Target cells for lithium in different forms within a heterogeneous hepatocarcinoma-29 population

Liver cancer is an aggressive and heterogeneous human tumor. Lithium compounds block proliferation and induce apoptosis of hepatocarcinoma cells, but cannot cause the death of an entire population of tumor cells. The aim of this study was to reveal morphological types of target cells for different lithium preparations on the basis of their action on hepatocarcinoma-29 cells. The viability of hepatocarcinoma-29 cells was assessed by the MTT test. A dose-dependent decrease in viability was revealed upon addition of native and nanosized lithium carbonate and citrate. Target cells for lithium salts were revealed based on the morphological criteria for five differentiation stages of hepatocarcinoma-29 cells. It was shown that hepatocarcinoma- 29 proliferating cells of differentiation stages I and II are the target cells for native and nanosized lithium citrate, while differentiated cells of differentiation stages III and IV are the target cells for nanosized lithium carbonate. It was revealed that hepatocarcinoma-29 cells are more sensitive to nanosized lithium salts rather than to their native forms. This makes it possible to affect tumor growth more effectively.     

Alkaline phosphatase reactivity in rabbit airway epithelium: a potentially useful marker for airway basal cells

The purpose of this study was to investigate alkaline phosphatase (ALPase) reactivity in rabbit airway epithelial cells. Acetone-fixed, methyl benzoate and xylene-cleared (AMeX-treated) paraffin sections of trachea, bronchus, and lung tissue were stained by an azo dye coupling method for ALPase and examined by light microscopy. Electron histochemical staining was also performed in order to study the sensitivity and specificity of reactivity in each cell type. ALPase reactivity at the light microscopic level was observed exclusively in trachco-bronchial basal cells, and not in bronchiolar basal cells. By electron microscopy, ALPase reactivity was noted in 97.9% of basal cells in the trachea, 97.0% of basal cells in the bronchus, and 94.5% of basal cells and 15.4% of Clara cells in the bronchiole. This was also true for dispersed tracheal epithelial cells. Reactivity was rarely observed in ciliated cells, non-goblet-type secretory cells, and undetermined cells. The reactivity was heatlabile, levamisole-sensitive, and of a non-specific type. These findings indicate that basal cells of rabbit trachea and bonchus have fairly high specificity for ALPase of a non-specific isozyme (92.2% and 95.6%, respectively). Therefore, ALPase is considered to be a useful marker for these cells.     

Preparation and properties of an antiplasma cell serum directed against a defined plasmacytoma cell population.

Heterologous antisera were prepared against a subpopulation of MOPC-104E tumor cells obtained by centrifugation on discontinuous BSA gradients as well as against cells from the whole tumor mass. The gradient-separated cells were more effective than the cells from the whole tumor mass in eliciting antisera not only higher titer, but also with greater specificity for plasmacytoma antigens. The unabsorbed antiserum prepared against the gradient-separated plasmacytoma population was cytotoxic for murine lymphoid cells, but not for murine kidney, liver, or brain cells. After in vitro absorption with murine thymocytes and removal of anti-immunoglobulin activity by affinity chromatography, the antiserum was found to be reactive against plasmacytoma cells, but was no longer cytotoxic for murine thymus or unstimulated spleen cells. This absorbed antiserum was also cytotoxic for LPS-, but not PHA- or Con A-stimulated normal murine spleen cells.     

