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 S&auml;llskapet, 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.

1. Preparing Reagents for Kidney Digestion
<ol>
	<li>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 enzymatic digest. Mix solution on a nutator for 10 minutes to dissolve Collagenase A. Add 100 mg of Pancreatin to dissolved Collagenase A solution and mix on a nutator for at least 2 hours. Alternatively, Collagenase A/Pancreatin digest solution can be mixed on a nutator overnight at 4&deg; C. It is recommended that you wear a mask, as Collagenase A and Pancreatin disperse easily into the atmosphere during weighing and can irritate nasal passages.</li>
	<li>Prepare a 5 % Fetal Bovine Serum (FBS) (v/v) solution in Hank's Buffered Saline solution (HBSS). Invert to mix. 1 ml for each tube of 8 kidneys will be necessary.</li>
	<li>Prepare culture medium by supplementing Keratinocyte serum free medium with 1 % glutamine (v/v) and 1 % Penicillin-Streptomycin (PenStrep) (v/v).</li>
	<li>Coat sterile flat bottom polystyrene tissue culture plates (96, 48, 24 or 6 well) with human fibronectin solution at a concentration of 5 mg per cm2 of growth surface. 5 mg of fibronectin powder is resuspended in 5 ml of sterile H2O. This is diluted 1:25 in sterile DPBS without calcium or magnesium and 250 ml is added to each well of a 24 well plate. Plates are then incubated with fibronectin solution at room temperature for 1 hour followed by 1 hour at 4&deg; C. Alternatively, plates can be incubated overnight at 4&deg; C. The solution is then aspirated in a laminar flow hood prior to addition of medium/cells.</li>
</ol>
2. Dissecting Kidneys &amp; Removing Capsules (Done At Room Temperature)
<ol>
	<li>At E17.5, sacrifice the mother using a procedure approved by your Institutional Animal Care and Use Committee, remove the uterus, and place it in a Petri dish containing DPBS with calcium and magnesium. Under a dissecting microscope, use watchmakers' forceps to remove each embryo from the uterus and decapitate each embryo. Once the entire litter is out of the uterus, transfer the embryos to a clean Petri dish containing enough DPBS with calcium and magnesium to entirely cover the embryos leaving behind as much blood and tissue debris as possible.</li>
	<li>Using forceps, make a circumferential incision in the abdominal wall of the embryo at the level of the umbilicus. Then place the embryo on its back, pin it down at the shoulders with one set of forceps, and remove the internal organs from the lungs down to the bladder using the other set of forceps. With practice this can be done in one motion. The kidneys should be attached to the back of the mass of organs. If the kidneys do not remain attached to the eviscerated organs, they can be carefully removed from the dorsal body wall. Kidneys that have been damaged during this dissection process should be discarded as they will contaminate the final culture with inappropriate cell types.</li>
	<li>Gently tease away the kidneys from the rest of the organs, taking care to keep the kidneys connected to each other. Hold the tissue between the kidneys with one set of forceps and grab the adrenal gland attached to one of the kidneys with the other forceps. The adrenal gland should be attached to the kidney capsule. Gently peel the adrenal gland and capsule away from around the outside of the kidney, similar to peeling a banana. Take care not to split the kidney in half while peeling, and be sure not to damage the kidney tissue underneath the capsule. Carefully remove any remaining capsule as well as the ureter if it is still attached. Repeat with the remaining kidney.</li>
	<li>Use a cut transfer pipet to place the kidneys into a 5 ml polystyrene tube of HBSS. Repeat steps 2.2-2.4 for each embryo. Kidneys may be pooled 8 per tube. With practice, dissection and capsule removal will take 2-3 minutes per embryo.</li>
</ol>
3. Enzymatic Digestion of Kidneys (All Steps At Room Temperature Unless Otherwise Indicated)
<ol>
	<li>Remove HBSS solution from 5 ml tubes containing kidneys with a pipettor while carefully avoiding contact with kidneys. Immediately add 1.5 ml of Collagenase A/Pancreatin digest solution to the 5 ml tube containing kidneys before moving to the next sample. Repeat for each tube of kidneys.</li>
	<li>Place tubes with enzyme/kidney digests on a nutator in a pre-warmed 37&deg; C incubator for 15 minutes. 2 minutes prior to completion of the digest, add 3 &mu;l of DNase (1 U/ml) to one fresh 5 ml polystyrene tube for each digest being performed.</li>
	<li>Quickly remove digests from incubator, add 75 &mu;l of FBS and invert 3 times to mix. Let stand on bench for 2 minutes.</li>
	<li>Transfer 1.4 ml of the cell suspension to the 5 ml polystyrene tube containing DNase (1 U/ml) while leaving the remaining 0.2 ml cell suspension and kidneys behind. The extra 0.2 mL of solution left behind will contain impurities such as extracellular matrix.</li>
	<li>Mix cell suspension/DNase on a nutator in a pre-warmed 37&deg; C incubator for 10 minutes.</li>
	<li>Quickly remove cell suspensions from incubator and transfer each to a 1.5 ml Eppendorf tube. Spin cell suspensions in microfuge at 325 g for 5 minutes.</li>
	<li>Remove supernatant from cell pellet and resuspend pellet in 1 ml of 5% FBS/HBSS, pipetting up and down 5 to 6 times. Cap each tube before moving to the next sample.</li>
	<li>Spin cell suspensions in a microcentrifuge at 325 g for 5 minutes.</li>
