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GC/MS-based Analysis of Volatile Metabolic Profile Along in vitro Differentiation of Human Induced Pluripotent Stem Cells
基于GC/MS的人诱导多能干细胞体外分化过程中挥发性化合物代谢谱分析   

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Abstract

Human induced pluripotent stem cells (hiPSCs) are a promising tool in cell-based therapies for degenerative diseases. A safe application of hiPSCs in vivo, requires the detection of the presence of residual undifferentiated pluripotent cells that can potentially cause the insurgence of teratomas. Several studies point out that metabolic products may provide an alternative method to identify the different steps of cells differentiation. In particular, the analysis of volatile organic compounds (VOCs) is gaining a growing interest in this context, thanks to its inherent noninvasiveness. Here, a protocol for VOCs analysis from human induced pluripotent stem cells (hiPSCs) is illustrated. It is based on Solid-Phase Microextraction (SPME) technique coupled with gas chromatography-mass spectrometry (GC/MS). The method is applied to measure the volatile metabolite modifications in cells headspace during cell reprogramming from chorionic villus samples (CVS) to hiPSCs, and along hiPSCs in vitro differentiation into early neural progenitors (NPs), passing through embryoid bodies (EBs) formation.

Keywords: Gas chromatography-mass spectrometry(气相色谱 - 质谱法), GC/MS(GC/MS), Human induced pluripotent stem cell(人诱导多能干细胞), hiPSC(hiPSC), Solid phase microextraction(固相微萃取), SPME(SPME), Volatile organic compounds(挥发性有机化合物), VOCs(VOCs), Metabolic profile(代谢谱)

Background

Cellular metabolism is proposed as an alternative to studying stem cells during the various steps of differentiation. Indeed, it is reasonable to suppose that the transition of stem cells from pluripotency to the complete differentiation, might give rise to a dramatic change of metabolic products. First evidence of this assumption was observed between induced pluripotent stem cells, parental fibroblasts, and embryonic stem cells (Meissen et al., 2012).

Within the metabolic products, the volatile organic compounds (VOCs) are attracting interest for the supposed simplicity of their collection, the intrinsic non-invasiveness and the wide availability of the analysis methods (Boots et al., 2015). To this regard, several studies show that the headspace of cancer cells exhibit a VOCs profile which is altered as compared to that of normal cells (Sponring et al., 2010; Peled et al., 2013; Filipiak et al., 2016).

Recently, we investigated the VOCs profiles of hiPSCs along the successive steps of differentiation (Capuano et al., 2017). Results support the hypothesis that the volatile fraction of the metabolic profile changes along the differentiation process as a reflection of the dramatic variations occurring in the cells.

GC/MS analysis evidences a number of compounds whose relative abundance can signal the difference between the various phases of the differentiation. Most of these compounds are aldehydes, alcohols, and alkanes. It is worth to remark that these compounds are detected because of their affinity with the chosen SPME and the GC/MS column. As a consequence, at this stage, it cannot be excluded that additional and even more discriminating volatile compounds might be found using different experimental setups. However, the outlined protocol is thoroughly valid even when different materials for SPME and GC/MS columns are selected. In other words, the protocol to sample volatile compounds from cell cultures and to analyze them with GC/MS is valid in general. Albeit, changes in the materials of the SPME fiber and the column modify the sensitivity respect to volatile compounds, then the use of different materials may highlight the presence of some classes of compounds and hinder the detection of others.

Materials and Reagents

  1. 60 mm tissue-culture sterile dish (Corning, Falcon®, catalog number: 353004 )
  2. 25-cm2 tissue-culture sterile flask (Corning, catalog number: 430639 )
  3. 35 mm tissue-culture sterile dish (Corning, Falcon®, catalog number: 353001 )
  4. Sterile cell scraper (Corning, Falcon®, catalog number: 353086 )
  5. 15-ml conical sterile tubes (Corning, Falcon, catalog number: 352096 )
  6. 50-ml conical sterile tubes (Corning, Falcon, catalog number: 352070 )
  7. 4-well tissue-culture sterile plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 176740 )
  8. 6-well tissue-culture sterile plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675 )
  9. 100 mm tissue-culture sterile dish (Corning, Falcon®, catalog number: 353003 )
  10. 60 mm ultra-low attachment culture sterile dishes (Corning, catalog number: 3261 )
  11. 5-ml plastic disposable sterile pipette (SARSTEDT, catalog number: 86.1253.001 )
  12. 10-ml plastic disposable sterile pipette (SARSTEDT, catalog number: 86.1254.001 )
  13. 25-ml plastic disposable sterile pipette (SARSTEDT, catalog number: 86.1685.001 )
  14. 0.22 µm sterile filter system (Corning, catalog number: 431097 )
  15. Sterile glass Pasteur pipettes (VWR, catalog number: HECH40567001)
    Manufacturer: Glaswarenfabrik Karl Hecht, catalog number: 40567001 .
  16. Sterile CryoTube Vials (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 375418 )
  17. 50/30 μm Divinylbenzene/Carboxen/PDMS (DVB/CAR/PDMS) SPME fiber assembly, needle size 24 ga, for use with manual holder (Sigma-Aldrich, catalog number: 57328-U )
  18. SPME Fiber Holder for use with manual sampling (Sigma-Aldrich, catalog number: 57330-U )
  19. Customized lids in polymethylmethacrylate (PMMA) for 60 mm tissue-culture dish (50 mm i.d x 60 mm h) having a suitable support for Solid Phase Micro-Extraction fiber insertion (1 mm i.d. x 40 mm h) (Figure 1). This system is made of three parts: a) the lid; b) the SPME fiber support and c) Shimadzu GC septa (Shimadzu, catalog number: 201-35584 ). Details are shown in Figure 2


    Figure 1. Customized lid in polymethylmethacrylate (PMMA). This system is suitable for headspace creation above 60 mm tissue-culture dishes and it is equipped with a support for Solid Phase Micro-Extraction fiber insertion to allow headspace VOC sampling.


    Figure 2. Details of the PMMA customized lid. A. The lid; B. The SPME fiber support; C. the Shimadzu GC septa (Shimadzu).

