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Ex vivo Culture of Fetal Mouse Gastric Epithelial Progenitors
胎鼠胃上皮祖细胞的离体培养   

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Abstract

Isolation and tridimensional culture of murine fetal progenitors from the digestive tract represents a new approach to study the nature and the biological characteristics of these epithelial cells that are present before the onset of the cytodifferentiation process during development. In 2013, Mustata et al. described the isolation of intestinal fetal progenitors growing as spheroids in the ex vivo culture system initially implemented by Sato et al. (2009) to grow adult intestinal stem cells. Noteworthy, fetal-derived spheroids have high self-renewal capacity making easy their indefinite maintenance in culture. Here, we report an adapted protocol for isolation and ex vivo culture and maintenance of fetal epithelial progenitors from distal pre-glandular stomach growing as gastric spheroids (Fernandez Vallone et al., 2016).

Keywords: ex vivo(离体), Spheroids(球体), Mouse fetal epithelium(胎鼠上皮), Progenitors(祖细胞), 3D(3D), Single cells(单细胞)

Background

Mouse adult stem cells from the glandular stomach can be grown ex vivo in a 3D matrigel as ‘mini-glands’ for indefinite periods of time (Barker et al., 2010). As compared to stem cells from the small intestine growing in presence of EGF, Noggin and R-spondin 1, adult gastric stem cells need to be further supplemented with Fgf10, Gastrin, Wnt3a and a higher concentration of R-spondin 1 to get productive long-term cultures. In contrast, little was known till recently about the fetal cells that line the pre-glandular epithelium during development. So far, their nature as well as their potential growth properties ex vivo were uncharacterized. Based on the previous study identifying the cells present in the fetal small intestine (Mustata et al., 2013), we report on the culture of mouse fetal gastric progenitors as spheroids (Fernandez Vallone et al., 2016). Gastric progenitors can be replated in the culture medium previously reported by Sato et al., 2009 to grow small intestinal adult stem cells and, contrary to adult-type gastric stem cells, they do not need extra growth factors supplementation (Fgf10, Wnt3a or Gastrin).

Materials and Reagents

  1. Disposable scalpels (Swan Morton, catalog number: 0510 )
  2. Petri dishes 92 x 16 mm with cams (SARSTEDT, catalog number: 82.1473 )
  3. Microcentrifuge tubes, 1.5 ml (VWR, catalog number: 212-0198 )
  4. Tubes 10 ml, 100 x 16 mm, PP (SARSTEDT, catalog number: 62.9924.284 )
  5. Tubes 50 ml, 30 x 115 mm, PP (Corning, Falcon®, catalog number: 352070 )
  6. 70 µm nylon filters (Corning, Falcon®, catalog number: 352350 )
  7. P6 well plate (VWR, catalog number: 734-2323 )
  8. 40 µm nylon filters (Corning, Falcon®, catalog number: 352340 )
  9. P12 well plate (VWR, catalog number: 734-2324 )
  10. Tips refill (VWR, catalog numbers: 89079-464 ; 89079-470 ; 89079-478 )
  11. Cryotubes 1 ml (Greiner Bio One, catalog number: 123263 )
  12. Syringe filter 0.2 µm (VWR, catalog number: 28145-477 )
  13. Serological pipets 5 ml, 10 ml and 25 ml (Corning, Falcon®, catalog numbers: 357543 ; 357551 ; 357535 )
  14. Mice (tested on RjOrl:SWISS and C57BL/6JRj backgrounds)
  15. Dulbecco’s phosphate-buffered saline (DPBS), CaCl2 free, MgCl2 free (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-094 )
  16. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270 )
  17. Stem Pro Accutase cell dissociation reagent (Thermo Fisher Scientific, GibcoTM, catalog number: A1110501 )
  18. Matrigel® basement membrane matrix (Corning, catalog number: 354234 )
  19. Liquid nitrogen (supplied from Air liquide)
  20. Ethanol 95-97% (VWR, TechniSolv®, catalog number: 84857.360 )
  21. Glucose (Merck Millipore, catalog number: 1083371000 )
  22. Leibovitz’s L-15 medium (Thermo Fisher Scientific, catalog number: 11415-049 )
  23. 500 mM EDTA (pH 8.0) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575-038 )
  24. Albumin from bovine serum (BSA) (Sigma-Aldrich, catalog number: A3294 )
  25. Advanced DMEM/F12 (Thermo Fisher Scientific, GibcoTM, catalog number: 12634-010 )
  26. Gentamycin 50 mg/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15750-037 )
  27. Penicillin-streptomycin cocktail 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  28. Amphotericin B 250 µg/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15290-026 )
  29. L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-081 )
  30. N-2 supplement 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 17502-048 )
  31. B-27 w/o vit. A 50x (Thermo Fisher Scientific, GibcoTM, catalog number: 12587-010 )
  32. 1 M HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630-056 )
  33. N-acetyl cysteine (Sigma-Aldrich, catalog number: A7250 )
  34. Growth factors
    Recombinant murine EGF (Peprotech, catalog number: 315-09 )
    Recombinant murine Noggin (Peprotech, catalog number: 250-38 )
    Recombinant murine CHO-derived R-spondin1 (R&D Systems, catalog number: 7150-RS/CF )
    Rho kinase inhibitor Y27632 (Sigma-Aldrich, catalog number: Y0503 )
  35. DMSO (Sigma-Aldrich, catalog number: D8418 )
  36. Propanol-2 (VWR, catalog number: 1.09634.9900 )
  37. 70% ethanol (see Recipes)
  38. 1 M glucose (see Recipes)
  39. Embryo’s medium (see Recipes)
  40. DPBS-EDTA 5 mM (see Recipes)
  41. DPBS-BSA 2%-EDTA 2 mM (see Recipes)
  42. Basal crypt medium (BCM) (see Recipes)
  43. ENR medium for initial seeding (see Recipes)
  44. ENR medium for maintenance (see Recipes)
  45. Freezing medium (see Recipes)
  46. De-freezing medium (see Recipes)