	<li>Remove supernatant and add 0.5 ml of Keratinocyte serum free medium (KSFM) with additives to each tube and cap tube before moving to the next sample. After medium has been added to all tubes, gently resuspend each cell pellet by pipetting up and down 5 to 6 times with a 1ml micropipette. Combine all cell suspensions in a fresh 5 ml polystyrene tube.</li>
	<li>Pre-wet two 40 micron cell-strainer caps placed on a 5 ml polystyrene round bottom tube with KSFM per sample. Pass cell suspensions through one cell strainer cap by transferring with a 1 ml micropipette. Avoid touching the filter with the pipette tip. Collect flow-through and repeat filtration through the second strainer cap. Remove the cell strainer cap and replace with a new cap from a normal 5 ml polystyrene tube.</li>
	<li>Count cells with a hemocytometer or other cell counting device and transfer cells to wells of a fibronectin coated culture plate at a density of 100,000-200,000 cells per cm2, diluting as required with KSFM with additives. Generally, we retrieve approximately 4.5x106 cells per 10 embryo litter.</li>
	<li>Incubate cells in a 37&deg; C humidified incubator with 5% CO2.</li>
	<li>Allow cells to recover and attach for 2-4 hours in medium prior to stimulation with additional reagents. Stimulate cells with compound of interest for 1 to 48 hours. Purify RNA for PCR analysis using the Qiagen RNeasy Micro Kit following the manufacturer's instructions or follow the instructions below to fluorescently immunostain the cells.</li>
</ol>
4. Preparing Reagents for Fixation of Cells and Immunostaining
<ol>
	<li>Prepare 4% PFA: Dissolve 4% paraformaldehyde in PBS (v/v) at 55&deg; C with regular agitation. Cool to room temperature before use. This solution is toxic and should be discarded in designated waste containers.</li>
	<li>Prepare permeabilization solution by adding Triton X-100 to PBS to obtain a 0.3% (v/v) solution.</li>
	<li>Prepare a 5 % blocking solution in PBS with serum from the same species as was used to raise the secondary antibody to be used. For example, if you will detect a rabbit antibody against your target antigen with a donkey anti-Rabbit Alexa Fluor 568, prepare a blocking solution containing 5% donkey serum. Store at 4&deg; C.</li>
	<li>Prepare primary antibody solution by adding the appropriate concentration of antibody to 5% blocking solution.</li>
	<li>Prepare secondary antibody solution immediately prior to use. Add fluorophore conjugated antibody at manufacturer's recommended concentration together with the nuclear counterstain DAPI and other organelle markers such as phalloidin to 5% blocking solution. If storage is required, keep solution in the dark at 4&deg; C until use.</li>
	<li>Prepare 50% glycerol/PBS solution (v/v) for storage of immunostained cells. Alternatively, Vectashield can be used.</li>
</ol>
5. Fixation of Cells and Immunostaining
<ol>
	<li>The following protocol is designed for fixation and staining of NZCs in a 24 well plate. Volumes given are for a single well. Reagents should be gently pipetted during all steps to avoid detachment of cells, and plate agitation is not recommended in the incubation steps.</li>
	<li>Gently remove medium with a 1 ml micropipette and add 0.5 ml of 4% PFA to attached cultured cells. Incubate for 15 minutes undisturbed at room temperature.</li>
	<li>Remove 4% PFA and add 0.5 ml per well of permeabilization solution. Incubate for 10 minutes at room temperature.</li>
	<li>Remove permeabilization solution and gently rinse twice with 0.5 ml of PBS per rinse.</li>
	<li>Add 0.5 ml of blocking solution and incubate at room temperature for 60 minutes.</li>
	<li>Remove blocking solution and add 0.225 ml primary antibody solution.</li>
	<li>Incubate at room temperature for 2 hours or overnight at 4&deg; C.</li>
	<li>Remove primary antibody solution and gently rinse twice with 0.5 ml of PBS per rinse.</li>
	<li>Add 0.225 ml secondary antibody solution. Incubate plate in the dark at room temperature for 60 minutes.</li>
	<li>Remove secondary antibody solution and gently rinse twice with 0.5 ml of PBS per rinse.</li>
	<li>Add 0.3 ml of 50% glycerol/PBS solution (v/v) or enough Vectashield to cover bottom of well surface.</li>
	<li>Image cells with epifluorescence microscope or store wrapped in foil at 4&deg; C.</li>
</ol>
6. Representative Results
<img src="/files/ftp_upload/2555/2555fig1.jpg" alt="Figure 1" /> 
Figure 1. NZCs were purified from E17.5 embryos, plated at 200,000 cells per cm2 and incubated at 37&deg; C for a total of 24 hours. Cells were imaged by phase contrast with a Leica DM IRB inverted microscope prior to immunofluorescent staining. (A) 20X phase contrast view of NZCs after 24 hours in culture. (B) Cells were fixed and stained using a rabbit anti-PAX2 antibody specific for nephron progenitor cells (red) and a secondary Alexa Fluor 568 conjugated donkey anti-Rabbit. Note blue nuclei counterstained with DAPI. (C) 40X phase contrast view of a cluster of nephron progenitors (centered) after 24 hours in culture. (D) 40X image of NZCs isolated from LacZ+ embryos of the Foxd1LacZ strain, which expresses &beta;-galactosidase under the control of the Foxd1 promoter. Foxd1 is expressed in the stromal cell population within the nephrogenic zone of the developing kidney. Cells were stained with the F-actin marker phalloidin (Oregon green primary conjugate), the nuclear stain DAPI (Blue) and rabbit anti-&beta;-galactosidase (secondary - Alexa Fluor donkey anti-Rabbit 568) for detection of Foxd1+ stromal cells (red).