  20. Gelatin from porcine skin, Type A (Sigma-Aldrich, catalog number: G1890 )
  21. Dulbecco’s phosphate buffered saline (D-PBS) (Mediatech, catalog number: 21-031-CV )
  22. Collagenase type XI (Sigma-Aldrich, catalog number: C7657 )
  23. 0.25% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  24. Trypsin-EDTA (CARLO ERBA Reagents, catalog number: FA30WL0940100 )
  25. Collagenase type IV (Sigma-Aldrich, catalog number: C5138 )
  26. Accutase (Merck, catalog number: SCR005 )
  27. Poly-L-ornithine solution (Sigma-Aldrich, catalog number: P4957 )
  28. 2-Ethyl-1-Hexanol (Sigma-Aldrich, catalog number: 08607 )
  29. Styrene (Sigma-Aldrich, catalog number: 45993 )
  30. Hexanal (Sigma-Aldrich, catalog number: 18109 )
  31. 3-Hexen-1-ol, propanoate, (Z)- (Sigma-Aldrich, catalog number: W393304 )
  32. Acetone (Sigma-Aldrich, catalog number: 48358 )
  33. o-Cymene (In NIST 127 and NIST 147 mass spectral libraries it appears with the alternative name: Benzene, 1-methyl-2-(1-methylethyl)-) (Sigma-Aldrich, catalog number: 255270 )
  34. 1,4-Cyclohexadiene, 1-methyl-4-(1-methylethyl)- (Sigma-Aldrich, catalog number: 86476 )
  35. Propanoic acid, 2-hydroxy-2-methyl-, ethyl ester (Sigma-Aldrich, catalog number: E31200 )
  36. Nonanal (Sigma-Aldrich, catalog number: 442719 )
  37. Vinyl butyrate (Sigma-Aldrich, catalog number: 19390 )
  38. Tridecane (Sigma-Aldrich, catalog number: 442713 )
  39. Decanal (Sigma-Aldrich, catalog number: 59581 )
  40. Propanoic acid, 2-methyl-, anhydride (Sigma-Aldrich, catalog number: 245771 )
  41. Butanoic acid, 2-methylpropyl ester (Sigma-Aldrich, catalog number: 94888 )
  42. Hexadecane (Sigma-Aldrich, catalog number: 442679 )
  43. Methanone, (4-aminophenyl)phenyl- (Sigma-Aldrich, catalog number: A41402 )
  44. Heptadecane (Sigma-Aldrich, catalog number: 51578 )
  45. 3,5-Dimethyl-4-octanone (Sigma-Aldrich, catalog number: S504696 )
  46. Butanoic acid, anhydride (Sigma-Aldrich, catalog number: 19270 )
  47. Mouse Laminin (Merck, catalog number: CC095 )
  48. CHANG MEDIUM C Lyophilized (Irvine Scientific, catalog number: T101-019 )
  49. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270 )
  50. Penicillin-streptomycin solution (CARLO ERBA Reagents, catalog number: FA30WL0022100 )
  51. L-Glutamine (CARLO ERBA Reagents, catalog number: FA30WX0550100 )
  52. Dulbecco’s modified Eagle’s medium–high glucose (DMEM) (Sigma-Aldrich, catalog number: D5671 )
  53. DMEM/F-12 1:1 (Sigma-Aldrich, catalog number: D6421 )
  54. Knockout SR Serum replacement (KOSR) (Thermo Fisher Scientific, GibcoTM, catalog number: 10828028 )
  55. MEM non essential amino acids (Thermo Fisher Scientific, GibcoTM, catalog number: 11140050 )
  56. 2-Mercaptoethanol (Thermo Fisher Scientific, GibcoTM, catalog number: 31350010 )
  57. Basic fibroblast growth factor (bFGF) (Thermo Fisher Scientific, GibcoTM, catalog number: PHG6015 )
  58. Retinoic acid (Sigma-Aldrich, catalog number: R2625 )
  59. Hedgehog pathway activator (Hh-Ag1.3) (Curis, Lexington, MA)
  60. Human BDNF (PeproTech, catalog number: 450-02 )
  61. Recombinant Human CNTF (PeproTech, catalog number: 450-13 )
  62. Human GDNF (PeproTech, catalog number: 450-10 )
  63. Chang medium (CVS medium) (see Recipes)
  64. MEF medium (see Recipes)
  65. hiPS cell medium (see Recipes)
  66. EB medium 1 (see Recipes)
  67. EB medium 2 (see Recipes)

Equipment

  1. CO2 incubator (37 °C) (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i )
  2. Inverted microscope (Nikon Instruments, model: Eclipse TE2000-S )
  3. Pipettes (20 µl, 200 µl and 1000 µl) (Gilson, catalog number: F167300 )
  4. Centrifuge (provided with TX-200 swinging bucket rotor (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 75003658 ) containing round buckets (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 75003659 ) suitable to process up to 20 x 15 ml conical tubes. Flexible capacity with adapters ranging from 15 to 50 ml. Maximum speed 5,500 rpm) (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM MultifugeTM X1 , catalog number: 75004210)
  5. Herasafe KS, Class II biological safety cabinet with UV surface disinfection irradiator (Thermo Fisher Scientific, Thermo ScientificTM, model: HerasafeTM KS , Class II, catalog number: 51022481)
  6. Zoom stereomicroscope (Nikon Instruments, model: SMZ100 )
  7. Gas chromatography-mass spectrometry (GC/MS) system (Shimadzu, model: GCMS-QP2010 )
  8. SPME liner for SPL injector, special SPME liner, without filling, Phenylmethyl-deactivated deactivated (Shimadzu, catalog number: 961-01482-01 )
  9. Equity-5 capillary column (poly(5% diphenyl/95% dimethyl siloxane) phase; 30 m length x 0.25 mm i.d.; film thickness: 0.25 μm) (Sigma-Aldrich, catalog number: 28089-U )
  10. Ultra-high purity helium (99.999%)

Software

  1. GCMS solution (version 2.4, Shimadzu Corporation)
  2. NIST 127 and NIST 147 mass spectral libraries (National Institute of Standards and Technology-http://www.nist.gov/)
  3. MATLAB® (version R2016 b, MathWorks)

Procedure

  1. Preparation of cell culture sample for volatile metabolite analysis
    1. Chorionic villus samples (CVS) from three independent samples
      1. 60 mm culture dishes are coated with 2 ml of 0.1% gelatin and incubated at 37 °C for at least 20 min.
      2. CVS cells are disaggregated after several PBS washes by serial incubation with collagenase XI at 37 °C for 30 min, followed by 0.25% trypsin-EDTA at 37 °C for 10 min.
      3. Aggregates are dissociated using gentle pipetting, centrifuged at 266 x g for 10 min and seeded on three gelatin-coated 60 mm culture dishes with 2 ml Chang medium (see Recipes) as previously described in Spitalieri et al., 2009.
      4. CVS cells are detached from 25 cm2 culture flasks using trypsin and seeded at 2.5 x 106 with 2 ml Chang medium (see Recipes) on three gelatin-coated 60 mm culture dishes.
      5. Cell culture dishes are incubated at 37 °C in an incubator with 5% CO2 for 24 h.
    2. Human induced pluripotent stem cells (hiPSCs)
      1. CVS cells are reprogrammed in human pluripotent stem cells (hiPSCs) using a single lentiviral ‘stem cell cassette’, flanked by loxP sites (hSTEMCCA-loxP), encoding for reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) in a single polycistronic vector (Spitalieri et al., 2015). Details are reported in Figure 3.
      2. Once derived, hiPS cells are expanded, using mechanical passage under the stereomicroscope when reach confluence (Please see Note 1) and placed on irradiated mouse embryonic fibroblast (MEF) feeder layers on until line is well established in 35 mm culture plates for at least five passages.
      3. hiPSC medium (see Recipes) is changed every day. Please see Note 2.
      4. Three independent hiPS cell lines with different genotype are passaged on irradiated mouse embryonic fibroblast (MEF) feeder layers with 2 ml hiPSC medium on 60 mm culture dish (4 x 106 cells/dish). Please see Note 2.
      5. Cell culture dishes are incubated at 37 °C in an incubator with 5% CO2 for 24 h.


        Figure 3. Timeline of GC/MS analysis in CVS cells and hiPSCs. CVS cells are isolated from human samples, analyzed for volatile metabolite by SPME linked to GC/MS and then reprogrammed in hiPSCs. Embryonic stem cell-like colonies are observed on feeder layers after 14 days of infection and after expansion using mechanical passage are subjected to VOCs analysis.

