This study was performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were approved by the Committee on the Use and Care of Animals at the Korea Atomic Energy Research Institute (KAERI; approval no. KAERI-IACUC-2023-004) and performed according to accepted veterinary standards set by the KAERI animal care center (RI-BIOMICS; Jeongeup, South Korea). Mice were euthanized by CO₂ inhalation, as specified by the KAERI Institutional Animal Care and Use Committee guidelines.

Most chemical reagents were sourced from Sigma-Aldrich (St. Louis, USA). Human embryonic stem cells (hESCs) and all reagents for the proliferation and culture of organoids were obtained from Organoid Science (Pangyo, South Korea) or STEMCELL Technologies (Vancouver, Canada). The bacterial strains used in this study were generously provided by Prof. Moon H. Nahm (University of Alabama at Birmingham). Pneumococcal strains were cultured in Todd Hewitt Yeast broth or agar at 37°C.

Specific pathogen-free female BALB/c mice, aged 6–7 weeks, were obtained from Orient Bio Inc. (Seoul, South Korea). Animal housing conditions, compliant with standards for specific pathogen-free animals, were provided by RI-BIOMICS (Jeongeup, South Korea) and approved by the Committee on the Use and Care of Animals at KAERI (Protocol 2023-003). The mice were housed in individually ventilated cages (Orient Bio Inc.) within a Biosafety Level 2 (BSL2) facility, maintained at 22–23°C with a 12-hour light/dark cycle. For experimental infection, mice (n=5) were challenged intranasally (i.n.) with pneumococci at 1×10⁸ colony forming unit (CFU)/100 μL to compare lung colonization and blood invasion. At 1, 2, and 3 days post-infection, lungs were excised and homogenized in 1.5 mL PBS using a 40-μm mesh strainer. Bacterial load was quantified by culturing serially diluted lung homogenates or blood samples on blood agar plates (BAPs).

To differentiate into definitive endoderm cells, the hESCs were seeded on a Matrigel-coated 6-well plate at 8 × 10⁵ cells/2 mL/well and cultured with RPMI1640 medium containing B27 supplement (2%), human activin A (100 ng/mL), CHIR99021 (1 μM), and sodium butyrate (0.125 mM) for 6 days. For anteriorization, the cells were differentiated using a DMEM/F12 plus Glutamax medium containing B27 supplement (2%), L-ascorbic acid (0.05 mg/mL), monothioglycerol (0.4 mM), human noggin (100 ng/mL), and SB431542 (10 μM) for 4 days. Then, the cells were treated with DMEM/F12 plus Glutamax medium containing B27 supplement (2%), L-ascorbic acid (0.05 mg/mL), monothioglycerol (0.4 mM), human BMP4 (20 ng/mL), ATRA (0.05 μM), and CHIR99021 (3 μM) for additional 4 days to differentiated into ventralized anterior foregut endoderms (VAFEs). VAFEs were stimulated with human FGF10 (10 ng/mL), human KGF (10 ng/mL), CHIR99021 (3 μM), and DAPT (20 μM) for 7 days. On differentiation day 21, the cells were detached with Accutase and seeded onto ultra-low attachment 96-well U-bottom plate at 2 × 10⁵ cells/250 μL/well to generate fibroblast-free spheroids. After 2 days, spheroids were gently collected and resuspened in 20 μL of human lung normal organoid medium (hLuN medium; Organoid Sciences) mixed with precooled Matrigel at a ratio of 1:1. The drop was embedded onto a 48-well plate to form a three-dimension culture environment and cultured with 400 μL of hLuN medium in the presence of Y27632 (10 μM). The hLuN medium was changed every 2 days. Embedded spheroids mature into human lung organoids (hLOs) by passaging every 5 - 7 days. Final organoids were sub-cultured more than three-times to remove cell debris and undifferentiated cells.

Organoids were collected and total RNA was isolated using the TRIzol Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Reverse transcription on 3 µg of total RNA was performed using random primers, dNTP mixture, and MMLV reverse transcriptase (Promega, Madison, WI, USA). Quantitative real-time PCR was performed using StepOne Real-Time PCR (Applied Biosystems, Foster City CA, USA) with the SYBR Green reagent (Takara, Tokyo, Japan). Primers were designed using Primer-BLAST, and the sequences are presented in Supplemental Table 1. The comparative Ct method was used, and relative mRNA expression was calculated based on the normalization to GAPDH expression. All experiments were repeated thrice.

