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May 2017

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FACS-based Isolation of Neural and Glioma Stem Cell Populations from Fresh Human Tissues Utilizing EGF Ligand
基于FACS利用EGF配体从新鲜人组织中分离神经和胶质瘤干细胞群   

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Abstract

Direct isolation of human neural and glioma stem cells from fresh tissues permits their biological study without prior culture and may capture novel aspects of their molecular phenotype in their native state. Recently, we demonstrated the ability to prospectively isolate stem cell populations from fresh human germinal matrix and glioblastoma samples, exploiting the ability of cells to bind the Epidermal Growth Factor (EGF) ligand in fluorescence-activated cell sorting (FACS). We demonstrated that FACS-isolated EGF-bound neural and glioblastoma populations encompass the sphere-forming colonies in vitro, and are capable of both self-renewal and multilineage differentiation. Here we describe in detail the purification methodology of EGF-bound (i.e., EGFR+) human neural and glioma cells with stem cell properties from fresh postmortem and surgical tissues. The ability to prospectively isolate stem cell populations using native ligand-binding ability opens new doors for understanding both normal and tumor cell biology in uncultured conditions, and is applicable for various downstream molecular sequencing studies at both population and single-cell resolution.

Keywords: EGF (EGF), EGFR (EGFR), FACS (FACS), Stem cells (干细胞), Neural (神经), Glioblastoma (胶质母细胞瘤), Germinal matrix (生发基质), Human (人)

Background

Understanding the intrinsic biology of human neural and glioma stem cells has been challenging, due to the lack of universal neural and glioma stem cell markers (Lathia et al., 2015) and the frequent reliance on cultured cells rather than those isolated directly from the tissue. The transmembrane glycoprotein Prominin, or CD133, is one of the best described and frequently used stem cell markers for the isolation of neural (Uchida et al., 2000) and glioma stem cells (GSC) (Singh et al., 2003; Singh et al., 2004; Lathia et al., 2015), with its utility being demonstrated in both acutely sorted human tissues and neurospheres. However, some recent studies have pointed out that CD133-negative cells isolated from human glioblastoma (GBM) also harbor stem cell properties (Beier et al., 2007; Wang et al., 2008; Tome-Garcia et al., 2017). Other cell surface markers used to isolate GSC include CD44 (Anido et al., 2010), CD15 (Son et al., 2009), A2B5 (Ogden et al., 2008), integrin alpha (Lathia et al., 2010) or EGFR (Mazzoleni et al., 2010), but these antibody-based methodologies have also lacked the ability to capture all sphere-forming populations or their use has been limited to cultured cells.

A direct comparison of the molecular phenotypes between non-neoplastic and tumoral stem cell niches can provide novel insight into the developmental pathways co-opted during tumor formation and may uncover more comprehensive stem cell markers. In the context of gliomagenesis, several developmental pathways important for the growth and proliferation of normal neural progenitors have been demonstrated to be aberrantly reactivated during gliomagenesis (Sanai et al., 2005; Canoll and Goldman, 2008; Chen et al., 2012; Tsankova and Canoll, 2014; Lathia et al., 2015). Among these is the Epidermal Growth Factor Receptor (EGFR) tyrosine kinase pathway. Expression of EGFR is high during human neural development, especially within the germinal matrix stem cell niche, diminishing significantly in adulthood (Weickert et al., 2000; Sanai et al., 2011; Erfani et al., 2015), and it is aberrantly re-activated in GBM (Verhaak et al., 2010; Brennan et al., 2013). Recently, we adapted a mouse fluorescence-activated cell sorting (FACS) strategy, which selects for EGFR+ cells based on their native binding to EGF ligand (Ciccolini et al., 2005; Pastrana et al., 2009; Codega et al., 2014), and isolated human EGFR+ populations from fresh germinal matrix (GM) dissections and GBM tissues. This allowed us to directly compare the functional properties and whole-transcriptome signatures in developing and neoplastic neural populations (Tome-Garcia et al., 2017). EGFR+ populations from both GM and GBM tissues captured all sphere-forming cells in vitro, displayed similar proliferative stem cell properties, and shared transcriptome signatures related to cell growth and cell-cycle regulation. EGFR+ GBM populations also displayed tumor initiation in vivo (Tome-Garcia et al., 2017). Below, we describe this prospective purification strategy for neural and glioblastoma populations with stem cell properties from primary human samples, detailing the steps of tissue dissociation, ligand/antibody incubation, FACS, and in vitro functional stem cell property analysis.

Materials and Reagents

  1. Pipette tips (Fisher Scientific, FisherbrandTM, catalog numbers: 02-707-404 ; 02-707-430 ; 02-707-432 )
  2. American Safety Razors PersonnaTM PalTM Single Edge Blade (AccuTec Blades, catalog number: 620177 )
  3. Falcon tissue culture dish (Corning, Falcon®, catalog number: 353002 )
  4. Falcon® 15 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352099 )
  5. 40 μm cell strainer (Corning, Falcon®, catalog number: 352340 )
  6. 1.7 ml Microcentrifuge snap-cap tubes (Corning, Costar®, catalog number: 3621 )
  7. Ultra Low attachment 96-well plates (Corning, catalog number: 3474 )
  8. 0.2 μm filter (Corning, catalog number: 431219 )
  9. 10 ml serological pipets (Corning, catalog number: 357530 )
  10. 60 ml syringes (BD, catalog number: 309653 )
  11. Micro cover glass (VWR, catalog number: 48393-081 )
  12. Papain (Worthington Biochemical, catalog number: LS003119 )
  13. 10x Red Blood Cells lysis buffer (Thermo Fisher Scientific, eBioscienceTM, catalog number: 00-4300-54 )
  14. Trypan blue solution, 0.4% (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  15. 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (Thermo Fisher Scientific, InvitrogenTM, catalog number: D1306 )
  16. Antibodies
    1. Anti-CD24 PE-conjugated (BD, BD Biosciences, catalog number: 560991 , working dilution 1:10)
    2. Anti-CD34 PE-conjugated (BD, BD Biosciences, catalog number: 550619 , working dilution 1:10)
    3. Anti-CD45 PE-conjugated (BD, BD Biosciences, catalog number: 555483 , working dilution 1:10)
    4. Donkey anti-Rabbit Cy3 conjugated (Jackson ImmunoResearch, catalog number: 711-165-152 , working dilution 1:250)
    5. Donkey anti-Rat AF647 conjugated (Jackson ImmunoResearch, catalog number: 712-605-153 , working dilution 1:500)
    6. EGF ligand-AF647 conjugated (Thermo Fisher Scientific, InvitrogenTM, catalog number: E35351 )
    7. Goat anti-Mouse-AF488 IgM, µ Chain Specific (Jackson ImmunoResearch, catalog number: 115-545-075 , working dilution 1:500)
    8. Mouse anti-O4 (Millipore Sigma, catalog number: MAB345 , working dilution 1:200)
    9. Rabbit anti-TuJ1 (Covance, catalog number: PRB-435P , working dilution 1:1,000)
    10. Rat anti-GFAP (Thermo Fisher Scientific, InvitrogenTM, catalog number: 13-0300 , working dilution 1:1,000)
  17. Laminin 1 mg/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 23017015 )
  18. Aqua-Poly/Mount (Polysciences, catalog number: 18606-20 )
  19. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: S392212 )
  20. JustPure PIPES (Fisher Scientific, catalog number: BP292450 )
    Note: This product has been discontinued.
  21. D-Glucose (Fisher Scientific, catalog number: D16-1 )
  22. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333-500G )
  23. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-1 )
  24. Ultrapure distilled water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977023 )
  25. Phenol red solution, 0.02% (Fisher Scientific, catalog number: S25464 )
  26. Antibiotic-Antimycotic 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 15240062 )
  27. L-Cysteine hydrochloride (Sigma-Aldrich, catalog number: C1276-10G )
  28. Ethylenediaminetetraacetic acid (EDTA) (Fisher Scientific, catalog number: BP118-500 )
  29. Deoxyribonuclease I (DNase I) (Worthington Biochemical, catalog number: LS002139 )
  30. Trypsin inhibitor, Ovomucoid (Sigma-Aldrich, catalog number: T9253-250MG )
  31. Phosphate buffered saline, (PBS) pH 7.4 (Fisher Scientific, catalog number: BP665-1 )
  32. Percoll (MP Biomedicals, catalog number: 0219536925 )
  33. Bovine serum albumin (BSA) (Fisher Scientific, catalog number: BP9704100 )
  34. 1x Hank’s balanced salt solution (HBSS), no calcium, no magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 14175103 )
  35. B-27 Supplement (50x), serum-free (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
  36. N-2 Supplement (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 17502048 )
  37. L-Glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030149 )
  38. 100x Insulin-Transferrin-Selenium (ITS-X) (Thermo Fisher Scientific, GibcoTM, catalog number: 51500056 )
  39. Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F-12) (Thermo Fisher Scientific, GibcoTM, catalog number: 11320082 )
  40. Human recombinant basic Fibroblast Growth Factor (bFGF) (STEMCELL Technologies, catalog number: 78003 )
  41. Human recombinant Epidermal Growth Factor (EGF) (STEMCELL Technologies, catalog number: 78006.1 )
  42. 16% paraformaldehyde (PFA) (Alfa Aesar, catalog number: 43368 )
  43. Normal donkey serum (NDS) (Jackson ImmunoResearch, catalog number: 017-000-121 )
  44. 1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  45. Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
  46. 10 N sodium hydroxide (NaOH) (see Recipes)
  47. 1 M PIPES (see Recipes)
  48. 10x sodium chloride (NaCl)/potassium chloride (KCl) (NaCl/KCl) salt solution (see Recipes)
  49. 30% D-glucose solution (see Recipes)
  50. 1 N NaOH (see Recipes)
  51. PIPES solution (see Recipes)
  52. Activating solution for papain (see Recipes)
  53. 10 mg/ml DNase I solution (see Recipes)
  54. 7 mg/ml ovomucoid (see Recipes)
  55. 10x phosphate buffered saline (PBS) (see Recipes)
  56. 22% Percoll solution (see Recipes)
  57. 10% bovine serum albumin (BSA) (see Recipes)
  58. 1% BSA/0.1% glucose/1x HBSS (see Recipes)
  59. 1x PBS (see Recipes)
  60. Neurosphere media (see Recipes)
  61. Epidermal Growth Factor (EGF) (20μg/ml) (see Recipes)
  62. Basic Fibroblast Growth Factor (bFGF) (20μg/ml) (see Recipes)
  63. 10% normal donkey serum (NDS)/0.5% Triton X-100 (see Recipes)
  64. 1% normal donkey serum (NDS)/0.25% Triton X-100 (see Recipes)
  65. 4% paraformaldehyde (PFA) (see Recipes)