<ol>
	<li>Blank, U., Brown, A., Adams, D. C., Karolak, M. J., Oxburgh, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP7+promotes+proliferation+of+nephron+progenitor+cells+via+a+JNK-dependent+mechanism.">BMP7 promotes proliferation of nephron progenitor cells via a JNK-dependent mechanism.</a> Development. 136, 3557-3566 (2009).</li><li>Brunskill, E. W., Aronow, B. J., Georgas, K., Rumballe, B., Valerius, M. T., Aronow, J., Kaimal, V., Jegga, A. G., Grimmond, S., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atlas+of+gene+expression+in+the+developing+kidney+at+microanatomic+resolution.">Atlas of gene expression in the developing kidney at microanatomic resolution.</a> Developmental Cell. 15, 781-791 (2008).</li><li>Godin, R. E., Takaesu, N. T., Robertson, E. J., Dudley, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+BMP7+expression+during+kidney+development.">Regulation of BMP7 expression during kidney development.</a> Development. 125, 3473-3482 (1998).</li><li>Grobstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inductive+epitheliomesenchymal+interaction+in+cultured+organ+rudiments+of+the+mouse.">Inductive epitheliomesenchymal interaction in cultured organ rudiments of the mouse.</a> Science. 118, 52-55 (1953).</li><li>Grobstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenetic+interaction+between+embryonic+mouse+tissues+separated+by+a+membrane+filter.">Morphogenetic interaction between embryonic mouse tissues separated by a membrane filter.</a> Nature. 172, 869-870 (1953).</li><li>Hatini, V., Huh, S. O., Herzlinger, D., Soares, V. C., Lai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+stromal+mesenchyme+in+kidney+morphogenesis+revealed+by+targeted+disruption+of+Winged+Helix+transcription+factor+BF-2.">Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2.</a> Genes and Development. 10, 1467-1478 (1996).</li><li>Oxburgh, L., Chu, G. C., Michael, S. K., Robertson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFb+superfamily+signals+are+required+for+morphogenesis+of+the+kidney+mesenchyme+progenitor+population.">TGFb superfamily signals are required for morphogenesis of the kidney mesenchyme progenitor population.</a> Development. 131, 4593-4605 (2004).</li><li>Srinivas, S., Watanabe, T., Lin, C. S., William, C. M., Tanabe, Y., Jessell, T. M., Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+reporter+strains+produced+by+targeted+insertion+of+EYFP+and+ECFP+into+the+ROSA26+locus.">Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.</a> BMC Developmental Biology. 1, 4-4 (2001).</li><li>Yu, J., Carroll, T. J., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+regulates+proliferation+and+differentiation+of+mesenchymal+cells+in+the+mouse+metanephric+kidney.">Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney.</a> Development. 129, 5301-5312 (2002).</li></ol>

