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PubMed1999

One thousand faces of Langerhans islets.

Studies with different strains of Syrian hamsters and Syrian golden hamsters have revealed the remarkable potential of islet cells to undergo orthotopic and heterotopic metaplasia. The most common spontaneous change included the development of hepatocytes in aged and malnourished hamsters. Of the many other alterations that occurred during carcinogenesis, most of the metaplastic changes originated within the islet periphery and progressed inside and outside the islets. The development of ductular structures within islets and their progression either to structures identical to human serous cystadenoma or to highly invasive adenocarcinomas were the most common alterations. The remarkably greater invasive potential of cancer cells arising within the islets contrasted sharply with the slow growth of the tumors developing within ducts (intraductal tumors). Studies in human tissue also showed development of malignant cells within islets, and, in some cases, transition of islet cells to malignant cells was suggested. The overall results, along with recent findings in other studies in cultured human and hamster islets, indicate the enormous potential of islet cells to differentiate and undergo malignant transformation. Whether the metaplastic and malignant cells derive from stem cells embedded within islets or from transdifferentiated islet cells remains to be seen.

Animals↗
PubMed2004

Stem cell plasticity, beyond alchemy.

Cell plasticity is a central issue in stem cell biology. Differentiated somatic nuclei have the flexibility to dedifferentiate when transferred into oocytes or when fused to pluripotent embryonic stem cells. Recent publications also claim that somatic stem cells can convert into developmentally unrelated cell types both in vivo and ex vivo without such drastic cell manipulations. Some of these claims are still controversial, making it difficult for us to determine the reality of somatic stem cell plasticity. Indeed, we have heard enough about the "potentials" of cell plasticity; how much do we know about mechanisms? A fundamental issue in current stem cell biology is to understand the mechanisms underlying cell plasticity. In this short review, we overview three research fields related to cell plasticity: nuclear transfer, transdifferentiation, and cell fusion, with an emphasis on studies of molecular mechanisms underlying cell plasticity.

Adult↗
PubMed2002

A new cell therapy using bone marrow cells to repair damaged liver.

Hepatic stem cells can transdifferentiate into hepatocytes, bile duct cells, pancreatic cells, or intestinal cells. Hepatic stem cells had been thought to be located in the canal of Hering in the liver, but recent work has demonstrated the existence of hepatic stem cells in bone marrow as well. Cell therapy using autologous bone marrow cells has few ethical problems and many applications for treating severe liver disease. Further in vitro and in vivo analysis is crucial to develop the therapy for clinical use.

Animals↗
PubMed2000

R-(+)-verapamil, S-(-)-verapamil, and racemic verapamil inhibit human retinal pigment epithelial cell contraction.

PURPOSE: To evaluate the effects of R-(+)-verapamil, S-(-)-verapamil, and the racemate on in vitro contraction of human retinal pigment epithelial cells. METHODS: RPE cells from human donor eyes were enzymatically dissociated and cultured on collagen gels. Transdifferentiated RPE cells (seventh and eighth passage) were used for experiments. The contraction assays were treated with different concentrations (10-1000 microM) of R-(+)-verapamil, S-(-)-verapamil, and racemic verapamil. RESULTS: Verapamil inhibited the gel contraction of human RPE cells. R-(+)-verapamil, S-(-)-verapamil, and racemic verapamil acted equally in a dose-dependent manner from 10-1000 microM. CONCLUSION: The results indicate the effectiveness of verapamil to inhibit the contraction of RPE cells. R-(+)-verapamil is recommended because it is associated with fewer systemic and local side effects.

Adolescent↗
PubMed2006

[Electrophysiological properties of stem cells].

