Single-cell analysis of progenitor cell dynamics and lineage specification of the human fetal kidney

Menon, Rajasree; Kokoruda, Austin; Zhou, Jian; Zhang, Zidong; Yoon, Euisik; Chen, Yu‐Chih; Troyanscaya, Olga; Spence, Jason R.; Kretzler, Matthias; Cebrián, Cristina

doi:10.1101/258798

Cited by 9 publications

(15 citation statements)

References 63 publications

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“…Of interest, mesenchymal-2 cells in organoids were enriched for MGP, GAS2, PRXX2 and FOXP2 , known markers of fetal mesangial cells in the developing kidney. Similarly, mesenchymal-5 cells in organoids were enriched for fetal stromal genes SULTIE1, DKK1 (Iglesias et al, 2007), NR2F1 (Schwab et al, 2003), ID3, ZEB2, COL6A3 (Menon et al, 2018) and DCN. In contrast, mesenchymal types that did not map to human kidney cell types included genes associated with cartilage formation (Ohba et al, 2015) (ACAN, SOX9, MATN4, LECT1, EPYC, COL9A3 and COL9A1)( Fig.…”

Section: Methods)mentioning

confidence: 98%

Kidney organoid reproducibility across multiple human iPSC lines and diminished off target cells after transplantation revealed by single cell transcriptomics

Subramanian

Sidhom

Emani

et al. 2019

Preprint

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Human iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we used single cell RNA-Seq (scRNA-Seq) to profile 415,775 cells to show that organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes were largely reproducible across iPSC lines, time points, protocols, and replicates, cell proportions were variable between different iPSC lines. Off-target cell proportions were the most variable. Prolonged in vitro culture did not alter cell types, but organoid transplantation under the mouse kidney capsule diminished offtarget cells. Our work shows how scRNA-seq can help score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells.

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Section: Methods)mentioning

confidence: 98%

Kidney organoid reproducibility across multiple human iPSC lines and diminished off target cells after transplantation revealed by single cell transcriptomics

Subramanian

Sidhom

Emani

et al. 2019

Preprint

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“…The 18 clusters expressed on average 12,900 genes -likely a good representation of each cell type's full transcriptome. Importantly, three-fold more clusters were detected for the S-shaped body compared to previously reported data (Lindström et al, 2018a;Menon et al, 2018;Hochane et al, 2019;Tran et al, 2019) and the depth and breadth of the resulting transcriptomic data enabled subtle gene expression changes to be visualized within scarce transitioning cell-states.…”

Section: Single-cell Transcriptomes Identified For Human Nephrogenesismentioning

confidence: 67%

“…In previous efforts to resolve the diversity of nephron precursors, we and others have applied single cell RNA sequencing (scRNA-seq) to human nephrogenesis (Lindström et al, 2018a;Menon et al, 2018;Hochane et al, 2019;Tran et al, 2019). When cell clusters are compared with in situ and immunolocalization studies (Lindström et al, 2018b(Lindström et al, , 2018a, distinct cell clusters are likely an amalgamation of multiple related precursor types.…”

Section: Introductionmentioning

confidence: 99%

Spatial Transcriptional Mapping of the Human Nephrogenic Program

Lindström

Sealfon

Chen

et al. 2020

Preprint

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Congenital abnormalities of the kidney and urinary tract are amongst the most common birth defects affecting 3% of newborns. The human kidney develops over a 30-week period in which a nephron progenitor pool gives rise to around a million nephrons. To establish a framework for human nephrogenesis, we spatially resolved a stereotypical process by which equipotent nephron progenitors generate a nephron anlagen, then applied data-driven approaches to construct three-dimensional protein maps on anatomical models of the nephrogenic program. Single cell RNA sequencing identified novel progenitor states which were spatially mapped to the nephron anatomy enabling the generation of functional gene-networks predicting interactions within and between nephron cell-types. Network mining identified known developmental disease genes and predicts new targets of interest. The spatially resolved nephrogenic program made available through the Human Nephrogenesis Atlas (https://sckidney.flatironinstitute.org/) will facilitate an understanding of kidney development and disease, and enhance efforts to generate new kidney structures.

