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Unlocking the Potential of Tracheal Epithelial Cells in Respiratory Research

Tracheal epithelial cells are a critical constituent of the respiratory system. It acts as a barrier for the pathogens and pollutants in the air we breathe. Although most of the tracheal airway functions are elucidated through in vivo/ex vivo models, understanding the cellular and physiological functions require physiologically relevant models for drug discovery and disease modeling.

Tracheal epithelial cells are part of the pseudostratified columnar epithelium, composed of ciliated cells, goblet cells and basal cells. The ciliated cells contain hair-like cilia which traps particles entering in the trachea. Goblet cells secrete mucus that traps dust, microbes and other foreign materials. Basal cells serve as progenitors for all other cell types of tracheae, thus maintaining the integrity of the epithelial layer.

Primary Tracheal Epithelial Cells

Isolation of tracheal epithelial cells from Biopsies
Figure 1: Isolation of tracheal epithelial cells from Biopsies (Image Source: PMID: 27539459)

For disease modeling, drug discovery and tissue engineering, researchers use various cell sources. Primary human tracheal epithelial cells directly isolated from patients or healthy donors’ tracheal tissue biopsies. Usage of primary cells are limited by donor availability, immune rejection and donor-to-donor variability. Maintaining the native differentiated state of these cells are challenging in vitro, as they tend to lose their functionality due to dedifferentiation over time. Induced pluripotent stem cells (iPSCs) based cell resource is a best suited alternative, although limited by development of robust differentiation recipe.

 

Induced Pluripotent Stem Cells (iPSCs) Derived Tracheal Epithelial Cells

iPSCs-derived tracheal epithelial cells
Figure 2: iPSCs-derived tracheal epithelial cells (Image Source: PMID: 28366587)

iPSC technology opens a new avenue for generating tracheal epithelial cells for tissue engineering and disease modeling. To generate tracheal epithelial cells, researchers mimic the embryonic development in vitro using a cocktail of specific growth factors and signaling molecules. This process first transforms iPSCs to progenitor cells, eventually forming mature tracheal epithelial cells. Successful differentiation is validated by expression of native tracheal epithelial cells markers, ciliary functions and mucus production.

Characterization of Tracheal Epithelial Cells:

Characterization of tracheal epithelial cells is crucial to confirm its functionality, molecular and cellular properties.

Morphological Characterization:

By microscopy, to observe cell morphology and confluence. Electron microscopy to observe ultra structures like cilia structure and other cell organelles.

Functional Characterization:

  • Ciliary Beating: Ciliary beating motion is analyzed by real time high speed video microscope. It also analyzes the beating frequency and pattern.
  • Mucus Production: Mucus producing cells are qualitatively analyzed by Periodic Acid-Schiff (PAS) staining and quantitative by ELISA of specific secretory proteins (e.g. MUC5AC) in spent media.

Molecular Characterization:

  • Immunocytochemistry (ICC) and Immunofluorescence: By analyzing presence and localization of specific markers like ciliated cells (Acetylated tubulin, EpCAM/pan-cytokeratin, DNAH5, integrin subunit α 6), Goblet Cells (MUC2, MUC5AC, MUC5B), Basal cells (p63, cytokeratin 5) etc.
  • Flow Cytometry: Quantitative analysis of surface and intracellular markers.
  • Western Blotting: Western blotting is generally used to detect mucins and ciliary proteins in tracheal epithelial cells.
  • Quantitative PCR (qPCR): Quantitative gene expression analysis of genes e.g. FOXJ1(ciliogenesis marker), MUC5AC (mucin marker), P53 etc.

Barrier Function:

Transepithelial Electrical Resistance (TEER) is a quantitative technique to measure integrity of tight junction dynamics of epithelial cell layer. High TEER values means strong barrier functions. Permeability assays measure the permeabilization of tracer molecules (e.g. FITC dextran) from one side of the cell layer to the other side. Low permeability means a tight barrier.

Secretory Function: 

By measuring secretion of cytokines and chemokines like IL6, IL8, TNF α etc. involved in immune and inflammatory response.

Genetic Characterization:

By karyotyping to analyze any genetic abnormality (generally observed in iPSC-derived tracheal epithelial cells). Whole genome sequencing for comprehensive genetic or mutation (if any) study.

