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Isolation of Mouse Cardiac Neural Crest Cells and Their Differentiation into Smooth Muscle Cells
小鼠心脏神经嵴细胞的分离及向平滑肌细胞的分化   

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Abstract

Cardiac neural crest cells (CNCCs) originate at the dorsal edge of the neural tube between the otic pit and the caudal edge of the 3rd somite, and migrate into the pharyngeal arches and the heart. We have shown that fibronectin (Fn1) plays an important role in the development of the CNCC by regulating the differentiation of CNCCs into vascular smooth muscle cells around pharyngeal arch arteries (Wang and Astrof, 2016). This protocol describes the isolation of CNCCs from the neural tube and from the caudal pharyngeal arches, and the differentiation of neural crest-derived cells into smooth muscle cells. This protocol was adapted from (Newgreen and Murphy, 2000; Pfaltzgraff et al., 2012).

Keywords: Cardiac neural crest(心脏神经嵴), Vascular smooth muscle cells(血管平滑肌细胞), Neural tube(神经管), Pharyngeal arch(咽弓), Differentiation(分化)

Background

Previous published protocols described the isolation of neural crest cells from the neural tube. However, neural crest cells in the region of the neural tube between the otic pit and the 3rd somite include neural crest cell populations that contribute to a number of different cell types; for example, vagal neural crest cells also originate from this region. In this protocol, we modified the conventional method for the isolation of cardiac neural crest cells. Instead of using the neural tube, we used the caudal pharyngeal arch region at embryonic day (E) 9.5 (22-25 somite stage). This is prior to differentiation of cardiac neural crest cells into vascular smooth muscle cells. It is common for neural crest cultures to contain contaminating mesenchymal cell types, which often express smooth muscle genes. To identify neural crest-derived cells, we isolated neural crest cells from embryos resulting from the following cross: Fn1flox/flox;ROSAmTmG/mTmG female mice x Fn1+/−;Tfap2αIRESCre/+ male mice. In 50% of the progeny from this cross, neural crest cells are lineage-labeled by the expression of GFP, so we could easily identify neural crest cells by their GFP expression without the need for cell sorting (Wang and Astrof, 2016). Additional Cre-expressing strains that can be used are Wnt1-Cre2 (Lewis et al., 2013) and P3ProCre (Li et al., 2000) transgenic strains, e.g., (Wang and Astrof, 2016). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University and conducted in accordance with federal guidelines for humane care of animals.

Materials and Reagents

  1. 12 mm round glass coverslips (Electron Microscopy Sciences, catalog number: 72231-01 )
  2. 24-well plates (Corning, Falcon®, catalog number: 353047 )
  3. Nunc 4-well dishes untreated (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 144444 )
  4. 35 cm Petri dish (Corning, Falcon®, catalog number: 353001 )
  5. 1.5 ml centrifuge tube
  6. Sterile transfer pipet (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: PP89SB )
  7. Glass pipet (Fisher Scientific, model: 13-678-6A )
  8. Parafilm
  9. Glass slide
  10. 0.2 μm syringe filter unit
  11. Pregnant mice
  12. Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 ), for dissection of embryos and for cell culture
  13. Trypsin (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  14. 4% PFA prepared in 1x PBS
  15. 0.1% Triton X-100 in 1x PBS
  16. Phosphate-buffered saline (PBS)
  17. Donkey serum (Sigma-Aldrich, catalog number: D9663-10ML )
  18. Anti-GFP antibody (Aves Labs, catalog number: GFP-1020 )
  19. Anti-αSMA antibody (Sigma-Aldrich, catalog number: SAB2500963 )
  20. Anti-calponin antibody (Abcam, catalog number: ab46794 )
  21. Donkey anti-goat IgG (H+L) secondary antibody, Alexa Fluor® 555 conjugate (Thermo Fisher Scientific, catalog number: A-21432 )
  22. Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor® 647 conjugate (Thermo Fisher Scientific, catalog number: A-31573 )
  23. Alexa Fluor® 488 AffiniPure F(ab’)2 fragment donkey anti-chicken IgY (IgG) (H+L) (Jackson ImmunoResearch, catalog number: 703-546-155 )
  24. ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, InvitrogeTM, catalog number: P36931 )
  25. Collagenase and dispase (Roche Diagnostics, catalog number: 10269638001 )
  26. Glacial acetic acid
  27. Collagen I (Corning, catalog number: 354249 )
  28. DMEM-low glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 11885076 )
  29. Neurobasal medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049 )
  30. Chicken embryo extract (MP Biomedicals, catalog number: 092850145 )
  31. Penicillin/Streptomycin (Pen/Strep) (Mediatech, catalog number: 30-001-CI )
  32. N2-supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 17502048 )
  33. B27-supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
  34. Retinoic acid (Sigma-Aldrich, catalog number: R2625 )
  35. Ethanol
  36. 2-Mercaptoethanol (Thermo Fisher Scientific, GibcoTM, catalog number: 21985023 )
  37. Fibroblast growth factor-basic (bFGF) (Sigma-Aldrich, catalog number: F0291 )
  38. Tris pH 7.6
  39. Insulin-like growth factor 1 (IGF-1) (R&D Systems, catalog number: 291-G1 )
  40. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-125 )
  41. DMEM-high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
  42. Collagenase/dispase stock solution (see Recipes)
  43. Collagen I working solution (see Recipes)
  44. Neural crest self-renewal medium (see Recipes)
  45. Differentiation medium (see Recipes)

