scholarly journals Single Cell Sequencing and Kidney Organoids Generated from Pluripotent Stem Cells

2020 ◽  
Vol 15 (4) ◽  
pp. 550-556 ◽  
Author(s):  
Haojia Wu ◽  
Benjamin D. Humphreys
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
High Throughput Screening ◽  
Kidney Development ◽  
Toxicity Testing ◽  
Human Kidney ◽  
Cell Types ◽  
Time Frame ◽  
Kidney Organoids

Methods to differentiate human pluripotent stem cells into kidney organoids were first introduced about 5 years ago, and since that time, the field has grown substantially. Protocols are producing increasingly complex three-dimensional structures, have been used to model human kidney disease, and have been adapted for high-throughput screening. Over this same time frame, technologies for massively parallel, single-cell RNA sequencing (scRNA-seq) have matured. Now, both of these powerful approaches are being combined to better understand how kidney organoids can be applied to the understanding of kidney development and disease. There are several reasons why this is a synergistic combination. Kidney organoids are complicated and contain many different cell types of variable maturity. scRNA-seq is an unbiased technology that can comprehensively categorize cell types, making it ideally suited to catalog all cell types present in organoids. These same characteristics also make scRNA-seq a powerful approach for quantitative comparisons between protocols, batches, and pluripotent cell lines as it becomes clear that reproducibility and quality can vary across all three variables. Lineage trajectories can be reconstructed using scRNA-seq data, enabling the rational adjustment of differentiation strategies to promote maturation of desired kidney cell types or inhibit differentiation of undesired off-target cell types. Here, we review the ways that scRNA-seq has been successfully applied in the organoid field and predict future applications for this powerful technique. We also review other developing single-cell technologies and discuss how they may be combined, using “multiomic” approaches, to improve our understanding of kidney organoid differentiation and usefulness in modeling development, disease, and toxicity testing.

scholarly journals A simple bioreactor-based method to generate kidney organoids from pluripotent stem cells

2017 ◽  
Author(s):  
Aneta Przepiorski ◽  
Veronika Sander ◽  
Tracy Tran ◽  
Jennifer A. Hollywood ◽  
Brie Sorrenson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Kidney Development ◽  
Kidney Tissue ◽  
Experimental System ◽  
Human Kidney ◽  
Cost Effective ◽  
Cost Effective Method ◽  
Induced Pluripotent ◽  
Kidney Organoids

SummaryKidney organoids generated from human pluripotent stem cells have the potential to revolutionize how kidney development and injury are studied. Current protocols are technically complex and suffer from poor reproducibility and high reagent costs restricting scalability. To overcome these issues, we have established a simple, inexpensive and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day (d) 8 and show optimal tissue morphology at d14. A comparison with fetal human kidney suggests that d14 organoid renal structures most closely resemble ‘capillary loop’ stage nephrons. We show that deletion of HNF1B, a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant human renal development.

scholarly journals Detection of Hematopoietic Stem Cell Transcriptome in Human Fetal Kidneys and Kidney Organoids Derived From Human Induced Pluripotent Stem Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Jin Wook Hwang ◽  
Christophe Desterke ◽  
Julien Loisel-Duwattez ◽  
Frank Griscelli ◽  
Annelise Bennaceur-Griscelli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Fetal Liver ◽  
Human Kidney ◽  
Kidney Cortex ◽  
Hematopoietic Stem ◽  
Induced Pluripotent ◽  
Cell Transcriptome ◽  
Kidney Organoids

BackgroundIn mammalians, hematopoietic stem cells (HSCs) arise in the dorsal aorta from the hemogenic endothelium, followed by their migration to the fetal liver and to the bone marrow. In zebrafish, the kidney is the site of primary hematopoiesis. In humans, the presence of HSCs in the fetal or adult kidney has not been established.MethodsWe analyzed the presence of HSC markers in the human fetal kidneys by analysis of single-cell datasets. We then analyzed in kidney organoids derived from induced pluripotent stem cells (iPSCs) the presence of hematopoietic markers using transcriptome analyses.ResultsTwelve clusters were identified as stromal, endothelial, and nephron cell type-specific markers in the two fetal stage (17 weeks) kidney datasets. Among these, the expression of hematopoietic cells in cluster 9 showed an expression of primitive markers. Moreover, whole transcriptome analysis of our iPSC-derived kidney organoids revealed induction of the primitive hematopoietic transcription factor RUNX1 as found in the human fetal kidney cortex.ConclusionThese finding support the presence of cells expressing HSC transcriptome in the human kidney. The mechanisms of the appearance of the cells with the same transcriptional features during iPSC-derived kidney organoid generation require further investigation.

