Nullipotent Teratocarcinoma Cells Acquire the Pluripotency for Differentiation by Fusion with Somatic Cells

Abstract. By fusion of nullipotent embryonal carcinoma F9 cells with certain somatic cells freshly collected from differentiated tissues such as thymus and lens, pluripotent hybrid cell lines were obtained. They exhibited a wide spectrum of differentiation, including neural tubes, cartilages, skeletal muscles, ciliated epithelia and others, in solid tumors formed after injection into syngeneic mice. Cells from these tumors differentiated into several cell types when cultured in vitro. A possibility of the introduction of genes to code the factors for regulating differentiation into F9 cells by fusion is suggested.     

In situ localization of T cell receptor beta chain in the murine thymus: changes in the intrathymic distribution of thymocytes expressing beta chain during fetal development

The expression of T cell receptor beta chain in the developing thymus was examined at the light and electron microscopic levels using the monoclonal antibody F23.1. Cells expressing cytoplasmic forms of beta chain were first observed at Day 16 of gestation, while thymocytes expressing cell surface beta chain were detected about a day later. Clustering of cortical F23.1+ cells was more pronounced in fetal thymus when compared to adult. The density of F23.1+ cells in the subcapsular areas of the thymus was initially lower than that in the rest of the cortex or the medulla. Within the subcapsular and cortical areas of the thymus there was an inverse relationship between the density of F23.1+ cells and cells labeled with the lectin from Dolichos bifloris, which binds to terminal alpha-linked N-acetylgalactosamine residues preferentially expressed by L3T4-/Lyt2- thymocytes. Although this pattern was less pronounced with increasing gestational age, it was still apparent at birth.     

Differentiation potential of intestinal mesenchyme and its interaction with epithelial cells: a study using beta-galactosidase-expressing fibroblast lines

Rat intestinal fibroblast lines (F1:G9 and A1:F1) differing in their potential to support intestinal mucosal development were marked with reporter genes to investigate their differentiation potential. The fibroblasts were transfected with plasmids expressing either beta-galactosidase (with or without a nuclear localisation signal) or green fluorescent protein (GFP). Transfection using Tfx50 or Fugene was more efficient than electroporation. The expression of beta-galactosidase was more stable and stronger than GFP. Cells were optimally labelled using the plasmid pL27B-GAL, and sub-clones with a strong and uniform nuclear expression of beta-galactosidase were isolated. These clones expressed beta-galactosidase even after prolonged passage in the absence of selection. The beta-galactosidase tagged lines (F1:G9gal and A1:F1gal) retained the morphological characteristics, viability and differentiation properties of the parental non-transfected lines. In co-culture with a colorectal tumour cell line Caco-2, the F1:G9gal and A1:F1gal cells differed in their morphological organisation but this did not change their expression of smooth muscle alpha-actin.     

Differential modulation of cell phenotype by different molecular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight forms

To study possible functional differences of the 18-kD and high molecular weight forms of basic fibroblast growth factor (bFGF), we have examined the effect of endogenous production of different bFGF forms on the phenotype of NIH 3T3 cells. Cells transfected with cDNAs coding for either 18-kD bFGF (18-kD bFGF) or all four molecular forms (18, 22, 22.5, 24 kD; wild type [WT] bFGF) exhibit increased migration and decreased FGF receptor number compared to parental cells. However, migration and FGF receptor number of cells transfected with a cDNA coding only for high molecular weight bFGF (22, 22.5, and 24 kD; HMW bFGF) were similar to that of parental cells transfected with vector alone. Cells expressing HMW, 18 kD, or WT bFGF grew to high saturation densities in 10% serum. However, only cells expressing HMW or WT bFGF grew in low serum. Cell surface or metabolic labeling of the different cell types followed by immunoprecipitation with anti-bFGF antibody showed primarily cell surface-associated 18-kD bFGF. In addition, when cells expressing exclusively HMW bFGF were transfected with a cDNA coding for 18-kD bFGF, migration was increased, bFGF receptors were down-regulated, and 18-kD bFGF was found on the cell surface. Cells expressing 18-kD bFGF transfected with a cDNA encoding FGF receptor-2 lacking the COOH-terminal domain (dominant negative bFGF receptor) exhibited a flat morphology and decreases in migration and saturation density. Cells expressing HMW bFGF transfected with the dominant negative bFGF receptor continued to grow to a high saturation density, proliferated in low serum, and exhibited no morphological changes. These results indicate that increased cell migration and FGF receptor down-regulation are mediated by the extracellular interaction of 18-kD bFGF with its cell surface receptor. Growth in low serum may be stimulated by the intracellular action of HMW bFGF through mechanisms independent of the presence of a cell surface receptor. Thus, the different molecular forms of bFGF may act through distinct but convergent pathways.     

