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Nov 2015
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Cobblestone Area-forming Cell Assay of Mouse Bone Marrow Hematopoietic Stem Cells
小鼠骨髓造血干细胞的鹅卵石样区域形成细胞测定   

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Abstract

Bone Marrow Hematopoietic Stem Cells (HSCs) require bone marrow microenvironment for their maintenance and proliferation. Culture of Bone Marrow Mesenchymal Stromal Cells (MSCs) provides appropriate environmental signals for HSCs survival in vitro. Here, we provide a detailed protocol that describes culture conditions for MSCs, flow cytometric isolation of HSCs from mouse bone marrow, and perform co-culture of MSCs and HSCs known as Cobblestone area-forming cell (CAFC) assay. Altogether, CAFC assays can be used as a high-throughput in vitro screening model where efforts are made to understand and develop therapies for complex bone marrow diseases. This protocol needs 3 to 4 weeks starting from culturing MSCs, isolating LSK cells (HSCs), and to performing limited dilution CAFC assay.

Keywords: Mesenchymal Stromal Cells (间充质基质细胞), Hematopoietic Stem Cells (造血干细胞), Co-culture assay (共培养测定), Cobblestone area-forming cell assay (鹅卵石样区域形成细胞测定)

Background

The proliferative, survival and differentiation potential of HSCs is very much dependent on its microenvironment also known as niche. The bone marrow MSCs support the HSCs to keep them in a quiescent state in the bone marrow niche. The intrinsic and extrinsic signals received by the niche contribute to the differentiation of HSCs into mature blood-cell lineages also known as hematopoiesis, without inducing aberrant expansion (Yoshihara et al., 2007; Spindler et al., 2014; Hu et al., 2016). The Cobblestone-Area-Forming Cell Assay (CAFC Assay) is an in vitro co-culture assay of long-term bone marrow HSCs and MSCs. While MSCs are cultured to complete confluence in a tissue culture dish, HSCs are plated over MSCs (de Haan and Ploemacher, 2002). CAFC assays are comparable to in vivo studies of bone marrow and can be used as a rapid screening assay to test the stem cell activity of HSCs and supportive activity of MSCs (Ploemacher et al., 1989). There is a high demand for high throughput screening models that reflect the complex physiology or pathology of the bone marrow microenvironment both in native state or disease models respectively. In this regard, large scale screening was made possible using the HSCs-stroma co-culture system to identify small-molecule inhibitors to develop an effective therapy for acute leukemia (Hartwell et al., 2013). Further, a co-culture system was applied as a model to study several diseases like Fanconi anemia (FA), where it was identified that FA MSCs produce elevated levels of metabolites like glycerophospholipids, which can skew the normal HSCs function (Amarachintha et al., 2015). Further, MSCs provided an impaired environment for HSCs proliferation in patients with aplastic anemia suffering with pancytopenia and in the patients who are in remission after immunosuppression (Schrezenmeier et al., 1996). Further, MSCs from T-cell lymphocytic leukemia mouse model showed adverse proliferation and differentiation capacity of HSCs (Lim et al., 2016). However, in Cord Blood transplants, Cord Blood-MSC co-culture holds promise for successful expansion of cord blood and surge the engraftment in recipients (Denning-Kendall et al., 2003; Robinson et al., 2006). Although several theories were proposed to characterize the isolation and culture of MSCs, ‘The International Society for Cellular Therapy’ has set the minimal criteria for defining ‘multipotent mesenchymal stromal cells’. MSCs derived from bone marrow must be plastic-adherent in standard culture conditions, express cell surface markers, and must differentiate to osteoblasts, adipocytes, and chondroblasts in vitro (Dominici et al., 2006; Keating, 2012). Adhering to these principles, we identified a simplistic approach to culture MSCs from mouse bone marrow and performed limited dilution CAFC assay to enable rapid screenings.

Materials and Reagents

  1. Pipette tips (USA Scientific, catalog number: 1126-7810 )
  2. 1,000 µl large orifice pipette tip (USA Scientific, catalog number: 1011-9000 )
  3. BD Precision glide needles (BD, catalog number: 305155 )
  4. BD Slip Tip Sterile Syringe 1 ml (BD, catalog number: 309659 )
  5. BD Single-use Needles 22 G (BD, catalog number: 305159 )
  6. Falcon 15 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352097 )
  7. Falcon standard tissue culture dishes (100 mm culture dish) (Corning, Falcon®, catalog number: 353003 )
  8. Falcon 35 mm TC-treated Easy-Grip style cell culture dish (Corning, Falcon®, catalog number: 353001 )
  9. Nunc Edge 2.0 96-well cell culture plates (Thermo Fischer Scientific, Thermo ScientificTM, catalog number: 167425 )
  10. 3-well chamber slide (IBIDI, catalog number: 80381 )
  11. Falcon Polystyrene Microplates 6-well plate TC-treated (Corning, Falcon®, catalog number: 353934 )
  12. C57BL/6J mice of age 4 to 8 weeks old (THE JACKSON LABORATORY, catalog number: 000664 )
  13. 70% ethanol
  14. Red blood cell lysis buffer (Sigma-Aldrich, Roche Diagnostics, catalog number: 11814389001 )
  15. Trypsin-EDTA (0.25%), phenol red (Thermo Fischer Scientific, GibcoTM, catalog number: 25200056 )
  16. Mouse MSC Functional Identification Kit containing antibodies Osteopontin, Fabp4, and Collagen II (R&D Systems, catalog number: SC010 )
  17. DPBS (10x), no calcium, no magnesium (Thermo Fischer Scientific, GibcoTM, catalog number: 14200075 )
  18. 16% formaldehyde (w/v), methanol-free (Thermo Fischer Scientific, Thermo ScientificTM, catalog number: 28906 )
  19. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  20. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
  21. 4’,6-Diamidino-2-phenylindole (DAPI) (Thermo Fischer Scientific, InvitrogenTM, catalog number: D1306 )
  22. VECTASHIELD Antifade mounting medium (Vector Laboratories, catalog number: H-1000 )
  23. Ficoll-Paque PLUS (GE Healthcare, catalog number: 17144002 )
  24. Streptavidin APC-Cy-7 (BD, BD Biosciences, catalog number: 554063 )
  25. PE rat anti-mouse Ly-6A/E (BD, BD Biosciences, catalog number: 561076 )
  26. Rat anti-mouse CD117 (BD, BD Biosciences, catalog number: 561074 )
  27. V450 mouse lineage antibody cocktail (BD, BD Biosciences, catalog number: 561301 )
  28. Fetal bovine serum, qualified, heat inactivated, USDA-approved (FBS) (Thermo Fischer Scientific, GibcoTM, catalog number: 10438034 )
  29. Iscove’s modified Dulbecco’s medium (Thermo Fischer Scientific, InvitrogenTM, catalog number: 12440053 )
  30. Bovine calf serum (GE Healthcare, Hyclone, catalog number: SH30072.03 )
  31. Epidermal growth factor (R&D Systems, catalog number: 2028-EG-200 )
  32. Platelet-Derived growth factor (R&D Systems, catalog number: 220-BB-010 )
  33. Penicillin-streptomycin (Thermo Fischer Scientific, GibcoTM, catalog number: 15140122 )
  34. 2-Mercaptoethanol (Thermo Fischer Scientific, GibcoTM, catalog number: 21985023 )
  35. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  36. Donkey serum (Sigma-Aldrich, catalog number: D9663 )
  37. Bone marrow wash buffer (see Recipes)
  38. MSCs media (see Recipes)
  39. DPBS blocking solution (see Recipes)

