Generation of a homozygous (MCRIi031-A-3) WT1 knockout human iPSC line.

The transcription factor WT1 plays a critical role in several embryonic developmental processes such as gonadogenesis, nephrogenesis, and cardiac development. We generated a homozygous (MCRIi031-A-3) WT1 knockout induced pluripotent stem cell (iPSC) line from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. The cells exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of WT1 in critical developmental processes.

Generation of heterozygous (MCRIi031-A-1) and homozygous (MCRIi031-A-2) SOX9 knockout human iPSC lines.

The transcription factor SOX9 plays a critical role in several embryonic developmental processes such as gonadogenesis, chrondrogenesis, and cardiac development. We generated heterozygous (MCRIi031-A-1) and homozygous (MCRIi031-A-2) SOX9 knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of SOX9 in critical developmental processes.

A genetically inducible endothelial niche enables vascularization of human kidney organoids with multilineage maturation and emergence of renin expressing cells.

Vascularization plays a critical role in organ maturation and cell-type development. Drug discovery, organ mimicry, and ultimately transplantation hinge on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcame this hurdle by combining a human induced pluripotent stem cell (iPSC) line containing an inducible ETS translocation variant 2 (ETV2) (a transcription factor playing a role in endothelial cell development) that directs endothelial differentiation in vitro, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive endothelialization with a cellular identity most closely related to human kidney endothelia. Endothelialized kidney organoids also show increased maturation of nephron structures, an associated fenestrated endothelium with de novo formation of glomerular and venous subtypes, and the emergence of drug-responsive renin expressing cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Thus, incorporation of an engineered endothelial niche into a previously published kidney organoid protocol allowed the orthogonal differentiation of endothelial and parenchymal cell types, demonstrating the potential for applicability to other basic and translational organoid studies.

Generation of heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2/COUP-TFII knockout human iPSC lines.

The NR2F2 gene encodes the transcription factor COUP-TFII, which is upregulated in embryonic mesoderm. Heterozygous variants in NR2F2 cause a spectrum of congenital anomalies including cardiac and gonadal phenotypes. We generated heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2-knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibited a normal karyotype, typical pluripotent cell morphology, pluripotency marker expression, and the capacity to differentiate into the three embryonic germ layers. These lines will allow us to explore the role of NR2F2 during development and disease.

Generation of a human ACTA1-tdTomato reporter iPSC line using CRISPR/Cas9 editing.

We used gene editing to introduce DNA sequences encoding the tdTomato fluorescent protein into the α -skeletal actin 1 (ACTA1) locus to develop an ACTA1-tdTomato induced pluripotent stem cell reporter line for monitoring differentiation of skeletal muscle. This cell line will be used to better understand skeletal muscle maturation and development in vitro as well as provide a useful tool for drug screening and the evaluation of novel therapeutics for the treatment of skeletal muscle disease.

Genetically engineering endothelial niche in human kidney organoids enables multilineage maturation, vascularization and de novo cell types.

Vascularization plays a critical role in organ maturation and cell type development. Drug discovery, organ mimicry, and ultimately transplantation in a clinical setting thereby hinges on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcome this hurdle by combining an inducible ETS translocation variant 2 (ETV2) human induced pluripotent stem cell (iPSC) line, which directs endothelial fate, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive vascularization by endothelial cells with an identity most closely related to endogenous kidney endothelia. Vascularized organoids also show increased maturation of nephron structures including more mature podocytes with improved marker expression, foot process interdigitation, an associated fenestrated endothelium, and the presence of renin+ cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Furthermore, this approach is orthogonal to native tissue differentiation paths, hence readily adaptable to other organoid systems and thus has the potential for a broad impact on basic and translational organoid studies.

Generation of iPSC lines from peripheral blood mononuclear cells from five healthy donors.

We describe the generation and characterisation of five human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) of healthy adult individuals. The PBMCs were reprogrammed using non-integrating Sendai viruses containing the reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC. The iPSC lines exhibited a normal karyotype, and pluripotency was validated by flow cytometry and immunofluorescence of pluripotency markers, and their differentiation into cells representative of the three embryonic germ layers. These iPSC lines can be used as controls in studying disease mechanisms.

