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Differentiation of Human Induced Pluripotent Stem Cells (iPS Cells) and Embryonic Stem Cells (ES Cells) into Dendritic Cell (DC) Subsets
诱导的人多能干细胞(iPS细胞)和胚胎干细胞(ES细胞)向树突状细胞(DC)亚型的分化   

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Stem Cells
Apr 2017

Abstract

Induced pluripotent stem cells (iPS cells) are engineered stem cells, which exhibit properties very similar to embryonic stem cells (ES cells; Takahashi and Yamanaka, 2016). Both iPS cells and ES cells have an extraordinary self-renewal capacity and can differentiate into all cell types of our body, including hematopoietic stem/progenitor cells and dendritic cells (DC) derived thereof. This makes iPS cells particularly well suited for studying molecular mechanisms of diseases, drug discovery and regenerative therapy (Grskovic et al., 2011; Bellin et al., 2012; Robinton and Daley, 2012).

DC are the major antigen presenting cells of the immune system and thus they are key players in modulating and directing immune responses (Merad et al., 2013). DC patrol peripheral and interface tissues (e.g., lung, intestine and skin) to detect invading pathogens, and upon activation they migrate to lymph nodes to activate and prime lymphocytes.

DC comprise a phenotypically heterogeneous family with functionally specialized subsets (Schlitzer and Ginhoux, 2014). Generally, classical DC (cDC) and plasmacytoid DC (pDC) are distinguished, exhibiting a classical and plasma cell-like DC morphology, respectively. cDC recognize a multitude of pathogens and secrete proinflammatory cytokines upon activation, while pDC are specialized to detect intracellular pathogens and secrete type I interferons (Merad et al., 2013; Schlitzer and Ginhoux, 2014). cDC are further divided into cross-presenting cDC1 and conventional cDC2, in the human system referred to as CD141+ Clec9a+ cDC1 and CD1c+ CD14- cDC2. Human pDC are characterized as CD303+ CD304+ (Jongbloed et al., 2010; Joffre et al., 2012; Swiecki and Colonna, 2015).

To investigate subset specification and function of human DC, we established a protocol to generate cDC1, cDC2 and pDC in vitro from human iPS cells (or ES cells) (Sontag et al., 2017). Therefore, we differentiated iPS cells (or ES cells), via mesoderm commitment and hemato-endothelial specification, into CD43+ CD31+ hematopoietic progenitors. Subsequently, those were seeded onto inactivated OP9 stromal cells with FLT3L, SCF, GM-CSF and IL-4 or FLT3L, SCF and GM-CSF to specify cDC1 and cDC2, or cDC1 and pDC, respectively.

Keywords: iPS cells (iPS细胞), ES cells (ES细胞), Hematopoiesis (造血), Hematopoietic differentiation (造血分化), Human dendritic cells (人树突状细胞), Dendritic cell differentiation (树突状细胞分化)

Background

DC and their development have mostly been studied in mice (Belz and Nutt, 2012; Merad et al., 2013; Schlitzer and Ginhoux, 2014). Owing to their rarity in non-lymphoid tissues and the restricted access to human lymphoid tissues, studying DC in humans is challenging (Jongbloed et al., 2010; Villadangos and Shortman, 2010). Yet, understanding developmental pathways, origins and mechanisms during DC subset specification is important in order to generate autologous DC subsets in vitro for therapeutic applications (e.g., anti-tumor agents). iPS cells (and ES cells) with their unlimited self-renewal and differentiation potential raise hopes that DC can be generated and modified (e.g., loaded with patient and disease specific antigens) in high numbers and quality in vitro to study DC development and function and to improve DC immunotherapies (Sontag et al., 2017).