Equipment

  1. Binocular (Motic, model: SMZ-168 )
  2. Cold light source (SCHOTT, model: KL1500 LCD )
  3. Scissors: straight sharp tip (Fine Science Tool, catalog numbers: 14090-09 and 14084-08 )
  4. Angled serrated tip forceps (Fine Science Tool, catalog number: 11080-02 )
  5. Standard (fine) tip forceps (Fine Science Tool, catalog number: 11251-20 )
  6. Micro-dissecting scissors (Fine Science Tool, catalog number: 15018-10 )
  7. Pipettors with Tip Ejector 20-200 µl and 100-1,000 µl (VWR, catalog numbers: 89079-970 and 89079-974 )
  8. Refrigerated centrifuge Refrigerated centrifuge (Beckman Coulter, model: Allegra X-15R )
  9. MaxQTM 4000 shaker with adaptable temperature (Thermo Fisher Scientific, Thermo ScientificTM, model: MaxQ TM 4000 )
  10. Biological safety cabinet (Esco Micro Pte, model: Class II Type A2 )
  11. Cell culture incubator (37 °C, 5% CO2) (BINDER, model: C150 )
  12. Inverted bright field microscope (Motic, model: AE31 )
  13. Nalgene Cryo ‘Mr Frosty’ freezing container (Thermo Fisher Scientific, Thermo ScientificTM, model: 5100-0050 )
  14. Ultra-low temperature upright freezer (Thermo Fisher Scientific, model: [该编号有问题请联系管理员])
  15. Cryostorage system K Series (Taylor-Wharton, model: 24K )

Procedure

Notes:

  1. General considerations regarding mice and pregnancies: Animals should be housed in a temperature (21 ± 1 °C) and humidity (55 ± 10%)-controlled room with a 12 h light/12 h dark cycle. Embryonic stage is calculated as embryonic day 0.5 (E0.5) the day the gestational plug is observed. It is important to remove the male from the mating cage the morning the plug is observed. Pregnancies can be followed by daily weighing of the females.
  2. Unless specified, steps are carried out at room temperature (RT).
  1. Dissection procedures and preparation of samples
    1. Isolation of gastric fetal epithelium for culture (Figure 1)


      Figure 1. Overall procedure schematic of step A1

      1. General preparations before starting the dissection
        Sterilize working area and dissection tools with 70% ethanol. It is not necessary to work under laminar flow, meanwhile all the steps are carried out carefully and in a clean disinfected area. It is recommended to maintain dissection tools in a glass with 70% ethanol during intermediate steps of the dissection protocol to minimize contamination.
        Equipment: binocular and cold light source (Figure 2).
        Dissection tools: scissors, curved forceps, standard tip forceps, scalpel, dissection bed.
        Box with ice, plastic Petri dishes, embryos medium (conserved on ice), tubes with ice cold DPBS (volume according to developmental stage and number of embryos pooled – see Table 1 below and Figure 2).

        Table 1. Recommended volumes (DPBS or DPBS-EDTA 5 mM or Stem Pro Accutase) for different amounts of processed tissues



        Figure 2. Example of equipment (a) and material and dissection tools (b). a. Binocular and cold light source; b. Material and dissection tools needed for embryos dissection: tubes with DPBS, Petri dish with embryos media, scalpels and straight thin tip forceps.

      2. Euthanize the pregnant female according to the local institutional guidelines at the desired developmental stage: E14.5-E18.5. Note that visual discrimination between proximal and distal stomach areas is difficult before E14.5. Beyond E18.5 stage, spheroid yield decreases.
      3. Lay the pregnant female on its back and fix her to the dissection bed. Spray the abdominal area with 70% ethanol in order to sterilize the area and minimize further contamination (Figure 3a).
      4. Grasp the skin with angled forceps and cut transversally through the skin and peritoneum at the level of the lower abdomen enough to visualize the uterus. Uterus can be recognized as a string of pearls. Dissect out with forceps the oviduct removing the entire uterus (Figures 3b-3h).
      5. Place the uterus with embryos on a Petri dish filled with ice-cold embryo’s medium (see Recipes section) maintained on ice (Figure 3i).
      6. Separate each embryo from the uterus with the intact yolk sac using angled serrated tip forceps (Figure 3j).
      7. Place one of the embryos on an empty Petri dish under the binocular with cold light source. Keep the rest in the medium on ice (Figure 3k). It is recommended to perform steps A1h-l under the binocular.
      8. Open the yolk sac using fine straight tip forceps. Be careful not to damage the embryo’s abdomen when separating the umbilical cord (especially at E14.5-15.5 stages), as sometimes this may pull out the internal digestive tract and compromise the rest of the dissection (Figure 3l). At this point, check that the morphological embryo’s developmental stage corresponds with the one estimated by the observed vaginal plug.
      9. Place the embryo on its left side up and open the abdomen pulling up the skin (be careful: at early developmental stages – E14-15 the tissue is very soft and might be easily damaged). Visualize space between ribs and legs. Recognize liver-intestine-stomach block and pull it out carefully with straight thin tip forceps (Figure 3m).
      10. Separate the stomach from the block by ‘cleaning away’ the other organs of no interest (intestine, liver, pancreas and esophagus) using ice-cold DPBS (Figure 3n).
      11. To obtain higher yield of fetal spheroids, it is recommended to remove the proximal area of the stomach and continue the protocol with the distal part (Figure 3o).
        Note: The elements growing in culture from the proximal part correspond to ‘squamous-like’ organoids, higher proportion of hollow fetal spheroids is obtained from the distal area (Fernandez Vallone et al., 2016).


        Figure 3. Representative pictures of embryonic stomach dissection at E18.5. Successive steps are detailed (a-o). Duo: duodenum, Em: embryo, Eso: esophagus, Liv: liver, Pan: pancreas, Per: peritoneum, SI: small intestine, Spl: spleen, Sto: stomach, Uc: umbilical cord, Ut: uterus, Ys: yolk sac. Scale bars: 0.5 cm (l-m); 0.1 cm (n-o).