Impaired Shc, Ras, and MAPK activation but normal Akt activation in FL5.12 cells expressing an insulin-like growth factor I receptor mutated at tyrosines 1250 and 1251

The Y1250F/Y1251F mutant of the insulin-like growth factor I receptor (IGF-IR) has tyrosines 1250 and 1251 mutated to phenylalanines and is deficient in IGF-I-mediated suppression of apoptosis in FL5.12 lymphocytic cells. To address the mechanism of loss of function in this mutant we investigated signaling responses in FL5.12 cells overexpressing either a wild-type (WT) or Y1250F/Y1251F (mutant) IGF-IR. Cells expressing the mutant receptor were deficient in IGF-I-induced phosphorylation of the JNK pathway and had decreased ERK and p38 phosphorylation. IGF-I induced phosphorylation of Akt was comparable in WT and mutant expressing cells. The decreased activation of the mitogen-activated protein kinase (MAPK) pathways was accompanied by greatly decreased Ras activation in response to IGF-I. Although phosphorylation of Gab2 was similar in WT and mutant cell lines, phosphorylation of Shc on Tyr(313) in response to IGF-I was decreased in cells expressing the mutant receptor, as was recruitment of Grb2 and Ship to Shc. However, phosphorylation of Shc on Tyr(239), the Src phosphorylation site, was normal. A role for JNK in the survival of FL5.12 cells was supported by the observation that the JNK inhibitor SP600125 suppressed IGF-I-mediated protection from apoptosis. Altogether these data demonstrate that phosphorylation of Shc, and assembly of the Shc complex necessary for activation of Ras and the MAPK pathways are deficient in cells expressing the Y1250F/Y1251F mutant IGF-IR. This would explain the loss of IGF-I-mediated survival in FL5.12 cells expressing this mutant and may also explain why this mutant IGF-IR is deficient in functions associated with cellular transformation and cell migration in fibroblasts and epithelial tumor cells.     

Involvement of bone marrow-derived vascular progenitor cells in neovascularization during formation of the corpus luteum in mice

Neovascularization is necessary for formation of the corpus luteum (CL) and includes angiogenesis and vasculogenesis. Vasculogenesis is the formation of new blood vessels by bone marrow-derived endothelial progenitor cells. Here we investigated whether vasculogenesis occurs in neovascularization during CL formation. Mice transplanted with bone marrow from transgenic mice expressing green fluorescent protein (GFP) were injected with equine chorionic gonadotropin and human chorionic gonadotropin (hCG) to induce ovulation and subsequent CL formation. Immunohistochemistry was performed on the ovaries obtained before hCG injection and at 6, 12, and 24 h after hCG injection using antibodies for CD34 or CD31 (an endothelial cell marker), platelet-derived growth factor receptor beta (PDGFR-beta, a pericyte marker), F4/80 (a macrophage marker), and GFP (a bone marrow-derived cell marker). Cells immunostained for CD34, PDGFR-beta, F4/80, and GFP were present in the theca cell layer of the preovulatory follicle before hCG injection. Each of these cell types invaded the granulosa cell layer after hCG injection, and a number of them were observed in the CL 24 h after hCG injection. Fluorescence-based immunohistochemistry or double immunohistochemical staining revealed that a few CD34/CD31-positive cells and PDGFR-beta-positive cells were also positive for GFP in the preovulatory follicle and CL, and that many of the GFP-positive cells recruited to the CL during CL formation were F4/80-positive macrophages. In conclusion, bone marrow-derived vascular progenitor cells and macrophages contribute to neovascularization during CL formation.     