    3. Floating EBs
      Days 1 to 6
      1. Neural induction is performed after the formation of cell aggregates called embryoid bodies (EBs) as reported, with some modifications, by Nakahama and Di Pasquale, 2016.
      2. hiPS cell lines are treated with collagenase IV/Accutase solution at 37 °C for 8 min and detached from the plate bottom using a cell scraper.
      3. The colonies are collected in a 15 ml conical tube, centrifuged at 17 x g for 5 min and plated in hiPSC medium without basic fibroblast growth factor (bFGF) on ultra-low attachment plates in 2:1 ratio (e.g., for two 35 mm dish of hiPSCs, use 1 dish of 60 mm ultra-low attachment dish). The cell pellet is gently resuspended, avoiding to break up clumps too much. Please see Note 3.
      4. hiPSC medium without bFGF is daily changed for the first 6 days, except for the day after passage. Please see Note 4.
      5. To change medium EBs are collected in a 15 ml conical tube, centrifuged at 17 x g for 5 min and gently resuspended in fresh medium.
      6. Three independent floating EBs samples (2 x 106 samples/dish) are placed in 2 ml hiPSC medium without basic fibroblast growth factor (bFGF) on 60 mm ultra-low attachment culture dishes.
      7. Cell culture dishes are incubated at 37 °C in an incubator with 5% CO2 for 24 h.

      Days 7 to 13
      1. On day 7, EBs are collected using a 10 ml pipette into a 15 ml conical tube under the stereomicroscope, centrifuged at 17 x g for 5 min and gently resuspended in EB medium 1 (see Recipe 4).
      2. Cell culture dishes are incubated at 37 °C in an incubator with 5% CO2 and EB medium 1 is daily changed for another week.

      Days 14 to 20
      1. On day 14, EB medium 1 is removed and replaced with EB medium 2 (see Recipe 5), following the same procedure reported above at step A3e.
      2. Cell culture dishes are incubated at 37 °C in an incubator with 5% CO2 and EB medium 2 is daily changed for another 7 days.

      Day 21
    4. Early Neural Progenitors (NPs)
      1. 60 mm Petri dishes are coated with 0.01% poly-L-ornithine overnight at 37 °C and then with 20 µg/ml laminin for 2 h at 37 °C. The plates are washed with PBS three times and left in a laminar flow hood to dry (Varga et al., 2014).
      2. Floating EBs are collected into a 15 ml conical tube, centrifuged at 17 x g for 5 min and gently resuspended and transferred onto 60 mm poly-L-ornithine/laminin-coated-plates (Varga et al., 2014) in 2 ml EB medium 2.
      3. Cell culture dishes are incubated at 37 °C in an incubator with 5% for 24 h. All samples preparation are performed as previously described in Capuano et al., 2017.
        Differentiation steps of hiPSCs into NPs and timeline of analysis are shown in Figure 4.


        Figure 4. Timeline of GC/MS sampling during NPs differentiation. Human induced pluripotent stem cells (hiPSCs) are differentiated into early neural progenitors (NPs) through the formation of floating embryoid bodies (EBs). VOCs analysis is carried out at different days of differentiation (floating EBs days 1-4, floating EBs days 9-11, floating EBs days 15-18 and early NPs 24 h after plating).

  2. Analysis of cell culture volatile metabolite by SPME linked to GC/MS
    1. This protocol is applied to the analysis of the following samples:
      1. CVS and hiPSCs after an incubation time of 24 h (after steps A1d and A2d respectively);
      2. Floating EBs at different differentiation steps on days 1 and 4, 9 and 11, 15 and 18 (during phases A3e, A3g, A3i respectively);
      3. Early Neural Progenitors (NPs) 24 h after plating (after step A4b);
      4. Chang medium;
      5. hiPSC medium;
      6. hiPSC medium without bFGF;
      7. EB medium 1;
      8. EB medium 2.
      Note: Cell culture media are always previously incubated for 24 h before VOC sampling. These are the ‘sample zero’ for the analysis of each cell line.
    2. The customized lid is sterilized under UV light in a biological hood for 20 min before any sampling procedure, in order to prevent culture contamination.
    3. Immediately after sterilization, the lid is put on the Petri dish containing the cell culture of interest cultivated in the incubator.
    4. SPME fiber holder is assembled (Figure 5).
      To ensure the reproducibility of the volatile compounds sampling, it is recommended to set the depth of the SPME needle at the same value. In this experiment, the sampling system required to set it to the 4-depth gauge setting on the plunger.
      Note: In order to prevent SPME fiber contamination, it is better to assemble this component immediately before its use. Twelve hours before any sampling procedure, SPME fibers have been conditioned following SUPELCO guidelines: DVB/CAR/PDMS fiber was baked at 270 °C for 1 h and then stored in its own box until use.


      Figure 5. SPME fiber equipment. Components (a), (b) and (c) constitute the SPME Fiber Holder for use with manual sampling; the element (d) is the needle system that contains and protects DVB/CAR/PDMS fiber.

    5. The needle protecting SPME fiber is inserted in the appropriate lid support and then the fiber is exposed to the cell culture headspace.
    6. Volatile metabolites are preconcentrated using Solid-Phase Microextraction (SPME) technique (Capuano et al., 2017). A 50/30 μm Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PSMS) fiber (SUPELCO) is inserted for 1 h to cell culture headspace. Samples are maintained in the incubator at a temperature of 37 °C and 5% CO2 during fiber exposure, in order to avoid any stress effect on cell growth (Capuano et al., 2017).
    7. After VOC adsorption, fiber is pulled inside the needle and removed from the holder to be placed in its box; fiber is stored at 4 °C until GC/MS analysis, performed within three hours.
    8. GC/MS analysis is carried out using a GCMS-QP2010 (Shimadzu) equipped with an Equity-5 (or equivalent) capillary column and a proper liner for SPME (for details see Equipment section). The volatile compounds adsorbed onto DVB/CAR/PDMS fiber are desorbed in splitless mode at 250 °C for 3 min in the GC injection port. High-purity helium is used as a carrier gas at 0.7 ml/min of column flow, total flow of 5.9 ml/min and pressure of 24.9 kPa. Analysis is performed working in linear velocity constant mode at 30.2 cm/sec. Inlet, ion source and transfer line temperatures are maintained at 250 °C. Initial oven temperature of 40 °C is maintained for 5 min, followed by temperature gradient to 220 °C at 7 °C/min, then increased by 15 °C/min to a final temperature of 300 °C, held for 3 min (total runtime: 39 min). The mass spectrometer is a single quadrupole analyzer operating in electron ionization mode, with electrons energy of 70 eV. Mass spectra are recorded from 40 to 450 m/z in full scan mode.
      Steps from B2 to B8 are summarized in Figure 6.


      Figure 6. Workflow of VOC analysis on cell culture by SPME coupled to gas chromatography-mass spectrometry

    9. Chromatogram acquisition and data elaboration are made with Shimadzu workstation software GCMS solution (version 2.4). Chemical identification of detected peaks is performed using both NIST 127 and NIST 147 libraries. Examples of GC/MS spectra are shown in Figures 7 and 8.


      Figure 7. GC/MS spectrum of the volatile metabolites of floating EBs cells culture at day 9, derived from total ion chromatograms. Data elaborated with the GCMS solution software. Volatile compound profile may differ from those shown here, in terms of composition and concentration, because of altered cells culturing conditions and/or different considered cell lines.


      Figure 8. Comparison of GC/MS spectra derived from total ion chromatograms of the volatile metabolic profiles of Chorionic villus (CVS) (red line), floating embryoid bodies (EBs) at day 9 (black line), human induced pluripotent stem cells (hiPSCs) (blue line) and early neural progenitors (NPs) (pink line). Data elaborated with the GCMS solution software. Volatile compounds profiles may differ from those shown here, in terms of composition and concentration, as a result of altered cell culturing conditions and/or different considered cell lines.