Equipment

  1. Autoclave
  2. Biological safety hood (Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series Class II, Type A2 )
  3. Scissors (Fisher Scientific, model: 13-804-6 )
  4. Forceps 9 cm (Fine Science Tool, model: 14060-09 )
  5. Forceps 0.1 x 0.06 mm (Fine Science Tool, model: 11251-23 )
  6. Forceps 11 cm (Fine Science Tool, model: 11254-20 )
  7. EdgeGARD® horizontal flow hood (The Baker Company, model: EdgeGARD® HF )
  8. Flat bench
  9. Dissection microscope and light source (Carl Zeiss, model: Stemi 2000-C )
  10. Humidified, 37 °C tissue culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i )
  11. Fluorescence microscope

Procedure

  1. Coat glass coverslips and 4-well/24-well plates with collagen I working solution (150 μg/ml)
    1. Autoclave round glass coverslips, 13 mm diameter, in advance.
    2. Roughly estimate the desired number of round glass coverslips, and place one glass coverslip within one well of a 24-well plate in a biological safety hood. Coat each coverslip with enough collagen I (~400 μl for each well) at room temperature (RT) for 2 h.
    3. Remove collagen I, rinse coverslips with sterile H2O twice and allow the coverslips to dry overnight.
    4. Store coated plates and plates with coated coverslips at 4 °C for no more than 2 weeks.

  2. Embryo preparation
    Note: This can be done on a lab bench.
    1. Timed pregnant mice are sacrificed by CO2. To isolate neural crest cells from the neural tube, dissect embryos at the embryonic day (E) 9.0 (12-15 somite stage). To isolate neural crest-derived cells form pharyngeal arches posterior to the 2nd arch, dissect embryos at E9.5 (24-26 somite stage).
    2. Place the sacrificed mouse on its back, and wet the lower abdomen with 70% ethanol. Open the peritoneal cavity with fine scissors, push gut aside and expose the uterus.
    3. Cut the uterus from the cervix with fine scissors. Hold the broken end of the uterus with forceps, pull the uterus straight, open a small hole on the uterus, insert a fine tip of scissors into the uterus, and slowly cut open the uterus on the vessel-free edge of the uterus.
    4. Pick up the embryos and transfer to a dish with DPBS at RT.

  3. Isolation of neural crest cells from neural tubes (see Movie in Pfaltzgraff et al., 2012)
    Neural tube dissection and explant culture:
    Note: Dissection should be done inside a horizontal flow hood.
    1. Prepare collagenase/dispase solution fresh for each experiment: add 50 μl of 100 mg/ml collagenase/dispase stock solution to 5 ml DPBS at RT. Pipet 500 μl into each 1.5 ml centrifuge tube. One tube will be used to digest one embryo.
    2. Under dissection microscope, isolate E9.0 embryos by stripping of decidua and membranes with fine forceps. Trim off the tail of the embryo.
    3. Wash each embryo once in DPBS at RT by transferring it into a new plate with fresh DPBS, and then transfer the embryo into a 1.5 ml centrifuge tube containing 500 μl of the collagenase/dispase solution by using sterile plastic transfer pipet. Incubate embryos at 37 °C water bath for 7 min. The incubation time is critical. It is difficult to separate the neural tube from the surrounding tissue if the tissue is not digested enough; however, if over-digested, the neural tube will be curled.
    4. While waiting for the digestion, pipet 180 μl self-renewal medium into each well of a 4-well Nunc plate coated with collagen I, and label 1.5 ml centrifuge tubes, which will be used to save the rest of the embryo for genotyping.
    5. Wash embryos with DPBS three times. And transfer embryos to a 35-mm dish in DPBS.
    6. Under dissection microscope, hold the head of the embryo with one pair of forceps, and peel the surface ectoderm overlaying the cardiac neural tube segment (from otic vesicle to the 3rd somite) with another pair of fine forceps. Insert the tips of forceps between the lateral neural tube and the somites, and carefully separate them by moving the tip back and forth. Do the same for the other side of the neural tube. If the digestion is ideal, it should be very easy to do this step. Cut the neural tube at the level of the otic vesicle and the upper edge of the 4th somite, and free the neural tube from ventral tissues.
    7. Rinse the neural tube with self-renewal medium (Bixby et al., 2002) and transfer neural tube segments to the center of wells containing 180 μl self-renewal medium. The neural tube will dry out if too little medium is used and will float in the medium if too much is used. Place the neural tube laterally, so the neural crest cells can migrate out and attach to the bottom of the dish. Save the rest of the embryo for genotyping.
    8. 12 h later, when the neural tube is well-attached to the bottom, carefully and slowly add 350 μl of fresh self-renewal medium pre-warmed to 37 °C into each well and culture 4 more days. Replace half of the medium with fresh self-renewal medium on the 3rd day.