Equipment

  1. ErgoOne 100-1,000 µl Single Channel Pipettes (USA Scientific, model: 7110-1000 )
  2. Eppendorf 5804 Benchtop Centrifuge (Eppendorf, model: 5804 )
  3. BD FACSCanto II to analyze the hematopoietic stem cells (BD, model: BD FACSCanto II )
  4. BD FACSAria II to sort the hematopoietic stem cells (BD, model: BD FACSAria II )
  5. NIKON Eclipse 90i microscope to capture immunofluorescence images (Nikon Instruments, model: Eclipse 90i )
  6. OLYMPUS IX53 Inverted Microscope to capture phase contrast images of cobblestone area (Olympus, model: IX53 )

Software

  1. BD FACSDiva v8.0.1 Software (BD Biosciences)
  2. FlowJo software (FLOW JO)
  3. CellSens software (Olympus)
  4. GraphPad Prism software

Procedure

  1. Isolation and culture of Mouse Bone Marrow Mesenchymal Stromal Cells
    1. Euthanize an adult C57BL/6 mouse by CO2 asphyxiation in accordance with Institutional Animal Care and Use Committee (IACUC) protocol. Spray the mouse with 70% ethanol to disinfect. Incise the skin at hind leg portion and expose the muscles. Cut the pelvic joint and remove the hind leg. Cut the knee joint to separate femur from the lower portion of the hind limb. Remove the muscles of the femur and cut the ends of the femur to enable flushing.
    2. Transfer the femur into ice-cold wash buffer and flush gently with ice-cold wash buffer using a 1 ml syringe with a 22 G needle until all the cells are removed and the bone appears white. Repeat the same procedure for the other hind limb and combine the cells from both femurs.
    3. Spin BM cells in a 15 ml conical tube at 500 x g for 5 min at room temperature.
    4. Lyse RBCs using red blood cell lysis buffer.
    5. To lyse RBCs, suspend the cells obtained from two femurs in 2 ml wash buffer and add 4 ml of red blood cell lysis buffer. Mix the cells by inverting few times every min for 10 min.
    6. Centrifuge the tube at 400 x g for 6 min at room temperature.
    7. Discard the supernatant and resuspend the BM cells in 10 ml wash buffer and centrifuge at 400 x g for 10 min to remove the lysed red blood cells. Repeat this process for one more time.
    8. Remove the excess wash buffer from the cell pellet and add 10 ml of MSCs media to the cell pellet. Pipet the media gently to evenly mix the cell pellet.
    9. Plate 1 x 107 cells in a 100 mm tissue culture dish with 10 ml of MSCs media.
    10. Incubate the cells at 37 °C with 5% CO2 in a standard humidified tissue culture room incubator.
    11. Mesenchymal stromal cells start to adhere to the plastic dish while the rest of the BM cells float in the media.
    12. Replace the media with fresh MSCs media after three days in culture and culture the cells for 10 to 12days.
    13. During the first media change, most of the suspending cells are removed and MSCs start to adhere to the bottom of the dish.
    14. Subculture the MSCs from one 100 mm dish into a 35 mm dish using trypsin-EDTA. The cells are devoid of any other types of BM cells by the second passage and are ready for the assay from passage 3 (Figure 1).
    15. Since MSCs do not have high dividing potential, split the cells into 1 to 2 ratios each time when sub-cultured to subsequent passages and maintain in 35 mm dishes until assay is performed. MSCs can be passaged up to five passages without losing MSC function.
    16. Cells can be subsequently passaged to 96-well flat-bottom plates when ready to perform the assay.


      Figure 1. Mouse Bone Marrow Mesenchymal stromal cells. Plastic adhered MSCs were shown in phase contrast image. Scale bar represents 50 µm.

