The centrosomal protein 83 (CEP83) regulates human pluripotent stem cell differentiation toward the kidney lineage
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Curated by eLife
Evaluation Summary:
This work will be of interest to the field of researchers that generate human stem cell-derived kidney organoids to model genetic kidney diseases. It describes a novel and crucial role of the protein CEP83 in mesoderm patterning, which further determines whether kidney tissues can form correctly. Using cutting-edge technologies the authors provide strong data, which support the key claims of this manuscript. This work is of high impact due to the relevance of CEP83 mutations in human kidney disease.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
During embryonic development, the mesoderm undergoes patterning into diverse lineages including axial, paraxial, and lateral plate mesoderm (LPM). Within the LPM, the so-called intermediate mesoderm (IM) forms kidney and urogenital tract progenitor cells, while the remaining LPM forms cardiovascular, hematopoietic, mesothelial, and additional progenitor cells. The signals that regulate these early lineage decisions are incompletely understood. Here, we found that the centrosomal protein 83 (CEP83), a centriolar component necessary for primary cilia formation and mutated in pediatric kidney disease, influences the differentiation of human-induced pluripotent stem cells (hiPSCs) toward IM. We induced inactivating deletions of CEP83 in hiPSCs and applied a 7-day in vitro protocol of IM kidney progenitor differentiation, based on timed application of WNT and FGF agonists. We characterized induced mesodermal cell populations using single-cell and bulk transcriptomics and tested their ability to form kidney structures in subsequent organoid culture. While hiPSCs with homozygous CEP83 inactivation were normal regarding morphology and transcriptome, their induced differentiation into IM progenitor cells was perturbed. Mesodermal cells induced after 7 days of monolayer culture of CEP83 -deficient hiPCS exhibited absent or elongated primary cilia, displayed decreased expression of critical IM genes ( PAX8 , EYA1 , HOXB7 ), and an aberrant induction of LPM markers (e.g. FOXF1 , FOXF2 , FENDRR , HAND1 , HAND2 ). Upon subsequent organoid culture, wildtype cells differentiated to form kidney tubules and glomerular-like structures, whereas CEP83 -deficient cells failed to generate kidney cell types, instead upregulating cardiomyocyte, vascular, and more general LPM progenitor markers. Our data suggest that CEP83 regulates the balance of IM and LPM formation from human pluripotent stem cells, identifying a potential link between centriolar or ciliary function and mesodermal lineage induction.
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Evaluation Summary:
This work will be of interest to the field of researchers that generate human stem cell-derived kidney organoids to model genetic kidney diseases. It describes a novel and crucial role of the protein CEP83 in mesoderm patterning, which further determines whether kidney tissues can form correctly. Using cutting-edge technologies the authors provide strong data, which support the key claims of this manuscript. This work is of high impact due to the relevance of CEP83 mutations in human kidney disease.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This manuscript describes a novel role of the centrosomal protein CEP83 in mesoderm patterning, specifically the balancing between the intermediate mesoderm (IM) and the lateral plate mesoderm (LPM). The authors nicely demonstrate that CEP83 is required for the accurate formation of the IM and differentiation into the kidney lineage. This is achieved by generating CEP83 knockout iPSC lines and differentiating these into mesoderm cultures and kidney organoids. The most obvious defects in the knockout situation are ciliary abnormalities (in the mesoderm monolayer as well as 3D organoid cultures), indicative of the critical role of the cilia in normal kidney formation. Strikingly, the CEP83-deficient cultures completely fail to undergo further differentiation into kidney epithelia as shown by …
Reviewer #1 (Public Review):
This manuscript describes a novel role of the centrosomal protein CEP83 in mesoderm patterning, specifically the balancing between the intermediate mesoderm (IM) and the lateral plate mesoderm (LPM). The authors nicely demonstrate that CEP83 is required for the accurate formation of the IM and differentiation into the kidney lineage. This is achieved by generating CEP83 knockout iPSC lines and differentiating these into mesoderm cultures and kidney organoids. The most obvious defects in the knockout situation are ciliary abnormalities (in the mesoderm monolayer as well as 3D organoid cultures), indicative of the critical role of the cilia in normal kidney formation. Strikingly, the CEP83-deficient cultures completely fail to undergo further differentiation into kidney epithelia as shown by immunohistochemistry for nephron and podocyte markers. Thorough transcriptomic and bioinformatic analyses provide insights into these defects on a molecular basis, i.e. the CEP83 knockout appears to favor the formation of the LPM at the expense of the IM, with LPM-specific marker genes strongly upregulated in the knockout. A possible key role for the transcription factor HAND2 is discussed.
Strengths and appraisal
A major strength of this work is the use of cutting-edge technologies, including the CEP83 knockout in iPSCs, mesoderm and kidney organoid cultures, and bulk and single cell transcriptomics. These techniques provide strong data, which support the conclusions of this study.Weaknesses
There are no major weaknesses.The impact of this work is strengthened by its relevance to human kidney disease, i.e. CEP83 mutations that manifest, amongst others, in the kidney. It is also interesting for the field of researchers that use kidney organoids from human pluripotent stem cells and their ongoing attempts of improving the organoid model to more accurately recapitulate human kidney pathologies.
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Reviewer #2 (Public Review):
The manuscript gives novel insight into the role of a ciliary protein in participating in cell fate decisions in the early embryo. Given the known role of CEP83 mutations in causing nephronophthisis, it is surprising that a full knockout leads to such dramatic effects on the progenitor tissue of the kidney. The authors use advanced transcriptomic and sc-RNA Seq to gain insight into the cellular identities and fate ad various time points. The use of 3 independent clonal knockout lines is solid and thus the interpretation of the results is well supported by the evidence.
It remains to be explored to what degree this phenotype also occurs in knockout organoids of other ciliary components and which signalling events drive this process.
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