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Isolation of THY1+ Undifferentiated Spermatogonia from Mouse Postnatal Testes Using Magnetic-activated Cell Sorting (MACS)
采用免疫磁珠分选法(MACS)分离小鼠出生后睾丸中的THY1+未分化精原细胞   

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Abstract

In mammals, homeostasis of many tissues rely on a subpopulation of cells, referred to as stem cells, to sustain an appropriate number of undifferentiated and differentiated cells. Spermatogonial stem cells (SSCs) provide the fundamental cellular source for spermatogenesis and are responsible for the lifelong maintenance of the germline pool in testes throughout the reproductive lifespan of males. To gain insight into germline stem cell biology and develop strategies for infertility treatment, several germ cell isolation methods have been reported in order to acquire good quality and quantity of undifferentiated spermatogonia. Among them, magnetic-activated cell sorting (MACS) is an efficient cell isolation method that requires less time and less initial cell numbers to obtain an enriched cell population using an antigen-antibody reaction. Thymus cell antigen 1 (THY1, CD90.2) is recognized as a surface marker of undifferentiated spermatogonia in mouse neonatal and adult testes. Here, we describe a protocol for the isolation of one-week-old THY1+ cells and four-week-old THY1+ cells from mouse testes. The isolation procedure consists of three steps: testis collection and single cell suspension, cell labeling using a biotin-conjugated anti-THY1 antibody and magnetic cell separation. Note, this isolation protocol should be completed within five hours to maximize the quality and the amount of living cells.

Keywords: Testis(睾丸), THY1+ spermatogonial stem cell(THY1 +精原干细胞), Magnetic-activated cell sorting(免疫磁珠分选法), Germ cell(生殖细胞)

Background

Co-existence of active and quiescent stem cells is observed in several adult tissues. Adequate balance between quiescence, self-renewal and differentiation is necessary to sustain an appropriate number of undifferentiated stem cells and to avoid premature stem cell exhaustion for the homeostasis of many tissues (Tseng et al., 2015; Wabik and Jones, 2015; Xin et al., 2016). Infertility has become an increasing problem for human couples and a significant portion of male-related infertility cases results from impaired undifferentiated spermatogonia (Boivin et al., 2007; Matzuk and Lamb, 2008). For this reason, SSCs in spermatogenesis, which is a well-characterized stem cell-dependent process (Oatley and Brinster, 2008), is a valuable model to study regulation of tissue homeostasis. Numerous studies have developed germ cell isolation methods in order to gain insight into the biological functions and regulatory networks of undifferentiated spermatogonia. However, in vivo undifferentiated spermatogonia are heterogeneous in their expression of markers including GFRA1, ID4, PLZF and THY1, and different experimental protocols have influences on the enrichment of subpopulations in specific cellular states (Buageaw et al., 2005; Chan et al., 2014; Costoya et al., 2004; Gassei and Orwig, 2013; Hermann et al., 2015; Kubota et al., 2003; Liao et al., 2014). For instance, a large proportion of PLZF+ and THY1+ undifferentiated spermatogonia is found in quiescent phase of the cell cycle in vivo, whereas PLZF+ and THY1+ undifferentiated spermatogonia cultured in mediums supplemented with serum and growth factors are inclined to the proliferation phase (Costoya et al., 2004; Kanatsu-Shinohara et al., 2011; Kubota et al., 2004a; Liao et al., 2014; Sada et al., 2009).

Here, we provide a step-by-step procedure for the isolation of relative quiescent THY1+ undifferentiated spermatogonia from pre-pubertal mice through a serum-free protocol using magnetic-activated cell sorting. This protocol also contains several important information, including cell numbers needed at each step for suitable enzymatic procedures for living germ cell isolation. This protocol should be a valuable tool to obtain large amount of high quality live undifferentiated spermatogonia with specific subpopulation enrichment through the use of antibodies against SSC surface antigens.

Materials and Reagents

  1. 100 mm Petri dish (Corning, Falcon®, catalog number: 351029 )
  2. Polypropylene tube:
    2 ml tube (Corning, Axygen®, catalog number: MCT-200-C )
    5 ml tube (Corning, Axygen®, catalog number: MCT-500-C )
  3. Centrifuge tubes:
    15 ml tube (Corning, Falcon®, catalog number: 352096 )
    50 ml tube (Corning, Falcon®, catalog number: 352070 )
  4. Cell strainer:
    5 ml polystyrene tube with cell strainer snap cap (35 µm nylon mesh) (Corning, Falcon®, catalog number: 352235 )
    70 µm nylon mesh (Corning, Falcon®, catalog number: 352350 )
  5. Columns for cell separation:
    MS columns (Miltenyi Biotec, catalog number: 130-042-201 )
    LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
  6. Poly-L-Lysine coated slides (Thermo Fisher Scientific, catalog number: 10143265 )
  7. ARTTM Barrier Hinged Rack pipette tips
    P1000 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2079-HR )
    P200 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2069-05-HR )
    P20 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2149-05-HR )
  8. C57BL/6N Mice (BioLASCO, catalog number: C57BL/6N )
  9. Hank’s balanced salt solution (HBSS), calcium, magnesium, no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 14025092 )
  10. Hank’s balanced salt solution (HBSS), no calcium, no magnesium, no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 14175095 )
  11. Type IV collagenase (Thermo Fisher Scientific, GibcoTM, catalog number: 17104019 )
  12. DNase I (RNase-free) (New England Biolabs, catalog number: M0303 )
  13. Antibodies:
    Biotin rat anti-mouse CD90.2 (Clone 30-H12) (BD, catalog number: 553011 )
    Biotin rat IgG2b, κ isotype control (Clone A95-1) (BD, catalog number: 553987 )
    Anti-CD90.2­Biotin antibody (Lot 5150211128) (Miltenyi Biotec, catalog number: 130-101-908 )
    Anti-CD49f antibody (BD, catalog number: 551129 )
    Isotype antibody (negative control) (BD, catalog number: 551066 )
    Anti-PLZF antibody (Santa Cruz Biotechnology, catalog number: sc-28319 )
    Anti-VASA antibody (Abcam, catalog number: ab13840 )
    AffiniPure Donkey Anti-Rabbit IgG (H+L) (Alexa Fluor® 488) (Jackson ImmunoResearch, catalog number: 711-545-152 )
    AffiniPure Donkey Anti-Mouse IgG (H+L) (Alexa Fluor® 488) (Jackson ImmunoResearch, catalog number: 715-545-151 )
  14. Anti-biotin microbeads (Miltenyi Biotec, catalog number: 130-090-485 )
  15. PFA (Sigma-Aldrich, catalog number: 158127 )
  16. Bovine serum albumin (BSA), IgG-free and protease-free (Jackson ImmunoResearch, catalog number: 001-000-162 )
  17. Normal donkey serum (Abcam, catalog number: ab7475 )
  18. EDTA, 0.5 M, pH 8.0 (Thermo Fisher Scientific, AnbionTM, catalog number: AM9260G )
  19. BSA
  20. Dulbecco’s phosphate-buffered saline (DPBS), no calcium, no magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
  21. Trypsin-EDTA, 0.25%, phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  22. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 16000044 )
  23. PE-Cy5-conjugated antibody
  24. Tween-20 (Sigma-Aldrich, catalog number: P9416 )
  25. E-HBSS (see Recipes)
  26. MACS buffer (see Recipes)
  27. Collagenase solution (see Recipes)
  28. Trypsin solution (see Recipes)
  29. F-MACS buffer (see Recipes)
  30. Staining buffer (see Recipes)
  31. FACS buffer (see Recipes)
  32. PBST (see Recipes)

