Organoids are not organs: Sources of variation and misinformation in organoid biology.

In the past decade, the term organoid has moved from obscurity to common use to describe a 3D in vitro cellular model of a tissue that recapitulates structural and functional elements of the in vivo organ it models. The term organoid is now applied to structures formed as a result of two distinct processes: the capacity for adult epithelial stem cells to re-create a tissue niche in vitro and the ability to direct the differentiation of pluripotent stem cells to a 3D self-organizing multicellular model of organogenesis. While these two organoid fields rely upon different stem cell types and recapitulate different processes, both share common challenges around robustness, accuracy, and reproducibility. Critically, organoids are not organs. This commentary serves to discuss these challenges, how they impact genuine utility, and shine a light on the need to improve the standards applied to all organoid approaches.

A Toolbox to Characterize Human Induced Pluripotent Stem Cell-Derived Kidney Cell Types and Organoids.

BACKGROUND: The generation of reporter lines for cell identity, lineage, and physiologic state has provided a powerful tool in advancing the dissection of mouse kidney morphogenesis at a molecular level. Although use of this approach is not an option for studying human development in vivo, its application in human induced pluripotent stem cells (iPSCs) is now feasible. METHODS: We used CRISPR/Cas9 gene editing to generate ten fluorescence reporter iPSC lines designed to identify nephron progenitors, podocytes, proximal and distal nephron, and ureteric epithelium. Directed differentiation to kidney organoids was performed according to published protocols. Using immunofluorescence and live confocal microscopy, flow cytometry, and cell sorting techniques, we investigated organoid patterning and reporter expression characteristics. RESULTS: Each iPSC reporter line formed well patterned kidney organoids. All reporter lines showed congruence of endogenous gene and protein expression, enabling isolation and characterization of kidney cell types of interest. We also demonstrated successful application of reporter lines for time-lapse imaging and mouse transplantation experiments. CONCLUSIONS: We generated, validated, and applied a suite of fluorescence iPSC reporter lines for the study of morphogenesis within human kidney organoids. This fluorescent iPSC reporter toolbox enables the visualization and isolation of key populations in forming kidney organoids, facilitating a range of applications, including cellular isolation, time-lapse imaging, protocol optimization, and lineage-tracing approaches. These tools offer promise for enhancing our understanding of this model system and its correspondence with human kidney morphogenesis.

Does Renal Repair Recapitulate Kidney Development?

Over a decade ago, it was proposed that the regulation of tubular repair in the kidney might involve the recapitulation of developmental pathways. Although the kidney cannot generate new nephrons after birth, suggesting a low level of regenerative competence, the tubular epithelial cells of the nephrons can proliferate to repair the damage after AKI. However, the debate continues over whether this repair involves a persistent progenitor population or any mature epithelial cell remaining after injury. Recent reports have highlighted the expression of Sox9, a transcription factor critical for normal kidney development, during postnatal epithelial repair in the kidney. Indeed, the proliferative response of the epithelium involves expression of several pathways previously described as being involved in kidney development. In some instances, these pathways are also apparently involved in the maladaptive responses observed after repeated injury. Whether development and repair in the kidney are the same processes or we are misinterpreting the similar expression of genes under different circumstances remains unknown. Here, we review the evidence for this link, concluding that such parallels in expression may more correctly represent the use of the same pathways in a distinct context, likely triggered by similar stressors.

Anlaysis of complementary expression profiles following WT1 induction versus repression reveals the cholesterol/fatty acid synthetic pathways as a possible major target of WT1.

The Wilms' tumour suppressor gene, WT1, encodes a zinc-finger protein that is mutated in Wilms' tumours and other malignancies. WT1 is one of the earliest genes expressed during kidney development. WT1 proteins can activate and repress putative target genes in vitro, although the in vivo relevance of such target genes often remains unverified. To better understand the role of WT1 in tumorigenesis and kidney development, we need to identify downstream target genes. In this study, we have expression profiled human embryonic kidney 293 cells stably transfected to allow inducible WT1 expression and mouse mesonephric M15 cells transfected with a WT1 antisense construct to abolish endogenous expression of all WT1 isoforms to identify WT1-responsive genes. The complementary overlap between the two cell lines revealed a pronounced repression of genes involved in cholesterol biosynthesis by WT1. This pathway is transcriptionally regulated by the sterol responsive element-binding proteins (SREBPs). Here, we provide evidence that the C-terminal end of the WT1 protein can directly interact with SREBP, suggesting that WT1 may modify the transcriptional function of SREBPs via a direct protein-protein interaction. Therefore, the tumour suppressor activities of WT1 may be achieved by repressing the mevalonate pathway, thereby controlling cellular proliferation and promoting terminal differentiation.

Coexpression of SCL and GATA3 in the V2 interneurons of the developing mouse spinal cord.

The differentiation of neural progenitors into the many classes of neurons that exist in the mature spinal cord is a process that relies heavily on the activation of precise combinations of transcription factors. Defining these transcription factor combinations is an important aspect of research in developmental neurobiology that promises to provide incredible insights into the structure, function, and pathology of the central nervous system. The present study aimed to investigate a possible role for the Stem Cell Leukemia (SCL) gene, a basic helix-loop-helix (bHLH) transcription factor gene, in the specification of a population of neural cells in the ventral neural tube. Section RNA in situ hybridisation revealed that SCL is transiently expressed within the V2 postmitotic domain of the developing mouse spinal cord between 10.5 and 13.5 days post coitum. Double-immunofluorescence experiments were subsequently carried out to directly compare the expression of SCL with other V2-specific markers at the cellular level. These experiments revealed that SCL is expressed in a medially restricted subpopulation of GATA-3 producing cells, suggesting a possible role for this factor in the differentiation of the GATA population of V2 interneurons.