  4. Isolation of cardiac neural crest-derived cells from pharyngeal arches (Figure 1 and Video 1)
    Pharyngeal arch dissection and culture:
    Note: Dissection should be done within the horizontal flow hood.


    Figure 1. Work-flow of isolation cardiac neural crest cells from E9.5 mouse embryo and their differentiation into smooth muscle cells

    1. Prepare collagenase/dispase solution.
    2. Dissect E9.5 embryos.
    3. Digest embryos with collagenase/dispase at 37 °C in a water bath for 4 min; if multiple embryos are processed, digest one embryo at a time.
    4. While waiting for the digestion to complete, pipet 500 μl self-renewal medium into each well of 24-well plate coated with collagen I, and label 1.5 centrifuge tubes that will be used to save a small part of each embryo for genotyping.
    5. Wash each embryo with DPBS three times, and transfer to a 35-mm dish in DPBS for dissection.
    6. Video 1 starts at this step:
      1. Under dissection microscope, hold the head of the embryo with one pair of forceps, chop off the embryo’s tail and peel the surface ectoderm off of pharyngeal region (PA 3-6) with another pair of fine forceps.
      2. Trim the head off from the upper edge of PA 3. Trim the heart off by cutting through the aortic sac.
      3. Detach the pharyngeal arches from the dorsal tissue by cutting through both dorsal aortae. The pharyngeal arch tissue is composed of cardiac neural crest cells and other cell types, such as mesodermal and endodermal cells.

        Video 1. Demonstration of dissection of pharyngeal arches 3-6 from E9.5 embryos

    7. Transfer the dissected piece of pharyngeal tissue to a 24-well plate. The tissue will sink and attach to the bottom of the well in 12 h. Save the rest of the embryo for genotyping.
    8. Culture pharyngeal arch tissue for 4 days. And replace half of the medium with fresh self-renewal medium on the 3rd day.

  5. Smooth muscle cell differentiation
    Note: This should be done within a biological safety hood.
    1. To replate neural crest cells that have migrated from neural tube explant (Figure 2A), the neural tube should be removed by pipetting the medium up and down in the wells for about 5 times (neural crest cells will not be affected because they are tightly attached to the bottom). This step is omitted for the pharyngeal arch culture.
    2. Wash the wells containing neural crest cells once with DPBS pre-warmed to 37 °C, and incubate with 150 μl 0.25% trypsin for 1 min at 37 °C or until all cells round up and appear to be nearly detached from the plate.
    3. Add 350 μl differentiation medium into each well, pipet up and down about 5 times, to dissociate neural crest cells into single cells. Together with trypsin, wells now have 500 μl of medium. Cells can now be plated for differentiation, as described in the next step. There are usually enough cells to split into 4 wells of a 24-well plate.
    4. For differentiation, plate neural crest cells onto collagen I-coated coverslips in a 24-well plate, and culture for 2 h. When cells settle down, replace the medium with fresh differentiation medium and culture for 2 days.