Equipment

  1. Biological safety cabinet (Labconco, model: Class II, Type A2 )
  2. P2 pipetman (Gilson, catalog number: F144801 )
  3. P20 pipetman (Gilson, catalog number: F123600 )
  4. P200 pipetman (Gilson, catalog number: F123601 )
  5. P1000 pipetman (Gilson, catalog number: F123602 )
  6. Motorized pipette controller (Accupet, catalog number: PH01 )
  7. Milligram balance (Sartorius, model: Entris 323 )
  8. Minidizer hybridization oven (UVP, model: HB-500 )
  9. 37 °C, 5% CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i )
  10. Refrigerated centrifuge (Eppendorf, model: 5804 R )
  11. Hemocytometer chamber (Hausser Scientific, catalog number: 3110 )
  12. Water bath (Fisher Scientific, model: Isotemp 2340 )
  13. Thermo ScientificTM NuncTM Lab-Tek® Chamber Slide System (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 178599 )
  14. Fluorescence-activated cell sorter (BD, model: FACSARIATM III )
  15. Inverted microscope (Motic, model: AE20 )
  16. Confocal microscope (ZEISS, model: LSM 710 )
  17. Beaker (Fisher Scientific, catalog numbers: FB10050 , FB100100 , FB100150 )
  18. Autoclave (Getinge, model: PACS 2000 )

Procedure

  1. Fresh brain tissue dissociation into single cell suspension
    Notes:
    1. Tissue processing and FACS are performed in a Biological Safety Level (BSL-2) laminar flow hood, using universal precautions for bloodborne human pathogens. All tissues are collected de-identified, under approved IRB protocols.
    2. It is recommended that tissues/cells are kept on ice as much as possible, unless otherwise noted for specific incubation steps.

    1. Collect fresh brain tissue (GM postmortem dissection or GBM surgical resection) in cold, freshly prepared PIPES solution (see Recipes).
    2. Dissolve papain in activating solution (1 ml of activating solution per 0.003 g papain for each 50-200 mg tissue) (see also Note 7).
      Note: The papain must be activated for at least 20 min but no more than 30 min at room temperature, prior to tissue incubation.
    3. Mechanical digestion
      While the papain is activating, mince the tissue with a razor blade in a small Petri dish, containing a small volume of cold PIPES solution (1-2 ml), until the tissue is finely minced (Figure 1A). The process of mincing should not last more than 5 min. Once the tissue is minced, the Petri dish should be placed on ice.


      Figure 1. Tissue dissociation from fresh human brain tissue. A. Mechanical tissue dissociation using a razor blade, prior to adding papain. B. Enzymatic tissue dissociation using papain, with resultant cell pellet after centrifugation. C. Cell suspension formed after trituration of dissociated tissue. D. Layering of cell suspension onto 22% Percoll solution. E. Separation of cells from extracellular debris after gentle centrifugation in 22% Percoll. Myelin and other extracellular debris are contained within the supernatant (top) while live cells are pelleted (bottom).

    4. Transfer the minced tissue into a 15 ml Falcon tube and add additional cold PIPES solution up to 9 ml. Add 1 ml of activated papain for a final volume of 10 ml (final concentration of papain 300 μg/ml).
      Note: Papain digestion solution (10 ml total) = 0.003 g papain + 1 ml activating solution + 50-200 mg tissue in 9 ml cold PIPES.
    5. Enzymatic digestion
      Incubate the tissue with activated papain, 13 min at 37 °C, in a rotating incubator (rocking speed: 12 rpm). After the first 5 min, add 50 μl of DNase I (10 mg/ml, see Recipes), mix the digesting tissue by shaking, and then re-incubate at 37 °C for the remaining 8 min.
    6. Centrifuge the digested tissue in a refrigerated centrifuge, 10 min at 310 x g at 4 °C.
    7. Stop the enzymatic digestion
      After centrifugation, there will be a pellet of cells (Figure 1B). Gently decant the supernatant, taking care not to disturb the pellet, which may be loose. Resuspend the cells in 1 ml of ice cold DMEM/F-12 with additional 50 μl of DNAse I (10 mg/ml) and 100 μl of papain/trypsin inhibitor (ovomucoid) (7 mg/ml, see Recipes).
    8. Single cell suspension
      Triturate the cell pellet continuously first with a P1000 pipette until no tissue chunks are visible (Video 1). Then, triturate with a P200 pipette, approximately 50 times, or until a murky, homogenous cell suspension is obtained without visible debris (Figure 1C) (Video 2). Avoid foaming.

      Video 1. Primary trituration of dissociated tissue suspension using P1000. After enzymatic digestion, the dissociated cell pellet is triturated continuously first with a P1000 pipette until no large tissue chunks are visible.

      Video 2. Secondary trituration of dissociated tissue suspension using P200. Subsequent trituration with a P200 pipette is also performed in order to obtain a homogenous cell suspension without any visible debris.

    9. Eliminate extracellular debris
      Add 4 ml of cold 22% Percoll solution to new, sterile 15 ml tubes (two tubes per sample). Take approximately half of the cell suspension (~650 μl) and layer it on top of the Percoll in a continuous drop-by-drop manner in order to prevent as much as possible mixing between the cell suspension and the Percoll solution (see Recipes and Note) (Video 3) (Figure 1D). Layer the remaining half of the cell suspension into another tube with 4 ml of cold 22% Percoll (split each cell suspension sample into two Percoll tubes).
      Note: Tilting the Percoll tube at 45° while adding the cell suspension may help to prevent mixing.

      Video 3. Elimination of extracellular debris using Percoll solution. The cell suspension is carefully layered onto a 22% cold Percoll solution in a continuous drop-by-drop manner, with the tube being tilted at 45°, minimizing contact between the Percoll solution and the cell suspension. This step allows elimination of myelin and other extracellular debris.
       
