Hepatic progenitor cells in human liver diseases

The canals of Hering and bile ductules in human liver contain hepatic progenitor cells that can differentiate towards the biliary and hepatocytic lineage. Proliferation and differentiation of hepatic progenitor cells is referred to as 'activation' and this process occurs to a variable degree in almost all human liver diseases. Several studies indicate that hepatic progenitor cell activation in diseased liver is regulated by neural and neuroendocrine factors such as the vagal innervation. Analogous to oval cells in animal liver, there is evidence that human hepatic progenitor cells may be able to give rise to hepatocellular carcinoma and other liver tumors.     

Aggregate cultures of foetal rat liver cells: development and maintenance of liver gene expression

Rotation-mediated aggregate cultures of foetal rat liver cells were prepared and grown in a chemically defined medium. Their capacity for cellular organisation and maturation was studied over a culture period of 3 wk by using both morphologic and biochemical criteria. It was found that within each aggregate, distinct liver cell types were present and attained their normal, differentiated phenotype. Parenchymal cells formed small acini with a central lumen. Within the first 2 wk in culture, albumin and ferritin mRNA levels were maintained, while the alpha-fetoprotein mRNA levels decreased, and tyrosine aminotransferase (TAT) gene expression increased. No significant response to glucocorticoids was observed in early cultures, whereas after 3 wk a marked increase in TAT mRNA levels was elicited by dexamethasone and glucagon (additive stimulatory effects). The results show that foetal rat liver cells cultured in a chemically defined medium are able to rearrange themselves into histotypic structures, and display a developmental pattern of gene expression comparable to that of perinatal rat liver in vivo. This culture system offers therefore a useful model to study the development and function of liver cells.     

Effect of human plasmalipoproteins on erythropoietic progenitor cells in serum-free cultures

The purpose of the present study was to investigate the influence of human lipoproteins on CFU-e and BFU-e proliferation from human bone marrow in a serum-free system. In our previously described miniaturized agar system the main lipoprotein-density-classes from human plasma, namely very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), high density lipoproteins2 (HDL2) and HDL3 and a mixture of all the five lipoproteins were added in rising concentrations (from 1/10 to normal human plasma concentration) to serum-free medium containing delipidated and deionized bovine serum albumin (BSA), iron saturated transferrin and erythropoietin. The results demonstrate that all lipoproteins markedly increased the CFU-e and BFU-e proliferation after 7 and 14 days of incubation, respectively. Moreover, the lipoproteins induced a shift towards a lower threshold concentration of erythropoietin. Serumlike conditions were obtained if LDL and the mixture of lipoproteins were added to serum-free medium. Furthermore, in the serum-free cultures a maturation to the mature erythrocyte could be found.     

Production of progenitor cells from primary human epithelial cell monolayer cultures

Primary keratinocytes derived from human epidermis are widely used in tissue engineering and regenerative medicine. An important aspect in clinical applications is the preservation of human skin keratinocyte stem cells. However, it is difficult to expand the number of human skin keratinocyte stem cells, which are undifferentiated and highly proliferative in culture by using standard cell culture methods. It is even more difficult to identify them, since universal specific markers for human skin keratinocyte stem cells have not been identified. In this paper, we show a method to produce a large number of primary progenitor human skin keratinocytes by using our novel culture techniques. Primary human skin keratinocyte monolayers are cultured using twice the volume of medium without serum and lacking essential fatty acids. Once the cells reach 70–80% confluence, they begin to float up into the overlying medium and are called “epithelial pop-up keratinocytes (ePUKs)” allowing the cells to be passaged without the use of trypsin. We analyzed the properties of ePUKs by cell size, cell viability, immunocytofluorescence biomarker staining, and cell cycle phase distribution by fluorescence-activated cell sorting (FACS). Our results showed that these ePUKs appear to be progenitor epithelial cells, which are small in size, undifferentiated, and have a high proliferative capacity. We believe that ePUKs are suitable for use in medical applications requiring a large number of primary human progenitor skin keratinocytes.     

