Cells of mouse submandibular salivary gland in culture in vitro

A cell culture that preserves its phenotype up to the 20th passage was obtained from mouse submandibular salivary glands. An analysis of the heterogeneous culture indicates the existence of several morphological types of cells, including small, densely packed cells of cuboidal or polygonal shapes and large, rounded cells. Epithelial cells of the submandibular gland cultured for several weeks were able to form tubular structures. Our studied cell culture of glandulocytes (cells of glandular epithelium) was represented by K19- and NGF-positive cells. It is important to note that, using both immunocytochemical staining and PCR, the expression of genes that encode the proinsulin and insulin proteins is revealed in the studied cell population.     

Elevated apoptosis in pre-mature neurons differentiated from mouse ES cells containing a single human chromosome 21

A decrease in the number and density of neurons is the most common phenotype in the brains of Down syndrome (DS) patients, causing mental retardation. Studies using primary cultured neurons from DS patients or from model mice have suggested that a defect in metabolism of reactive oxygen species, or diminished levels of glutathione, causes mitochondrial and caspase-mediated neuronal apoptosis in vitro. However, it is not well documented whether neuronal apoptosis also occurs in immature DS neurons, owing to the difficulty in isolating or identifying neuronal stem cells in human or mouse fetuses. Here we utilized an in vitro model system for neuronal differentiation, with mouse embryonic stem cells containing human chromosome 21 (TT2F/hChr.21) to examine the effect of an additional hChr.21 on the early phases of neurogenesis. The differentiation profile of TT2F/hChr.21 cells was essentially the same as those of parental TT2F ES cells. In differentiations of both TT2F and TT2F/hChr.21 cells, high level of apoptosis was observed in neuronal stem cells, but the rate of apoptosis in TT2F/hChr.21 cells was significantly higher than that of parental cells. These results suggest that quantitative changes in the level of apoptosis in DS neuronal stem cells may account for the reduction of neuronal number and density in the DS brain.     

Study of cell culture of mouse submandibular salivary gland in vitro

Cell culture keeping its phenotype till the 20th passage was obtained from mouse submandibular salivary glands. The analysis of the heterogeneous culture showed that there were some morphological types of cells: densely packed small cells that had cuboidal or polygonal form and also large round cells. Epithelial cells of submandibular gland cultured during several weeks were able to form tubular strucures. It was shown that glandulocyte culture was presented by K19-positive and NGF-positive cells. It is important to note that expression of genes coding proinsulin and insulin was detected by immunocytochemical staining and by PCR as well.     

Potassium release from submandibular salivary gland in vitro

Rat submandibular gland slices, incubated in continuously-gassed Krebs-Ringer bicarbonate buffer, were shown to release K⁺ in response to α-adrenergic and muscarinic cholinergic stimulation. The system employed the specific α-, β-adrenergic and cholinergic receptor-blocking agents phentolamine, propranolol and atropine, respectively, in combination with the agonists l-epinephrine and carbamylcholine both of which required the presence of Ca²⁺ for their effect. The introduction of Ca²⁺ into the cell via the ionophore A23187, with all neurotransmitter receptors blocked, resulted in K⁺ release. Ouabain also allowed extensive K⁺ release which was in addition to, and hence independent of, that elicited by epinephrine and carbamylcholine. Ethacrynic acid, a potent inhibitor of salivary secretion in vivo, had no influence on K⁺ movement. K⁺ was released by both physalaemin and an eledoisin-related peptide independently of normal neurotransmitter receptors. The activity of the eledoisin-related peptide did not require the presence of extracellular Ca²⁺. The implication of cyclic GMP at some stage of K⁺ release was suggested by experiments with a phosphodiesterase inhibitor.The results support an hypothesis where the initial stimulus at either α-adrenergic or muscarinic cholinergic receptors causes an immediate permeability change such that Ca²⁺ enters the cells resulting in K⁺ release. The loss of K⁺ is quickly countered by the ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$) ATPase which would be activated by the lowered intracellular K⁺ levels.     