    10. Centrifuge the sample, 10 min at 594 x g in a refrigerated centrifuge at 4 °C, with ‘no brake’ or ‘free deceleration’ setup.
    11. After centrifugation, myelin and other extracellular and fibrillar debris will be contained within the Percoll supernatant, while intact cells will be pelleted at the bottom of the tube (Figure 1E). Carefully decant the entire supernatant and resuspend each pellet in 450 μl of cold 1% BSA/0.1% glucose/1x HBSS (see Recipes). Re-combine every two resuspended pellets (that were split during Step A9) into a new 15 ml Falcon tube.
    12. Eliminate red blood cells:
      Add 100 μl (10x) of Red blood cell lysis (RBL) buffer and incubate at room temperature, 10 min.
    13. Wash the RBL
      After erythrocyte lysis, add 9 ml of cold 1% BSA/0.1% glucose/1x HBSS and centrifuge, 5 min at 310 x g at 4 °C.
    14. Count live cells
      Decant the supernatant and resuspend the cell pellet in 550 μl of cold 1% BSA/0.1% glucose/1x HBSS. Count the number of live cells using trypan blue exclusion in a hemocytometer. Mix 5 μl of trypan blue with 5 μl of sample, and load in a hemocytometer chamber to count live cells, which exclude trypan blue, under the microscope. Calculate the number of live cells in the stock solution.
    15. Ligand and antibody incubation for FACS:
      1. Aliquot out 5% of the total cell suspension for each single color/no color controls (approximately 30 μl for each) and use the remaining cell suspension for the experimental sample.
      2. For the experimental sample, add EGF-Alexa Fluor 647 ligand (5 μg/106 live cells) and exclusion antibody markers (anti CD24-PE, 1:10; anti CD34-PE, 1:10; anti CD45-PE, 1:10) to the remaining cell suspension in the same 15-ml tube (approximately 450 μl). Mix gently by flicking the tube.
      3. For single color controls, add EGF-Alexa Fluor 647 ligand only or CD24-PE/CD34-PE/CD45-PE only to each 30 μl (5%) cell suspension aliquot in a new 15-ml tube, using the same ligand and antibody dilution used for the experimental sample. For the no color (DAPI only) control, incubate the 30 μl aliquot on ice without adding anything and add DAPI only during Step A18.
    16. Incubate on ice, 30 min, in darkness. Re-mix gently by flicking the tube after the first 15 min.
    17. Wash unbound antibodies/ligand
      Wash all tubes with 10 ml of cold 1% BSA/0.1% glucose/1x HBSS and centrifuge, 5 min at 310 g at 4 °C.
    18. Decant the supernatant and resuspend the cell pellet in 1 ml of cold 1% BSA/0.1% glucose/1x HBSS with DAPI (1:1,000).
    19. Achieve single cell suspension and minimize clumping:
      Pass the solution through a 40 μm filter.

  2. Fluorescence-Activated Cell Sorting (FACS)
    Note: Technical support during sorting is highly recommended for untrained personnel.
    1. Gate and sort cells following gating procedure described in Tome-Garcia et al. (2017) and in Figure 2. No color (DAPI only), single color, and fluorescence minus one color (if using more than two colors) controls should be performed with each experiment to establish negative/positive cut-off values for the specific cell populations tested, and to ensure consistency.


      Figure 2. Isolation of EGFR+ cells from freshly dissociated GM and GBM tissues by FACS. Representative examples of sequential gating during FACS in order to eliminate tissue debris (A) (GBM sample), exclude cell doublets (B and C) (GBM sample), remove dead/dying cells using DAPI (D and F) (GBM and GM samples, respectively), and exclude ependymal/neuroblasts/endothelial/inflammatory cells using CD24/34/45 antibody cocktail (E and G) (GBM and GM samples, respectively, see also Note 6). In GBM, but not in GM sorts, a subset of live cells displays a shift in DAPI fluorescence (DAPIlow) (D). The EGFR+ DAPIlow GBM population contains the majority of cells with stem cell properties (Tome-Garcia et al., 2017) (see also Notes). Violet-A, PE-A, and APC-A light filters were used to visualize DAPI, bound CD24/34/45-PE antibodies, and bound EGF-AF647 ligand, respectively.

    2. After proper gating, collect EGF-bound (i.e., EGFR+) live (DAPI- and DAPIlow) cells for molecular or functional downstream analysis. For cell culture, collect cells in a 1.5 ml microcentrifuge tube containing sterile and freshly prepared neurosphere media (see Recipes) supplemented with EGF (20 ng/ml) and bFGF (20 ng/ml).
      Note: Neurosphere growth is also observed in the absence of ligand supplementation.

  3. Functional validation of in vitro stem cell behavior
    Note: Culture validation is recommended for all GBM tumors, since in rare cases, tumors without a well-defined EGFR+ population may show sphere growth in both positive and negative fractions (Tome-Garcia et al., 2017) (see also Note 4).
    1. Seed the cells in 96-well low-attachment plates at a clonal density of 10 cells/μl (200 μl/well), (Pastrana et al., 2011), in triplicate wells at minimum. Let the cells form neurospheres for one week and then change media every three days (replace only ~1/3-1/2 of the media volume, removing it carefully from the edge of the well without disturbing the neurospheres). Neurosphere formation can be checked under an inverted microscope as soon as 1 week after seeding (Figures 3A-3B). Consider letting cells grow for 12-21 days or until neurospheres reach at least 40 μm diameter before passaging them into secondary neurospheres or testing their multipotency for differentiation (Figure 3).


      Figure 3. In vitro functional analysis of stem cell properties of acutely isolated EGFR+ and EGFR- GM and GBM cells. A-B. FACS-isolated EGFR+ and EGFR- primary cells are seeded at clonal density (10 cells/μl). Neurosphere formation, assessed at day 6 of seeding, in GM EGFR+/EGFR- populations (A), and at day 12 in GBM EGFR+/EGFR- populations (B). C. Illustration of trilineage differentiation of primary GM neurospheres using immunofluorescence, with EGFR+ cells showing differentiation into astrocytic (GFAP+,  white); neuronal (TuJ1+, red) and oligodendroglial (O4+, green) lineages. DAPI (blue) is used to counterstain cell nuclei. Magnification of neurosphere pictures, 10x. Pictures have been adapted from Tome-Garcia et al., 2017. Scale bars = 20 μm.

    2. To differentiate neurospheres, pick up single neurospheres with a 200 μl pipette and seed them on laminin-coated chambers. Let the neurospheres attach to the laminin for 4-5 h in a 37 °C cell incubator. Once attached, remove the old neurosphere media, and add new neurosphere media without B27, EGF, bFGF growth factors supplement. Let the neurospheres differentiate in a 37 °C cell incubator for 1 week with no change of media.
      Note: Prior to neurosphere seeding, Lab-Tek® chambers should be pre-coated with laminin (10 μg/ml), incubated overnight at 37 °C, and then washed three times with 1x PBS.
    3. After 1 week of differentiation, fix the cells using 4% PFA solution (see Recipes), 10 min at room temperature. Wash the cells with 1x PBS (see Recipes), three times.
    4. To perform immunofluorescence studies, incubate the differentiated cells with blocking solution (10% normal donkey serum (NDS)/0.5% Triton X-100) (see Recipes), 1 h at room temperature, and then incubate with primary antibodies in 1% NDS/0.25% Triton X-100 (see Recipes), overnight at 4 °C. Perform three washes with 1x PBS, 5 min each, and incubate with species-appropriate fluorochrome-conjugated secondary antibody in 1% NDS/0.25% Triton X-100 (see Recipes), 4 h at room temperature. Wash the secondary antibody with 1x PBS, 5 min each, counterstain with DAPI (1:1,000), and mount with Aqua-Poly/Mount aqueous mounting medium.
    5. Specifically, for O4, immunofluorescence is performed on live, unfixed cells. Incubate cells with primary anti-O4 antibody, 40 min at 4 °C, perform three washes with DMEM/F-12 media, 5 min each at room temperature, and incubate with anti-mouse IgM secondary antibody, 40 min at 4 °C. Wash three times with DMEM/F-12 media at room temperature, 5 min each time, and fix cells for 10 min with 4% PFA solution.
    6. Check immunoreactivity for GFAP (astrocytes), TuJ1 (neurons) and O4 (oligodendrocytes) under a confocal or epifluorescence microscope (Figure 3C).

Data analysis

To ensure statistical significance and reproducibility in the neurosphere and differentiation assays, all experiments were performed in three technical replicates and were repeated at least three independent times. To determine statistical significance for the number of neurospheres between EGFR+ and EGFR- cell populations, unpaired two-tailed Student’s t-test was applied.