Kidney Organoids Generated Using an Allelic Series of NPHS2 Point Variants Reveal Distinct Intracellular Podocin Mistrafficking.

BACKGROUND: NPHS2 variants are the most common cause of steroid-resistant nephrotic syndrome in children >1 month old. Missense NPHS2 variants were reported to cause mistrafficking of the encoded protein, PODOCIN, but this conclusion was on the basis of overexpression in some nonpodocyte cell lines. METHODS: We generated a series of human induced pluripotent stem cell (iPSC) lines bearing pathogenic missense variants of NPHS2 , encoding the protein changes p.G92C, p.P118L, p.R138Q, p.R168H, and p.R291W, and control lines. iPSC lines were also generated from a patient with steroid-resistant nephrotic syndrome (p.R168H homozygote) and a healthy heterozygous parent. All lines were differentiated into kidney organoids. Immunofluorescence assessed PODOCIN expression and subcellular localization. Podocytes were transcriptionally profiled and PODOCIN-NEPHRIN interaction interrogated. RESULTS: All variant lines revealed reduced levels of PODOCIN protein in the absence of reduced transcription. Although wild-type PODOCIN localized to the membrane, distinct variant proteins displayed unique patterns of subcellular protein trafficking, some unreported. P118L and R138Q were preferentially retained in the endoplasmic reticulum (ER); R168H and R291W accumulated in the Golgi. Podocyte profiling demonstrated minimal disease-associated transcriptional change. All variants displayed podocyte-specific apoptosis, which was not linked to ER stress. NEPHRIN-PODOCIN colocalization elucidated the variant-specific effect on NEPHRIN association and hence NEPHRIN trafficking. CONCLUSIONS: Specific variants of endogenous NPHS2 result in distinct subcellular PODOCIN localization within organoid podocytes. Understanding the effect of each variant on protein levels and localization and the effect on NEPHRIN provides additional insight into the pathobiology of NPHS2 variants. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_01_05_JASN2022060707.mp3.

Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids.

While pluripotent stem cell-derived kidney organoids are now being used to model renal disease, the proximal nephron remains immature with limited evidence for key functional solute channels. This may reflect early mispatterning of the nephrogenic mesenchyme and/or insufficient maturation. Here we show that enhanced specification to metanephric nephron progenitors results in elongated and radially aligned proximalised nephrons with distinct S1 - S3 proximal tubule cell types. Such PT-enhanced organoids possess improved albumin and organic cation uptake, appropriate KIM-1 upregulation in response to cisplatin, and improved expression of SARS-CoV-2 entry factors resulting in increased viral replication. The striking proximo-distal orientation of nephrons resulted from localized WNT antagonism originating from the organoid stromal core. PT-enhanced organoids represent an improved model to study inherited and acquired proximal tubular disease as well as drug and viral responses.

Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids.

While pluripotent stem cell-derived kidney organoids are now being used to model renal disease, the proximal nephron remains immature with limited evidence for key functional solute channels. This may reflect early mispatterning of the nephrogenic mesenchyme and/or insufficient maturation. Here we show that enhanced specification to metanephric nephron progenitors results in elongated and radially aligned proximalised nephrons with distinct S1 - S3 proximal tubule cell types. Such PT-enhanced organoids possess improved albumin and organic cation uptake, appropriate KIM-1 upregulation in response to cisplatin, and improved expression of SARS-CoV-2 entry factors resulting in increased viral replication. The striking proximo-distal orientation of nephrons resulted from localized WNT antagonism originating from the organoid stromal core. PT-enhanced organoids represent an improved model to study inherited and acquired proximal tubular disease as well as drug and viral responses.

DevKidCC allows for robust classification and direct comparisons of kidney organoid datasets.