Comparison of dark- and light-adapted carp retinas with NADPH diaphorase staining

The carp retina was examined by NADPH diaphorase histochemistry to determine if the staining pattern of retinal cells was changed depending on the adaptation state of the retina. When dark-adapted for 5 h, ellipsoids of inner segments of both rods and cones and some horizontal cells were heavily stained. Staining was also found in subpopulations of amacrine cells and ganglion cells. In addition, Muller cells were strongly positive for NADPH diaphorase. When light-adapted for 5h, ellipsoids of photoreceptors and ganglion cells were less intensely stained, whereas Muller cells and horizontal cells became negative for NADPH diaphorase. Furthermore, rod ON-center bipolar cells were clearly stained. The difference of staining of amacrine cells between dark- and light-adapted retinas was not significant. The differences in diaphorase-staining pattern between dark- and light-adapted retinas suggest that Muller cells, some horizontal cells and rod ON-center bipolar cells contain inducible nitric oxide synthase,     

The effects of wild-type and mutant HFE expression upon cellular iron uptake in transfected human embryonic kidney cells

In order to measure the effects of HFE (haemochromatosis) upon iron uptake, stable expression of wild-type and C282Y, H63D and S65C mutant HFE cDNA was established in HEK 293 cells. Control cells were transfected with empty vector. Expression of HFE mRNA and protein was detected in the cell lines transfected with HFE cDNA, but not in the control cell line. The ferritin concentration in wild-type cells cultured in 40 microM ferric ammonium citrate was 69% of that in control cells and 81% of that in C282Y cells. The ferritin concentration in H63D cells was intermediate between wild-type and C282Y and the ferritin concentration in S65C cells was similar to wild-type cells. Uptake of transferrin-iron in wild-type, C282Y and control cells was measured over 45 min. The Hill coefficients for transferrin-iron uptake were similar. The V(max) for transferrin-iron uptake in wild-type cells was 59.5% of control cells and 69.5% of C282Y cells. Estimates of K(m) were 232 nM for wild-type cells, 338 nM for C282Y cells and 570 nM for controls. Transferrin receptor levels were lowered, but not significantly, in the HFE transfected cells. The results show that HFE reduces transferrin-iron uptake, probably as an uncompetitive inhibitor.     

Apoptosis of mouse embryonic stem cells induced by single cell suspension

Embryonic stem cells (ES cells) are pluripotential, and are therefore used to construct gene knock-out mice. We found that the apoptosis of mouse ES cells was induced when the cells were dispersed as single cells, whereas this process was suppressed when they proliferated in aggregates. The apoptosis of ES cells was repressed when the cells were cultured on feeders prepared from STO cells, a cell line established from embryonic fibroblasts. Culture supernatants from STO cells did not block the apoptosis of ES cells, which suggests that a direct interaction between ES cells and STO cells is required for the suppression of apoptosis. The viability of ES cells examined by the trypan blue exclusion test or by the MTT ((3-4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay decreased dramatically when the cells were dispersed in phosphate-buffered saline PBS. Cellular activity was restored by the addition of culture medium for ES cells. Glucose in the medium was found to be a major factor responsible for the restoration. Amino acids also restored the decrease in reduction of MTT. Suspension of the ES cells in PBS(-) caused leakage of the nucleosome into cytoplasm. Results indicate that the single cell suspension of ES cells leads to leakage of substrates for oxidative phosphorylation from the mitochondria, and that these cells finally become committed to apoptosis.     

Suppression of interleukin-2 production by human concanavalin A-induced suppressor cells

When PHA-activated normal responder cells (R cells) were cocultured with mononuclear cells (MN cells) which had been preincubated for 48 hr in medium alone (C cells) an enhanced proliferative response was observed. This enhancement was only obtained when the R cells were cultured with allogeneic C cells or when PHA was in the cocultures for the entire culture period. This effect was due to greater production of interleukin 2 (IL-2) by irradiated C cells in the presence of allogeneic or mitogenic stimulation. Con A-treated mononuclear cells (S cells) cultured with PHA-activated allogeneic or autologous responder cells showed reduced [3H]thymidine incorporation and IL-2 production as compared to activated R cells alone. Glutaraldehyde-treated S cells (which retained the ability to absorb IL-2) did not affect the proliferative response or IL-2 production by the R cells, indicating that passive absorption of IL-2 was not entirely responsible for suppression induced by S cells. S cells, pretreated with IL-2, still inhibited R-cell activity. These results show that Con A-treated MN cells suppressed or prevented [3H]thymidine incorporation by actively inhibiting IL-2 production.     

