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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.


Fluorescent image of branching structures.(A) Nucleus image, (B) F-actin, (C) composite image of (A–B). (D) Cross section of the X-X plane, in which the hollow structure can be observed (E) Cross section of the Y-Y plane. Scale bars 100 μm.
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f2: Fluorescent image of branching structures.(A) Nucleus image, (B) F-actin, (C) composite image of (A–B). (D) Cross section of the X-X plane, in which the hollow structure can be observed (E) Cross section of the Y-Y plane. Scale bars 100 μm.

Mentions: Fig. 1F shows cellular growth over time. Compared to the homogeneously distributed NHBE cell culture, the cells grew larger in the case of co-culture with HUVEC and MSC. This indicates HUVEC and MSC produce morphogens to grow NHBE or stimulated NHBE. In any case, cells derived from the clots grew much faster. From day 3, the branch length was more than 3-fold greater than that observed when NHBE cells were co-cultured with HUVECs and MSCs (p = 0.027), and branch length reached a maximum of 700 μm by day 8. The variation of the branch length from a clot at day 7 was relatively large (100 μm to 700 μm) due to the out-of-round shape of clot but even shorter branch length exceeded size of co-cultured NHBE. Figure 2 shows the fluorescent images of a 3D branching structure derived from NHBE cell clot. Nucleus and F-actin of NHBE were stained to analyze the branching structure. The inside of the branch formed a hollow structure and the branches exhibited tubular patterns. Based on nucleus counting from the 3D image, the developed branch was consist of approximately 70 cells per 100 μm and the tubular was consist of 10–25 cells depending on the sections.


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 image of branching structures.(A) Nucleus image, (B) F-actin, (C) composite image of (A–B). (D) Cross section of the X-X plane, in which the hollow structure can be observed (E) Cross section of the Y-Y plane. Scale bars 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Fluorescent image of branching structures.(A) Nucleus image, (B) F-actin, (C) composite image of (A–B). (D) Cross section of the X-X plane, in which the hollow structure can be observed (E) Cross section of the Y-Y plane. Scale bars 100 μm.
Mentions: Fig. 1F shows cellular growth over time. Compared to the homogeneously distributed NHBE cell culture, the cells grew larger in the case of co-culture with HUVEC and MSC. This indicates HUVEC and MSC produce morphogens to grow NHBE or stimulated NHBE. In any case, cells derived from the clots grew much faster. From day 3, the branch length was more than 3-fold greater than that observed when NHBE cells were co-cultured with HUVECs and MSCs (p = 0.027), and branch length reached a maximum of 700 μm by day 8. The variation of the branch length from a clot at day 7 was relatively large (100 μm to 700 μm) due to the out-of-round shape of clot but even shorter branch length exceeded size of co-cultured NHBE. Figure 2 shows the fluorescent images of a 3D branching structure derived from NHBE cell clot. Nucleus and F-actin of NHBE were stained to analyze the branching structure. The inside of the branch formed a hollow structure and the branches exhibited tubular patterns. Based on nucleus counting from the 3D image, the developed branch was consist of approximately 70 cells per 100 μm and the tubular was consist of 10–25 cells depending on the sections.

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.