N-cadherin is a useful marker for the progenitor of cardiomyocytes differentiated from mouse ES cells in serum-free condition

Cardiomyocytes are known to differentiate spontaneously from embryonic stem (ES) cells when they formed aggregates, so called "embryoid bodies", in the presence of serum. In this study, we explored the induction of cardiomyocytes from mouse ES cells in chemically defined serum-free medium by using a mesoderm-inducing factor, BMP4. Comparing the different inductive methods, we found a candidate cell surface marker, N-cadherin, for cardiomyocyte progenitors from ES cells. N-cadherin-positive cells highly expressed cardiogenic markers, Nkx2.5, Tbx5, and Isl1, and showed a high differentiation rate into cardiomyocyte lineage. These results indicate that N-cadherin can be a useful cell surface marker for the progenitors of cardiomyocyte differentiated from ES cells in the serum-free culture.     

Isolation and maintenance of differentiated exocrine gland acinar cells in vitro

Summary Methods have been developed for isolating and maintaining differentiated rat exorbital lacrimal, parotid, and pancreatic acinar cells for up to 1 month in culture. The dissociated cells retained their differentiated morphology when cultured as suspension cultures at 35°C with the appropriate secretagogue (exorbital lacrimal, 10⁻⁶ M carbamyl choline; pancreas 10⁻⁵ M carbamyl choline; parotid, 10⁻⁶ M isoproterenol). Under these conditions the cells remained viable and differentiated for up to 4 weeks in culture and continued to incorporate³H-leucine at rates similar to those of freshly isolated cells. If secretagogue was omitted from the medium, the cells rapidly degenerated. These results indicate that differentiated from the medium, the cells rapidly degenerated. These results indicate that differentiated exocrine gland acinar cells may be maintained in vitro and utilized as a model system for the study of secretory processes.     

Transfer into the mouse of mast cells differentiated in vitro

A population of mast cells can be derived in vitro by culturing normal spleen cells from C57BL/6 mice with the factor Interleukin 3. These mast cells share the morphological and histochemical features of mast cells at different stages of maturation. We have labelled these in vitro produced mast cells with 111In-Ox and injected them i. v. into normal syngeneic mice. The localisation of labelled cells has been determined 24 and 96 hours after the injection in the spleen, thymus and lymph-nodes.     

Collagenase inhibitor stimulates cleft formation during early morphogenesis of mouse salivary gland

A collagenase inhibitor obtained from the culture medium of bovine dental pulp markedly enhanced the cleft formation of mouse embryonic salivary gland epithelium when the inhibitor was included in the culture medium for 12-day and 13-day salivary glands. Determination of collagenase activity using [3H]collagen as substrate indicated that there was a latent collagenase activity in 12-day glands. In addition, a highly purified Clostridial collagenase freed from protease and hyaluronidase activities, strongly inhibited initiation of the cleft formation of the 12-day epithelium. Scanning electron microscopic observation showed that abundant collagen-like fibrils were seen on the epithelium in the collagenase-inhibitor-treated glands compared to those in the control. These findings suggest that during early morphogenesis tissue collagenase may regulate the cleft formation in the epithelium.     

Endocrine cells develop within pancreatic bud-like structures derived from mouse ES cells differentiated in response to BMP4 and retinoic acid

We have examined factors affecting the in vitro differentiation of Pdx1^GFP/w ESCs to pancreatic endocrine cells. Inclusion of Bone Morphogenetic Protein 4 (BMP4) during the first four days of differentiation followed by a 24-hour pulse of retinoic acid (RA) induced the formation of GFP⁺ embryoid bodies (EBs). GFP expression was restricted to E-cadherin⁺ tubes and GFP bright (GFP^br) buds, reminiscent of GFP⁺ early foregut endoderm and GFP^br pancreatic buds observed in Pdx1^GFP/w embryos. These organoid structures developed without further addition of exogenous factors between days 5 and 12, suggesting that day 5 EBs contained a template for the subsequent phase of development. EBs treated with nicotinamide after day 12 of differentiation expressed markers of endocrine and exocrine differentiation, but only in cells within the GFP^br buds. Analysis of Pdx1^GFP/w ESCs modified by targeting a dsRed1 gene to the Ins1 locus (Pdx1^GFP/wIns1^RFP/w ESCs) provided corroborating evidence that insulin positive cells arose from GFP^br buds, mirroring the temporal relationship between pancreatic bud development and the formation of endocrine cells in the developing embryo. The readily detectable co-expression of GFP and RFP in grafts derived from transplanted EBs demonstrated the utility of Pdx1^GFP/wIns1^RFP/w ESCs for investigating pancreatic differentiation in vitro and in vivo.     

