In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.
Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering naïve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated http://www.gudmap.org/, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.
In this protocol we describe a method to isolate and culture cells from the nephrogenic zone of the embryonic kidney. It is the development of a method initially published as part of a study of the effects of BMP7 treatment on cells of the nephrogenic zone (Blank et al., 2009). In the initial study, a series of experiments to characterize the cell types represented within the NZC population were conducted. Briefly, purification of NZCs from genetic reporters for the cortical stroma (Foxd1+/lacZ</e…
The authors have nothing to disclose.
This work was supported by R01 DK078161 from NIDDK (LO), postdoctoral fellowships from the American Heart Association (AB), the Wenner-Gren Foundations of Sweden and Kungliga Fysiografiska Sällskapet, Lund, Sweden (UB). Additional support was provided by Maine Medical Center Research Institute core facilities for Histopathology, Bioinformatics and FACS (supported by 2P20RR18789-06) and the MMCRI Animal Facility.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Watchmaker’s forceps #5 | Roboz | RS-4905 | 2 pairs required | |
Collagenase A | Roche | 10 103 578 001 | Wear mask | |
Porcine Pancreatin | Sigma | P8096 | Wear mask | |
DPBS w/ Ca, Mg | Lonza | 17-513F | With Mg & Ca | |
Fetal bovine serum (FBS) | BioWhittaker | 14-901E | ||
HBSS | Gibco | 14175 | ||
KSFM | Gibco | 10724-011 | without added growth factors | |
L-Glutamine 200mM | Sigma | G7513 | Use @ 1% v/v | |
PenStrep 10,000 U penicillin / ml 10 mg streptomycin / ml | Sigma | P4333 | Use @ 1% v/v | |
Fibronectin | BD Biosciences | 356008 | Supplied as powder | |
24 well culture plate | Thermo Scientific | 142485 | Nunclon | |
DPBS w/o Mg & Ca | Lonza | 17-512F | ||
5mL polystyrene tubes | BD Biosciences | 352235 | ||
DNase (1 U/μl) | Invitrogen | 18068-015 | 4 μl per 1.5 ml | |
40 micron cell-strainer | BD Biosciences | 352235 | w/cap and tube | |
Paraformaldehyde | Sigma | P6148 | Use @ 4 % w/v | |
Triton X100 | VWR | VW3929-2 | Use @ 0.3 % v/v | |
PBS | Sigma | P3813 | Supplied as powder | |
Donkey serum | Jackson ImmunoResearch | 017-000-121 | Use @ 5 % v/v | |
Rabbit anti-PAX2 | Invitrogen | 71-6000 | Dilute 1:100 | |
Rabbit anti-β-gal | MP Biomedicals | 559762 | Dilute 1:200 | |
Oregon Green 488 phalloidin | Invitrogen | O7466 | Dilute 1:200 | |
Donkey anti-rabbit Alexa Fluor 568 | Invitrogen | A10042 | Dilute 1:200 | |
DAPI | Invitrogen | D1306 | Dilute 1:5000 | |
Vectashield | Vector Laboratories | H-1000 | ||
Glycerol | EMD | GX0185-6 | Mix 50 % with H2O | |
RNeasy Micro Kit | Qiagen | 74004 | Use “DNase on column” protocol | |
Transfer pipettes, 3ml | BD Falcon | 357575 |