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Aug 2021
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Assessing the Presence of Hematopoietic Stem and Progenitor Cells in Mouse Spleen
评估小鼠脾脏中造血干细胞和祖细胞的存在   

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Abstract

Transplantation of hematopoietic material into recipient mice is an assay routinely used to determine the presence and function of hematopoietic stem and progenitor cells (HSPCs) in vivo. The principle of the method is to transplant donor cells being tested for HSPCs into a recipient mouse following bone marrow ablation and testing for reconstitution of hematopoiesis. Congenic mouse strains where donor and recipient differ by a distinct cell surface antigen (commonly CD45.1 versus CD45.2) are used to distinguish between cells derived from the donor and any residual recipient cells. Typically, the transplantation is performed using bone marrow cells, which are enriched for HSPCs. Here, we describe an analogous procedure using hematopoietic material from spleen, allowing detection of functional progenitors and/or stem cells in the spleen that can occur under certain pathologies. Key to the success of this procedure is the prior removal of mature T cells from the donor sample, to minimize graft versus host reactions. As such, this protocol is highly analogous to standard bone marrow transplant procedures, differing mainly only in the source of stem cells (spleen rather than bone marrow) and the recommendation for T cell depletion to avoid potential immune incompatibilities.


Graphical abstract:



Schematic overview for assessment of stem cells in spleen by transplantation.
Single cell suspensions from spleens are depleted of potentially pathogenic mature T lymphocytes by magnetic bead immunoselection using biotinylated antibodies against CD4 and CD8, followed by streptavidin magnetic beads, which are subsequently removed by using a magnet (MojoSort, Biolegend). Successful T cell depletion is then evaluated by Fluorescence Activated Cell Sorting (FACS). T-cell depleted cell suspension is injected intravenously through the retro-orbital sinus into lethally irradiated recipients. Recipients are analyzed for successful engraftment by FACS analysis for the presence of donor-derived mature hematopoietic lineages in the peripheral blood. A second serial transplantation can be used to document the presence of long-term reconstituting stem cells in the periphery of the original donor mice.


Keywords: Spleen transplantation (脾移植), Hematopoiesis (造血), Hematopoietic stem cells (造血干细胞), Hematopoietic progenitor cells (造血祖细胞), T cell depletion (T 细胞耗竭), Inflammatory disease (炎症性疾病)

Background

The hematopoietic system of mice can be regenerated from hematopoietic stem and progenitor cells (HSPCs). In adult mice, HSPCs largely reside in the bone marrow, where they are maintained in a largely quiescent state. However, during some inflammatory pathologies, HSPCs become mobilized and can colonize various peripheral lymphoid organs, such as spleen. Definitive assessment of the presence and function of HSPCs is accomplished by observing their ability to reconstitute the full hematopoietic system of an irradiated recipient mouse (so the HSPCs of the recipient mouse are depleted) following transplantation. Committed progenitors will give rise to only a subset of hematopoietic lineages in this assay, while true hematopoietic stem cells (HSCs) will be capable of reconstituting all mature blood lineages. Stem cells with long-term reconstitution ability can be distinguished from short-term stem cells by secondary transplantation. While short-term HSCs are capable of reconstituting hematopoiesis in an initial transplant, only long-term HSCs will give rise to pluripotent stem cells that are capable of reconstituting a secondary recipient.


The present protocol, adapted from a recent publication (Marié et al., 2021), describes a method for assessing the presence of HSPCs in mouse spleens by serial engraftment in recipient mice whose hematopoietic system has been ablated by x-ray irradiation. Antigenic differences between donor and recipients allow the detection of donor cells following engraftment by a simple flow cytometry analysis. Flow cytometry is also used to assess the presence of major hematopoietic lineages derived from donor cells through analysis of characteristic cell surface markers. Following primary reconstitution, the presence of short-term versus long-term HSPCs can be assessed by conducting a secondary engraftment and observing the reconstitution of hematopoietic lineages in the secondary recipients. Moreover, the ability of the donor HSPCs to home to the bone marrow versus secondary lymphoid organs can be assessed by performing secondary reconstitutions with cells derived from either bone marrow or spleen. In all transplantations involving cells from peripheral organs, it is essential to deplete mature T lymphocytes to minimize graft versus host reactions. A potential limitation of this technique is that its sensitivity has not yet been strictly defined. At present, the minimum threshold of stem cell abundance in the spleen capable of triggering a successful transplant needs to be defined. This limitation could be addressed by including serial dilution studies.

Materials and Reagents

  1. 35 mm culture dish (Falcon, catalog number: 3001)

  2. 3 mL syringe (BD Biosciences, catalog number: 305270)

  3. 1 mL syringes (BD, catalog number: 320933)

  4. 15 mL conical tubes (BD Biosciences, Falcon®, catalog number: 352196)

  5. 50 mL conical tubes (BD Biosciences, Falcon®, catalog number: 430829)

  6. 70 μM nylon strainers (BD Biosciences, Falcon®, catalog number: 352350)

  7. Eppendorf tubes 1.5 mL (Axygen®, catalog number: MCT-175-C)

  8. FACS tubes 5 mL (BD Biosciences, catalog number: 352054)

  9. C57BL6 (CD45.2) donor mice (bred in our laboratory by mating a male and a female both CD45.2)

  10. C57BL6 (CD45.1) recipient mice (bred in our laboratory by mating a male and a female both CD45.1)

  11. Antibiotic suspension of Sulfamethoxazole (40 mg/mL) and Trimethoprim (8 mg/mL) in 0.03% ethanol, Ani Pharmaceuticals

