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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

NHBE cell morphogenesis following homogeneous distribution of cells in Matrigels.(A) Phase contrast images of homogeneously distributed NHBE cells cultured with various cell concentrations at day 7. Even when cells were seeded at high concentration, no branching was observed under homogeneous seeding conditions. Scale bar: 100 μm. (B) Result of reaction-diffusion model calculations with various cell concentrations. Cells were distributed homogeneously in a confined space. Y distribution indicates normalized cells positions and size. H distribution indicates normalized inhibitor concentration distributions produced by cells themselves. At higher cell concentrations, the inhibitor was occupied in a confined space and inhibited cell growth. Parameters: c = 0.04 ± 5%, μ = 0.12, ν = 0.04, ρA = 0.014, ρH = 0.00014, c0 = 0.02, γ = 0.02, ε = 0.087, d = 0.0013, e = 0.1, f = 10, DA = 0.015, DH = 0.18, DS = 0.06.
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f4: NHBE cell morphogenesis following homogeneous distribution of cells in Matrigels.(A) Phase contrast images of homogeneously distributed NHBE cells cultured with various cell concentrations at day 7. Even when cells were seeded at high concentration, no branching was observed under homogeneous seeding conditions. Scale bar: 100 μm. (B) Result of reaction-diffusion model calculations with various cell concentrations. Cells were distributed homogeneously in a confined space. Y distribution indicates normalized cells positions and size. H distribution indicates normalized inhibitor concentration distributions produced by cells themselves. At higher cell concentrations, the inhibitor was occupied in a confined space and inhibited cell growth. Parameters: c = 0.04 ± 5%, μ = 0.12, ν = 0.04, ρA = 0.014, ρH = 0.00014, c0 = 0.02, γ = 0.02, ε = 0.087, d = 0.0013, e = 0.1, f = 10, DA = 0.015, DH = 0.18, DS = 0.06.

Mentions: Next, we examined the spatial requirements for branching morphogenesis. NHBE were filled in culturing area and distributed homogeneously in 100 μl Matrigel with various cell concentrations (Fig. 4A). Cells formed spherical colonies, but no branches were observed at any concentrations at day 7 although branches were developed rapidly with higher cell concentration when cells were confined to a limited area in the Matrigel (as described above). This observation was supported by another RD model after 2000 iterations of calculations (Fig. 4B). Cells were set homogeneously over the calculation area with concentrations of 50%, 12.5%, 3.1%, and 0.8%. Y distribution shows the actual cell positions and H distribution shows the inhibitor concentration distribution over the calculation area. At higher coverage concentration (50%, 12.5%, 3.1%), the inhibitor filled the entire space, since its diffusion rate is much faster than that of the activator3031. Thus, cells were prevented from developing branching structures. On the other hand, while cells seeded at lower coverage (0.8%) had more space to grow, the amount of activator they encountered locally was insufficient to produce branching; this is because the distance between neighboring cells was too far. This indicates the importance of proximity for activator-induced branching, as some of the cells were accidentally close to each other, and thus elongated in small spaces.


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)

NHBE cell morphogenesis following homogeneous distribution of cells in Matrigels.(A) Phase contrast images of homogeneously distributed NHBE cells cultured with various cell concentrations at day 7. Even when cells were seeded at high concentration, no branching was observed under homogeneous seeding conditions. Scale bar: 100 μm. (B) Result of reaction-diffusion model calculations with various cell concentrations. Cells were distributed homogeneously in a confined space. Y distribution indicates normalized cells positions and size. H distribution indicates normalized inhibitor concentration distributions produced by cells themselves. At higher cell concentrations, the inhibitor was occupied in a confined space and inhibited cell growth. Parameters: c = 0.04 ± 5%, μ = 0.12, ν = 0.04, ρA = 0.014, ρH = 0.00014, c0 = 0.02, γ = 0.02, ε = 0.087, d = 0.0013, e = 0.1, f = 10, DA = 0.015, DH = 0.18, DS = 0.06.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: NHBE cell morphogenesis following homogeneous distribution of cells in Matrigels.(A) Phase contrast images of homogeneously distributed NHBE cells cultured with various cell concentrations at day 7. Even when cells were seeded at high concentration, no branching was observed under homogeneous seeding conditions. Scale bar: 100 μm. (B) Result of reaction-diffusion model calculations with various cell concentrations. Cells were distributed homogeneously in a confined space. Y distribution indicates normalized cells positions and size. H distribution indicates normalized inhibitor concentration distributions produced by cells themselves. At higher cell concentrations, the inhibitor was occupied in a confined space and inhibited cell growth. Parameters: c = 0.04 ± 5%, μ = 0.12, ν = 0.04, ρA = 0.014, ρH = 0.00014, c0 = 0.02, γ = 0.02, ε = 0.087, d = 0.0013, e = 0.1, f = 10, DA = 0.015, DH = 0.18, DS = 0.06.
Mentions: Next, we examined the spatial requirements for branching morphogenesis. NHBE were filled in culturing area and distributed homogeneously in 100 μl Matrigel with various cell concentrations (Fig. 4A). Cells formed spherical colonies, but no branches were observed at any concentrations at day 7 although branches were developed rapidly with higher cell concentration when cells were confined to a limited area in the Matrigel (as described above). This observation was supported by another RD model after 2000 iterations of calculations (Fig. 4B). Cells were set homogeneously over the calculation area with concentrations of 50%, 12.5%, 3.1%, and 0.8%. Y distribution shows the actual cell positions and H distribution shows the inhibitor concentration distribution over the calculation area. At higher coverage concentration (50%, 12.5%, 3.1%), the inhibitor filled the entire space, since its diffusion rate is much faster than that of the activator3031. Thus, cells were prevented from developing branching structures. On the other hand, while cells seeded at lower coverage (0.8%) had more space to grow, the amount of activator they encountered locally was insufficient to produce branching; this is because the distance between neighboring cells was too far. This indicates the importance of proximity for activator-induced branching, as some of the cells were accidentally close to each other, and thus elongated in small spaces.

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