      12. Cut into small pieces the distal part of the stomach with the scalpel.
      13. Transfer the tissue to a tube containing sterile ice cold DPBS and place it on ice.
      14. Continue the dissection of the rest of the embryos always keeping the tissue already processed on ice.
      15. Centrifuge the tubes at 230 x g for 5 min, 4 °C.
      16. Re-suspend the pellet of each embryo or pool with the adequate volume of sterile 5 mM DPBS-EDTA (see Table 1).
      17. Place the tubes laid down on ice and incubate them in the shaker for 30 min with 75 rpm agitation speed.
      18. Centrifuge the tubes at 230 x g for 5 min, 4 °C.
      19. From this step onward, the protocol should be carried out under a tissue culture hood.
      20. Remove supernatant by aspiration and re-suspend each pellet in the adequate volume of sterile DPBS (see Table 1).
      21. Pipet up and down samples using FBS-coated micropipette (P1000) to disrupt the tissue and further separate the epithelium from the basal layer in contact with mesenchyme. The number of up and down pipetting times depends on the tissue developmental stage and on the number of embryos processed per sample. For example, tissues from E14-E15 individual embryos are pipetted 12 times whereas tissues from E16-E18 individual embryos or pools of embryos are pipetted between 15 and 20 times.
      22. Pass each suspension through a 70 µm filter into a new tube.
      23. Centrifuge the tubes at 300 x g for 5 min, 4 °C.
      24. Remove supernatant. Pellet is ready to be seeded in culture (see step B).

Note: These steps allow isolating gastric fetal epithelium as clumps or group of cells that will ultimate give rise to gastric fetal spheroids in culture. However, gastric fetal spheroids can also be obtained from isolated single cells after FACS for example. In that case the user of this protocol should follow step A2 instead of step A1 (see below).

  1. Isolation of gastric fetal epithelium as single cells (Figure 4)


    Figure 4. Overall procedure schematic of step A2

    1. Follow protocol described in section A from step 1a to step 1o.
    2. Re-suspend the pellet in Stem Pro Accutase cell dissociation reagent, using volumes as described in Table 1.
    3. Transfer each suspension to a P6 well plate and incubate it at 37 °C with 75 rpm agitation. Time of incubation varies according to the quantity of initial material (between 30 min to 2 h). It is recommended to check the dissociation every 15 min under the inverted microscope and to help this process with mechanical up and down pipetting (micropipette P1000).
    4. When single cell suspension is reached, pass it through a 40 µm filter into a new tube.
    5. Centrifuge the tubes at 300 x g for 5 min, 4 °C.
    6. Remove supernatant and re-suspend the pellets in 2 ml ice cold 2 mM DPBS-BSA 2%-EDTA solution to wash.
    7. Repeat step A2f twice.
    8. Finally re-suspend the single cell preparation in 1 ml 2 mM DPBS-BSA 2%-EDTA. Maintain the cell suspension on ice and proceed with staining steps or direct sorting in case of fluorescent protein expression. It is recommended to pass the suspension through a 40 µm filter once again before sorting.
    9. Single cells of interest can finally be centrifuged at 300 x g for 5 min, 4 °C. Remove supernatant and if desired, proceed with sample seeding for ex vivo culture.

  1. Ex vivo culture – 3D
    1. General preparations before starting
      Defreeze Matrigel aliquots on ice and always keep them on ice, small changes in the temperature might accelerate undesired polymerization.
    2. The surface of plating and amount of Matrigel will be chosen for each sample based on the size of the pellet obtained in step A. For individual samples obtained from embryos at E14-18 or sorted samples, it is recommended to use 1 well of 12 wells plate and 100 µl of Matrigel per pellet. For pooled samples obtained after step A1, 1 well of 6 wells plate and 240 µl Matrigel per pellet will be preferred. All these estimations may change according to the user’s plans and needs.
    3. Re-suspend the pellet in the tube with the adequate amount of Matrigel and homogenize the suspension on ice.
    4. Transfer the mix sample/Matrigel suspension to the plate as a drop. Stretch the drop from the center until the bottom of the well is covered without touching the walls (use a tip for this purpose) (Figure 5a).
    5. Place the plate in an incubator at 37 °C for 10 min until the mix polymerizes.
    6. Distribute the Spheroids-ENR medium for initial seeding (see Recipes section): 700 µl per well (12 well plate) and 1.4 ml per well (6 well plate).
    7. Place the plate in the incubator.
    8. Medium should be fully changed every other day with ENR medium for maintenance (see Recipes section).

      Note: All steps, including the decision of plating according to density of sample should be followed by observation under inverted bright field microscope.


      Figure 5. Spheroids initial seeding. a. Scheme showing side and upper view of the 3D culture; b. Representative pictures of gastric spheroids culture at day 6, arrows show dead cells inside the spheroids (b). Scale bars = 100 µm.

  2. Maintenance of gastric spheroids
    1. General preparations before starting
      After 5 to 7 days in culture (depending on the rate of growth), when few dead cells start to accumulate inside the spheroids, they should be replated (Figure 5b). This can be done by ‘picking’ selected elements (amplify selected population) (step C2) or by harvesting a complete part of the well (just culture maintenance) (step C3). See below details (Figure 6) for each case.


      Figure 6. Procedure for maintenance of gastric spheroids. Fully grown spheroids are isolated from the Matrigel, mechanically dissociated into small groups of cells, which are then mixed with a new Matrigel aliquot and seeded. The ENR seeding medium is added after Matrigel polymerization.

    2. Replating by element selection
      1. Select elements to be picked under the inverted microscope.
      2. Prepare one tube per sample with 1 ml DPBS at room temperature, pick the elements from the well (1 ml DPBS for 12 well plate) with a micropipette (P200) and transfer them to the tube with DPBS. Repeat the process for all the desired elements.
      3. Centrifuge the tube at 300 x g for 5 min.
      4. Remove supernatant and re-suspend the elements in 0.3 ml DPBS.
      5. Disrupt the elements mechanically by pipetting up and down with a micropipette (P200) until visual disappearance of big pieces.
      6. Add 1 ml DPBS to further wash the suspension
      7. Centrifuge the tube at 300 x g for 5 min.
      8. Remove supernatant and re-suspend the pellet in the adequate volume of Matrigel as described in step B.
      9. Repeat steps B3-B7.
    3. Replating without selection
      1. Remove the medium from the well by aspirating.
      2. Add 1 ml DPBS to the well (P6 or P12 well plate) and harvest the whole Matrigel with the elements embedded by pipetting up and down with a micropipette (P1000) until homogeneous suspension is reached.
      3. Take 0.25 ml from the suspension (¼) and transfer it to a new tube. It is not recommended to replate the whole well unless specific need for spheroid amplification. A too high density of replating would result in too much material and debris, ending with a bad quality of replating.
      4. Add 1 ml DPBS to the tube and centrifuge it at 300 x g for 5 min.
      5. Remove supernatant and re-suspend the elements in 0.3 ml DPBS.
      6. Disrupt the elements mechanically by pipetting up and down with a micropipette (P200) until visual disappearance of big pieces.
      7.  Add 1 ml DPBS to further wash the suspension.
      8. Centrifuge the tube at 300 x g for 5 min.
      9. Remove supernatant and re-suspend the pellet in the adequate volume of Matrigel as described in step B.
      10. Repeat steps B3-B7.