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“…Several technologies are generating gene expression data at bulk, regional and single cell level for comprehensive coverage of the transcriptome for the reference (relatively healthy or pathologically normal) and disease atlases (AKI and CKD subtypes). Single nucleus (sn) (UCSD/WU) and single cell (sc) (UCSF and Premiere -Michigan/Broad/Princeton sites) RNA-seq technologies are being used for cataloging molecularly-defined cell populations for the generation a single cell kidney atlas (13)(14)(15). Each of these technologies uses different approaches in sample preparation, dissociation or processing that have unique advantages and disadvantages (see TIS protocol on https://kpmp.org/researcher-resources/).…”

Section: Overview Of Kpmp Technologies: Creating a Molecular And Funcmentioning

confidence: 99%

A Multimodal and Integrated Approach to Interrogate Human Kidney Biopsies with Rigor and Reproducibility: The Kidney Precision Medicine Project

El-Achkar

Eadon

Menon

et al. 2019

Preprint

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Comprehensive and spatially mapped molecular atlases of organs at a cellular level are a critical resource to gain insights into pathogenic mechanisms and therapies tailored to the disease of each patient. Obtaining rigorous and reproducible results from disparate methods and at different sites to interrogate biomolecules at a single cell level or in 3-dimensional (3D) space is a significant challenge that can be a futile exercise if not well controlled. The Kidney Precision Medicine Project (KPMP) is an endeavor to generate 3D molecular atlases of healthy and diseased kidney biopsies using multiple state-of-the-art OMICS and imaging technologies across several institutions. We describe a pipeline for generating a reliable and authentic single cell/region 3D molecular atlas of human adult kidney with emphasis on quality assurance, quality control, validation and harmonization across different OMICS and imaging methods. Our “follow the tissue” approach encompasses sample procurement to data generation, analysis, data sharing, while standardizing, and harmonizing procedures at sample collection, processing, storage and shipping. We provide key features of preanalytical parameters, bioassays, post analyses, reference standards and data depositions. A pilot experiment from a common source tissue processed and analyzed at different institutions was executed to identify potential sources of variation, feasibility of multimodal analyses and unique and redundant features of the macromolecules being characterized by each technology. An important outcome was identification of limitations and strengths of the different technologies, and how composite information can be leveraged for clinical application. A peer review system was established to critically review quality control measures and the reproducibility of data generated by each technology before granting approval to work on clinical biopsy specimens. This unique pipeline establishes a process that economizes the use of valuable biopsy tissue for multi-OMICS and imaging analysis with stringent quality control to ensure rigor and reproducibility of results and which can serve as a model for similar personalized medicine projects.Author ContributionsMTE, RM, BBL, TS, TA, AS, SP, CRA, DD, EAO, SW, GZ, MJ and KD performed the ground work for the quality control group, wrote the KPMP TIS manual of procedures and generated figures. HH, JZ, RS, RM, TS, EAO, MTE, TME, PH, SP, MK, ZL and SJ generated the initial working reference marker list. CEA, TME, JBH, JL, MK and SJ led the Pilot 1 protocol. TME, VD, LB, JG, CEA, ZL, SJ and JBH developed the pathology QC tissue qualification and tissue processing criteria. JBH organized and executed the Pilot tissue collection and distribution. JC designed the SpecTrack system. CP prepared and organized the TIS manual of procedures and performed data organization services. YH led ontology development for QC metadata and knowledge standardization. BS and EA organized data integration efforts and data authentication in the data hub. KS and MS led the OMICS discussion group. SJ led the quality control group. TME and CEA led the tissue processing group. MTE and SJ led the Molecular and Pathology Integration group. MTE and SJ conceived and led the TISAC process. RI, OGT KZ, ZL, PH, BR, PCD, KS, MS, JBH, CEA, LB, JG, TME, MK and SJ conceived the integrated TIS pipeline and QC vision. TME and SJ wrote the initial draft of the paper. All authors contributed to the writing and editing of the manuscript.

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Single-cell analysis of progenitor cell dynamics and lineage specification of the human fetal kidney

Cited by 9 publications

References 63 publications

Kidney organoid reproducibility across multiple human iPSC lines and diminished off target cells after transplantation revealed by single cell transcriptomics

Kidney organoid reproducibility across multiple human iPSC lines and diminished off target cells after transplantation revealed by single cell transcriptomics

Spatial Transcriptional Mapping of the Human Nephrogenic Program

A Multimodal and Integrated Approach to Interrogate Human Kidney Biopsies with Rigor and Reproducibility: The Kidney Precision Medicine Project

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