Equipment

  1. Sterilized forceps and scissors
  2. Dissecting microscope (Leica MZ16F Stereomicroscope)
  3. P1000 pipette
  4. Centrifuge
  5. Hemocytometer
  6. Magnetic separator
    OctoMACSTM separator for MS columns (Miltenyi Biotec, catalog number: 130-042-109 )
    MidiMACSTM separator for LS columns (Miltenyi Biotec, catalog number: 130-042-302 )
  7. Beckman Coulter FC500 cytometer (Beckman Coulter, model: FC500 )
  8. Leica TCS SP5 II confocal microscope (Leica Microsystems, model: Leica TCS SP5 II )

Procedure

  1. Testis collection and single cell suspension
    1. Remove testes from mice using sterilized forceps and scissors. Collect the testes in a 100 mm Petri dish containing 10 ml of ice-cold HBSS.
    2. Remove tunica albuginea from the testes to expose seminiferous tubules using sterilized forceps under a dissecting microscope (Figure 1A, upper panel). Transfer seminiferous tubule clumps to a new 100 mm Petri dish containing 10 ml of ice-cold HBSS. Keep the seminiferous tubules on ice.
    3. Wash seminiferous tubule clumps using ice-cold HBSS to remove debris and suspension cells.
    4. In order to estimate the rough volume of seminiferous tubule clumps, we transfer these clumps to a 2 ml polypropylene tube to calculate the volumes. Then, transfer seminiferous tubule clumps to a new Petri dish containing at least 3-fold seminiferous tubule clump volumes of room temperature collagenase solution, supplemented with 1 mg/ml type IV collagenase and 5 U/ml DNase I to remove interstitial Leydig cells, blood cells and peritubular myoid cells, and to digest genomic DNA from dead cells, respectively (see Note 1).
      Note: We normally collect 0.2-0.3 ml seminiferous tubule clumps from five one-week-old mice and add 1 ml collagenase solution, and obtain 1-1.5 ml seminiferous tubule clumps from five 4-week-old mice and add 6 ml collagenase solution.
    5. Loosen seminiferous tubules using forceps under a dissecting microscope within 20 min at room temperature (~25 °C) to avoid prolonged enzyme incubation (Figure 1A, lower panel).
      The procedures of testis collection, tunica albuginea removal and loosening of seminiferous tubules is demonstrated in Video 1.

      Video 1. Testis collection, tunica albuginea removal and loosening of seminiferous tubules from 4-week-old mice (steps A1-A5)

    6. Wash and clean the dispersed seminiferous tubules twice with at least 3-fold volumes of room temperature E-HBSS for 20 sec with gentle horizontal shaking at 30 rpm.
    7. Remove E-HBSS, add 3-fold volumes of collagenase solution and incubate at 37 °C for 20 min.
    8. Discard supernatant and wash dispersed seminiferous tubules by adding 3-fold volumes of room temperature E-HBSS for 20 sec with gentle horizontal shaking at 30 rpm.
    9. Repeat this wash process thoroughly five times with room temperature E-HBSS buffer for 20 sec with gentle horizontal shaking at 30 rpm to remove suspension cells.
    10. Transfer seminiferous tubules to a 2 ml polypropylene tube (or to 5 ml polypropylene tube when executing large number of samples). Add 5-fold volumes of room temperature trypsin solution supplemented with 5 U/ml DNase I. Pipette up and down using a P1000 pipette with cut tip to mechanically mince seminiferous tubules in order to obtain single cell suspension (Figure 1B).
    11. Incubate the tissues at 37 °C for 3 min.
      Note: It is important to execute this reaction (steps A10 and A11) within 10 min, since prolonged trypsin solution incubation may cause a proteolytic cleavage of the cell surface protein (see Note 2).
    12. Add 3-fold volumes of room temperature F-MACS to quench the trypsin reaction (see Note 3).
    13. Transfer suspension cells to a new 50 ml centrifuge tubes.
    14. Remove residual tissue and filter the suspension using a 70 µm nylon cell strainer. Wash cell strainer with 2-fold volumes of room temperature MACS buffer after adding cell suspension.
    15. Centrifuge cell suspension at 300 x g for 10 min at 4 °C.
    16. Re-suspend cells in ice-cold MACS buffer and count the number of cells with a hemocytometer (see Note 4).

  2. Cell labeling and magnetic separation of one-week-old testicular cells
    1. Centrifuge again at 300 x g for 10 min at 4 °C and aspirate supernatant. Re-suspend cells in 100 µl ice-cold MACS buffer and add 10 µl biotin-conjugated anti-CD90.2 (THY1) antibody per 107 cells isolated from one-week-old mice. We use approximately 5 µg antibody per 100 µl ice-cold MACS buffer.
    2. Mix well by gentle pipetting and incubate at 4 °C for 15 min with gentle rotation at 30 rpm (see Note 5).
    3. Wash by adding equal volume ice-cold MACS buffer to remove the unbound primary antibody.
    4. Centrifuge cell suspension at 300 x g for 10 min at 4 °C.
    5. Re-suspend cells and wash again with 2 ml ice-cold MACS buffer per 107 cells and centrifuge at 300 x g for 10 min at 4 °C.
    6. Aspirate supernatant completely and re-suspend cells in 80 µl MACS buffer and add 20 µl microbeads (Miltenyi Biotec) per 107 cells.
    7. Mix well by gentle pipetting and incubate for 15 min at 4 °C with gentle rotation at 30 rpm.
    8. Wash by adding 2 ml MACS buffer per 107 cells and centrifuge at 300 x g for 10 min at 4 °C.
    9. Aspirate supernatant completely and re-suspend up to 108 cells in 1 ml ice-cold MACS buffer.
    10. Pass testicular cells through a 35 µm nylon cell strainer to remove cell clumps. Approximate 10% cells are lost compared with the cell number after trypsin treatment (step A16).
    11. Place MS column on magnetic separator. Prepare MS column (Miltenyi Biotec) for cell separation by rinsing with 500 µl ice-cold MACS buffer.
    12. Apply cell suspension onto the prepared MS columns (see Note 6) and pipette gently every 30 sec to avoid sedimentation and aggregation of the cells before they pass through the column. This ensures the individual cells will pass through the column fluently and effectively.
    13. Collect the unbound cells that pass through and wash the column three times with 500 µl ice-cold MACS buffer. These cells can serve as a sample to test the efficiency of removing THY1+ cells from the subpopulation, or as a THY1-negative control group.
    14. Remove MS column from magnetic separator and place it over a new 15 ml centrifuge tube.
    15. Add 1 ml ice-cold MACS buffer into MS column in order to elute the THY1+ cells.
    16. Collect the magnetically labeled THY1+ cells by using the plunger supplied with the column (see Notes 5 and 7; Figures 1E-1M for germ cell purity and live/dead cell ratio).