New concepts for treatment of myocardial infarction include the implantation of adult stem cells for regeneration of damaged muscle tissue. Several clinical trials have demonstrated a small, but significant improvement of ventricular function. Transdifferentiation of stem cells into cardiomyocytes, formation of new vessels and paracrine factors have been discussed as putative mechanisms for the therapeutic effect. Several types of stem cells have been used clinically including myoblasts derived from skeletal muscle satellite cells, bone marrow-derived stem cells or blood-derived mononuclear progenitor cells. In addition, multiple organs were shown to contain a small number of stem cells that could differentiate into cardiomyocytes. Embryonic stem cells differentiate into spontaneously beating cells that have varying electrophysiological properties. Their action potentials resemble those of cardiac pacemaker cell, atrial or ventricular myocytes (Figure 1) suggesting true differentiation into cardiomyocytes. Beating cells derived from a newly described population of skeletal muscle-derived cells ("skeletal precursors of cardiomyocytes" [SPOCs]) also exhibit spontaneous action potentials, however, unlike cardiac pacemaker cells, their electrical activity is suppressed with the sodium channel blocker tetrodotoxin (Figure 2). Undifferentiated bone marrow-derived mesenchymal stem cells are not electrically excitable. Nevertheless, they express functional ion channels like L-type Ca(2+) channels, albeit not in every cell. Co-culturing stem cells with neonatal rat ventricular myocytes induces good electrical contacts between cells via gap junction formation. Excitatory wave fronts spread evenly in the co-culture. By contrast, gap junctions fail to form when myoblasts are co-cultured with neonatal cardiomyocytes and reentry arrhythmias develop. This pathomechanism could serve as an explanation for the enhanced clinical risk of arrhythmia after transplantation of myoblasts into the infarcted hearts.

Action Potentials↗
PubMed2003

[Genetic risk factors for hepatic fibrosis in chronic liver diseases].

Hepatic fibrosis is the result of chronic viral, toxic, autoimmune, or cholestatic liver injury. During the complex fibrotic response, hepatic stellate cells transdifferentiate into matrix-producing myofibroblasts, and interactions between multiple cytokines, receptors and extracellular matrix components modulate the fibrotic phenotypes. Of note, course and extent of hepatic fibrosis display significant variability among individual patients. These well-known differences in progression of hepatic fibrosis have been attributed to age, sex, and exogenous factors, e. g., coinfections or alcohol consumption. However, host genetic factors are likely to play key roles in the modulation of hepatic fibrosis and to contribute to the overall variability in disease progression. In recent years, different genetic polymorphisms that may influence the progression of hepatic fibrosis have been identified in animal models and human case-control studies. These findings indicate that variants of genes encoding immunoregulatory proteins as well as pro- and anti-inflammatory cytokines determine liver fibrosis in patients with chronic hepatitis C virus infection, alcoholic liver disease, and autoimmune liver diseases. However, the results of these studies are mixed and restricted to selected known genes. Therefore, definition of consensus criteria for association studies and multicenter studies are needed to increase statistical power. Genome-wide scans in experimental animal crosses should be employed to identify unknown gene loci that confer susceptibility to hepatic fibrosis (Hfib loci). In conclusion, genetic association studies have a great potential for identification of fibrogenic genes, but large-scale, well-designed studies are required to clarify their actual relevance and to provide a solid framework for future therapeutic strategies.

Animals↗
PubMed2004

Metabolic adaptations to chronic glucose infusion in rats.

AIMS/HYPOTHESIS: Several studies have employed the chronic glucose infusion protocol to quantify the metabolic adaptations associated with a prolonged glucose challenge. However, the limited number of indices and time points reported by these studies has generated an incomplete picture of this process. In this study we aimed to generate an integrative and dynamic picture of the physiological adaptations that occur during chronic glucose infusion. METHODS: Sprague-Dawley rats were infused with either 50% dextrose or saline (2 ml/h) for a period of between 0 and 6 days. Glucose, insulin and NEFA dynamics were determined from daily blood samples. Subsets of animals were killed daily for histological determination of beta cell mass, size and replication rates. The mathematical model of coupled beta cell mass, insulin and glucose (the betaIG model) was used to estimate insulin sensitivity, beta cell function and net neogenesis from this data. RESULTS: Glucose-infused rats displayed transient hyperglycaemia, persistent hyperinsulinaemia and unchanged NEFA levels. Insulin sensitivity decreased by approximately 80% during the first day of glucose infusion, but had returned to pre-infusion levels by Day 3. Beta cell function was four to six times higher than in control rats throughout the experiment. Beta cell mass doubled over the 6 days of glucose infusion due to three phases of adaptation: (i) neogenesis; (ii) hypertrophy and hyperplasia; and (iii) continued hyperplasia coupled to a second wave of neogenesis. CONCLUSIONS/INTERPRETATION: Contrary to the results reported for perfused pancreas and in vitro experiments, we found that chronic glucose infusion elevated beta cell function. The prediction of a second wave of beta cell neogenesis, coupled with our previous report of "focal areas" on Day 3, suggests the existence of delayed acinar-to-islet transdifferentiation.