    10. The identity of those compounds in Table 1 which are commercially available was validated comparing the retention times and the mass spectra registered in sample chromatograms with those obtained from the analysis of a solution composed by standard compounds and culture media. The VOC collection and the analysis have been performed following the same procedure used for the samples (see steps B6-B8). The abundance of a subset of these compounds was found statistically different among the cells lines (Capuano et al., 2017).

      Table 1. List of compounds found in the chromatogram of all samples and for which identification has been validated with an analytical standard


    11. Styrene and 2-ethyl-1-hexanol are identified and quantified by comparing the retention times and the mass spectra with those obtained with analytical standards. Three concentrations of Styrene and 1-hexenol-2-ethyl are prepared diluting 1, 10, 100 times the pure compounds in 2 ml of different culture media in 60 mm Petri dishes. VOCs in the headspace are collected by DVB/CAR/PDMS-SPME fiber exposed for 1 h to the sample. The concentration (Cx) of Styrene and 2-ethyl-1-hexanol in the headspace of standard solutions is estimated by Antoine’s law (considered in parts per billion by volume (ppbv)):



      where, P is the vapor pressure (bar) and T the working temperature (K). Parameters A, B and C are empirical constants depending on the nature of the substance using the parameters available, in NIST database (http://webbook.nist.gov/chemistry). The amount (Ax) of styrene and 2-ethyl-1-hexanol (in ppbv) in the cells culture headspace (CVS, hiPSCs, EBs at different stage, and early NPs) and their culture media is calculated from the peak area in the sample (PAx) and the peak area of the standard (PSx), as follows:



      Note: This procedure is valid for any compound identified by an analytical standard.

Data analysis

  1. Each kind of cell line and culture medium is replicated three times in independent samples.
  2. Measured chromatograms are integated and the absolute area of each peak is considered to estimate VOC abundance.
  3. Chromatographic peak alignment is performed manually, considering retention time and mass spectra of peaks in the several chromatograms.
  4. GC/MS VOC abundance data are arranged in matrices where each row corresponds to a sample and columns contain area under the curve of each integrated peaks. Data have been analyzed with multivariate techniques such as Principal Component Analysis (PCA) (Capuano et al., 2017).
  5. The statistical significance of VOC abundances among different cell lines are evaluated by a parametric Kruskal-Wallis rank sum test applied for binary comparison among different cell lines (Table 2). The same test is applied to culture media related data, in order to eliminate VOCs characterizing different media rather than cell metabolism.

    Table 2. Cell culture couples considered for binary comparison using Kruskal-Wallis rank sum test


  6. Significant compounds in culture media discrimination, resulting from Kruskal-Wallis rank sum test, are not considered for multivariate data analysis.
  7. The abundance of all compounds selected by the Kruskal-Wallis rank sum test (P < 0.05) forms the pattern of cell headspace. Not normalized data are analyzed using the principal component analysis (PCA). This multivariate method allows comparing the different cell line data in order to represent differences and similarities between them graphically. Results of PCA are thoroughly discussed in Capuano et al., 2017.
  8. All data analysis is performed using MATLAB.

Notes

  1. Mechanical passage of hiPS cells has to be performed using a stereomicroscope under a regular laminar flow hood. Cells should be passaged approximately every 5 days, at around 60-75% confluence, without the colony of cells touching each other, removing differentiating areas with pulled glass Pasteur pipettes.
  2. hiPS cell medium must be renewed every day for optimal growth, however, occasional double feeding (adding twice the required volume of medium during one feed) is possible without affecting culture quality. For instance, it is possible to perform a double feed on a Friday, with the next medium change on Sunday. hiPS cell medium must be pre-warmed at room temperature and protected from light, suggesting to thaw and add growth factor to the medium just prior to use.
  3. Avoid over-pipetting to break up the colonies too much, checking the size under the stereomicroscope.

Recipes

  1. Chang medium
    CHANG MEDIUM C Lyophilized containing the following supplements:
    10% fetal bovine serum (FBS)
    100 U/ml penicillin
    100 mg/ml streptomycin
    2 mM L-glutamine
  2. MEF medium
    D-MEM containing the following supplements:
    10% fetal bovine serum (FBS)
    100 U/ml penicillin
    100 mg/ml streptomycin
    2 mM L-glutamine
  3. hiPS cell medium
    DMEM/F-12 containing the following supplements:
    20% KnockOut Serum Replacement (KOSR)
    100 U/ml penicillin
    100 mg/ml streptomycin
    2 mM L-glutamine
    1% MEM non essential amino acids
    0.1 mM β-mercaptoethanol
    Finally, the medium is sterilized using a 0.22 μm bottle-top vacuum filter and supplemented with 10 ng/ml basic fibroblast growth factor (bFGF)
  4. EB medium 1
    hiPS cell medium without bFGF supplemented with:
    1 µM RA (retinoic acid)
    500 mM Hh-Ag1.3 (sonic-hedgehog pathway agonist)
  5. EB medium 2
    hiPS cell medium minus bFGF supplemented with:
    1 µM RA
    500 mM Hh-Ag1.3 (a Sonic Hedgehog pathway agonist)
    20 ng/ml Ciliary neurotrophic factor (CNTF)
    10 ng/ml brain-derived neurotrophic factor (BDNF)
    10 ng/ml glial cell line-derived neurotrophic factor (GDNF)

Acknowledgments

This is a detailed protocol of the analyses reported in Capuano et al. (2017), adapted from previous works (Spitalieri et al., 2015; Murdocca et al., 2016a and 2016b). The authors declare that they have no competing interests.

References

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  7. Nakahama, H. and Di Pasquale, E. (2016). Generation of cardiomyocytes from pluripotent stem cells. Methods Mol Biol 1353: 181-190.
  8. Peled, N., Barash, O., Tisch, U., Ionescu, R., Broza, Y. Y., Ilouze, M., Mattei, J., Bunn, P. A. Jr, Hirsch, F. R., Haick, H (2013). Volatile fingerprints of cancer specific genetic mutations. Nanomedicine 9(6): 758-66.
  9. Spitalieri, P., Cortese, G., Pietropolli, A., Filareto, A., Dolci, S., Klinger, F. G., Giardina, E., Di Cesare, S., Bernardini, L., Lauro, D., Scaldaferri, M. L., Citro, G., Novelli, G., De Felici, M. and Sangiuolo, F. (2009). Identification of multipotent cytotrophoblast cells from human first trimester chorionic villi. Cloning Stem Cells 11(4): 535-556.
  10. Spitalieri, P., Talarico, R. V., Botta, A., Murdocca, M., D'Apice, M. R., Orlandi, A., Giardina, E., Santoro, M., Brancati, F., Novelli, G. and Sangiuolo, F. (2015). Generation of human induced pluripotent stem cells from extraembryonic tissues of fetuses affected by monogenic diseases. Cell Reprogram 17(4): 275-287.
  11. Sponring, A., Filipiak, W., Ager, C., Schubert, J., Miekisch, W., Amann, A. and Troppmair, J. (2010). Analysis of volatile organic compounds (VOCs) in the headspace of NCI-H1666 lung cancer cells. Cancer Biomark 7(3): 153-161.
  12. Varga, E., Nemes, C., Davis, R. P., Ujhelly, O., Klincumhom, N., Polgar, Z., Muenthaisong, S., Pirity M. K. and Dinnyes, A. (2014). Generation of transgene-free mouse induced pluripotent stem cells using an excisable lentiviral system. Exp Cell Res 322(2): 335-44.