  2. Characterization of Mouse Bone Marrow Mesenchymal stromal cells
    1. To obtain pure MSCs, sub-culture plastic adherent cells for at least three passages. MSCs are multipotent cells and can be induced to differentiate into osteoblasts, adipocytes, and chondrocytes. However, MSCs in culture consist of a mix of osteoblasts, adipocytes, and chondrocytes and express markers unique to them. It is critical to evaluate the functionality of MSCs before performing CAFC since the loss of a gene or exposure to various environments can give rise to differences in differentiation of MSCs.
    2. To identify the multipotential of MSCs, plate cells on chamber slides and stain with antibodies Osteopontin, Fatty acid binding protein 4 (Fabp4), and Collagen II to identify osteoblasts, adipocytes, chondroblasts respectively using the Mouse MSC Functional Identification Kit.
    3. Immunofluorescence of MSCs: Rinse MSCs once with 1x DPBS and fix with 4% paraformaldehyde (PFA) diluted in 1x DPBS for 10 min at room temperature.
    4. Permeabilize the cells with 0.2% Triton X-100 made in 1x DPBS for 3 min and block with blocking buffer DPBS blocking solution for 30 min.
    5. Gently remove the blocking solution and follow overnight incubation at 4 °C with primary antibody. Adipocyte marker: Goat Anti-Mouse FABP4 Antigen-affinity Purified Polyclonal Antibody. Chondrocyte marker: Sheep Anti-Mouse Collagen II Antigen-affinity Purified Polyclonal Antibody. Osteocyte marker: Goat Anti-Mouse Osteopontin Antigen-affinity Purified Polyclonal Antibody. Dilute the primary antibodies to 1:500 in 1x DPBS containing 0.1% BSA.
    6. Wash the slides with 1x DPBS containing 0.1% BSA for three times at room temperature with each wash for 10 min. Incubate with secondary antibodies diluted to 1:1,000 in 1x DPBS containing 0.1% BSA for at least 2 h at room temperature. Use Donkey Anti-Goat and Donkey Anti-Sheep IgG secondary antibodies.
    7. Wash the slides with 1x DPBS containing 0.1% BSA for three times at room temperature with each wash for 10 min.
    8. After the final wash, add the nuclear dye DAPI at 1:2,000 in 1x DPBS for 5 min to evaluate nuclear morphology.
    9. Wash further 3 times with 1x DPBS for 5 min each wash.
    10. Remove excess DPBS and mount slides with Vectashield mounting media.
    11. Acquire images using any immunofluorescence microscope at 20x objective (Figure 2).
    12. The ratio of adipocytes:chondrocytes:osteocytes from a wildtype C57BL/6J mice is 60:20:20. 


      Figure 2. Representative images of Mouse BM MSCs stained with antibodies Fabp4, Collagen II, and Osteopontin to identify adipocytes, chondroblasts, and osteoblasts lineage cells respectively present in bone marrow derived mesenchymal stromal cells. Scale bar represents 10 µm.

  3. Isolation of Mouse Bone Marrow Hematopoietic stem cells (HSCs)
    1. Isolate HSCs from Bone marrow mononuclear cells (BMMCs) using flow cytometry after staining with cell surface markers negative for Lineage cocktail and positive for Sca-1, c-Kit (Lineage Negative, Sca-1 Positive, c-Kit Positive, LSK). HSCs are also termed as LSKs.
    2. Obtain BM cells from mouse femurs by gently flushing cells out the bones using wash buffer.
    3. Wash BM cells twice with wash buffer by centrifugation at 500 x g for 5 min at room temp.
    4. Resuspend the cells with 3 ml of wash buffer and gently overlay on 6 ml of Ficoll-Paque PLUS in a 15 ml conical tube.
    5. Carefully layer the diluted cell sample onto the Ficoll-Paque PLUS solution and take precautions not to mix the cells and Ficoll.
    6. Centrifuge at 400 x g for 30 min at room temperature with slow acceleration and brake turned off.
    7. Pipette out upper layer containing plasma and platelets using a sterile pipette, leaving the BMMCs layer undisturbed at the interface.
    8. Transfer the layer of BMMCs to a sterile centrifuge tube using a sterile pipette.
    9. Wash the BMMCs with wash buffer once.
    10. Pellet BMMCs and first stain with a lineage (Lin) cocktail of antibodies (biotinylated anti-mouse antibodies directed against CD3e, CD11b, CD45R/B220, Gr-1, and Ter119). Add 2 μl of each antibody to stain one million BMMCs. Incubate the cells with antibodies at 4 °C for 20 min.
    11. Wash the cells with bone marrow wash buffer one time and pellet the cells.
    12. Further, add 5 μl of fluorochrome-conjugated Streptavidin to bind to lineage antibodies.
    13. Wash BMMCs once with buffer and pellet the cells. Further, add 2 μl of Sca-1 and c-kit conjugated with fluorochrome to stain one million BMMCs.
    14. Wash Triple (Lineage, Sca-1, c-kit) stained BMMCs couple times and suspend one million cells in 1 ml of wash buffer for flow sorting.
    15. Flow sort on a BD FACS Aria II with a FACSDiva software.
    16. Analyze the Flow cytometric data to check the quality of LSK cells using the FlowJo software as displayed in pseudo color plot (Figure 3).


      Figure 3. Gating strategy to identify and isolate Hematopoietic stem cells from Mouse Bone Marrow. Bone Marrow cells flushed from mouse femurs were subjected to Ficoll-Paque PLUS separation to isolate Bone Marrow Mono Nuclear Cells and then stained with HSC cell surface markers to identify and cell-sort using the flow cytometer.

