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Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney
Published on: April 22, 2011
Identification of multipotent progenitors in the embryonic mouse kidney by a novel colony-forming assay
Kenji Osafune1, Minoru Takasato, Andreas Kispert
1Division of Stem Cell Regulation, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
Abstract:
Renal stem or progenitor cells with a multilineage differentiation potential remain to be isolated, and the differentiation mechanism of these cell types in kidney development or regeneration processes is unknown. In an attempt to resolve this issue, we set up an in vitro culture system using NIH3T3 cells stably expressing Wnt4 (3T3Wnt4) as a feeder layer, in which a single renal progenitor in the metanephric mesenchyme forms colonies consisting of several types of epithelial cells that exist in glomeruli and renal tubules. We found that only cells strongly expressing Sall1 (Sall1-GFP(high) cells), a zinc-finger nuclear factor essential for kidney development, form colonies, and that they reconstitute a three-dimensional kidney structure in an organ culture setting. We also found that Rac- and JNK-dependent planar cell polarity (PCP) pathways downstream of Wnt4 positively regulate the colony size, and that the JNK pathway is also involved in mesenchymal-to-epithelial transformation of colony-forming progenitors. Thus our colony-forming assay, which identifies multipotent progenitors in the embryonic mouse kidney, can be used for examining mechanisms of renal progenitor differentiation.
Insights
Researchers identified multipotent renal progenitors in embryonic mouse kidneys using a novel in vitro culture system. These progenitors, expressing Sall1, can form kidney structures and their differentiation is regulated by Wnt4-dependent pathways.
Area of Science:
- Nephrology
- Developmental Biology
- Stem Cell Biology
Background:
- Isolation of multipotent renal stem/progenitor cells remains challenging.
- Mechanisms governing renal progenitor differentiation in kidney development and regeneration are largely unknown.
Purpose of the Study:
- To develop an in vitro system for isolating and characterizing multipotent renal progenitors.
- To investigate the differentiation mechanisms of these progenitors during kidney development.
Main Methods:
- Established an in vitro culture system using NIH3T3 cells expressing Wnt4 (3T3Wnt4) as a feeder layer.
- Utilized Sall1-GFP(high) cells, identified as key progenitors, for colony formation assays.
- Employed organ culture settings to assess the reconstitution of three-dimensional kidney structures.
Main Results:
- A single renal progenitor from the metanephric mesenchyme formed colonies of epithelial cells found in glomeruli and tubules.
- Only Sall1-GFP(high) cells formed colonies and could reconstitute kidney structures.
- Rac- and JNK-dependent planar cell polarity (PCP) pathways, downstream of Wnt4, positively regulated colony size.
- The JNK pathway was implicated in the mesenchymal-to-epithelial transformation of these progenitors.
Conclusions:
- Developed a functional colony-forming assay to identify multipotent progenitors in embryonic mouse kidneys.
- Demonstrated that Sall1 expression is crucial for the self-renewal and differentiation of renal progenitors.
- Revealed the role of Wnt4-mediated PCP signaling in regulating renal progenitor proliferation and differentiation.
