Distal Lung Organoid Model Lecture Notes

Flashcards covering the development, methodology, and applications of a distal lung organoid model derived from human pluripotent stem cells as described in the provided research protocol.

Distal lung organoid model

A strategy for directed differentiation of human pluripotent stem cells (hPSCs) that sequentially specifies cells into definitive endoderm, anterior foregut endoderm, ventral anterior foregut endoderm, lung bud organoids, and finally lung organoids.

Human pluripotent stem cells (hPSCs)

The starting biological material for the protocol, including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), used to generate three-dimensional lung structures.

Lung bud organoids (LBOs)

Small organoids generated in suspension culture starting around day 8 that express patterns associated with in vivo lung buds and consist of folding epithelial sheets interspersed with mesenchymal cells.

Branching morphogenesis

The developmental process where the lung epithelium undergoes outward budding and forms complex structures, which is recapitulated when LBOs are embedded in Matrigel.

Definitive endoderm (DE)

The first lineage specification in the protocol, verified via flow cytometry by the high expression ($>90\%$) of markers $EPCAM$, $cKIT$, and $CXCR4$.

Anterior foregut endoderm (AFE)

A stage achieved by inhibiting $TGF-\beta$, $BMP$, and $WNT$ signaling using molecules like $SB\,431542$, $Noggin$, and $IWP2$.

Alveolar epithelial type 2 (AT2) cells

Surfactant-producing cells in the lung alveoli that arise at the organoid tips, containing lamellar bodies and expressing markers such as $SFTPC$, $SFTPB$, $ABCA3$, and $NAPSA$.

Alveolar epithelial type 1 (AT1) cells

Cells in the alveoli responsible for gas exchange, which express specific markers that have not yet been successfully induced in this organoid model in vitro, despite potential being evident in vivo.

Pseudoglandular stage

A stage of lung development occurring between $5-17\,\text{post-conception weeks}$ ($pcw$) in humans ($E12.5\text{--}E16.5$ in mice) characterized by stereotyped branching.

Canalicular stage

The fetal lung developmental stage ($16\text{--}26\,pcw$) where specialization of the airway epithelium occurs and cell cycle activity decreases.

Saccular stage

The development stage ($26\text{--}38\,pcw$) where canaliculi widen into sacculations to form primitive alveoli.

Alveolar stage

The final stage of lung development occurring from $38\,pcw$ to roughly $21$ years of age in humans, involving maturation and secondary septation.

Interstitial lung diseases (ILDs)

A heterogeneous group of diseases affecting the lung interstitium, such as idiopathic pulmonary fibrosis ($IPF$), which can be modeled using this organoid system due to the presence of mesenchyme.

Hermansky–Pudlak syndrome (HPS)

A genetic disorder affecting lysosome-related organelles; organoids with mutations in $HPS1$, $HPS2$, or $HPS4$ are used to model HPS-associated interstitial pneumonia ($HPSIP$).

Pulmonary mesenchyme

A key feature of this organoid model expressing genes like $WNT2B$ and $TBX4$, allowing for the study of epithelial–mesenchymal interactions.

$NKX2.1$ (TTF1)

A critical transcription factor and marker for lung progenitors and epithelium.

$CPM$ (Carboxypeptidase M)

A specific surface marker identified for the isolation and enrichment of $NKX2.1+$ lung progenitors.

Matrigel

An extracellular matrix used to embed LBOs to support branching morphogenesis and long-term expansion for more than $6$ months.

Serum-free conditions

A characteristic of this protocol that avoids the use of uncharacterized fetal bovine serum ($FBS$) to ensure the proper development and maintenance of distal lung organoids.

$$SFTPC$$

A marker for mature $AT2$ cells, the expression of which is often late-appearing (around day $170$) and highly variable in organoid cultures.

Following injury, how does the lung react to repair the gas-exchange sacs?

AT2 cells self-renew and change into alveolar type 1 (AT1) cells to repair the gas-exchange sacs (as AT1 cells are the ones responsible for gas exchange in the alveoli). This transition stretches the cube shape AT2 cell to a flat, squamous shape to allow for proper breathing and blood-gas exchange

Before becoming an AT1 cell, what happens to the AT2 cell?

The AT2 cells changes into a temporary, transitional cell called an ADI cells to repair (Kr8+ alveolar differentiation intermediate)

What are AT1-specific genes?

Ager and Hopx

How does the transition from AT2 cells to AT1 cells relate to IPF?

In IPF, this transition gets stuck (as opposed to being smooth in healthy lungs)

• The ADI cells stall and don’t make it to become mature AT1 cells, and instead build up in the lungs, becoming scar tissue and inducing inflammation

• creates a chronic, pro-fibrotic driver

Which dysregulated signaling pathways block ADI cells from completing terminal differentiation in IPF?

• Hyperactive TGF-Beta (Transforming Growth Factor-Beta) signaling

What is the significance of the TGF-Beta pathway?

• fundamental signaling pathway that regulates cell growth, differentiation, apoptosis, and immune homeostasis

• For terminal AT1 cells to be differentiated normally, TGF-Beta must be deactivated

• So, if this pathway is consistently on, then the cells cannot be differentiated to fully AT1 cells

What is MUC5B?

also known as Mucin-5B

is a protein that in humans is encoded by the MUC5B

one of the five gel-forming mucin (primary structural component that gives mucus its gel-like, lubricating properties

What is the CAV1 gene responsible for?

codes for making a key membrane protein that is the principal structure component of caveolae (tiny, flask-shaped pouches on the cell surface)

• essential for organizing lipid rafts, transporting molecules into cells, and regulating chemical signaling pathways

What is the significance