  4. Cobble stone area forming cell assay
    1. Prior to isolation of LSK cells using flow cytometry, culture MSCs to complete confluence in a 35 mm culture dish or a 6-well plate.
    2. MSCs between passages 3 to 6 should be used for plating LSK cells.
    3. On the day of assay, sort LSK cells from Bone marrow as described (Procedure C) using flow cytometry.
    4. Pellet the isolated LSK cells and resuspend with MSCs media. Replace the media in the culture dish growing MSCs in media suspended with LSK cells. Approximately, plate 2 x 103 LSK cells on confluent MSCs in a 35 mm dish.
    5. Co-culture the cells at 37 °C, 5% CO2 in a humidified incubator to allow the precursor cells to form hematopoietic clones under the stromal layers.
    6. Cobblestone areas start to appear as early as 5 days and can be predominantly seen by 7 days.
    7. Feed the culture two times a week by replacing half of the medium. Image the phase-dark hematopoietic clones using a phase contrast microscope (Figure 4) at 10x objective and installed with software like CellSens software from Olympus.


      Figure 4. Representative image of Cobblestone area forming cell assay. HSCs are cultured on a confluent layer of MSCs to form the cobblestone areas. Yellow arrow–phase dull cells (true stem cells growing under the MSCs layer), Blue arrow–phase bright cells (stem progenitor cells suspended or loosely attached to MSCs), and Red arrow–MSCs (Confluent cell layer adhered to the plastic dish). Scale bar represents 50 µm.

  5. CAFC limited dilution assay
    1. Limiting dilution assay (LDA) is performed in 96-well flat-bottom plates with confluent MSCs and dilutions of LSK cells (0, 10, 30, 90, 270, and 810) differing with a factor of 3 in each well. Plate MSCs in 10 wells in a flat-bottom 96 well plate per each dilution (Figure 5A). No LSK cells are added to 0 dilution wells and they serve as negative control.
    2. Perform three different LDA experiments with independently derived MSCs from bone marrows of mice.
    3. Score the well as ‘positive’ if it contains one or more cobblestone areas and ‘negative’ if contains no cobblestone areas.
    4. Cobblestone area is at least 6 cells (in proximity to each other) growing underneath the stroma. Although cobblestone-like cells appear as phase dark, these cells appear as nonrefractile in 96-well plates because of the deflection of light.
    5. Only dilutions with both negative and positive wells are informative for frequency analysis (Figure 5B).


      Figure 5. CAFC Limited dilution assay. A. CAFC assay performed in a 96-well flat-bottom plate. Flow sorted LSK cells were counted and plated on confluent MSCs in a flat-bottom 96-well plate. Each assay was performed in triplicates. Representative images of the cobblestones formed in wells with no LSK cells, 90 and 270 LSK cells. Scale bars represent 100 µm. A portion of the cobblestone area was magnified; Scale bar represents 50 µm. Phase dull cells were identified in a dotted circle while phase bright cells were identified in a solid circle. Both phase dull and phase bright cells need to be present to call it a true cobblestone area. B. Phase dull cell areas were measured in each well as shown in above panel. Percentage of the well covered with cobblestone areas was counted and plotted against the no of LSK cells plated in each well. Cultures were maintained for two weeks and the areas were counted at the end of each week.

Data analysis

Plot the charts and analyze data using GraphPad Prism software. Compare the means of two groups in LDA using t-test.

Notes

  1. MSCs do not have high recovery rate when cryopreserved. So always culture and perform the assay with fresh MSCs.
  2. To prevent rupture of MSCs and LSK cells while suspending into media, use large orifice pipette tips.
  3. Perform CAFC assay only when MSCs are grown to complete confluence.
  4. Add LSK cell surface markers to BMMCs suspended into 200 µl or less volume of buffer.

Recipes

  1. Bone marrow wash buffer
    Dulbecco’s phosphate-buffered saline made to 1x concentration (1x DPBS)
    10% FBS
  2. MSCs media
    Iscove’s modified Dulbecco’s medium
    500 ml
    Bovine calf serum
    20%
    Epidermal growth factor (rmEGF)
    10 ng/ml
    Platelet-Derived growth factor (rhPDGF)
    200 ng/μl
    Penicillin-streptomycin
    1%
    2-Mercaptoethanol
    10-4 mol/L
  3. DPBS blocking solution
    1x DPBS
    1% Triton X-100
    1% DMSO
    1% BSA
    1% Serum (Serum from the animal in which the secondary antibodies were raised)

Acknowledgments

This protocol was adapted from previous work published in Hematopoietic Stem Cell Protocols in 2002 by de Haan and Ploemacher. Cell sorting for LSK cells was performed at Research Flow Cytometry Core at Cincinnati Children’s Hospital Medical Center. This investigation was supported by NIH grants R01 HL076712, R01 CA157537 and T32 HL091805. Qishen Pang is supported by a Leukemia and Lymphoma Scholar award.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Contribution: S. A. designed and performed research, analyzed data, and wrote the paper; Q. P. designed research, contributed vital new reagents, analyzed data.

References

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简介

骨髓造血干细胞(HSC)需要骨髓微环境来维持和增殖。 骨髓间充质基质细胞(MSC)的培养为体外HSC存活提供适当的环境信号。 在这里,我们提供了描述MSCs培养条件的详细方案,从小鼠骨髓中流式细胞术分离HSCs,并进行称为鹅卵石区域形成细胞(CAFC)分析的MSC和HSC的共培养。 总而言之,CAFC分析可用作高通量体外筛选模型,其中努力了解和开发复杂骨髓疾病的治疗方法。 该方案需要培养MSC,分离LSK细胞(HSC)和执行有限稀释CAFC测定3至4周。