BACKGROUND: While single-cell transcriptional profiling has greatly increased our capacity to interrogate biology, accurate cell classification within and between datasets is a key challenge. This is particularly so in pluripotent stem cell-derived organoids which represent a model of a developmental system. Here, clustering algorithms and selected marker genes can fail to accurately classify cellular identity while variation in analyses makes it difficult to meaningfully compare datasets. Kidney organoids provide a valuable resource to understand kidney development and disease. However, direct comparison of relative cellular composition between protocols has proved challenging. Hence, an unbiased approach for classifying cell identity is required. METHODS: The R package, scPred, was trained on multiple single cell RNA-seq datasets of human fetal kidney. A hierarchical model classified cellular subtypes into nephron, stroma and ureteric epithelial elements. This model, provided in the R package DevKidCC ( github.com/KidneyRegeneration/DevKidCC ), was then used to predict relative cell identity within published kidney organoid datasets generated using distinct cell lines and differentiation protocols, interrogating the impact of such variations. The package contains custom functions for the display of differential gene expression within cellular subtypes. RESULTS: DevKidCC was used to directly compare between distinct kidney organoid protocols, identifying differences in relative proportions of cell types at all hierarchical levels of the model and highlighting variations in stromal and unassigned cell types, nephron progenitor prevalence and relative maturation of individual epithelial segments. Of note, DevKidCC was able to distinguish distal nephron from ureteric epithelium, cell types with overlapping profiles that have previously confounded analyses. When applied to a variation in protocol via the addition of retinoic acid, DevKidCC identified a consequential depletion of nephron progenitors. CONCLUSIONS: The application of DevKidCC to kidney organoids reproducibly classifies component cellular identity within distinct single-cell datasets. The application of the tool is summarised in an interactive Shiny application, as are examples of the utility of in-built functions for data presentation. This tool will enable the consistent and rapid comparison of kidney organoid protocols, driving improvements in patterning to kidney endpoints and validating new approaches.

Forward steps in organoid-based forward screening.

In this issue of Cell Stem Cell, Ungricht et al. (2022) perform a temporally controlled CRISPR/Cas9-based genome-wide screen in kidney organoids to uncover key gene networks important for the specification of kidney cell types from human pluripotent stem cells, thus furthering our understanding of human kidney development and disease.

Determining lineage relationships in kidney development and disease.

The lineage relationships of cells provide information about the origins of component cell types during development and repair as well as the source of aberrant cells during disease. Genetic approaches to lineage tracing applied in the mouse have revealed much about how the mammalian kidney forms, including the identification of key progenitors for the nephrons and stromal compartments. Inducible Cre systems have also facilitated lineage tracing studies in the postnatal animal that illustrate the changes in cellular fate that can occur during kidney injury. With the advent of single-cell transcriptional profiling and trajectory analyses, predictions of cellular relationships across development are now being made in model systems, such as the mouse, as well as in human fetal kidney. Importantly, these approaches provide predictions of lineage relationships rather than definitive evidence. Although genetic approaches to the study of lineage have not previously been possible in a human setting, the application of CRISPR-Cas9 gene editing of pluripotent stem cells is beginning to teach us about human lineage relationships.

Generating an iPSC line (with isogenic control) from the PBMCs of an ACTA1 (p.Gly148Asp) nemaline myopathy patient.

To produce an in vitro model of nemaline myopathy, we reprogrammed the peripheral blood mononuclear cells (PBMCs) of a patient with a heterozygous p.Gly148Asp mutation in exon 3 of the ACTA1 gene to iPSCs. Using CRISPR/Cas9 gene editing we corrected the mutation to generate an isogenic control line. Both the mutant and control show a normal karyotype, express pluripotency markers and could differentiae into the three cell states that represent embryonic germ layers (endoderm, mesoderm and neuroectoderm) and the dermomyotome (precursor of skeletal muscle). When differentiated these cell lines will be used to explore disease mechanisms and evaluate novel therapeutics.

In vivo survival and differentiation of Friedreich ataxia iPSC-derived sensory neurons transplanted in the adult dorsal root ganglia.

Friedreich ataxia (FRDA) is an autosomal recessive disease characterized by degeneration of dorsal root ganglia (DRG) sensory neurons, which is due to low levels of the mitochondrial protein Frataxin. To explore cell replacement therapies as a possible approach to treat FRDA, we examined transplantation of sensory neural progenitors derived from human embryonic stem cells (hESC) and FRDA induced pluripotent stem cells (iPSC) into adult rodent DRG regions. Our data showed survival and differentiation of hESC and FRDA iPSC-derived progenitors in the DRG 2 and 8 weeks post-transplantation, respectively. Donor cells expressed neuronal markers, including sensory and glial markers, demonstrating differentiation to these lineages. These results are novel and a highly significant first step in showing the possibility of using stem cells as a cell replacement therapy to treat DRG neurodegeneration in FRDA as well as other peripheral neuropathies.