  12. Phosphate Buffered Saline (PBS) (without Ca2+ and Mg2+) (Sigma-Aldrich, catalog number: D8537)

  13. BV711 mouse anti-mouse CD45.1 (cloneA20) (Biolegend, catalog number: 110739) 0.2 mg/mL

  14. BV605 mouse anti-mouse CD45.2 (clone104) (Biolegend, catalog number: 109841) 0.2 mg/mL

  15. Biotin rat anti-mouse CD4 (clone RM4-5) (Biolegend, catalog number:100508) 0.5 mg/mL

  16. Biotin rat anti-mouse CD8a (clone 53-6.7) (Biolegend, catalog number: 100704) 0.5 mg/mL

  17. PE Cy5 rat anti-mouse CD4 (clone RM4-5) (Biolegend, catalog number: 100514) 0.2 mg/mL

  18. APC rat anti-mouse CD8a (clone 53-6.7) (Biolegend, catalog number: 100712) 0.2 mg/mL

  19. PE Cy7 rat anti-mouse CD11b (M1/70) (Biolegend, catalog number: 101215) 0.2 mg/mL

  20. APC-eFluor 780 rat anti-mouse B220 (RA3-6B2) (eBiosciences, catalog number: 47-0452-82) 0.2 mg/mL

  21. Fetal Bovine Serum (FBS) (Gibco, catalog number: 10437-028)

  22. Red Blood Cell (RBC) lysing buffer (BD PharmLyseTM lysing buffer, BD Biosciences, catalog number: 555899)

  23. MojoSortTM Buffer (Biolegend, catalog number: 480017)

  24. DMEM medium (Corning, Cellgro, catalog number: 10-013-CV)

  25. MojoSortTM Streptavidin nanobeads (Biolegend, catalog number: 480016)

  26. Needles (27G1/2) (BD Biosciences, catalog number: 305109)

  27. Goldenrod animal lancet 5mm (Medipoint)

  28. BD EDTA-coated Microtainer (BD Biosciences, catalog number: 365974)

  29. FACS buffer (see Recipes)

Equipment

  1. Forceps and sharp scissors

  2. Inverted microscope

  3. Ice bucket

  4. Hemacytometer (Sigma-Aldrich, catalog number: Z359629-1EA)

  5. Centrifuge (low-speed for 15 and 50 mL tubes)

  6. Centrifuge for microcentrifuge tubes (1.5 mL)

  7. MojoSort Magnet (Biolegend, catalog number: 480019), cooled at 4°C the day before

  8. Mice Irradiation machine (Faxitron MR350 X-ray irradiator)

  9. BD LSR II flow cytometer

Software

  1. FACSDiva software

  2. FlowJo software

Procedure

Part I: Transplantation of spleen cells


The following protocol has been adapted from the more traditional bone marrow transplantation protocol (Duran-Struuck and Dysko, 2009). Bone marrow transplantation is routinely used to assess the presence and efficiency of progenitors/stem cells in a given bone marrow sample. While normally virtually absent in the spleen, HSPCs may accumulate in the spleen during some pathological processes, for example, during inflammation, splenomegaly, or extramedullary hematopoiesis. Our protocol aims at detecting the presence of functional progenitors and/or stem cells in the spleen.

It is important to note that spleens contain more reactive T cells than bone marrow, which can lead to adverse effects during engraftment, such as graft versus host disease (Baker et al., 1996). To avoid such potential problems, mature T cells are depleted before transplantation.

  1. Irradiation of host mice

    The hematopoietic system of recipient mice is ablated by lethal irradiation applied in two fractional doses separated by 4 h.

    1. Irradiate C57BL6 (CD45.1) 8- to 12-week-old host mice, 20 to 25 g, sex irrelevant (10 mice per transplantation group) with a first dose of 4.5 Gy total body irradiation (TBI), (Faxitron MR350 X-ray irradiator using SnCuAl filter) using a mouse irradiation pie cage (Figure 1). Following irradiation, supplement the drinking water with 1:200 dilution of antibiotic suspension of Sulfamethoxazole and Trimethoprim (Materials and Reagents #11).

    2. Irradiate host mice with a second dose of 4.5 Gy 4 h later, just before transplantation. In the meantime, proceed to the preparation of donor material.



    Figure 1. Mouse irradiation pie cage.

    Mice are placed in an irradiation pie cage prior to exposure to x-ray irradiation.


  2. Preparation of donor material

    The whole procedure has to be carried out under sterile conditions
    1. Sacrifice C57BL6 donor mice (CD45.2) by CO2 euthanasia. Collect the spleen (Figure 2A) and place in PBS on ice. Trim away any remaining connective tissue or fat from the spleen.



      Figure 2. Mouse bleeding, injection, and spleen recovery procedures.

      (A) Illustration of post-mortem spleen collection. (B) Fluid injection into the retro-orbital sinus. (C) Puncturing the submandibular vein with a lance. (D) Blood collection from a pierced submandibular vein.


    2. Transfer the spleen into a sterile 35 mm culture dish containing 5 mL of FACS buffer (see Recipes below) or DMEM medium containing 2% FBS.

    3. Remove the plunger from a 3 mL syringe. Use the flat rubber end of the plunger to crush the spleen by using gentle circular motions. This will disrupt the pulp and release the splenocytes.

    4. Pass the 5 mL of cell suspension through a 70 μm nylon strainer fitted on top of a 50 mL conical tube to obtain a uniform single-cell suspension. Gently help the suspension pass through the strainer by pressing in a circular manner with the syringe plunger. A video presentation of this method can be seen at https://www.stemcell.com/prepare-single-cell-suspension-from-mouse-spleen.html.

    5. Wash the 35 mm culture dish with 2 mL of FACS buffer or DMEM medium containing 2% FBS

      and pass through the strainer. Repeat this step twice.

    6. Centrifuge the tube at 300 × g for 10 min at 4°C.

    7. Discard the supernatant and flick the tube to loosen the pellet. Resuspend the pellet in a volume of 200 μL to 1 mL of medium containing 3% FBS, depending on the spleen size, and store on ice.

    8. Count the cells after diluting an aliquot of the cell suspension 1:100 with PBS. Count all nine squares of the hemacytometer under an inverted light microscope. One normal spleen (approximately 100 mg) typically produces around 100 million cells. We recommend using a live dye such as trypan blue (1:1 v/v) to exclude potential dead cells (Figure 3).