【背景】HSC的增殖,存活和分化潜力非常依赖于其微环境,也被称为小生境。骨髓MSC支持HSC以使其在骨髓龛中保持静止状态。由生态位接收的内在和外在信号有助于将HSC分化为也称为造血的成熟血细胞谱系,而不诱导异常扩增(Yoshihara等人,2007; Spindler等人 ,2014; Hu等人,2016)。鹅卵石区域形成细胞试验(CAFC试验)是长期骨髓HSC和MSC的体外共培养试验。当培养MSC在组织培养皿中完成融合时,将HSC铺在MSC上(de Haan和Ploemacher,2002)。 CAFC测定与骨髓的体内研究相当,并且可用作快速筛选测定以测试HSC的干细胞活性和MSC的支持活性(Ploemacher等人, 1989)。对高通量筛选模型的需求很高,这些模型分别反映了天然状态或疾病模型中骨髓微环境的复杂生理学或病理学。在这方面,使用HSCs-基质共培养系统进行大规模筛选以鉴定小分子抑制剂以开发用于急性白血病的有效疗法(Hartwell等人,2013)。此外,共培养系统被用作模型来研究几种疾病,如范可尼贫血(FA),其中确认FA MSCs产生升高水平的代谢物如甘油磷脂,其可以使正常HSC功能偏斜(Amarachintha em> et al。,2015)。此外,MSCs为患有全血细胞减少症的再生障碍性贫血患者和免疫抑制后缓解的患者提供了HSC增殖受损的环境(Schrezenmeier等人,1996)。此外,来自T细胞淋巴细胞白血病小鼠模型的MSC表现出HSC的不利增殖和分化能力(Lim等人,2016)。然而,在脐带血移植中,脐带血 - MSC共培养有望成功扩张脐带血并刺激接受者的植入(Denning-Kendall等人,2003; Robinson等人,2006)。尽管提出了几种理论来描述MSCs的分离和培养特征,但“国际细胞治疗学会”已经为确定'多潜能间充质基质细胞'设定了最低标准。来源于骨髓的MSC必须在标准培养条件下是塑性粘附的,表达细胞表面标记,并且必须在体外分化成成骨细胞,脂肪细胞和成软骨细胞(Dominici 等,2006; Keating,2012)。遵循这些原则,我们确定了一种从小鼠骨髓培养MSCs的简单方法,并进行有限稀释CAFC检测以实现快速筛查。

关键字:间充质基质细胞, 造血干细胞, 共培养测定, 鹅卵石样区域形成细胞测定

材料和试剂

  1. 移液器吸头(USA Scientific,目录号:1126-7810)
  2. 1000μl大孔移液器吸头(USA Scientific,目录号:1011-9000)
  3. BD精密滑翔针(BD,目录号:305155)
  4. BD Slip Tip无菌注射器1 ml(BD,目录号:309659)
  5. BD一次性针22 G(BD,目录号:305159)
  6. Falcon 15ml锥形离心管(Corning,Falcon ,目录号:352097)
  7. Falcon标准组织培养皿(100mm培养皿)(Corning,Falcon ,目录号:353003)
  8. Falcon 35毫米TC处理的Easy-Grip样式细胞培养皿(Corning,Falcon ,目录号:353001)
  9. Nunc Edge 2.0 96孔细胞培养板(Thermo Fischer Scientific,Thermo Scientific TM,目录号:167425)
  10. 3孔室幻灯片(IBIDI,目录号:80381)
  11. Falcon聚苯乙烯微孔板TC处理的6孔板(Corning,Falcon ,产品目录号:353934)
  12. 4至8周龄的C57BL / 6J小鼠(THE JACKSON LABORATORY,目录号:000664)
  13. 70%乙醇
  14. 红细胞裂解缓冲液(Sigma-Aldrich,Roche Diagnostics,目录号:11814389001)
  15. 胰蛋白酶-EDTA(0.25%),酚红(Thermo Fischer Scientific,Gibco TM,目录号:25200056)
  16. 包含抗体骨架蛋白,Fabp4和胶原蛋白II(R& D Systems,目录号:SC010)的小鼠MSC功能鉴定试剂盒
  17. DPBS(10x),不含钙,不含镁(Thermo Fischer Scientific,Gibco,产品目录号:14200075)
  18. 16%甲醛(w / v),不含甲醇(Thermo Fischer Scientific,Thermo Scientific TM,目录号:28906)
  19. Triton X-100(Sigma-Aldrich,目录号:T8787)
  20. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9418)
  21. 4',6-二脒基-2-苯基吲哚(DAPI)(Thermo Fischer Scientific,Invitrogen TM,目录号:D1306)
  22. VECTASHIELD Antifade固定介质(Vector Laboratories,目录号:H-1000)
  23. Ficoll-Paque PLUS(GE Healthcare,目录号:17144002)
  24. 链霉亲和素APC-Cy-7(BD,BD Biosciences,目录号:554063)
  25. PE大鼠抗小鼠Ly-6A / E(BD,BD Biosciences,目录号:561076)
  26. 大鼠抗小鼠CD117(BD,BD Biosciences,目录号:561074)
  27. V450小鼠谱系抗体鸡尾酒(BD,BD Biosciences,目录号:561301)

  28. 胎牛血清,合格,热灭活,USDA认可(FBS)(Thermo Fischer Scientific,Gibco TM,目录号:10438034)
  29. Iscove改良的Dulbecco's培养基(Thermo Fischer Scientific,Invitrogen TM,目录号:12440053)
  30. 牛小牛血清(GE Healthcare,Hyclone,目录号:SH30072.03)
  31. 表皮生长因子(R& D Systems,目录号:2028-EG-200)
  32. 血小板衍生生长因子(R& D Systems,目录号:220-BB-010)
  33. 青霉素 - 链霉素(Thermo Fischer Scientific,Gibco TM,目录号:15140122)
  34. 2-巯基乙醇(Thermo Fischer Scientific,Gibco TM,目录号:21985023)
  35. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  36. 驴血清(Sigma-Aldrich,目录号:D9663)
  37. 骨髓洗涤缓冲液(见食谱)
  38. MSCs媒体(见食谱)
  39. DPBS阻断解决方案(请参阅食谱)

