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

Summary

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.

Abstract

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 cells^1-3.

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 factors^5,6. 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 SESC^2,3.

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.

Protocol

1. Preparation of Feeder Cells

Note that all reagents described below should be prepared in sterile fashion (see Tables 1 & 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.

1. 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.
2. Remove medium from confluent JK1 feeder cell monolayer.
3. Wash cell layer once with PBS without calcium and magnesium.
4. Add pre-warmed trypsin/EDTA, 1x (0.05% trypsin/0.53 mM EDTA; Corning Cellgro, catalog #25-051-CI), and incubate at 37 °C for 5-10 min.
5. 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.
6. 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.
7. Plate ~250 cells per mm² in gelatin-coated cell culture plate (e.g. 48-well or 6-well format). Incubate culture at 37 °C for 16 to 24 hr.
8. Remove culture medium from wells. CAUTION: To inactivate cell growth, add a fresh solution of mitomycin-C at 10 μg/ml diluted in DMEM to each well (e.g. 200 μl/well for a 48 well plate). Mitomycin-C is toxic. Handle and dispose of it with care. Incubate at 37 °C for 4 hr.
9. Remove mitomycin-C solution from cells. Wash 3 times with DMEM prior to plating stem cells.
10. Remove DMEM from feeder cells wells and add enough stem cell medium (100 μl/ 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.

2. Dissociation of Mouse Testis Tissue to Obtain Donor Germ Cells

1. 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.
2. 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 ~½-¾ 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.
3. Rapidly mince tubules with fine spring scissors at least 3 min per testis, while keeping plate on ice.
4. Collect the tubule fragments in a 50 ml conical tube and wash in ~40 ml of chilled PBS/1% bovine serum albumin.
5. Centrifuge at 60 x g for 10 min to separate tubule fragments from spermatozoa and debris. Discard supernatant.
6. 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).
7. Place the conical tube horizontally in a rack in a shaker set to 150 rpm at 37 °C for 15 min to maximize agitation.
8. Spin down at 60 x g for 10 min to separate single cells from undissociated chunks.
9. 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.
10. Repeat step 2.6 to 2.7 with the saved pellet from step 2.9 to re-digest chunks remaining after step 2.8.
11. Add an equal volume of DMEM/10% FBS to neutralize dissociation buffer.
12. Recombine suspension from step 2.11 with saved cell suspension from step 2.9. Centrifuge for 5 min at 300 x g.
13. Resuspend pellet in stem cell medium in a volume of 16 ml per pair of testes (see Table 3).

3. Plating of Testicular Cell Suspension

1. 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 °C in 5% CO₂.
2. After 48 hr, aspirate medium, add 2 ml fresh medium and return to incubator.
3. After 48 hr, add 0.5 ml fresh stem cell medium to each well and return to incubator.
4. After 48 hr, subsequently feed cells three times per week thereafter as follows until large, discreet colonies (>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.

4. Colony Picking for First Passage

1. Remove medium from wells containing colonies to be passaged and add fresh stem cell medium.
2. 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 μm 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).
3. Using a 200 μl pipette tip, with the pipetteman set to 50 μl, 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 μl of medium to dislodge the colony by suction into the tip (Figure 1B).
4. Expel the medium containing the colony into one well of a 48-well plate containing inactivated feeder cells with 100 μl of stem cell medium (Figure 1C).
5. Repeat step 4.3 and add up to 8 colonies to the same well without exceeding 500 μl per well.
6. Repeat steps 4.3 to 4.5 to prepare additional wells of the 48 well plate with passage one SSC colonies.
7. Wells will be ready to split in 7-14 days or after large clumps of up to 500 cells have emerged (Figure 1D).

5. Subsequent Expansion and Passaging of SSCs by Trituration

1. 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.
2. 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.
3. Aspirate supernatant and re-suspend in fresh stem cell medium by thoroughly pipetting up and down to disrupt colonies.
4. Plate SSCs on freshly prepared feeder cells inactivated with mitomycin-C as described in section 1.
5. 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 >98% germ cells (i.e., <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.

Results

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 μm 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 prepa...

Discussion

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 employed^2,3,7,14. 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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Corning	10-013	Diluent for dissociation buffer
Trypsin/EDTA	Mediatech	25-051-CI
Stem cell base medium (StemPro-34)	Life Technologies	10639-011	Requires supplementation as per Shinohara et al. (2003)*
Stem cell medium supplements	various	see Table 3	Requires supplementation as per Shinohara et al. (2003)*
JK1 cells	Cell Biolabs, Inc.	CBA-315	Can substitute with adult testicular stromal cells as per Seandel et al. (2007)
mitomycin-C (CAUTION)	Sigma-Aldrich	M4287	Toxic; Handle with care.
Gelatin	Sigma-Aldrich	G1890	0.4% solution in water
EVOS xl digital inverted microscope	Advanced Microscopy Group	-
Table 1. Specific reagents and equipment. *See Table 3
DMEM	Corning	10-013	Diluent for dissociation buffer
trypsin (1:250)	Life Technologies	27250-018	Dissociation buffer: Final 0.05% wt/vol
collagenase, type I, 235 U/ml	Worthington	CLS1 235	Dissociation buffer: Final 0.03% wt/vol
DNAse I	Sigma-Aldrich	DN25	Dissociation buffer: Final 80 U/ml
bovine serum albumin	ICP Bio	ABRE-100g	Dissociation buffer: Final 0.5% wt/vol
Table 2. Dissociation buffer
StemPro-34 SFM	Life Technologies	10639-011
StemPro-34 Nutrient supplement	Life Technologies	10639-011
Additional supplements**
Non-essential amino acids	Sigma-Aldrich	M7145	1X
MEM Vitamin solution	Life Technologies	11120-052	1X
L-glutamine	Mediatech	25-005	2 mM
bovine serum albumin	ICP Bio	ABRE	0.50%
Antibiotic-Antimycotic Solution	Mediatech	30-004-CI	1X
D(+)glucose	Sigma-Aldrich	G8769	6 mg/ml
β-estradiol	Sigma-Aldrich	E2758	30 ng/ml
progesterone	Calbiochem	5341	60 ng/ml
fetal bovine serum	variable	n/a	1%
bovine holo-transferrin	Sigma-Aldrich	T1283	100 μg/ml
insulin	Gemini Bio-Products	700-112P	25 μg/ml
human GDNF	Life Technologies	PHC7041	10 ng/ml
human bFGF	Life Technologies	PHG0023	10 ng/ml
mouse EGF	Life Technologies	PHG0313	20 ng/ml
putrescine	Research Organics	0778P	60 μM
sodium Selenite	Sigma-Aldrich	S5261	30 nM
pyruvic acid	Alfa Aesar	A13875	30 μg/ml
DL-lactic acid	J.T. Baker	0196-04	1 μg/ml
β-mercapt–thanol	Life Technologies	21985-023	50 μM
ascorbic acid	Sigma-Aldrich	A4544	100 μM
D-biotin	Sigma-Aldrich	B4639	10 μg/ml
Table 3. Stem cell medium
*Note: Add supplements below before using medium. Filter sterilize and keep it at 4 °C. The medium is stable for at least 2 weeks.
**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.