  3. Cryopreservation of gastric spheroids
    1. Freezing protocol
      1. Remove the medium from the well by aspirating.
      2. Add 1 ml DPBS to the well and harvest the whole Matrigel with the elements embedded pipetting up and down with a micropipette (P1000) until homogeneous suspension is reached.
      3. Centrifuge the tube at 300 x g for 5 min.
      4. Wash the pellet in 2 ml DPBS.
      5. Centrifuge the tube at 300 x g for 5 min.
      6. Re-suspend the pellet in 1 ml of freezing medium (see Recipes section).
      7. Transfer the suspension to a labeled cryotube and place it in the Cryo freezing container (filled with isopropanol at RT). Put the container in a -80 °C freezer.
      8. After 48 h, cryotubes can be stored in a cryostorage system in liquid nitrogen.
    2. De-freezing protocol
      1. Warm at 37 °C a tube with 2 ml of de-freezing medium (see Recipes section).
      2. Take out from liquid nitrogen the selected cryotube and de-freeze the sample by pipetting up and down with pre-warmed de-freezing medium.
      3. Transfer all the spheroid suspension to the pre-warmed tube and centrifuge at 300 x g for 5 min.
      4. Remove supernatant and wash the pellet in 2 ml of BCM medium twice and centrifuge it at 300 x g for 5 min.
      5. Remove supernatant and re-suspend the elements in 0.3 ml DPBS.
      6. Disrupt the elements mechanically by pipetting up and down 20 times with a micropipette (P200).
      7. Add 1 ml DPBS to further wash the suspension.
      8. Centrifuge the tube at 300 x g for 5 min.
      9. Remove supernatant and re-suspend the pellet in the adequate volume of Matrigel as described in step B.

Data analysis

Details of replicates are provided in the original research paper published in free access (Fernandez Vallone et al., 2016).

Notes

  1. The yield of spheroid production obtained upon initial seeding is highly dependent on the developmental stage, with higher efficiency at E14-E15, and progressive decrease over time. At later developmental stages, a low proportion of clear spheroids can be obtained among the grown elements. In this case, replating of gastric spheroids will require selective picking of the clearest elements from the initial plate, the rest of the elements (usually dark spheroid-like structures) correspond to adult-type stem cells, which cannot efficiently grow in the ENR medium.
  2. In order to improve cell viability after cell sorting, single sorted cells are collected in the BCM medium containing 10 µM Y27632.
  3. This protocol can be similarly used to efficiently grow mouse fetal progenitors of the small intestine, a protocol initially described by Mustata et al. (2013).

Recipes

  1. 70% ethanol
    70% ethanol (v/v) in distilled water
  2. 1 M glucose
    Dissolve 180 g glucose in 1 L distilled water
    Pass the solution through a 0.22 µm filter
  3. Embryo’s medium
    Add 17.5 ml 1 M glucose (sterile) to 500 ml Leibovitz’s l-15 medium
  4. DPBS-EDTA (5 mM)
    0.5 ml 500 mM EDTA in DPBS
    Final volume: 50 ml
  5. DPBS-BSA 2%-EDTA (2 mM)
    1 g BSA
    0.1 ml 500 mM EDTA in DPBS
    Final volume: 50 ml
    Pass the solution through a 0.22 µm filter
  6. Basal crypt medium (BCM)
    500 ml Advanced DMEM/F12 supplemented with:
    0.4 ml gentamycin
    5 ml penicillin-streptomycin cocktail stock
    5 ml amphotericin B
    5 ml L-glutamine (final concentration: 2 mM)
  7. ENR medium for initial seeding
    BCM supplemented with:
    0.5 ml N-2
    1 ml B-27 w/o vit. A
    0.5 ml 10 mM HEPES
    0.1 ml 1 mM N-acetyl cysteine
    Growth factors at a final concentration of: 50 ng/ml for EGF, 100 ng/ml for Noggin and 100 ng/ml for CHO-derived R-spondin 1, 10 µM Rho kinase inhibitor (Y-27632)
    Final volume: 50 ml
  8. ENR medium for maintenance
    BCM supplemented with:
    0.5 ml N-2
    1 ml B-27 w/o vit. A
    0.5 ml 10 mM HEPES
    0.1 ml 1 mM N-acetyl cysteine
    Growth factors at a final concentration of: 50 ng/ml for EGF, 100 ng/ml for Noggin and 100 ng/ml for CHO-derived R-spondin1
    Final volume: 50 ml
  9. Freezing medium
    BCM
    1 ml FBS
    1 ml DMSO
    Final volume: 10 ml
  10. De-freezing medium
    BCM
    1 ml FBS
    Final volume: 10 ml

Acknowledgments

This work was supported by the Interuniversity Attraction Poles Programme-Belgian State-Belgian Science Policy (6/14), the Fonds de la Recherche Scientifique Médicale of Belgium, the Walloon Region (program CIBLES) and the non-for-profit Association Recherche Biomédicale et Diagnostic. This protocol was adapted from previous work for sample preparation (Mustata et al., 2013) and the initial report of Sato et al. (2009) for ex vivo culture conditions.

References

  1. Barker, N., Huch, M., Kujala, P., van de Wetering, M., Snippert, H. J., van Es, J. H., Sato, T., Stange, D. E., Begthel, H., van den Born, M., Danenberg, E., van den Brink, S., Korving, J., Abo, A., Peters, P. J., Wright, N., Poulsom, R. and Clevers, H. (2010). Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6(1): 25-36.
  2. Fernandez Vallone, V., Leprovots, M., Strollo, S., Vasile, G., Lefort, A., Libert, F., Vassart, G. and Garcia, M. I. (2016). Trop2 marks transient gastric fetal epithelium and adult regenerating cells after epithelial damage. Development 143(9): 1452-1463.
  3. Mustata, R. C., Vasile, G., Fernandez-Vallone, V., Strollo, S., Lefort, A., Libert, F., Monteyne, D., Perez-Morga, D., Vassart, G. and Garcia, M. I. (2013). Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium. Cell Rep 5(2): 421-432.
  4. Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., van Es, J. H., Abo, A., Kujala, P., Peters, P. J. and Clevers, H. (2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244): 262-265.