Pigment recovery from encapsulated-dehydrated Vaccinium pahalae (ohelo) cryopreserved cells

Pigment producing in vitro cells of Vaccinium pahalae (ohelo) were tested for their ability to survive cryopreservation and retain pigment-production capacity after encapsulation-dehydration. Preculture of cells for 6 to 8 days in a medium containing 1.0 M sucrose was essential before dehydration. Reduction of bead water content before quenching in liquid nitrogen was highly correlated (r = 0.94) with increased survival rate in cells after cryopreservation. Dehydration of beads for 4 h was satisfactory for survival of cells. After quenching in liquid nitrogen, colored cells became pale, but pigment content was recovered once cells resumed growth. After three subcultures, cells regained their maximum capacity for pigment accumulation. The percentage of colored-to-total cell volume was not influenced by cryopreservation. Encapsulation-dehydration and cryopreservation did not diminish the capacity of cells to produce anthocyanins and other flavonoids. This revised version was published online in June 2006 with corrections to the Cover Date.     

Varicella-zoster virus glycoprotein I is essential for growth of virus in Vero cells.

Varicella-zoster virus (VZV) encodes at least six glycoproteins. Glycoprotein I (gI), the product of open reading frame 67, is a 58- to 62-kDa glycoprotein found in VZV-infected cells. We constructed two VZV gI deletion mutants. Immunoprecipitation of VZV gE from infected cells indicated that cells infected with VZV deleted for gI expressed a gE that was larger (100 kDa) than that expressed in cells infected with the parental virus (98 kDa). Cell-associated or cell-free VZV deleted for gI grew to lower titers in melanoma cells than did parental VZV. While VZV deleted for gI replicated in other human cells, the mutant virus replicated to very low titers in primary guinea pig and monkey cells and did not replicate in Vero cells. When compared with the parental virus, rescued viruses, in which the gI deletion was restored with a wild-type allele, showed a similarly sized gE and comparable growth patterns in melanoma and Vero cells. VZV deleted for gI entered Vero cells; however, viral DNA synthesis was impaired in these cells. The VZV gI mutant was slightly impaired for adsorption to human cells. Thus, VZV gI is required for replication of the virus in Vero cells, for efficient replication of the virus in nonhuman cells, and for normal processing of gE.     

Cytochemical and biochemical characterization of testicular peritubular myoid cells

Testicular peritubular myoid cells secrete a paracrine factor that is a potent modulator of Sertoli cell functions involved in the maintenance of spermatogenesis. These cells also play an integral role in maintaining the structural integrity of the seminiferous tubule. To better understand this important testicular cell type, studies were initiated to characterize cultured peritubular cells using biochemical and histochemical techniques. The electrophoretic pattern of radiolabeled secreted proteins was similar for primary and subcultured peritubular cells and was unique from that of Sertoli cells. Morphologic differences between Sertoli cells and peritubular cells were noted and extended with histochemical staining techniques. Desmin cytoskeletal filaments were demonstrated immunocytochemically in peritubular cells, both in culture and in tissue sections, but were not detected in Sertoli cells. Desmin is proposed to be a marker for peritubular cell differentiation as well as a marker for peritubular cell contamination in Sertoli cell cultures. Peritubular cells and Sertoli cells were also stained histochemically for the presence of alkaline phosphatase. Staining for the alkaline phosphatase enzyme was associated with peritubular cells but not with Sertoli cells. Alkaline phosphatase is therefore an additional histochemical marker for peritubular cells. Biochemical characterization of peritubular cells relied on cell-specific enzymatic activities. Creatine phosphokinase activity, a marker for contractile cells, was found to be associated with peritubular cells, while negligible activity was associated with Sertoli cells. Alkaline phosphatase activity assayed spectrophotometrically was found to be a useful biochemical marker for peritubular cell function and was utilized to determine the responsiveness of primary and subcultured cells to regulatory agents. Testosterone stimulated alkaline phosphatase activity associated with primary cultures of peritubular cells, thus supporting the observation that peritubular cells provide a site of androgen action in the testis. Retinol increased alkaline phosphatase activity in subcultured peritubular cells. Alkaline phosphatase activity increased in response to dibutyryl cyclic adenosine monophosphate (AMP) in both primary and subcultured peritubular cell cultures. Observations indicate that the ability of androgens and retinoids to regulate testicular function may be mediated, in part, through their effects on peritubular cells. This provides additional support for the proposal that the mesenchymal-epithelial cell interactions between peritubular cells and Sertoli cells are important for the maintenance and control of testicular function. Results imply that the endocrine regulation of tissue function may be mediated in part through alterations in mesenchymal-epithelial cell interactions.     

