Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Summary

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.

Abstract

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.

Protocol

1. Preparing Reagents for Kidney Digestion

1. 1.5 ml of 0.25 % Collagenase A (w/v) and 1 % Pancreatin digest (w/v) solution will be needed for extraction of nephrogenic zone cells (NZCs) from 8 E17.5 kidneys. Because Pancreatin takes approximately 2 hours to dissolve at room temperature, enough digest solution for the largest number of embryonic kidneys expected should be made prior to the experiment. Prepare digest solution by first adding 25 mg of Collagenase A to 10 ml Dulbecco's Phosphate Buffered Saline (DPBS) in a clear 15 ml, sterile centrifuge tube. It is critical that the DPBS does NOT contain calcium or magnesium as this will interfere with the enz....

Discussion

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Watchmaker’s forceps #5	Roboz Surgical Instruments Co.	RS-4905	2 pairs required
Collagenase A	Roche Group	10 103 578 001	Wear mask
Porcine Pancreatin	Sigma-Aldrich	P8096	Wear mask
DPBS w/ Ca, Mg	Lonza Inc.	17-513F	With Mg & Ca
Fetal bovine serum (FBS)	BioWhittaker	14-901E
HBSS	GIBCO, by Life Technologies	14175
KSFM	GIBCO, by Life Technologies	10724-011	without added growth factors
L-Glutamine 200mM	Sigma-Aldrich	G7513	Use @ 1% v/v
PenStrep 10,000 U penicillin / ml 10 mg streptomycin / ml	Sigma-Aldrich	P4333	Use @ 1% v/v
Fibronectin	BD Biosciences	356008	Supplied as powder
24 well culture plate	Thermo Fisher Scientific, Inc.	142485	Nunclon
DPBS w/o Mg & Ca	Lonza Inc.	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-Aldrich	P6148	Use @ 4 % w/v
Triton X100	VWR international	VW3929-2	Use @ 0.3 % v/v
PBS	Sigma-Aldrich	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 Millipore	GX0185-6	Mix 50 % with H2O
RNeasy Micro Kit	Qiagen	74004	Use "DNase on column" protocol
Transfer pipettes, 3ml	BD Biosciences	357575