Detection of T-precursor cells in human bone marrow and foetal liver.

Human lymphoid cells that have been incubated with conditioned medium from confluent monolayers of human thymic epithelium (HTCM) show an increase in cells forming rosettes with sheep erythrocytes at 4 degrees C (E-rosettes). Those cells demonstrating this in vitro conversion have been interpreted to be T-cell precursors. Separation of human bone marrow cells on discontinuous bovine serum albumin (BSA) gradients, and on a single-step 23% BSA gradient showed enrichment of these T-precursor cells, not only in bone marrow, but also in human foetal liver cells. Bone marrow precursor cells from a patient with Wiskott-Aldrich syndrome (T-cell deficiency) showed a normal in vitro response to HTCM, but no response was seen in cells from a patient with severe combined immunodeficiency disease.     

Side population cells in developing human liver are primarily haematopoietic progenitor cells

Side population (SP) cells have recently been identified in a number of tissues although their phenotype and functional abilities are poorly understood. Surface marker characterisation and functional assessment of developing liver SP cells might allow for their isolation and manipulation using clinically relevant techniques. It was hypothesised that SP cells are present early during human liver development and contribute to haematopoietic and epithelial lineage generation. Whilst the SP population remained positive for CD34 during the 1st and 2nd trimester, 1st trimester SP cells were more highly enriched for haematopoietic and epithelial progenitor activity than those from the 2nd trimester in vitro. Marker expression and functional similarities indicate that SP cells in developing human liver may share a temporal relationship with oval/progenitor cells, responsible for liver regeneration after massive or chronic hepatic injury. Furthermore, modification of SP integrin expression during development suggests a potential adaptive interaction with niche components such as fibronectin. Improved understanding of developing human liver SP cells will contribute to the generation of novel cell-based therapies for liver disease.     

Characterization of the double-stranded RNA responses in human liver progenitor cells

Human HepaRG cells are liver progenitors which possess hepatocyte-like functionality. We investigated the effects of double-stranded (ds) RNA on interferon (IFN)-β and chemokine (CK) expression in these cells. By microarray and ELISA, we showed strong induction of CXCL10 and interleulin (IL)-8 besides IFN-β and other CK ligands. RNA interference directed silencing of TLR3, RIG-I, IRF3, NFκB or MAP kinases (p38, ERK, JNK) was carried out. Knockdown of all these molecules, except ERK and JNK, blocked IFN-β production. Both TLR3 and RIG-I are required for CXCL10 expression. Silencing of TLR3 completely impaired the IL-8 expression. dsRNA-conditioned medium from HepaRG cells exerted a drastic antiviral effect in HCV replicons, and in the JFH-1-based HCV production cell culture system. The IFN-β knockdown in HepaRG cells removed this antiviral effect but did not enhance their capacity to initiate HCV RNA replication. We conclude that dsRNA induces antiviral and pro-inflammatory status in HepaRG cells.     

Divergent action of transforming growth factor beta on DNA synthesis in human foetal liver cells

Purified human transforming growth factor beta (TGF beta) was found to be a potent inhibitor of DNA synthesis in human foetal hepatocytes. Half-maximal inhibition occurred at 0.5-1 pM and was reversible, with an increase in DNA synthesis within 24 h following removal of TGF beta. By contrast, in the same cultures, 'fibroblast-like' non-hepatocytes retained the ability to synthesize DNA in the presence of up to 200 fold higher doses of TGF beta. This differential response to TGF beta suggests that it may act as an important cell growth regulator in the human foetal liver.     

