This work was supported by the New York State Department of Health (C026878). M.S. was a New York Stem Cell Foundation-Druckenmiller Fellow. Supported in Part by Research Grant No. 5-FY11-571 from the March of Dimes Foundation.

1. Preparation of Feeder Cells
Note that all reagents described below should be prepared in sterile fashion (see Tables 1 &amp; 2). This protocol employs the JK1 cell line (Cell Biolabs, Inc., catalog #CBA-315) as feeders which is a transformed derivative of adult mouse testicular somatic cells and has been described elsewhere3. Note also that all animal procedures should be performed in accordance with institutional guidelines and regulations.
<ol>
 <li>JK1 cells can be maintained in culture and used successfully as feeders up to passage 39. Culture JK1 cells in a 100 mm cell culture dish (BD Falcon, catalog #353003) with 10 ml of filter-sterilized feeder growth medium (DMEM supplemented with 10% fetal bovine serum [FBS], 2 mM L-glutamine and antibiotics). When the culture reaches 95% confluence, split the cells 1:6 -1:10 ratio.</li>
 <li>Remove medium from confluent JK1 feeder cell monolayer.</li>
 <li>Wash cell layer once with PBS without calcium and magnesium.</li>
 <li>Add pre-warmed trypsin/EDTA, 1x (0.05% trypsin/0.53 mM EDTA; Corning Cellgro, catalog #25-051-CI), and incubate at 37 &deg;C for 5-10 min.</li>
 <li>Inactivate trypsin with an equal volume of feeder growth medium. Collect cells and centrifuge at 300 x g for 5 min. Re-suspend cells in feeder growth medium.</li>
 <li>Pre-coat multi-well cell culture plates by adding enough 0.4% gelatin solution to cover the well and remove after 5 min at room temperature.</li>
 <li>Plate ~250 cells per mm2 in gelatin-coated cell culture plate (e.g. 48-well or 6-well format). Incubate culture at 37 &deg;C for 16 to 24 hr.</li>
 <li>Remove culture medium from wells. CAUTION: To inactivate cell growth, add a fresh solution of mitomycin-C at 10 &mu;g/ml diluted in DMEM to each well (e.g. 200 &mu;l/well for a 48 well plate). Mitomycin-C is toxic. Handle and dispose of it with care. Incubate at 37 &deg;C for 4 hr.</li>
 <li>Remove mitomycin-C solution from cells. Wash 3 times with DMEM prior to plating stem cells.</li>
 <li>Remove DMEM from feeder cells wells and add enough stem cell medium (100 &mu;l/ well of a 48 well plate; see Table 3) to prevent desiccation of the feeders during passaging. Add SSCs the same day, as described below in sections 3.1 or 3.4, using indicated sizes of wells containing feeders.</li>
</ol>
2. Dissociation of Mouse Testis Tissue to Obtain Donor Germ Cells
<ol>
 <li>Harvest adult mouse testes in sterile fashion and temporarily store the testes in a covered Petri dish (without any liquid added). The dish must placed immediately on ice to prevent dessication and maintain cell viability.</li>
 <li>Using sterile fine forceps and a fine scissor in a cell culture hood, make a transverse incision in the tunica albuginea without completely transecting the testis (i.e., cut ~&frac12;-&frac34; distance through) and use forceps to squeeze out seminiferous tubules into a corner of the plate; discard tunica and attached tissue. Keep plate on ice throughout to keep tissue chilled, thereby maintaining its integrity and preventing evaporation.</li>
 <li>Rapidly mince tubules with fine spring scissors at least 3 min per testis, while keeping plate on ice.</li>
 <li>Collect the tubule fragments in a 50 ml conical tube and wash in ~40 ml of chilled PBS/1% bovine serum albumin.</li>
 <li>Centrifuge at 60 x g for 10 min to separate tubule fragments from spermatozoa and debris. Discard supernatant.</li>
 <li>In a 15 ml conical tube, resuspend the pellet in 3 ml per testis of pre-warmed dissociation buffer (DMEM, 0.05% Trypsin, 0.03% Collagenase type I, 80 U/ml of DNAse I and 0.5% bovine serum albumin; See Table 2 for more specific details of dissociation buffer).</li>
 <li>Place the conical tube horizontally in a rack in a shaker set to 150 rpm at 37 &deg;C for 15 min to maximize agitation.</li>
 <li>Spin down at 60 x g for 10 min to separate single cells from undissociated chunks.</li>
 <li>Save the pellet containing chunks until the next step. Collect supernatant from Step 2.8 and add 3 ml DMEM/10% fetal bovine serum to neutralize dissociation buffer. Place tube on ice temporarily.</li>
 <li>Repeat step 2.6 to 2.7 with the saved pellet from step 2.9 to re-digest chunks remaining after step 2.8.</li>