Acclimatization↗
PubMed2004

In vitro generation of insulin-producing beta cells from adult exocrine pancreatic cells.

AIMS/HYPOTHESIS: Transplantation of insulin-producing beta cells from donors can cure diabetes, but they are available in insufficient quantities. In this study, we investigated the possibility of generating insulin-producing cells from adult rat exocrine cells cultured in the presence of growth factors. METHODS: Rat exocrine pancreatic cells were isolated and treated in vitro with epidermal growth factor (EGF) and leukaemia inhibitory factor (LIF). Analysis was performed by immunocytochemistry, DNA measurement and radioimmunoassay. Cells were transplanted to alloxan-treated (70 mg/kg) nude mice and glycaemia was monitored for 21 days. Nephrectomy was performed on day 15. RESULTS: In a 3-day culture period, addition of LIF plus EGF to the medium resulted in an 11-fold increase of the beta cell mass. This could not be attributed to the very low mitotic activity of contaminating beta cells. Furthermore, when contaminating beta cells were initially destroyed with alloxan, this effect was even more pronounced. The newly formed cells secreted insulin in response to glucose and were immunoreactive for C-peptide-I, Pdx-1 and GLUT-2, which are characteristics of mature beta cells. Electron microscopy showed that they also contained insulin-immunoreactive secretory granules. Some insulin-positive cells were immunoreactive for amylase and cytokeratin-20, or were binucleated, which are characteristics of exocrine cells. The cells were able to restore normoglycaemia when transplanted to alloxan-diabetic mice, and hyperglycaemia recurred upon removal of the graft. CONCLUSIONS/INTERPRETATION: Our study shows that functional beta cells can be generated from exocrine tissue by transdifferentiation and thereby may offer a new perspective for beta cell therapy.

Animals↗
PubMed2005

Expression and function of leukaemia inhibitory factor and its receptor in normal and regenerating rat pancreas.

AIMS/HYPOTHESIS: It was recently reported that culturing adult exocrine cells in the presence of epidermal growth factor and leukaemia inhibitory factor (LIF) resulted in their transdifferentiation into endocrine beta cells. The aim of this study was to examine the expression and function of LIF in the pancreas. MATERIALS AND METHODS: We studied the expression of LIF and its receptor components, LIF-receptor-beta and gp130, by immunohistochemistry, western blotting and RT-PCR in normal rat pancreas, pancreas with duct ligation-induced islet neogenesis, and in pancreatic cell cultures. Isolated duct fragments were cultured in the presence of LIF and a janus kinase 2 (JAK2) inhibitor. RESULTS: LIF was detected by immunohistochemistry, western blot and RT-PCR in the ducts of the normal pancreas. Both LIF-receptor-beta and gp130 were detected by RT-PCR in the pancreas. Immunostaining revealed gp130 exclusively in the ducts and centro-acinar cells. After duct ligation-induced tissue injury, upregulation of LIF and its receptor occurred in rat pancreas. Metaplastic exocrine cells also started to express LIF and this was increased after alloxan treatment. Signalling via LIF-receptor-beta/gp130 involves the JAK/signal transducer and activator of transcription (STAT) pathway. LIF induced increased activation of STAT3 in pancreatic cells. In isolated duct fragments, addition of LIF resulted in a significant increase in duct cell proliferation, while a specific inhibitor of the JAK/STAT signalling pathway inhibited proliferation. CONCLUSION/INTERPRETATION: Our observations show that LIF and its receptor are expressed in cells from pancreatic ducts. The cytokine plays a role in pancreatic physiology, controls duct cell proliferation and is involved in repair processes following pancreatic injury.

Animals↗
PubMed1999

The role of chondrocytes in intramembranous and endochondral ossification during distraction osteogenesis in the rabbit.