Growth kinetics of progenitor cell-enriched hematopoietic cell populations in long-term liquid cultures under continuous removal of mature cells

BACKGROUND: During long-term culture of primitive hematopoietic cells large numbers of mature cells are generated that, on the one hand, consume nutrients and cytokines present in the medium and, on the other hand, may produce or elicit the production of soluble factors that limit the growth of primitive cells. Thus it is possible that under standard culture conditions hematopoietic stem and progenitor cells are unable to display their true proliferation and expansion potentials. METHODS: Hematopoietic cell populations, enriched for CD34+ cells, were obtained from both umbilical cord blood (UCB) and mobilized peripheral blood (MPB), and cultured in cytokine-supplemented liquid culture, under continuous removal of mature cells by means of weekly re-selection of primitive, lineage-negative (Lin-) cells. Proliferation and expansion capacities of such cells were determined weekly for a 42-day culture period. RESULTS: As expected, based on our previous studies in standard liquid cultures, throughout the culture period there was a continuous decrease in the proportion of progenitor cells; however, after every re-selection on days 7, 14 and 21, there was a significant enrichment for both CD34+ cells and colony-forming cells (CFC). As a result of such an enrichment, the cumulative increase in the numbers of total cells and CFC in cultures with two, three or four selections was significantly higher than the increments observed in standard cultures, in which only a single selection was performed on day 0. Cultures of UCB cells showed consistently higher levels of both total cells and CFC than cultures of MPB cells. DISCUSSION: Taken together, these results indicate that continuous removal of mature cells from liquid cultures of primitive progenitors results in higher increments in the levels of both total cells and CFC.     

Hepatotropin mRNA expression in human foetal liver development and in liver regeneration

A 569 bp probe against the β-chain of hepatotropin was used to examine expression of RNA for this growth factor in human adult and foetal liver, foetal kidney and pancreas, and rat liver after partial hepatectomy. Low level expression of a 6kb RNA occurred in human adult and normal rat liver. 70% hepatectomy increased expression, peaking at 10 h and returning to near normal levels 24 h after resection. The 6 kb band was strongly expressed in human foetal liver, as compared with adult, but not in foetal kidney or pancreas, suggesting a major role for hepatotropin in both foetal development and regeneration of the liver.     

Portal venous endothelium in developing human liver contains haematopoietic and epithelial progenitor cells

Future treatments for chronic liver disease are likely to involve manipulation of liver progenitor cells (LPCs). In the human, data characterising the regenerative response is limited and the origin of adult LPCs is unknown. However, these remain critical factors in the design of cell-based liver therapies. The developing human liver provides an ideal model to study cell lineage derivation from progenitors and to understand how foetal haematopoiesis and liver development might explain the nature of the adult LPC population. In 1st trimester human liver, portal venous endothelium (PVE) expressed adult LPC markers and markers of haematopoietic progenitor cells (HPCs) shared with haemogenic endothelium found in the embryonic dorsal aorta. Sorted PVE cells were able to generate hepatoblast-like cells co-expressing CK18 and CK19 in addition to Dlk/pref-1, E-cadherin, albumin and fibrinogen in vitro. Furthermore, PVE cells could initiate haematopoiesis. These data suggest that PVE shares phenotypical and functional similarities both with adult LPCs and embryonic haemogenic endothelium. This indicates that a temporal relationship might exist between progenitor cells in foetal liver development and adult liver regeneration, which may involve progeny of PVE.     

The production of terminal deoxynucleotidyl transferase (TdT) positive cells in cultures of human pluripotent hemopoietic progenitor cells

A transient increase in terminal deoxynucleotidyl transferase positive (TdT+) cells was observed during the early phase of (less than or equal to day 5) cultures supporting the growth of pluripotent myeloid progenitor cells (CFU-mix). T-cell growth-promoting medium and erythropoietin were not required. The rapidity with which TdT+ cells appeared in cultures and the results of cultures where TdT+ cells were high initially (greater than 800 cells/culture) were not consistent with their having been produced by proliferation of pre-existing TdT+ cells from the bone marrow inoculum. The results suggest production of TdT+ cells from a TdT-negative precursor either by altered enzyme expression or by production of TdT+ progeny.