Suppression of the in vitro secondary response to syngeneic tumor and of in vivo tumor therapy with immune cells by culture-induced suppressor cells.

Mice with advanced disseminated syngeneic tumor can be successfully treated with a combination of chemotherapy and adoptively transferred syngeneic immune cells. We have previously demonstrated that in vivo primed cells secondarily sensitized in vitro became more effective in tumor therapy, whereas primed cells cultured for 5 days without tumor stimulation became less effective than an equal number of uncultured fresh primed cells. Therefore, we examined stimulated and unstimulated cultures of tumor-primed cells for the presence of culture-induced suppressor cells, and determined whether in vivo tumor therapy with immune cells could be inhibited by concurrent inoculation of immune effector cells and cultured normal spleen cells, which contain culture-induced suppressor cells but are devoid of additional effector cells. The in vitro primary allogeneic response was suppressed by cultured normal spleen cells, or tumor-primed spleen cells previously cultured for 5 days with or without tumor stimulation. In vitro secondary sensitization to syngeneic tumor was suppressed by normal or tumor-primed cells that had previously been cultured for 5 days without stimulation. The majority of this suppression was mediated by T cells in the cultured populations. The efficacy of fresh tumor-primed cells, as well as primed cells secondarily sensitized in vitro, in adoptive chemoimmunotherapy of advanced tumor was diminished by concurrent inoculation of cultured normal cells. The cells mediating suppression of in vivo therapy required previous in vitro culture for induction, and were radiation sensitive.     

Amplification of sodium- and potassium-activated adenosinetriphosphatase in HeLa cells by ouabain step selection

A multistep selection for ouabain resistance was used to isolate a clone of HeLa S3 cells that overproduces the plasma membrane sodium, potassium activated adenosinetriphosphatase (Na+,K+-ATPase). Measurements of specific [3H]ouabain-binding to the resistant clone, C+, and parental HeLa cells indicated that C+ cells contain 8-10 X 10(6) ouabain binding sites per cell compared with 8 X 10(5) per HeLa cell. Plasma membranes isolated from C+ cells by a vesiculation procedure and analyzed for ouabain-dependent incorporation of [32P]phosphate into a 100,000-mol-wt peptide demonstrated a ten- to twelvefold increase in Na+,K+-ATPase catalytic subunit. The affinity of the enzyme for ouabain on the C+ cells was reduced and the time for half maximal ouabain binding was increased compared with the values for the parental cells. The population doubling time for cultures of C+ cells grown in dishes was increased and C+ cells were unable to grow in suspension. Growth of C+ cells in ouabain-free medium resulted in revertant cells, C-, with biochemical and growth properties identical with HeLa. Karyotype analysis revealed that the ouabain-resistant phenotype of the C+ cells was associated with the presence of minute chromosomes which are absent in HeLa and C- cells. This suggests that a gene amplification event is responsible for the Na+,K+-ATPase increase in C+ cells.     