简介

来自消化道的鼠胎儿祖细胞的分离和三维培养代表了研究在发育过程中细胞分化过程开始前存在的这些上皮细胞的性质和生物学特征的新方法。在2013年,Mustata等人描述了在最初由佐藤等人实施的离体培养系统中分离作为球体生长的肠胎细胞祖细胞。 >(2009)增长成年肠干细胞。值得注意的是,胎儿衍生的球体具有较高的自我更新能力,使其在文化中的无限期维护变得容易。在这里,我们报告了用于分离和远离前胃腺胃生长为胃球体的胎儿上皮祖细胞的分离和离体培养和维持的修改方案(Fernandez Vallone等人, 2016)。

背景 来自腺体的小鼠成体干细胞可以在3D matrigel中离体生长,作为“迷你腺体”无限期(Barker等人,2010) 。与在EGF,Noggin和R-spondin 1存在下生长的小肠的干细胞相比,成年胃干细胞需要进一步补充Fgf10,胃泌素,Wnt3a和更高浓度的R-spondin 1以获得生产性 - 文化。相比之下,到目前为止,很少知道在发育期间排列前腺上皮细胞的胎儿细胞。到目前为止,它们的性质以及其离体的潜在生长特性未明确。基于以前的研究,确定胎儿小肠(Mustata等人,2013年)中存在的细胞,我们报告了作为球体的小鼠胎儿胃祖细胞的培养(Fernandez Vallone et al。 。,2016)。可以在2009年由佐藤等人先前报道的培养基中重复胃祖细胞以生长小肠成体干细胞,与成人型胃干细胞相反,它们不需要额外的生长因子补充(Fgf10,Wnt3a或胃泌素)。

关键字:离体, 球体, 胎鼠上皮, 祖细胞, 3D, 单细胞

材料和试剂

  1. 一次性手术刀(Swan Morton,目录号:0510)
  2. 带凸轮的培养皿92 x 16毫米(SARSTEDT,目录号:82.1473)
  3. 微量离心管,1.5毫升(VWR,目录号:212-0198)
  4. 管10 ml,100 x 16 mm,PP(SARSTEDT,目录号:62.9924.284)
  5. 管50毫升,30×115毫米,PP(康宁,猎鹰,目录号:352070)
  6. 70μm尼龙过滤器(Corning,Falcon ®,目录号:352350)
  7. P6孔板(VWR,目录号:734-2323)
  8. 40μm尼龙过滤器(Corning,Falcon ®,目录号:352340)
  9. P12孔板(VWR,目录号:734-2324)
  10. 提示补充(VWR,目录号:89079-464; 89079-470; 89079-478)
  11. 冷冻筒1ml(Greiner Bio One,目录号:123263)
  12. 注射器过滤器0.2μm(VWR,目录号:28145-477)
  13. 血清学移液管5ml,10ml和25ml(Corning,Falcon ,目录号:357543; 357551; 357535)
  14. 小鼠(在RjOrl:SWISS和C57BL/6JRj背景上测试)
  15. Dulbecco的磷酸盐缓冲盐水(DPBS),游离CaCl 2,不含MgCl 2(Thermo Fisher Scientific,Gibco< sup>,目录号:14190- 094)
  16. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270)
  17. Stem Pro Accutase细胞解离试剂(Thermo Fisher Scientific,Gibco TM,目录号:A1110501)
  18. Matrigel ®基底膜基质(Corning,目录号:354234)
  19. 液氮(由液化气供应)
  20. 乙醇95-97%(VWR,TechniSolv ,目录号:84857.360)
  21. 葡萄糖(Merck Millipore,目录号:1083371000)
  22. Leibovitz的L-15培养基(Thermo Fisher Scientific,目录号:11415-049)
  23. 500mM EDTA(pH8.0)(Thermo Fisher Scientific,Invitrogen TM,目录号:15575-038)
  24. 来自牛血清(BSA)的白蛋白(Sigma-Aldrich,目录号:A3294)
  25. 高级DMEM/F12(Thermo Fisher Scientific,Gibco TM ,目录号:12634-010)
  26. 庆大霉素50mg/ml(Thermo Fisher Scientific,Gibco TM,目录号:15750-037)
  27. 100x(Thermo Fisher Scientific,Gibco TM,目录号:15140-122)的青霉素 - 链霉素混合物
  28. 两性霉素B250μg/ml(Thermo Fisher Scientific,Gibco TM,目录号:15290-026)
  29. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030-081)
  30. N-2补充100x(Thermo Fisher Scientific,Gibco TM,目录号:17502-048)
  31. B-27 w/o vit 50x(Thermo Fisher Scientific,Gibco TM ,目录号:12587-010)
  32. 1 M HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:15630-056)
  33. 乙酰半胱氨酸(Sigma-Aldrich,目录号:A7250)
  34. 生长因子
    重组鼠EGF(Peprotech,目录号:315-09)
    重组鼠Noggin(Peprotech,目录号:250-38)
    重组鼠CHO衍生的R-spondin1(R& D Systems,目录号:7150-RS/CF)
    Rho激酶抑制剂Y27632(Sigma-Aldrich,目录号:Y0503)
  35. DMSO(Sigma-Aldrich,目录号:D8418)
  36. 丙醇-2(VWR,目录号:1.09634.9900)
  37. 70%乙醇(见食谱)
  38. 1 M葡萄糖(见食谱)
  39. 胚胎的中等(见食谱)
  40. DPBS-EDTA 5 mM(参见食谱)
  41. DPBS-BSA 2%-EDTA 2mM(参见食谱)
  42. 基础隐窝培养基(BCM)(见食谱)
  43. 用于初始播种的ENR培养基(参见食谱)
  44. ENR用于维护的介质(参见配方)
  45. 冷冻介质(参见食谱)
  46. 脱冻介质(参见食谱)

