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In vitro reconstruction of branched tubular structures from lung epithelial cells in high cell concentration gradient environment.

Hagiwara M, Peng F, Ho CM - Sci Rep (2015)

Bottom Line: However, homogeneous high cell concentration does not make a branching structure.Spatial distributions of morphogens, such as BMP-4, play important roles in the pattern formation.This simple yet robust system provides an optimal platform for the further study and understanding of branching mechanisms in the lung airway, and will facilitate chemical and genetic studies of lung morphogenesis programs.

View Article: PubMed Central - PubMed

Affiliation: 1] Nanoscience and Nanotechnology Research Center, Research Organization for the 21st Century, Osaka Prefecture University, 1-2, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan [2] Mechanical and Aerospace Engineering Department, University of California Los Angeles, 420 Westwood Plaza, Los Angeles, CA 90095, USA.

ABSTRACT
We have succeeded in developing hollow branching structure in vitro commonly observed in lung airway using primary lung airway epithelial cells. Cell concentration gradient is the key factor that determines production of the branching cellular structures, as optimization of this component removes the need for heterotypic culture. The higher cell concentration leads to the more production of morphogens and increases the growth rate of cells. However, homogeneous high cell concentration does not make a branching structure. Branching requires sufficient space in which cells can grow from a high concentration toward a low concentration. Simulation performed using a reaction-diffusion model revealed that long-range inhibition prevents cells from branching when they are homogeneously spread in culture environments, while short-range activation from neighboring cells leads to positive feedback. Thus, a high cell concentration gradient is required to make branching structures. Spatial distributions of morphogens, such as BMP-4, play important roles in the pattern formation. This simple yet robust system provides an optimal platform for the further study and understanding of branching mechanisms in the lung airway, and will facilitate chemical and genetic studies of lung morphogenesis programs.

No MeSH data available.


Related in: MedlinePlus

Fluorescent imaging result of BMP-4 expression on the branch.(A) Fluorescent images of nucleus and BMP-4 on developed branches from NHBE clot. BMP-4 expression was observed over the cell clot and branches. Scale bars 100 μm. (B) Comparison of fluorescent intensity of BMP-4 expression from homogeneously distributed NHBE and from NHBE cell clot with branches after day 7. The intensity from cell clot is higher than from homogeneously distributed NHBE (p = 0.014). Exposure time 300 ms. (n = 6).
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f5: Fluorescent imaging result of BMP-4 expression on the branch.(A) Fluorescent images of nucleus and BMP-4 on developed branches from NHBE clot. BMP-4 expression was observed over the cell clot and branches. Scale bars 100 μm. (B) Comparison of fluorescent intensity of BMP-4 expression from homogeneously distributed NHBE and from NHBE cell clot with branches after day 7. The intensity from cell clot is higher than from homogeneously distributed NHBE (p = 0.014). Exposure time 300 ms. (n = 6).

Mentions: It is considered in many studies that one of the potential activator, inhibitor and substrate for the lung airway are bone morphogenetic protein 4 (BMP-4), matrix GLA protein, and fibroblast growth factor 10 (FGF10) respectively1532333435. Fig. 5A shows the fluorescent image result by applying anti-BMP-4 antibody to the developed branches from NHBE clot. BMP-4 was expressed over the branches as well as the clot area. Next, fluorescent intensity of BMP-4 expression was measured in both cases of NHBE clots and homogeneously distributed NHBE culture after day 7 (Fig. 5B). The exposure time of UV light was set as 300 ms and the maximum fluorescent intensity was measured. Compared to the homogeneously distributed NHBE cell culture, the fluorescent intensities from cell clots were higher (p = 0.014). These experimental results indicate that cell clot produce much more activator (BMP-4) in a certain area.


In vitro reconstruction of branched tubular structures from lung epithelial cells in high cell concentration gradient environment.

Hagiwara M, Peng F, Ho CM - Sci Rep (2015)

Fluorescent imaging result of BMP-4 expression on the branch.(A) Fluorescent images of nucleus and BMP-4 on developed branches from NHBE clot. BMP-4 expression was observed over the cell clot and branches. Scale bars 100 μm. (B) Comparison of fluorescent intensity of BMP-4 expression from homogeneously distributed NHBE and from NHBE cell clot with branches after day 7. The intensity from cell clot is higher than from homogeneously distributed NHBE (p = 0.014). Exposure time 300 ms. (n = 6).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306969&req=5

f5: Fluorescent imaging result of BMP-4 expression on the branch.(A) Fluorescent images of nucleus and BMP-4 on developed branches from NHBE clot. BMP-4 expression was observed over the cell clot and branches. Scale bars 100 μm. (B) Comparison of fluorescent intensity of BMP-4 expression from homogeneously distributed NHBE and from NHBE cell clot with branches after day 7. The intensity from cell clot is higher than from homogeneously distributed NHBE (p = 0.014). Exposure time 300 ms. (n = 6).
Mentions: It is considered in many studies that one of the potential activator, inhibitor and substrate for the lung airway are bone morphogenetic protein 4 (BMP-4), matrix GLA protein, and fibroblast growth factor 10 (FGF10) respectively1532333435. Fig. 5A shows the fluorescent image result by applying anti-BMP-4 antibody to the developed branches from NHBE clot. BMP-4 was expressed over the branches as well as the clot area. Next, fluorescent intensity of BMP-4 expression was measured in both cases of NHBE clots and homogeneously distributed NHBE culture after day 7 (Fig. 5B). The exposure time of UV light was set as 300 ms and the maximum fluorescent intensity was measured. Compared to the homogeneously distributed NHBE cell culture, the fluorescent intensities from cell clots were higher (p = 0.014). These experimental results indicate that cell clot produce much more activator (BMP-4) in a certain area.

Bottom Line: However, homogeneous high cell concentration does not make a branching structure.Spatial distributions of morphogens, such as BMP-4, play important roles in the pattern formation.This simple yet robust system provides an optimal platform for the further study and understanding of branching mechanisms in the lung airway, and will facilitate chemical and genetic studies of lung morphogenesis programs.

View Article: PubMed Central - PubMed

Affiliation: 1] Nanoscience and Nanotechnology Research Center, Research Organization for the 21st Century, Osaka Prefecture University, 1-2, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan [2] Mechanical and Aerospace Engineering Department, University of California Los Angeles, 420 Westwood Plaza, Los Angeles, CA 90095, USA.

ABSTRACT
We have succeeded in developing hollow branching structure in vitro commonly observed in lung airway using primary lung airway epithelial cells. Cell concentration gradient is the key factor that determines production of the branching cellular structures, as optimization of this component removes the need for heterotypic culture. The higher cell concentration leads to the more production of morphogens and increases the growth rate of cells. However, homogeneous high cell concentration does not make a branching structure. Branching requires sufficient space in which cells can grow from a high concentration toward a low concentration. Simulation performed using a reaction-diffusion model revealed that long-range inhibition prevents cells from branching when they are homogeneously spread in culture environments, while short-range activation from neighboring cells leads to positive feedback. Thus, a high cell concentration gradient is required to make branching structures. Spatial distributions of morphogens, such as BMP-4, play important roles in the pattern formation. This simple yet robust system provides an optimal platform for the further study and understanding of branching mechanisms in the lung airway, and will facilitate chemical and genetic studies of lung morphogenesis programs.

No MeSH data available.


Related in: MedlinePlus