Binding of diphtheria toxin to CHO-K1 and Vero cells is dependent on cell density

We studied the binding of 125I-labeled diphtheria toxin (DTX) to receptors on monolayer cultures of Chinese hamster ovary cells (CHO-K1) and Vero cells. The number of DTX receptors detected on the cell surface was shown to be dependent on the cell density (number of cells per unit area). Cells at low density (less than 23,000 cells per cm2 for CHO-K1 cells; less than 80,000 cells per cm2 for Vero cells) had more receptors for DTX than cells at higher densities. The difference in receptor number between low- and high-density cells was 33-fold for CHO-K1 cells and 19-fold for Vero cells. We estimated the maximum number of DTX receptors on low-density CHO-K1 and Vero cells to be 50,000 and 370,000 per cell, respectively. The cell density at which the binding of DTX was reduced to 50% of maximum was considerably lower for CHO-K1 cells than for Vero cells (33,000 vs. 220,000 cells per cm2, respectively). Vero cells grown on a surface that had been conditioned by high-density cells bound less DTX, suggesting that interaction of these cells with the underlying extracellular matrix might regulate the number of cell surface receptors for DTX. Low-density cells were more sensitive to DTX than high-density cells, suggesting that low-density cells possessed an increased number of functional receptors that actively transported DTX to the cytosol. CHO-K1 and Vero cells were equally protected by SITS (4-Acetamido-4'-Isothiocyano-Stilbene-2,2'-disulfonic Acid), a compound that has been shown to inhibit the binding and entry of DTX in Vero cells, suggesting that intoxication of CHO-K1 and Vero cells is mediated by a similar mechanism. The data illustrate the importance of taking into account the cell density when measuring the number of DTX receptors on adherent cells.     

On the importance of the level of glutathione and the activity of the pentose phosphate pathway in heat sensitivity and thermotolerance

Heating of Ehrlich ascites tumour (EAT) cells and mouse fibroblast LM cells to 43 or 44 degrees C respectively, results in an increased level of reduced glutathione (GSH). The maximum elevation in GSH was to 140 per cent for LM cells and to 120 per cent for EAT cells. No increase of GSH in EAT cells was observed after heating at 44 degrees C. LM cells were treated with diethylmaleate (DEM) and the EAT cells with buthionine-sulphoximine (BSO) at non-toxic doses to deplete the levels of GSH. No effect on thermosensitivity or on the development of thermotolerance was observed when the DEM and BSO treatments were chosen such that the lowering of GSH was just down to the level of detection (about 5 per cent of control). When higher concentrations of DEM were used, thermal sensitization was observed. The activity of the pentose phosphate pathway (PPP) was also investigated because of its importance in supplying NADPH for the regeneration of GSH from GSSG and for the endogenous production of polyols. Hyperthermia was found to enhance markedly the flux of glucose through the PPP. While the DEM treatment inhibited glucose oxidation through the PPP, BSO addition to the cells resulted in a slightly increased activity of the PPP. The PPP activity of thermotolerant cells was lower (fibroblasts) or hardly affected (EAT cells) compared to control cells. The extent of PPP activation by hyperthermia was comparable for thermotolerant and control cells. For the two cell lines studied neither a high level of GSH nor an active PPP is a prerequisite for the development of thermotolerance.     

Suppressor cell activity of ovine caruncular and intercaruncular tissues during the placentation period

We assessed the suppression of lymphocyte proliferation ovine endometrial cells recovered during each trimester (Days 45, 90, and 135) of pregnancy. We evaluated fractionated and unfractionated caruncular (C) and intercaruncular (IC) cells for suppression of cocultured peripheral blood lymphocytes (PBL) in phytohemagglutinin (PHA)-treated cultures. We also evaluated cells for the release of the suppressor factor into the culture medium and tested the factor for transforming growth factor-beta (TGF-beta) activity. Suppression of PHA-induced proliferation of PBL was evident for C and IC cells recovered from each day of pregnancy, and the activity was predominantly attributed to the population(s) of low-density (1.006-1.054 g/ml) cells. The activity was greater for unfractionated C than for IC cells on Day 45, whereas the pattern was reversed by Day 135 of pregnancy. For the C cells, the activity was greatest on Day 45 and least by Day 135. Although suppressor factor was released into the medium from cultured C and IC cells, its activity was not apparently mediated by TGF-beta. In conclusion, we observed a temporal pattern in suppressor activity for unfractionated endometrial cells during pregnancy. Suppression was predominately mediated by a population(s) of low-density cells. In addition, the cells released a soluble suppressor factor that seems to be unrelated to TGF-beta. The suppressor cells may provide immunological protection to the fetoplacental unit by suppressing specific lymphocyte responses directed toward conceptus tissues.     

