Human embryonic stem cell (hESC) derived 3D human lung organoids (HLOs) provide a promising model to study human lung development and disease. HLOs containing proximal or/and immature distal airway epithelial cells have been successfully generated in vitro, such as early staged alveolar type 2 (AT2) cells (SPC⁺/SOX9⁺) and immature alveolar type 1 (AT1) cells (HOPX⁺/SOX9⁺). When HLOs were transplanted into immunocompromised mice for further differentiation in vivo, only few distal epithelial cells could be observed. In this study, we transplanted different stages of HLOs into immunocompromised mice to assess whether HLOs could expand and mature in vivo. We found that short-term transplanted HLOs contained lung progenitor cells (NKX2.1⁺, SOX9⁺, and P63⁺), but not SPC⁺ AT2 cells or AQP5⁺ AT1 cells. Meanwhile, long-term engrafted HLOs could differentiate into lung distal bipotent progenitor cells (PDPN⁺/SPC⁺/SOX9⁺), AT2 cells (SPC⁺, SPB⁺), and immature AT1 cells (PDPN⁺, AQP5^-). However, HLOs at late in vitro stage turned into mature AT1-like cells (AQP5⁺/SPB^-/SOX9^-) in vivo. Immunofluorescence staining and transmission electron microscopy (TEM) results revealed that transplanted HLOs contained mesenchymal cells (collagen I⁺), vasculature (ACTA2⁺), neuroendocrine-like cells (PGP9.5⁺), and nerve fiber structures (myelin sheath structure). Together, these data reveal that hESC-derived HLOs would be useful for human lung development modeling, and transplanted HLOs could mimic lung organ-like structures in vivo by possessing vascular network and neuronal network.

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