 <li>Add an equal volume of DMEM/10% FBS to neutralize dissociation buffer.</li>
 <li>Recombine suspension from step 2.11 with saved cell suspension from step 2.9. Centrifuge for 5 min at 300 x g.</li>
 <li>Resuspend pellet in stem cell medium in a volume of 16 ml per pair of testes (see Table 3).</li>
</ol>
3. Plating of Testicular Cell Suspension
<ol>
 <li>Plate 2 ml of cell suspension per well in a 6-well plate containing feeder cells mitotically-inactivated with mitomycin-C and place in a cell culture incubator at 37 &deg;C in 5% CO2.</li>
 <li>After 48 hr, aspirate medium, add 2 ml fresh medium and return to incubator.</li>
 <li>After 48 hr, add 0.5 ml fresh stem cell medium to each well and return to incubator.</li>
 <li>After 48 hr, subsequently feed cells three times per week thereafter as follows until large, discreet colonies (&gt;50 cells) appear (within 7 to 21 days). For first and second feeding each week (e.g. Monday and Wednesday) remove ~50% of the medium and add back 50% by volume of fresh medium. For third feeding (e.g. Friday), aspirate all medium and replace with 2 ml fresh medium. This approach is based on the rationale that avoidance of complete medium changes minimizes fluctuations in concentrations of paracrine signals that are secreted into the culture medium9.</li>
</ol>
4. Colony Picking for First Passage
<ol>
 <li>Remove medium from wells containing colonies to be passaged and add fresh stem cell medium.</li>
 <li>Identify colonies using a 10x objective. With the microscope slightly de-focused, SSC colonies at the edge of the well will appear as homogeneous bright clumps, comprised of many 11-12 &mu;m diameter cells, in which it is difficult or impossible to discern the individual cell borders, since the cells appear fused together (Figure 1A). Non-SSC colonies may appear darker, more granular, or less homogenous, with discernable borders (Figure 1A, inset).</li>
 <li>Using a 200 &mu;l pipette tip, with the pipetteman set to 50 &mu;l, first take up medium from the well and expel to wet the tip. Then, gently nudge the colony with the tip before rapidly withdrawing 50 &mu;l of medium to dislodge the colony by suction into the tip (Figure 1B).</li>
 <li>Expel the medium containing the colony into one well of a 48-well plate containing inactivated feeder cells with 100 &mu;l of stem cell medium (Figure 1C).</li>
 <li>Repeat step 4.3 and add up to 8 colonies to the same well without exceeding 500 &mu;l per well.</li>
 <li>Repeat steps 4.3 to 4.5 to prepare additional wells of the 48 well plate with passage one SSC colonies.</li>
 <li>Wells will be ready to split in 7-14 days or after large clumps of up to 500 cells have emerged (Figure 1D).</li>
</ol>
5. Subsequent Expansion and Passaging of SSCs by Trituration
<ol>
 <li>Cells at passage one should be subcultured onto fresh feeders after up to 14 days at a split ratio of 1:2 for the first 3 passages and then 1:4 to 1:6 (every 7 days) thereafter as follows. Gently triturate colonies off a semi-confluent well (e.g. ~300,000 SSCs/well of 6-well plate) with a 1 ml pipette tip by washing medium across the cells. Colonies can be seen detaching. Too much force with the stream of liquid will cause unwanted feeder cells to come off too.</li>
 <li>Collect triturated cells in a conical tube and centrifuge at 300 x g for 5 min. Note: A trypsinization step may be safely added here if desired by the investigator.</li>
 <li>Aspirate supernatant and re-suspend in fresh stem cell medium by thoroughly pipetting up and down to disrupt colonies.</li>
 <li>Plate SSCs on freshly prepared feeder cells inactivated with mitomycin-C as described in section 1.</li>
 <li>Feed cells three times per week as follows. For first and second feeding (e.g. Monday and Wednesday) add 50% by volume of fresh medium. For third feeding (e.g. Friday), aspirate all medium and replace with 2 ml fresh medium. Plates will become confluent in 7-10 days. Cultures should be comprised of &gt;98% germ cells (i.e., &lt;1% contaminating somatic cells) after 5-7 passages, based on immunostaining (e.g. using the murine germ cell marker GCNA) to distinguish germ cells from somatic cells10. Note: These methods were developed in compliance with regulations and requirements set forth by the Institutional Animal Care and Use Committee at Weill Cornell Medical College.</li>
</ol>

