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Biology

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

Published: February 25th, 2013

DOI:

10.3791/50017

1Department of Surgery, 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.

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.

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.

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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 pr.......

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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.......

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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.

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Name Company Catalog Number Comments
Name of Reagent/ Material 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-mercaptoethanol 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.

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