Cutting edge: Role of Toll-like receptor 9 in CpG DNA-induced activation of human cells

Unmethylated CpG motifs present in bacterial DNA stimulate a rapid and robust innate immune response. Human cell lines and PBMC that recognize CpG DNA express membrane-bound human Toll-like receptor 9 (hTLR9). Cells that are not responsive to CpG DNA become responsive when transfected with hTLR9. Expression of hTLR9 dramatically increases uptake of CpG (but not control) DNA into endocytic vesicles. Upon cell stimulation, hTLR9 and CpG DNA are found in the same endocytic vesicles. Cells expressing hTLR9 are stimulated by CpG motifs that are active in primates but not rodents, suggesting that evolutionary divergence between TLR9 molecules underlies species-specific differences in the recognition of bacterial DNA. These findings indicate that hTLR9 plays a critical role in the CpG DNA-mediated activation of human cells.     

The Actin Binding Domain of the Epidermal Growth Factor Receptor Is Required for EGF-Stimulated Tissue Invasion

NIH-3T3 fibroblasts expressing epidermal growth factor receptors (EGFRs) lacking the actin binding domain (ABD) were analyzed for their EGF-induced capacity to invade a bone marrow stromal cell (BMSC) monolayer. The fibroblasts display a reduction in the percentage of cytoskeleton-associated EGFRs. Furthermore, EGF-induced tyrosine kinase activity is unaffected by the mutation. Cells expressing the mutant EGFRs hardly invade a BMSC monolayer upon EGF stimulation in contrast to cells expressing wild-type EGFRs. Using the same cells no difference was observed in PDGF-induced invasion, which ligand was as potent in both cell types as EGF was in wild-type cells. Inhibition of both the phosphatidyl inositol-3-kinase (PI-3-K) and lipoxygenase pathways in wild-type cells mimicked the effect of the ABD deletion. Our results point to an important role for the ABD of the EGFR in EGF-induced tissue invasion.     

The growth and morphological characteristics of undifferentiated and differentiated cells of the F9 mouse teratocarcinoma line

Differentiation of the F9 cell line was induced by treating the cells with retinoic acid (10(-6) M) and dibutyryl cycloadenosinemonophosphate (10(-4) M). The population doubling time and the portion of cells in G1-phase increase and saturation density falls as the result of this treatment. Differentiated F9 cells demonstrate a decreased capacity of forming colonies in the soft agar, lose their capacity of proliferating at the clonal density, and acquire the limited life-span in culture after reseeding at a high density. Some cells in the differentiated population retain their capacity of forming colonies in the soft agar and (or) of binding antibodies against the stem cell marker SSEA-1. Cells with the stem cell morphology were found in the course of passaging of differentiated cells after reseeding at a high density. These cells were able to differentiate after the standard procedure of the induction of differentiation with retinoic acid and dibutyryl cAMP. Causes of the rising and supporting of heterogeneity of the differentiated F9 cells are discussed.     

A morphological study of cells isolated from the perfused gill of dab and Atlantic salmon

Single cell suspensions were derived from the gills of dab Limanda limanda and Atlantic salmon Salmo salar which were free from blood cell contamination. Gills were perfused with heparinized saline before the cells were liberated from their resident position using a procedure employing chelating agents and collagenase digestion. Cells were characterized using light and electron microscopy as well as histochemical staining. The results indicate that the cell types resident in the gill tissue should be capable of expressing local immune competence. Small and large lymphocytes, macrophages, neutrophils, eosinophilic granule cells, goblet cells, chloride cells and cells of unusual morphology were identified especially the presence of goblet cells apparently within epithelial-like cells.     