  6. Immunostaining
    To identify neural crest-derived smooth muscle cells, neural crest cultures were stained for GFP (neural crest cells are GFP+) and smooth muscle cell markers, such as αSMA and Calponin.
    1. Rinse cells with DPBS and fix them at RT with 4% PFA pre-warmed to RT for 20 min, then rinse with DPBS 3 times, 5 min each, before proceeding for immunostaining.
    2. Permeabilize cells with 500 μl 0.1% Triton X-100 in PBS (PBST) for 10 min at RT.
    3. Get coverslips from wells, and place them in a flat dish covered with Parafilm. The dish should be shallow, and should be topped with a lid. Tuck a few wet Kimwipes near sides of the dish to create a humidified environment when the dish is closed. Make sure the side of the coverslip with cells faces up. Add 50 μl of blocking buffer (5% normal donkey serum in PBST) for 30 min at RT. This set up minimizes the volume of antibody solutions used for staining, only 30 μl solution is required to cover cells on the coverslip.
    4. Remove the blocking buffer by aspiration, and add 30 μl of primary antibody solution in blocking buffer for 2 h at RT. The antibody concentrations: anti-GFP 1:500; anti-αSMA 1:300; anti-calponin 1:150.
    5. Return coverslips to wells in a multi-well plate and wash with PBST 3 times, changing to fresh PBST every 10 min.
    6. Get coverslips from the wells and place them on Parafilm in the staining chamber, add secondary antibodies and incubate for 1 h at RT: anti-chicken, anti-goat, anti-rabbit conjugated to Alexa 488, Alexa 555, Alexa 647, all diluted at 1:300.
    7. Put coverslips back into the wells of a multi-well plate, and wash with PBST 3 times, changing to fresh PBST every 10 min.
    8. Place 10 μl of anti-fade mounting reagent (with DAPI) on a glass slide, then put the coverslip upside down on the reagent.
    9. Observe slides using a fluorescence microscope.

Data analysis

The percentage of neural crest-derived smooth muscle cells is determined by the number of αSMA+ calponin+ cells expressing GFP divided by the total number of GFP positive cells (Figures 2B-2B’’’). Detailed results can be found in our previous publication (Wang and Astrof, 2016).


Figure 2. Neural crest cell migration from neural tube explant and staining of cultured cardiac neural crest cells for smooth muscle cell markers. A. Neural crest cells migrate from the neural tube explant on glass coverslips. B-B’’’. Representative images of cardiac neural crest cells isolated from Fnf/+; ROSAmTmG; TFAP2αIRESCre/+ embryos. Cells were stained with antibodies to GFP (to identify neural crest-derived cells), αSMA, and calponin. DAPI was used to stain nuclei.

Recipes

  1. Collagenase/dispase stock solution (100 mg/ml)
    500 mg collagenase/dispase powder
    Dissolve to 5 ml Ca2+/Mg2+-free PBS
    Filter through a 0.2 μm syringe filter unit
  2. Collagen I working solution (150 μg/ml)
    58 μl glacial acetic acid
    50 ml ddH2O
    Filter through a 0.2 μm filter unit
    Add 0.817 ml collagen I concentrate (9.18 mg/ml)
  3. Neural crest self-renewal medium (100 ml)
    50 ml DMEM-low glucose
    30 ml neurobasal medium
    15 ml chick embryo extract
    1 ml Pen/Strep (P/S)
    1 ml N2
    2 ml B27
    100 μl retinoic acid (3.5 mg/ml, dissolve in 100% ethanol)
    100 μl 2-mercaptoethanol
    80 μl bFGF (25 μg/ml, dissolve in 5 mM Tris pH 7.6)
    40 μl IGF-1 (50 μg/ml, dissolve in PBS)
    680 μl ddH2O
  4. Differentiation medium
    500 μl FBS and 50 μl Pen/Strep
    Add to 4,450 μl DMEM-high glucose
    Get a final concentration of 10% FBS and 1% P/S

Acknowledgments

This work was supported by the funding from the National Institutes of Health [NHLBI RO1 HL103920 to S.A.], X.W. was supported by an American Heart Association Postdoctoral Fellowship [12POST11750033 to X.W.].

References

  1. Bixby, S., Kruger, G. M., Mosher, J. T., Joseph, N. M. and Morrison, S. J. (2002). Cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity. Neuron 35(4): 643-656.
  2. Lewis, A. E., Vasudevan, H. N., O'Neill, A. K., Soriano, P. and Bush, J. O. (2013). The widely used Wnt1-Cre transgene causes developmental phenotypes by ectopic activation of Wnt signaling. Dev Biol 379(2): 229-234.
  3. Li, J., Chen, F. and Epstein, J. A. (2000). Neural crest expression of Cre recombinase directed by the proximal Pax3 promoter in transgenic mice. Genesis 26(2): 162-164.
  4. Newgreen, D. F. and Murphy, M. (2000). Neural crest cell outgrowth cultures and the analysis of cell migration. Methods Mol Biol 137: 201-211.
  5. Pfaltzgraff, E. R., Mundell, N. A. and Labosky, P. A. (2012). Isolation and culture of neural crest cells from embryonic murine neural tube. J Vis Exp (64): e4134.
  6. Wang, X. and Astrof, S. (2016). Neural crest cell-autonomous roles of fibronectin in cardiovascular development. Development 143(1): 88-100.