简介

人诱导的多能干细胞(hiPSC)是用于退化性疾病的基于细胞的疗法中的有前景的工具。 hiPSCs在体内的安全应用需要检测残留未分化多能细胞的存在,这可能会导致畸胎瘤的爆发。几项研究指出,代谢产物可能提供了另一种方法来确定细胞分化的不同步骤。特别是挥发性有机化合物(VOCs)的分析由于其固有的非侵入性而在这方面越来越受到关注。在这里,说明了从人诱导的多能干细胞(hiPSC)分析VOC的方案。它基于固相微萃取(SPME)技术与气相色谱 - 质谱联用(GC / MS)。该方法用于测量从绒毛膜样品(CVS)到hiPSC的细胞重编程期间和沿着hiPSC体外分化成早期神经祖细胞(NP)的细胞顶空中的挥发性代谢物修饰,穿过胚状体机构(EBs)的形成。

【背景】提出细胞代谢作为在分化的各个步骤期间研究干细胞的替代物。事实上,假设干细胞从多能性向完全分化的转变可能引起代谢产物的剧烈变化是合理的。在诱导的多能干细胞,亲本成纤维细胞和胚胎干细胞之间观察到了这种假设的第一个证据(Meissen等人,2012)。

在代谢产物中,挥发性有机化合物(VOC)吸引了人们对其收集的简单性,内在的非侵入性和广泛的分析方法的广泛关注(Boots et。,2015 )。就此而言,一些研究显示癌细胞的顶部空间显示与正常细胞相比改变的VOCs分布(Sponring等人,2010; Peled等人, / em>,2013; Filipiak 等,2016)。

最近,我们调查了hiPSCs沿着分化的连续步骤(Capuano et。,2017)的VOCs概况。结果支持这样的假设,即代谢谱的挥发性分数随着分化过程而改变,作为细胞中发生剧烈变化的反映。

GC / MS分析证明许多化合物的相对丰度可以表示分化的各个阶段之间的差异。大多数这些化合物是醛,醇和烷烃。值得一提的是,这些化合物是由于它们与选定的SPME和GC / MS色谱柱的亲和性而被检测到的。因此,在这个阶段,不能排除使用不同的实验装置可能会发现另外的甚至更多的挥发性化合物。但是,即使选择了不同的SPME和GC / MS柱材料,概述的方案也是完全有效的。换句话说,从细胞培养物中采样挥发性化合物并用GC / MS分析的方案通常是有效的。尽管SPME纤维和柱子的材料的变化改变了对挥发性化合物的敏感性,但是使用不同的材料可能突出显示一些类别化合物的存在并阻碍其他化合物的检测。

关键字:气相色谱 - 质谱法, GC/MS, 人诱导多能干细胞, hiPSC, 固相微萃取, SPME, 挥发性有机化合物, VOCs, 代谢谱

材料和试剂

  1. 60mm组织培养灭菌皿(Corning,Falcon ,产品目录号:353004)
  2. 25cm2组织培养无菌瓶(Corning,目录号:430639)
  3. 35毫米组织培养灭菌皿(Corning,Falcon ,目录号:353001)
  4. 无菌细胞刮刀(Corning,Falcon ,目录号:353086)

  5. 15毫升锥形无菌管(康宁,猎鹰,目录号:352096)
  6. 50毫升锥形无菌管(康宁,猎鹰,目录号:352070)
  7. 4孔组织培养无菌板(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:176740)
  8. 6孔组织培养无菌板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:140675)
  9. 100mm组织培养无菌皿(Corning,Falcon ,目录号:353003)
  10. 60毫米超低附着培养无菌培养皿(康宁,目录号:3261)
  11. 5毫升塑料一次性无菌吸管(SARSTEDT,目录号:86.1253.001)
  12. 10毫升塑料一次性无菌移液器(SARSTEDT,目录号:86.1254.001)

  13. 25毫升塑料一次性无菌移液器(SARSTEDT,目录号:86.1685.001)
  14. 0.22μm无菌过滤系统(Corning,目录号:431097)
  15. 无菌玻璃巴斯德移液管(VWR,目录号:HECH40567001)
    制造商:Glaswarenfabrik Karl Hecht,目录号:40567001。
  16. 无菌CryoTube样品瓶(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:375418)
  17. 50/30μm二乙烯基苯/ Carboxen / PDMS(DVB / CAR / PDMS)与手动固定器(Sigma-Aldrich,目录号:57328-U)一起使用的SPME纤维组件,
  18. 用于手工取样(西格玛奥德里奇,目录号:57330-U)的SPME光纤支架
  19. 定制的聚甲基丙烯酸甲酯(PMMA)盖子有60mm的组织培养皿(50mm i.d x 60mm h),具有适合于固相微萃取纤维插入(1mm i.d.×40mm h)的载体(图1)。该系统由三部分组成:a)盖子; b)SPME纤维支持物和c)岛津GC隔片(Shimadzu,目录号:201-35584)。详情见图2


    图1.聚甲基丙烯酸甲酯(PMMA)中的定制盖子。 该系统适用于60 mm以上组织培养皿的顶空生成,并配备了固相微萃取纤维插入支持,以实现顶空VOC采样。


    图2. PMMA定制盖子的细节。 :一种。盖子; B. SPME纤维支持; C.岛津GC隔片(岛津)。

  20. A型(Sigma-Aldrich,目录号:G1890)猪皮明胶
  21. 达尔伯克磷酸盐缓冲盐水(D-PBS)(Mediatech,目录号:21-031-CV)
  22. XI型胶原酶(Sigma-Aldrich,目录号:C7657)
  23. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  24. 胰蛋白酶-EDTA(CARLO ERBA试剂,目录号:FA30WL0940100)
  25. IV型胶原酶(Sigma-Aldrich,目录号:C5138)
  26. Accutase(Merck,产品目录号:SCR005)
  27. 聚-L-鸟氨酸溶液(Sigma-Aldrich,目录号:P4957)
  28. 2-乙基-1-己醇(Sigma-Aldrich,目录号:08607)
  29. 苯乙烯(Sigma-Aldrich,目录号:45993)
  30. 己醛(Sigma-Aldrich,目录号:18109)
  31. 3-己烯-1-醇,丙酸酯,(Z) - (Sigma-Aldrich,目录号:W393304)
  32. 丙酮(Sigma-Aldrich,目录号:48358)
  33. o-Cymene(在NIST 127和NIST 147质谱文库中,以替代品名称:苯,1-甲基-2-(1-甲基乙基) - )(Sigma-Aldrich,目录号:255270)
  34. 1,4-环己二烯,1-甲基-4-(1-甲基乙基) - (Sigma-Aldrich,目录号:86476)
  35. 丙酸,2-羟基-2-甲基 - 乙酯(Sigma-Aldrich,目录号:E31200)
  36. 壬醛(Sigma-Aldrich,目录号:442719)
  37. 丁酸乙烯酯(Sigma-Aldrich,目录号:19390)
  38. 十三烷(Sigma-Aldrich,目录号:442713)
  39. Decanal(Sigma-Aldrich,目录号:59581)
  40. 丙酸,2-甲基 - ,酸酐(Sigma-Aldrich,目录号:245771)
  41. 丁酸2-甲基丙酯(Sigma-Aldrich,目录号:94888)
  42. 十六烷(Sigma-Aldrich,目录号:442679)
  43. 甲酮,(4-氨基苯基)苯基 - (Sigma-Aldrich,目录号:A41402)
  44. 十七烷(Sigma-Aldrich,目录号:51578)
  45. 3,5-二甲基-4-辛酮(Sigma-Aldrich,目录号:S504696)
  46. 丁酸酐(Sigma-Aldrich,目录号:19270)
  47. 小鼠层粘连蛋白(Merck,目录号:CC095)
  48. CHANG MEDIUM C冻干(Irvine Scientific,目录号:T101-019)
  49. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco,产品目录号:10270)
  50. 青霉素 - 链霉素溶液(CARLO ERBA试剂,目录号:FA30WL0022100)
  51. L-谷氨酰胺(CARLO ERBA试剂,目录号:FA30WX0550100)
  52. Dulbecco改良的Eagle's中高葡萄糖(DMEM)(Sigma-Aldrich,目录号:D5671)
  53. DMEM / F-12 1:1(Sigma-Aldrich,目录号:D6421)
  54. Knockout SR血清替代品(KOSR)(Thermo Fisher Scientific,Gibco TM,产品目录号:10828028)
  55. MEM非必需氨基酸(Thermo Fisher Scientific,Gibco TM,目录号:11140050)。
  56. 2-巯基乙醇(Thermo Fisher Scientific,Gibco TM,目录号:31350010)
  57. 碱性成纤维细胞生长因子(bFGF)(Thermo Fisher Scientific,Gibco TM,目录号:PHG6015)
  58. 视黄酸(Sigma-Aldrich,目录号:R2625)
  59. 刺猬通路激活剂(Hh-Ag1.3)(Curis,Lexington,MA)
  60. 人类BDNF(PeproTech,目录号:450-02)
  61. 重组人CNTF(PeproTech,目录号:450-13)
  62. 人类GDNF(PeproTech,目录号:450-10)
  63. 张中(CVS中)(见食谱)
  64. MEF介质(见食谱)
  65. hiPS细胞培养基(见食谱)
  66. EB介质1(见食谱)
  67. EB介质2(见食谱)