Neural precursors derived from human embryonic stem cells

Before the successful isolation of human embryonic stem (hES) cells, many investigations had shown that mouse embryonic stem (mES) cells can be induced to differentiate into neural precursors which could be purified and differentiated to mature dopamine, motor, serotonin, GABA neurons, and oligodendrocytes and astrocytes in vitro[1―3]. mES cell-derived dopamine neurons have been shown capable of integrating into host brains after transplanting to the rodents of Park-inson’s disease model …     

Neural precursors derived from human embryonic stem cells

Human embryonic stem (hES) cells provide a promising supply of specific cell types for transplantation therapy. We presented here the method to induce differentiation of purified neural precursors from hES cells. hES cells (Line PKU-1 and Line PKU-2) were cultured in suspension in bacteriological Petri dishes, which differentiated into cystic embryoid bodies (EBs). The EBs were then cultured in N₂ medium containing bFGF in poly-L-lysine-coated tissue culture dishes for two weeks. The central, small cells with 2–3 short processes of the spreading outgrowth were isolated mechanically and replated. The resulting neurospheres were cultured in suspension for 10 days, then dissociated into single cell suspension with a Pasteur pipette and plated. Cells grew vigorously in an attached way and were passed every 4–5 days. Almost all the cells were proved nestin positive by immunostaining. Following withdrawal of bFGF, they differentiated into neurons expressing β-tubulin isotype_III, GABA, serotonin and synaptophysin. Through induction of PDGF-AA, they differentiated into astrocytes expressing GFAP and oligodendrocytes expressing O₄. The results showed that hES cells can differentiate into typical neural precursors expressing the specific marker nestin and capable of generating all three cell types of the central nervous system (CNS)in vitro.     

SLC26A9 stimulates CFTR expression and function in human bronchial cell lines

We investigated the possible functional‐ and physical protein‐interactions between two airway Cl^? channels, SLC26A9 and CFTR. Bronchial CFBE41o‐ cell lines expressing CFTR_WT or CFTR_ΔF508 were transduced with SLC26A9. Immunoblots identified a migrating band corresponding to SLC26A9 present in whole‐cell lysates as on apical membrane of cells grown on polarized filters. CFTR levels were increased by the presence of SLC26A9 in both CFTR_WT and CFTR_ΔF508 cell lines. In CFBE41o‐ cells and CFBE41o‐/CFTR_WT cells transduced with SLC26A9, currents associated to the protein expression were not detected. However, the forskolin (FK)‐stimulated currents were enhanced in SLC26A9‐transduced cells compared to control cells. Therefore, the presence of SLC26A9 resulted in an increase in CFTR activity (same % of CFTR_(inh)‐172 or GlyH‐101 inhibition in both groups). In CFBE41o‐/CFTR_ΔF508 cells transduced with SLC26A9 (at 27°C), a current associated to the protein expression was also lacking. FK‐stimulated currents and level of CFTR_(inh)‐172 inhibition were not different in both groups. The presence of SLC26A9 in Xenopus oocytes expressing CFTR also enhanced the FK‐stimulated currents as compared to oocytes expressing CFTR alone. This stimulation was mostly linked to CFTR. An enhancement of FK‐stimulated currents was not found in oocytes co‐expressing SLC26A9 and CFTR_ΔF508. In conclusion, in both protein expression systems used, SLC26A9 stimulates CFTR activity but not that of CFTR_ΔF508. Our co‐immunoprecipitation studies demonstrate a physical interaction between both anion channels. We propose as an alternative hypothesis (not exclusive) to the known SLC26A9‐STAS domain/CFTR interaction, that SLC26A9 favors the biogenesis and/or stabilization of CFTR, leading to stimulated currents. J. Cell. Physiol. 226: 212–223, 2010. © 2010 Wiley‐Liss, Inc.     

Water fluxes through aquaporin-9 prime epithelial cells for rapid wound healing

Cells move along surfaces both as single cells and multi-cellular units. Recent research points toward pivotal roles for water flux through aquaporins (AQPs) in single cell migration. Their expression is known to facilitate this process by promoting rapid shape changes. However, little is known about the impact on migrating epithelial sheets during wound healing and epithelial renewal. Here, we investigate and compare the effects of AQP9 on single cell and epithelial sheet migration. To achieve this, MDCK-1 cells stably expressing AQP9 were subjected to migration assessment. We found that AQP9 facilitated cell locomotion at both the single and multi-cellular level. Furthermore, we identified major differences in the monolayer integrity and cell size upon expression of AQP9 during epithelial sheet migration, indicating a rapid volume-regulatory mechanism. We suggest a novel mechanism for epithelial wound healing based on AQP-induced swelling and expansion of the monolayer.     