设备

  1. ErgoOne 100-1,000μl单通道移液器(USA Scientific,型号:7110-1000)
  2. Eppendorf 5804台式离心机(Eppendorf,型号:5804)
  3. BD FACSCanto II分析造血干细胞(BD,型号:BD FACSCanto II)
  4. BD FACSAria II分选造血干细胞(BD,型号:BD FACSAria II)
  5. NIKON Eclipse 90i显微镜捕捉免疫荧光图像(Nikon Instruments,型号:Eclipse 90i)
  6. OLYMPUS IX53倒置显微镜捕捉鹅卵石区域的相位对比图像(奥林巴斯,型号:IX53)

软件

  1. BD FACSDiva v8.0.1软件(BD Biosciences)
  2. FlowJo软件(FLOW JO)
  3. CellSens软件(奥林巴斯)
  4. GraphPad Prism软件

程序

  1. 小鼠骨髓间充质干细胞的分离培养
    1. 根据机构动物护理和使用委员会(IACUC)协议,通过CO 2窒息将成人C57BL / 6小鼠安乐死。用70%乙醇喷洒小鼠进行消毒。在后腿部分切开皮肤并暴露肌肉。切开骨盆关节并取下后腿。切开膝关节以将股骨与下肢下部分开。去除股骨的肌肉并切割股骨的末端以使其能够冲洗。
    2. 将股骨转移至冰冷的清洗缓冲液中,用带有22 G针头的1 ml注射器,用冰冷的清洗缓冲液轻轻冲洗,直至所有细胞都被去除,骨骼呈白色。对另一只后肢重复同样的程序,并将来自两只股骨的细胞合并。
    3. BM细胞在室温下在500mlxg的15ml锥形管中旋转5分钟。
    4. 溶血红细胞使用红细胞裂解缓冲液。
    5. 为了裂解红细胞,将从两个股骨获得的细胞悬浮在2ml洗涤缓冲液中并加入4ml红细胞裂解缓冲液。每分钟颠倒几次混合细胞10分钟。

    6. 在室温下将试管在400×g下离心6分钟。
    7. 弃去上清液并用10ml洗涤缓冲液重悬BM细胞,并在400×g下离心10分钟以除去裂解的红血细胞。重复此过程一次。
    8. 从细胞沉淀中除去多余的洗涤缓冲液,并向细胞沉淀中加入10ml的MSC培养基。
      轻轻吸取培养基均匀混合细胞沉淀。
    9. 在含有10ml MSCs培养基的100mm组织培养皿中平板1×10 7个细胞。
    10. 在标准湿润的组织培养室培养箱中,用37℃的5%CO 2孵育细胞。

    11. 间充质基质细胞开始粘附在塑料培养皿上,而其余的BM细胞漂浮在培养基中。

    12. 培养三天后,用新鲜MSCs培养基更换培养基并培养细胞10至12天。
    13. 在第一次媒体更换期间,大多数悬浮细胞被移除并且MSC开始粘附到盘底部。
    14. 使用胰蛋白酶-EDTA将MSC从一个100mm培养皿继代培养到35mm培养皿中。第二代细胞没有任何其他类型的BM细胞,并准备好从第3代开始进行测定(图1)。
    15. 由于MSC不具有高的分裂潜能,每次分次培养至随后的传代时将细胞分成1比2的比例并保持在35mm培养皿中直至进行测定。
      可以将MSCs传至五代而不会失去MSC功能。

    16. 当准备好进行测定时,细胞可以随后传代到96孔平底板

      图1.小鼠骨髓间充质基质细胞塑料粘附的MSCs显示在相衬图像中。比例尺代表50微米。

  2. 小鼠骨髓间充质干细胞的鉴定
    1. 为了获得纯的MSC,亚培养塑料贴壁细胞至少三代。 MSC是多能细胞,可被诱导分化成成骨细胞,脂肪细胞和软骨细胞。然而,培养的MSC由成骨细胞,脂肪细胞和软骨细胞的混合物组成,并表达它们独特的标志物。在进行CAFC之前评估MSC的功能至关重要,因为丢失基因或暴露于各种环境可能会导致MSCs分化的差异。
    2. 为了鉴定MSCs的多能性,在平板上铺板细胞并用抗体骨桥蛋白,脂肪酸结合蛋白4(Fabp4)和胶原II染色,分别用小鼠MSC功能鉴定试剂盒鉴定成骨细胞,脂肪细胞,成软骨细胞。
    3. 间充质干细胞的免疫荧光:用1×DPBS漂洗间充质干细胞一次,并在室温下用在1×DPBS中稀释的4%多聚甲醛(PFA)固定10分钟。
    4. 用在1x DPBS中制备的0.2%Triton X-100对细胞透化3分钟并用封闭缓冲液DPBS封闭溶液封闭30分钟。
    5. 轻轻地去除封闭溶液,并在4°C与第一抗体孵育过夜。脂肪细胞标志物:山羊抗小鼠FABP4抗原亲和纯化的多克隆抗体。软骨细胞标记:绵羊抗小鼠胶原II抗原亲和纯化的多克隆抗体。骨髓细胞标记物:山羊抗小鼠骨桥蛋白抗原亲和纯化的多克隆抗体。在含有0.1%BSA的1x DPBS中稀释一抗至1:500。
    6. 用含有0.1%BSA的1x DPBS在室温下洗涤载玻片三次,每次洗涤10分钟。在室温下用含有0.1%BSA的1x DPBS稀释至1:1,000的二抗孵育至少2小时。使用驴抗山羊和驴抗羊IgG二抗。
    7. 用含有0.1%BSA的1x DPBS在室温下洗涤载玻片三次,每次洗涤10分钟。
    8. 最后一次洗涤后,将核染料DAPI以1:2,000在1x DPBS中加入5分钟以评估核形态。

    9. 用1x DPBS洗涤3次,每次5分钟
    10. 删除多余的DPBS并使用Vectashield安装介质安装幻灯片。
    11. 使用任何免疫荧光显微镜以20倍物镜采集图像(图2)。