Functional Assays:

  • Air-Liquid Interface (ALI) Cultures: By mimicking in vivo airway epithelial functions, mucus formation and ciliary beating are studied using ALI cultures.
  • Lung Infection Models: Lung infections are one of the leading causes of death worldwide. Three-dimensional lung models are validated by infecting it with various lung disease-causing viruses.

Applications of tracheal epithelial cells:

Disease modeling:

To study disease mechanisms, progression and potential treatments of respiratory diseases like cystic fibrosis, asthma, chronic obstructive pulmonary disease (COPD) etc.

Drug Discovery:

To study efficacy and safety of new drugs for respiratory diseases.

Regenerative Medicine:

Bioengineered tracheal tissues are used for treatment of patients with severe tracheal injuries or defects.

Personalized Medicine:

Using iPSCs-derived tracheal epithelial cells personalized therapies are provided to the patients.

Toxicology Studies:

To study the effects of pollutants on respiratory health.

Conclusion

Tracheal epithelial cells play a crucial role in disease modeling, drug discovery, tissue engineering, personalized medicines and regenerative medicines. These are useful in the discovery of new treatments for respiratory diseases. In Kosheeka, a branch of Advancells group, we provide ready-to-use tracheal epithelial cells, amenable to high-throughput drug screening, disease modeling, toxicology studies and various other applications. 

Frequently Asked Questions (FAQs)

What are Tracheal Epithelial Cells?

Tracheal epithelial cells are the main component of the respiratory system. Pseudostratified epithelium is composed of ciliated cells, goblet cells and basal cells. These cells act as a physical barrier to pathogens and other pollutants in the air we breathe.

What are the Major Role of Different Cell Types of Tracheal Epithelial Cells?

Ciliated Cells contain hair-like structures called cilia that trap foreign particles entering the trachea. Goblet Cells secrete mucus to trap foreign particles. Basal cells serve as progenitor cells for all cell types of tracheae, thus maintaining the integrity of the epithelial layer.

How to Isolate Primary Tracheal Epithelial Cells?

Primary human tracheal epithelial cells are isolated directly from tracheal tissue biopsies of patients or healthy donors. These can be isolated by explant culture or by enzymatic treatment, further maintained in specific culture media, containing growth factors and small molecules.

What are the Challenges Associated with in Vitro Culture of Primary Tracheal Epithelial Cells?

Primary tracheal epithelial cells are prone to lose their native differentiated state and functionality in vitro. Although these challenges can be addressed by using specified culture media and following specified culture conditions and robust protocols.

What are the Advantages of iPSC-Derived Tracheal Epithelial Cells?

Induced pluripotent stem cells (iPSCs) provide an unlimited resource of virtually all cell types. iPSCs are differentiated into tracheal epithelial cells using specific growth factors and signaling molecules. This method offers a more consistent and scalable source of cells.

How to Confirm the Functionality of Tracheal Epithelial Cells?

  • Morphological Characterization: By microscopy, Tracheal epithelial cells are spindle-shaped cells.
  • Functional Characterization: By analyzing ciliary beating, mucus production, and other cellular functions. By measuring barrier functions by transepithelial electrical resistance (TEER) and permeability assays.
  • Molecular Characterization: By analyzing specific markers by immunocytochemistry, flow cytometry, Western blotting, and quantitative PCR. e.g. ciliated cells (Acetylated tubulin, EpCAM/pan-cytokeratin, DNAH5, integrin subunit α 6), Goblet Cells (MUC2, MUC5AC, MUC5B), Basal cells (p63, cytokeratin 5) etc.

What are the Applications of Tracheal Epithelial Cells?

  • Disease Modeling: Studying mechanisms, progression, and treatments of respiratory diseases like cystic fibrosis, asthma, and COPD.
  • Drug Discovery: Evaluating the efficacy and safety of new drugs for respiratory diseases.
  • Regenerative Medicine: Using bioengineered tracheal tissues to treat severe tracheal injuries or defects.
  • Personalized Medicine: Developing therapies tailored to individual patients using iPSC-derived tracheal epithelial cells.
  • Toxicology Studies: Assessing the impact of pollutants on respiratory health.

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