The radiation response of cells recovering after chronic hypoxia

Experiments were performed to study the influence of hypoxic pretreatment on the radiation response of A431 human squamous carcinoma cells. Reaeration for 10 min after chronic hypoxia (greater than 2 h) was found to enhance the radiosensitivity of A431 cells, and the maximal effect was seen for those cells reaerated after 12 h of hypoxia. The radiosensitivity enhancement for reaerated cells after 12 h of hypoxia was maximized by 5 min after the return to aerobic conditions and reached the control level by 12 h of reaeration. This enhanced radiosensitive state was characterized by a reduced shoulder region and increased slope of the radiation dose-response curve for cells in both the exponential and plateau phases of growth. There was a slight increase in the number of G1 and decrease in the number of S and G2 + M cells for both exponential- and plateau-phase cultures following 12 h hypoxic treatment. Although growth inhibition induced by 12 h of hypoxia was seen for cells in the exponential phase, there was no cell number change in the plateau-phase culture after hypoxia. Plating efficiency (PE) of cells in both growth phases was reduced by 30% after hypoxia. Furthermore, in the exponential-phase culture, the extent of reduction in PE after hypoxia was similar among cells in different phases of the cell cycle. Although S-phase cells in exponentially growing cultures were relatively more resistant to radiation than G1 and G2 + M cells, the cell age-response pattern was the same whether the cells had been aerobic or hypoxic before reaeration and irradiation. Furthermore, the enhancement ratio associated with reaeration after 12 h of hypoxia for these three subpopulations of cells was 1.3. Our results indicate that the increase in radiosensitivity due to reaeration after chronic hypoxia is unlikely to be related to the changes of cell cycle stage and growth phase during hypoxic treatment.     

Isolation of two distinct populations of cells from rat kidney cortex and their use in the study of chemical-induced toxicity

Procedures for the isolation and enrichment of cell populations from suspensions of rat kidney cortical cells were developed. Using Percoll density-gradient centrifugation, two populations of cells were obtained; marker enzymes [alkaline phosphatase and gamma-glutamyltransferase for proximal tubular (PT) cells and hexokinase for distal tubular (DT) cells] and functional responses (stimulation of PT cell oxygen consumption by succinate and inhibition of DT cell oxygen consumption by amiloride) were then employed to identify and assess the purity of the two fractions. The PT cell fraction was estimated to contain 97% PT cells and the DT cell fraction was estimated to contain 88% DT cells. Staining with toluidine blue and light microscopy showed that PT cells contained a brush border, were larger than DT cells, and had more intensely staining nuclei than DT cells. To demonstrate the usefulness of these cell preparations in the study of biochemical mechanisms of renal cell injury, time- and concentration-dependent effects of the PT cell-specific nephrotoxin cephaloridine (CPH) on PT and DT cell trypan blue exclusion were examined. CPH was toxic in PT cells but not in DT cells; viability of PT cells incubated with 0.1 or 1 mM CPH for 2 h was 57 or 34%, respectively, compared to 81% for control cells; viability of DT cells incubated with 0.1 or 1 mM CPH for 2 h was 74 or 71%, respectively, compared to 74% for control cells. This method thus provides highly enriched preparations of freshly isolated PT and DT cells that retain their unique properties and are suitable for studies of biochemical mechanisms of chemical toxicity and nephron heterogeneity.     