  3. Cell labeling and magnetic separation of 4-week-old testicular cells
    1. Centrifuge again at 300 x g for 10 min at 4 °C and aspirate supernatant. Re-suspend cells in 1 ml ice-cold MACS buffer and add 20 µl biotin-conjugated anti-CD90.2 (THY1) antibody per 108 cells isolated from 4-week-old mice. There is approximately 10 microgram antibody per 1 ml ice-cold MACS buffer.
    2. Mix well by gentle pipetting and incubate at 4 °C for 15 min with gentle rotation at 30 rpm (see Note 5).
    3. Wash by adding equal volume ice-cold MACS buffer and centrifuge cell suspension at 300 x g for 10 min at 4 °C.
    4. Re-suspend cells and wash again with 2 ml ice-cold MACS buffer per 108 cells and centrifuge at 300 x g for 10 min at 4 °C.
    5. Aspirate supernatant completely and re-suspend cells in 1 ml ice-cold MACS buffer and add 40 µl microbeads (Miltenyi Biotec) per 108 cells.
    6. Mix well by gentle pipetting and incubate 15 min at 4 °C with gentle rotation at 30 rpm.
    7. Wash with 2 ml ice-cold MACS buffer per 108 cells and centrifuge at 300 x g for 10 min at 4 °C.
    8. Aspirate supernatant completely and re-suspend up to 108 cells in 1 ml ice-cold MACS buffer.
    9. Pass cells through a 35 µm nylon cell strainer to remove cell clumps.
    10. Place LS column on magnetic separator. Prepare LS column (Miltenyi Biotec) by rinsing with 2 ml ice-cold MACS buffer.
    11. Apply cell suspension onto the prepared LS columns (see Note 6) and pipette gently every 30 sec to avoid sedimentation and aggregation of the cells before they pass through the column. This ensures the individual cells will pass through the column fluently and effectively.
    12. Collect unlabeled cells that pass through and wash the column three times with 2 ml ice-cold MACS buffer. These cells can serve as a sample to test the efficiency of removing THY1+ cells from the subpopulation, or as a THY1-negative control group.
    13. Remove LS column from the magnetic separator and place it over a new 15 ml centrifuge tube.
    14. Add 2 ml ice-cold MACS buffer into the LS column in order to elute the THY1+ cells.
    15. Collect the magnetically labeled THY1+ cells by using the plunger supplied with the column (see Notes 5 and 7; Figures 1E-1M for germ cell purity and live/dead cell ratio)

Data analysis

  1. All analytical results published (Liao et al., 2014; Tseng et al., 2015) with samples isolated by the exact protocol described here were calculated from three to eight independent experiments.
  2. Most of the isolated 8 dpp THY1+ cells (~80%) display perinuclear PLZF distribution (Tseng et al., 2015), suggesting this subpopulation is relatively quiescence (Buaas et al., 2004; Costoya et al., 2004).
  3. More than 85% of total isolated cells from this protocol exhibit PLZF and VASA signals. PLZF is recognized as a marker for undifferentiated spermatogonia and VASA is expressed in all germ cells. The high purity of SSC-enriched primary germ cells (without culture) via this isolation protocol is suitable for genome wide high throughput transcriptome and epigenomic analysis (Liao et al., 2014; Tseng et al., 2015).
  4. For quality control of immunostaining, samples from the luxoid (ZBTB16lu/lu) mutant mice were used as a negative control to validate the specificity of the PLZF antibody. VASA expressing germ cells and non-VASA expressing somatic cells (mouse embryonic fibroblasts) are used as positive and negative controls to validate the specificity of anti-VASA antibody. The strongest signal intensity from IgG control (secondary antibody only) was used as the cutoff line for background noise.

Representative data



Figure 1. Germ cell enrichment from postnatal mouse testes using magnetic-activated cell sorting. A. Dispersed seminiferous tubules of a testis for an efficient removal of somatic cells outside of seminiferous tubules. Upper panel reveals seminiferous tubule clumps without tunica albuginea. Lower panel indicates dispersed seminiferous tubules. B. A single cell suspension using efficient trypsin solution (pink color, upper panel). Lower panel displays single cell suspension after trypsin treatment under a microscope. C. Each batch of isolated THY1+ cells was treated with trypan blue dye to calculate the percentage of cell death. Unstained viable and round cells (for instance, yellow arrow) are recognized in a phase-contrast microscopy. Red arrow indicates the black color of dead cell. D. Flow cytometry analysis of isolated 8 dpp THY1+ cells labeled with an isotype antibody as negative control (upper panel) and with a PE-Cy5-conjugated anti-CD49f antibody (lower panel) (see Note 8). E-G. Immunostaining of isolated 8 dpp THY1+ cells using a monoclonal anti-PLZF antibody. Green, PLZF; blue, Hoechst 33342. Scale bar = 60 µm. H-J. The PLZF (green) subcellular localization pattern in 80% of isolated 8 dpp THY1+ cells. Green, PLZF; blue, Hoechst 33342. Scale bar = 2.5 µm. K-M. Immunostaining of isolated 4-week-old THY1+ cells using an anti-VASA antibody. VASA predominantly localizes in the cytoplasm. Green, VASA; blue, Hoechst 33342. Scale bar = 25 µm (see Note 9).

Notes

  1. THY1 is exhibited in various cell types including hematopoietic cells, epithelial cells and fibroblasts. In testes, cells display THY1 expression outside of seminiferous tubules (Kubota et al., 2004b; Rege and Hagood, 2006). Therefore, a thorough removal of somatic THY1+ cells outside of seminiferous tubules is important for the purity of isolated THY1+ undifferentiated spermatogonia (steps A4-A9).
  2. Trypsin treatment may lead to proteolytic cleavage of surface proteins. To minimize this disadvantage, samples are separated into several groups when there are a large number of testes. F-MACS-treated single cell suspension is kept on ice after being filtered through a 70 µm nylon cell strainer. Add more trypsin solution if cells are clumped or if the reaction solution turns from pink to yellow color, and pipette again to achieve a single cell suspension (Figure 1B).
  3. Pre-warm the F-MACS buffer to room temperature to minimize cell clumping.
  4. When five postnatal mice were used for each experiment, testicular cells obtained by this protocol is approximately 1-1.3 x 106 cells per testis at 8 dpp and around 0.7-1 x 107 per testis for 4-week-old mice. The date of birth was defined as 0 dpp.
  5. Longer antibody incubation at 4 °C for 30 min can increase the recovery rate of THY1+ cells; however, it also increases non-specific binding of anti-CD90.2 antibody to somatic cells (Liao et al., 2014). Based on our experience, the isolation efficiency and purity of undifferentiated spermatogonia varied significantly with different Lot of biotin-conjugated anti-CD90.2 antibodies. For instance, in experienced hands, the ratio of CD49f+ or PLZF+ germ cells is more than 85% of total isolated cells when using Lot 25136 and Lot 80806 of biotin-conjugated anti-CD90.2 antibodies (BD Pharmingen; Figures 1D-1G), whereas the PLZF+ ratio of total isolated cells is between 60% and 85% with Lot 3032695, Lot 5152843 and Lot 6110644 of anti-CD90.2 antibodies (BD Pharmingen) as well as anti-CD90.2­Biotin antibody (Miltenyi Biotec, Lot 5150211128). The reduced ratio of germ cells when applying less preferable batches of anti-CD90.2 antibodies may be due to a somatic cell contamination based on the increased VASA-negative cells after immunostaining using an anti-VASA antibody (germ cell marker) (Figures 1K-1M).
    In addition, there may be a disadvantage in applying this protocol to isolation of THY1+ undifferentiated spermatogonia from adult mice since there are more THY1+ somatic cells presented in adult testes (Kubota and Brinster, 2008).
  6. In order to perform efficient cell isolation, the typical cell number for MS column should be less than 5 x 107 testicular cells per column and the cell number for a LS column should be less than 5 x 108 testicular cells.
  7. Dead cells are generally less than 10% of total isolated THY1+ cells determined by trypan blue staining (Figure 1C). An optional centrifugation at 300 x g for 10 min at 4 °C can efficiently remove the dead cells.
  8. Magnetically isolated THY1+ cells were analyzed through a flow cytometry. A PE-Cy5-conjugated anti-CD49f antibody and an isotype antibody were used for this analysis. THY1+ cells were re-suspended and incubated on ice for 20 min in the dark in staining buffer. After being washed twice with FACS buffer and filtered through a 35 µm nylon cell strainer, PE-Cy5-conjugated cells were analyzed through a Beckman Coulter FC500 Cytometer (Figure 1D).
  9. For immunocytochemical analysis, isolated THY1+ cells were placed onto Poly-L-Lysine coated slides (Thermo Scientific) and fixed in 4% PFA for 10 min at room temperature. After being washed five times with PBST for 10 min at room temperature, the cells were blocked with 2% BSA and 5% donkey serum for 2 h at room temperature and incubated at 4 °C overnight with the following primary antibody incubation: anti-PLZF antibody or anti-VASA antibody. The cells were washed three times with PBST containing 1% BSA. After incubation with secondary antibodies for 1 h at room temperature, the slides were washed three times in PBST, counterstained with Hoechst 33342 and mounted with mounting medium. The slides were analyzed through a Leica TCS SP5 II confocal microscope (Figures 1E-1M).