Notes

  1. Due to potentially low cell viability in tissues with longer surgical ischemia or postmortem times, samples should be immersed in freshly made (same day) cold PIPES solution immediately after being removed from the brain, and the PIPES-immersed sample should be kept cold (on ice) until it can be processed in the laboratory.
  2. Human germinal matrix specimens display a wide range of viable cell yield (8.9%-96% DAPI- live cells out of total cells) and EGFR+ cells (1%-25% of total viable cells), depending on gestational age and postmortem time. Human GBM samples also display a wide range of viable cell yield (36%-99%) and EGFR+ cells (1.3%-77% of total viable cells), which we attribute to intra- and intertumoral molecular heterogeneity of glioblastoma samples, in addition to post-surgical ischemia time. For detailed information on percent EGFR+ cell yield under the different abovementioned variables, refer to Supplemental Figure 1 in the original report (Tome-Garcia et al., 2017).
  3. Gating of EGFR+ and EGFR- populations can affect the selectivity of stem cell isolation. While we recommend following the gates illustrated in Figure 2, some modifications may be necessary for individual samples. Particularly, for GBM sorts, the proportion of DAPI- and DAPIlow cells can be quite variable, depending on cell aneuploidy, post-surgical ischemia time, and the percentage of glioma cells with stem cell properties in the tissue sample. In our experience, the majority of tumor cells with stem cell properties are captured in the DAPIlow (EGFR+) population, although occasional DAPI- (EGFR+) cells also showed sphere formation in some tumor samples. Thus, depending on whether specificity or sensitivity is a priority for the downstream analysis, the user may choose to sort DAPI- separately from DAPIlow, or to combine both as illustrated in Figure 2.
  4. The use of EGF ligand for FACS isolation of sphere-forming human glioma populations has been validated for most de novo glioblastoma resections, both IDH-wildtype and IDH-mutant, more than 90% of which show expression of EGFR, even in cases when EGFR amplification is not detected (Erfani et al., 2015; Tome-Garcia et al., 2017). This methodology may not be applicable for GBMs with strong amplification of other receptor tyrosine kinases, such as the platelet-derived growth factor receptor alpha (PDGFRalpha) seen in ~10% of GBMs (Brennan et al., 2013), especially if the tumor lacks significant EGFR expression altogether (2/19 GBMs tested in our cohort) (Tome-Garcia et al., 2017 [Figure S2]). This methodology has not been validated for recurrent GBMs and lower-grade gliomas. 
  5. In germinal matrix tissues of early gestation age (< 18 weeks), rare small sphere formations have been observed in the EGFR- population, which were unable to undergo trilineage differentiation (Tome-Garcia et al., 2017 [Figure S2]).
  6. In the outlined experiments above and previously published (Tome-Garcia et al., 2017), CD24 was used as an exclusion marker of neuroblasts and ependymal cells in GM sorts, and was also included in GBM sorts in order to mimic FACS conditions between GM and GBM. Inclusion of CD24 in GBM sorts is optional, but omitting it has not been fully tested.
  7. The weight of tissue processed can vary, depending on GM gestational age and the extent of GBM resection. In our experiments, we calibrated the papain concentration based on a range of 50-200 mg of tissue. If more than 200 mg of tissue was used, we digested the additional tissue in a separate papain solution (0.003 g papain per 50-200 mg of additional tissue). Most of our processed samples had a starting weight of ~300 mg. For processing of tissue less than 50 mg, papain concentration may need to be optimized.

Recipes

  1. 10 N sodium hydroxide (NaOH)
    20 g of NaOH pellets
    Bring the volume to 50 ml with ultrapure distilled water
    Note: As a precaution use a beaker and keep it on ice during the process.
  2. 1 M piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES)
    1. Dissolve 30.22 g of PIPES in 90 ml of ultrapure distilled water
    2. Add NaOH pellets until reaching pH 6.7
    3. Add 10 N NaOH dropwise to reach pH 6.9
    4. Bring the volume to 100 ml with ultrapure distilled water
    5. Filter with a 0.2 μm filter
  3. 10x sodium chloride (NaCl)/potassium chloride (KCl) (NaCl/KCl) salt solution
    3.5 g sodium chloride (NaCl)
    0.186 g potassium chloride (KCl)
    Bring the volume to 50 ml with ultrapure distilled water
    Filter with a 0.2 μm filter
  4. 30% D-glucose solution
    15 g D-glucose
    Bring the volume to 50 ml with ultrapure distilled water
    Filter with a 0.2 μm filter and store at 4 °C
  5. 1 N NaOH
    2 g of NaOH pellets
    Bring the volume to 50 ml with ultrapure distilled water
    Note: As a precaution use a beaker and keep it on ice during the process.
  6. PIPES solution
    1 ml 1 M PIPES
    5 ml 10x NaCl/KCl salt solution
    750 μl 30% glucose
    1,250 μl 0.02% phenol red
    500 μl 100x antibiotic/antimycotic
    Bring the volume to 50 ml with ultrapure distilled water
    Add 75 μl 1 N NaOH to turn the solution from salmon to pink (pH 7.4, if it turns magenta-to-purple, start over)
    Filter with a 0.2 μm filter
  7. Activating solution for papain
    0.00878 g Cysteine HCl
    0.0093 g EDTA
    Bring the volume to 50 ml with ultrapure distilled water
    Filter with a 0.2 μm filter and store at 4 °C
  8. 10 mg/ml DNase I solution
    10 mg DNase I
    Bring the volume to 1,000 μl with ultrapure distilled water
    Aliquot and keep frozen at -20 °C
  9. 7 mg/ml ovomucoid
    7 mg ovomucoid
    Bring the volume to 1,000 μl with ultrapure distilled water
    Aliquot and keep frozen at -20 °C
  10. 10x phosphate buffered saline (PBS)
    98.9 g 10x concentrated powder PBS
    Bring the volume to 1 L with ultrapure distilled water
    Dissolve and autoclave
  11. 22% Percoll solution
    11 ml Percoll
    5 ml 10x PBS
    Bring the volume to 34 ml with ultrapure distilled water
    Filter with 0.2 μm filter and store at 4 °C
  12. 10% bovine serum albumin (BSA)
    5 g BSA
    Bring the volume to 50 ml with 1x HBSS
    Store in the fridge until it dissolves completely
    Filter with 0.2 μm filter and store at 4 °C
  13. 1% BSA/0.1% glucose/1x HBSS
    50 ml 10% BSA
    1.67 ml 30% D-glucose
    448.3 ml 1x HBSS
    Filter with a 0.2 μm filter and store at 4 °C
  14. 1x PBS
    100 ml 10x PBS
    900 ml ultrapure distilled water
  15. Neurosphere media
    500 μl N-2 Supplement (100x)
    1,000 μl B-27 Supplement (50x)
    500 μl 200 mM L-glutamine
    500 μl 100x antibiotic-antimycotic
    500 μl 100x Insulin-Transferrin-Selenium (ITS-X)
    750 μl 1 M HEPES
    1 ml 30% D-glucose
    Bring the volume to 50 ml with DMEM/F12
  16. Epidermal Growth Factor (EGF) (20 μg/ml)
    1. Resuspend the lyophilized product in enough volume of ultrapure distilled water to have a final stock concentration of 20 μg/ml
    2. Dilute 1:1,000 in Neurosphere media to reach a working concentration of 20 ng/ml
  17. Basic Fibroblast Growth Factor (bFGF) (20 μg/ml)
    1. Resuspend the lyophilized product in enough volume of ultrapure distilled water to have a final stock concentration of 20 μg/ml
    2. Dilute 1:1,000 in Neurosphere media to reach a working concentration of 20 ng/ml
  18. 10% normal donkey serum (NDS)/0.5% Triton X-100
    100 μl NDS
    5 μl Triton X-100
    895 μl 1x PBS
  19. 1% normal donkey serum (NDS)/0.25% Triton X-100
    10 μl NDS
    2.5 μl Triton X-100
    987.5 μl 1x PBS
  20. 4% paraformaldehyde (PFA)
    Dilute 10 ml of 16% PFA with 30 ml of 1x PBS

Acknowledgments

These experimental procedures have been previously published and are herein adapted and modified from Tome-Garcia et al., 2017. The work was partially supported by R03NS104669 (N.T.). We thank members of the Pathology department and Biorepository Core at the Icahn School of Medicine at Mount Sinai (ISMMS) for facilitating tissue collection and procurement and the ISMMS Flow Cytometry CORE for expert advice and accommodation of fresh human tissue sorts 24 h/day. The research was supported by Mount Sinai seed grant funds. The authors declare no conflict of interest.

