Three-dimensional Gel Culture Research Articles

Developmental time rather than local environment regulates the schedule of epithelial polarization in the zebrafish neural rod

BackgroundMorphogenesis requires developmental processes to occur both at the right time and in the right place. During neural tube formation in the zebrafish embryo, the generation of the apical specializations of the lumen must occur in the center of the neural rod after the neural cells have undergone convergence, invagination and interdigitation across the midline. How this coordination is achieved is uncertain. One possibility is that environmental signaling at the midline of the neural rod controls the schedule of apical polarization. Alternatively, polarization could be regulated by a timing mechanism and then independent morphogenetic processes ensure the cells are in the correct spatial location.ResultsEctopic transplantation demonstrates the local environment of the neural midline is not required for neural cell polarization. Neural cells can self-organize into epithelial cysts in ectopic locations in the embryo and also in three-dimensional gel cultures. Heterochronic transplants demonstrate that the schedule of polarization and the specialized cell divisions characteristic of the neural rod are more strongly regulated by time than local environmental signals. The cells’ schedule for polarization is set prior to gastrulation, is stable through several rounds of cell division and appears independent of the morphogenetic movements of gastrulation and neurulation.ConclusionsTime rather than local environment regulates the schedule of epithelial polarization in zebrafish neural rod.

Laminin 5 Regulates Polycystic Kidney Cell Proliferation and Cyst Formation

Renal cyst formation is the hallmark of autosomal dominant polycystic kidney disease (ADPKD). ADPKD cyst-lining cells have an increased proliferation rate and are surrounded by an abnormal extracellular matrix (ECM). We have previously shown that Laminin 5 (Ln-5, a alpha(3)beta(3)gamma(2) trimer) is aberrantly expressed in the pericystic ECM of ADPKD kidneys. We report that ADPKD cells in primary cultures produce and secrete Ln-5 that is incorporated to the pericystic ECM in an in vitro model of cystogenesis. In monolayers, purified Ln-5 induces ERK activation and proliferation of ADPKD cells, whereas upon epidermal growth factor stimulation blocking endogenously produced Ln-5 with anti-gamma(2) chain antibody reduces the sustained ERK activation and inhibits proliferation. In three-dimensional gel culture, addition of purified Ln-5 stimulates cell proliferation and cyst formation, whereas blocking endogenous Ln-5 strongly inhibits cyst formation. Ligation of alpha(6)beta(4) integrin, a major Ln-5 receptor aberrantly expressed by ADPKD cells, induces beta(4) integrin phosphorylation, ERK activation, cell proliferation, and cyst formation. These findings indicate that Ln-5 is an important regulator of ADPKD cell proliferation and cystogenesis and suggest that Ln-5 gamma(2) chain and Ln-5-alpha(6)beta(4) integrin interaction both contribute to these phenotypic changes.

Fibronectin induces ureteric bud cells branching and cellular cord and tubule formation

The extracellular matrix (ECM) protein fibronectin is involved in several stages of embryogenesis. Fibronectin exerts its effect through interaction with cellular integrin and nonintegrin receptors. We investigated the effect of fibronectin on branching and tubulogenesis of ureteric bud cells in a three-dimensional gel culture system. Primary ureteric bud cells from mouse embryos at gestation 11 days (E11) were isolated and established in culture. Fibronectin and integrin subunits were localized using immunoperoxidase staining. In three-dimensional collagen type I gel culture of ureteric bud cell, fibronectin dose dependently induces cord and tubule formation. Both ureteric bud cells and ureteric bud branches in embryonic kidney express the same multiple integrin subunits that include beta(1), beta(3), alpha(3), alpha(4) and alpha(v). Embryonic kidneys examined at E12, E14, and E16 days of gestation express fibronectin in the undifferentiated mesenchyme especially next to ureteric bud branches and in the interstitium around glomerulotubular structures and blood vessels. Fibronectin expression was similar at the tips and stalks of branching ureteric bud. Fibronectin expression is maximum at E12 and decreases with advanced gestation. Cultured ureteric bud cells also express fibronectin. RGD peptides inhibit cord and tubular formation in the three-dimensional gel. Anti-alpha(3)beta(1) antibody partially inhibits fibronectin-induced cord and tubule formation. Hepatocyte growth factor (HGF), fibroblast growth factor (FGF), and glial cell line-derived neurotrophic factor (GDNF) induce ureteric bud cell cord formation in three-dimensional gel. The effects of growth factors are delayed and quantitatively less compared to the effect of fibronectin. Fibronectin induces ureteric bud cells branching and tubulogenesis through interaction with multiple integrin receptors. Cultured ureteric bud cells express fibronectin and the origin of fibronectin at mesenchyme-ureteric bud interface is likely both the metanephric mesenchyme and ureteric bud epithelium. Addition of individual neutralizing antibodies to beta(1), beta(3), alpha(3), alpha(4,)alpha(6) and alpha(v) integrin subunits does not block the effect of fibronectin. Only an antibody to alpha(3)beta(1) integrin substantially blocks the effect of fibronectin. Other mechanisms, including unidentified integrins, are likely involved in fibronectin-induced cord and tubule formation.

