[23-2 KIST Europe] Lung Alveolar Organoid with Hybrid Culturing

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Component of Lung Epithelium

① Airway Epithelium

including trachea(기관), bronchi(기관지), bronchiole(세기관지)

Ciliated Cell: Move mucus and trapped particles out of the airways

Goblet Cell: Produce and secrete mucus

Basal Cell: Act as progenitor cells, differentiating into other types of airway epithelial cells

Club (Clara) Cell: Secrete various substances, including surfactant components, detoxify harmful substances

Pulmonary Neuroendocrine Cell (PNEC): Release signaling molecules for regulating airway tone & immune response

Ionocyte: Transport chloride ions; Results maintaining the hydration of airway

② Alveolar Epithelium

Type Ⅰ Alveolar Cell (AT1): Facilitates gas exchange between the air in alveolar cell and blood, 혈관과 직접 접촉

 

Type Ⅱ Alveolar Cell (AT2): Produce & secrete surfactant; Acting as progenitor cell, when weak AT1s are damaged differentiate its own to regenerate AT1; 폐 표면활성제 단백질을 분비하여 폐포의 표면장력 유지

 

Alveolar Macrophages: Engulf & digest bacteria, dust, and other particles

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Culturing Method

Air-liquid Interface (ALI) Culturing

 

In vitro inhalation model

Combined with an air-liquid exposure system to aerosolize a substance or chemical of interest

ALI Culturing vs 3D Culturing

	2D Air-liquid Interface (ALI) Culturing	3D Culturing
PRO	Physiological Relevance Closely mimics the natural environment of alveolar cells in the lungs (inhalation model) Improved Cell Differentiation & Function including surfactant production and proper barrier function for alveolar cells	Cell-Cell & Cell-Matrix Interactions Allow for complex interactions Longer-Term Culturing support the growth of multiple cell types, reflecting the cellular diversity
CON	Limited Cell Expansion Since finite lifespan, undergoes only a limited # of cell divisions before stopping proliferating; Limited differentiation into AT1 cells Lengthy Differentiation ALI cultures often grow slowly compared to other in vitro culture systems	Lack of Mimicking in Vivo Environment Alveolar cells are exposed to air on one side and fluid on the other
Figure

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Motivation for the research

 

The existing cell organoid culture protocol had the drawback of limited differentiation into AT1 cells which hindered effective gas exchange when using the ALI method, and has an in vivo similarity issue when the alveolar organoid is cultured in the 3D method. To address these issues, an effort was made to create a protocol that integrates the strengths of both ALI culture and 3D culture.

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Prior Research & Application

Article	Result	Apply
(1)	Culture alveolar organoid using hiPSC in a submerged way for 24 days, and maintain it in ALI for 3 days	Use as the main protocol of culturing alveolar organoids with ALI
(2)	Approximating in-vivo environment, necessary signals supplied by fibroblast-derived factors, Possible to maintain max 120 days	Co-culture with fibroblast for longer maintenance period, efficient differentiation
(3)	R-spondin2 protein, Amlexanox role as an activator of the canonical WNT/β-Catenin signaling pathway	Add R-spondin2 protein, and Amlexanox in a culture medium
(4)	Dissociation of 3D spheres organoid into 2D air-liquid culturing, maintained 5-14 days	Introduce methodology into the main protocol

 

(1) In vitro modeling of alveolar repair at the air-liquid interface using alveolar epithelial cells derived from hiPSC, Riet S, et al. (2020)
(2) Long-term expansion of alveolar stem cells derived from human iPS cells in organoids, Yamamoto Y, et al. (2017)
(3) A drug screen with approved compounds identifies Amlexanox as a novel Wnt/β-catenin activator, Costa R, et al (2021)
(4) Generating 3D Spheres and 2D Air-Liquid Interface Cultures of hiPSC-derived Type 2 Alveolar Epithelial Cells, Werder R, et al. (2022)

 

Background

 

① Fibroblast

Ubiquitous presence; Support, healing, and interaction with immune cell

By co-culturing alveolar organoids with fibroblast, it can activate Paracrine Signaling, Extracellular Matrix (ECM) Remodeling, TGF-β Signaling Pathway, Notch Signaling Pathway, WNT Signaling Pathway, Hypoxia-Induced Factors, … (which has way more effect than just using growth factor in the existing protocol)

 

② R-spondin2

Consists of secreted proteins known for their role as an activator of the canonical WNT/β-Catenin signaling pathway interacting with LGR4-6 receptors (LGR4: airway, LGR5: branching points of the airway, LGR6: distal airways & alveoli)

Effect on the lung: Lung Development and Regeneration, Stem/Progenitor Cell Regulation especially club cells and AT2 cells, and Interaction with the WNT/β-Catenin signaling pathway

 

③ Amlexanox

Small-molecule compound with pharmacological properties, activating Wnt/β-Catenin Signaling, and Inhibition of IKKε/TBK1 (which is the enzymes that interact and regulate β-catenin)

 

④ WNT Signaling

 Role: Early Lung Development; Regulation of Gene Expression, Lung Epithelial Cell Differentiation (AT2 → AT1), Stem Cell Maintenance; Repair and Regeneration, and Interaction with Other Signaling Pathways

Mechanism: Canonical (β-Catenin-Dependent) Pathway regulates gene expression related to cell fate, and Non-Canonical Pathways regulate cell movement and organization

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Hybrid Culturing in Alveolar Organoid

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