No conflicts of interest declared.

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...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Watchmaker&#x2019;s forceps #5</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-4905</td>
<td>2 pairs required</td>
</tr>
<tr>
<td>Collagenase A</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10 103 578 001</td>
<td>Wear mask</td>
</tr>
<tr>
<td>Porcine Pancreatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8096</td>
<td>Wear mask</td>
</tr>
<tr>
<td>DPBS w/ Ca, Mg</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-513F</td>
<td>With Mg &amp; Ca</td>
</tr>
<tr>
<td>Fetal bovine serum (FBS)</td>
<td>&nbsp;</td>
<td>BioWhittaker</td>
<td>14-901E</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14175</td>
<td>&nbsp;</td>
<tr>
<td>KSFM</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>10724-011</td>
<td>without added growth factors</td>
</tr>
<tr>
<td>L-Glutamine 200mM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7513</td>
<td>Use @ 1% v/v</td>
</tr>
<tr>
<td>PenStrep 10,000 U penicillin / ml 10 mg streptomycin / ml</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4333</td>
<td>Use @ 1% v/v</td>
</tr>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>356008</td>
<td>Supplied as powder</td>
</tr>
<tr>
<td>24 well culture plate</td>
<td>&nbsp;</td>
<td>Thermo Scientific </td>
<td>142485</td>
<td>Nunclon</td>
</tr>
<tr>
<td>DPBS w/o Mg &amp; Ca</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-512F</td>
<td>&nbsp;</td>
<tr>
<td>5mL polystyrene tubes</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352235</td>
<td>&nbsp;</td>
<tr>
<td>DNase (1 U/&mu;l)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18068-015</td>
<td>4 &mu;l per 1.5 ml</td>
</tr>
<tr>
<td>40 micron cell-strainer </td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352235</td>
<td>w/cap and tube</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P6148</td>
<td>Use @ 4 % w/v</td>
</tr>
<tr>
<td>Triton X100</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>VW3929-2</td>
<td>Use @ 0.3 % v/v</td>
</tr>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P3813</td>
<td>Supplied as powder</td>
</tr>
<tr>
<td>Donkey serum</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>017-000-121</td>
<td> Use @ 5 % v/v</td>
</tr>
<tr>
<td>Rabbit anti-PAX2 </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>71-6000</td>
<td>Dilute 1:100</td>
</tr>
<tr>
<td>Rabbit anti-&beta;-gal</td>
<td>&nbsp;</td>
<td>MP Biomedicals</td>
<td>559762</td>
<td>Dilute 1:200</td>
</tr>
<tr>
<td>Oregon Green 488 phalloidin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>O7466</td>
<td>Dilute 1:200</td>
</tr>
<tr>
<td>Donkey anti-rabbit Alexa Fluor 568</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A10042</td>
<td>Dilute 1:200</td>
</tr>
<tr>
<td>DAPI</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D1306</td>
<td>Dilute 1:5000</td>
</tr>
<tr>
<td>Vectashield</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>H-1000</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>GX0185-6</td>
<td>Mix 50 % with H2O</td>
</tr>
<tr>
<td>RNeasy Micro Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>74004</td>
<td>Use "DNase on column" protocol</td>
</tr>
<tr>
<td>Transfer pipettes, 3ml</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357575</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation and culture of cells from the nephrogenic zone of the embryonic mouse kidney

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 na&iuml;ve 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 <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, 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.

Watch this Scientific Journal Video about Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney at JoVE.com

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

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 ...

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17.9K Views.  Maine Medical Center Research Institute. 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.

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

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...