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A2898 - The Role of Retinoid and Hedgehog Signaling in the Derivation of Lung Specific Mesenchymal Progenitors from iPS Cells
Author Block: H. A. Marquez1, W. Shi2, D. N. Kotton3; 1Center for Regenerative Medicine and Pulmonary, Boston University School of Medicine, Boston, MA, United States, 2Childrens Hosp Los Angeles, Los Angeles, CA, United States, 3Center for Regenerative Medicine, Boston Univ Sch of Med, Boston, MA, United States.
Rationale:
The use of induced pluripotent stem cells (iPSCs) is becoming a powerful tool to study early development as well as a platform for in vitro disease modeling, drug testing, and cell-based reconstituting therapies. Recent literature has demonstrated successful derivation of lung epithelial linages from iPSCs in vitro. To date, there has been no literature of lung-specific mesenchymal lineages derived from iPSCs. These lineages would be valuable for the study of mesenchymal diseases such as pulmonary fibrosis, pulmonary vascular diseases, and to better understand the epithelial-mesenchymal cross-talk in development and disease. Recent animal studies have shown the importance of retinoid and hedgehog (Hh) signaling in the development of lung mesenchyme. Using an iPSC model, we sought to study retinoic acid (RA) and Hh signaling in the development of lung-specific mesenchymal progenitors.
Method:
We established an iPSC line from a triple transgenic mouse carrying an inducible GFP reporter cassette (mTmG) under the regulatory control of the lung mesenchyme specific Tbx4 lung enhancer region (Tbx4 enhancer Hsp68-rtTA x TetOCre; Tbx4-LER) which in vivo is able to lineage trace the majority of lung-specific mesenchymal lineages in early development. We differentiated this engineered line towards mesoderm and tested conditions with RA and purmorphamine (a smoothened agonist) for the induction of the Tbx4-LER reporter in order to generate putative lung-specific mesenchyme in vitro.
Results:
Using our current directed differentiation protocol, this iPSC line was first differentiated towards a Flk1+ mesodermal progenitor stage. Next a combination of RA and purmorphamine induced expression of the Tbx4-LER lineage trace within a subset of cells, and the efficiency of Tbx4-LER induction was significantly lower in cells stimulated with either RA or purmorphamine alone. The reporter positive cells expressed higher gene expression levels of Foxf1, Wnt2, Ptch1, and Gli1 as compared to reporter negative cells.
Conclusion:
We differentiated the Tbx4-LER iPSCs line via a mesodermal intermediate into cells expressing a putative lung mesenchymal fluorochrome tracer enriched in expression of Foxf1 (lateral plate mesoderm marker) as well as Wnt2 (a lung mesenchyme-enriched marker). The combined induction of RA and Hh signaling appears to promote this differentiation. Ongoing work is focused on optimizing the induction of putative lung-specific mesenchyme in vitro from iPSCs which could be used as a model to study lung mesenchymal development and disease.