Recipes

  1. E-HBSS
    2 mM EDTA in HBSS, no calcium, no magnesium, no phenol red
  2. MACS buffer
    2 mM EDTA, 0.5% BSA in DPBS
  3. Collagenase solution
    1 mg/ml type IV collagenase and 5 U/ml DNase I in HBSS
  4. Trypsin solution
    5 U/ml DNase I in 0.25% trypsin-EDTA, phenol red
  5. Freshly prepared F-MACS buffer
    10% FBS in MACS buffer
    Note: Keep the F-MACS on ice and pre-warm the F-MACS buffer to room temperature (~25 °C) before use.
  6. Staining buffer
    1x DPBS
    2 mM EDTA
    2% heat-inactivated FBS
    PE-Cy5-conjugated antibody (1:1000 dilution)
  7. FACS buffer
    1x DPBS
    2 mM EDTA
  8. PBST
    1x DPBS
    0.2% Tween-20

Acknowledgments

The authors would like to express their sincere gratitude to Mr. Pei-Lung Lee, Miss Hung-Chun Tung and Miss Yi-Chun Chen for useful discussions. This work was supported by grants from Ministry of Science and Technology, Taiwan (MOST 103-2321-B-002-099 and MOST 104-2321-B-002-043) and National Taiwan University (NTU-105R4000).

References

  1. Boivin, J., Bunting, L., Collins, J. A. and Nygren, K. G. (2007). International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care. Hum Reprod 22(6): 1506-1512.
  2. Buaas, F. W., Kirsh, A. L., Sharma, M., McLean, D. J., Morris, J. L., Griswold, M. D., de Rooij, D. G. and Braun, R. E. (2004). Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet 36(6): 647-652.
  3. Buageaw, A., Sukhwani, M., Ben-Yehudah, A., Ehmcke, J., Rawe, V. Y., Pholpramool, C., Orwig, K. E. and Schlatt, S. (2005). GDNF family receptor alpha1 phenotype of spermatogonial stem cells in immature mouse testes. Biol Reprod 73(5): 1011-1016.
  4. Chan, F., Oatley, M. J., Kaucher, A. V., Yang, Q. E., Bieberich, C. J., Shashikant, C. S. and Oatley, J. M. (2014). Functional and molecular features of the Id4+ germline stem cell population in mouse testes. Genes Dev 28(12): 1351-1362.
  5. Costoya, J. A., Hobbs, R. M., Barna, M., Cattoretti, G., Manova, K., Sukhwani, M., Orwig, K. E., Wolgemuth, D. J. and Pandolfi, P. P. (2004). Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet 36(6): 653-659.
  6. Gassei, K. and Orwig, K. E. (2013). SALL4 expression in gonocytes and spermatogonial clones of postnatal mouse testes. PLoS One 8(1): e53976.
  7. Hermann, B. P., Mutoji, K. N., Velte, E. K., Ko, D., Oatley, J. M., Geyer, C. B. and McCarrey, J. R. (2015). Transcriptional and translational heterogeneity among neonatal mouse spermatogonia. Biol Reprod 92(2): 54.
  8. Kanatsu-Shinohara, M., Inoue, K., Ogonuki, N., Morimoto, H., Ogura, A. and Shinohara, T. (2011). Serum- and feeder-free culture of mouse germline stem cells. Biol Reprod 84(1): 97-105.
  9. Kubota, H., Avarbock, M. R. and Brinster, R. L. (2003). Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells. Proc Natl Acad Sci U S A 100(11): 6487-6492.
  10. Kubota, H., Avarbock, M. R. and Brinster, R. L. (2004a). Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biol Reprod 71(3): 722-731.
  11. Kubota, H., Avarbock, M. R. and Brinster, R. L. (2004b). Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 101(47): 16489-16494.
  12. Kubota, H. and Brinster, R. L. (2008). Culture of rodent spermatogonial stem cells, male germline stem cells of the postnatal animal. Methods Cell Biol 86: 59-84.
  13. Liao, H. F., Chen, W. S., Chen, Y. H., Kao, T. H., Tseng, Y. T., Lee, C. Y., Chiu, Y. C., Lee, P. L., Lin, Q. J., Ching, Y. H., Hata, K., Cheng, W. T., Tsai, M. H., Sasaki, H., Ho, H. N., Wu, S. C., Huang, Y. H., Yen, P. and Lin, S. P. (2014). DNMT3L promotes quiescence in postnatal spermatogonial progenitor cells. Development 141(12): 2402-2413.
  14. Matzuk, M. M. and Lamb, D. J. (2008). The biology of infertility: research advances and clinical challenges. Nat Med 14(11): 1197-1213.
  15. Oatley, J. M. and Brinster, R. L. (2008). Regulation of spermatogonial stem cell self-renewal in mammals. Annu Rev Cell Dev Biol 24: 263-286.
  16. Rege, T. A. and Hagood, J. S. (2006). Thy-1 as a regulator of cell-cell and cell-matrix interactions in axon regeneration, apoptosis, adhesion, migration, cancer, and fibrosis. FASEB J 20(8): 1045-1054.
  17. Sada, A., Suzuki, A., Suzuki, H. and Saga, Y. (2009). The RNA-binding protein NANOS2 is required to maintain murine spermatogonial stem cells. Science 325(5946): 1394-1398.
  18. Tseng, Y. T., Liao, H. F., Yu, C. Y., Mo, C. F. and Lin, S. P. (2015). Epigenetic factors in the regulation of prospermatogonia and spermatogonial stem cells. Reproduction 150(3): R77-91.
  19. Wabik, A. and Jones, P. H. (2015). Switching roles: the functional plasticity of adult tissue stem cells. EMBO J 34(9): 1164-1179.
  20. Xin, T., Greco, V. and Myung, P. (2016). Hardwiring stem cell communication through tissue structure. Cell 164(6): 1212-1225.

