High throughput single cell RNA-seq of developing mouse kidney and human kidney organoids reveals a roadmap for recreating the kidney

AbstractRecent advances in our capacity to differentiate human pluripotent stem cells to human kidney tissue are moving the field closer to novel approaches for renal replacement. Such protocols have relied upon our current understanding of the molecular basis of mammalian kidney morphogenesis. To date this has depended upon population based-profiling of non-homogenous cellular compartments. In order to improve our resolution of individual cell transcriptional profiles during kidney morphogenesis, we have performed 10x Chromium single cell RNA-seq on over 6000 cells from the E18.5 developing mouse kidney, as well as more than 7000 cells from human iPSC-derived kidney organoids. We identified 16 clusters of cells representing all major cell lineages in the E18.5 mouse kidney. The differentially expressed genes from individual murine clusters were then used to guide the classification of 16 cell clusters within human kidney organoids, revealing the presence of distinguishable stromal, endothelial, nephron, podocyte and nephron progenitor populations. Despite the congruence between developing mouse and human organoid, our analysis suggested limited nephron maturation and the presence of ‘off target’ populations in human kidney organoids, including unidentified stromal populations and evidence of neural clusters. This may reflect unique human kidney populations, mixed cultures or aberrant differentiation in vitro. Analysis of clusters within the mouse data revealed novel insights into progenitor maintenance and cellular maturation in the major renal lineages and will serve as a roadmap to refine directed differentiation approaches in human iPSC-derived kidney organoids.

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

10.1101/516807 ◽

2019 ◽

Cited By ~ 4

Author(s):

Ayshwarya Subramanian ◽

Eriene-Heidi Sidhom ◽

Maheswarareddy Emani ◽

Nareh Sahakian ◽

Katherine Vernon ◽

...

Keyword(s):

Single Cell ◽

Human Kidney ◽

Cell Types ◽

Mouse Kidney ◽

Target Cells ◽

Rna Seq ◽

Kidney Capsule ◽

Human Ipsc ◽

Kidney Organoids

AbstractHuman 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 off-target 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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Single cell census of human kidney organoids shows reproducibility and diminished off-target cells after transplantation

Nature Communications ◽

10.1038/s41467-019-13382-0 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 17

Author(s):

Ayshwarya Subramanian ◽

Eriene-Heidi Sidhom ◽

Maheswarareddy Emani ◽

Katherine Vernon ◽

Nareh Sahakian ◽

...

Keyword(s):

Single Cell ◽

Single Cells ◽

Human Kidney ◽

Mouse Kidney ◽

Target Cells ◽

Rna Seq ◽

Kidney Capsule ◽

Time Points ◽

Human Ipsc ◽

Kidney Organoids

AbstractHuman 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 profile four human iPSC lines for a total of 450,118 single cells to show how organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes are largely reproducible across time points, protocols, and replicates, we detect variability in cell proportions between different iPSC lines, largely due to off-target cells. To address this, we analyze organoids transplanted under the mouse kidney capsule and find diminished off-target cells. Our work shows how single cell RNA-seq (scRNA-seq) can 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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Graft immaturity and safety concerns in transplanted human kidney organoids

Experimental & Molecular Medicine ◽

10.1038/s12276-019-0336-x ◽

2019 ◽

Vol 51 (11) ◽

pp. 1-13 ◽

Cited By ~ 2

Author(s):

Sun Ah Nam ◽

Eunjeong Seo ◽

Jin Won Kim ◽

Hyung Wook Kim ◽

Hong Lim Kim ◽

...

Keyword(s):

