Branching morphogenesis of immortalized human bronchial epithelial cells in three-dimensional culture - PubMed (original) (raw)
Branching morphogenesis of immortalized human bronchial epithelial cells in three-dimensional culture
Aadil Kaisani et al. Differentiation. 2014 Mar-Apr.
Abstract
While mouse models have contributed in our understanding of lung development, repair and regeneration, inherent differences between the murine and human airways requires the development of new models using human airway epithelial cells. In this study, we describe a three-dimensional model system using human bronchial epithelial cells (HBECs) cultured on reconstituted basement membrane. HBECs form complex budding and branching structures on reconstituted basement membrane when co-cultured with human lung fetal fibroblasts. These structures are reminiscent of the branching epithelia during lung development. The HBECs also retain markers indicative of epithelial cell types from both the central and distal airways suggesting their multipotent potential. In addition, we illustrate how the model can be utilized to understand respiratory diseases such as lung cancer. The 3D novel cell culture system recapitulates stromal-epithelial interactions in vitro that can be utilized to understand important aspects of lung development and diseases.
Keywords: Branching; Bronchial epithelial cells; Differentiation; Distal airways; Fibroblasts.
Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.
Figures
Figure 1. HBEC3 KT cells exhibit different morphologies under different culture conditions
HBEC3 KT cells cultured in 2D and 3D conditions exhibit different morphologies. (A) Under 2D culture conditions on gelatin coated plates, HBEC3 KTs grow in a monolayer with a cobblestone appearance. (B) When seeded on top of Matrigel® in the presence of IMR90s seeded as a monolayer, (C) HBEC3 KT cells aggregate into tubular structures which bud and branch (Day 10). (D) In the absence of IMR90s, HBEC3 KTs fail to form complex branching structures when cultured on Matrigel®. (E) IMR90s seeded as a monolayer under the Matrigel® culture do not form any structures or invade the Matrigel®. (F) The phenotype of HBEC3 KTs on Matrigel® is not limited to IMR90s and can be recapitulated with other primary lung fibroblast cells. Scale bar 100 μm.
Figure 2. HBEC3 KT cells form complex branching structures when co-cultured with IMR90s on Matrigel®
When co-cultured with IMR90s on Matrigel®, (A) HBEC3 KTs aggregate and form tubule-like structures after 1 day. This phenotype is very similar to what is observed in 3D cultures of endothelial cells. (B) Small budding structures emerge from the initial tubule-like structure by day 3. (C) Initial budding structures start to branch by day 6. The aggregated structures continue to grow and (D) develop more complex branching by day 10. (E) E-cadherin immunostaining shows formation of cell-cell junctions and organization within the HBEC3 KT structures. (F) HBEC3 KT cells in the buds are organized and exhibit a columnar morphology by day 8 as indicated by the H&E stain. (G) Cells in the tubule part of the structure are also organized along the length of the structure by day 8. Scale bar 100μm (A–D, F, G), 10μm (E).
Figure 3. Branching HBEC3 KTs structures express basal cell markers
HBEC3 KT cells cultured on Matrigel® were immunostained for expression of basal cell markers (Day 8). (A) Nuclear staining with DAPI is shown in blue. p63 expression is seen in all the nuclei within the branching structures. (B) Keratin 14, another basal cell marker is expressed in the cytoplasm of all the cells within the branching structure. Scale bar 10μm
Figure 4. HBEC3 KTs express markers of distal lung lineages when cultured on Matrigel®
HBEC 3KT cells cultured on Matrigel® were immunostained for markers of cell types present in the distal lung (Day 8). (A) Nuclear stain with DAPI is shown in blue. CCSP (red) is expressed in HBEC3 KTs in 3D culture. The protein is localized within the cytoplasm of the cells in the branching structure. (B) Immunostaining with antibody against SP-A (red), a marker for type II pneumocytes cells. The expression of the protein is localized towards the apical side of the buds of the branching structures. (C) TTF1 is expressed in HBEC3 KTs cultured on top of Matrigel®. The TTF1 protein is expressed in the cytoplasm in a majority of the cells with weak nuclear expression within a few cells. Scale bar 10μm (A–C). (D–E) Transmission EM analysis on branching structures after 8 days in culture show the presence of lamellar-like bodies within the HBEC3 KT cells in 3D culture. Scale bar 0.5μm (D–E).
Figure 5. The in vitro 3D culture system can be utilized for understanding lung cancer progression
The 3D system can be used to understand different respiratory diseases by analyzing the branching phenotype on Matrigel®. The branching characteristic that HBEC 3KTs demonstrate on Matrigel® can be recapitulated by other HBECs. (A) HBEC13 KTs exhibit cobblestone monolayer appearance in 2D. (B) When cultured on top of Matrigel® in the presence of IMR90s, HBEC13 KTs forms complex branching structures. (C) Mutant K-rasv12 or (E) both K-rasv12 and stable TP53 knockdown HBECs retain normal epithelial morphology in 2D. (D, F) These partially transformed cells also form branching structures when cultured on Matrigel®. (G) HBEC3 KTs expressing K-rasv12, c-myc and knockdown TP53 display a mesenchymal morphology in 2D culture. (H) These HBECs fail to form branching structures when cultured on top of Matrigel®. The cells invade through the reconstituted basement membrane (pointed by the arrows). Scale bar 100μm
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