简介

在哺乳动物中,许多组织的体内平衡依赖于称为干细胞的细胞亚群,以维持适量的未分化细胞和分化细胞。精原细胞干细胞(SSC)为精子发生提供了基本的细胞来源,并且负责终生维持雄性生殖期间睾丸中的种系池。为了获得对种系干细胞生物学的了解并制定不孕症治疗策略,已经报道了几种生殖细胞分离方法,以获得良好质量和数量的未分化精原细胞。其中,磁激活细胞分选(MACS)是一种有效的细胞分离方法,其需要较少的时间和较少的初始细胞数以使用抗原 - 抗体反应来获得富集的细胞群体。胸腺细胞抗原1(THY1,CD90.2)被认为是小鼠新生儿和成年睾丸中未分化精原细胞的表面标志物。在这里,我们描述了一个用于分离来自小鼠睾丸的一周龄THY1 + 细胞和四周龄THY1 + 细胞的方案。分离过程包括三个步骤:睾丸收集和单细胞悬浮,使用生物素缀合的抗THY1抗体的细胞标记和磁性细胞分离。注意,这种隔离方案应在五小时内完成,以最大限度地提高活细胞的质量和数量。

背景 在几个成人组织中观察到活性和静止干细胞的共存。静止,自我更新和分化之间的充分平衡对于维持适当数量的未分化干细胞是必要的,并且避免过早的干细胞耗尽许多组织的体内平衡(Tseng等人,2015; Wabik和Jones,2015; Xin等人,2016)。不孕不育已经成为人类夫妇越来越多的问题,男性相关的不育症的很大一部分是由于未分化的精原细胞受损(Boivin et al。,2007; Matzuk和Lamb,2008)。因此,精子发生中的SSCs是一种良好表征的干细胞依赖性过程(Oatley和Brinster,2008),是研究组织体内平衡调节的有价值的模型。许多研究已经开发了生殖细胞分离方法,以便了解未分化精原细胞的生物学功能和调节网络。然而,体内未分化的精原细胞在其包括GFRA1,ID4,PLZF和THY1的标记物的表达中是异质的,并且不同的实验方案对特定细胞状态的亚群的富集有影响(Buageaw等人,2005年; Chan等人,2014年; Costoya等人,2004; Gassei和Orwig,2013; Hermann等人,2015;久保田等人,2003;廖等人,2014)。例如,PLZF + 和THY1 + 未分化精原细胞的大部分发现于体内细胞周期的静止期,而PLZF 在补充有血清和生长因子的培养基中培养的未分化的精原细胞在增殖阶段倾向于增殖期(Costoya等,2004; Kanatsu ,Shinohara等人,2011; Kubota等人,2004a; Liao等人,2014; Sada等人, / em>。,2009)。
 这里,我们提供了一个逐步的步骤,通过使用磁激活细胞分选的无血清方案从前青春期小鼠中分离相对静止的THY1 + 未分化精原细胞。该协议还包含几个重要信息,包括每个步骤所需的细胞编号,用于生殖细胞分离的合适的酶程序。该方案应该是通过使用针对SSC表面抗原的抗体获得大量具有特定亚群富集的高质量活的未分化精原细胞的有价值的工具。

关键字:睾丸, THY1 +精原干细胞, 免疫磁珠分选法, 生殖细胞

材料和试剂

  1. 100mm培养皿(Corning,Falcon ®,目录号:351029)
  2. 聚丙烯管:
    2ml管(Corning,Axygen ,目录号:MCT-200-C)
    5 ml管(Corning,Axygen ,目录号:MCT-500-C)
  3. 离心管:
    15 ml管(Corning,Falcon ®,目录号:352096)
    50ml管(Corning,Falcon ®,目录号:352070)
  4. 细胞过滤器:
    5 ml聚苯乙烯管,带细胞过滤器扣帽(35μm尼龙网)(Corning,Falcon ®,目录号:352235)
    70μm尼龙网(Corning,Falcon ®,目录号:352350)
  5. 细胞分离柱:
    MS柱(Miltenyi Biotec,目录号:130-042-201)
    LS列(Miltenyi Biotec,目录号:130-042-401)
  6. 聚-L-赖氨酸涂层载玻片(Thermo Fisher Scientific,目录号:10143265)
  7. ART TM 屏障铰链机架吸头提示
    P1000(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:2079-HR)
    P200(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:2069-05-HR)
    P20(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:2149-05-HR)
  8. C57BL/6N小鼠(BioLASCO,目录号:C57BL/6N)
  9. Hank的平衡盐溶液(HBSS),钙,镁,无酚红(Thermo Fisher Scientific,Gibco TM,目录号:14025092)
  10. 汉克平衡盐溶液(HBSS),无钙,无镁,无酚红(Thermo Fisher Scientific,Gibco TM,目录号:14175095)
  11. IV型胶原酶(Thermo Fisher Scientific,Gibco TM,目录号:17104019)
  12. DNase I(不含RNase)(New England Biolabs,目录号:M0303)
  13. 抗体:
    生物素大鼠抗小鼠CD90.2(克隆30-H12)(BD,目录号:553011)
    生物素大鼠IgG2b,κ同种型对照(克隆A95-1)(BD,目录号:553987)
    抗CD90.2生物素抗体(批号5150211128)(Miltenyi Biotec,目录号:130-101-908)
    抗CD49f抗体(BD,目录号:551129)
    同种型抗体(阴性对照)(BD,目录号:551066)
    抗PLZF抗体(Santa Cruz Biotechnology,目录号:sc-28319)
    抗VASA抗体(Abcam,目录号:ab13840)
    AffiniPure驴抗兔IgG(H + L)(Alexa Fluor) 488)(Jackson ImmunoResearch,目录号:711-545-152)
    AffiniPure驴抗小鼠IgG(H + L)(Alexa Fluor 488)(Jackson ImmunoResearch,目录号:715-545-151)
  14. 抗生物素微珠(Miltenyi Biotec,目录号:130-090-485)
  15. PFA(Sigma-Aldrich,目录号:158127)
  16. 牛血清白蛋白(BSA),无IgG和无蛋白酶(Jackson ImmunoResearch,目录号:001-000-162)
  17. 正常驴血清(Abcam,目录号:ab7475)
  18. EDTA,0.5M,pH 8.0(Thermo Fisher Scientific,Anbion TM,目录号:AM9260G)
  19. BSA
  20. Dulbecco的磷酸盐缓冲盐水(DPBS),不含钙,无镁(Thermo Fisher Scientific,Gibco TM,目录号:14190144)
  21. 胰蛋白酶-EDTA,0.25%,酚红(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  22. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:16000044)
  23. PE-Cy5缀合的抗体
  24. 吐温-20(Sigma-Aldrich,目录号:P9416)
  25. E-HBSS(见配方)
  26. MACS缓冲区(见配方)
  27. 胶原酶溶液(参见食谱)
  28. 胰蛋白酶溶液(参见食谱)
  29. F-MACS缓冲区(见配方)
  30. 染色缓冲液(见配方)
  31. FACS缓冲区(见配方)
  32. PBST(见配方)