Therapeutic Option ◽

Kidney Tissue ◽

Human Kidney ◽

Ips Cells ◽

Attractive Potential ◽

Transplanted Kidney ◽

Filtration Barrier ◽

Kidney Organoids

AbstractFor chronic kidney disease, regeneration of lost nephrons with human kidney organoids derived from induced pluripotent stem (iPS) cells is proposed to be an attractive potential therapeutic option. It remains unclear, however, whether organoids transplanted into kidneys in vivo would be safe or functional. Here, we purified kidney organoids and transplanted them beneath the kidney capsules of immunodeficient mice to test their safety and maturity. Kidney organoid grafts survived for months after transplantation and became vascularized from host mouse endothelial cells. Nephron-like structures in grafts appeared more mature than kidney organoids in vitro, but remained immature compared with the neighboring mouse kidney tissue. Ultrastructural analysis revealed filtration barrier-like structures, capillary lumens, and tubules with brush border in the transplanted kidney organoids, which were more mature than those of the kidney organoids in vitro but not as organized as adult mammalian kidneys. Immaturity was a common feature of three separate differentiation protocols by immunofluorescence analysis and single cell RNA sequencing. Stroma of transplanted kidney organoid grafts were filled with vimentin-positive mesenchymal cells, and chondrogenesis, cystogenesis, and stromal expansion were observed in the long term. Transcription profiles showed that long-term maintenance after kidney organoid transplantation induced transcriptomic reprogramming with prominent suppression of cell-cycle-related genes and upregulation of extracellular matrix organization. Our data suggest that kidney organoids derived from iPS cells may be transplantable but strategies to improve nephron differentiation and purity are required before they can be applied in humans as a therapeutic option.

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Revisiting The Role of Notch in Nephron Segmentation: Notch is required for proximal, not distal fate selection during mouse and human nephrogenesis

10.1101/2021.12.14.472634 ◽

2021 ◽

Author(s):

Kathryn Duvall ◽

Lauren Bice ◽

Alison J Perl ◽

Naomi Pode Shakked ◽

Praneet Chaturvedi ◽

...

Keyword(s):

Notch Signaling ◽

Human Kidney ◽

Distal Tubule ◽

Rna Seq ◽

Early Markers ◽

Nephron Segmentation ◽

Human Ipsc ◽

Kidney Organoids ◽

In Cells

Notch signaling promotes maturation of nephron epithelia, but its proposed contribution to nephron segmentation into proximal and distal domains has been called into doubt. We leveraged single cell and bulk RNA-seq, quantitative immunofluorescent lineage/fate tracing, and genetically modified human iPSC to revisit this question in developing mouse kidneys and human kidney organoids. We confirmed that Notch signaling is needed for maturation of all nephron lineages, and thus mature lineage markers fail to detect a fate bias. By contrast, early markers identified a distal fate bias in cells lacking Notch2, and a concomitant increase in early proximal and podocyte fates in cells expressing hyperactive Notch1 was observed. Orthogonal support for a conserved role for Notch signaling in the distal/proximal axis segmentation is provided by the ability of Nicastrin-deficient hiPSCs-derived organoids to differentiate into TFA2B+ distal tubule and CDH1 connecting segment progenitors, but not into HNF4A+ or LTL+ proximal progenitors.

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Fate-mapping within human kidney organoids reveals conserved mammalian nephron progenitor lineage relationships

10.1101/432161 ◽

2018 ◽

Cited By ~ 1

Author(s):

Sara E Howden ◽

Jessica M Vanslambrouck ◽

Sean B Wilson ◽

Ker Sin Tan ◽

Melissa H Little

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Kidney Development ◽

Collecting Duct ◽

Kidney Tissue ◽

Human Kidney ◽

Lineage Tracing ◽

Nephron Progenitor ◽

Kidney Organoids

AbstractWhile mammalian kidney morphogenesis has been well documented, human kidney development is poorly understood. Here we combine reprogramming, CRISPR/Cas9 gene-editing and organoid technologies to study human nephron lineage relationships in vitro. Early kidney organoids contained a SIX2+ population with a transcriptional profile akin to human nephron progenitors. Lineage-tracing using gene-edited induced pluripotent stem cell (iPSC) lines revealed that SIX2-expressing cells contribute to nephron formation but not to the putative collecting duct epithelium. However, Cre-mediated temporal induction of the SIX2+ lineage revealed a declining capacity for these cells to contribute to nephron formation over time. This suggests human kidney organoids, unlike the developing kidney in vivo, lack a nephron progenitor niche capable of both self-renewal and ongoing nephrogenesis. Nonetheless, human iPSC-derived kidney tissue maintains previously identified lineage relationships supporting the utility of pluripotent stem cell-derived kidney organoids for interrogating the molecular and cellular basis of early human development.

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YAP1 Overexpression Is Associated with Kidney Dysfunction in Lupus Nephritis

Pathobiology ◽

10.1159/000517575 ◽

2021 ◽

pp. 1-12

Author(s):

Ying Xie ◽

Yuanyuan Ruan ◽

Huimei Zou ◽

Yixin Wang ◽

Xin Wu ◽

...