References

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  9. Lathia, J. D., Gallagher, J., Heddleston, J. M., Wang, J., Eyler, C. E., Macswords, J., Wu, Q., Vasanji, A., McLendon, R. E., Hjelmeland, A. B. and Rich, J. N. (2010). Integrin alpha 6 regulates glioblastoma stem cells. Cell Stem Cell 6(5): 421-432.
  10. Lathia, J. D., Mack, S. C., Mulkearns-Hubert, E. E., Valentim, C. L. and Rich, J. N. (2015). Cancer stem cells in glioblastoma. Genes Dev 29(12): 1203-1217.
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简介

从新鲜组织中直接分离人类神经和胶质瘤干细胞允许其在没有事先培养的情况下进行生物学研究,并且可以在其天然状态中捕获其分子表型的新方面。最近,我们展示了前瞻性地从新鲜人类生发基质和胶质母细胞瘤样品中分离干细胞群的能力,利用细胞在荧光激活细胞分选(FACS)中结合表皮生长因子(EGF)配体的能力。我们证明FACS分离的EGF结合的神经和成胶质细胞瘤细胞群体在体外包含球体形成的集落,并且能够自我更新和多向分化。在此我们详细描述了具有来自新鲜死亡和手术组织的干细胞特性的EGF-结合(即EGFR +)人类神经和胶质瘤细胞的纯化方法。利用天然配体结合能力前瞻性分离干细胞群的能力为了解非培养条件下的正常和肿瘤细胞生物学打开了新的门,并且适用于在种群和单细胞分辨率下的各种下游分子测序研究。

【背景】由于缺乏通用的神经和神经胶质瘤干细胞标志物(Lathia et al。,2015)以及频繁依赖于培养的细胞,理解人神经和胶质瘤干细胞的内在生物学一直是一个挑战比那些直接从组织分离的。跨膜糖蛋白Prominin或CD133是分离神经(Uchida等,2000)和神经胶质瘤干细胞(GSC)(Singh等,2000)的最好描述和经常使用的干细胞标记物之一。等人,2003; Singh等人,2004; Lathia等人,2015),其效用在两个急剧排序人体组织和神经球。然而,最近的一些研究已经指出,从人胶质母细胞瘤(GBM)中分离的CD133阴性细胞也具有干细胞特性(Beier et al。,2007; Wang等人 。,2008; Tome-Garcia等人,2017)。用于分离GSC的其他细胞表面标志物包括CD44(Anido等,2010),CD15(Son等,2009),A2B5(Ogden等,et al。 ,2008),整联蛋白α(Lathia等人,2010)或EGFR(Mazzoleni等人,2010),但是这些基于抗体的方法也缺乏捕获所有形成球体的群体的能力,或者它们的使用仅限于培养的细胞。
非肿瘤和肿瘤干细胞生态位之间的分子表型的直接比较可以提供肿瘤形成过程中选择的发育途径的新颖见解,并且可以揭示更全面的干细胞标志物。在神经胶质瘤形成的背景下,已经证明对于正常神经祖细胞的生长和增殖重要的若干发育途径在神经胶质瘤形成过程中异常地被再活化(Sanai et al。,2005; Canoll and Goldman,2008; Chen ,2012; Tsankova和Canoll,2014; Lathia等人,2015年)。其中包括表皮生长因子受体(EGFR)酪氨酸激酶途径。在人类神经发育过程中,尤其是在生发基质干细胞生态位内EGFR的表达高,在成年期显着减少(Weickert et al。,2000; Sanai et al。 2011; Erfani et al。,2015),并且在GBM中被异常地重新激活(Verhaak et al。,2010; Brennan等人 >,2013)。最近,我们调整了小鼠荧光激活细胞分选(FACS)策略,其基于其与EGF配体的天然结合选择EGFR +细胞(Ciccolini等人,2005; Pastrana等人2009年; Codega等人,2014年),以及从新鲜生发基质(GM)解剖和GBM组织中分离的人类EGFR +群体。这使我们能够直接比较发育和赘生性神经群体的功能特性和全转录组特征(Tome-Garcia et al。,2017)。来自GM和GBM组织的EGFR +群体在体外捕获所有球形细胞,显示出相似的增殖干细胞特性,并且共享与细胞生长和细胞周期调控相关的转录组签名。 EGFR + GBM群体也在体内显示肿瘤起始(Tome-Garcia等人,2017)。下面,我们描述了具有来自原始人类样品的干细胞特性的神经和成胶质细胞瘤细胞群的这种前瞻性纯化策略,详述了组织解离,配体/抗体孵育,FACS和体外功能性干细胞特性分析。

关键字:EGF, EGFR, FACS, 干细胞, 神经, 胶质母细胞瘤, 生发基质, 人

材料和试剂

  1. 移液器吸头(Fisher Scientific,Fisherbrand TM,目录号:02-707-404; 02-707-430; 02-707-432)
  2. 美国安全剃刀Personna™TM Pal单刃刀片(AccuTec刀片,目录号:620177)
  3. Falcon组织培养皿(Corning,Falcon ,目录号:353002)
  4. Falcon15ml锥形离心管(Corning,Falcon ,产品目录号:352099)
  5. 40μm细胞过滤器(Corning,Falcon ,目录号:352340)
  6. 1.7毫升微量离心管卡扣管(Corning,Costar ®,目录号:3621)
  7. 超低附件96孔板(康宁,目录号:3474)
  8. 0.2μm过滤器(Corning,目录号:431219)
  9. 10毫升血清移液器(康宁,目录号:357530)
  10. 60毫升注射器(BD,目录号:309653)
  11. 微盖玻璃(VWR,目录号:48393-081)
  12. 木瓜蛋白酶(Worthington Biochemical,产品目录号:LS003119)
  13. 10x红血细胞裂解缓冲液(Thermo Fisher Scientific,eBioscience TM,产品目录号:00-4300-54)
  14. 台盼蓝溶液,0.4%(Thermo Fisher Scientific,Gibco TM,产品目录号:15250061)
  15. 4',6-二脒基-2-苯基吲哚二盐酸盐(DAPI)(Thermo Fisher Scientific,Invitrogen TM,目录号:D1306)
  16. 抗体
    1. 抗CD24 PE-缀合的(BD,BD Biosciences,目录号:560991,工作稀释度1:10)
    2. 抗CD34 PE-缀合的(BD,BD Biosciences,目录号:550619,工作稀释度1:10)
    3. 抗CD45 PE-缀合的(BD,BD Biosciences,目录号:555483,工作稀释度1:10)
    4. 驴抗兔Cy3结合(杰克逊免疫研究,目录号:711-165-152,工作稀释度1:250)
    5. 驴抗大鼠AF647结合(杰克逊免疫研究,目录号:712-605-153,工作稀释度1:500)
    6. EGF配体-AF647缀合(Thermo Fisher Scientific,Invitrogen TM,目录号:E35351)。
    7. 山羊抗小鼠-AF488 IgM,μ链特异性(Jackson ImmunoResearch,目录号:115-545-075,工作稀释度1:500)
    8. 小鼠抗O4(Millipore Sigma,目录号:MAB345,工作稀释度1:200)
    9. 兔抗TuJ1(Covance,目录号:PRB-435P,工作稀释度1:1,000)
    10. 大鼠抗GFAP(Thermo Fisher Scientific,Invitrogen TM,产品目录号:13-0300,工作稀释度1:1,000)。
  17. 层粘连蛋白1mg / ml(Thermo Fisher Scientific,Gibco TM,目录号:23017015)
  18. Aqua-Poly / Mount(Polysciences,目录号:18606-20)
  19. 氢氧化钠(NaOH)(Fisher Scientific,目录号:S392212)
  20. JustPure PIPES(Fisher Scientific,目录号:BP292450)
    注:此产品已停产。
  21. D-葡萄糖(Fisher Scientific,目录号:D16-1)
  22. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333-500G)
  23. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-1)
  24. 超纯蒸馏水(Thermo Fisher Scientific,Invitrogen TM,目录号:10977023)
  25. 酚红溶液,0.02%(Fisher Scientific,目录号:S25464)
  26. 抗生素 - 抗真菌药100x(Thermo Fisher Scientific,Gibco TM,目录号:15240062)
  27. L-半胱氨酸盐酸盐(Sigma-Aldrich,目录号:C1276-10G)
  28. 乙二胺四乙酸(EDTA)(Fisher Scientific,目录号:BP118-500)
  29. 脱氧核糖核酸酶I(DNase I)(Worthington Biochemical,目录号:LS002139)
  30. 胰蛋白酶抑制剂卵类粘蛋白(Sigma-Aldrich,目录号:T9253-250MG)
  31. 磷酸盐缓冲盐水(PBS)pH7.4(Fisher Scientific,目录号:BP665-1)
  32. Percoll(MP Biomedicals,目录号:0219536925)
  33. 牛血清白蛋白(BSA)(Fisher Scientific,目录号:BP9704100)
  34. 1x Hank平衡盐溶液(HBSS),不含钙,不含镁(Thermo Fisher Scientific,Gibco TM,产品目录号:14175103)
  35. B-27补充物(50x),无血清(Thermo Fisher Scientific,Gibco TM,产品目录号:17504044)
  36. N-2补充物(100x)(Thermo Fisher Scientific,Gibco TM,目录号:17502048)
  37. L-谷氨酰胺(200mM)(Thermo Fisher Scientific,Gibco TM,目录号:25030149)
  38. 100x胰岛素 - 转铁蛋白 - 硒(ITS-X)(Thermo Fisher Scientific,Gibco TM,目录号:51500056)
  39. Dulbecco改良的Eagle培养基/营养混合物F-12(DMEM / F-12)(Thermo Fisher Scientific,Gibco TM,产品目录号:11320082)
  40. 人重组碱性成纤维细胞生长因子(bFGF)(STEMCELL Technologies,目录号:78003)
  41. 人重组表皮生长因子(EGF)(STEMCELL Technologies,目录号:78006.1)
  42. 16%多聚甲醛(PFA)(Alfa Aesar,目录号:43368)
  43. 正常驴血清(NDS)(Jackson ImmunoResearch,目录号:017-000-121)
  44. 将1M HEPES(4-(2-羟乙基)-1-哌嗪乙磺酸)(Thermo Fisher Scientific,Gibco TM,目录号:15630080)
  45. Triton X-100(Fisher Scientific,目录号:BP151-500)
  46. 10N氢氧化钠(NaOH)(见食谱)
  47. 1 M PIPES(见食谱)
  48. 10倍氯化钠(NaCl)/氯化钾(KCl)(NaCl / KCl)盐溶液(见食谱)
  49. 30%的D-葡萄糖溶液(见食谱)
  50. 1 N NaOH(见食谱)
  51. PIPES解决方案(请参阅食谱)
  52. 激活木瓜蛋白酶的解决方案(见食谱)
  53. 10毫克/毫升DNase I溶液(见食谱)
  54. 7毫克/毫升卵类粘蛋白(见食谱)
  55. 10倍磷酸盐缓冲盐水(PBS)(见食谱)
  56. 22%Percoll解决方案(见食谱)
  57. 10%牛血清白蛋白(BSA)(见食谱)
  58. 1%BSA / 0.1%葡萄糖/ 1倍HBSS(见食谱)
  59. 1x PBS(见食谱)
  60. 神经球媒体(见食谱)
  61. 表皮生长因子(EGF)(20μg/ ml)(见食谱)
  62. 碱性成纤维细胞生长因子(bFGF)(20μg/ ml)(见食谱)
  63. 10%正常驴血清(NDS)/0.5% Triton X-100(见食谱)
  64. 1%正常驴血清(NDS)/0.25% Triton X-100(见食谱)
  65. 4%多聚甲醛(PFA)(见食谱)