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

2019 ◽  
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.

scholarly journals Defining totipotency using criteria of increasing stringency

2020 ◽  
Author(s):  
Eszter Posfai ◽  
John Paul Schell ◽  
Adrian Janiszewski ◽  
Isidora Rovic ◽  
Alexander Murray ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Differentiated Cells ◽  
Totipotent Cell

AbstractTotipotency is the ability of a single cell to give rise to all the differentiated cells that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies upon a variety of assays of variable stringency. Here we describe criteria to define totipotency. We illustrate how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in the mouse, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbor increased totipotent potential relative to conventional embryonic stem cells under in vivo conditions.

scholarly journals Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics

2019 ◽  
Author(s):  
Esther Liu ◽  
Behram Radmanesh ◽  
Byungha H. Chung ◽  
Michael D. Donnan ◽  
Dan Yi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kidney Disease ◽  
Single Cell ◽  
Human Kidney ◽  
Cell Types ◽  
Genomic Locus ◽  
Glomerular Epithelial Cell ◽  
Risk Variants ◽  
Ifn Γ ◽  
Kidney Organoids

ABSTRACTBackgroundDNA variants in APOL1 associate with kidney disease, but the pathophysiological mechanisms remain incompletely understood. Model organisms lack the APOL1 gene, limiting the degree to which disease states can be recapitulated. Here we present single-cell RNA sequencing (scRNA-seq) of genome-edited human kidney organoids as a platform for profiling effects of APOL1 risk variants in diverse nephron cell types.MethodsWe performed footprint-free CRISPR-Cas9 genome editing of human induced pluripotent stem cells (iPSCs) to knock in APOL1 high-risk G1 variants at the native genomic locus. iPSCs were differentiated into kidney organoids, treated with vehicle, IFN-γ, or the combination of IFN-γ and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared to isogenic G0 controls.ResultsBoth G0 and G1 iPSCs differentiated into kidney organoids containing nephron-like structures with glomerular epithelial cells, proximal tubules, distal tubules, and endothelial cells. Organoids expressed detectable APOL1 only after exposure to IFN-γ. scRNA-seq revealed cell type-specific differences in G1 organoid response to APOL1 induction. Additional stress of tunicamycin exposure led to increased glomerular epithelial cell dedifferentiation in G1 organoids.ConclusionsSingle-cell transcriptomic profiling of human genome-edited kidney organoids expressing APOL1 risk variants provides a novel platform for studying the pathophysiology of APOL1-mediated kidney disease.SIGNIFICANCE STATEMENTGaps persist in our mechanistic understanding of APOL1-mediated kidney disease. The authors apply genome-edited human kidney organoids, combined with single-cell transcriptomics, to profile APOL1 risk variants at the native genomic locus in different cell types. This approach captures interferon-mediated induction of APOL1 gene expression and reveals cellular dedifferentiation after a secondary insult of endoplasmic reticulum stress. This system provides a human cellular platform to interrogate complex mechanisms and human-specific regulators underlying APOL1-mediated kidney disease.

scholarly journals Human pluripotent stem cells for the modelling and treatment of respiratory diseases

2021 ◽  
Vol 30 (161) ◽  
pp. 210042
Author(s):  
Pien A. Goldsteen ◽  
Christina Yoseif ◽  
Amalia M. Dolga ◽  
Reinoud Gosens
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
High Throughput Screening ◽  
Respiratory Diseases ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Human Pluripotent Stem Cells ◽  
Medical Need ◽  
New Chemical Entities

Respiratory diseases are among the leading causes of morbidity and mortality worldwide, representing a major unmet medical need. New chemical entities rarely make it into the clinic to treat respiratory diseases, which is partially due to a lack of adequate predictive disease models and the limited availability of human lung tissues to model respiratory disease. Human pluripotent stem cells (hPSCs) may help fill this gap by serving as a scalable human in vitro model. In addition, human in vitro models of rare genetic mutations can be generated using hPSCs. hPSC-derived epithelial cells and organoids have already shown great potential for the understanding of disease mechanisms, for finding new potential targets by using high-throughput screening platforms, and for personalised treatments. These potentials can also be applied to other hPSC-derived lung cell types in the future. In this review, we will discuss how hPSCs have brought, and may continue to bring, major changes to the field of respiratory diseases by understanding the molecular mechanisms of the pathology and by finding efficient therapeutics.