We have used a rabbit leg-lengthening model for detailed studies of the histology of distraction osteogenesis. Some unusual features of the endochondral ossification that occurs during the rapid transition of cartilage to bone in the regenerate were observed. Histological staining techniques together with immunohistochemistry and nonradioactive in situ mRNA hybridization for cartilage and bone-related molecules have been used to document the presence of an overlapping cartilage-bone phenotype in cells of the cartilage-bone transitional region. In those particular areas, some chondrocytes appeared to be directly transformed into newly formed bone trabeculae which are surrounded by bone matrix. Acid phosphatases were found within the cartilage matrix in some of the cartilage/bone transitional regions and type I collagen mRNA and type II collagen protein were found together in some of the marginal hypertrophic chondrocytes. This study indicates an unusual role of chondrocytes in the process of ossification at a distraction rate of 1.3 mm/day in the rabbit. Further direct evidence is required to prove the hypothesis that the hypertrophic chondrocytes may transdifferentiate into bone cells in this model.

Acid Phosphatase↗
PubMed2004

[Transplantation of iris pigment epithelium].

Transplantation of iris pigment epithelial (IPE) cells to the subretinal space has been attempted as a therapeutic modality for the treatment of age-related macular degeneration (AMD). IPE cells are used because autologous cells are readily available and because IPE and RPE cells share a common embryonic origin, possess the capacity of transdifferentiation into other ocular cells, and share common morphological and functional characteristics. Once the technique of IPE cell transplantation was established in an animal mode, several clinical studies analyzed the behavior of IPE cell suspensions transplanted to the subretinal space of patients with AMD following surgical membrane extraction. In our experience, as well as that of other investigators, transplantation of IPE cells to the subretinal space of AMD patients prevents the recurrence of the subretinal neovascularization and stabilizes but does not improve visual acuity. Since IPE cells transplanted as a cell suspension do not appear to form a cell monolayer in the subretinal space, the transplantation of preformed IPE or RPE cell monolayers is being investigated as the development of an functional cell monolayer is mandatory if functional success, i.e., recovery of vision in AMD patients, is the ultimate goal of IPE cell transplantation.

Aged↗
PubMed2005

Differential responses of human lens epithelial cells to intraocular lenses in vitro: hydrophobic acrylic versus PMMA or silicone discs.

BACKGROUND: The purpose of this study was to determine the influence of different materials of intraocular lenses (IOLs) on human lens epithelial cell behavior, including adhesion, migration, proliferation, apoptosis, and epithelial-mesenchymal transdifferentiation (EMT) in vitro. METHODS: Human lens epithelial cells (SRA 01/04) were grown on hydrophobic acrylic (Acrysof), polymethylmethacrylate (PMMA), and silicone IOLs. Cellular adhesion, migration, proliferation, and apoptotic assays were performed to assess cell behavior. The expression of EMT markers (fibronectin and type I collagen) produced by cells on IOLs was determined by immunoblotting and immunocytochemistry. RESULTS: Human lens epithelial cells exhibited preferred adhesion and reduced apoptosis when cultured on acrylic IOLs, in comparison to PMMA and silicone IOLs. Cells grown on acrylic lenses formed a confluent epithelial monolayer. Migration of lens epithelial cells under the acrylic lens was substantially blocked in an in vitro assay. In contrast, cells grown on PMMA and silicone lenses displayed a spindle-shaped, myofibroblast-like morphology, increased apoptosis, reduced adhesion, and enhanced production of EMT proteins such as fibronectin and type I collagen. The migration of lens epithelial cells under PMMA and silicone IOLs was substantial in the in vitro assay. CONCLUSION: This report demonstrates that hydrophobic acrylic lenses are more capsular biocompatible than PMMA and silicone lenses. The in vitro assays are reliable measurements for evaluating the responses of human lens epithelial cells to different IOL materials, and could advance our understanding of the preferential capsular opacification conferred by different IOL materials.

Apoptosis↗
PubMed2004

Early signs of lung fibrosis after in vitro treatment of rat lung slices with CdCl2 and TGF-beta1.