Keyword(s):

Lupus Nephritis ◽

Epithelial Mesenchymal Transition ◽

Kidney Tissue ◽

Mouse Kidney ◽

Experimental Comparison ◽

Control Group ◽

Mrna Levels ◽

Mesenchymal Transition ◽

Protein Levels

Objective: The goal of the present study was to determine the expression of yes-associated protein 1 (YAP1) in renal tissues of mice with lupus nephritis (LN) and elucidate its role in the progression of renal fibrosis. Methods: C57BL/6 mice and MRL/lpr mice were selected for experimental comparison. Mouse kidney tissues were removed and sectioned for hematoxylin and eosin staining, Masson’s trichome staining, Sirius staining, and immunohistochemistry. The mRNA and protein levels of YAP1 in mouse kidney tissues were detected, and the correlation between YAP1 and fibronectin (FN) mRNA levels was analyzed. Mouse renal epithelial cells were used for in vitro experiments. After transfection and stimulation, the cells were divided into 4 groups, namely the C57BL/6 serum group (group 1), the MRL/lpr serum group (group 2), the MRL/lpr serum + siRNA-negative control group (group 3), and the MRL/lpr serum + siRNA-YAP1 group (group 4). Epithelial-mesenchymal transition (EMT) markers in each group were detected by Western blotting and immunofluorescence staining. Serum creatinine, blood urea nitrogen, and urinary protein levels were detected and assessed for their correlation with YAP1 mRNA levels by Spearman’s analysis. Results: Compared to C57BL/6 mice, MRL/lpr mice exhibited obvious changes in fibrosis in renal tissues. In addition, YAP1 expression was significantly higher in the renal tissues of MRL/lpr mice than in those of C57BL/6 mice, and YAP1 mRNA levels were positively correlated with those of FN. YAP1 silencing in lupus serum-stimulated cells could effectively relieve serum-induced EMT. Finally, we observed that YAP1 mRNA levels in mouse kidney tissue were significantly and positively correlated with the degree of renal function injury. Conclusion: YAP1 expression in the kidney tissues of LN mice was higher than that observed in normal mice, indicating that YAP1 may play an important role in the occurrence and development of LN.

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Single cell analysis reveals the impact of age and maturation stage on the human oocyte transcriptome

10.1101/2020.09.25.309658 ◽

2020 ◽

Author(s):

Silvia Llonch ◽

Montserrat Barragán ◽

Paula Nieto ◽

Anna Mallol ◽

Marc Elosua-Bayes ◽

...

Keyword(s):

Single Cell ◽

Chromosome Segregation ◽

Maternal Age ◽

Maturation Stage ◽

P Value ◽

Rna Seq ◽

Transcript Levels ◽

Human Oocytes ◽

The Impact

AbstractStudy questionTo which degree does maternal age affect the transcriptome of human oocytes at the germinal vesicle (GV) stage or at metaphase II after maturation in vitro (IVM-MII)?Summary answerWhile the oocytes’ transcriptome is predominantly determined by maturation stage, transcript levels of genes related to chromosome segregation, mitochondria and RNA processing are affected by age after in vitro maturation of denuded oocytes.What is known alreadyFemale fertility is inversely correlated with maternal age due to both a depletion of the oocyte pool and a reduction in oocyte developmental competence. Few studies have addressed the effect of maternal age on the human mature oocyte (MII) transcriptome, which is established during oocyte growth and maturation, and the pathways involved remain unclear. Here, we characterize and compare the transcriptomes of a large cohort of fully grown GV and IVM-MII oocytes from women of varying reproductive age.Study design, size, durationIn this prospective molecular study, 37 women were recruited from May 2018 to June 2019. The mean age was 28.8 years (SD=7.7, range 18-43). A total of 72 oocytes were included in the study at GV stage after ovarian stimulation, and analyzed as GV (n=40) and in vitro matured oocytes (IVM-MII; n=32).Participants/materials, setting, methodsDenuded oocytes were included either as GV at the time of ovum pick-up or as IVM-MII after in vitro maturation for 30 hours in G2™ medium, and processed for transcriptomic analysis by single-cell RNA-seq using the Smart-seq2 technology. Cluster and maturation stage marker analysis were performed using the Seurat R package. Genes with an average fold change greater than 2 and a p-value < 0.01 were considered maturation stage markers. A Pearson correlation test was used to identify genes whose expression levels changed progressively with age. Those genes presenting a correlation value (R) >= |0.3| and a p-value < 0.05 were considered significant.Main results and the role of chanceFirst, by exploration of the RNA-seq data using tSNE dimensionality reduction, we identified two clusters of cells reflecting the oocyte maturation stage (GV and IVM-MII) with 4,445 and 324 putative marker genes, respectively. Next we identified genes, for which RNA levels either progressively increased or decreased with age. This analysis was performed independently for GV and IVM-MII oocytes. Our results indicate that the transcriptome is more affected by age in IVM-MII oocytes (1,219 genes) than in GV oocytes (596 genes). In particular, we found that genes involved in chromosome segregation and RNA splicing significantly increase in transcript levels with age, while genes related to mitochondrial activity present lower transcript levels with age. Gene regulatory network analysis revealed potential upstream master regulator functions for genes whose transcript levels present positive (GPBP1, RLF, SON, TTF1) or negative (BNC1, THRB) correlation with age.Limitations, reasons for cautionIVM-MII oocytes used in this study were obtained after in vitro maturation of denuded GV oocytes, therefore, their transcriptome might not be fully representative of in vivo matured MII oocytes.The Smart-seq2 methodology used in this study detects polyadenylated transcripts only and we could therefore not assess non-polyadenylated transcripts.Wider implications of the findingsOur analysis suggests that advanced maternal age does not globally affect the oocyte transcriptome at GV or IVM-MII stages. Nonetheless, hundreds of genes displayed altered transcript levels with age, particularly in IVM-MII oocytes. Especially affected by age were genes related to chromosome segregation and mitochondrial function, pathways known to be involved in oocyte ageing. Our study thereby suggests that misregulation of chromosome segregation and mitochondrial pathways also at the RNA-level might contribute to the age-related quality decline in human oocytes.Study funding/competing interest(s)This study was funded by the AXA research fund, the European commission, intramural funding of Clinica EUGIN, the Spanish Ministry of Science, Innovation and Universities, the Catalan Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) and by contributions of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership and to the “Centro de Excelencia Severo Ochoa”.The authors have no conflict of interest to declare.

