biostudies-arrayexpressXiangning DongHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14975We performed micro-dissections of developing human lungs during the late pseudoglandular stage and performed single nuclear RNA-sequencing (n = 4; biological replicates from 103-122 PCD) on trachea, bronchi, non-cartilaginous airways, and distal lung.biostudies-arrayexpressSample Collection - Human lung tissue research was reviewed and approved by The University of Michigan Institutional Review Board (IRB). Normal, de-identified human fetal lung tissue was obtained from the University of Washington Laboratory of Developmental Biology. Tissue was shipped overnight in UW-Belzer’s solution (Thermo Fisher, Cat#NC0952695) on ice and was processed for experiments or fixation within 24h. Normal, de-identified, donor human adult lungs not eligible for transplant were obtained from Gift of Life Michigan Donor Care Center. Specific tissue regions were dissected according to anatomic region and snap-frozen in liquid nitrogen for further downstream analysis.Library Construction - Immediately after tissue dissociation, single nucleus libraries were prepared on the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 16 rxns PN-1000283 was used with a target of 7500 cells.Nucleic Acid Extraction - Preparation was performed as described previously in accordance with 10X Genomics’ protocols (CG000375 Rev B for fetal tissue). Briefly, snap-frozen tissue was minced into smaller fragments using a scalpel and then added to lysis buffer (Thermo Fisher Scientific, Cat#PI28324). Tissue was homogenized using a pellet pestle 15 times then incubated for 5 minutes in lysis buffer. The cell suspension was filtered through a 33 μm strainer. Suspension was centrifuged at 500g for 5 mins at 4°C. Supernatant was removed and the pellet was washed with PBS + 1% BSA, centrifuged at 500g for 5 mins at 4°C; pellet was washed and centrifuged again. An added debris removal column step was used for the adult tissue. Permeabilization for ATAC-seq samples was performed by incubating the pellet in 0.1X lysis buffer and incubated for 2 minutes. The suspension was then centrifuged at 500g for 5 mins at 4°C; supernatant was removed and pellet was resuspended in diluted nuclei buffer.Sequencing - All single-nucleus RNA-sequencing was performed using Illumina Novaseq 6000 by the University of Michigan Advanced Genomics Sequencing Core.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Data matrices for further analysis were generated using the CellRanger pipeline (v) under standard parameters by the University of Michigan Advanced Genomics Sequencing Core.UnknownTranscriptomicsGenomicsProteomicsN/AChromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle,Illumina NovaSeq 600010X Genomics reagent kit<h4>ABSTRACT</h4> Organs are composed of diverse cell types that change across space and time during development. To interrogate this diversity, we micro-dissected developing human lungs along the proximal-distal axis during the late pseudoglandular stage and generated an integrated analysis of single-nucleus sequencing and spatial transcriptomics, creating a cellularly-resolved atlas of the lung. These rich datasets revealed positional niches and cellular heterogeneity along the proximal-distal axis, including the identification of a unique population of TP63 + basal cells, the primary stem cell of the airway, marked by expression of LGR5 and LGR6 . Analysis of the LGR5+ basal cell niche and functional experiments with primary organoid models suggest a tonic level of WNT pathway activity in LGR5 + basal cells that is potentiated by mesenchyme-derived R-SPONDIN. We found that basal cell self-renewal is enhanced by WNT activity, suggesting that the WNT pathway plays a previously unappreciated but critical role in airway stem cell maintenance during human development. These results enhance our fundamental understanding of the positional and cellular heterogeneity in the developing human lungs and begin to reveal unique niches that maintain homeostasis throughout the lung.single nucleus RNA sequencingHomo sapiensA spatially-resolved blueprint of the developing human lung reveals a WNT-driven niche for basal stem cellsPeggy P. Hsu, Ansley S. Conchola, Tristan Frum, Xiangning Dong, Lila Tudrick, Varun Ponnusamy, Michael S. Downey, Manqi Wu, Mengkun Yang, Yusoo Lee, Emma Niestroy, Yu-Hwai Tsai, Angeline Wu, Sha Huang, Ian A. Glass, Sofia D. Merajver, Jason R. SpenceXiangning DongTristan FrumAnsley ConcholaPeggy HsuJason SpencefalseSingle-nucleus RNA sequencing of human fetal lung from 4 anatomic regionsWe performed micro-dissections of developing human lungs during the late pseudoglandular stage and performed single nuclear RNA-sequencing (n = 4; biological replicates from 103-122 PCD) on trachea, bronchi, non-cartilaginous airways, and distal lung.2025-08-01T00:00:00Z2025-03-26T11:20:19.608Z2025-03-26T11:20:19.608ZE-MTAB-14975ERP170847EFO_0002944EFO_0004170EFO_0009809EFO_0005518EFO_0003816EFO_000418410.1101/2024.10.01.612096