Deoxyglucose transport of uninfected, murine sarcoma virus-transformed, and murine leukemia virus-infected mouse cells

The initial rates of deoxy-D-glucose transport by cultures of growing and density-inhibited mouse embryo cells and lines of mouse cells transformed spontaneously or after infection by murine leukemia virus or murine sarcoma virus were investigated as a function of the deoxyglucose concentration. The apparent K_m for deoxyglucose transport was about the same for all types of cells (1–2 mM). The V_max of secondary cultures of mouse embryo cells decreased from 6 nmoles/10⁶ cells/minute for sparse cultures to less than 1 nmole/10⁶ cells/minute for density-inhibited cultures. The V_max was about the same whether estimated in monolayer culture or in suspensions of cells dispersed by treatment with trypsin. The V_max for deoxyglucose transport by the established cells, whether transformed spontaneously or by virus infection, was 4 to 25 times higher than that for density-inhibited mouse embryo cells and was independent of the cell density of the cultures. Deoxyglucose transport was competitively inhibited by Cytochalasin B, Persantin, glucose and 3-O-methyl-D-glucose and the apparent K_i values of inhibition were similar for the mouse embryo cells and the various cell lines. Similarly, the sensitivity of the glucose transport systems to inactivation by p-chloromercuribenzoate was about the same for all types of cells. The results suggest that the glucose transport system of the normal mouse embryo cells and the cells of the various established lines is qualitatively the same, but that the number of functional transport sites differs for the various cell lines and decreases markedly in mouse embryo cells with an increase in cell density of the cultures.     

Development and retroviral transduction of porcine neonatal pancreatic islet cells in monolayer culture

To learn more about the potential of neonatal porcine pancreatic duct and islet cells for xenotransplantation, the development of these cells when cultured as monolayers was evaluated. Immunostaining for islet hormones and cytokeratin-7 revealed that day eight monolayers consisted of approximately 70% duct cells and less than 10% beta cells. Using Ki-67 immunostaining as a proliferation marker, the fraction of beta cells in the cell cycle was shown to decrease from 20% at day three to 10% at day eight, and for duct cells from 36 to 19%. Insulin secretion increased 2.4-fold upon glucose stimulation, and 38-fold when 10 mm theophylline was added, showing the responsiveness of the neonatal beta cells. Reaggregated monolayers consisted mostly of duct cells, but 4 weeks after transplantation, grafts contained predominantly endocrine cells, with duct cells being almost absent, suggesting in vivo differentiation of duct cells to endocrine cells. Monolayer susceptibility to retroviral transduction was also investigated using a Moloney Murine Leukemia Virus-based vector. Approximately 60% of duct cells but less than 5% of beta cells expressed the transgene, indicating that precursor duct cells are better targets for transgene expression. These results show that porcine neonatal pancreatic cells can be cultured as monolayers in preparation for transplantation. Furthermore, in such a culture setting, precursor duct cells have a high rate of proliferation and are more efficiently transduced with a retrovirus-based reporter gene than are beta cells.     

Monoclonal side population progenitors isolated from human fetal pancreas

The side population (SP) phenotype might represent a common molecular feature for a wide variety of stem cells. The aim of this study was to investigate whether monoclonal SP progenitor cells were established from human fetal pancreas. Islet-like cell clusters (ICCs) were isolated from human fetal pancreas. Monolayer epithelium-like cells were obtained from the ICCs and passaged thereafter. Single SP or non-SP cells were sorted from these cells at the sixth passage. The rate of clone formation was about 2.7% for the SP cells, whereas there was no clone formation for the non-SP cells. The SP cell clones were further expanded for more than 15 passages and induced for differentiation into cells with characteristics of pancreatic beta-cells. We show for the first time that the monoclonal SP progenitors are established from human fetal pancreas. Therefore, this study may offer a novel method to purify pancreatic progenitor cells from human tissues.