设备

  1. CO 2孵育器(37℃)(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heracell TM 150i)
  2. 倒置显微镜(尼康仪器,型号:Eclipse TE2000-S)
  3. 移液器(20μl,200μl和1000μl)(Gilson,目录号:F167300)
  4. 离心机(配备有带圆桶(Thermo Fisher Scientific,Thermo Scientific TM)的TX-200摆动桶式转子(Thermo Fisher Scientific,Thermo Scientific TM,目录号:75003658)目录号:75003659)适用于加工多达20 x 15 ml的锥形管,适配器的灵活容量范围为15至50 ml,最高转速为5,500 rpm(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Multifuge TM X1,目录号:75004210)
  5. 带有UV表面消毒辐照器(Thermo Fisher Scientific,Thermo Scientific TM,型号:Herasafe TM KS,Class II,目录号:51022481)的Herasafe KS,II类生物安全柜
  6. 变焦立体显微镜(尼康仪器,型号:SMZ100)
  7. 气相色谱 - 质谱(GC / MS)系统(岛津,型号:GCMS-QP2010)
  8. 用于SPL注射器的SPME衬垫,特殊的SPME衬垫,无填充,苯基甲基钝化失活(Shimadzu,目录号:961-01482-01)
  9. 等量-5毛细管柱(聚(5%二苯基/ 95%二甲基硅氧烷)相; 30m长x 0.25mm i.d .;膜厚度:0.25μm)(Sigma-Aldrich,目录号:28089-U)
  10. 超高纯度氦气(99.999%)

软件

  1. GCMS解决方案(版本2.4,岛津公司)
  2. NIST 127和NIST 147质谱库(美国国家标准与技术研究院 - http://www.nist.gov/
  3. MATLAB ®(版本R2016 b,MathWorks)

程序

  1. 用于挥发性代谢物分析的细胞培养样品的制备
    1. 来自三个独立样品的绒毛膜绒毛样品(CVS)

      1. 60毫米的培养皿用2毫升的0.1%明胶包被,并在37°C孵育至少20分钟。
      2. 经过几次PBS洗涤后,通过与胶原酶XI在37℃下连续温育30分钟,然后在37℃下0.25%胰蛋白酶-EDTA 10分钟,使CVS细胞解聚。
      3. 使用轻轻移液将集料解离,在266xg离心10分钟并如先前在Spitalieri等人所述用2ml Chang培养基(参见食谱)接种于三个涂有明胶的60mm培养皿上,2009年。
      4. 使用胰蛋白酶将CVS细胞从25cm 2培养瓶上分离,并用2mlChang培养基(参见食谱)以2.5×10 6接种于三个明胶包被的60mm培养物菜肴。
      5. 将细胞培养皿在37℃,含有5%CO 2的培养箱中温育24小时。
    2. 人诱导多能干细胞(hiPSC)
      1. CVS细胞在人多能干细胞(hiPSC)中使用单个慢病毒“干细胞盒”重编程,两侧是loxP位点(hSTEMCCA-loxP),其编码重编程因子(OCT4,SOX2,KLF4和c-MYC)单个多顺反子载体(Spitalieri等人,2015)。详情见图3.
      2. 一旦获得,hiPS细胞被扩增,当达到汇合时使用立体显微镜下的机械通道(请参见注释1),并放置在辐射的小鼠胚胎成纤维细胞(MEF)饲养层上,直到在35mm培养板中良好建立至少5通道。
      3. hiPSC培养基(见食谱)每天更换。请参阅注释2.
      4. 将具有不同基因型的三个独立的hiPS细胞系在60mm培养皿(4×10 6个细胞/培养皿)上用2ml hiPSC培养基辐射的小鼠胚胎成纤维细胞(MEF)饲养层进行传代。请参阅注释2.
      5. 将细胞培养皿在37℃,含有5%CO 2的培养箱中温育24小时。


        图3.CVS细胞和hiPSC中GC / MS分析的时间线。从人类样品中分离CVS细胞,通过与GC / MS连接的SPME分析挥发性代谢物,然后在hiPSC中重新编程。在感染14天后在饲养层上观察到胚胎干细胞样集落,并且在利用机械通道的膨胀之后进行VOC分析。

    3. 浮动EBs
      第1至6天
      1. 根据Nakahama和Di Pasquale在2016年报告的一些修改,形成被称为胚状体(EB)的细胞聚集体后,进行神经诱导。
      2. 将hiPS细胞系用胶原酶IV / Accutase溶液在37℃下处理8分钟,并使用细胞刮刀从平板底部分离。
      3. 将菌落收集在15ml锥形试管中,在17℃下离心5分钟,并以2:1的比率(平板)接种在不含碱性成纤维细胞生长因子(bFGF)的hiPSC培养基上例如,对于两个35mm的hiPSC培养皿,使用1个60mm超低附着培养皿的培养皿)。细胞沉淀物轻轻地重新悬浮,避免破坏团块太多。请参阅注3
      4. 在没有bFGF的hiPSC培养基中,除了通过后的第一天,每天更换6天。请参阅注释4.
      5. 将改变的培养基EB收集在15ml锥形管中,在17℃下离心5分钟,轻轻地重新悬浮在新鲜培养基中。
      6. 将3个独立的浮动EB样品(2×10 6个样品/培养皿)置于2ml不含碱性成纤维细胞生长因子(bFGF)的hiPSC培养基上,在60mm超低附着培养皿中。
      7. 将细胞培养皿在37℃,含有5%CO 2的培养箱中温育24小时。

      第7至13天
      1. 在第7天,使用10ml移液管将EB收集到立体显微镜下的15ml锥形管中,在17gxg下离心5分钟并轻轻地重悬在EB培养基1中(参见配方4) br />
      2. 将细胞培养皿在37℃下在具有5%CO 2的培养箱中孵育,并且EB培养基1每天更换一周。