设备

  1. 生物安全柜(Labconco,型号:Class II,Type A2)
  2. P2 pipetman(Gilson,目录号:F144801)
  3. P20 pipetman(Gilson,目录号:F123600)
  4. P200移液器(Gilson,目录号:F123601)
  5. P1000移液器(吉尔森,目录号:F123602)
  6. 电动移液器控制器(Accupet,目录号:PH01)
  7. 毫克平衡(Sartorius,型号:Entris 323)
  8. Minidizer杂交炉(UVP,型号:HB-500)
  9. 37℃,5%CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heracell TM 150i)
  10. 冷冻离心机(Eppendorf,型号:5804 R)
  11. 血细胞计数器室(Hausser Scientific,目录编号:3110)
  12. 水浴(Fisher Scientific,型号:Isotemp 2340)
  13. Thermo Scientific TM Nunc TM Lab-Tek室幻灯片系统(Thermo Fisher Scientific,Thermo Scientific TM,目录号号码:178599)
  14. 荧光激活细胞分选仪(BD,型号:FACSARIA TM III)
  15. 倒置显微镜(Motic,型号:AE20)
  16. 共聚焦显微镜(蔡司,型号:LSM 710)
  17. 烧杯(Fisher Scientific,产品目录号:FB10050,FB100100,FB100150)

  18. 高压灭菌器(Getinge,型号:PACS 2000)

程序

  1. 新鲜脑组织解离成单细胞悬液
    注意:
    1. 组织处理和FACS在生物安全级别(BSL-2)层流罩中进行,对于血源人类病原体使用通用预防措施。在批准的IRB协议下,收集所有的组织并进行鉴定。
    2. 建议将组织/细胞尽可能保存在冰上,除非特定的孵化步骤另有说明。
    1. 在冷的新鲜制备的PIPES溶液中收集新鲜的脑组织(GM尸体解剖或GBM手术切除)(见食谱)。
    2. 将木瓜蛋白酶溶解在活化溶液中(每50-200mg组织用0.003g木瓜蛋白酶溶解1ml活化溶液)(参见注释7)。
      注意:在组织培养之前,木瓜蛋白酶必须在室温下活化至少20分钟但不超过30分钟。
    3. 机械消化
      在木瓜蛋白酶活化的同时,用含有少量冷PIPES溶液(1-2ml)的小培养皿中的刀片将组织切碎,直至组织细碎(图1A)。切碎的过程不应超过5分钟。一旦组织被切碎,培养皿应放置在冰上。


      图1.新鲜人脑组织解离A.在加入木瓜蛋白酶之前使用剃刀刀片机械组织解离。 B.使用木瓜蛋白酶进行酶解组织解离,离心后产生细胞沉淀。 C.游离组织研磨后形成的细胞悬液。 D.将细胞悬液分层到22%Percoll溶液上。 E.在22%Percoll中温和离心后从细胞外碎片中分离细胞。
      髓鞘和其他细胞外碎片包含在上清液(上),而活细胞沉淀(下)。

    4. 将切碎的组织转移到15ml Falcon管中,并添加额外的冷PIPES溶液至9ml。加入1毫升活化的木瓜蛋白酶,终体积为10毫升(木瓜蛋白酶终浓度为300微克/毫升)。
      注意:木瓜蛋白酶消化液(总共10毫升)= 0.003克木瓜蛋白酶+ 1毫升活化液+ 50-200毫克组织在9毫升冷PIPES。
    5. 酶消化
      用旋转的培养箱(摇动速度:12rpm)在37℃下用活化的木瓜蛋白酶孵育组织13分钟。在第一个5分钟后,加入50μlDNase I(10mg / ml,参见食谱),通过摇动混合消化组织,然后在37℃重新孵育剩余的8分钟。
    6. 在冷藏离心机中离心消化组织,10分钟,310℃×4℃。
    7. 停止酶消化
      离心后,将有一个细胞颗粒(图1B)。轻轻倒出上清液,注意不要打扰可能松散的颗粒。用另外的50μlDNAseI(10mg / ml)和100μl木瓜蛋白酶/胰蛋白酶抑制剂(卵类粘蛋白)(7mg / ml,参见食谱)将细胞重悬于1ml冰冷的DMEM / F-12中。 />
    8. 单细胞悬液
      首先用P1000移液管连续研磨细胞团,直到没有可见的组织块(视频1)。然后,用P200移液管研磨约50倍,或直到获得没有可见碎片的黑色均质细胞悬液(图1C)(视频2)。避免起泡。

      视频1

      视频2

    9. 消除细胞外碎片
      添加4毫升冷的22%Percoll溶液到新的无菌15毫升管(每个样品两管)。取大约一半的细胞悬液(〜650μl)并将其铺在Percoll的顶部,以连续的逐滴方式进行,以尽可能地防止细胞悬液和Percoll溶液之间的混合(参见食谱和注)(视频3)(图1D)。将剩余的一半细胞悬液与4ml冷的22%Percoll(将每个细胞悬液样品分成两个Percoll管)一起放入另一个管中。
      注意:在添加细胞悬液时倾斜Percoll管45°可能有助于防止混合。