scholarly journals Comparative analysis of kidney organoid and adult human kidney single cell and single nucleus transcriptomes

2017 ◽  
Author(s):  
Haojia Wu ◽  
Kohei Uchimura ◽  
Erinn Donnelly ◽  
Yuhei Kirita ◽  
Samantha A. Morris ◽  
...  
Keyword(s):  
Single Cell ◽  
Human Kidney ◽  
Cell Types ◽  
Great Promise ◽  
Full Potential ◽  
Diverse Range ◽  
Adult Human ◽  
Cell Diversity ◽  
Single Nucleus ◽  
Kidney Organoids

AbstractKidney organoids differentiated from human pluripotent stem cells hold great promise for understanding organogenesis, modeling disease and ultimately as a source of replacement tissue. Realizing the full potential of this technology will require better differentiation strategies based upon knowledge of the cellular diversity and differentiation state of all cells within these organoids. Here we analyze single cell gene expression in 45,227 cells isolated from 23 organoids differentiated using two different protocols. Both generate kidney organoids that contain a diverse range of kidney cells at differing ratios as well as non-renal cell types. We quantified the differentiation state of major organoid kidney cell types by comparing them against a 4,259 single nucleus RNA-seq dataset generated from adult human kidney, revealing immaturity of all kidney organoid cell types. We reconstructed lineage relationships during organoid differentiation through pseudotemporal ordering, and identified transcription factor networks associated with fate decisions. These results define impressive kidney organoid cell diversity, identify incomplete differentiation as a major roadblock for current directed differentiation protocols and provide a human adult kidney snRNA-seq dataset against which to benchmark future progress.

scholarly journals Retinal organoids derived from rhesus macaque iPSCs undergo accelerated differentiation compared to human stem cells

2021 ◽  
Author(s):  
Antonio Jacobo Lopez ◽  
Sangbae Kim ◽  
Xinye Qian ◽  
Jeffrey Rogers ◽  
J. Timothy Stout ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Human Embryonic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Human Stem Cells ◽  
Retinal Differentiation ◽  
Optic Vesicles

Purpose: To compare the timing and efficiency of the development of non-human primate (NHP) derived retinal organoids in comparison to those derived from human embryonic stem cells. Methods: Human embryonic stem cells (hESCs) and induced-pluripotent stem cells (rhiPSCs) derived from non-human primates (Macaca mulatta) were differentiated into retinal organoids by using an established differentiation protocol. Briefly, embryoid bodies were formed from pluripotent stem cells and induced into a neural lineage with neural induction media with the addition of BMP4. Thereafter, self-formation of optic vesicles was allowed to form in a 2D culture in retinal differentiation media (RDM). Optic vesicles were then manually harvested and cultured in suspension in 3D-RDM media until analysis. Differences in the timing of differentiation and efficiency of retinal organoid development were assessed by light microscopy, electron microscopy, immunocytochemistry, and single-cell transcriptomics. Results: Generation of retinal organoids was achieved from both human and several NHP pluripotent stem cells lines. All rhiPSC lines resulted in retinal differentiation with the formation of optic vesicle-like structures similar to what has been observed in hESC retinal organoids. NHP retinal organoids had laminated structure and were composed of mature retinal cell types including cone and rod photoreceptors. Single cell RNA sequencing was conducted at two time points, which allowed identification of cell types and characterization of developmental trajectory in the developing organoid. Important differences between rhesus and human cells were measured regarding the timing and efficiency of retinal organoid differentiation. While the culture of NHP-derived iPSCs is relatively difficult compared to human stem cells, the generation of retinal organoids is feasible and may be less time consuming due to an intrinsically faster timing of retinal differentiation. Conclusions: Retinal organoids produced from iPSCs derived from Rhesus monkey using established protocols differentiate through the stages of organoid development faster than those derived from human stem cells. The production of NHP retinal organoids may be advantageous to reduce experimental time and cost for basic biology studies in retinogenesis as well as for preclinical trials in NHPs studying retinal allograft transplantation.

scholarly journals Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 876
Author(s):  
Raquel Bernad ◽  
Cian J. Lynch ◽  
Rocio G. Urdinguio ◽  
Camille Stephan-Otto Attolini ◽  
Mario F. Fraga ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cell ◽  
Normal Karyotype ◽  
Cell Types ◽  
Dna Demethylation ◽  
Starting State ◽  
Teratoma Formation

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture.

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