<ol>
	<li>Kanatsu-Shinohara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+proliferation+in+culture+and+germline+transmission+of+mouse+male+germline+stem+cells.">Long-term proliferation in culture and germline transmission of mouse male germline stem cells.</a> Biol. Reprod. 69, 612-616 (2003).</li><li>Seandel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+multipotent+adult+stem+cells+from+GPR125++germline+progenitors.">Generation of functional multipotent adult stem cells from GPR125+ germline progenitors.</a> Nature. 449, 346-350 (2007).</li><li>Kim, J., Seandel, M., Falciatori, I., Wen, D., Rafii, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD34++testicular+stromal+cells+support+long-term+expansion+of+embryonic+and+adult+stem+and+progenitor+cells.">CD34+ testicular stromal cells support long-term expansion of embryonic and adult stem and progenitor cells.</a> Stem Cells. 26, 2516-2522 (2008).</li><li>Ogawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+morphological+characterization+of+mouse+spermatogonial+stem+cell+lines.">Derivation and morphological characterization of mouse spermatogonial stem cell lines.</a> Arch. Histol. Cytol. 67, 297-306 (2004).</li><li>Schmidt, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+aging+is+detrimental+to+mouse+spermatogonial+stem+cell+function.">In vivo and in vitro aging is detrimental to mouse spermatogonial stem cell function.</a> Biology of Reproduction. 84, 698-706 (2011).</li><li>Zhang, X., Ebata, K. T., Robaire, B., Nagano, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+of+male+germ+line+stem+cells+in+mice.">Aging of male germ line stem cells in mice.</a> Biology of Reproduction. 74, 119-124 (2006).</li><li>Hobbs, R. M., Seandel, M., Falciatori, I., Rafii, S., Pandolfi, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plzf+regulates+germline+progenitor+selfrenewal+by+opposing+mTORC1.">Plzf regulates germline progenitor selfrenewal by opposing mTORC1.</a> Cell. 142, 468-479 (2010).</li><li>Nagano, M., Ryu, B. Y., Brinster, C. J., Avarbock, M. R., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+mouse+male+germ+line+stem+cells+in+vitro.">Maintenance of mouse male germ line stem cells in vitro.</a> Biol. Reprod. 68, 2207-2214 (2003).</li><li>Csaszar, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+expansion+of+human+hematopoietic+stem+cells+by+automated+control+of+inhibitory+feedback+signaling.">Rapid expansion of human hematopoietic stem cells by automated control of inhibitory feedback signaling.</a> Cell Stem Cell. 10, 218-229 (2012).</li><li>Enders, G. C., May, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmentally+regulated+expression+of+a+mouse+germ+cell+nuclear+antigen+examined+from+embryonic+day+11+to+adult+in+male+and+female.">Developmentally regulated expression of a mouse germ cell nuclear antigen examined from embryonic day 11 to adult in male and female.</a> 163, 331-340 (1994).</li><li>Oatley, J. M., Brinster, R. L. <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+spermatogonial+stem+cell+self-renewal+in+mammals.">Regulation of spermatogonial stem cell self-renewal in mammals.</a> Annu Rev Cell Dev Biol. 24, 263-286 (2008).</li><li>Brinster, R. L., Zimmermann, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis+following+male+germ-cell+transplantation.">Spermatogenesis following male germ-cell transplantation.</a> Proc Natl Acad Sci U.S.A. 91, 11298-11302 (1994).</li><li>Tang, L., Rodriguez-Sosa, J. R., Dobrinski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+Cell+Transplantation+and+Testis+Tissue+Xenografting+in+Mice.">Germ Cell Transplantation and Testis Tissue Xenografting in Mice.</a> J. Vis. Exp. (60), e3545 (2012).</li><li>Arnold, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox2(+)+adult+stem+and+progenitor+cells+are+important+for+tissue+regeneration+and+survival+of+mice.">Sox2(+) adult stem and progenitor cells are important for tissue regeneration and survival of mice.</a> Cell Stem Cell. 9, 317-329 (2011).</li><li>Yeh, J. R., Zhang, X., Nagano, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+short-term+in+vitro+assay+for+mouse+spermatogonial+stem+cells.">Establishment of a short-term in vitro assay for mouse spermatogonial stem cells.</a> Biology of Reproduction. 77, 897-904 (2007).</li></ol>