      Note: Since the cell suspension has not undergone red blood cell lysis, it is important to make sure that the red blood cells (smaller than the white blood cells) are not counted (Figure 3).

    9. The cell count per mL is the number of cells counted × dilution factor × 104 (the dilution factor being 100 in this case).



      Figure 3. Peripheral blood cell counting with a hematocytometer.

      Viable leukocytes are counted on a hematocytometer grid. Red arrow points to a red blood cell, and black arrow points to a trypan blue-stained dead cell, neither of which are included in the leukocyte count.


    T cell depletion
    1. Transfer 108 cells resuspended in 500 μL of MojoSort Buffer into a 5 mL (12 × 75 mm) polystyrene tube. Add 5 μL each of biotin-anti CD4 and biotin-anti CD8a to the tube, mix well, and incubate on ice for 15 min.

      Note: Keep a small aliquot (106 cells) of starting material prior to depletion (step B7) on ice, to be used from step 20 onward, to assess the extent of the depletion after the procedure.

    2. Wash the cells by adding 3.5 mL of MojoSort buffer and centrifuge at 300 × g for 5 min at 4°C.

    3. Discard the supernatant and resuspend in 500 μL of MojoSort Buffer.

    4. Resuspend the Streptavidin Nanobeads by vortexing, at maximum speed, five touches. Add 50 μL of beads to the cell suspension, mix well, and incubate for 15 min on ice.

    5. Wash the cells by adding 3 mL of MojoSort Buffer; Centrifuge at 300 × g for 5 min at 4°C, discard the supernatant and resuspend in 3 mL of MojoSort Buffer.

    6. Place the tube in a cold MojoSort magnet (see Equipment) for 5 min.

    7. Collect the cells by pouring out the liquid into a 15 mL conical tube. These are your cells of interest, depleted of T cells that are retained on the magnet.

    8. To increase yield if needed, add 3 mL of MojoSort Buffer to the beads, repeat steps 16 and 17, and pool the flow-through fractions.

    9. Count the cells obtained after depletion as indicated in steps B8 to B10.


    Assessment of T cell depletion by FACS
    1. Take an aliquot containing 100,000 to 500,000 cells from the sample set aside before T cell depletion (step B7) and from the T cell-depleted sample (step B18). Set up three additional tubes, one for unstained control and two for single color controls containing each 50,000 to 100,000 cells per tube.

    2. Centrifuge the pre- and post- depletion samples, as well as the two single color controls at 200 × g for 5 min at 4°C. Resuspend the pre- and post- depletion samples in 50 μL of staining solution containing PeCy5 anti-mouse CD4 (diluted 1:250) and APC anti-mouse CD8a (diluted 1:250) in FACS buffer.

    3. Resuspend one single color control in 50 μL of PeCy5 anti-mouse CD4 (diluted 1:250) and the other single color control in 50 μL of APC anti-mouse CD8a (diluted 1:250).

    4. Incubate on ice for 15 to 30 min, protected from light.

    5. Wash all tubes with 300 μL of FACS buffer by centrifugation.

    6. Resuspend in 200 μL of FACS buffer and transfer to FACS tubes by passing through a mesh filter to remove any clumps of cells.

    7. Analyze all tubes on a flow cytometer.

    8. On the BD LSR II software, select 'New Experiment'.

    9. Select for Area, Height, and Width for FSC and SSC; select for Log and Area for the colors Pe-Cy5 and APC.

    10. Create compensation controls and adjust the gating for unstained blood cells (adjust FSC and SSC voltages as necessary).

    11. Adjust each color of single stains in the voltage panel so that the positive peak is at the 104 mark.

    12. Analyze single color controls.

    13. Record the desired voltages after any adjustments for each of the single stains and then calculate compensation controls.

    14. Events are now ready to be recorded. Set up to collect 5,000–10,000 events per sample for each pre- and post-depletion sample.

    Note: Typically, the procedure depletes over 95% of CD4 and CD8 T cells. The overall depletion procedure is illustrated in the following video: https://www.youtube.com/watch?v=cH6rlIFNVp0.


  3. Transplantation into irradiated mice

    1. Centrifuge the cell suspension of T cell-depleted splenocytes for 5 min at 200 × g at 4°C, remove supernatant, and resuspend in sterile PBS containing 2% of FBS at a cell concentration of 2 × 106 per 100 μL. Keep on ice.

    2. Set up an isoflurane anesthesia machine.

    3. Place an irradiated mouse within the anesthesia chamber.

    4. Turn on the oxygen to flow at 1–2 L/min and the isoflurane gauge to 2–5%.

    5. The animal should become anesthetized within a few minutes.

    6. Load 100 μL of spleen cell suspension into a 1 mL syringe fitted with a 27 G needle.

    7. When the mouse is anesthetized, remove it from the chamber. Promptly, place the animal on absorbent paper, head tilted sideways. With one hand, retract the fur around the eye to make it protrude a little. With the other hand, insert the needle at a 45° angle in the inner corner of the eye to penetrate the retro-orbital sinus (Yardeni et al., 2011). See Figure 2B.

    8. Slowly and smoothly inject the 100 μL of spleen cell suspension (approx. 5 s) and return the mouse to the cage as soon as it starts waking up.

    9. Repeat the procedure for each irradiated animal to be transplanted.

    10. Following injection, the recipient mice are left to recover for 4–6 weeks. Antibiotics can be removed from drinking water after two weeks. Monitor the viability of the transplanted mice every day. The presence of adequate stem cells in the test sample being evaluated should result in greater than 80% recipient survival.

    11. Remember to have proper irradiation controls. These mice are comparable to the ones used for the transplant (same age and sex) that will be irradiated with the experimental mice, but they will not be transplanted. They should become moribund after 10–14 days, at which point they are euthanized.



Part II: Blood reconstitution analysis

First analysis of total blood donor reconstitution is done between 4–6 weeks following cell transplantation. This analysis aims to determine whether donor cells have engrafted by assessing the presence of CD45.2 cells (from the donor) in the recipient blood.