Constitutive c-myc expression enhances proliferation of differentiating F9 teratocarcinoma cells

The c-myc protooncogene is expressed in many tumor cells as well as during normal development. In order to study the role of c-myc in differentiation, proliferation and tumorigenicity of F9 mouse teratocarcinoma cells, the pSVmyc1 plasmid constitutively expressing an active c-myc oncogene was introduced into F9 stem cells by cotransfection with the selectable marker RSVneo. Enhanced expression of c-myc did not alter the properties of F9 stem cells. Prolonged proliferation during retinoic acid induced differentiation was observed in cell clones constitutively expressing c-myc. In contrast, as determined by morphology, by immunocytochemistry for markers specific for stem cells and differentiated derivatives, and by Northern hybridization for mRNAs specific for differentiated cells, differentiation was neither inhibited nor delayed by constitutive c-myc expression. Tumorigenicity of stem cells as well as retinoic acid-treated cells--as measured by soft agar cloning efficiency and tumor formation in syngenic mice--was not altered by SVmyc1. We conclude that in F9 teratocarcinoma cells down-regulation of c-myc is related to arrest of proliferation rather than differentiation.     

Characterization of cells in the developing human liver

Human hepatic progenitor cells (HPCs) have been shown to co-express the hematopoietic stem cell (HSC) markers, CD117 and CD34. These cells differentiate not only into hepatocytes and cholangiocytes but also into pancreatic ductal and acinar cells under certain conditions. The fetal liver (FL) is rich in precursor/stem cells; however, little is known about (i) the markers expressed by liver cells during fetal development and (ii) whether an equivalent to the adult liver stem-like progenitors exists in the FL. Here, (i) FL tissue obtained from human 5-18-week-old fetuses were evaluated by means of flow cytometry, immunocyto-, and histochemistry for the emergence of cells expressing and co-expressing known hematopoietic, hepatic, and pancreatic cell markers, and (ii) isolated putative HPCs were phenotypically and molecularly characterized. We report that (i) red blood and endothelial cell precursors were most abundant in early gestation. Cells expressing HSC and pancreatic markers were found in the first trimester, while cells expressing hepatic markers appeared in the second trimester. Very few committed cells were present in FLs obtained early in the first trimester. In addition, cells expressing pancreatic markers co-expressed the HSC marker CD117. (ii) Isolated CD117+/CD34+/CD90- cells in vitro expressed both the genes and proteins for the hepatic markers such as albumin, alpha feto protein (AFP), alpha1-antitrypsin, and cytokeratin 19 (CK19). Our study suggests that hepatoblast and ductal plate/bile duct development mainly occurs during the second trimester. FLs in gestation weeks 5-9 had the highest numbers of precursor cells and the least committed cells. Cells that differentiate into Alb+ or CK19+ can be isolated from early FLs and may be appropriate progenitors for establishing novel systems to investigate basic mechanisms for cell therapy.     

Analysis of the differentiation-promoting potential of inducible c-fos genes introduced into embryonal carcinoma cells.

To investigate the differentiation-promoting potential of c-fos in embryonal carcinoma cells (EC cells) we have designed various human metallothionein promoter-mouse-c-fos gene constructs containing also the selectable SV40 promoter-driven neo gene. Upon transfection into F9 EC cells and selection for neo resistance, the following results were obtained. (i) With each of the constructs, colonies of morphologically altered and differentiated (i.e., TROMA-1 and TROMA-3 expressing) cells were identified. (ii) Expression of c-fos was required to affect the differentiation state of F9 cells to a significant extent, but a low level was sufficient; no enhancement of differentiation was noticeable even after 100-fold induction of c-fos expression by cadmium. (iii) F9 cell clones were isolated which, in spite of very high levels of exogenous c-fos expression, had stem cell morphology. These cells, however, continuously generated morphologically altered and differentiated cells upon subculturing. (iv) In other EC cell lines, which resemble stem cells more closely than the 'partially differentiated' F9 cells, c-fos expression showed either a less pronounced (P19 cells) or no differentiation-promoting effect at all (PC13 cells). Our results suggest that the c-fos gene product acts in concert with other, probably 'spontaneously' occurring events to promote differentiation of certain EC cell lines.