简介

心脏神经嵴细胞(CNCC)起源于神经管的背部边缘,位于第3个体节的耳穴和尾缘之间,并迁移到咽弓和心脏。 我们已经表明,纤连蛋白(Fn1)通过调节CNCCs到咽弓动脉周围的血管平滑肌细胞的分化,在CNCC的发展中起重要作用(Wang and Astrof,2016)。 该方案描述了CNCC与神经管和尾尾弓的分离,以及神经嵴衍生细胞分化成平滑肌细胞。 该方案从(Newgreen和Murphy,2000; Pfaltzgraff等人,2012)改编。
【背景】以前发表的方案描述了从神经管分离神经嵴细胞。然而,在耳孔和第三体细胞之间的神经管区域中的神经嵴细胞包括有助于许多不同细胞类型的神经嵴细胞群体;例如,迷走神经嵴细胞也来自该区域。在该方案中,我们修改了用于分离心脏神经嵴细胞的常规方法。而不是使用神经管,我们在胚胎期(E)9.5(22-25个体节期)使用尾部咽部弓形区。这是在将心脏神经嵴细胞分化为血管平滑肌细胞之前。神经嵴培养物通常含有污染性间充质细胞,通常表达平滑肌基因。为了鉴定神经嵴衍生细胞,我们从以下交叉产生的胚胎中分离出神经嵴细胞:Fn1flox / flox; ROSAmTmG / mTmG雌性小鼠×Fn1 +/-;Tfap2αIRESCre/ +雄性小鼠。在这个十字架的50%的后代,神经嵴细胞被GFP的表达谱系标记,所以我们可以很容易地通过GFP表达来识别神经嵴细胞,而不需要细胞分选(Wang and Astrof,2016)。可以使用的另外的Cre表达菌株是Wnt1-Cre2(Lewis等人,2013)和P3ProCre(Li等人,2000)转基因株,例如(Wang和Astrof,2016)。所有实验程序均由托马斯·杰斐逊大学的机构动物护理和使用委员会批准,并按照联邦人类动物护理指南进行。

关键字:心脏神经嵴, 血管平滑肌细胞, 神经管, 咽弓, 分化

材料和试剂

  1. 12毫米圆形玻璃盖玻片(电子显微镜科学,目录号:72231-01)
  2. 24孔板(Corning,Falcon ®,目录号:353047)
  3. 未处理的Nunc 4孔培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:144444)
  4. 35厘米培养皿(Corning,Falcon ®,目录号:353001)
  5. 1.5 ml离心管
  6. 无菌转移吸管(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:PP89SB)
  7. 玻璃移液管(Fisher Scientific,型号:13-678-6A)
  8. 石蜡玻璃
  9. 玻璃幻灯片
  10. 0.2μm注射器过滤器单元
  11. 怀孕的老鼠
  12. Dulbecco的磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190144),用于胚胎和细胞培养的分离
  13. 胰蛋白酶(Thermo Fisher Scientific,Gibco TM ,目录号:25200056)
  14. 4%PFA在1x PBS中制备
  15. 0.1%Triton X-100在1x PBS中
  16. 磷酸盐缓冲盐水(PBS)
  17. 驴血清(Sigma-Aldrich,目录号:D9663-10ML)
  18. 抗GFP抗体(Aves Labs,目录号:GFP-1020)
  19. 抗αSMA抗体(Sigma-Aldrich,目录号:SAB2500963)
  20. 抗钙调蛋白抗体(Abcam,目录号:ab46794)
  21. 驴抗山羊IgG(H + L)二抗,Alexa Fluor 555结合物(Thermo Fisher Scientific,目录号:A-21432)
  22. 驴抗兔IgG(H + L)二抗,Alexa Fluor 6470缀合物(Thermo Fisher Scientific,目录号:A-31573)
  23. Alexa Fluor 488 AffiniPure F(ab')2片段驴抗鸡IgY(IgG)(H + L)(Jackson ImmunoResearch,目录号:703-546-155)
  24. ProLong Gold Antifade Mountant with DAPI(Thermo Fisher Scientific,Invitroge TM ,目录号:P36931)
  25. 胶原酶和分散素(Roche Diagnositcs,目录号:10269638001)
  26. 冰醋酸
  27. 胶原蛋白I(康宁,目录号:354249)
  28. DMEM-低葡萄糖(Thermo Fisher Scientific,Gibco TM,目录号:11885076)
  29. Neurobasal培养基(Thermo Fisher Scientific,Gibco TM,目录号:21103049)
  30. 鸡胚提取物(MP Biomedicals,目录号:092850145)
  31. 青霉素/链霉素(Pen / Strep)(Mediatech,目录号:30-001-CI)
  32. N2补充剂(Thermo Fisher Scientific,Gibco TM,目录号:17502048)
  33. B27补充剂(Thermo Fisher Scientific,Gibco TM,目录号:17504044)
  34. 视黄酸(Sigma-Aldrich,目录号:R2625)
  35. 乙醇
  36. 2-巯基乙醇(Thermo Fisher Scientific,Gibco TM,目录号:21985023)
  37. 成纤维细胞生长因子碱性(bFGF)(Sigma-Aldrich,目录号:F0291)
  38. Tris pH 7.6
  39. 胰岛素样生长因子1(IGF-1)(R& D Systems,目录号:291- G1)
  40. 胎牛血清(FBS)(Gemini Bio-Products,目录号:100-125)
  41. DMEM-高葡萄糖(Thermo Fisher Scientific,Gibco TM,目录号:11965092)
  42. 胶原酶/分散液储备溶液(参见食谱)
  43. 胶原蛋白I工作方案(见食谱)
  44. 神经嵴自我更新培养基(见食谱)
  45. 分化培养基(参见食谱)