      视频3
    10. 在4°C的冷冻离心机中以594克xg离心10分钟,同时设置“不制动”或“自由减速”。
    11. 离心后,髓鞘和其他细胞外和纤维状碎片将被包含在Percoll上清液中,而完整的细胞将在管底部沉淀(图1E)。仔细滗析整个上清液,并重悬在450微升冷1%BSA / 0.1%葡萄糖/ 1倍HBSS(见食谱)每粒。将每两颗重新悬浮的颗粒(在步骤A9期间分开)重新组合成新的15ml Falcon管。
    12. 消除红血细胞:
      加入100μl(10x)红血细胞溶解(RBL)缓冲液并在室温下孵育10分钟。
    13. 清洗RBL
      红细胞裂解后,加入9ml冷的1%BSA / 0.1%葡萄糖/ 1x HBSS,并在4℃以310×g离心5分钟。
    14. 计数活细胞
      滗析上清液并用550μl冷的1%BSA / 0.1%葡萄糖/ 1×HBSS重悬细胞沉淀。在血细胞计数器中使用台盼蓝排除计数活细胞的数量。将5μl台盼蓝与5μl样品混合,并在血细胞计数器室中加载,以在显微镜下计数排除台盼蓝的活细胞。计算原液中活细胞的数量。
    15. 用于FACS的配体和抗体孵育:
      1. 将每种单一颜色/无颜色对照(每种约30μl)分出总细胞悬液的5%,并将剩余的细胞悬液用于实验样品。
      2. 对于实验样品,添加EGF-Alexa Fluor 647配体(5μg/ 10 6活细胞)和排除抗体标志物(抗CD24-PE,1:10;抗CD34-PE,1:10 ;抗CD45-PE,1:10)到相同15-ml管中的剩余细胞悬液(约450μl)中。轻轻地搅拌,轻轻搅拌。
      3. 对于单一颜色的对照,只使用相同的配体,在每个30微升(5%)的细胞悬液中加入EGF-Alexa Fluor 647配体或CD24-PE / CD34-PE / CD45-用于实验样品的抗体稀释液。对于无颜色(仅限DAPI)的对照,将30μl等分试样在冰上孵育而不添加任何物质,仅在步骤A18期间添加DAPI。
    16. 冰上孵育30分钟,在黑暗中。
      在第一个15分钟后,轻轻地轻轻搅拌
    17. 洗涤未结合的抗体/配体
      用10毫升冷的1%BSA / 0.1%葡萄糖/ 1倍HBSS清洗所有试管,并在310℃4℃下离心5分钟。
    18. 滗析上清液,并将细胞沉淀重悬于1ml含1%DBSI(1:1,000)的冷1%BSA / 0.1%葡萄糖/ 1x HBSS中。
    19. 实现单细胞悬浮,并尽量减少结块:
      将溶液通过一个40微米的过滤器。

  2. 荧光激活细胞分选(FACS)
    注意:分类过程中的技术支持强烈建议未经培训的人员。
    1. 按照Tome-Garcia et al 中描述的门控程序门和分选细胞。 (2017)和图2中。每个实验都应该执行没有颜色(仅限DAPI),单色和荧光减去一种颜色(如果使用两种以上颜色)的对照,以建立特定的阴性/阳性截止值细胞群体测试,并确保一致性。


      图2.通过FACS从新分离的GM和GBM组织中分离EGFR +细胞为了消除组织碎片(A)(GBM样品),在FACS期间连续选通的代表性实例排除细胞双联体(B和C)(GBM样品),使用DAPI(D和F)(分别为GBM和GM样品)去除死亡细胞,并使用CD24 / 34/45抗体混合物(E和E)排除室管膜/成神经细胞/内皮/ G)(GBM和GM样本,分别见附注6)。在GBM中,但不是在GM中,活细胞的子集显示DAPI荧光(DAPI low )(D)的转变。 EGFR + DAPI低位GBM群体含有大部分具有干细胞特性的细胞(Tome-Garcia等人,2017)(另见注释)。使用紫色-A,PE-A和APC-A滤光片分别显现DAPI,结合的CD24 / 34/45-PE抗体和结合的EGF-AF647配体。

    2. 在适当的门控之后,收集EGF-结合的(即,EGFR +)活的(DAPI-和DAPI低)细胞用于分子或功能下游分析。对于细胞培养,将细胞收集在含有补充有EGF(20ng / ml)和bFGF(20ng / ml)的无菌和新鲜制备的神经球培养基(参见配方)的1.5ml微量离心管中。
      注意:在没有配体补充的情况下,也会观察到神经球的生长。

  3. 体外干细胞行为的功能验证
    注意:对于所有的GBM肿瘤,建议进行培养验证,因为在极少数情况下,没有明确定义的EGFR +群体的肿瘤可能在正面和负面部分显示球体生长(Tome-Garcia等,2017) em>
    1. 将细胞以10个细胞/μl(200μl/孔)的克隆密度(Pastrana et al。,2011)在96孔低附着板中以最少的三个重复孔进行接种。让细胞形成神经球一周,然后每隔三天更换一次培养基(仅替换介质体积的〜1 / 3-1 / 2,小心地从孔的边缘去除而不干扰神经球)。接种1周后,可在倒置显微镜下检查神经球形成情况。考虑让细胞生长12-21天或直到神经球达到至少40微米的直径,然后将它们传递到次级神经球或测试其分化的多能性(图3)。


      图3.急性分离的EGFR +和EGFR-GM和GBM细胞的干细胞特性的体外功能分析A-B。 FACS分离的EGFR +和EGFR-原代细胞以克隆密度(10个细胞/μl)接种。 (B)在GM EGFR + / EGFR-群(A)中和第12天,在接种的第6天评估神经球形成。 C.使用免疫荧光,使EGFR +细胞显示分化成星形胶质细胞(GFAP +,白色)的原代GM神经球的三系分化的说明;神经元(TuJ1 +,红色)和少突神经胶质(O4 +,绿色)谱系。 DAPI(蓝色)用于复染细胞核。神经球图片放大倍数为10倍。图片已经从Tome-Garcia et al。,2017改编。比例尺= 20微米。

    2. 为了区分神经球,用200μL移液管拿起单个神经球,并将其种在层粘连蛋白涂层的室上。让神经球粘附层粘连蛋白4-5小时在37°C细胞培养箱。一旦连接,删除旧的神经球媒体,并添加新的神经球媒体没有B27,EGF,bFGF生长因子补充。让神经球在37°C细胞培养箱中分化1周,不改变培养基。
      注意:在神经球接种之前,Lab-Tek室应该用层粘连蛋白(10μg/ ml)预包被,在37℃下孵育过夜,然后用1x PBS。
    3. 分化1周后,用4%PFA溶液(参见食谱)固定细胞,室温下10分钟。用1x PBS洗细胞(见食谱),三次。
    4. 为了进行免疫荧光研究,用封闭溶液(10%正常驴血清(NDS)/0.5% Triton X-100)(见食谱)孵育分化细胞,室温1小时,然后与1%NDS中的一抗/0.25% Triton X-100(见食谱),在4℃过夜。用1x PBS洗涤3次,每次5分钟,并在室温下4小时,在1%NDS / 0.25%Triton X-100(参见配方)中与种类合适的荧光染料结合的二抗孵育。用1x PBS清洗二次抗体,每次5分钟,用DAPI(1:1,000)复染,并用Aqua-Poly / Mount水性固定介质进行安装。
    5. 具体而言,对于O4,在活的未固定的细胞上进行免疫荧光。孵育细胞与初级抗O4抗体,在4°C 40分钟,用DMEM / F-12培养基洗三次,每次室温5分钟,并与抗小鼠IgM二抗孵育,在4°C孵育40分钟。用DMEM / F-12培养基在室温下每次洗涤5次,每次5分钟,用4%PFA溶液固定细胞10分钟。
    6. 检查GFAP(星形胶质细胞),TuJ1(神经元)和O4(少突胶质细胞)在共聚焦或落射荧光显微镜下(图3C)的免疫反应性。

数据分析

为了确保神经球和分化测定中的统计学显着性和重现性,所有实验以三个技术重复进行,并且重复至少三次独立的时间。为了确定EGFR +和EGFR-细胞群体之间的神经球数量的统计学显着性,应用未配对的双尾Student's检验。

