Claus Kordes

StellaTUM: current consensus and discussion on pancreatic stellate cell research

The field of pancreatic stellate cell (PSC) biology is very young, as the essential in-vitro tools to study these cells (ie, methods to isolate and culture PSC) were only developed as recently as in 1998. Nonetheless, there has been an exponential increase in research output in this field over the past decade, with numerous research groups around the world focusing their energies into elucidating the biology and function of these cells. It is now well established that PSC are responsible for producing the stromal reaction (fibrosis) of two major diseases of the pancreas—chronic pancreatitis and pancreatic cancer. Despite exponentially increasing data, the methods for studying PSC remain variable. Although within individual laboratories methods are consistent, different methodologies used by various research groups make it difficult to compare results and conclusions. This article is not a review article on the functions of PSC. Instead, members of the Pancreatic Star Alliance (http://www.pancreaticstaralliance.com) discuss here and consolidate current knowledge, to outline and delineate areas of consensus or otherwise (eg, with regard to methodological approaches) and, more importantly, to identify essential directions for future research. Hepatic stellate cells (HSC) were first described by Karl von Kupffer in 1876; however, similar cells in the pancreas were first observed in the 1980s.1–3 In 1998, Apte et al 4 and Bachem et al 5 isolated and cultured PSC.4 5 In the normal pancreas, PSC are located in close proximity to the basal aspect of pancreatic acinar cells. In sections immunostained for the marker desmin (a cytoskeletal protein), quiescent PSC can be seen as cells with a central cell body and long cytoplasmic projections extending along the base of adjacent acinar cells similar to that of pericytes in the mammary gland. …

The niche of stellate cells within rat liver.

It is well‐accepted that hepatic stellate cells (HSCs) can develop into myofibroblast‐like cells that synthesize extracellular matrix proteins and contribute to liver fibrosis. Recently, molecular markers of stem/progenitor cells were discovered in HSCs of rats. Moreover, the cells displayed the capacity to differentiate and to participate in liver regeneration. In addition, stellate cells possess signaling pathways important for maintenance of stemness and cell differentiation such as hedgehog and β‐catenin–dependent Wnt signaling. All these properties are congruently found in stem/progenitor cells. Stem cells require a special microenvironment, the so‐called stem cell niche, to maintain their characteristics. Thus, we investigated if the space of Disse, where stellate cells reside in the liver innervated by the sympathetic nervous system and surrounded by sinusoidal endothelial cells and parenchymal cells, exhibits similarities with known stem cell niches. The present study describes the niche of stellate cells within the liver of rats that is composed of sinusoidal endothelial cells, which release stromal cell–derived factor‐1 to attract stellate cells via the cysteine‐X‐cysteine receptor 4, basal lamina proteins (laminin and collagen type IV), and parenchymal cells, which synthesize β‐catenin–dependent Wnt ligands and Jagged1. Conclusion: The space of Disse shows analogies to typical stem cell niches comprising of basal lamina components, sympathetic innervation, and adjacent cells that constitute a milieu by paracrine factors and direct physical interactions to retain HSCs at this site and to influence their cellular fate. The space of Disse serves as a niche of stellate cells, which is a novel function of this unique organ structure. (HEPATOLOGY 2009.)

Hepatic stellate cells contribute to progenitor cells and liver regeneration

Retinoid-storing hepatic stellate cells (HSCs) have recently been described as a liver-resident mesenchymal stem cell (MSC) population; however, it is not clear whether these cells contribute to liver regeneration or serve as a progenitor cell population with hepatobiliary characteristics. Here, we purified HSCs with retinoid-dependent fluorescence-activated cell sorting from eGFP-expressing rats and transplanted these GFP(+) HSCs into wild-type (WT) rats that had undergone partial hepatectomy in the presence of 2-acetylaminofluorene (2AAF) or retrorsine, both of which are injury models that favor stem cell-based liver repair. Transplanted HSCs contributed to liver regeneration in host animals by forming mesenchymal tissue, progenitor cells, hepatocytes, and cholangiocytes and elevated direct bilirubin levels in blood sera of GUNN rats, indicating recovery from the hepatic bilirubin-handling defect in these animals. Transplanted HSCs engrafted within the bone marrow (BM) of host animals, and HSC-derived cells were isolated from BM and successfully retransplanted into new hosts with injured liver. Cultured HSCs transiently adopted an expression profile similar to that of progenitor cells during differentiation into bile acid-synthesizing and -transporting hepatocytes, suggesting that stellate cells represent a source of liver progenitor cells. This concept connects seemingly contradictory studies that favor either progenitor cells or MSCs as important players in stem cell-based liver regeneration.