hLOs were fixed with either 10% neutral formalin or Carnoy’s solution for 2 days and embedded in paraffin. Histology sections (5 μm) were collected. Formalin-fixed lung organoid sections were deparaffinized prior to performing antigen retrieval in sodium citrate buffer (10mM Sodium citrate, 0.05% Tween 20, pH 6.0). Sections were permeabilized with 0.2% Triton X-100 in PBS for 30 min, then incubated in a blocking buffer (5% BSA in PBS) for 1 h. Clara cell, AEC-I, AEC-II, and goblet cells were stained using anti-uteroglobin (CC10) rabbit monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti-sodium potassium ATPase mouse monoclonal antibody (Abcam, Cambridge, UK), anti-surfactant protein B (SP-B) mouse monoclonal antibody (Santa Cruz Biotechnology), anti-mucin 5Ac (MUC5Ac) rabbit monoclonal antibody (Abcam) in a blocking buffer overnight at 4°C. Goat anti-mouse or rabbit secondary antibody conjugated to Alexa-488 or Alexa-568 were used according to manufacturer’s instructions (Thermo Fisher) for 1 h RT in blocking buffer. DAPI was used to stain DNA. Sections were mounted using coverslips and Prolong Glass Antifade Mountant (Thermo Fisher). Fluorescence images were taken using a Zeiss LSM 700 confocal fluorescence microscope (Carl Zeiss; Oberkochen, Germany). In a separate experiment, S. pneumoniae serotype ST23F or ST4 (4 × 10⁸ CFU/mL) were stained by CellTrace CFSE (Thermo Fisher) and injected in hLOs using a FemtoJet 4i microinjector (Eppendorf, Hamburg, Germany) at a pressure of 200 hPA for 4 seconds. At 2 hours post-infection, the hLOs were fixed and treated with DAPI to stain nuclei. Fluorescence images were taken using a Zeiss LSM 700 confocal fluorescence microscope (Carl Zeiss).

All experiments were repeated twice unless indicated otherwise. Data are presented as the mean ± SD of a representative experiment. Statistical significance was determined using one-way or two-way analysis of variance with Tukey’s multiple comparison post-hoc test. Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Prism Software, San Diego, CA, USA).

Four pneumococcal serotypes (ST4, ST6B, ST14, and ST23F) were selected to assess their virulence using a mouse pneumonia model. As reported previously, ST4 is highly virulent, ST6B shows moderate virulence, while ST14 and ST23F are low virulence in mice (11). Mice were intranasally inoculated with same numbers of pneumococci, and the bacterial load in the lungs and blood was counted from Day 1 to Day 3. Fig. 1A shows that all serotypes were present in the lungs on Day 1, with their CFU declining daily. In note, ST23F was rapidly cleared from the lungs, and disappearing by Day 3. Although there were slight differences, the colonization and clearance patterns of the other serotypes in the lungs were similar. To assess pneumococcal invasion from the lungs, bacterial numbers in the spleen were measured by plating on BAPs. As shown in Fig. 1B, no invasive pneumococci were detected in the spleen of mice infected with ST14 and ST23F. ST6B was detected on Day 1 but cleared by Day 2. In contrast, ST4 showed a high bacterial load from Day 1 through Day 3. In conclusion, ST4 exhibited high pathogenicity and ST6B showed moderate pathogenicity, indicating that both serotypes can effectively colonize the lungs and invade the bloodstream. ST14 demonstrated similar lung colonization patterns to ST6B but lacked the ability to invade the blood. Conversely, ST23F showed neither lung colonization nor blood invasion. Thus, ST14 is of low pathogenicity, and ST23F is non-pathogenic in mice.

hESC-derived lung alveolar organoid was generated by following previous report (18). hESCs were sequentially differentiated by adding appropriate growth factors and differentiation factors to progress through the definitive endoderm (DE) stage (Day 2), anterior foregut endoderm (AFE) stage (Day 8), ventral anterior foregut endoderm (VAFE) stage (Day 12), lung progenitor stage (Day 18), and finally into lung organoids (Day 33) (Fig. 2A). hLOs were developed by cultured in the expansion media and monitored for growth characteristics by brightfield microscopy. They were cultured with consistent phenotypes until passage 10 and beyond, and passage 3–8 hLOs were used for experiments. To confirm the presence of major constituent cells in hLOs, we analyzed cell-specific markers using quantitative RT-PCR (qRT-PCR) and immunofluorescence. mRNA expression of various cell markers of constituent cells in hLOs was significantly higher in the organoids than other cell type (Fig. 2B), and immunostaining verified the presence of AEC-I markers (podoplanin; PDPN), AEC-II markers (surfactant protein A1; SFTPA1), goblet cell markers (MUC5AC), and club cell markers (uteroglobin; CC10) (Fig. 2C). These data suggested that ESCs were stably differentiated into cells that make up the lung through several stages by various growth factors.

Banked hLOs were sub-cultured in expansion media for 7-10 days. Once the hLOs had formed, more than 100 were microinjected with CFSE-stained ST23F or ST4 in PBS (4×106 CFU per hLO) (Fig. 3A and 3B). As shown in Fig. 3C, ST23F effectively invaded the epithelial cells of the hLOs which was relatively similar with hLOs infected with ST4. Interestingly, both ST23F and ST4 seems to be localized in specific epithelial cells (Fig. 3C), suggesting that they may primarily interact with AEC-II cells indicated as previous reports (19). To assess whether ST23F interaction with epithelial cells of hLOs leads to pro-inflammatory cytokine production, we performed qRT-PCR (Fig. 4). While no pro-inflammatory cytokines (TNF-α, IL-1β) were detected in the lung BALF of mice infected with ST23F (Fig. 5), hLOs infected with ST23F showed significant upregulation of all tested pro-inflammatory cytokines (TNF-α, IL-6, IFN-β, IFN-γ) comparable to the response observed in ST4-infected hLOs. CXCL10 and CCL2 chemokines were also upregulated in hLOs infected with either ST23F or ST3. But both serotypes were not induced the expression of IL-8 and TGF- β. These findings indicate that ST23F, a serotype non-virulent in mice, exhibits similar hLO infectivity to ST4, a highly virulent serotype in mice. This highlights the hLO model’s potential as a valuable tool for studying pneumococcal pathogenicity and evaluating pneumococcal vaccines.