In vitroinduction andin vivoengraftment of lung bud tip progenitor cells derived from human pluripotent stem cells

Abstract

SummaryThe bud tip epithelium of the branching mouse and human lung contains multipotent progenitors that are able to self-renew and give rise to all mature lung epithelial cell types. The current study aimed to understand the developmental signaling cues that regulate bud tip progenitor cells in the human fetal lung, which are present during branching morphogenesis, and to use this information to induce a bud tip progenitor-like population from human pluripotent stem cells (hPSCs) in vitro. We identified that FGF7, CHIR-99021 and RA maintained isolated human fetal lung epithelial bud tip progenitor cells in an undifferentiated state in vitro, and led to the induction of a 3-dimensional lung-like epithelium from hPSCs. 3-dimensional hPSC-derived lung tissue was initially patterned, with airway-like interior domains and bud tip-like progenitor domains at the periphery. Epithelial bud tip-like domains could be isolated, expanded and maintained as a nearly homogeneous population by serial passaging. Comparisons between human fetal lung epithelial bud tip cells and hPSC-derived bud tip-like cells were carried out using immunostaining, in situ hybridization and transcriptome-wide analysis, and revealed that in vitro derived tissue was highly similar to native lung. hPSC-derived epithelial bud tip-like structures survived in vitro for over 16 weeks, could be easily frozen and thawed and maintained multi-lineage potential. Furthermore, hPSC-derived epithelial bud tip progenitors successfully engrafted in the proximal airways of injured immunocompromised NSG mouse lungs, where they persisted for up to 6 weeks and gave rise to several lung epithelial lineages.

Long-term Reconstruction of Human Airway Epithelium-like Structurein Vivowith hESCs-derived organoid cells

“…In the present study, we reconstructed human airway epithelium-like structure in vivo by using hESCs-derived lung organoid cells. Compared with the previous report that hPSC-derived epithelial bud tip organoid cells could form patch structures along the airway epithelium [6], in our study, engrafted hESCsderived lung organoid cells could survival, differentiate and long-term reconstruct airway epithelium-like structure. It has been reported that CD47 high cells were NKX2.1+ cells [8], and our RNA-seq data indicated that CD47 high cells in D25 lung organoids were lung lineage determined cells.…”

“…Immune deficient B-NSG (NOD-Prkdcscid IL2rgtm1/Bcgen) mice were purchased from Beijing Biocytogen Co., Ltd. (Beijing, China). Naphthalene (NA) (200mg/ml) was intraperitoneal injected once as previous described [6].…”

“…Human pluripotent stem cells (hPSCs) derived lung organoids as a promising tool for human lung development study and disease modeling, as well as cell source for lung regeneration [1][2][3][4][5]. Although hPSC-derived epithelial bud tip organoids can engraft into an injured mouse airway and undergo multilineage differentiation, reconstruction of sequential airways epithelium structure is absent [6]. In the present study, we long-term reconstruction of sequential airways epithelium-like structure in naphthalene (NA-induced acute lung injury mice by intratracheal transplantation of lung organoid cells derived from human embryonic stem cells (hESCs).…”

“…It was reported that damaging the lung epithelium promoted engraftment of adult human lung epithelial cells [7], and that long-term engraftment (6W) of hPSCs derived bud tip organoids cells possessed differentiation capability and generated patch structures in airways of NA-injured immunocompromised NSG mice [6]. We tried to investigated differentiation and airway epithelium structure reconstruction by using our previously reported modified hESCs-derived lung organoid (HLO) differentiation protocol [1].…”

Human induced pluripotent stem cell-derived lung organoids in an ex vivo model of the congenital diaphragmatic hernia fetal lung

“…There are more than 20 different genetic mutations that have been described in nonsyndromic CDH, and a laboratory gene panel does not currently exist to screen for these genes 10 . Third, it is possible that alternative directed differentiation protocols, as we and others have described, 85‐87 may yield different results within our ex vivo compression system. Fourth, unlike hypoplastic fetal rodent lung explants used in our laboratory and elsewhere, 73 this organoid model is not suitable for studying aberrant CDH branching morphogenesis 86 and the subsequent instructional role that airway smooth muscle may play during this process 88 .…”