Hepatic stem cell niches

Stem cell niches are special microenvironments that maintain stem cells and control their behavior to ensure tissue homeostasis and regeneration throughout life. The liver has a high regenerative capacity that involves stem/progenitor cells when the proliferation of hepatocytes is impaired. In recent years progress has been made in the identification of potential hepatic stem cell niches. There is evidence that hepatic progenitor cells can originate from niches in the canals of Hering; in addition, the space of Disse may also serve as a stem cell niche during fetal hematopoiesis and constitute a niche for stellate cells in adults.

Hepatic stellate cells support hematopoiesis and are liver-resident mesenchymal stem cells.

Background/Aims: Hematopoiesis can occur in the liver, when the bone marrow fails to provide an adequate environment for hematopoietic stem cells. Hepatic stellate cells possess characteristics of stem/progenitor cells, but their contribution to hematopoiesis is not known thus far. Methods: Isolated hepatic stellate cells from rats were characterized with respect to molecular markers of bone marrow mesenchymal stem cells (MSC) and treated with adipocyte or osteocyte differentiation media. Stellate cells of rats were further co-cultured with murine stem cell antigen-1+ hematopoietic stem cells selected by magnetic cell sorting. The expression of murine hematopoietic stem cell markers was analyzed by mouse specific quantitative PCR during co-culture. Hepatic stellate cells from eGFP+ rats were transplanted into lethally irradiated wild type rats. Results: Desmin-expressing stellate cells were associated with hematopoietic sites in the fetal rat liver. Hepatic stellate cells expressed MSC markers and were able to differentiate into adipocytes and osteocytes in vitro. Stellate cells supported hematopoietic stem/progenitor cells during co-culture similar to bone marrow MSC, but failed to differentiate into blood cell lineages after transplantation. Conclusion: Hepatic stellate cells are liver-resident MSC and can fulfill typical functions of bone marrow MSC such as the differentiation into adipocytes or osteocytes and support of hematopoiesis.

Hepatic and pancreatic stellate cells in focus.

Abstract Stellate cells are vitamin A-storing cells of liver and pancreas and have been described in all vertebrates ranging from lampreys (primitive fish) to humans, demonstrating their major importance. This cell type is thought to contribute to fibrosis, a condition characterized by an excess deposition of extracellular matrix proteins. Recently, the expression of stem/progenitor cell markers, such as CD133 (prominin-1) and Oct4, was discovered in hepatic stellate cells (HSCs) of rats. Moreover, HSCs possess signaling pathways important for maintenance of stemness and cell differentiation, such as hedgehog, β-catenin-dependent Wnt, and Notch signaling, and are resistant to CD95-mediated apoptosis. In analogy to a stem cell niche, some characteristics of quiescent HSC are maintained by aid of a special microenvironment located in the space of Dissé. Finally, stellate cells display a differentiation potential as investigated in vitro and in vivo. Collectively all these properties are congruently found in stem/progenitor cells and support the concept that stellate cells are undifferentiated cells, which might play an important role in liver regeneration. The present review highlights findings related to this novel aspect of stellate cell biology.

Differential and synergistic effects of platelet-derived growth factor-BB and transforming growth factor-beta1 on activated pancreatic stellate cells.

Objective: The cytokines platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-β1 are major factors influencing the transformation from the quiescent to the activated phenotype of pancreatic stellate cells (PSC), a process involved in the pathogenesis of chronic pancreatitis. Albeit much effort has been made to study the effects of PDGF and TGF-β1 on PSCs, their interaction is still unclear, because these cytokines show both differential and synergistic effects as outlined by this study. Methods: Culture-activated PSCs of rats were treated with PDGF-BB and TGF-β1. Subsequent changes of cell proliferation and migration were determined by cell counting, (+)-bromo-2′-deoxyuridine enzyme-linked immunosarbant assay (ELISA), and migration assay. Gene expression, synthesis of proteins, and activation of kinases were further studied by reverse transcription-polymerase chain reaction, real-time polymerase chain reaction, ELISA, and Western blot. Results: PDGF-BB increased PSC proliferation and migration, accompanied by elevated expression of matrix metalloproteinases (MMP)-13 and MMP-3. The mRNA amount of procollagen α2(I), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)-1, and TGF-β1 was also increased by PDGF-BB. In contrast, PDGF-BB reduced collagen type I in culture medium and synthesis of α-SMA. Treatment of PSC with TGF-β1 decreased proliferation, had no significant effect on migration and MMP expression, but increased expression and synthesis of procollagen α2(I) and α-SMA. Both cytokines induced phosphorylation of extracellular signal regulated kinase (ERK)-1/2 and p38MAPK, but only PDGF-BB activated the protein kinase B signaling pathway. Conclusion: PDGF-BB augments effects of TGF-β1 on the mRNA level presumably because of up-regulation of TGF-β1 synthesis and common signaling pathways of the 2 cytokines. However, at the protein level, PDGF-BB impairs typical TGF-β1 effects such as increased synthesis of collagen (type I) and α-SMA. Moreover, PDGF-BB facilitates degradation of extracellular matrix proteins by enhancement of MMP synthesis, but MMP activity was probably limited because of elevated tissue inhibitor of metalloproteinase 1 expression.