设备

  1. 灭菌镊子和剪刀
  2. 解剖显微镜(Leica MZ16F立体显微镜)
  3. P1000移液器
  4. 离心机
  5. 血细胞计数器
  6. 磁分离器
    用于MS列的OctoMACS TM分隔符(Miltenyi Biotec,目录号:130-042-109)
    用于LS列的MidiMACS TM分隔符(Miltenyi Biotec,目录号:130-042-302)
  7. Beckman Coulter FC500细胞仪(Beckman Coulter,型号:FC500)
  8. Leica TCS SP5 II共焦显微镜(Leica Microsystems,型号:Leica TCS SP5 II)

程序

  1. 睾丸收集和单细胞悬浮液
    1. 使用灭菌镊子和剪刀从小鼠中取出睾丸。在含有10毫升冰冷HBSS的100毫米培养皿中收集睾丸。
    2. 从解剖显微镜(见图1A,上图),从睾丸中取出白膜以暴露生精小管。将生精小管块转移到含有10ml冰冷HBSS的新的100mm培养皿中。将生精小管置于冰上。
    3. 使用冰冷的HBSS清洗生精小管团块,以清除碎片和悬浮细胞
    4. 为了估计生精小管团的粗大体积,我们将这些团块转移到2ml聚丙烯管中以计算体积。然后,将生精小管块转移到含有至少3倍生精小管的体积的室温胶原酶溶液的新培养皿中,补充有1mg/ml IV型胶原酶和5U/ml DNase I以除去间质Leydig细胞,血液细胞和管周细胞,分别从死细胞中消化基因组DNA(见注1) 注意:我们通常从五只一周龄的小鼠中收集0.2-0.3ml生精小管团块,加入1ml胶原酶溶液,从五只4周龄小鼠中获得1-1.5ml生精小管团块,并加入6 ml胶原酶溶液。
    5. 在室温(〜25°C)的20分钟内,用解剖显微镜在镊子内松开生精小管,以避免长时间的酶孵育(图1A,下图)。
      视频1中显示了睾丸采集,精液清除和生精小管松动的程序。

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    6. 使用至少3倍体积的室温E-HBSS洗涤并清洁分散的生精小管20秒,同时以30rpm的温和水平摇动。
    7. 去除E-HBSS,加入3倍体积的胶原酶溶液,并在37℃下孵育20分钟。
    8. 丢弃上清液并通过加入3倍体积的室温E-HBSS洗涤分散的生精小管20秒,同时以30rpm的温和水平摇动。
    9. 用室温E-HBSS缓冲液重复该洗涤过程五次,持续20秒,以30rpm的温和水平摇动除去悬浮细胞。
    10. 将生精小管转移到2毫升聚丙烯管(或执行大量样品时为5毫升聚丙烯管)。加入5倍体积的补充有5U/ml DNA酶I的室温胰蛋白酶溶液。使用具有切割尖端的P1000移液管上下移液以机械切碎生精小管以获得单细胞悬浮液(图1B)。
    11. 在37℃下孵育组织3分钟 注意:在10分钟内执行该反应(步骤A10和A11)很重要,因为延长的胰蛋白酶溶液孵育可能导致细胞表面蛋白质的蛋白水解切割(见注2)。
    12. 加入3倍体积的室温F-MACS以猝灭胰蛋白酶反应(见注3)
    13. 将悬浮细胞转移到新的50ml离心管中
    14. 使用70μm尼龙细胞过滤器去除残留组织并过滤悬浮液。在加入细胞悬液后,用2倍体积的室温MACS缓冲液洗涤细胞过滤器。
    15. 在4℃下以300×g离心细胞悬液10分钟。
    16. 将细胞重新悬浮在冰冷的MACS缓冲液中,并用血细胞计数器计数细胞数(见注4)。

  2. 一周龄睾丸细胞的细胞标记和磁分离
    1. 在4℃下再次以300×g离心10分钟并吸出上清液。将细胞重新悬浮在100μl冰冷的MACS缓冲液中,并从一周龄小鼠分离的每10个细胞中加入10μl生物素缀合的抗CD90.2(THY1)抗体。我们每100μl冰冷的MACS缓冲液使用约5μg抗体。
    2. 通过温和移液进行良好的混合,并在30℃下以4℃静置15分钟(见附注5)。
    3. 通过加入等体积冰冷的MACS缓冲液洗去未结合的一抗
    4. 在4℃以300×g离心细胞悬浮液10分钟。
    5. 重新悬浮细胞并再次用2ml冰冷的MACS缓冲液/10μg/ml细胞洗涤并在4℃下以300×g离心10分钟。
    6. 完全吸出上清液,并将细胞重新悬浮在80μlMACS缓冲液中,每10个细胞添加20μl微珠(Miltenyi Biotec)。
    7. 通过温和移液混匀并在4℃下温和旋转30分钟温和旋转15分钟。
    8. 通过加入2ml MACS缓冲液/10μg/ml细胞洗涤并在4℃下以300×g离心10分钟。
    9. 完全吸出上清液,并在1ml冰冷的MACS缓冲液中再悬浮至10μg/ml细胞。
    10. 通过35μm尼龙细胞过滤器通过睾丸细胞去除细胞团。与胰蛋白酶处理后的细胞数相比,大约10%的细胞丢失(步骤A16)。
    11. 将MS柱放在磁选机上。通过用500μl冰冷的MACS缓冲液冲洗,准备MS柱(Miltenyi Biotec)进行细胞分离。
    12. 将细胞悬浮液应用于制备的MS色谱柱(见附注6),并每30秒轻轻移液一次,以避免细胞在通过色谱柱之前沉降和聚集。这样可以确保各个细胞能够流畅地通过色谱柱。
    13. 收集通过的未结合的细胞,并用500μl冰冷的MACS缓冲液洗涤柱子三次。这些细胞可以作为样本,以测试从亚群中除去THY1 + 细胞的效力,或作为THY1阴性对照组。
    14. 从磁选机中取出MS柱,并将其置于新的15 ml离心管上
    15. 将1ml冰冷的MACS缓冲液加入MS柱中,以洗脱THY1 + 细胞。
    16. 通过使用柱提供的柱塞收集磁性标记的THY1 + 细胞(参见注释5和7;图1E-1M,用于生殖细胞纯度和活/死细胞比)。

  3. 4周龄睾丸细胞的细胞标记和磁分离
    1. 在4℃下再次以300×g离心10分钟并吸出上清液。将细胞重新悬浮在1ml冰冷的MACS缓冲液中,并从4周龄小鼠分离的每10个细胞添加20μl生物素缀合的抗CD90.2(THY1)抗体。每1毫升冰冷的MACS缓冲液中有约10微克抗体。
    2. 通过轻轻移液进行良好的混合,并以30rpm的温和旋转在4℃温育15分钟(见注5)。
    3. 通过加入等体积冰冷的MACS缓冲液并在300℃下离心细胞悬浮液在4℃下洗涤10分钟进行洗涤。
    4. 重新悬浮细胞并再次用2ml冰冷的MACS缓冲液每108个细胞洗涤并在4℃下以300×g离心10分钟。
    5. 完全吸出上清液,并将细胞重新悬浮在1ml冰冷的MACS缓冲液中,每10个细胞添加40μl微珠(Miltenyi Biotec)。
    6. 通过轻轻移液混匀,并在4℃温育15分钟,以30rpm的温和旋转。
    7. 用2ml冰冷的MACS缓冲液/10μg/ml细胞洗涤,并在4℃以300×g离心10分钟。
    8. 将上清液完全吸入,并在1ml冰冷的MACS缓冲液中重新悬浮至10μg/ml细胞。
    9. 将细胞通过35μm尼龙细胞过滤器,以去除细胞团块
    10. 将LS柱放在磁选机上。用2ml冰冷的MACS缓冲液冲洗,制备LS柱(Miltenyi Biotec)
    11. 将细胞悬浮液应用到制备的LS柱上(见附注6),每30秒轻轻移液一次,以避免细胞在通过柱子之前沉降和聚集。这样可以确保各个细胞能够流畅地通过色谱柱。
    12. 收集未通过标记的细胞,并用2ml冰冷的MACS缓冲液洗涤柱子三次。这些细胞可以作为样本,以测试从亚群中除去THY1 + 细胞的效力,或作为THY1阴性对照组。
    13. 从磁选机中取出LS柱,放在新的15 ml离心管上
    14. 将2ml冰冷的MACS缓冲液加入LS柱,以洗脱THY1 + 细胞。
    15. 通过使用柱提供的柱塞收集磁性标记的THY1 + 细胞(参见注释5和7;图1E-1M,用于生殖细胞纯度和活/死细胞比)