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Single cell RNA sequencing of calvarial and long bone endocortical cells

10.1101/849224 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ugur M. Ayturk ◽

Joseph P. Scollan ◽

Alexander Vesprey ◽

Christina M. Jacobsen ◽

Paola Divieti Pajevic ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cultured Cells ◽

Neutralizing Antibody ◽

Long Bone ◽

Appendicular Skeleton ◽

Rna Seq ◽

Isolated Cells

ABSTRACTSingle cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

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Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classifier DIEM

10.1101/786285 ◽

2019 ◽

Cited By ~ 4

Author(s):

Marcus Alvarez ◽

Elior Rahmani ◽

Brandon Jew ◽

Kristina M. Garske ◽

Zong Miao ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cell Types ◽

Supervised Machine Learning ◽

Data Sets ◽

Rna Seq ◽

Novel Approach ◽

Single Nucleus ◽

Downstream Analysis

AbstractSingle-nucleus RNA sequencing (snRNA-seq) measures gene expression in individual nuclei instead of cells, allowing for unbiased cell type characterization in solid tissues. Contrary to single-cell RNA seq (scRNA-seq), we observe that snRNA-seq is commonly subject to contamination by high amounts of extranuclear background RNA, which can lead to identification of spurious cell types in downstream clustering analyses if overlooked. We present a novel approach to remove debris-contaminated droplets in snRNA-seq experiments, called Debris Identification using Expectation Maximization (DIEM). Our likelihood-based approach models the gene expression distribution of debris and cell types, which are estimated using EM. We evaluated DIEM using three snRNA-seq data sets: 1) human differentiating preadipocytes in vitro, 2) fresh mouse brain tissue, and 3) human frozen adipose tissue (AT) from six individuals. All three data sets showed various degrees of extranuclear RNA contamination. We observed that existing methods fail to account for contaminated droplets and led to spurious cell types. When compared to filtering using these state of the art methods, DIEM better removed droplets containing high levels of extranuclear RNA and led to higher quality clusters. Although DIEM was designed for snRNA-seq data, we also successfully applied DIEM to single-cell data. To conclude, our novel method DIEM removes debris-contaminated droplets from single-cell-based data fast and effectively, leading to cleaner downstream analysis. Our code is freely available for use at https://github.com/marcalva/diem.

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CellMap: Characterizing the types and composition of iPSC-derived cells from RNA-seq data

10.1101/2021.05.24.445360 ◽

2021 ◽

Author(s):

Zhengyu Ouyang ◽

Nathanael Bourgeois ◽

Eugenia Lyashenko ◽

Paige Cundiff ◽

Patrick F Cullen ◽

...

Keyword(s):

Single Cell ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Model Systems ◽

Rna Seq ◽

Cell Type ◽

Fine Grained ◽

Single Nucleus ◽

Induced Pluripotent

Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

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