satisfaction assessed on the SAM scale. Contribution to the research field: This study presents a mixed-method approach for the validation of a sensitive client satisfaction measure in healthcare management and public health research using the example of a measure for the Swiss homecare sector