Stellate Cells from Rat Pancreas Are Stem Cells and Can Contribute to Liver Regeneration

The identity of pancreatic stem/progenitor cells is still under discussion. They were suggested to derive from the pancreatic ductal epithelium and/or islets. Here we report that rat pancreatic stellate cells (PSC), which are thought to contribute to pancreatic fibrosis, have stem cell characteristics. PSC reside in islets and between acini and display a gene expression pattern similar to umbilical cord blood stem cells and mesenchymal stem cells. Cytokine treatment of isolated PSC induced the expression of typical hepatocyte markers. The PSC-derived hepatocyte-like cells expressed endodermal proteins such as bile salt export pump along with the mesodermal protein vimentin. The transplantation of culture-activated PSC from enhanced green fluorescent protein-expressing rats into wild type rats after partial hepatectomy in the presence of 2-acetylaminofluorene revealed that PSC were able to reconstitute large areas of the host liver through differentiation into hepatocytes and cholangiocytes. This developmental fate of transplanted PSC was confirmed by fluorescence in situ hybridization of chromosome Y after gender-mismatched transplantation of male PSC into female rats. Transplanted PSC displayed long-lasting survival, whereas muscle fibroblasts were unable to integrate into the host liver. The differentiation potential of PSC was further verified by the transplantation of clonally expanded PSC. PSC clones maintained the expression of stellate cell and stem cell markers and preserved their differentiation potential, which indicated self-renewal potential of PSC. These findings demonstrate that PSC have stem cell characteristics and can contribute to the regeneration of injured organs through differentiation across tissue boundaries.

The Epigenetic Regulation of Stem Cell Factors in Hepatic Stellate Cells

The epigenetic regulation by DNA methylation is an important mechanism to control the expression of stem cell factors as demonstrated in tumor cells. It was recently shown that hepatic stellate cells (HSC) express stem/progenitor cell factors and have a differentiation potential. The aim of this work was to investigate if the expression of stem cell markers is regulated by DNA methylation during activation of rat HSC. It was found that CD133, Notch1, and Notch3 are regulated via DNA methylation in HSC, whereas Nestin shows no DNA methylation in HSC and other undifferentiated cells such as embryonic stem cells and umbilical cord blood stem cells from rats. In contrast to this, DNA methylation controls Nestin expression in differentiated cells like hepatocytes and the hepatoma cell line H4IIE. Demethylation by 5-Aza-2-deoxycytidine was sufficient to induce Nestin in H4IIE cells. In quiescent stellate cells and embryonic stem cells, the Nestin expression was suppressed by histone H3 methylation at lysine 9, which is another epigenetic mechanism. Apart from the known induction of Nestin in cultured HSC, this intermediate filament protein was also induced after partial hepatectomy, indicating activation of HSC during liver regeneration. Taken together, this study demonstrates for the first time that the expression of stem cell-associated factors such as CD133, Notch1, and Notch3 is controlled by DNA methylation in HSC. The regulation of Nestin by DNA methylation seems to be restricted to differentiated cells, whereas undifferentiated cells use different epigenetic mechanisms such as histone H3 methylation to control Nestin expression.

Effects of angiotensin II on rat pancreatic stellate cells.

The aim of the study was to identify pancreatic stellate cells (PSCs) as a potential target of angiotensin II (ATII) action because recently a local renin-angiotensin system (RAS) has been described in the pancreas. PSCs were isolated from male Wistar rats and investigated for ATII receptor expression and ATII-induced calcium transients, contractions, proliferation, and &agr;-smooth muscle actin expression. Quiescent and activated PSCs expressed the ATII receptor subtype AT1 but not AT2. Addition of ATII led to a rapid elevation of intracellular calcium ([Ca2+]i). The sensitivity toward ATII with respect to calcium transients did not change during the transdifferentiation process. In activated PSCs, ATII dose dependently induced PSC cell contraction. Furthermore, ATII induced an activation of the c-Jun-N-terminal kinase (JNK) and extracellular regulated kinase (Erk), which was inhibited after intracellular calcium chelation by BAPTA-AM. The p38 mitogen-activated protein kinase (p38MAPK) was also activated by ATII. BAPTA-AM itself induced p38MAPK activation, which was not further enhanced by ATII. ATII stimulated PSC proliferation, while PSC transdifferentiation, as indicated by &agr;-smooth muscle actin expression and collagen type I secretion, was not enhanced. The data suggest that PSCs are targets of ATII action with potential pathophysiological relevance.