  1. Obtaining total white blood cells for flow cytometry

    1. Blood collection is performed by puncture of the facial vein that runs from the submandibular vein across the cheek (Regan et al., 2016). See Figure 2C and 2D.

    2. Hold the mouse firmly by the scruff in a vertical position and puncture skin at the rear of the jawbone, at the intersection of two virtual lines, one originating at the external corner of the eye and the other one at the hairless spot close to the mouth, with a Goldenrod animal lancet.

    3. Collect blood into an EDTA-coated BD microtainer; 200 µL is sufficient (approximately four drops).

    4. Keep doing the same for all the recipient mice of the experiment. In addition, bleed a non-transplanted CD45.2 mouse and a non-transplanted CD45.1 mouse to perform the controls for flow cytometry.

    5. Transfer blood samples into 15 mL conical tubes. Perform red blood cell lysis by adding 1mL of BD PharmLyse lysing solution diluted 1:10 in water. Gently vortex each tube and incubate on ice, protected from light, for 15 min.

    6. Centrifuge the tubes at 200 × g for 5 min at 4°C. Carefully aspirate the supernatant without disturbing the pellet (which should appear almost white) and add 2 mL of 1× PBS containing 1% heat-inactivated FBS and 0.1% sodium azide.


  2. Labeling cells for flow cytometry

    1. Centrifuge the tubes at 200 × g for 5 min at 4°C. Carefully aspirate the supernatant without disturbing the pellet and resuspend in 50 µL of staining solution made of a mix of BV711 anti-mouse CD45.1 antibody (diluted 1:200) and BV605 anti-mouse CD45.2 antibody (diluted 1:200) in FACS buffer.

    2. Prepare two samples for single color controls by adding 50 µL of BV711 anti-mouse CD45.1 antibody (diluted 1:200) in the CD45.1 blood sample and 50 µL of BV605 anti-mouse CD45.2 antibody (diluted 1:200) in the CD45.2 blood and retain one sample as unstained.

    3. Incubate on ice for 15 to 30 min, protected from light.

    4. Wash with 300 µL of FACS buffer. Centrifuge the cells, 200 × g, 5 min at 4°C. Aspirate the supernatant.

    5. Resuspend in 200 µL of FACS buffer and transfer to FACS tubes through a mesh filter to remove clumps of cells.

    6. Analyze on Flow cytometer as previously described (steps B27 to B33).


Part III: Long-term engraftment data analysis

This analysis aims at determining whether the engrafted transplanted cells gave rise to all lineages of white blood cells and is performed at least 4 months following transplantation.

  1. Bleed animals and perform red blood cell lysis as previously described (steps A1 to A6).

  2. Stain blood with a staining mix composed of PE-Cy5 anti-mouse CD4 (diluted 1:200), APC anti-mouse CD8a (diluted 1:200), PE-Cy7 anti-mouse CD11b (diluted 1:500), APC-eFluor 780 anti-mouse B220 (diluted 1:200), and BV605 anti-mouse CD45.2 (diluted 1:200) in FACS buffer.

  3. Proceed as detailed in part II-B.

    This analysis will determine the percentage of CD4 and CD8 T cells, B cells, and myeloid cells originating from the CD45.2-positive transplanted cells. The presence of all major lineages indicates the presence of HSPCs in the donor material, while the presence of only myeloid cells of donor origin indicates that only committed progenitors were present in the experimental spleen.


    To firmly assert that multipotent stem cells were present in the donor spleen, we recommend performing a secondary transplant using the spleen and/or bone marrow of the primary recipients and recapitulating the entire procedure. The presence of blood cell lineages from donor cells in recipients following secondary transplant provides definitive evidence for the presence of totipotent hematopoietic stem cells in the spleens of the primary animals. Depending on the experimental system being studied, the long-term HSCs engrafted during the primary transplant may reside exclusively in the bone marrow or may have also been homed to peripheral organs, which can be evaluated by performing secondary engraftments using either bone marrow or spleen cells.

      It will be of interest to apply this protocol to additional secondary lymphoid organs other than spleen. In particular, this technique could be applied to measure the potential presence of hematopoietic stem cells in lymph nodes or other tissues with abundant hematopoietic cells, such as the small and large intestine.

Recipes

  1. FACS buffer

    Bovine Serum Albumin (BSA) 0.5% (w/v)

    1 mM EDTA

    0.05% sodium azide (w/v)

    Dissolved in PBS

    Filter sterilize using a 0.2 μm filter

Acknowledgments

The protocol was adapted from the previously published paper: Marié et al. (2021). This work was supported in part by National Institutes of Health grants R01AI28900 and Lupus Research Alliance grant 579817 to DEL, and by the Laura and Isaac Perlmutter Comprehensive Cancer Center support grant P30CA016087 from the National Cancer Institute.

Competing interests

The authors declare no competing interests.

Ethics

All work in the development of this protocol was approved by the Institutional Animal Care and Use Committee of NYU Grossman School of Medicine.

References

  1. Baker, M. B., Altman, N. H., Podack, E. R. and Levy, R. B. (1996). The role of cell-mediated cytotoxicity in acute GVHD after MHC-matched allogeneic bone marrow transplantation in mice. J Exp Med 183(6): 2645-2656.
  2. Duran-Struuck, R. and Dysko, R. C. (2009). Principles of bone marrow transplantation (BMT): providing optimal veterinary and husbandry care to irradiated mice in BMT studies. J Am Assoc Lab Anim Sci 48(1): 11-22.
  3. Marié, I. J., Brambilla, L., Azzouz, D., Chen, Z., Baracho, G. V., Arnett, A., Li, H. S., Liu, W., Cimmino, L., Chattopadhyay, P., et al. (2021). Tonic interferon restricts pathogenic IL-17-driven inflammatory disease via balancing the microbiome. Elife 10: e68371.
  4. Regan, R. D., Fenyk-Melody, J. E., Tran, S. M., Chen, G. and Stocking, K. L. (2016). Comparison of Submental Blood Collection with the Retroorbital and Submandibular Methods in Mice (Mus musculus). J Am Assoc Lab Anim Sci 55(5): 570-576.
  5. Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M. and Hoogstraten-Miller, S. (2011). Retro-orbital injections in mice. Lab Anim (NY) 40(5): 155-160.