数据分析

  1. 通过这里描述的精确方案分离的样品的所有分析结果(廖等人,2014; Tseng等人,2015)分别由三到八个独立实验。
  2. 大多数分离的8dpp THY1 + 细胞(〜80%)显示出核周PLZF分布(Tseng等人,2015),表明该亚群是相对静止的(Buaas < ,2004年; Costoya等人,2004)。
  3. 来自该协议的全部隔离细胞的85%以上表现出PLZF和VASA信号。 PLZF被认为是未分化精原细胞的标记,VASA在所有生殖细胞中表达。通过该分离方案,富含SSC的初级生殖细胞(无培养)的高纯度适用于全基因组高通量转录组和表观基因组分析(Liao等人,2014; Tseng等人。,2015)。
  4. 为了免疫染色的质量控制,使用来自 luxoid(ZBTB16 )突变小鼠的样品作为阴性对照以验证PLZF抗体的特异性。 VASA表达生殖细胞和非VASA表达体细胞(小鼠胚胎成纤维细胞)用作阳性和阴性对照以验证抗VASA抗体的特异性。使用来自IgG对照的最强信号强度(仅二次抗体)作为背景噪声的截止线

代表数据




图1.使用磁激活细胞分选的产后小鼠睾丸的生殖细胞富集A.睾丸分散的生精小管,用于有效去除生精小管外的体细胞。上图显示无精囊白细胞的生精小管团块。下图显示分散的生精小管。 B.使用高效胰蛋白酶溶液的单细胞悬浮液(粉红色,上图)。在显微镜下,下面板显示胰蛋白酶处理后的单细胞悬液。 C.用台盼蓝染料处理每批分离的THY1 + 细胞以计算细胞死亡的百分比。在相差显微镜下可以识别未染色的活细胞(例如黄色箭头)。红色箭头表示死细胞的黑色。 D.用同种型抗体标记的分离的8dpp THY1 + 细胞的流式细胞术分析(上图)和用PE-Cy5缀合的抗CD49f抗体(下图) 8)。例如。使用单克隆抗PLZF抗体免疫染色分离的8dpp THY1 + 细胞。绿色,PLZF;蓝色,Hoechst 33342.刻度棒=60μm。 H-J。在80%分离的8dpp THY1 + 细胞中的PLZF(绿色)亚细胞定位模式。绿色,PLZF;蓝色,Hoechst 33342.刻度棒=2.5μm。 K-M。使用抗VASA抗体免疫染色分离的4周龄THY1 + 细胞。 VASA主要位于细胞质中。绿色,VASA;蓝色,Hoechst 33342.刻度棒= 25μm(见注9)。

笔记

  1. THY1表现在各种细胞类型,包括造血细胞,上皮细胞和成纤维细胞。在睾丸中,细胞在生精小管外显示THY1表达(Kubota等人,2004b; Rege和Hagood,2006)。因此,彻底去除生精小管之外的体细胞THY1 + 细胞对于分离的THY1 未分化精原细胞的纯度是重要的(步骤A4-A9)。
  2. 胰蛋白酶处理可能导致表面蛋白质的蛋白水解切割。为了最小化这个缺点,当有大量的睾丸时,将样品分成几组。将F-MACS处理的单细胞悬浮液通过70μm尼龙细胞过滤器过滤后保存在冰上。如果细胞结块或如果反应溶液从粉红色变成黄色,再添加更多的胰蛋白酶溶液,再次移液以达到单细胞悬浮液(图1B)。
  3. 将F-MACS缓冲液预温至室温以最小化细胞团聚。
  4. 当每个实验使用五个产后小鼠时,通过该方案获得的睾丸细胞在8dpp处约为1-1.3×10 6个细胞/睾丸,约0.7-1×10 7个/每个睾丸为4周龄小鼠。出生日期定义为0 dpp。
  5. 在4℃下更长的抗体孵育30分钟可以提高THY1 + 细胞的回收率;然而,它也增加抗CD90.2抗体与体细胞的非特异性结合(Liao等人,2014)。根据我们的经验,未分化精原细胞的分离效率和纯度随着不同批次的生物素缀合的抗CD90.2抗体而显着变化。例如,在有经验的手中,当使用Lot 25136和Lot 80806的生物素标记时,CD49f + 或PLZF + 生殖细胞的比例大于总分离细胞的85%共轭抗CD90.2抗体(BD Pharmingen;图1D-1G),而总分离细胞的PLZF + 比率为60%至85%,批号为3032695,Lot 5152843和Lot 6110644为抗CD90.2抗体(BD Pharmingen)以及抗CD90.2Biotin抗体(Miltenyi Biotec,Lot 5150211128)。当应用较不优选批次的抗CD90.2抗体时,生殖细胞的减少的比例可能是由于使用抗VASA抗体(生殖细胞标记)免疫染色后基于增加的VASA阴性细胞的体细胞污染(图1K -1M)。
    另外,由于在成年睾丸中存在更多的THY1 + 体细胞,所以将该方案应用于从成年小鼠中分离THY1 + 未分化精原细胞可能存在缺点(久保田和布林斯特,2008)。
  6. 为了进行有效的细胞分离,MS柱的典型细胞数量应该小于每列5×10 7个睾丸细胞,LS柱的细胞数应小于5×10 7 sup> 8 睾丸细胞。
  7. 死细胞通常小于通过台盼蓝染色确定的总分离的THY1 + 细胞的10%(图1C)。在4℃下以300×g可选离心10分钟可有效去除死细胞。
  8. 通过流式细胞术分析磁性分离的THY1 + 细胞。使用PE-Cy5缀合的抗CD49f抗体和同种型抗体进行该分析。重新悬浮THY1 + 细胞,并在黑色染色缓冲液中在冰上孵育20分钟。用FACS缓冲液洗涤两次并通过35μm尼龙细胞过滤器过滤后,通过Beckman Coulter FC500 Cytometer分析PE-Cy5缀合的细胞(图1D)。
  9. 对于免疫细胞化学分析,将分离的THY1 + 细胞置于聚-L-赖氨酸包被的载玻片(Thermo Scientific)上,并在室温下固定在4%PFA中10分钟。在室温下用PBST洗涤5次10分钟后,将细胞用2%BSA和5%驴血清在室温下封闭2小时,并在4℃下孵育过夜,随后进行一次抗体孵育:抗-PLZF抗体或抗VASA抗体。细胞用含有1%BSA的PBST洗涤三次。在室温下与二次抗体孵育1小时后,将载玻片在PBST中洗涤三次,用Hoechst 33342重新染色,并安装有载置介质。通过Leica TCS SP5 II共焦显微镜分析载玻片(图1E-1M)。