Precision-cut rat lung slices have been employed in combination with an extensive immunohistochemistry of paraffin-embedded slices for monitoring of early pathohistological changes after exposure to CdCl(2)/TGF-beta(1). Three days of CdCl(2) exposure in combination with TGF-beta(1) seem to be sufficient to induce lung injury with alterations similar to changes observed in early lung fibrogenesis: (1) extracellular matrix accumulation and myofibroblast transdifferentiation (Sirius red staining, collagen type IV, alpha-smooth muscle actin), (2) type I cell injury with loss of type I cell antigens (T1alpha antigen, aquaporin-5, RAGE), (3) increased apoptosis of pulmonary cells (active caspase-3, vimentin cleavage product V1 of caspase-9), and (4) activation of microvascular endothelial cells (podocalyxin, caveolin-1). Western blot analysis confirmed the increasing amount of alpha-smooth muscle actin, the loss of T1alpha antigen, and the increase in caveolin-1 immunoreactivity. The explant culture using CdCl(2)/TGF-beta(1) provides a suitable tool for the study of other factors involved in pulmonary pathology including transcription factors, cytokines, and other metabolites involved in early stages of fibrogenesis.

Actins↗
PubMed2006

Switch of cadherin expression from E- to N-type during the activation of rat hepatic stellate cells.

The activation of hepatic stellate cell (HSC) is a common pathway leading to hepatic fibrosis. However, the molecular mechanisms underlying HSC activation remain obscure. To elucidate the nature of the HSC activation, we investigated the expression of E-cadherin and its switch to N-cadherin during rat HSC activation, in vivo and in vitro. Immunohistochemical and immunocytochemical staining were performed to identify the expressions of E-cadherin, N-cadherin, and beta-catenin in rat HSCs, in vivo and in vitro. Serial changes in the expressions of these adhesion molecules during the spontaneous activation of cultured rat HSCs were also demonstrated by RT-PCR and by immunoblotting. E-cadherin and beta-catenin were expressed on opposing cell membranes of GFAP-positive rat HSCs and adjacent hepatocytes in vivo, and between desmin-positive rat HSCs in vitro. With the progression of rat HSC activation in tissue and in culture, E-cadherin disappeared gradually, whereas N-cadherin appeared at the cell periphery. The results of RT-PCR and immunoblotting were concordant with immunocytochemistry findings. In conclusion, resting rat HSCs express E-cadherin and beta-catenin both in vivo and in vitro, and E-cadherin switches to N-cadherin during HSC activation. These results suggest that HSC activation represents transdifferentiation from an epithelial to a mesenchymal phenotype.

Animals↗
PubMed2000

Spatial and temporal organization of TrkB expression in the developing musculature of the mouse esophagus.

TrkB expression was investigated immunocytochemically in the developing musculature of mouse esophagus using conventional and confocal laser scanning microscopy. To demonstrate spatial relationships of TrkB immunoreactive cells to striated and smooth muscle fibers we combined TrkB immunocytochemistry with fluorochrome-tagged alpha-bungarotoxin for labeling of nicotinic acetylcholine receptors, and alpha-smooth muscle actin for labeling of smooth muscle cells. At developmental stages E15 to P7, TrkB immunoreactive cells transiently occurred in a transformation zone where striated intermingled with smooth muscle fibers. This transformation zone started in the rostral esophagus at E15, moved caudally, and disappeared between P7 and P10 in the caudal esophagus. The first TrkB-immunoreactive cells appeared in the outer muscle layer at E15. No TrkB-positive cells exhibited acetylcholine receptor clusters or were positive for alpha-smooth muscle actin. A few showed slight alpha-bungarotoxin staining over their entire surface. Taken together, the appearance of TrkB-expressing cells in the transformation zone suggest a role in muscle transdifferentiation. Alternatively, these results, together with recent in vitro data, suggest that TrkB is expressed in a subpopulation of myoblasts in which acetylcholine receptor clustering may be inhibited through a TrkB-mediated pathway.

Aging↗
PubMed2004

Phenotypic characterization of multi-functional somatotropes, mammotropes and gonadotropes of the mouse anterior pituitary.