笔记

  1. 由于在手术缺血或死后时间较长的组织中细胞活力可能较低,应将样品从大脑中取出后立即浸入新鲜制备的(同一天)冷PIPES溶液中,浸入PIPES的样品应保持冷冻(在冰),直到它可以在实验室处理。
  2. 根据胎龄和死后时间,人胚胎基质标本显示出广泛的活细胞产量(总细胞中有8.9%-96%的DAPI-活细胞)和EGFR +细胞(总有活力细胞的1%-25%)。此外,人类GBM样品还表现出广泛的活细胞产率(36%-99%)和EGFR +细胞(总活细胞的1.3%-77%),这也归因于胶质母细胞瘤样品的内和分子间异质性到术后缺血时间。有关上述不同变量下EGFR +细胞百分比的详细信息,请参阅原始报告(Tome-Garcia et al。2017)中的补充图1。
  3. EGFR +和EGFR-群体的门控可影响干细胞分离的选择性。尽管我们建议遵循图2所示的门,但是对于单个样品可能需要进行一些修改。特别是,对于GBM分选,DAPI-和DAPI低细胞的比例可以是相当可变的,这取决于细胞非整倍性,手术后缺血时间以及具有干细胞特性的神经胶质瘤细胞的百分比组织样本。根据我们的经验,大多数具有干细胞特性的肿瘤细胞被捕获在DAPI低(EGFR +)群体中,尽管偶尔的DAPI-(EGFR +)细胞也在一些肿瘤样品中显示球形成。因此,根据特异性或灵敏度是否是下游分析的优先级,用户可以选择将DAPI与DAPI low分开,或者将两者组合如图2所示。
  4. 用于FACS分离形成球形的人胶质瘤群体的EGF配体的使用已经很好地用于从头部胶质母细胞瘤的切除,其中大多数甚至当未检测到EGFR扩增时显示EGFR的表达。该方法可能不适用于复发肿瘤以及缺乏显着EGFR表达的那些肿瘤(Tome-Garcia等,2017 [图S2]),并且尚未在低级别胶质瘤中进行检测。
  5. 在早孕期(<18周)的生发基质组织中,已经在EGFR-群体中观察到罕见的小球形成,其不能进行三系分化(Tome-Garcia等人, 2017 [图S2])。
  6. 在以上和先前公开的(Tome-Garcia等人,2017)上述概述的实验中,CD24被用作GM分类中的成神经细胞和室管膜细胞的排除标志物,并且还被包括在GBM中为了模仿GM和GBM之间的FACS条件。在GBM排序中包含CD24是可选的,但省略它还没有完全测试。
  7. 处理组织的重量可能会有所不同,这取决于GM孕龄和GBM切除的程度。在我们的实验中,我们基于50-200mg组织的范围校准了木瓜蛋白酶浓度。如果使用超过200mg的组织,我们在另外的木瓜蛋白酶溶液(每50-200mg另外的组织0.003g木瓜蛋白酶)中消化另外的组织。我们大多数加工的样品起始重量约为300毫克。为了处理小于50毫克的组织,木瓜蛋白酶浓度可能需要优化。

食谱

  1. 10N氢氧化钠(NaOH)
    20克NaOH颗粒

    用超纯蒸馏水将体积加到50毫升 注意:作为一项预防措施,使用烧杯并在此过程中保持在冰上。
  2. 1 M哌嗪-N,N'-双(2-乙磺酸)(PIPES)
    1. 将30.22克PIPES溶于90毫升超纯蒸馏水中
    2. 加入NaOH颗粒直至pH值达到6.7 逐滴添加10 N NaOH达到pH 6.9。
    3. 用超纯蒸馏水将体积加到100毫升
    4. 使用0.2μm过滤器过滤
    5. 10倍氯化钠(NaCl)/氯化钾(KCl)(NaCl / KCl)盐溶液
      3.5克氯化钠(NaCl)
      0.186克氯化钾(KCl)

      用超纯蒸馏水将体积加到50毫升 用0.2μm过滤器过滤
    6. 30%D-葡萄糖溶液
      15克D-葡萄糖

      用超纯蒸馏水将体积加到50毫升 使用0.2μm过滤器进行过滤,并在4°C下保存
    7. 1 N NaOH
      2克NaOH颗粒

      用超纯蒸馏水将体积加到50毫升 注意:作为一项预防措施,使用烧杯并在此过程中保持在冰上。
    8. PIPES解决方案
      1毫升1 M PIPES
      5毫升10倍氯化钠/氯化钾盐溶液
      750微升30%的葡萄糖
      1,250μl0.02%酚红
      500μl100x抗生素/抗真菌剂

      用超纯蒸馏水将体积加到50毫升 加入75μl1N氢氧化钠将溶液从鲑鱼变成粉红色(pH值为7.4,如果变成紫红色,则重新开始)
      用0.2μm过滤器过滤
    9. 激活木瓜蛋白酶解决方案
      0.00878克半胱氨酸盐酸盐
      0.0093g EDTA

      用超纯蒸馏水将体积加到50毫升 使用0.2μm过滤器进行过滤,并在4°C下保存
    10. 10毫克/毫升DNase I溶液
      10毫克脱氧核糖核酸我

      用超纯蒸馏水将容量调至1000μl 分装并保存在-20°C
    11. 7毫克/毫升卵类粘蛋白
      7毫克ovomucoid

      用超纯蒸馏水将容量调至1000μl
      在-20°C分装并保持冷冻
    12. 10倍磷酸盐缓冲盐水(PBS)
      98.9克10倍浓缩PBS PBS
      用超纯蒸馏水将体积加到1L 溶解和高压灭菌
    13. 22%Percoll解决方案
      11毫升Percoll
      5毫升10x PBS

      用超纯蒸馏水将体积加到34毫升 过滤0.2微米过滤器,并在4°C存储
    14. 10%牛血清白蛋白(BSA)
      5克BSA
      用1x HBSS将体积加到50ml
      存放在冰箱里,直到完全溶解
      过滤0.2微米过滤器,并在4°C存储
    15. 1%BSA / 0.1%葡萄糖/ 1x HBSS
      50毫升10%BSA
      1.67毫升30%D-葡萄糖
      448.3毫升1x HBSS
      使用0.2μm过滤器进行过滤,并在4°C下保存
    16. 1x PBS
      100毫升10倍PBS
      900毫升超纯蒸馏水
    17. 神经球媒体
      500μlN-2补充(100x)
      1,000微升B-27补充(50x)
      500μl200mM L-谷氨酰胺
      500μl100x抗生素 - 抗真菌剂
      500μl100x胰岛素 - 转铁蛋白 - 硒(ITS-X)
      750μl1 M HEPES
      1毫升30%D-葡萄糖
      使用DMEM / F12
      将体积加到50毫升
    18. 表皮生长因子(EGF)(20微克/毫升)
      1. 用足够体积的超纯蒸馏水重悬冻干产品,使终浓度为20μg/ ml
      2. 在Neurosphere培养基中稀释1:1,000以达到20 ng / ml的工作浓度
    19. 碱性成纤维细胞生长因子(bFGF)(20μg/ ml)
      1. 用足够体积的超纯蒸馏水重悬冻干产品,使终浓度为20μg/ ml。
      2. 在Neurosphere培养基中稀释1:1,000以达到20 ng / ml的工作浓度
    20. 10%正常驴血清(NDS)/0.5% Triton X-100
      100μlNDS
      5μlTriton X-100
      895μl1x PBS
    21. 1%正常驴血清(NDS)/0.25% Triton X-100
      10μlNDS
      2.5微升Triton X-100
      987.5μl1x PBS
    22. 4%多聚甲醛(PFA)

      用30ml 1x PBS稀释10ml 16%PFA

    致谢

    这些实验过程已经在之前发表过,并在本文中由Tome-Garcia等人于2017年进行修改和修改。我们感谢西奈山伊坎医学院的病理科和生物储存库核心成员( ISMMS),用于促进组织收集和采购,以及ISMMS流式细胞仪核心技术,以获得24小时/天的新鲜人体组织分类的专家建议和调节。这项研究得到了西奈山种子基金的支持。作者宣称没有利益冲突。

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tome-Garcia, J., Doetsch, F. and Tsankova, N. M. (2017). FACS-based Isolation of Neural and Glioma Stem Cell Populations from Fresh Human Tissues Utilizing EGF Ligand. Bio-protocol 7(24): e2659. DOI: 10.21769/BioProtoc.2659.
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