Functional Genetic Targeting of Embryonic Kidney Progenitor Cells Ex Vivo

Junttila, Sanna; Saarela, Ulla; Halt, Kimmo; Manninen, Aki; Pärssinen, Heikki; Lecca, M. Rita; Brändli, André W.; Sims‐Lucas, Sunder; Skovorodkin, Ilya; Vainio, Seppo

doi:10.1681/asn.2013060584

Cited by 40 publications

(64 citation statements)

References 50 publications

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“…The organoids are known as cellular aggregates containing more than one cell type typical for the organ they replicate [18]. Such organoids have successfully been constructed with the use of primary kidney cells [19–21]. …”

Section: Introductionmentioning

confidence: 99%

Exosomes as secondary inductive signals involved in kidney organogenesis

Krause

Rak-Raszewska

Naillat

et al. 2018

J of Extracellular Vesicle

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The subfraction of extracellular vesicles, called exosomes, transfers biological molecular information not only between cells but also between tissues and organs as nanolevel signals. Owing to their unique properties such that they contain several RNA species and proteins implicated in kidney development, exosomes are putative candidates to serve as developmental programming units in embryonic induction and tissue interactions. We used the mammalian metanephric kidney and its nephron-forming mesenchyme containing the nephron progenitor/stem cells as a model to investigate if secreted exosomes could serve as a novel type of inductive signal in a process defined as embryonic induction that controls organogenesis. As judged by several characteristic criteria, exosomes were enriched and purified from a cell line derived from embryonic kidney ureteric bud (UB) and from primary embryonic kidney UB cells, respectively. The cargo of the UB-derived exosomes was analysed by qPCR and proteomics. Several miRNA species that play a role in Wnt pathways and enrichment of proteins involved in pathways regulating the organization of the extracellular matrix as well as tissue homeostasis were identified. When labelled with fluorescent dyes, the uptake of the exosomes by metanephric mesenchyme (MM) cells and the transfer of their cargo to the cells can be observed. Closer inspection revealed that besides entering the cytoplasm, the exosomes were competent to also reach the nucleus. Furthermore, fluorescently labelled exosomal RNA enters into the cytoplasm of the MM cells. Exposure of the embryonic kidney-derived exosomes to the whole MM in an ex vivo organ culture setting did not lead to an induction of nephrogenesis but had an impact on the overall organization of the tissue. We conclude that the exosomes provide a novel signalling system with an apparent role in secondary embryonic induction regulating organogenesis.

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Section: Introductionmentioning

confidence: 99%

Exosomes as secondary inductive signals involved in kidney organogenesis

Krause

Rak-Raszewska

Naillat

et al. 2018

J of Extracellular Vesicle

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“…Indeed, there is substantial experimental evidence supporting the notion that the sorting of NP cells between the tip and corner regions is established by cell-cell adhesion differences and both inductive and chemotactic molecular signalling from the ureteric bud epithelia (Brown et al, 2013;Karner et al, 2011;Lefevre et al, 2017). Previous studies identified cellcell adhesion molecules such as cadherins, to drive cell sorting (Junttila et al, 2015;Lefevre et al, 2017). Extracellular signals inducing NP cell commitment involve the secretion of WNT11, BMP7, FGF9, and WNT9B, which upregulates Wnt4 (Bohnenpoll and Kispert, 2014).…”

Section: Discussionmentioning

confidence: 99%

Computational Modelling of Nephron Progenitor Cell Movement and Aggregation during Kidney Organogenesis

Tikka

Mercker

Skovorodkin

et al. 2020

Preprint

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During early kidney organogenesis, nephron progenitor (NP) cells move from the tip to the corner region of the ureteric bud (UB) branches in order to form the pretubular aggregate, the early structure giving rise to nephron formation. Chemotaxis and cell-cell adhesion differences are believed to drive cell patterning during this critical period of organogenesis, but the spatiotemporal organization of this process is incompletely understood.We applied a Cellular Potts model to explore to how these processes contribute to directed cell movement and aggregation. Model parameters were estimated based on fitting to experimental data obtained in ex vivo kidney explant and dissociation-reaggregation organoid culture studies.Our simulations indicated that optimal enrichment and aggregation of NP cells in the UB corner niche requires chemoattractant secretion from both the UB epithelial cells and the NP cells themselves, as well as differences in cell-cell adhesion energies. Furthermore, NP cells were observed, both experimentally and by modelling, to move at higher speed in the UB corner as compared to the tip region where they originated. The existence of different cell speed domains along the UB was confirmed using self-organizing map analysis.In summary, we demonstrated the suitability of a Cellular Potts Model approach to simulate cell movement and patterning during early nephrogenesis. Further refinement of the model should allow us to recapitulate the effects of developmental changes of cell phenotypes and molecular crosstalk during organ development. Author SummaryThe emergence of tissue patterns during vertebrate development is a major interest of both experimental research and biocomputational modelling. In this study, we established a Cellular Potts Model to explore cellular processes during early kidney development. The goal was to elucidate movements and aggregations of nephron progenitor cells. These precursor cells derive from mesenchymal cells around the ureteric buds and eventually form the epithelial structure of the nephron. Moreover, we wanted to explore computationally the mechanisms how these cells segregate from metanephric mesenchyme and move towards the location where the nephron will be formed. Utilizing the Compucell3D simulation software, we developed a model which assumes that nephron progenitor movement and aggregation is governed by only two mechanisms, i.e. cell-cell adhesion differences between cell types and nephron progenitor cell chemotaxis in response to chemoattractant secretion from two sources. These sources were either the epithelial cells of a static ureteric bud and/or the nephron progenitor cells themselves. The simulations indicated faster average cell speeds near the ureteric bud corner, the target region of cell movement and aggregation, and slower speeds near the place of origin, the tip of ureteric bud. The results were validated by comparison of the model predictions with experimental data from two ex vivo embryonic kidney models and a computational optimization protocol.

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“…In our experiments we used organoids derived from primary cell cultures isolated from embryonic metanephric mesenchyme, as described by Junttila et al (2015), except that 10 µM BIO (6-bromoindirubin-3′-oxime, Sigma) was used for induction.…”

Section: Methodsmentioning

confidence: 99%

Novel fixed Z-dimension (FiZD) kidney primordia and an organoid culture system for time-lapse confocal imaging

et al. 2017

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Tissue, organ and organoid cultures provide suitable models for developmental studies, but our understanding of how the organs are assembled at the single-cell level still remains unclear. We describe here a novel fixed z-direction (FiZD) culture setup that permits high-resolution confocal imaging of organoids and embryonic tissues. In a FiZD culture a permeable membrane compresses the tissues onto a glass coverslip and the spacers adjust the thickness, enabling the tissue to grow for up to 12 days. Thus, the kidney rudiment and the organoids can adjust to the limited z-directional space and yet advance the process of kidney morphogenesis, enabling long-term time-lapse and high-resolution confocal imaging. As the data quality achieved was sufficient for computer-assisted cell segmentation and analysis, the method can be used for studying morphogenesis ex vivo at the level of the single constituent cells of a complex mammalian organogenesis model system.

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Functional Genetic Targeting of Embryonic Kidney Progenitor Cells Ex Vivo

Cited by 40 publications

References 50 publications

Exosomes as secondary inductive signals involved in kidney organogenesis

Exosomes as secondary inductive signals involved in kidney organogenesis

Computational Modelling of Nephron Progenitor Cell Movement and Aggregation during Kidney Organogenesis

Novel fixed Z-dimension (FiZD) kidney primordia and an organoid culture system for time-lapse confocal imaging

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