设备

  1. 高压灭菌器
  2. 生物安全罩(Thermo Fisher Scientific,Thermo Scientific TM,型号:1300 Series II,A2型)
  3. 剪刀(Fisher Scientific,型号:13-804-6)
  4. 镊子9厘米(精细科学工具,型号:14060-09)
  5. 镊子0.1 x 0.06 mm(精细科学工具,型号:11251-23)
  6. 镊子11厘米(精细科学工具,型号:11254-20)
  7. EdgeGARD ®水平流动罩(贝克公司,型号:EdgeGARD ® HF)
  8. 平板凳
  9. 解剖显微镜和光源(Carl Zeiss,型号:Stemi 2000-C)
  10. 加湿的37℃组织培养培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heracell TM 150i)
  11. 荧光显微镜

程序

  1. 涂层玻璃盖玻片和具有胶原蛋白I工作溶液(150μg/ ml)的4孔/ 24孔板
    1. 高压灭菌玻璃盖玻片,直径13毫米,提前。
    2. 大概估计所需数量的圆形玻璃盖玻片,并将一个玻璃盖玻片放在生物安全罩中的24孔板的一个孔内。在室温(RT)下,每个盖玻片用足够的胶原蛋白I(每孔约400μl)涂覆2小时。
    3. 去除胶原蛋白I,用无菌H 2 O 2冲洗盖玻片两次,并允许盖玻片干燥过夜。
    4. 将涂有盖玻片的涂层板和板在4°C下放置不超过2周。

  2. 胚胎准备
    注意:这可以在实验台上完成。
    1. 定时怀孕的小鼠被CO 2处死。从神经管中分离神经嵴细胞,在胚胎期(E)9.0(12-15个体节期)切割胚胎。将神经嵴衍生细胞分离形成2> nd> form form form form arch at at at at at at at at at at at at。。。。。。。。。。。。。。。。。。。。。
    2. 将牺牲的小鼠放在其背上,并用70%乙醇湿下腹部。用精细的剪刀打开腹腔,将肠子推开,露出子宫。
    3. 用精细的剪刀从子宫颈切下子宫。用钳子握住子宫的断端,直接拉子宫,在子宫上打开一个小洞,将一小把剪刀插入子宫,慢慢切开子宫无血管边缘的子宫。 />
    4. 拿起胚胎并转移到带有DPBS的盘子上。

  3. 从神经管中分离神经嵴细胞(见Pfaltzgraff等人的电影,2012)
    神经管解剖和外植体培养:
    注意:解剖应在水平流动罩内进行。
    1. 为每个实验新鲜制备胶原酶/分散液:在室温下,加入50μl100 mg / ml胶原酶/分散液储存液至5ml DPBS。在每个1.5 ml离心管中吸取500μl。一条管将用于消化一个胚胎。
    2. 在解剖显微镜下,通过用细镊子剥离蜕膜和膜分离E9.0胚胎。修剪胚胎的尾巴。
    3. 通过将新鲜DPBS转移到新板中,每个胚胎在DPBS中洗涤一次,然后通过使用无菌塑料转移移液管将胚胎转移到含有500μl胶原酶/分散液的1.5ml离心管中。在37℃水浴孵育胚胎7分钟。孵化时间至关重要。如果组织不能消化足够的话,难以将神经管与周围组织分离;然而,如果过度消化,神经管将卷曲。
    4. 在等待消化的同时,将180μl自我更新培养基移液到涂有胶原蛋白I的4孔Nunc板的每个孔中,并标记1.5 ml离心管,用于保存胚胎的其余部分进行基因分型。
    5. 用DPBS洗涤胚胎三次。并将胚胎转移到DPBS中的35mm盘中。
    6. 在解剖显微镜下,用一对镊子握住胚胎的头部,并用另一对精镊子剥离覆盖心脏神经管段(从耳囊到3 rd somite)的表面外胚层。在侧向神经管和体节之间插入镊子的尖端,并通过前后移动尖端来仔细分离镊子。对神经管的另一侧做同样的事情。如果消化是理想的,那么这个步骤应该很容易。将神经管切割在耳囊水平和4 体细胞的上边缘,并从腹侧组织释放神经管。
    7. 用自我更新培养基(Bixby等人,2002)冲洗神经管,并将神经管段转移到含有180μl自我更新培养基的孔的中心。如果使用的介质太少,神经管会变干,并且如果使用太多,它将漂浮在培养基中。放置神经管横向,所以神经嵴细胞可以移出并附着在盘的底部。保存胚胎的其余部分进行基因分型。
    8. 12小时后,当神经管附着在底部时,仔细缓慢加入350μl新鲜自我更新培养基,预热至37°C,每孔培养4天。在3小时以上的时候,用新鲜的自我更新介质取代一半的培养基。

