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Lerou, Paul

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Lerou

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Paul

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Lerou, Paul
Author's Publications: search.filters.isAuthorOfPublication.75960e43-aa01-4f4d-b30c-08441741546c

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    Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids
    (Nature Pub. Group, 2015) Freedman, Benjamin S.; Brooks, Craig R.; Lam, Albert; Fu, Hongxia; Morizane, Ryuji; Agrawal, Vishesh; Saad, Abdelaziz F.; Li, Michelle; Hughes, Michael R.; Werff, Ryan Vander; Peters, Derek T.; Lu, Junjie; Baccei, Anna; Siedlecki, Andrew; Valerius, M. Todd; Musunuru, Kiran; McNagny, Kelly M.; Steinman, Theodore; Zhou, Jing; Lerou, Paul; Bonventre, Joseph
    Human-pluripotent-stem-cell-derived kidney cells (hPSC-KCs) have important potential for disease modelling and regeneration. Whether the hPSC-KCs can reconstitute tissue-specific phenotypes is currently unknown. Here we show that hPSC-KCs self-organize into kidney organoids that functionally recapitulate tissue-specific epithelial physiology, including disease phenotypes after genome editing. In three-dimensional cultures, epiblast-stage hPSCs form spheroids surrounding hollow, amniotic-like cavities. GSK3β inhibition differentiates spheroids into segmented, nephron-like kidney organoids containing cell populations with characteristics of proximal tubules, podocytes and endothelium. Tubules accumulate dextran and methotrexate transport cargoes, and express kidney injury molecule-1 after nephrotoxic chemical injury. CRISPR/Cas9 knockout of podocalyxin causes junctional organization defects in podocyte-like cells. Knockout of the polycystic kidney disease genes PKD1 or PKD2 induces cyst formation from kidney tubules. All of these functional phenotypes are distinct from effects in epiblast spheroids, indicating that they are tissue specific. Our findings establish a reproducible, versatile three-dimensional framework for human epithelial disease modelling and regenerative medicine applications.
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    Multi-Scale Imaging and Informatics Pipeline for In Situ Pluripotent Stem Cell Analysis
    (Public Library of Science, 2014) Gorman, Bryan R.; Lu, Junjie; Baccei, Anna; Lowry, Nathan C.; Purvis, Jeremy E.; Mangoubi, Rami S.; Lerou, Paul
    Human pluripotent stem (hPS) cells are a potential source of cells for medical therapy and an ideal system to study fate decisions in early development. However, hPS cells cultured in vitro exhibit a high degree of heterogeneity, presenting an obstacle to clinical translation. hPS cells grow in spatially patterned colony structures, necessitating quantitative single-cell image analysis. We offer a tool for analyzing the spatial population context of hPS cells that integrates automated fluorescent microscopy with an analysis pipeline. It enables high-throughput detection of colonies at low resolution, with single-cellular and sub-cellular analysis at high resolutions, generating seamless in situ maps of single-cellular data organized by colony. We demonstrate the tool's utility by analyzing inter- and intra-colony heterogeneity of hPS cell cycle regulation and pluripotency marker expression. We measured the heterogeneity within individual colonies by analyzing cell cycle as a function of distance. Cells loosely associated with the outside of the colony are more likely to be in G1, reflecting a less pluripotent state, while cells within the first pluripotent layer are more likely to be in G2, possibly reflecting a G2/M block. Our multi-scale analysis tool groups colony regions into density classes, and cells belonging to those classes have distinct distributions of pluripotency markers and respond differently to DNA damage induction. Lastly, we demonstrate that our pipeline can robustly handle high-content, high-resolution single molecular mRNA FISH data by using novel image processing techniques. Overall, the imaging informatics pipeline presented offers a novel approach to the analysis of hPS cells that includes not only single cell features but also colony wide, and more generally, multi-scale spatial configuration.
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    The Distribution of Genomic Variations in Human iPSCs Is Related to Replication-Timing Reorganization during Reprogramming
    (Elsevier BV, 2014) Lu, Junjie; Hu, Ming; Li, Hu; Sasaki, Takayo; Baccei, Anna; Gilbert, David M.; Liu, Jun; Collins, James J.; Lerou, Paul
    Cell fate change involves significant genome reorganization, including change in replication timing, but how these changes are related to genetic variation has not been examined. To study how change in replication timing that occurs during reprogramming impacts the copy number variation (CNV) landscape, we generated genome-wide replication timing profiles of induced pluripotent stem cells (iPSCs) and their parental fibroblasts. A significant portion of the genome changes replication timing as a result of reprogramming, indicative of overall genome reorganization. We found that early and late replicating domains in iPSCs are differentially affected by copy number gains and losses, and that in particular CNV gains accumulate in regions of the genome that change to earlier replication during the reprogramming process. This differential relationship was present irrespective of reprogramming method. Overall, our findings reveal a functional association between reorganization of replication timing and the CNV landscape that emerges during reprogramming.