设备

  1. 双目(Motic,型号:SMZ-168)
  2. 冷光源(肖特,型号:KL1500 LCD)
  3. 剪刀:直尖尖(Fine Science Tool,目录号:14090-09和14084-08)
  4. 斜角锯齿镊子(精细科学工具,目录号:11080-02)
  5. 标准(精细)尖端镊子(精细科学工具,目录号:11251-20)
  6. 微解剖刀(精细科学工具,目录号:15018-10)
  7. 带喷嘴的移液器20-200μl和100-1,000μl(VWR,目录号:89079-970和89079-974)
  8. 冷冻离心机冷冻离心机(Beckman Coulter,型号:Allegra X-15R)
  9. MaxQ TM 具有适应温度的摇床(Thermo Fisher Scientific,Thermo Scientific TM,型号:MaxQ TM 4000)
  10. 生物安全柜(Esco Micro Pte,型号:Class II Type A2)
  11. 细胞培养箱(37℃,5%CO 2)(BINDER,型号:C150)
  12. 倒立式明视野显微镜(Motic,型号:AE31)
  13. Nalgene Cryo先生Frosty先生的冷冻容器(Thermo Fisher Scientific,Thermo Scientific TM ,型号:5100-0050)
  14. 超低温立式冷冻机(Thermo Fisher Scientific,型号:Thermo Scientificifc Queue Basic)
  15. 冷冻系统K系列(泰勒沃顿,型号:24K)

程序

注意:

  1. 关于小鼠和妊娠的一般考虑:动物应在12小时光/12小时黑暗循环的温度(21±1℃)和湿度(55±10%)的控制室内饲养。胚胎期计算为胚胎第0.5天(E0.5),观察妊娠期的日子。早晨,请先从插座上取出男性,这一点非常重要。妊娠之后可以每天称重女性。
  2. 除非另有规定,步骤在室温(RT)下进行。
  1. 解剖程序和样品的制备
    1. 分离培养的胃胎上皮(图1)

      图1.步骤A1的总体程序原理图

      1. 开始解剖前的一般准备工作
        用70%乙醇灭菌工作区域和解剖工具。不需要在层流下工作,同时所有的步骤都要在干净的消毒区进行。建议在解剖方案的中间步骤时,将70%乙醇玻璃中的夹层工具保持在最低限度。
        设备:双目和冷光源(图2) 解剖工具:剪刀,弯钳,标准镊子,手术刀,解剖床。
        包含冰,塑料的培养皿,胚胎培养基(冰上保存),冰冷的DPBS(根据发育阶段的体积和合并的胚胎数量 - 见下表1和图2)。

        表1.不同量的加工组织的推荐体积(DPBS或DPBS-EDTA 5mM或Stem Pro Accutase)



        图2.设备(a)和材料和解剖工具(b)的示例。 a。双目冷光源; b。胚胎解剖所需的材料和解剖工具:带有DPBS的管,具有胚胎培养基的培养皿,解剖刀和直的薄尖端镊子。

      2. 在所期望的发展阶段,根据当地的制度准则对孕妇进行安乐死:E14.5-E18.5。请注意,在E14.5之前,近端和远端胃区域之间的视觉辨别是困难的。超越E18.5阶段,球体产量下降
      3. 将怀孕的女性放在背部,并将其固定在解剖床上。用70%乙醇喷洒腹部以消毒该区域,并减少进一步的污染(图3a)。
      4. 用倾斜的镊子抓住皮肤,并通过下腹部的皮肤和腹膜横向切割,足以使子宫可视化。子宫可以被认为是一串珍珠。用镊子解剖输卵管去除整个子宫(图3b-3h)。
      5. 将子宫用胚胎放在装有冰冷胚胎培养基的培养皿(见食谱部分),保持在冰上(图3i)。
      6. 用完整的卵黄囊将每个胚胎与子宫分开,使用角度锯齿尖端镊子(图3j)。
      7. 将一个胚胎放在双筒望远镜上的空白培养皿中,用冷光源。将其余部分放在冰上的介质中(图3k)。建议在双目镜下执行A1h-l步骤。
      8. 使用细直的镊子打开卵黄囊。分离脐带时尤其要注意不要损伤胚胎的腹部(特别是在E14.5-15.5阶段),有时这可能会拉出内部消化道并损害其余的解剖(图31)。在这一点上,检查形态胚胎的发育阶段是否符合观察到的阴道栓子估计的发展阶段。
      9. 将胚胎放在左侧,打开腹部拉起皮肤(注意:在早期发育阶段 - E14-15,组织非常柔软,可能容易损坏)。可视化肋骨和腿之间的空间。识别肝 - 肠 - 胃块,并用直薄的镊子仔细拉出(图3m)。
      10. 通过使用冰冷的DPBS(图3n)"清除"其他不感兴趣的器官(肠,肝,胰腺和食管)将胃与块分开。
      11. 为了获得更高的胎儿球体产量,建议去除胃的近端区域并继续与远端部分的协议(图3o)。
        注意:从近端部分生长在培养物中的元素对应于"鳞状"的有机体,从远端区域获得较高比例的中空胎儿球体(Fernandez Vallone等,2016)。 br />

        图3. E18.5胚胎胃解剖的代表性图片。连续步骤详细(a-o)。二重奏:十二指肠,胚胎,Eso:食道,肝脏,肝,胰:胰腺,Per:腹膜,SI:小肠,Spl:脾,Sto:胃,Uc:脐带,Ut:子宫,Ys:卵黄囊。刻度棒:0.5cm(1-m); 0.1厘米(n-o)
      12. 用手术刀切成小块远端胃部。
      13. 将组织转移到含有无菌冰冷DPBS的管中,并将其置于冰上
      14. 继续解剖剩余的胚胎,始终保持组织已经在冰上加工。
      15. 将管以230×g离心5分钟,4℃。
      16. 用足够体积的无菌5mM DPBS-EDTA重新悬浮每个胚胎或游泳池的沉淀(见表1)。
      17. 将管放置在冰上,并将其在振荡器中孵育30分钟,并以75rpm的搅拌速度
      18. 将管以230×g离心5分钟,4℃。
      19. 从这一步开始,协议应该在组织培养罩下进行。
      20. 通过抽吸去除上清液,并将每个沉淀物重新悬浮在足够体积的无菌DPBS中(见表1)
      21. 使用FBS涂覆的微量移液管(P1000)吸取上下样品,以破坏组织,并进一步将上皮与基底层分离,与间质接触。上下移液次数取决于组织发育阶段和每个样品处理的胚胎数量。例如,来自E14-E15个体胚胎的组织被吸取12次,而来自E16-E18个体胚胎或胚胎池的组织被吸移15至20次。
      22. 将每个悬浮液通过70μm过滤器通入新管中
      23. 将管以300×g离心5分钟,4℃。
      24. 去除上清液。颗粒准备种植在文化中(见步骤B)。

