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A3824 - Human Lung Organoids In Vivo Using iPS Cell-Derived Alveolar Epithelial Type 2 Cells
Author Block: Y. Yamamoto1, S. Gotoh2, Y. Korogi1, S. Ikeo1, N. Sone1, K. Tamai1, K. Satoshi1, T. Hirai1; 1Department of Respiratory Medicine, Kyoto University, Kyoto, Japan, 2Department of Respiratory Medicine, Department of Drug Discovery for Lung Diseases, Kyoto University, Kyoto, Japan.
RATIONALE: Alveolar epithelial type 2 (AT2) cells function as stem cells in alveoli. Elucidating behavior of human AT2 cells in alveologenesis and process of damage repair is challenging due to difficulty in obtaining and expanding primary AT2 cells from fetus as well as adults. To overcome these hurdles, we have recently established methods of generation and long-term expansion of human iPS cell-derived AT2 cells (hiAT2 cells) in organoids. Although we achieved stable expansion of hiAT2 cells in vitro, their proliferative ability and behavior in vivo are remained unclear. Then, we tried to engraft hiAT2 cells in immunodeficient mice to ask about their organoid-forming ability in vivo. METHODS: ""Alveolar organoids"" (AOs) were efficiently generated by co-culturing stepwise differentiated and ""preconditioned"" CPMhigh ventralized anterior foregut endoderm cells (VAFECs) derived from human iPS cells and human fetal lung fibroblasts (HFLFs). AOs were then dissociated enzymatically and hiAT2 cells were isolated by FACS. Cell aggregates were formed by co-culturing the isolated hiAT2 cells, HFLFs and human umbilical vein endothelial cells (HUVECs) in non-adherent well plates. After 48 to 72 hours, cells were engrafted in murine renal subcapsular space. One to four months post-engraftment, mice were sacrificed and kidneys were excised for analysis. RESULTS: Immunocytochemistry showed that cell aggregates involved SFTPC+ cells, vimentin+ cells, and CD31+ cells, separately. Three-dimensional structures were observed macroscopically in the murine renal subcapsular space after engraftment. Immunostaining of harvested kidney showed that renal subcapsular cells consisted of human cells. These cells involved NKX2-1+ cells, although SFTPC or SFTPB were negative. CONCLUSION: We successfully engrafted cell aggregates derived from each human lineage cells involving hiAT2 in mice, although engrafted hiAT2 cells lost their specific lineage markers in these organoid-like structures. Further studies for reconstituting the ""niche"" for maintaining the property of AT2 cells are needed to control the quality of human lung organoids in vivo.