简介

[摘要] 将造血材料移植到受体小鼠体内是一种常规用于确定体内造血干细胞和祖细胞 (HSPC) 的存在和功能的检测方法。该方法的原理是在骨髓消融和造血重建测试后,将接受 HSPC 测试的供体细胞移植到受体小鼠中。供体和受体因不同的细胞表面抗原(通常是 CD45.1 与 CD45.2)而不同的同类小鼠品系用于区分来自供体的细胞和任何残留的受体细胞。通常,移植是使用富含 HSPC 的骨髓细胞进行的。在这里,我们描述了使用来自脾脏的造血材料的类似程序,允许检测脾脏中可能在某些病理下发生的功能性祖细胞和/或干细胞。该程序成功的关键是事先从供体样本中去除成熟的 T 细胞,以尽量减少移植物与宿主的反应。因此,该协议与标准的骨髓移植程序高度相似,主要区别在于干细胞的来源(脾脏而不是骨髓)和建议去除 T 细胞以避免潜在的免疫不相容性。

图形概要:

通过移植评估脾脏中干细胞的示意图。
来自脾脏的单细胞悬液通过磁珠免疫选择去除潜在致病性成熟 T 淋巴细胞,使用针对 CD4 和 CD8 的生物素化抗体,然后是链霉亲和素磁珠,随后使用磁铁将其去除( MojoSort , Biolegend )。然后通过荧光激活细胞分选 (FACS) 评估成功的 T 细胞消耗。将 T 细胞耗尽的细胞悬液通过眶后窦静脉注射到受致命照射的受体中。通过 FACS 分析外周血中是否存在来自供体的成熟造血谱系,分析受体的成功植入。第二次连续移植可用于记录原始供体小鼠周围长期重组干细胞的存在。


[背景] 小鼠的造血系统可以从造血干细胞和祖细胞(HSPCs)再生。在成年小鼠中,HSPCs 主要存在于骨髓中,在那里它们基本上处于静止状态。然而,在一些炎症病理过程中,HSPC 会被调动起来,并可以定植各种外周淋巴器官,例如脾脏。 HSPCs 的存在和功能的最终评估是通过观察它们在移植后重建受照射受体小鼠的完整造血系统的能力(因此受体小鼠的 HSPCs 被耗尽)来完成的。在该测定中,承诺的祖细胞将仅产生一部分造血谱系,而真正的造血干细胞 (HSC) 将能够重建所有成熟的血液谱系。具有长期重建能力的干细胞可以通过二次移植与短期干细胞区分开来。虽然短期 HSC 能够在初始移植中重建造血功能,但只有长期 HSC 才能产生能够重建二级受体的多能干细胞。
本议定书改编自最近的出版物(玛丽 et al ., 2021 ) 描述了一种通过在造血系统已被 X 射线照射消融的受体小鼠中连续植入来评估小鼠脾脏中 HSPC 存在的方法。供体和受体之间的抗原差异允许通过简单的流式细胞术分析检测移植后的供体细胞。流式细胞术还用于通过分析特征性细胞表面标志物来评估源自供体细胞的主要造血谱系的存在。在初次重建后,可以通过进行二次植入并观察二次受体中造血谱系的重建来评估短期与长期 HSPC 的存在。此外,可以通过对源自骨髓或脾脏的细胞进行二次重组来评估供体 HSPCs 归巢骨髓与次级淋巴器官的能力。在涉及来自外周器官的细胞的所有移植中,必须消耗成熟的 T 淋巴细胞以尽量减少移植物抗宿主反应。该技术的一个潜在限制是其敏感性尚未得到严格定义。目前,需要确定能够触发成功移植的脾脏中干细胞丰度的最低阈值。这种限制可以通过包括连续稀释研究来解决。

关键字:脾移植, 造血, 造血干细胞, 造血祖细胞, T 细胞耗竭, 炎症性疾病



材料和试剂


1. 35 mm培养皿(Falcon,目录号:3001)
2. 3 mL注射器(BD Biosciences,目录号:305270)
3. 1 mL注射器(BD,目录号:320933)
4. 15 mL锥形管(BD Biosciences,Falcon ® ,目录号:352196)
5. 50 mL锥形管(BD Biosciences,Falcon ® ,目录号:430829)
6. 70μM尼龙过滤器(BD Biosciences,Falcon ® ,目录号:352350 )
7. Eppendorf管1.5 mL( Axygen ® ,目录号:MCT-175-C)
8. FACS管5 mL(BD Biosciences,目录号:352054)
9. C57BL6 (CD45.2) 供体小鼠(在我们实验室通过将 CD45.2 的雄性和雌性交配而繁殖)
10. C57BL6 (CD45.1) 受体小鼠(在我们实验室通过将 CD45.1 的雄性和雌性交配而繁殖)
11. 磺胺甲恶唑 (40 mg/mL) 和甲氧苄啶 (8 mg/mL) 在 0.03% 乙醇中的抗生素悬浮液,Ani Pharmaceuticals
12. 磷酸盐缓冲盐水(PBS)(不含Ca 2+和Mg 2+ )(Sigma-Aldrich,目录号:D8537)
13. BV711小鼠抗小鼠CD45.1(cloneA20)( Biolegend ,目录号:110739)0.2 mg/mL
14. BV605小鼠抗小鼠CD45.2(clone104)( Biolegend ,目录号:109841)0.2 mg/mL
15. 生物素大鼠抗小鼠CD4(克隆RM4-5)( Biolegend ,目录号:100508)0.5 mg/mL
16. 生物素大鼠抗小鼠CD8a(克隆53-6.7)( Biolegend ,目录号:100704)0.5 mg/mL
17. PE Cy5大鼠抗小鼠CD4(克隆RM4-5)( Biolegend ,目录号:100514)0.2 mg/mL
18. APC大鼠抗小鼠CD8a(克隆53-6.7)( Biolegend ,目录号:100712)0.2 mg/mL
19. PE Cy7大鼠抗小鼠CD11b(M1 / 70)( Biolegend ,目录号:101215)0.2 mg / mL
20. APC - eFluor 780 大鼠抗小鼠 B220(RA3-6B2)( eBiosciences ,目录号:47-0452-82)0.2 mg/mL
21. 胎牛血清(FBS)(Gibco,目录号:10437-028)
22. 红细胞 (RBC) 裂解缓冲液 (BD PharmLyse TM 裂解缓冲液,BD Biosciences,目录号:555899)
23. MojoSort TM Buffer( Biolegend ,目录号:480017)
24. DMEM 培养基(Corning, Cellgro ,目录号:10-013-CV)
25. MojoSort TM链霉亲和素纳米珠( Biolegend ,目录号:480016)
26. 针头(27G1/2)(BD Biosciences,目录号:305109)
27. Goldenrod 动物柳叶刀 5mm ( Medipoint )
28. BD EDTA 涂层微量容器(BD Biosciences,目录号:365974)
29. FACS 缓冲液(见配方)