注意:这些步骤允许将胃胎儿上皮分离成最终在培养物中产生胃胎儿球体的团块或一组细胞。然而,例如,在FACS之后,也可以从分离的单细胞获得胃胎儿球体。在这种情况下,该协议的用户应遵循步骤A2而不是步骤A1(见下文)。

  1. 将胃胎儿上皮分离为单细胞(图4)


    图4.步骤A2的总体程序原理图

    1. 遵循从步骤1a到步骤1o的部分A中描述的协议。
    2. 使用表1中所述的体积,在Stem Pro Accutase细胞解离试剂中重新悬浮沉淀。
    3. 将每个悬浮液转移到P6孔板,并在37℃下以75rpm搅拌孵育。孵育时间根据初始材料的数量(30分钟至2小时)而变化。建议在倒置显微镜下每15分钟检查解离,并通过机械上下移液(微量移液管P1000)帮助该过程。
    4. 当达到单细胞悬浮液时,将其通过40μm的过滤器通入新的管中
    5. 将管以300×g离心5分钟,4℃。
    6. 除去上清液并将沉淀重新悬浮在2ml冰冷的2mM DPBS-BSA 2%-EDTA溶液中以洗涤。
    7. 重复步骤A2f两次。
    8. 最后将单细胞制备物重新悬浮在1ml 2mM DPBS-BSA 2%-EDTA中。将细胞悬浮液保持在冰上,并进行染色步骤或在荧光蛋白表达的情况下进行直接分选。建议在分拣前再次将悬浮液通过40μm过滤器。
    9. 感兴趣的单细胞最终可以在300×g下离心5分钟,4℃。去除上清液,如果需要,进行离体培养的样品种子。

  1. 文化 - 3D
    1. 开始前的一般准备工作
      防冻基质胶在冰上等分,并始终保持在冰上,微小的温度变化可能加速不期望的聚合。
    2. 将根据步骤A中获得的颗粒的大小为每个样品选择电镀表面和基质胶量。对于从E14-18的胚胎或分选样品获得的各个样品,建议使用12孔的1孔平板和100微升Matrigel每粒。对于在步骤A1之后获得的合并样品,优选6孔板的1孔和每粒子240μl的Matrigel。所有这些估算可能会根据用户的计划和需求而改变。
    3. 用足够量的基质胶重新悬浮在管中,并将悬浮液均匀化在冰上
    4. 将混合样品/Matrigel悬浮液以一滴水转移到板上。从中心向下拉伸,直到井底不被接触到墙壁(为此目的使用尖端)(图5a)。
    5. 将板放在37℃的培养箱中10分钟,直到混合物聚合。
    6. 分配用于初始播种的球状体ENR培养基(参见食谱部分):每孔700μl(12孔板)和1.4ml /孔(6孔板)。
    7. 将板放在培养箱中。
    8. 应每隔一天使用ENR培养基进行全面的维护(参见食谱部分)。

      注意:所有步骤,包括根据样品密度进行电镀的决定,应在倒置的明视野显微镜下进行观察。


      图5.球体初始播种。 a。显示3D文化的侧视图和上视图; b。在第6天,胃球体培养的代表性图片,箭头显示球体内的死细胞(b)。刻度棒=100μm。

  2. 维持胃球体
    1. 开始前的一般准备工作
      培养5〜7天后(取决于生长速率),当细胞内几乎没有死细胞开始积聚在球体内时,应重新进行(图5b)。这可以通过"选择"所选择的元素(放大选定的群体)(步骤C2)或者通过收获井的完整部分(仅仅是文化维护)来完成(步骤C3)。有关每种情况,请参见以下详细信息(图6)。


      图6.维持胃球体的程序从Matrigel中分离完全生长的球体,机械解离成小组细胞,然后将其与新的Matrigel等分试样混合并接种。在基质胶聚合后加入ENR播种培养基
    2. 按元素选择进行替换
      1. 选择要在倒置显微镜下挑选的元素
      2. 每个样品在室温下用1ml DPBS制备一个管,用微量移液管(P200)从孔中挑取元素(1ml DPBS,12孔板),并用DPBS转移到管中。对所有需要的元素重复此过程。
      3. 以300×g离心管5分钟。
      4. 去除上清液并将元件重新悬浮在0.3ml DPBS中。
      5. 通过用微量吸管(P200)上下移动来机械地破碎元件,直到大块的视觉消失。
      6. 加入1ml DPBS以进一步清洗悬浮液
      7. 以300×g离心管5分钟。
      8. 除去上清液,并将沉淀物重新悬浮在足够体积的Matrigel中,如步骤B所述
      9. 重复步骤B3-B7。
    3. 无需选择即可
      1. 通过吸出将介质从井中取出。
      2. 向孔(P6或P12孔板)加入1ml DPBS,并用微量移液管(P1000)上下移动嵌入的元件收获整个基质胶,直到达到均匀的悬浮液。
      3. 从悬浮液(¼)取0.25ml,并将其转移到新管中。除非特别需要球形放大,否则不建议复制整个孔。太高的重复密度会导致太多的材料和碎片,结果是质量不佳。
      4. 向管中加入1ml DPBS,并以300×g离心5分钟。
      5. 去除上清液并将元件重新悬浮在0.3ml DPBS中。
      6. 通过用微量吸管(P200)上下移动来机械地破碎元件,直到大块的视觉消失。
      7. 加入1ml DPBS进一步清洗悬浮液。
      8. 以300×g离心管5分钟。
      9. 除去上清液,并将沉淀物重新悬浮在足够体积的Matrigel中,如步骤B所述
      10. 重复步骤B3-B7。