  4. 从咽部拱门分离心脏神经嵴衍生细胞(图1和视频1)
    咽弓解剖与文化:
    注意:解剖应在水平流动罩内进行。


    图1.来自E9.5小鼠胚胎的分离心脏神经嵴细胞的工作流程及其分化成平滑肌细胞

    1. 准备胶原酶/分散液。
    2. 解剖E9.5胚胎。
    3. 用胶原酶/分解酶在37℃在水浴中消化4分钟;如果多个胚胎被处理,一次消化一个胚胎。
    4. 在等待消化完成时,将500μl自我更新培养基移入每个涂有胶原蛋白I的24孔板的每个孔中,并标记1.5个离心管,将用于保存每个胚胎的一小部分进行基因分型。 />
    5. 用DPBS洗涤每个胚胎三次,并转移到DPBS中的35mm皿中进行解剖。
    6. 视频1从此步骤开始:
      1. 在解剖显微镜下,用一对镊子握住胚胎的头部,切下胚胎的尾巴,用另一对精镊从咽部区域(PA 3-6)剥离表皮外胚层。
      2. 将头部从PA 3的上边缘修剪掉。通过切开主动脉囊来调整心脏。
      3. 通过切开背侧主动脉将背腹组织分离出来。咽组织由心脏神经嵴细胞和其他细胞类型组成,如中胚层和内胚层细胞。

        Video 1. Demonstration of dissection of pharyngeal arches 3-6 from E9.5 embryos

        To play the video, you need to install a newer version of Adobe Flash Player.

        Get Adobe Flash Player


    7. 将解剖的咽部组织转移到24孔板上。组织将在12小时内下沉并附着在井的底部。保存胚胎的其余部分进行基因分型。
    8. 文化咽组织4天。在3日上午,您可以用新鲜的自我更新介质取代一半的介质。

  5. 平滑肌细胞分化
    注意:这应该在生物安全罩内完成。
    1. 为了复制从神经管外植体迁移的神经嵴细胞(图2A),应通过在孔中上下移动培养基约5次来除去神经管(神经嵴细胞不会受到影响,因为它们紧密连接至底部)。咽部文化遗漏这一步骤。
    2. 将含有神经嵴细胞的孔用DPBS预温至37℃洗涤一次,并在37℃下用150μl0.25%胰蛋白酶孵育1分钟,或直到所有细胞四舍五入,似乎几乎与板分离。 br />
    3. 向每个孔中加入350μl分化培养基,上下移动5次,将神经嵴细胞分离成单个细胞。与胰蛋白酶一起,现在有500微升的培养基。现在可以将细胞电镀以进行分化,如下一步所述。通常有足够的细胞分解成24孔板的4口井。
    4. 为了分化,将板神经嵴细胞置于24孔板中的胶原I包被的盖玻片上,并培养2小时。当细胞沉降时,用新鲜分化培养基更换培养基并培养2天
  6. 免疫染色
    为了鉴定神经嵴衍生的平滑肌细胞,将神经嵴培养物染色GFP(神经嵴细胞是GFP + )和平滑肌细胞标记物,例如αSMA和Calponin。
    1. 用DPBS冲洗细胞,并用预热至RT的4%PFA在室温下固定20分钟,然后用DPBS冲洗3次,每次5分钟,然后进行免疫染色。
    2. 在PBS(PBST)中将500μl0.1%Triton X-100在室温下将细胞渗透10分钟。
    3. 从井中取出盖玻片,将它们放在用Parafilm覆盖的平盘上。该盘应较浅,并应盖上盖子。在盘子的两侧附近几个湿的Kimwipes,在菜肴关闭时创造一个潮湿的环境。确保盖玻片的一面与细胞面朝上。在室温下加入50μl封闭缓冲液(PBST中5%正常驴血清)30分钟。这种设置使用于染色的抗体溶液的体积最小化,只需要30μl溶液来覆盖盖玻片上的细胞。
    4. 通过抽吸去除封闭缓冲液,并在室温下在封闭缓冲液中加入30μl一抗溶液2小时。抗体浓度:抗GFP 1:500;抗αSMA1:300;抗calponin 1:150。
    5. 将盖玻片返回到多孔板中的孔中并用PBST洗涤3次,每10分钟更换为新鲜的PBST。
    6. 从孔中取盖玻片并将其置于染色室中的Parafilm上,加入二抗并在室温孵育1小时:抗鸡,抗山羊,与Alexa 488缀合的抗兔,Alexa 555,Alexa 647,全部稀释在1:300
    7. 将盖玻片放回多孔板的孔中,用PBST洗涤3次,每10分钟更换成新鲜的PBST。
    8. 将10μl抗褪色安装试剂(带DAPI)放在玻璃片上,然后将盖玻片倒置在试剂上。
    9. 使用荧光显微镜观察载玻片。