Recessive NOS1AP variants impair actin remodeling and cause glomerulopathy in humans and mice.

Nephrotic syndrome (NS) is a leading cause of chronic kidney disease. We found recessive NOS1AP variants in two families with early-onset NS by exome sequencing. Overexpression of wild-type (WT) NOS1AP, but not cDNA constructs bearing patient variants, increased active CDC42 and promoted filopodia and podosome formation. Pharmacologic inhibition of CDC42 or its effectors, formin proteins, reduced NOS1AP-induced filopodia formation. NOS1AP knockdown reduced podocyte migration rate (PMR), which was rescued by overexpression of WT Nos1ap but not by constructs bearing patient variants. PMR in NOS1AP knockdown podocytes was also rescued by constitutively active CDC42Q61L or the formin DIAPH3 Modeling a NOS1AP patient variant in knock-in human kidney organoids revealed malformed glomeruli with increased apoptosis. Nos1apEx3-/Ex3- mice recapitulated the human phenotype, exhibiting proteinuria, foot process effacement, and glomerulosclerosis. These findings demonstrate that recessive NOS1AP variants impair CDC42/DIAPH-dependent actin remodeling, cause aberrant organoid glomerulogenesis, and lead to a glomerulopathy in humans and mice.

Plasticity of distal nephron epithelia from human kidney organoids enables the induction of ureteric tip and stalk.

During development, distinct progenitors contribute to the nephrons versus the ureteric epithelium of the kidney. Indeed, previous human pluripotent stem-cell-derived models of kidney tissue either contain nephrons or pattern specifically to the ureteric epithelium. By re-analyzing the transcriptional distinction between distal nephron and ureteric epithelium in human fetal kidney, we show here that, while existing nephron-containing kidney organoids contain distal nephron epithelium and no ureteric epithelium, this distal nephron segment alone displays significant in vitro plasticity and can adopt a ureteric epithelial tip identity when isolated and cultured in defined conditions. "Induced" ureteric epithelium cultures can be cryopreserved, serially passaged without loss of identity, and transitioned toward a collecting duct fate. Cultures harboring loss-of-function mutations in PKHD1 also recapitulate the cystic phenotype associated with autosomal recessive polycystic kidney disease.

Cellular extrusion bioprinting improves kidney organoid reproducibility and conformation.

Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here, we apply extrusion-based three-dimensional cellular bioprinting to deliver rapid and high-throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate the relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, three-dimensional bioprinting enables precise manipulation of biophysical properties, including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitates the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production delivers improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.

Generating Kidney Organoids from Human Pluripotent Stem Cells Using Defined Conditions.

The ultimate goal of regenerative medicine is to have access to an unlimited supply of specific cell types on demand, which can be used as effective therapies for a wide range of intractable disorders. With the availability of human pluripotent stem cells (hPSCs) and greatly improved protocols for their directed differentiation into specific cell types, including kidney, this prospect could soon become a reality. We have previously described the generation of kidney organoids from hPSCs. This chapter describes our latest differentiation protocol for generating kidney tissue, which uses a cost-effective and completely defined, xeno-free medium. As with our previous protocol, these complex, multicellular three-dimensional structures are composed of all anticipated kidney cell types including nephrons segmented into the glomerulus, proximal and distal tubule as well as an extensive endothelial network, and renal interstitium. As such, kidney organoids provide useful tools for understanding human development, disease modeling, drug screening/toxicology studies and tissue engineering applications, and may facilitate the development of transplantable hPSC-derived kidney tissue for regenerative medicine purposes in the future.

Particle-mediated delivery of frataxin plasmid to a human sensory neuronal model of Friedreich's ataxia.

Increasing frataxin protein levels through gene therapy is envisaged to improve therapeutic outcomes for patients with Friedreich's ataxia (FRDA). A non-viral strategy that uses submicrometer-sized multilayered particles to deliver frataxin-encoding plasmid DNA affords up to 27 000-fold increase in frataxin gene expression within 2 days in vitro in a stem cell-derived neuronal model of FRDA.