设备


1. 镊子和锋利的剪刀
2. 倒置显微镜
3. 冰桶
4. 血细胞计数器(Sigma-Aldrich,目录号:Z359629-1EA)
5. 离心机(用于 15 和 50 mL 管的低速离心机)
6. 用于微量离心管 (1.5 mL) 的离心机
7. MojoSort Magnet( Biolegend ,目录号:480019),前一天冷却至4 °C
8. 小鼠辐照机( Faxitron MR350 X 射线辐照器)
9. BD LSR II 流式细胞仪


软件


1. FACSDiva软件
2. FlowJo软件


程序


第一部分:脾细胞移植


以下方案改编自更传统的骨髓移植方案( Duran -Struuck和Dysko ,2009 年)。骨髓移植通常用于评估给定骨髓样本中祖细胞/干细胞的存在和效率。虽然通常脾脏中几乎不存在 HSPC,但在某些病理过程中,例如,在炎症、脾肿大或髓外造血过程中,HSPC 可能会在脾脏中积聚。我们的方案旨在检测脾脏中功能性祖细胞和/或干细胞的存在。
值得注意的是,脾脏含有比骨髓更多的反应性 T 细胞,这可能导致移植期间的不良反应,例如移植物抗宿主病 ( Baker et al ., 1996 )。为了避免此类潜在问题,成熟的 T 细胞在移植前被耗尽。


A. 宿主小鼠的辐照
受体小鼠的造血系统通过以相隔 4 小时的两个分数剂量施加的致死辐射消融。
1. 照射 C57BL6 (CD45 .1) 8 至 12 周龄宿主小鼠,20 至 25 g,性别无关(每个移植组 10 只小鼠),首剂剂量为 4.5 Gy全身照射 (TBI),(使用SnCuAl过滤器的Faxitron MR350 X 射线照射器)使用小鼠照射饼笼(图 1)。照射后,用 1:200 稀释的磺胺甲恶唑和甲氧苄啶(材料和试剂 #11)的抗生素悬浮液补充饮用水。
2. 用 4.5 的第二剂量照射宿主小鼠 Gy 4 小时后,移植前。与此同时,继续准备供体材料。


 
图 1. 小鼠辐照饼笼。 
在暴露于 X 射线辐照之前,将小鼠置于辐照饼笼中。


B. 供体材料的制备
整个过程必须在无菌条件下进行
1. 2安乐死牺牲 C57BL6 供体小鼠 (CD45.2) 。收集脾脏 (图 2A) 并放在冰上的 PBS 中。从脾脏中修剪掉任何剩余的结缔组织或脂肪。


 
图 2. 小鼠出血、注射和脾脏恢复程序。 
(A) 验尸脾脏收集的插图。 (B) 将液体注入眶后窦。 (C) 用长矛刺穿颌下静脉。 (D) 从刺穿的下颌下静脉采集血液。


2. 将脾脏转移到含有 5 mL FACS 缓冲液(参见下面的食谱)或含有 2% FBS 的 DMEM 培养基的无菌 35 mm 培养皿中。
3. 从 3 mL 注射器中取出柱塞。使用柱塞的扁平橡胶端通过温和的圆周运动来压碎脾脏。这将破坏牙髓并释放脾细胞。
4. 将 5 mL 的细胞悬浮液通过安装在 50 mL 锥形管顶部的 70 μm尼龙过滤器,以获得均匀的单细胞悬浮液。用注射器柱塞以圆形方式按压,轻轻帮助悬浮液通过过滤器。这种方法的视频演示可以在https://www.stemcell.com/prepare-single-cell-suspension-from-mouse-spleen.html看到。
5. 用 2 mL 的 FACS 缓冲液或含有 2% FBS 的 DMEM 培养基清洗 35 mm 培养皿
并通过过滤器。重复此步骤两次。
6. 在 4°C 下以 300 × g离心管10 分钟。
7. 丢弃上清液并轻弹管以松开颗粒。根据脾脏大小,将颗粒重新悬浮在 200 μL至 1 mL 的含有 3% FBS 的培养基中,并储存在冰上。
8. 用 PBS 以 1:100 的比例稀释细胞悬浮液的等分试样后对细胞进行计数。在倒置光学显微镜下计数血细胞计数器的所有九个方块。一个正常的脾脏(大约 100 毫克)通常会产生大约 1 亿个细胞。我们建议使用台盼蓝 (1:1 v/v) 等活染料来排除潜在的死细胞(图 3)。
注意:由于细胞悬液未经历红细胞裂解,因此确保不计算红细胞(小于白细胞)非常重要(图 3)。
9. 每毫升的细胞数是计数的细胞数×稀释倍数× 10 4 (本例中的稀释倍数为 100)。