  3. 胃球蛋白冷冻保存
    1. 冻结协议
      1. 通过吸出将介质从井中取出。
      2. 向孔中加入1ml DPBS并收集整个Matrigel,将元件用微量移液管(P1000)上下移动,直到达到均匀悬浮液。
      3. 以300×g离心管5分钟。
      4. 在2ml DPBS中洗涤沉淀。
      5. 以300×g离心管5分钟。
      6. 将沉淀重新悬浮在1ml冷冻介质中(见食谱部分)。
      7. 将悬浮液转移到标记的冷冻管中,并将其放入冷冻冷冻容器中(在室温下装满异丙醇)。将容器放在-80°C冰箱中。
      8. 48小时后,冷冻管可以储存在液氮中的冷冻保存系统中。
    2. 去冻协议
      1. 在37°C的温度下,加入2 ml冷冻培养基(见食谱部分)
      2. 从液氮中取出选定的冷冻管,并用预先加热的冷冻介质上下移动来冻干样品。
      3. 将所有的球体悬浮液转移到预热管中,并以300 x g离心5分钟。
      4. 除去上清液,并将沉淀物在2ml BCM培养基中洗涤两次,并以300×g离心5分钟。
      5. 去除上清液并将元件重新悬浮在0.3ml DPBS中。
      6. 用微量移液管(P200)上下移动20次,从而机械地破碎元件。
      7. 加入1ml DPBS以进一步洗涤悬浮液。
      8. 以300×g离心管5分钟。
      9. 除去上清液并将沉淀重新悬浮在足够体积的基质胶中,如步骤B中所述。

数据分析

在免费访问的原始研究论文(Fernandez Vallone等人,2016年)中提供了重复的细节。

笔记

  1. 初次播种时获得的球形产量的产量高度依赖于发育阶段,E14-E15效率较高,随着时间的推移逐渐降低。在后来的发育阶段,可以在生长的元素中获得低比例的透明球体。在这种情况下,胃球体的再次需要从初始板选择性地挑选最清楚的元件,其余部分(通常是黑色球状结构)对应于在ENR培养基中不能有效生长的成体型干细胞。
  2. 为了提高细胞分选后的细胞活力,将单个分选的细胞收集在含有10μMY27632的BCM培养基中。
  3. 该方案可以类似地用于有效地生长小肠的小鼠胎儿祖细胞,这是最初由Mustata等人(2013)描述的方案。

食谱

  1. 70%乙醇
    70%乙醇(v/v)在蒸馏水中
  2. 1 M葡萄糖
    将180克葡萄糖溶于1升蒸馏水中 将溶液通过0.22μm过滤器
  3. 胚胎中等
    将17.5ml 1M葡萄糖(无菌)加入到500ml Leibovitz的1-15培养基中
  4. DPBS-EDTA(5mM)
    DPBS中0.5ml 500mM EDTA 最终体积:50 ml
  5. DPBS-BSA 2%-EDTA(2mM)
    1克BSA
    在DPBS中0.1ml 500mM EDTA 最终体积:50 ml
    将溶液通过0.22μm过滤器
  6. 基底隐窝培养基(BCM)
    500 ml Advanced DMEM/F12补充:
    0.4 ml庆方霉素 5毫升青霉素 - 链霉素鸡尾酒库存
    5 ml两性霉素B
    5ml L-谷氨酰胺(终浓度:2mM)
  7. 初始播种的ENR培养基
    BCM补充:
    0.5 ml N-2
    1 ml B-27 w/o vit A
    0.5 ml 10 mM HEPES
    0.1ml 1mM N-乙酰半胱氨酸
    终浓度为50ng/ml的生长因子,EGF为50ng/ml,Noggin为100ng/ml,CHO-衍生的R-spondin为10μMRho激酶抑制剂(Y-27632)为100ng/ml。 最终体积:50 ml
  8. 用于维护的ENR介质
    BCM补充:
    0.5 ml N-2
    1 ml B-27 w/o vit A
    0.5 ml 10 mM HEPES
    0.1ml 1mM N-乙酰半胱氨酸
    生长因子终浓度为:EGF为50ng/ml,Noggin为100ng/ml,CHO为衍生的R-spondin1为100ng/ml。 最终体积:50 ml
  9. 冷冻介质
    BCM
    1 ml FBS
    1 ml DMSO
    最终体积:10 ml
  10. 脱冻介质
    BCM
    1 ml FBS
    最终体积:10 ml

致谢

这项工作得到了比利时国家比利时科学政策(6/14),比利时Fonds de la Recherche科学院,瓦隆地区(计划CIBLES)和非营利协会RechercheBiomédicale的支持et诊断。该协议是从以前的样品制备工作(Mustata等人,2013)和Sato等人的初步报告中进行的。 (2009)for ex emitation 培养条件。

参考文献

  1. Barker,N.,Huch,M.,Kujala,P.,van de Wetering,M.,Snippert,HJ,van Es,JH,Sato,T.,Stange,DE,Begthel,H.,van den Born,M ,Danenberg,E.,van den Brink,S.,Korving,J.,Abo,A.,Peters,PJ,Wright,N.,Poulsom,R.and Clevers,H.(2010)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/20085740"target ="_ blank"> Lgr5 + ve 干细胞驱动自我更新在胃中并在体外构建长寿命胃单位。细胞干细胞 6(1):25-36。
  2. Fernandez Vallone,V.,Leprovots,M.,Strollo,S.,Vasile,G.,Lefort,A.,Libert,F.,Vassart,G.and Garcia,MI(2016)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26989172"target ="_ blank"> Trop2标记了上皮损伤后的过渡性胃上皮细胞和成年再生细胞。 em>发展 143(9):1452-1463。
  3. Mustata,RC,Vasile,G.,Fernandez-Vallone,V.,Strollo,S.,Lefort,A.,Libert,F.,Monteyne,D.,Perez-Morga,D.,Vassart,G.and Garcia, MI(2013)。识别Lgr5独立球体 - 产生小鼠胎儿肠上皮的祖细胞。 Cell Rep 5(2):421-432。
  4. Sato,T.,Vries,RG,Snippert,HJ,van de Wetering,M.,Barker,N.,Stange,DE,van Es,JH,Abo,A.,Kujala,P.,Peters,PJ和Clevers, H.(2009)。单个Lgr5干细胞构建隐窝没有间充质生态位的体外结构。自然 459(7244):262-265。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Vallone, V. F., Leprovots, M., Vassart, G. and Garcia, M. (2017). Ex vivo Culture of Fetal Mouse Gastric Epithelial Progenitors. Bio-protocol 7(1): e2089. DOI: 10.21769/BioProtoc.2089.
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