The existence of bihormonal anterior pituitary (AP) cells co-storing growth hormone and either prolactin (mammosomatotrope) or gonadotropins (somatogonadotrope) has been described. These cells have been proposed to be involved in "paradoxical" secretion [secretion of an AP hormone induced by a non-related hypothalamic releasing factor (HRH) and transdifferentiation (a phenotypic switch between different cell types without cell division]. Here we combine calcium imaging (to assess HRH responsiveness) and multiple sequential immunoassay of the six AP hormones to perform a single-cell phenotypic study of multifunctional somatotropes, mammotropes and gonadotropes in the normal male and female mouse pituitaries. AP cell phenotypes differed from the classic view, showing multiple HRH-receptor expression and/or hormone storage. Mammosomatotropes represented only 5-6% of somatotropes and were poorly responsive to HRHs, suggesting that their contribution to paradoxical secretion should be very limited. Somatogonadotropes were present only in females and contained adrenocorticotropic hormone. They responded to growth hormone-releasing hormone but failed to respond to gonadotropin-releasing hormone (LHRH). Other polyhormonal cells identified include (1) gonadocorticotropes, restricted to females, where they make up more than 50% of all the gonadotropes and contain other AP hormones; (2) gonadomammotropes, which are present preferentially in female cells and respond to LHRH; and (3) gonadothyrotropes, which are present similarly in male and female pituitaries.

Animals↗
PubMed2002

Activity of cell-wall degradation associated with differentiation of isolated mesophyll cells of Zinnia elegans into tracheary elements.

Cell walls were prepared from cultured mesophyll cells of Zinnia elegans L. that were transdifferentiating into tracheary elements and incubated in a buffer to undergo autolysis. The rate of autolysis of cell walls was determined by measuring the amount of carbohydrate released from the cell walls into the buffer during incubation. During the course of culture of mesophyll cells, the autolysis rate increased markedly at the time when thickenings of secondary cell walls characteristic of tracheary elements became visible (after 48-72 h of culture), and thereafter the rate remained at a high level. Comparative studies on the autolysis rate of cell walls using various control cultures, in which tracheary element differentiation did not take place, revealed a close relationship between the autolysis rate around the 60th hour of culture and differentiation. Sugar analysis by colorimetric assays and gas chromatography of carbohydrates released from the cell walls detected uronic acid, arabinose, galactose, glucose, xylose, rhamnose, fucose, and mannose. Among these sugars, uronic acid was the most abundant, and accounted for approximately half of the total released sugars. The decrease of acidic polysaccharides in the primary cell walls during tracheary element differentiation was visualized by staining cultured cells with alcian blue at pH 2.5. These results suggest that active degradation of components of primary cell walls, including pectin, is integrated into the program of tracheary element differentiation.

Alcian Blue↗
PubMed2001

Involvement of local intercellular communication in the differentiation of zinnia mesophyll cells into tracheary elements.

The transdifferentiation of isolated mesophyll cells of zinnia (Zinnia elegans L.) into tracheary elements (TEs) has been well studied as a model of plant cell differentiation. In order to investigate intercellular communication in this phenomenon, two types of culture method were developed, in which mesophyll cells were embedded in a thin sheet of agarose gel and cultured on solid medium, or embedded in microbeads of agarose gel and cultured in liquid medium. A statistical analysis of the two-dimensional distribution of TEs in the thin-sheet cultures demonstrated their aggregation. In the microbead cultures, the frequency of TE differentiation was shown to depend on the local cell density (the cell density in each microbead): TE differentiation required local cell densities of more than 10(5) cells ml(-1). These results suggest that TE differentiation involves cell-cell communication mediated by a locally acting diffusible factor. This presumptive factor was characterized by applying a modified version of the sheet culture, which used two sheets of different cell densities, a low-density sheet and a high-density sheet. Differentiation of TEs in the former could be induced only by bringing it into contact with the latter. Insertion of a 25-kDa-cutoff membrane between the high-density and low-density sheets severely suppressed such induction of TEs in the low-density sheet while a 300-kDa-cutoff membrane suppressed induction only slightly. Insertion of agarose sheets containing immobilized pronase E or trypsin also interfered with the induction of TEs in the low-density sheets. Thus, a proteinaceous macromolecule of 25-300 kDa in molecular weight was assumed to mediate the local intercellular communication required for TE differentiation. This substance was designated "xylogen" with reference to its xylogenic activity. The time of requirement for xylogen during TE differentiation was assessed by experiments in which cells in the low-density sheet were separated from xylogen produced in the high-density sheet at various times by insertion of a 25-kDa-cutoff membrane between the two sheets, and was estimated to be from the 36th hour to the 60th hour of culture (12-36 h before visible thickening of secondary cell walls of TEs).

Asteraceae↗