
The special characteristics of stem cells (i.e., self-renewal capacity and pluripotency) that allow them to differentiate into any of three germ layer lineages (1, 2) can be regulated by a unique microenvironment called the stem cell niche, which consists of physicochemical and physiological niches, and the extracellular matrix (ECM) (3, 4). To date, the defined manipulation of physicochemical and physiological niches has been limited in its ability to precisely and specifically guide the fate of stem cells (5, 6). Therefore, the importance of niches based on ECM has been emphasized as an alternative to overcoming the difficulty of regulating the fate of stem cells.
The ECM is a non-cellular three-dimensional macromolecular network composed of a variety of fibrous ECM proteins, carbohydrates, and several glycoproteins (7, 8), which not only provide a physical scaffold but also signals to induce cellular responses including proliferation, migration, attachment, spreading, differentiation, survival, homeostasis, and morphogenesis (9). The integrins, a group of cell adhesion molecules that exist as heterodimers with various combinations of
Among the diverse stem cell types, spermatogonial stem cells (SSCs) are the only male germline stem cells that differentiate into sperm through spermatogenesis in the seminiferous tubule of the testis (13, 14). Their self-renewal is infinitely maintained on the seminiferous tubule basement membrane (STBM) with defined ECM components
Outbred stocks of mice are widely used in toxicology, pharmacology, and fundamental biomedical research (20), although most such studies can be done much more effectively with inbred strains. Particularly, their usage in genetic studies has been very successful as a base population for selection to produce new or improved humanized mouse models (21), and for genetic fine mapping of quantitative trait loci (QTL) (22). In addition, their better breeding performance compared to inbred strains makes it easy to develop and maintain genetically modified mice (23). However, despite their usefulness, to date, there have been no reports of
Accordingly, as a step towards developing a precisely defined non-cellular niche engineering integrin signaling to promote self-renewal of SSCs derived from outbred mice, we examined the types of integrin heterodimers on the surface of outbred ICR mouse SSCs in the undifferentiated state. The types of integrin subunits expressed in undifferentiated SSCs were identified at the transcriptional and translational levels, and the combinations of integrin
Three-week-old male ICR mice were purchased from DBL (Eumseong, Korea) and used as SSC donors. All of the animal housing, handling, and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (IACUC approval No. KW-130307-1) and conducted in accordance with the Animal Care and Use Guidelines of Kangwon National University.
The tunica albuginea and epididymis were removed from the mouse testes, and the seminiferous tubules were digested by incubation for 20 min in Dulbecco’s Modified Eagle’s Medium (DMEM; Welgene Inc., Daegu, Korea) supplemented with 0.5 mg/ml type IV collagenase (Worthington Biochemical, Lakewood, CA) at 37℃. Subsequently, the fragmented seminiferous tubules were washed with DMEM containing 10% (v/v) heat-inactivated fetal bovine serum (FBS; Welgene), and dissociated with 0.25% trypsin-EDTA (Welgene) for 5 min at 37℃. The dispersed testicular cells were filtered through a 70
Total mRNA was extracted from the cells using a Dynabeads mRNA DirectTM Kit (Ambion, Austin, TX), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA remover kit (Toyobo, Osaka, Japan) in accordance with the respective manufacturer’s instructions. Subsequently, specific gene expression was quantified with THUNDERBIRDTM SYBRⓇ qPCR Mix (Toyobo) using a 7500 Real-time PCR system (Applied Biosystems, Foster City, CA). PCR specificity was determined by analyzing the melting curve data. The mRNA levels are presented as 2−ΔCt where Ct=threshold cycle for target amplification and ΔCt=Cttarget gene (specific genes for each sample) – Ctinternal reference (
Cells fixed with 4% (v/v) paraformaldehyde (Junsei, Tokyo, Japan) for 10 min were washed twice with Dulbecco’s phosphate-buffered saline (DPBS; Welgene). The fixed cells were stained with fluorescence-conjugated integrin
Three thousand three hundred of cells fixed in 4% (v/v) paraformaldehyde (Junsei) for 10 min were rinsed twice with DPBS. Subsequently, the fixed cells were stained for 2 h at room temperature with FITC-conjugated anti-mouse integrin
First, 96-well tissue culture plates were coated with the following concentrations of purified ECM proteins overnight at 4℃: 0, 40, and 80
The wells of 96-well tissue culture plates were coated with 40
The SAS program was used for statistical analysis of the numerical data shown in each experiment. Comparisons among treatment groups were performed by the least-squares or DUNCAN method, and the significance of the main effects was determined by analysis of variance (ANOVA) in the SAS package. In all of the analyses, p< 0.05 was taken to indicate statistical significance.
A MACS technique based on antibody detecting Thy1 expressed on the surface of undifferentiated mouse SSCs did not show high yield in the isolation of undifferentiated SSCs from outbred ICR mouse testes. As shown in Supplementary Fig. S1, SSCs stained positively with GFR
In transcriptional analyses of genes encoding a total of 25 integrin subunits, significantly higher levels of expression were observed for integrin
Translational regulation of integrin
Subsequently, levels of integrin
Based on the results regarding integrin
In constructing niches for driving the self-renewal or differentiation of SSCs, information regarding the extracellular signals in the microenvironments around undifferentiated or differentiated SSCs is essential. Particularly, extracellular signals derived from ECM proteins composing STBM that interact directly with SSCs in the seminiferous tubule (25) play an important role in determining the fate of these cells. However, intracytoplasmic transduction of these ECM-derived signals is mediated by cell-surface receptors belonging to the integrin family (26). Therefore, information on integrins present in undifferentiated or differentiated SSCs should be reflected in fabricating niches customized to each type of SSC. Here, we confirmed the types of integrin heterodimers expressed on the surface of undifferentiated SSCs from outbred ICR mice. Through transcriptional analysis of 24 integrin subunits, followed by confirmation of their expression at the translational level, attachment to ECM proteins, and inhibition with blocking antibodies, integrin
The basement membrane in the seminiferous tubule consists of fibronectin (27), laminin (28), collagen IV (29, 30), entactin (31, 32), and perlecan (33). Accordingly, the expression of integrin
In conclusion, transcriptional, translational, and functional screening of integrins demonstrated the expression of integrin
Supplementary data including two tables and four figures can be found with this article online at http://pdf.medrang.co.kr/paper/pdf/IJSC/IJSC-13-s20061.pdf.
This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bioindustry Technology development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (IPET1170 42-3).
The authors have no conflicting financial interest.
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