M.S. receives royalties from Weill Cornell Medical College through a license to Cell Biolabs, Inc. to distribute JK1 cells.

This method for deriving adult SSCs using adult testis-derived feeder cells is robust and has succeeded when common genetic backgrounds (e.g. FVB, C57Bl6 and mixed 129SV/C57Bl/6) and different mutant strains were employed2,3,7,14. In fact, the microenvironment created by the culture system is sufficient to overcome some of the genetic barriers to maintaining adult SSCs in vivo (e.g. in the case of plzf-/- animals)7. While we employ JK1 cells as feeders, non-tran...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/ Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
	 </tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-013</td>
			<td>Diluent for dissociation buffer</td>
	 </tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Mediatech</td>
			<td>25-051-CI</td>
			<td></td>
	 </tr>
		<tr>
			<td>Stem cell base medium (StemPro-34)</td>
			<td>Life Technologies</td>
			<td>10639-011</td>
			<td>Requires supplementation as per Shinohara et al. (2003)*</td>
	 </tr>
		<tr>
			<td>Stem cell medium supplements</td>
			<td>various</td>
			<td>see Table 3</td>
			<td>Requires supplementation as per Shinohara et al. (2003)*</td>
	 </tr>
		<tr>
			<td>JK1 cells</td>
			<td>Cell Biolabs, Inc.</td>
			<td>CBA-315</td>
			<td>Can substitute with adult testicular stromal cells as per Seandel et al. (2007)</td>
	 </tr>
		<tr>
			<td>mitomycin-C (CAUTION)</td>
			<td>Sigma-Aldrich</td>
			<td>M4287</td>
			<td>Toxic; Handle with care.</td>
	 </tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890</td>
			<td>0.4% solution in water</td>
	 </tr>
		<tr>
			<td>EVOS xl digital inverted microscope</td>
			<td>Advanced Microscopy Group</td>
			<td>-</td>
			<td></td>
	 </tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Specific reagents and equipment. 
			*See Table 3</td>
	 </tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-013</td>
			<td>Diluent for dissociation buffer</td>
	 </tr>
		<tr>
			<td>trypsin (1:250)</td>
			<td>Life Technologies</td>
			<td>27250-018</td>
			<td>Dissociation buffer: Final 0.05% wt/vol</td>
	 </tr>
		<tr>
			<td>collagenase, type I, 235 U/ml</td>
			<td>Worthington</td>
			<td>CLS1 235</td>
			<td>Dissociation buffer: Final 0.03% wt/vol</td>
	 </tr>
		<tr>
			<td>DNAse I</td>
			<td>Sigma-Aldrich</td>
			<td>DN25</td>
			<td>Dissociation buffer: Final 80 U/ml</td>
	 </tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>ICP Bio</td>
			<td>ABRE-100g</td>
			<td>Dissociation buffer: Final 0.5% wt/vol</td>
	 </tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Dissociation buffer</td>
	 </tr>
		<tr>
			<td>StemPro-34 SFM</td>
			<td>Life Technologies</td>
			<td>10639-011</td>
		 <td></td>
	 </tr>
		<tr>
			<td>StemPro-34 Nutrient supplement</td>
			<td>Life Technologies</td>
			<td>10639-011</td>
		 <td></td>
	 </tr>
		<tr>
			<td>Additional supplements**</td>
			<td></td>
			<td></td>
		 <td></td>
	 </tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M7145</td>
		 <td>1X</td>
	 </tr>
		<tr>
			<td>MEM Vitamin solution</td>
			<td>Life Technologies</td>
			<td>11120-052</td>
		 <td>1X</td>
	 </tr>
		<tr>
			<td>L-glutamine</td>
			<td>Mediatech</td>
			<td>25-005</td>
		 <td>2 mM</td>
	 </tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>ICP Bio</td>
			<td>ABRE</td>
		 <td>0.50%</td>
	 </tr>
		<tr>
			<td>Antibiotic-Antimycotic Solution</td>
			<td>Mediatech</td>
			<td>30-004-CI</td>