Expression profiling of placentomegaly associated with nuclear transplantation of mouse ES cells

Transplantation of nuclei (NT) from engineered mouse ES cells is a potentially powerful and rapid route to create knockout mice, obviating the need for matings to obtain germ-line chimeras. However, such an application is currently impossible, because NT often results in abnormalities in embryo and placenta. Although the epigenetic instability of several imprinted genes in ES cells and ES-derived NT mice has been demonstrated, it is not clear yet what causes the abnormalities. To gain perspective on the extent and types of changes, we have done gene expression profiling for mouse placentas produced by NT of ES cells and compared them with the expression profiles of placentas produced by NT of one-cell embryos. Based on microarray studies with the NIA 15K mouse cDNA collection, we report five principal aberrant events: (1) inappropriate expression of imprinted genes; (2) altered expression of regulatory genes involved in global gene expression, such as DNA methyltransferase and histone acetyltransferase; (3) increased expression of oncogenes and growth promoting genes; (4) overexpression of genes involved in placental growth, such as Plac1; and (5) identification of many novel genes overexpressed in ES-derived NT mouse placentas, including Pitrm1, a new member of the metalloprotease family. The results indicate that placentomegaly in ES-derived NT mice is associated with large-scale dysregulation of normal gene expression patterns. The study also suggests the presence of two regulatory pathways that may lead to histologically discernable placentomegaly. The discovery of groups of genes with altered expression may provide potential targets for intervention to mimic natural regulation more faithfully in NT mice.     

In vitro enhanced differentiation of neural networks in ES gut-like organ from mouse ES cells by a 5-HT4-receptor activation

Using an embryoid body (EB) culture system, we developed a functional organ-like cluster, a “gut”, from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited various types of spontaneous movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (ICC) (c-kit, a transmembrane receptor that has tyrosine kinase activity, positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified, but enteric neural networks were not identified. In the present study, we succeeded in forming dense enteric neural networks by a 5-HT₄-receptor (SR4) agonist, mosapride citrate (1–10 μM) added only during EB formation. Addition of an SR4-antagonist, GR113808 (10 μM) abolished the SR4-agonist-induced formation of enteric neural networks. The SR4-agonist (1 μM) up-regulated the expression of mRNA of SR4 and the SR4-antagonist abolished this upregulation. 5-HT per se exerted similar effects to those of SR4-agonist, though less potent. These results suggest SR4-agonist differentiated enteric neural networks, mediated via activation of SR4 in the ES gut.     

Development of an optimized 5-stage protocol for the in vitro preparation of insulin-secreting cells from mouse ES cells

In order to produce insulin-secreting cells with a high value of glucose-stimulated insulin secretion (GSIS) from mouse embryonic stem cells, we have developed an optimized 5-stage protocol by referring to culture conditions so far reported elsewhere. This protocol is characterized by 4 points: (1) use of an activin-free medium in the first stage, (2) use of gelatin/fibronectin coated culture dishes in 1–4 stages throughout, (3) removal of undifferentiated cells by cell sorter at the end of 4th stage, and (4) sedimental culture in the 5th stage. GSIS value of the produced cells reached 2.4, that was at a higher rank of those so far reported. The produced cells were transplanted in diabetes model mice but no remedy effect was observed. Then transplantation was conducted in pre-diabetes model mice, in which GSIS was impaired without affecting insulin producing function. The transplantation of 5 × 10⁶ cells resulted in a marked improvement of glucose tolerance within 20 days. This effect decreased but was still observed at 120 days post-transplantation. This demonstrates the feasibility of the novel optimized protocol.