Cigarette smoke inhibits osteogenic differentiation and proliferation of human osteoprogenitor cells in monolayer and three-dimensional collagen gel culture

Cigarette smoke is a risk factor not only for emphysema but also for other disorders characterized by deficient tissue repair, including osteoporosis. We hypothesized, therefore, that smoke might directly impair bone cell repair processes. To evaluate this, bone marrow osteoprogenitor cells were isolated from normal subjects and cultured in monolayer and in three-dimensional type I collagen gel culture. Human osteoprogenitor cells could be induced to differentiate toward osteoblast-like cells in both culture conditions by osteogenic supplements. Under both culture conditions, cigarette smoke extract (CSE) inhibited the proliferation of osteoprogenitor cells in a concentration-dependent manner. CSE also inhibited differentiation of osteoprogenitor cells toward osteoblast-like cells as assayed by alkaline phosphatase activity and calcium incorporation into cell layer. Cells in monolayer culture were more sensitive to the effect of smoke than cells in three-dimensional gel culture. Similar results were obtained with osteoblast-like cells derived from osteosarcomas. This study, therefore, demonstrates that cigarette smoke may affect bone progenitor cells directly and in this manner may contribute to the development of osteoporosis. (J Lab Clin Med 2001;137:208-19)

Retardation of Phenotypic Transition of Rabbit Arterial Smooth Muscle Cells in Three-Dimensional Primary Culture

Three-dimensional gel culture systems represent conditions that mimic the differentiated state of mesenchymal cellsin vivo.We examined gel contraction, cell growth, and phenotypic modulation of rabbit arterial SMC in three-dimensional gel culture. The gel contraction rate was dependent on the collagen type; that is, the contraction by freshly isolated SMC was faster and more pronounced in type I collagen than in type III collagen. In contrast, the phenotypic modulation of SMC was independent of collagen type. The major portion of cells in both type I and III collagens with growth factors underwent transition from a contractile (G0 phase) to a synthetic phenotype (G1B phase), but this transition was clearly delayed compared with that on collagens. The cells had hardly begun DNA synthesis in either collagen type and failed to proliferate even after 10 days of culture. These results indicate that collagen type is important in gel contraction by vascular SMC, while the organization of collagen fibrils (two-dimensional vs three-dimensional) is more critical in the phenotypic transition and proliferation of these cells. However, the more specific organization of extracellular matrix than the collagen gel culture system may be necessary to maintain the contractile phenotype of SMC.

Interleukin-6 expression by fibroblasts grown in three-dimensional gel cultures

We investigated the expression and biological activity of interleukin-6 (IL-6) by human fibroblasts cultured as monolayers and within three-dimensional type I collagen lattices. In the course of contracting the gel to a dense tissue-like structure, the cells upregulated their levels of IL-6 mRNA as well as IL-6 biological activity. While there was little mRNA and protein activity (6,500 U/ml) in monolayer cultures, fibroblasts in the 3D system showed a 13-fold increase in IL-6 mRNA on day 3. IL-6 protein was increased 6-fold (38,000 U/ml) on day 4. Stimulation of fibroblast cultures with IL-1α resulted in enhanced IL-6 production in both systems, but the fibroblasts embedded into the 3D network continued to exhibit higher levels.