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.
<o...

<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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13.7K vistas.  Weill Cornell Medical College. Un método sencillo para obtener y mantener la madre de espermatogonias y las líneas de células progenitoras de ratones adultos se presenta aquí. El método utiliza las células de alimentación se originan en el compartimiento de células somáticas de los testículos de ratón adulto. Esta técnica es aplicable a las cepas de ratones comunes, incluyendo transgénicos, knock-out y knock-en ratones.