Sara E. Howden

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common dominant muscular dystrophies and remains without an approved disease modifying therapy. Caused by the aberrant expression of the cytotoxic gene DUX4, FSHD is typically diagnosed in adulthood, however clinical onset in children (DUX4 mRNA expression, muscle atrophy, and weakness, which correlate with the individuals’ specific disease severity. Next, we assessed the efficacy of adenine base editing (ABE) as a potential gene therapy approach to treat FSHD. DUX4 -targeted ABE reduced DUX4 mRNA expression, improved muscle area and force generation in the most severe individual. Together this work supports the use of iPSC-derived skeletal muscle models as a less invasive method to study childhood-onset FSHD and establishes targeted DUX4 gene editing therapies as a potential treatment approach.

The utility of human pluripotent stem cells (hPSCs) is greatly enhanced by the ability to introduce precise, site-specific genetic modifications with minimal off-target effects. Although Cas9 endonuclease is an exceptionally efficient gene-editing tool, its propensity for generating biallelic modifications often limits its capacity for introducing heterozygous variants. Here, we use prime editing (PE) to install heterozygous edits in over 10 distinct genetic loci, achieving knock-in efficiencies of up to 40% without the need for subsequent purification or drug selection steps. Moreover, PE enables the precise introduction of heterozygous edits in paralogous genes that are otherwise extremely challenging to achieve using endonuclease-based editing approaches. We also show that PE can be successfully combined with reprogramming to derive heterozygous induced pluripotent stem cell clones directly from human fibroblasts and peripheral blood mononuclear cells. Our findings highlight the utility of PE for generating hPSCs with complex edits and represent a powerful platform for modeling disease-associated dominant mutations and gene-dosage effects in an entirely isogenic context.

Human pluripotent stem cell-derived kidney organoids replicate embryonic nephrogenesis in a 3D-culture system. Recent advances suggest that refining the culture environment to replicate spatiotemporal cues present during embryonic organogenesis improves patterning. Here, this paradigm was applied to FGF signalling, a key regulator of embryonic nephron progenitor maintenance, nephrogenesis and ureteric branching. Both FGF8b and FGF10 signalling is sufficient to support nephrogenesis, with each having distinct effects on nephron patterning. FGF10 enhanced the initial WT1+ mesenchymal population, leading to proximally biased nephrons, while FGF8b biased toward early distal patterning, leading to the formation of cells with connecting segment identity. The addition of both FGF8b and FGF10 together had an additive effect, leading to a balance of proximal and distal patterning. This differential patterning was retained in tissue transplanted under the murine renal capsule, with FGF8b-treated organoids displaying increased distal/connecting segments. These findings highlight plasticity during organoid nephrogenesis that can be modulated by FGF signalling and identify an approach to refine nephrogenesis toward key cell types.

SUMMARY: The utility of human pluripotent stem cells (hPSCs) is greatly enhanced by the ability to introduce precise, site-specific genetic modifications with minimal off-target effects. Although Cas9 endonuclease is an exceptionally efficient gene-editing tool, its propensity for generating biallelic modifications often limits its capacity for introducing heterozygous variants. Here, we use prime editing (PE) to install heterozygous edits in over ten distinct genetic loci, achieving knock-in efficiencies of up to 40% without the need for subsequent purification or drug selection steps. Moreover, PE enables the precise introduction of heterozygous edits in paralogous genes that are otherwise extremely challenging to achieve using endonuclease-based editing approaches. We also show that PE can be successfully combined with reprogramming to derive heterozygous iPSC clones directly from human fibroblasts and peripheral blood mononuclear cells. Our findings highlight the utility of PE for generating hPSCs with complex edits and represents a powerful platform for modelling disease-associated dominant mutations and gene-dosage effects in an entirely isogenic context.

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. Translational Statement: Developing therapies for patients with kidney diseases relies on a morphologically and physiologically representative in vitro model. Human kidney organoids are an attractive model to recapitulate kidney physiology, however, they are limited by the absence of a vascular network and mature cell populations. In this work, we have generated a genetically inducible endothelial niche that, when combined with an established kidney organoid protocol, induces the maturation of a robust endothelial cell network, induces a more mature podocyte population, and induces the emergence a functional renin population. This advance significantly increases the clinical relevance of human kidney organoids for etiological studies of kidney disease and future regenerative medicine strategies. Graphical Genetically engineered endothelial niche induces mature cell populations in human 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.

Kidney organoids provide a valuable resource to understand kidney development and disease. Clustering algorithms and marker genes fail to accurately and robustly classify cellular identity between human pluripotent stem cell (hPSC)-derived organoid datasets. Here we present a new method able to accurately classify kidney cell subtypes, a hierarchical machine learning model trained using comprehensive reference data from single cell RNA-sequencing of human fetal kidney (HFK). We demonstrate the tool’s ( DevKidCC ) performance by application to all published kidney organoid datasets and a novel dataset. DevKidCC is available on Github and can be used on any kidney single cell RNA-sequence data.

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.

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.