Sr. Associate University of Rochester University of Rochester Rochester, New York, United States
Background: While studies of animal models have defined molecular mechanisms controlling cell diversity during lung morphogenesis, data 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.
Objective:
Design/Methods: Single cell RNA sequencing generated transcriptional profiles of 5500 cells obtained from two newborn human lungs received 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 v2.4, and cellular annotation was performed using the ToppGene functional analysis tool (ToppFun). Single cell sequence data from 32,000 mouse lung cells at postnatal day (PN) 1, 3, 7 and 10 generated by Cincinnati Children’s Hospital Medical Center was integrated with the human data. In situ hybridization was used to confirm the spatial location of cellular phenotypes.
Results: Transcriptional analysis of newborn human lung cells identified clusters of closely related cells, consistent with distinct populations of endothelial, epithelial, fibroblasts, pericytes and smooth muscle cells (Figure 1). Epithelial cells were under-represented, but expressed known markers (SFTPC, HOPX, NKX2-1). Multiple, distinct populations of immune cells, including macrophages and lymphocytes (both B and T cells), were identified in the newborn human lung. Neonatal mouse and newborn human lung cells were highly coherent, facilitating the identification of distinct cell populations, 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, matrix fibroblasts could be separated into those representative of younger cells, or older cells. Cells with each molecular profile were spatially resolved within newborn human lung tissue (Figure 2). Conclusion(s): This is a comprehensive molecular map of the cellular landscape of neonatal human lung, including biomarkers for cells at distinct states of maturity. Integrated single cell RNA profiling of human and mouse lungs show that maturation states of newborn human cells, even though largely in the expected range of 4 to 9 murine postnatal days, differ by cell type.
Figure 1:Single cell RNA-seq identifies cell populatons in human fetal lung. Shown is unsupervised graph-based clustering of scRNA-seq data, visualized using t-distributed stochastic neighbor embedding (tSNE). Each point represents a single cell, and individual clusters are annotated based on cell type associations derived from Toppfun.
Figure 2:Estimating maturity of human cells. (a) Distribution of the estimated ages of the human cells derived from post -natal age (PND) of 100 nearest mouse cells to each of the human cells. (b) Proportion of cells of individual human cell types at each stage of maturity defined in terms of estimated post-natal day age of mouse. (c) Fluorescent in situ hybridization (FISH) combined with immunofluorescence of Immature Matrix Fibroblast marker HES1 (red), Non-Mesenchymal Cell Markers, PECAM1 or CDH1 cyan), and Mesenchymal Cell Markers COL6A3 or TCF21 (green) on newborn human lung sections from a donor lung of 1 day of age. Pink arrows indicated the presence of immature matrix fibroblasts shown by co-localization of HES1 (red) and Mesenchymal Cell Markers COL6A3 or TCF21 (green).
Authors/Institutions: Soumyaroop Bhattacharya, University of Rochester, Rochester, New York, United States; Jacquelyn Myers, University of Rochester, Fairport, New York, United States; Cameron Baker, University of Rochester, Fairport, New York, United States; Minzhe Guo, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States; Soula Danopoulos, Children`s Hospital Los Angeles, Los Angeles, California, United States; Jason Myers, University of Rochester, Fairport, New York, United States; Gautam Bandyopadhyay, University of Rochester Medical Center, Rochester, New York, United States; Stephen Romas, University of Rochester, Rochester, New York, United States; Heidie Huyck, University of Rochester, Rochester, New York, United States; Ravi Misra, University of Rochester, Rochester, New York, United States; Jennifer Dutra, University of Rochester, Rochester , New York, United States; Jeanne Holden-Wiltse, University of Rochester, Rochester , New York, United States; Andrew McDavid, University of Rochester, Rochester, New York, United States; John Ashton, University of Rochester, Rochester, New York, United States; Denise Al Alam, Children`s Hospital Los Angeles, Los Angeles, California, United States; Steven Potter, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States; Jeffrey Whitsett, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States; YAN XU, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States; Gloria Pryhuber, University of Rochester, Rochester, New York, United States; Thomas Mariani, University or Rochester, Rochester, New York, United States