 
图 3. 使用血细胞计数器进行外周血细胞计数。
在血细胞计数器网格上对活的白细胞进行计数。红色箭头指向红细胞,黑色箭头指向台盼蓝染色的死细胞,这两种细胞都不包括在白细胞计数中。


T细胞耗竭
10. 悬于 500 μL MojoSort缓冲液中的10 8个细胞转移到 5 mL (12 × 75 mm) 聚苯乙烯管中。在试管中加入生物素抗 CD4 和生物素抗 CD8a 各 5 μL ,混合均匀,在冰上孵育 15 分钟。
注意:在消耗(步骤 B7)之前将一小部分起始材料(10 6 个细胞)放在冰上,从步骤 20 开始使用,以评估手术后消耗的程度。
11. MojoSort缓冲液清洗细胞,并在 4°C 下以 300 × g离心 5 分钟。
12. 弃去上清液并重悬于 500 μL的MojoSort缓冲液中。
13. 通过以最大速度涡旋五次接触来重悬链霉亲和素纳米珠。在细胞悬液中加入 50 μL珠子,充分混合,在冰上孵育 15 分钟。
14. MojoSort缓冲液清洗细胞; 300 离心机 × g在 4°C 下 5 分钟,弃去上清液并重悬于 3 mL MojoSort缓冲液中。
15. 将试管放入冷的MojoSort磁铁(参见设备)中 5 分钟。
16. 通过将液体倒入 15 mL 锥形管中收集细胞。这些是您感兴趣的细胞,已耗尽保留在磁体上的 T 细胞。
17. 如果需要,要增加产量,请向珠子中添加 3 mL 的MojoSort缓冲液,重复步骤 16 和 17,并汇集流通馏分。
18. 如步骤B8 至 B10所示,计算耗尽后获得的细胞。


通过 FACS 评估 T 细胞耗竭
19. 从 T 细胞耗尽前留出的样品(步骤 B7)和 T 细胞耗尽的样品(步骤 B18)中取出含有 g 100,000 至 500,000 个细胞的等分试样。设置三个额外的试管,一个用于未染色对照,两个用于单色对照,每管包含 50,000 至 100,000 个细胞。
20. 在 4°C 下以 200 × g离心去除前和去除后的样品以及两个单色对照5 分钟。在 FACS 缓冲液中重新悬浮含有 PeCy5 抗小鼠 CD4(稀释 1:250)和 APC 抗小鼠 CD8a(稀释 1:250)的50 μL染色溶液中耗尽前和耗尽后的样品。
21. μL PeCy5 抗小鼠 CD4(稀释 1:250)中重新悬浮一个单色对照,在50 μL APC 抗小鼠 CD8a(稀释 1:250)中重新悬浮另一个单色对照。
22. 在冰上孵育 15 至 30 分钟,避光。
23. 用 300 μL的 FACS 缓冲液通过离心清洗所有管。
24. 悬浮在 200 μL的 FACS 缓冲液中,并通过网状过滤器转移到 FACS 管中,以去除任何细胞团块。
25. 分析流式细胞仪上的所有管。
26. 在 BD LSR II 软件上,选择“新实验”。
27. 选择 FSC 和 SSC 的面积、高度和宽度;为颜色 Pe-Cy5 和 APC 选择 Log 和 Area。
28. 创建补偿控制并调整未染色血细胞的门控(根据需要调整 FSC 和 SSC 电压)。
29. 调整电压面板中单个污点的每种颜色,使正峰值在 10 4 标记。
30. 分析单色控件。
31. 在对每个单一污点进行任何调整后记录所需的电压,然后计算补偿控制。
32. 现在可以记录事件了。设置为为每个耗尽前和耗尽后样本收集每个样本5,000 – 10,000 个事件。
注意:通常,该过程会消耗超过 95% 的 CD4 和 CD8 T 细胞。以下视频说明了整个消耗过程: https ://www.youtube.com/watch?v=cH6rlIFNVp0 。


C. 移植到受辐射的小鼠体内
1. 在4°C 下以200 × g将 T 细胞耗尽的脾细胞的细胞悬液离心 5 分钟,去除上清液,然后重悬于含有 2% FBS 的无菌 PBS 中,细胞浓度为每 100 μL 2 × 10 6 。继续冰上。
2. 设置异氟醚麻醉机。
3. 在麻醉室内放置一只辐照鼠标。
4. 打开氧气以 1-2 升/分钟的速度流动,异氟醚计为2-5 %。
5. 动物应该在几分钟内被麻醉。
6. 100 μL的脾细胞悬浮液装入装有 27  G 针头的 1 mL 注射器中。
7. 当鼠标被麻醉后,将其从腔室中取出。立即将动物放在吸水纸上,头部侧向倾斜。用一只手缩回眼睛周围的皮毛,使其突出一点。另一只手以45°角将针头插入内眼角,以穿透眶后窦( Yardeni 等人,2011 年)。参见图 2B。
8. 缓慢平稳地注入 100 μL的脾细胞悬液(约 5 s),并在鼠标开始醒来后立即将其放回笼子。
9. 对要移植的每只辐照动物重复该程序。
10. 注射后,让受体小鼠恢复4-6周。两周后可以从饮用水中去除抗生素。每天监测移植小鼠的生存能力。在被评估的测试样本中存在足够的干细胞应导致接受者存活率超过 80%。
11. 记住要有适当的辐照控制。这些小鼠与用于移植的小鼠(相同年龄和性别)相当,这些小鼠将接受实验小鼠的照射,但它们不会被移植。它们应该在 10到14 天后变得垂死,此时它们将被安乐死。