		 <td>1X</td>
	 </tr>
		<tr>
			<td>D(+)glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8769</td>
		 <td>6 mg/ml</td>
	 </tr>
		<tr>
			<td>&beta;-estradiol</td>
			<td>Sigma-Aldrich</td>
			<td>E2758</td>
		 <td>30 ng/ml</td>
	 </tr>
		<tr>
			<td>progesterone</td>
			<td>Calbiochem</td>
			<td>5341</td>
		 <td>60 ng/ml</td>
	 </tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>variable</td>
			<td>n/a</td>
		 <td>1%</td>
	 </tr>
		<tr>
			<td>bovine holo-transferrin</td>
			<td>Sigma-Aldrich</td>
			<td>T1283</td>
		 <td>100 &mu;g/ml</td>
	 </tr>
		<tr>
			<td>insulin</td>
			<td>Gemini Bio-Products</td>
			<td>700-112P</td>
		 <td>25 &mu;g/ml</td>
	 </tr>
		<tr>
			<td>human GDNF</td>
			<td>Life Technologies</td>
			<td>PHC7041</td>
		 <td>10 ng/ml</td>
	 </tr>
		<tr>
			<td>human bFGF</td>
			<td>Life Technologies</td>
			<td>PHG0023</td>
		 <td>10 ng/ml</td>
	 </tr>
		<tr>
			<td>mouse EGF</td>
			<td>Life Technologies</td>
			<td>PHG0313</td>
		 <td>20 ng/ml</td>
	 </tr>
		<tr>
			<td>putrescine</td>
			<td>Research Organics</td>
			<td>0778P</td>
		 <td>60 &mu;M</td>
	 </tr>
		<tr>
			<td>sodium Selenite</td>
			<td>Sigma-Aldrich</td>
			<td>S5261</td>
		 <td>30 nM</td>
	 </tr>
		<tr>
			<td>pyruvic acid</td>
			<td>Alfa Aesar</td>
			<td>A13875</td>
		 <td>30 &mu;g/ml</td>
	 </tr>
		<tr>
			<td>DL-lactic acid</td>
			<td>J.T. Baker</td>
			<td>0196-04</td>
		 <td>1 &mu;g/ml</td>
	 </tr>
		<tr>
			<td>&beta;-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
		 <td>50 &mu;M</td>
	 </tr>
		<tr>
			<td>ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544</td>
		 <td>100 &mu;M</td>
	 </tr>
		<tr>
			<td>D-biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4639</td>
		 <td>10 &mu;g/ml</td>
	 </tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3. Stem cell medium </td>
	 </tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>*Note: Add supplements below before using medium. Filter sterilize and keep it at 4 &deg;C. The medium is stable for at least 2 weeks.</td>
	 </tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>**We have employed different manufacturers, formulations, and/or lot numbers of these reagents without any apparent deleterious effects. In general, cell culture grade reagents should be employed.</td>
	 </tr>
	</tbody>
</table>

serial enrichment of spermatogonial stem and progenitor cells (sscs) in culture for derivation of long-term adult mouse ssc lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

The appearance of passage zero wild type, adult SSC colonies after 7 days is shown in Figure 1. Three-dimensional colonies are comprised of a layer of flat cells attached to the feeders or underling extracellular matrix deposited by the feeders with multiple layers of SSCs growing on top. While healthy SSCs are brightly refractile and uniformly 11-12 &mu;m diameter, the cell borders are difficult to distinguish and the size of the colonies may be highly variable, both within the well and between wells pr...

Watch this Scientific Journal Video about Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines at JoVE.com

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

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13.8K Views.  Weill Cornell Medical College. A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Video: Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

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 ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...