食谱

  1. E-HBSS
    2mM EDTA在HBSS中,无钙,无镁,无酚红
  2. MACS缓冲区
    2mM EDTA,DPBS中0.5%BSA
  3. 胶原酶溶液
    1mg/ml IV型胶原酶和5U/ml DNase I在HBSS中
  4. 胰蛋白酶溶液
    5 U/ml DNase I在0.25%胰蛋白酶-EDTA,苯酚红色
  5. 新鲜准备的F-MACS缓冲区
    MACS缓冲区中的10%FBS
    注意:将F-MACS放在冰上,然后将F-MACS缓冲液预热至室温(〜25°C),然后再使用。
  6. 染色缓冲液
    1x DPBS
    2 mM EDTA
    2%热灭活FBS
    PE-Cy5缀合的抗体(1:1000稀释)
  7. FACS缓冲区
    1x DPBS
    2 mM EDTA
  8. PBST
    1x DPBS
    0.2%Tween-20

致谢

作者衷心感谢李培龙先生,陈婉娴小姐及陈奕臣小姐进行有意见的讨论。这项工作得到台湾科技部(MOST 103-2321-B-002-099和MOST 104-2321-B-002-043)和台湾大学(NTU-105R4000)的资助。

参考文献

  1. Boivin,J.,Bunting,L.,Collins,JA和Nygren,KG(2007)。  国际对不孕不育流行率和待遇要求的估计:对不孕不育医疗的潜在需求和需求。 um Reprod 22(6):1506-1512。
  2. Buaas,FW,Kirsh,AL,Sharma,M.,McLean,DJ,Morris,JL,Griswold,MD,de Rooij,DG和Braun,RE(2004)。< a class ="ke-insertfile"href = "http://www.nature.com/ng/journal/v36/n6/abs/ng1366.html"target ="_ blank">成年雄性生殖细胞需要Plzf进行干细胞自我更新。 Nat Genet 36(6):647-652。
  3. Buageaw,A.,Sukhwani,M.,Ben-Yehudah,A.,Ehmcke,J.,Rawe,VY,Pholpramool,C.,Orwig,KE和Schlatt,S。(2005)。< a class =在未成熟小鼠睾丸中的精原细胞干细胞的GDNF家族受体α1表型的ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/16014811" <生物复制 73(5):1011-1016。
  4. Chan,F.,Oatley,MJ,Kaucher,AV,Yang,QE,Bieberich,CJ,Shashikant,CS and Oatley,JM(2014)。  小鼠睾丸中Id4 + 种系干细胞群体的功能和分子特征。 Genes Dev 28(12):1351-1362。
  5. Costoya,JA,Hobbs,RM,Barna,M.,Cattoretti,G.,Manova,K.,Sukhwani,M.,Orwig,KE,Wolgemuth,DJ和Pandolfi,PP(2004)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15156143"target ="_ blank"> Plzf在维持精原干细胞中的重要作用。 Genet 36(6):653-659。
  6. Gassei,K.和Orwig,KE(2013)。  SALL4表达在产后小鼠睾丸的细胞和精原细胞克隆中。 PLoS One 8(1):e53976。
  7. Hermann,BP,Mutoji,KN,Velte,EK,Ko,D.,Oatley,JM,Geyer,CB和McCarrey,JR(2015)。< a class ="ke-insertfile"href ="http: www.ncbi.nlm.nih.gov/pubmed/25568304"target ="_ blank">新生儿小鼠精原细胞的转录和翻译异质性。 92(2):54. < br />
  8. Kanatsu-Shinohara,M.,Inoue,K.,Ogonuki,N.,Morimoto,H.,Ogura,A.and Shinohara,T。(2011)。< a class ="ke-insertfile"href ="http ://www.ncbi.nlm.nih.gov/pubmed/20844279"target ="_ blank">小鼠种系干细胞的无血清和无饲养细胞的培养物。生物再生培养 84 (1):97-105。
  9. Kubota,H.,Avarbock,MR和Brinster,RL(2003)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/12738887"target = "_blank">精原干细胞与其他干细胞共享一些但不是全部的表型和功能特征。美国国家科学院院刊100(11):6487-6492。 />
  10. Kubota,H.,Avarbock,MR和Brinster,RL(2004a)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15115718"target = "_blank">文化条件和单一生长因子影响小鼠精原干细胞的命运测定。生物学报71 /(71):722-731。
  11. Kubota,H.,Avarbock,MR和Brinster,RL(2004b)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15520394"target = "_blank">小鼠精原干细胞的自我更新和扩增所必需的生长因子。 Proc Natl Acad Sci USA 101(47):16489-16494。
  12. Kubota,H。和Brinster,RL(2008)。  啮齿动物精原干细胞的培养,出生后动物的雄性生殖系干细胞。方法细胞周期 86:59-84。
  13. 廖,高,陈,WS,陈,YH,高,TH,Tseng,YT,Lee,CY,Chiu,YC,Lee,PL,Lin,QJ,Ching,YH,Hata,K.,Cheng,WT,Tsai ,MH,Sasaki,H.,Ho,HN,Wu,SC,Huang,YH,Yen,P。和Lin,SP(2014)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/24850856"target ="_ blank"> DNMT3L在产后精原细胞祖细胞中促进静止。发展 141(12):2402-2413。
  14. Matzuk,MM and Lamb,DJ(2008)。  不孕不育生物学:研究进展和临床挑战。 Nat Med 14(11):1197-1213。
  15. Oatley,J.M。和Brinster,R.L。(2008)。 哺乳动物中精原干细胞自我更新的调节。  Annu Rev Cell Dev Biol 24:263-286。
  16. Rege,TA和Hagood,JS(2006)。  Thy-1作为轴突再生,细胞凋亡,粘附,迁移,癌症和纤维化中细胞和细胞 - 基质相互作用的调节因子。 FASEB J 20(8):1045 -1054。
  17. Sada,A.,Suzuki,A.,Suzuki,H。和Saga,Y。(2009)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/19745153"target ="_ blank"> RNA结合蛋白NANOS2是维持鼠精原干细胞所必需的。 325(5946):1394-1398。 >
  18. Tseng,YT,Liao,HF,Yu,CY,Mo,CF and Lin,SP(2015)。  繁殖和精原干细胞调节中的表观遗传因素。生殖 150(3):R77-91。
  19. Wabik,A. and Jones,PH(2015)。  切换角色:成人组织干细胞的功能可塑性。 EMBO J 34(9):1164-1179。
  20. Xin,T.,Greco,V.和Myung,P。(2016)。  通过组织结构进行硬连线干细胞通讯。 细胞 164(6):1212-1225。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liao, H., Kuo, J., Lin, H. and Lin, S. (2016). Isolation of THY1+ Undifferentiated Spermatogonia from Mouse Postnatal Testes Using Magnetic-activated Cell Sorting (MACS). Bio-protocol 6(24): e2072. DOI: 10.21769/BioProtoc.2072.
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