    12. 来自野生型C57BL / 6J小鼠的脂肪细胞:软骨细胞:骨细胞的比率是60:20:20。


      图2.用抗体Fabp4,胶原蛋白II和骨桥蛋白染色的小鼠BM MSC的代表性图像,以鉴定存在于骨髓衍生的间充质基质细胞中的脂肪细胞,成软骨细胞和成骨细胞谱系细胞。比例尺代表10 μm。

  3. 小鼠骨髓的分离造血干细胞(HSC)
    1. 用细胞表面标志物染色后,使用流式细胞术从骨髓单核细胞(BMMCs)中分离HSC,所述细胞表面标志物对Lineage鸡尾酒阴性并且对Sca-1,c-Kit(谱系阴性,Sca-1阳性,c-Kit阳性,LSK)呈阳性。 HSC也被称为LSK。

    2. 使用洗涤缓冲液将细胞从骨骼中轻轻冲洗出来,从小鼠股骨获得BM细胞。
    3. 通过在室温下以500μgxg离心5分钟,用洗涤缓冲液洗涤BM细胞两次。
    4. 用3ml洗涤缓冲液重悬细胞并轻轻覆盖在6ml Ficoll-Paque PLUS的15ml锥形管中。
    5. 小心地将稀释的细胞样本铺在Ficoll-Paque PLUS溶液上,并采取预防措施,避免混合细胞和Ficoll。
    6. 在室温下400×g离心30分钟,缓慢加速并关闭制动器。

    7. 用无菌吸管吸出含血浆和血小板的上层,使BMMC层不受干扰。

    8. 使用无菌移液器将BMMC层转移到无菌离心管中。

    9. 用清洗缓冲液清洗BMMC
    10. 沉淀BMMC并用抗体谱系(Lin)混合物(针对CD3e,CD11b,CD45R / B220,Gr-1和Ter119的生物素化的抗小鼠抗体)进行第一次染色。加入2μl的每种抗体染色一百万个BMMCs。将细胞与抗体在4°C孵育20分钟。
    11. 用骨髓洗涤缓冲液洗一次并沉淀细胞。
    12. 此外,加入5μL荧光标记的链霉亲和素以结合谱系抗体。
    13. 用缓冲液清洗BMMC一次并沉淀细胞。此外,加入2μlSca-1和c-kit与荧光染料结合染色一百万个BMMC。
    14. Wash Triple(Lineage,Sca-1,c-kit)将BMMC偶联染色一次,并将1百万个细胞悬浮在1 ml洗涤缓冲液中进行流动分选。
    15. 使用FACSDiva软件对BD FACS Aria II进行流式分类。
    16. 使用FlowJo软件分析流式细胞术数据以检查LSK细胞的质量,如伪色图(图3)中所示。


      图3.从小鼠骨髓鉴定和分离造血干细胞的门控策略从小鼠股骨冲洗的骨髓细胞经Ficoll-Paque PLUS分离以分离骨髓单核细胞,然后用使用流式细胞仪鉴别和细胞分选HSC细胞表面标记。

  4. 鹅卵石区域形成细胞分析
    1. 在使用流式细胞术分离LSK细胞之前,培养MSC以在35mm培养皿或6孔板中完成融合。

    2. 第3至第6代之间的MSC应该用于电镀LSK细胞
    3. 在测定当天,使用流式细胞术对所述骨髓LSK细胞进行分选(程序C)。
    4. 沉淀分离的LSK细胞并用MSC培养基重悬。在悬浮有LSK细胞的培养基中更换培养皿中培养MSC的培养基。大约在35mm培养皿中融合的MSC上铺2×10 3 LSK细胞。
    5. 在37℃,5%CO 2下在潮湿的培养箱中共培养细胞以使前体细胞在基质层下形成造血克隆。

    6. 。鹅卵石区域早在5天就开始出现,可以在7天内出现
    7. 通过替换一半的培养基每周喂养培养物两次。使用相差显微镜(图4)以10倍物镜拍摄相干黑暗造血克隆,并使用奥林巴斯cellSens软件等软件安装。


      图4.鹅卵石区域形成细胞分析的代表性图像 HSCs在融合的MSC层上培养形成鹅卵石区域。黄色箭头相消化细胞(在MSCs层下生长的真干细胞),蓝色箭头相明亮细胞(干细胞悬浮或松散地附着于MSC)和红色箭头-MSC(汇合细胞层粘附于塑料盘) 。比例尺代表50微米。

  5. CAFC有限稀释法
    1. 限制性稀释测定法(LDA)在具有汇合MSC的96孔平底培养板中进行,LSK细胞的稀释液(0,10,30,90,270和810)在每个孔中以3倍差异进行。在每个稀释度的平底96孔板中的10个孔中平板MSC(图5A)。
      没有LSK细胞加入到0稀释孔中,它们作为阴性对照。
    2. 用来自小鼠骨髓的独立衍生的MSC进行三个不同的LDA实验。
    3. 如果它包含一个或多个鹅卵石区域,则评价为“正面”,如果不包含鹅卵石区域则评价为“负面”。
    4. 鹅卵石区至少有6个细胞(彼此靠近)生长在基质下。虽然鹅卵石样细胞呈现为黑暗相,但由于光线的偏转,这些细胞在96孔板中表现为无瑕疵。

    5. 只有阴性和阳性的稀释液才能用于频率分析(图5B)

      图5. CAFC有限稀释测定法A.在96孔平底板中进行CAFC测定。计数流动分选的LSK细胞并在平底96孔板中铺板在融合的MSC上。每个测定一式三份进行。在没有LSK细胞,90和270LSK细胞的孔中形成鹅卵石的代表性图像。比例尺表示100μm。一部分鹅卵石区域被放大了;比例尺表示50微米。在虚线圆圈中鉴定相消隐细胞,而在实心圆圈中鉴定相明亮细胞。相位暗淡和相位明亮的细胞需要在场才能称之为真正的鹅卵石区域。 B.如上图所示,在每个孔中测量相消隐细胞面积。对铺满鹅卵石区域的孔的百分比进行计数,并将其与每孔中铺板的LSK细胞的数量作图。
      文化保持两周,并在每周结束时对这些区域进行计数。