第二部分:血液重建分析


细胞移植后4至6 周之间进行总献血者重建的首次分析。该分析旨在通过评估受体血液中 CD45.2 细胞(来自供体)的存在来确定供体细胞是否已移植。


A. 获取用于流式细胞术的总白细胞
1. 通过穿刺从下颌下静脉穿过脸颊的面部静脉进行血液采集( Regan等人,2016 年)。请参见图 2C 和 2D。
2. 垂直握住鼠标的颈背,在下颌骨后部皮肤,两条虚拟线的交点处,一条从外眼角开始,另一条在靠近嘴巴的无毛点处。 ,用一枝黄花动物柳叶刀。
3. 将血液收集到涂有 EDTA 的 BD 微量容器中; 200 µL 就足够了(大约四滴)。
4. 继续对实验的所有受体小鼠做同样的事情。此外,对未移植的CD45.2 小鼠和未移植的 CD45.1 小鼠进行放血,以执行流式细胞术的控制。
5. 将血液样本转移到 15 mL 锥形管中。加入 1mL 的 BD PharmLyse裂解液,在水中按 1:10 稀释,进行红细胞裂解。轻轻涡旋每个管并在冰上孵育 15 分钟,避光。
6. 离心管在 200 × g在 4°C 下保持 5 分钟。小心吸出上清液,不要干扰颗粒(应该看起来几乎是白色的),并加入 2 mL 的 1 × PBS,其中含有 1%热灭活FBS 和 0.1%叠氮化钠。


B. 标记细胞用于流式细胞术
1. 离心管在 200 × g在 4°C 下保持 5 分钟。小心吸出上清液,不要干扰沉淀,并重悬于 50 µL 由 BV711 抗小鼠 CD45.1 抗体(1:200 稀释)和 BV605 抗小鼠 CD45.2 抗体(1:200 稀释)混合而成的染色溶液中) 在 FACS 缓冲区中。
2. 通过在 CD45.1 血液样本中加入 50 μL BV711 抗小鼠 CD45.1 抗体(稀释 1:200)和 50 μL BV605 抗小鼠 CD45.2 抗体(稀释 1:200)制备两个样品用于单色对照) 在 CD45.2 血液中,并保留一份未染色的样品。
3. 在冰上孵育 15 至 30 分钟,避光。
4. 用 300 μL 的 FACS 缓冲液清洗。在 4°C 下以200 × g离心细胞5 分钟。吸出上清液。
5. 在 200 μL 的 FACS 缓冲液中重新悬浮,并通过网状过滤器转移到 FACS 管中,以去除细胞团块。
6. 如前所述在流式细胞仪上分析(步骤 B27 至 B33)。


第三部分:长期植入数据分析


该分析旨在确定移植的移植细胞是否产生了所有谱系的白细胞,并在移植后至少 4 个月进行。
1. 如前所述d 对动物放血并进行红细胞裂解(步骤 A1 至 A6)。
2. 用由 PE-Cy5 抗小鼠 CD4(1:200 稀释)、APC 抗小鼠 CD8a(1:200 稀释)、PE-Cy7 抗小鼠 CD11b(1:500 稀释)、APC-组成的染色混合物对血液进行染色eFluor 780 抗小鼠 B220(1:200 稀释)和 BV605 抗小鼠 CD45.2(1:200 稀释)在 FACS 缓冲液中。
3. 按照第 II-B 部分中的详细说明进行操作。
该分析将确定源自 CD45.2 阳性移植细胞的 CD4 和 CD8 T 细胞、B 细胞和骨髓细胞的百分比。所有主要谱系的存在表明供体材料中存在 HSPC,而仅存在供体来源的骨髓细胞表明实验脾脏中仅存在定型祖细胞。


为了坚定地断言多能干细胞存在于供体脾脏中,我们建议使用主要受体的脾脏和/或骨髓进行二次移植,并概括整个过程。二次移植后受者体内存在来自供体细胞的血细胞谱系,这为原代动物脾脏中存在全能造血干细胞提供了明确的证据。根据正在研究的实验系统,初次移植期间移植的长期 HSC 可能仅驻留在骨髓中,也可能已归巢于外周器官,这可以通过使用骨髓或脾细胞进行二次移植来评估.
将此协议应用于脾脏以外的其他次级淋巴器官将是有意义的。特别是,该技术可用于测量淋巴结或其他具有丰富造血细胞的组织(如小肠和大肠)中造血干细胞的潜在存在。


食谱


1. 流式细胞仪缓冲器
牛血清白蛋白 (BSA) 0.5% (w/v)
1 毫米乙二胺四乙酸
0.05%叠氮化钠(w/v)
溶于 PBS
使用 0.2 μ m过滤器过滤除菌


致谢


该协议改编自之前发表的论文: Marié 等。 (2021 年) 。这项工作得到了美国国立卫生研究院赠款 R01AI28900 和狼疮研究联盟向 DEL 赠款 579817 的部分支持,以及来自国家癌症研究所的劳拉和艾萨克·珀尔马特综合癌症中心支持赠款 P30CA016087 。


利益争夺


作者声明没有竞争利益。


伦理


该协议开发中的所有工作都得到了纽约大学格罗斯曼医学院机构动物护理和使用委员会的批准。


参考


1. Baker, MB, Altman, NH, Podack , ER 和 Levy, RB (1996)。小鼠 MHC 匹配的异基因骨髓移植后细胞介导的细胞毒性在急性 GVHD 中的作用。 J Exp Med 183(6):2645-2656。
2. -Struuck , R. 和Dysko , RC (2009)。骨髓移植 (BMT) 的原则:在 BMT 研究中为受辐射的小鼠提供最佳的兽医和畜牧业护理。 J Am Assoc 实验室动画科学48(1):11-22。
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Copyright Marie et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Marie, I. J., Brambilla, L. and Levy, D. E. (2022). Assessing the Presence of Hematopoietic Stem and Progenitor Cells in Mouse Spleen. Bio-protocol 12(11): e4438. DOI: 10.21769/BioProtoc.4438.
  2. Marié, I. J., Brambilla, L., Azzouz, D., Chen, Z., Baracho, G. V., Arnett, A., Li, H. S., Liu, W., Cimmino, L., Chattopadhyay, P., et al. (2021). Tonic interferon restricts pathogenic IL-17-driven inflammatory disease via balancing the microbiome. Elife 10: e68371.
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