      第14至20天
      1. 在第14天,按照上面在步骤A3e中报告的相同程序,将EB培养基1移出并用EB培养基2代替(参见方案5)。
      2. 将细胞培养皿在37℃下在具有5%CO 2的培养箱中孵育,并且EB培养基2每天更换另外的7天。

      第21天
    4. 早期神经祖细胞(NPs)
      1. 60mm培养皿在37℃下用0.01%聚-L-鸟氨酸包被过夜,然后用20μg/ ml层粘连蛋白在37℃包被2小时。用PBS洗板3次,留在层流罩中干燥(Varga et al。,2014)。
      2. 将漂浮的EB收集到15ml锥形管中,在17℃下离心5分钟,轻轻地重悬浮并转移到60mm聚-L-鸟氨酸/层粘连蛋白包被的平板(Varga等,2014)在2ml EB培养基2中。
      3. 细胞培养皿在37℃,5%培养箱中孵育24小时。所有的样品制备都按照Capuano等人在2017年描述的方法进行。
        图4显示了hiPSC分化为NPs的时间表和分析时间表。


        图4. NPs分化过程中GC / MS采样的时间线人类诱导的多能干细胞(hiPSCs)通过形成浮游胚状体(EB)而分化成早期神经祖细胞(NPs)。挥发性有机化合物的分析是在分化的不同日子进行的(浮动EBs第1-4天,浮动EBs第9-11天,浮动EBs第15-18天和早期NPs电镀后24小时)。

  2. GC / MS联用分析SPME对挥发性代谢物的影响
    1. 该协议适用于以下样本的分析:
      1. 在24小时的孵育时间后(分别在步骤A1d和A2d后)CVS和hiPSC;
      2. 在第1天,第4天,第9天,第11天,第15天和第18天(分别在A3e,A3g,A3i阶段)进行不同分化阶段的浮动EB;
      3. 早期神经祖细胞(NPs)在电镀24小时后(步骤A4b之后);
      4. 张中;
      5. hiPSC中等;
      6. 不含bFGF的hiPSC培养基;
      7. EB介质1;
      8. EB介质2。
      注意:在VOC采样之前,细胞培养基总是预先孵育24小时。这些是分析每个细胞系的“样品零点”。
    2. 为了防止培养物污染,定制的盖子在生物罩的紫外线下进行20分钟的灭菌处理。
    3. 消毒后立即将盖子放在含有在培养箱中培养的感兴趣的细胞培养物的培养皿中。
    4. SPME光纤支架已经组装(图5)。
      为确保挥发性化合物取样的重复性,建议将SPME针的深度设定为相同的值。在这个实验中,采样系统需要将其设置在柱塞上的4深度量规上。
      注意:为防止SPME纤维污染,最好在使用前立即组装。在任何取样程序之前的12个小时,SPME纤维已经按照SUPELCO指导原则进行了调整:DVB / CAR / PDMS纤维在270℃下烘烤1小时,然后储存在自己的盒子中直到使用。


      图5. SPME光纤设备。组分(a),(b)和(c)构成用于手工取样的SPME光纤夹持器;元素(d)是包含和保护DVB / CAR / PDMS光纤的针系统。

    5. 将针头保护的SPME纤维插入适当的盖子支架,然后将纤维暴露于细胞培养顶部空间。
    6. 挥发性代谢物使用固相微萃取(SPME)技术(Capuano等人,2017)预浓缩。将50/30微米的二乙烯基苯/ Carboxen /聚二甲基硅氧烷(DVB / CAR / PSMS)纤维(SUPELCO)插入细胞培养顶部空间1小时。为了避免对细胞生长的任何应力作用,将样品在培养器中在37℃和5%CO 2的温度下维持在培养器中(Capuano 等,2017)。
    7. 挥发性有机化合物吸附后,将纤维拉入针内并从支架上取下放入其中,纤维储存在4°C直到GC / MS分析,在三个小时内进行。
    8. 使用配备Equity-5(或等效)毛细管色谱柱和SPME(适用于SPME)的适当衬管的GCMS-QP2010(Shimadzu)进行GC / MS分析(详情请参阅设备章节)。吸附在DVB / CAR / PDMS纤维上的挥发性化合物在GC注射口中在250℃下以不分流模式解吸3分钟。使用高纯度氦气作为载气,流速为0.7毫升/分钟,总流量为5.9毫升/分钟,压力为24.9千帕。以30.2厘米/秒的线速度恒定模式进行分析。进口,离子源和传输线的温度保持在250°C。保持40℃的初始烘箱温度5分钟,然后以7℃/分钟升温至220℃,然后以15℃/分钟升温至300℃的最终温度,保持3分钟总运行时间:39分钟)。质谱仪是以电子电离模式操作的单四极杆分析仪,具有70eV的电子能量。质谱在全扫描模式下从40到450m / z记录。
      图6总结了从B2到B8的步骤。


      图6. SPME与气相色谱 - 质谱联用分析细胞培养物的VOC分析工作流程

    9. 使用Shimadzu工作站软件GCMS解决方案(版本2.4)进行色谱图采集和数据处理。检测到的峰的化学鉴定使用NIST 127和NIST 147文库进行。图7和图8显示了GC / MS谱图的实例。


      图7.浮游EB细胞在第9天培养的挥发性代谢物的GC / MS谱图来源于总离子色谱图GCMS解决方案软件详细阐述的数据。由于细胞培养条件改变和/或细胞系不同,挥发性化合物的分布可能与组成和浓度有所不同。

      “”src
      图8.绒毛膜绒毛(CVS)(红线),浮动胚状体(EB)在第9天(黑线)的挥发性代谢谱,人诱导的多能性的总离子色谱图干细胞(hiPSCs)(蓝线)和早期神经祖细胞(NPs)(粉红线)。使用GCMS解决方案软件详细阐述数据。由于改变细胞培养条件和/或不同的细胞系,挥发性化合物的分布可能与组成和浓度不同。

    10. 通过比较样品色谱图中记录的保留时间和质谱与由标准化合物和培养基组成的溶液分析得到的保留时间和质谱,证实了表1中可商购的那些化合物的特性。 VOC收集和分析按照与样品相同的步骤进行(参见步骤B6-B8)。这些化合物的子集的丰度在细胞系中被发现有统计学差异(Capuano et al。,2017)。

      表1.在所有样品的色谱图中发现的化合物列表,并且已经通过分析标准对其进行了鉴定


    11. 苯乙烯和2-乙基-1-己醇通过比较保留时间和质谱与用分析标准得到的质谱鉴定和定量。制备三种浓度的苯乙烯和1-己烯醇-2-乙基,在60毫米培养皿中稀释1,10,100倍于2毫升不同培养基中的纯化合物。顶空挥发性有机化合物通过DVB / CAR / PDMS-SPME纤维在样品中暴露1小时进行收集。苯乙烯和2-乙基-1-己醇在标准溶液顶部空间中的浓度(Cx)按照安托万定律(以十亿分之几体积(ppbv)计算):



      其中,P是蒸气压(bar),T是工作温度(K)。参数A,B和C是NIST数据库中使用可用参数的物质性质的经验常数( http: //webbook.nist.gov/chemistry )。在细胞培养顶部空间(CVS,hiPSC,不同阶段和早期NP中的EB)中的苯乙烯和2-乙基-1-己醇(以ppbv计)的量(A x)是从样品中的峰面积(PA )和标准(PS )的峰面积计算的,如下所示:



      注意:该程序适用于任何由分析标准鉴定的化合物。

数据分析


  1. 各种细胞系和培养基在独立样本中复制三次
  2. 测量的色谱图被整合,并且每个峰的绝对面积被认为是估计VOC丰度。
  3. 色谱峰校准是手动进行的,考虑到保留时间和几个色谱图中峰的质谱。
  4. GC / MS VOC丰度数据排列在矩阵中,其中每行对应于样品,列中每个积分峰的曲线下包含区域。数据已经用多元分析技术进行分析,如主成分分析(PCA)(Capuano等人,2017)。
  5. 通过应用于不同细胞系之间的二元比较的参数Kruskal-Wallis秩和检验评估不同细胞系中VOC丰度的统计显着性(表2)。对培养基相关数据进行相同的测试,以消除表征不同培养基的VOC而不是细胞代谢。

    表2.使用Kruskal-Wallis秩和检验考虑进行二元比较的细胞培养夫妇



  6. 在多元数据分析中不考虑由Kruskal-Wallis秩和检验得出的文化媒体歧视中的重要化合物。
  7. 通过Kruskal-Wallis秩和检验选择的所有化合物的丰度( p <0.05)形成了细胞顶空的模式。未使用主成分分析(PCA)分析归一化数据。这种多变量方法允许比较不同的细胞系数据,以便以图形方式表示它们之间的差异和相似性。
    在Capuano 等,2017年对PCA的结果进行了充分的讨论。
  8. 所有数据分析都使用MATLAB进行。

笔记

  1. hiPS细胞的机械通道必须在常规层流罩下使用立体显微镜进行。细胞应该大约每5天传代一次,大约在60-75%的汇合度,细胞不接触,使用拉式玻璃巴斯德吸管移除分化区域。
  2. hiPS细胞培养基必须每天更新以获得最佳生长,然而,偶尔的双重喂养(在一次饲喂期间添加所需量的培养基两倍)是可能的,而不影响培养质量。例如,可以在星期五执行双重输入,下个媒体在星期天更改。 hiPS细胞培养基必须在室温下预先加热,避光,建议在使用前解冻并在培养基中添加生长因子。

  3. 。避免过度移液,过分分解菌落,检查立体显微镜下的大小。

食谱

  1. 张中等
    CHANG MEDIUM C冻干含有以下补充剂:
    10%胎牛血清(FBS)
    100U / ml青霉素
    100毫克/毫升链霉素
    2mM L-谷氨酰胺
  2. MEF中等
    含有以下补充剂的D-MEM:
    10%胎牛血清(FBS)
    100U / ml青霉素
    100毫克/毫升链霉素
    2mM L-谷氨酰胺
  3. hiPS细胞培养基
    包含以下补充的DMEM / F-12:
    20%KnockOut血清替代品(KOSR)
    100U / ml青霉素
    100毫克/毫升链霉素
    2mM L-谷氨酰胺
    1%MEM非必需氨基酸
    0.1 mMβ-巯基乙醇
    最后,使用0.22μm瓶顶真空过滤器并补充10ng / ml碱性成纤维细胞生长因子(bFGF)来对培养基进行灭菌。
  4. EB介质1
    不含bFGF的hiPS细胞培养基补充有:
    1μMRA(视黄酸)
    500mM Hh-Ag1.3(声波刺猬蛋白通路激动剂)
  5. EB介质2
    hiPS细胞培养基减去补充有:
    的bFGF 1μMRA
    500 mM Hh-Ag1.3(Sonic Hedgehog途径激动剂)
    20 ng / ml睫状神经营养因子(CNTF)
    10 ng / ml脑源性神经营养因子(BDNF)
    10 ng / ml胶质细胞源性神经营养因子(GDNF)

致谢

这是Capuano等人(2017)报道的分析的详细方案,根据以前的工作(Spitalieri等人,2015; Murdocca等人。2016a; Murdocca等人,2016b)。作者声明他们没有竞争利益。

参考

  1. Boots,A.W.,Bos,L.D.,van der Schee,M.P.,van Schooten,F.J。和Sterk,P.J。(2015)。 诊断和监测中的呼气分子指纹识别:验证易变的许诺 Trends Mol Med 21(10):633-644。
  2. Capuano,R.,Spitalieri,P.,Talarico,RV,Domakoski,AC,Catini,A.,Paolesse,R.,Martinelli,E.,Novelli,G.,Sangiuolo,F.and Di Natale, )。 人体多能干细胞沿体外的挥发性代谢物的初步分析 em> Sci Rep 7(1):1621.
  3. Filipiak,W.,Mochalski,P.,Filipiak,A.,Ager,C.,Cumeras,R.,Davis,C.E.,Agapiou,A.,Unterkofler,K.和Troppmair,J。(2016)。 人类细胞系释放的挥发性有机化合物(VOCs)纲要 Curr Med Chem 23(20):2112-31。
  4. Meissen,J.K。,Yuen,B.T.,Kind,T.,Riggs,J.W.,Barupal,D.K。,Knoepfler,P.S。和Fiehn,O。(2012)。 诱导多能干细胞在多不饱和磷脂酰胆碱和初级代谢中显示与胚胎干细胞的代谢组学差异
  5. Murdocca,M.,Ciafre,S.A.,Spitalieri,P.,Talarico,R.V.,Sanchez,M.,Novelli,G。和Sangiuolo,F。(2016a)。人类iPSC衍生的运动神经元表现出微扰的分化和降低的miR-335-5p表达。
    。 Int J Mol Sci 17(8):1231.
  6. Murdocca,M.,Mango,R.,Pucci,S.,Biocca,S.,Testa,B.,Capuano,R.,Paolesse,R.,Sanchez,M.,Orlandi,A.,di Natale,C. ,Novelli,G。和Sangiuolo,F。(2016b)。 凝集素样氧化型低密度脂蛋白受体-1:一种新的结肠直肠癌潜在分子靶点。 a> Oncotarget 7(12):14765-14780。
  7. Nakahama,H.和Di Pasquale,E。(2016)。 从多能干细胞生成心肌细胞方法Mol Biol 1353:181-190。
  8. Peled,N.,Barash,O.,Tisch,U。,Ionescu,R.,Broza,Y.Y.,Ilouze,M.,Mattei,J.,Bunn,P.A.Jr,Hirsch,F.R.,Haick,H(2013)。 癌症特异性基因突变的挥发性指纹 纳米医学 9 (6):758-66。
  9. Spitalieri,P.,Cortese,G.,Pietropolli,A.,Filareto,A.,Dolci,S.,Klinger,FG,Giardina,E.,Di Cesare,S.,Bernardini,L.,Lauro,D., Scaldaferri,ML,Citro,G.,Novelli,G.,De Felici,M.和Sangiuolo,F。(2009)。 人绒毛膜绒毛绒毛细胞多能细胞滋养细胞的鉴定 克隆茎细胞 11(4):535-556。
  10. Spitalieri,P.,Talarico,RV,Botta,A.,Murdocca,M.,D'Apice,MR,Orlandi,A.,Giardina,E.,Santoro,M.,Brancati,F.,Novelli, Sangiuolo,F。(2015)。 受单基因疾病影响胎儿的胚外组织产生人类诱导性多能干细胞 细胞重编程 17(4):275-287。
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引用:Capuano, R., Talarico, R. V., Spitalieri, P., Roberto, P., Giuseppe, N., Sangiuolo, F. and Di Natale, C. (2017). GC/MS-based Analysis of Volatile Metabolic Profile Along in vitro Differentiation of Human Induced Pluripotent Stem Cells. Bio-protocol 7(23): e2642. DOI: 10.21769/BioProtoc.2642.
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