Background: Current in vitro human lung epithelial cell models derived from adult tissues may not accurately represent all attributes that define homeostatic and disease mechanisms relevant to the pediatric lung. Methods: We report methods for growing and differentiating primary Pediatric Human Lung Epithelial (PHLE) cells from organ donor infant lung tissues. We use immunohistochemistry, flow cytometry, quantitative RT-PCR, and single cell RNA sequencing (scRNAseq) analysis to characterize the cellular and transcriptional heterogeneity of PHLE cells. Results: PHLE cells can be expanded in culture up to passage 6, with a doubling time of approximately 4 days, and retain attributes of highly enriched epithelial cells. PHLE cells can form resistant monolayers, and undergo differentiation when placed at air-liquid interface. When grown at Air-Liquid Interface (ALI), PHLE cells expressed markers of airway epithelial cell lineages. scRNAseq suggests the cultures contained 4 main sub-phenotypes defined by expression of FOXJ1, KRT5, MUC5B and SFTPB. These cells are available to the research community through the Developing Lung Molecular Atlas Program Human Tissue Core. Conclusion: Our data demonstrate that PHLE cells provide a novel in vitro human cell model that represents the pediatric airway epithelium, which can be used to study perinatal developmental and pediatric disease mechanisms.

The osteogenic effect of a composite electrospun core-shell nanofiber membrane encapsulated with Emdogain ® (EMD) was evaluated. The membrane was developed through coaxial electrospinning using polycaprolactone as the shell and polyethylene glycol as the core. The effects of the membrane on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) were examined using Alizarin Red S staining and qRT-PCR. Characterization of the nanofiber membrane demonstrated core-shell morphology with a mean diameter of ~1 µm. Examination of the release of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) from core-shell nanofibers over a 22-day period showed improved release profile of encapsulated proteins as compared to solid nanofibers. When cultured on EMD-containing core-shell nanofibers, PDLSCs showed significantly improved osteogenic differentiation with increased Alizarin Red S staining and enhanced osteogenic gene expression, namely OCN, RUNX2, ALP, and OPN. Core-shell nanofiber membranes may improve outcomes in periodontal regenerative therapy through simultaneous mechanical barrier and controlled drug delivery function.

While animal model studies have extensively defined mechanisms controlling cell diversity in the developing mammalian lung, the limited data available from late stage human lung development represents a significant knowledge gap. The NHLBI Molecular Atlas of Lung Development Program (LungMAP) seeks to fill this gap by creating a structural, cellular and molecular atlas of the human and mouse lung. Single cell RNA sequencing generated transcriptional profiles of 5500 cells obtained from two newborn human lungs from the LungMAP Human Tissue Core Biorepository. Frozen single cell isolates were captured, and library preparation was completed on the Chromium 10X system. Data was analyzed in Seurat, and cellular annotation was performed using the ToppGene functional analysis tool. Single cell sequence data from an additional 32000 postnatal day 1 through 10 mouse lung cells generated by the LungMAP Cincinnati Research Center was integrated with the human data. Transcriptional interrogation of newborn human lung cells identified distinct clusters representing multiple populations of epithelial, endothelial, fibroblasts, pericytes, smooth muscle, and immune cells and signature genes for each of these population. Computational integration of newborn human and postnatal mouse lung development cellular transcriptomes facilitated the identification of distinct epithelial lineages including AT1, AT2 and ciliated epithelial cells. Integration of the newborn human and mouse cellular transcriptomes also demonstrated cell type-specific differences in maturation states of newborn human lung cells.In particular, newborn human lung matrix fibroblasts could be separated into those representative of younger cells (n=393), or older cells (n=158). Cells with each molecular profile were spatially resolved within newborn human lung tissue. This is the first comprehensive molecular map of the cellular landscape of neonatal human lung, including biomarkers for cells at distinct states of maturity.

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A developmental pattern of lung cell maturation known to occur in late gestation and postnatal life remains poorly understood in humans. This study demonstrates sub‐population enriched, as well as single cell, transcriptome analyses with and without surface protein cell‐indexing, to assess cell specific expression in developing lungs obtained from neonatal and pediatric donors.MethodsWe isolated four major lung cell types (epithelial [EPI], endothelial [END] and non‐endothelial mesenchymal [MES] cells and leukocytes [MIC]) using fluorescence‐activated cell sorting from donor lungs. Next‐generation sequencing libraries were prepared from sorted cell RNA. Similarly, transcriptional profiles of 5500 newborn human lung cells were generated using single cell RNA sequencing (sc‐RNAseq) and CITE‐seq techniques. Cell ranger v2.2.0 and CITE‐seq‐Count (v1.3.1a) were used to process transcriptional and surface protein data for each cell. Downstream analysis was performed within Seurat (v2.3) and the R 3.5.0 environment. Low quality cells were removed including if >12.5% mitochondrial transcription or < 500 UMI/cell. Hyper variable genes were detected and cells were visualized using a tSNE plot. Clusters were identified and annotated based on cell‐type cluster specific marker genes associated with the Lung GENS database (Thorax. 2015; 70:1092).ResultsBulk RNAseq confirmed developmental cell‐specific patterns of gene expression. On single cells, overlaying surface protein quantification onto cell type annotated clusters in tSNE space shows good RNA and protein signature agreement. For example, CD31 marks an endothelial cluster with probable substructure based on differential gene expression of VWF, HPGD, and CDH5. Likewise, EpCAM was restricted to a tight cluster consistent with lung EPIs, a portion of which co‐expressed podoplanin, consistent with type I alveolar EPIs. In addition, CD45+ marked leukocyte clusters. Defining a cell as CD45+ if surface protein quantification for CD45 was >2, created a subset of 1,138 cells further delineated by RNA into those consistent with NK T, B and T cells and a second larger myeloid cluster. Based on co‐expression of genes used to identify pulmonary macrophages, CD11b(ITGAM), CD206(MRC1), CD169(SIGLEC1) and CD15(FUT4), 10% of the myeloid cluster were consistent with monocytes (FUT4− ITGAM+MRC1−SIGLEC−), 4% interstitial macrophages (FUT4−ITGAM+MRC1+ SIGLEC−) and 0.01% alveolar macrophages (FUT4−ITGAM+MRC1+SIGLEC+).ConclusionsWe have demonstrated surface marker indexing of neonatal human lung cell subpopulations, consistent with non‐biased single cell transcriptome content, identifying differentiated antigen presenting cells and great potential to finely identify further cell subsets in early postnatal lung.Support or Funding InformationFunding: NHLBI U01HL122700This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.