Minilungs from hESCs to study the interaction of Streptococcus pneumoniae with the respiratory tract

The new generation of organoids derived from human pluripotent stem cells holds a promising strategy for modeling host-bacteria interaction studies. Organoids recapitulate the composition, diversity of cell types and, to some extent, the functional features of the native organ. We have generated lung bud organoids derived from human embryonic stem cells to study the interaction of Streptococcus pneumoniae (pneumococcus) with the alveolar epithelium. Invasive pneumococcal disease is an important health problem that may occur as a result of the spread of pneumococcus from the lower respiratory tract to sterile sites. We show here an efficient experimental approach to model the main events of the pneumococcal infection that occur in the human lung exploring bacterial adherence to the epithelium, internalization, and triggering of an innate response that includes the interaction with the surfactant and the expression of representative cytokines and chemokines. Thus, this model, based on human minilungs, can be used to study pneumococcal virulence factors, the pathogenesis of different serotypes and it will allow therapeutic interventions in a reliable human context. IMPORTANCE Streptococcus pneumoniae is responsible for high morbidity and mortalities rates worldwide affecting mainly children and adults older than 65 years. Pneumococcus is also the most common etiologic agent of bacterial pneumonia, non-epidemic meningitis, and a frequent cause of bacterial sepsis. Although the advent of pneumococcal vaccines has decreased the burden of the diseases caused by pneumococcus, the emerging of antibiotic-resistant strains and non-vaccine types by serotype replacement, is worrisome. To study the biology of pneumococcus and to establish a reliable human model for pneumococcal pathogenesis, we have generated human minilungs from embryonic stem cells. The results show that these organoids can be used to model some events occurring during the interaction of pneumococcus with the lung such as adherence, internalization, and the initial alveolar innate response. This model also represents a great alternative to study virulence factors involved in pneumonia, drug screening, and other therapeutic interventions. * AFE : Anterior Foregut Endoderm ATI : Alveolar Type I Cells ATII : Alveolar Type II Cells : BMP4 Bone Morphogenic Protein 4 BSA : Bovine serum albumin CCL20 : C-C motif chemokine ligand 20 CXCL5 : C-X-C motif chemokine ligand 5 EBs : Embryoid Bodies FBS : Fetal bovine serum FGF : Fibroblast Growth Factor FOXA2 : Forkhead box A2 hbFGF : Human basic fibroblast growth factor hESCs : Human Embryonic Stem Cells IL6 : Interleukin 6 IL8 : CXC8 , C-X-C motif chemokine ligand 8 KGF : Keratinocyte growth factor LBOs : Lung bud organoids MEFs : Mouse Embryonic Fibroblasts NANOG : Nanog homeobox NKX2-1 : NK2 homeobox 1 OCT3/4A : POU5F1 , POU class 5 homeobox 1 PBS : Phosphate-buffered saline PDPN : Podoplanin RT-qPCR : Quantitative Real-Time RT-PCR (Reverse Transcription Polymerase Chain Reaction) SEM : Standard error of the mean SFD : Serum-free differentiation SFTPA : Surfactant Protein A SFTPB : Surfactant Protein B SFTPC : Surfactant Protein C SFTPD : Surfactant Protein D SOX2 : SRY (Sex Determining Region Y)-box 2 STING1 : Stimulator of interferon response cGAMP interactor 1 TLR2 : Toll-like receptor 2 TNFα : TNF , tumor necrosis factor μm : Micrometer