数据分析

神经嵴衍生的平滑肌细胞的百分比由表达GFP的αSMA + / sup> calponin + 细胞数除以GFP阳性细胞总数确定(图2B- 2B ''')。详细的结果可以在我们以前的出版物(Wang和Astrof,2016)中找到。


图2.来自神经管外植体的神经嵴细胞迁移和用于平滑肌细胞标记物的培养的心脏神经嵴细胞的染色 A.神经嵴细胞从玻璃盖玻片上的神经管外植体迁移。 B-B”””。从fn分离的心脏神经嵴细胞的代表性图像 ROSA mTmG ;胚胎的TFAP2α IRESCre / + 胚胎。细胞用GFP抗体(鉴定神经嵴衍生的细胞),αSMA和calponin染色。 DAPI用于染色细胞核。

食谱

  1. 胶原酶/分散液储备溶液(100mg / ml)
    500毫克胶原酶/分散粉末
    溶解至5ml Ca 2+,无磷PBS
    通过0.2μm注射器过滤器单元过滤
  2. 胶原蛋白I工作溶液(150μg/ ml)
    58μl冰醋酸
    50ml ddH 2 O
    通过0.2μm过滤器单元过滤
    加入0.817ml胶原蛋白I浓缩物(9.18mg / ml)
  3. 神经嵴自我更新培养基(100 ml)
    50毫升DMEM低葡萄糖
    30 ml神经巴氏培养基 15毫升鸡胚提取物
    1 ml Pen / Strep(P / S)
    1 ml N2
    2 ml B27
    100μl视黄酸(3.5mg / ml,溶于100%乙醇) 100μl2-巯基乙醇
    80μlbFGF(25μg/ ml,溶于5mM Tris pH 7.6) 40μlIGF-1(50μg/ ml,溶于PBS) 680μlddH 2 O O
  4. 分化培养基
    500μlFBS和50μlPen / Strep
    加入4,450μlDMEM-high葡萄糖 最终浓度为10%FBS和1%P / S

致谢

这项工作得到了美国国家卫生研究院(NHLBI RO1 HL103920至S.A.),X.W.的资助。得到了美国心脏病学会博士后研究资助[12POST11750033给X.W.]。

参考

  1. Bixby,S.,Kruger,GM,Mosher,JT,Joseph,NM and Morrison,SJ(2002)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih。 gov / pubmed / 12194865“target =”_ blank“>来自周围神经系统不同区域的干细胞之间的细胞固有差异调节神经多样性的产生。神经元 35(4) :643-656。
  2. Lewis,AE,Vasudevan,HN,O'Neill,AK,Soriano,P.和Bush,JO(2013)。  广泛使用的Wnt1-Cre 转基因通过异位激活Wnt信号传导引起发育表型。 Dev Biol 379(2):229-234。
  3. Li,J.,Chen,F.and Epstein,JA(2000)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/10686619”target =“_ blank”>由转基因小鼠中的近端Pax3启动子引导的Cre重组酶的神经嵴表达。 26(2):162-164。
  4. Newgreen,DF and Murphy,M。(2000)。  神经嵴细胞生长培养和细胞迁移分析。方法Mol Biol 137:201-211。
  5. Pfaltzgraff,ER,Mundell,NA和Labosky,PA(2012)。  来自胚胎鼠神经管的神经嵴细胞的分离和培养。 (64):e4134。
  6. Wang,X.和Astrof,S。(2016)。纤维连接蛋白在心血管发育中的神经嵴细胞自主作用。 143(1):88-100。
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引用:Wang, X. and Astrof, S. (2017). Isolation of Mouse Cardiac Neural Crest Cells and Their Differentiation into Smooth Muscle Cells. Bio-protocol 7(17): e2530. DOI: 10.21769/BioProtoc.2530.
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