Простой метод для получения и поддержания сперматогониальные ствола и линии клеток-предшественников из взрослых мышей представлена здесь. Метод использует фидерных клеток, происходящих из отсека соматической клетки взрослой семенников мышей. Этот метод применим к общим линий мышей, в том числе трансгенных, нокаут, и забивные мышей.
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.
Этот способ для получения взрослого КСЭ использовании семенников взрослых клеток, полученных из питателя является надежной и удалось, когда общий генетический фон (например, FVB, C57BL6 и смешанных 129SV/C57Bl/6) и различные мутантные штаммы были заняты 2,3,7,14. В самом деле, микросреда с?…
The authors have nothing to disclose.
Эта работа была поддержана штата Нью-Йорк Департамент здравоохранения (C026878). MS был в Нью-Йорке Stem Cell Foundation-Дракенмиллер Fellow. При частичной поддержке исследовательского гранта № 5-FY11-571 с марта Dimes Foundation.
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 |
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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. |