数据分析

使用GraphPad Prism软件绘制图表并分析数据。使用 t -test比较LDA中两组的平均值。

笔记

  1. 当冷冻保存时,MSC不具有高回收率。所以总是培养并用新鲜的MSC进行检测。
  2. 为了防止悬浮在培养基中的MSCs和LSK细胞破裂,请使用大孔径移液器吸头。
  3. 只有当MSC生长到完全融合时才进行CAFC检测。
  4. 将LSK细胞表面标记添加到悬浮于200μl或更少体积缓冲液的BMMC中。

食谱

  1. 骨髓冲洗缓冲液
    Dulbecco的磷酸盐缓冲盐水制成1倍浓度(1x DPBS)
    10%FBS
  2. MSCs媒体
    Iscove的改良Dulbecco的媒介
    500毫升
    牛小牛血清
    20%
    表皮生长因子(rmEGF)
    10 ng / ml
    血小板衍生的生长因子(rhPDGF)
    200 ng /μl
    青霉素 - 链霉素
    1%
    2-巯基乙醇
    10 -4 mol / L
  3. DPBS阻止解决方案
    1x DPBS
    1%Triton X-100
    1%DMSO
    1%BSA
    1%血清(来自二次抗体产生的动物的血清)
  4. 致谢

    该协议是根据de Haan和Ploemacher于2002年发表在“造血干细胞方案”(Hematopoietic Stem Cell Protocols)中的以前的工作改编的。 LSK细胞的细胞分选在Cincinnati儿童医院医疗中心的研究流式细胞核心处进行。这项调查得到了美国国立卫生研究院拨款R01 HL076712,R01 CA157537和T32 HL091805的支持。

    是一个由白血病和淋巴瘤学者奖提供支持的Qishen Pang 利益冲突披露:作者声明没有竞争的财务利益。
    贡献:S.A.设计并进行研究,分析数据并撰写论文; Q. P.设计的研究,贡献了重要的新试剂,分析了数据。

    参考

    1. Amarachintha,S.,Sertorio,M.,Wilson,A.,Li,X.和Pang,Q.(2015)。 范可尼贫血间充质基质细胞衍生的甘油磷脂通过Toll样受体信号传导阻碍造血干细胞分化。 / a> 干细胞 33(11):3382-3396。
    2. de Haan,G.和Ploemacher,R.(2002)。 鹅卵石区域形成细胞检测在:造血干细胞方案。在:Klug,C.A。和Jordan,C.T。(Eds)中。 Humana Press ,Totowa 143-151。
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    4. Dominici,M.,Le Blanc,K.,Mueller,I.,Slaper-Cortenbach,I.,Marini,F.,Krause,D.,Deans,R.,Keating,A.,Prockop,D。和Horwitz, E.(2006)。 确定多潜能间充质基质细胞的最低标准。国际细胞治疗协会立场声明。细胞疗法 8(4):315-317。
    5. Hartwell,KA,Miller,PG,Mukherjee,S.,Kahn,AR,Stewart,AL,Logan,DJ,Negri,JM,Duvet,M.,Jaras,M.,Puram,R.,Dancik,V.,Al -Thalor,F.,Kindler,T.,Tothova,Z.,Chattopadhyay,S.,Hasaka,T.,Narayan,R.,Dai,M.,Huang,C.,Shterental,S.,Chu,LP, Haydu,JE,Shieh,JH,Steensma,DP,Munoz,B.,Bittker,JA,Shamji,AF,Clemons,PA,Tolliday,NJ,Carpenter,AE,Gilliland,DG,Stern,AM,Moore,MAS,Scadden ,DT,Schreiber,SL,Ebert,BL和Golub,TR(2013)。 基于小生境的筛查可鉴定白血病干细胞的小分子抑制剂。 Nat Chem Biol 9(12):840-848。
    6. Hu,X.,Garcia,M.,Weng,L.,Jung,X.,Murakami,J.L.,Kumar,B.,Warden,C.D。,Todorov,I和Chen,C.C。(2016)。 鉴定成人造血龛的常见间充质基质祖细胞 Nat Commun 7:13095.
    7. Keating,A。(2012)。 间充质干细胞:新方向 干细胞 10(6):709-716。
    8. Lim,M.,Pang,Y.,Ma,S.,Hao,S.,Shi,H.,Zheng,Y.,Hua,C.,Gu,X.,Yang,F.,Yuan,W。和Cheng,T。(2016)。 改变骨髓间充质细胞阻碍白血病骨髓中正常造血祖细胞的生成 Leukemia 30(1):154-162。
    9. Ploemacher,R.E.,van der Sluijs,J.P.,Voerman,J.S。和Brons,N.H。(1989)。 长期重建造血干细胞的体外极限稀释测定法鼠标中的细胞。 血液 74(8):2755-2763。
    10. Robinson,SN,Ng,J.,Niu,T.,Yang,H.,McMannis,JD,Karandish,S.,Kaur,I.,Fu,P.,Del Angel,M.,Messinger,R.,Flagge ,F.,de Lima,M.,Decker,W.,Xing,D.,Champlin,R。和Shpall,EJ(2006)。 高级体外脐血扩张与骨髓共培养后,骨髓移植 37(4):359-366。
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引用:Amarachintha, S. and Pang, Q. (2018). Cobblestone Area-forming Cell Assay of Mouse Bone Marrow Hematopoietic Stem Cells. Bio-protocol 8(9): e2824. DOI: 10.21769/BioProtoc.2824.
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