185 - Role of Amphiregulin in Human Lung Development

Publication Number: 185.6330

Daisy L. Reinoso, The Mount Sinai Kravis Children's Hospital, new York, NY, United States; Phoebe May, Icahn School of Medicine at Mount Sinai, New York, NY, United States; Susan Kim, The Mount Sinai Kravis Children's Hospital, New York, NY, United States; Min-Chi Yang, Icahn School of Medicine at Mount Sinai, New York, NY, United States; Jaden J. A.. Hastings, Icahn School of Medicine at Mount Sinai, New York, NY, United States; Ya-Wen Chen, Icahn School of Medicine at Mount Sinai, New York, NY, United States; Caterina Tiozzo, Icahn Schol of Medicine at Mount Sinai, New York, NY, United States

Background: Amphiregulin (AREG), an epidermal growth factor receptor (EGFR) ligand, is implicated in lung pathologies, including bronchopulmonary dysplasia (BPD). While various cell types, such as endothelial, epithelial, and mesenchymal cells in adult human lungs, are reported to synthesize AREG, its role in human lung development remains largely unknown.

Objective: To elucidate AREG's expression level in human lung development using lung organoids and fetal lung samples.

Design/Methods: To investigate AREG's role in lung development, we used human pluripotent stem cell (hPSC)-derived lung organoids (hPSC-LOs) that mimic human lung development and then validated our findings using human fetal lung samples. hPSC-LOs were generated and samples were harvested from day 4 to day 80 of differentiation, representing gastrulation up to the second trimester, and subjected for single-cell RNA sequencing using 10X platform. Fetal human lung samples (14 to 23 weeks of gestational age (GA) were obtained from the Developmental Origins of Health and Disease Biorepository at Icahn School of Medicine at Mount Sinai. Frozen lung samples were weighed, grounded under chilled conditions, and lysed in an ice-cold RIPA buffer. The homogenate was agitated at 4°C for 2 hours and centrifuged at 13,000g for 20 minutes to separate cellular debris. Supernatants were harvested and total protein levels measured using Bicinchoninic Acid protein assay. Equal protein amounts were used to measure AREG levels with enzyme-linked immunosorbent assay. Samples were grouped by two/three-week GA increments. A nomogram of AREG expression levels across lung developmental stages was created.

Results: AREG mRNA levels in hPSC-LOs significantly rose starting at day 40 aligning with the onset of vasculogenesis in the human lung during early second trimester (Fig.1 panel A). We further validated our results using human fetal samples. As expected, AREG levels were significantly upregulated in human fetal lungs after 15-week GA. (Fig.1 panel B).

Conclusion(s): The increasing levels of AREG in hPSC-LOs and fetal lung tissue after the second trimester suggest its potential role in lung vascular development. We are currently expanding the sample size in each group to five fetal human samples to further validate our findings and assess AREG levels in fetal human samples from the first trimester (GA 6 to 13 weeks). Additionally, we will use immunofluorescence to colocalize AREG and EGFR antibodies with markers for vascular as well as proximal and distal epithelial/mesenchymal lung cells. This will identify both the cells that produce AREG and those targeted by its action.

Panel A. AREG Expression in Lung Organoids Panel B. AREG level during fetal human lung development
AREG tables.pdfPanel A: AREG expression at different time points during lung differentiation of hPSCs lung organoids. D= days
Panel B: AREG expression in Human fetal lung samples at different gestational age. n=3 per group. *p < 0.05.