Several groups have generated monocyte derived DC from iPS cells (and ES cells) with conventional granulocyte macrophage colony stimulating factor (GM-CSF)/interleukin 4 (IL-4) protocols, but such DC represent inflammatory DC and are not subset specific (Senju et al., 2007; Tseng et al., 2009; Choi et al., 2011; Senju et al., 2011; Belz and Nutt, 2012; Rossi et al., 2012; Yanagimachi et al., 2013; Li et al., 2014). From mouse studies, it is known that fms-like tyrosine kinase ligand (FLT3L) is the key cytokine for DC development and that the combination of FLT3L and GM-CSF signaling specifies DC subsets in vivo (Gilliet et al., 2002; Schmid et al., 2010). Recently, human cDC1, cDC2 and pDC were generated from cord blood (CB) with FLT3L, stem cell factor (SCF) and GM-CSF on MS5 stromal cells (Lee et al., 2015). Additionally, Poulin et al. (2010) reported on the generation of cDC1 from CB with FLT3L, SCF, GM-CSF and IL-4 in a feeder-free environment (Poulin et al., 2010). In contrast, Silk et al. (2012) described the differentiation of cDC1 in a feeder-free GM-CSF/IL-4 system (Silk et al., 2012). However, recent genome wide transcriptional profiling studies highlight the impact of microenvironmental cues during DC development, indicating that feeder support is important (Lundberg et al., 2013). Therefore, we used FLT3L, SCF, GM-CSF and IL-4 (referred to as FSG4) and FLT3L, SCF and GM-CSF (referred to as FSG) in combination with OP9 stromal cells to generate cDC1, cDC2 and pDC from iPS cell (or ES cell) derived hematopoietic progenitors.

Here we describe a two-step protocol: First, human iPS cells (or ES cells) are differentiated into hematopoietic progenitors (adapted from Kennedy et al., 2012). Second, these hematopoietic stem/progenitor cells are then further differentiated into cDC1, cDC2 and pDC (Sontag et al., 2017).

Materials and Reagents

  1. 6-well tissue culture plates (TPP Techno Plastic Products, catalog number: 92006 )
  2. 10 cm microbiology grade Petri dishes (SARSTEDT, catalog number: 82.1473.001 )
  3. 50 ml Falcon tubes (Corning, Falcon®, catalog number: 352070 )
  4. 70 µm cell strainer (Greiner Bio One International, catalog number: 542070 )
  5. 40 µm cell strainer (Greiner Bio One International, catalog number: 542040 )
  6. Top bottle filter (TPP Techno Plastic Products, catalog number: 99505 )
  7. 5 ml Serological pipettes (Corning, Falcon®, catalog number: 357543 )
  8. 10 ml Serological pipettes (Corning, Falcon®, catalog number: 357551 )
  9. 25 ml Serological pipettes (Corning, Falcon®, catalog number: 357525 )
  10. Pipette tips 10 µl (STARLAB INTERNATIONAL, catalog number: S1180-3810 )
  11. Pipette tips 20 µl (STARLAB INTERNATIONAL, catalog number: S1120-1810 )
  12. Pipette tips 200 µl (STARLAB INTERNATIONAL, catalog number: S1120-8810 )
  13. Pipette tips 1,000 µl (STARLAB INTERNATIONAL, catalog number: S1126-7810 )
  14. Collagenase IV (Thermo Fisher Scientific, GibcoTM, catalog number: 17104019 )
  15. Knockout-Dulbecco’s modified Eagle medium (KO-DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 10829018 )
  16. Gelatin (Sigma-Aldrich, catalog number: G1890 )
  17. 1-Thiogylcerol (Sigma-Aldrich, catalog number: M1753 )
  18. L-ascorbic acid (Sigma-Aldrich, catalog number: A4403 )
  19. Bovine serum albumin (BSA) low endotoxin (PAA, catalog number: K31-011 )
  20. 1x Dulbecco’s phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190094 )
  21. HumanKine bone morphogenic protein 4 (BMP4) research grade (Miltenyi Biotec, catalog number: 130-110-921 )
  22. Recombinant human basic fibroblast growth factor (bFGF) (PeproTech, catalog number: 100-18B )
  23. Recombinant human FLT3L (PeproTech, catalog number: 300-19 )
  24. Recombinant human GM-CSF (PeproTech, catalog number: 300-03 )
  25. Recombinant human insulin-like growth factor 1 (IGF1) (PeproTech, catalog number: 100-11 )
  26. Human interleukin 3 (IL-3) research grade (Miltenyi Biotec, catalog number: 130-094-193 )
  27. Human IL-4 research grade (Miltenyi Biotec, catalog number: 130-095-373 )
  28. Human SCF research grade (Miltenyi Biotec, catalog number: 130-096-693 )
  29. Human thrombopoietin (TPO) research grade (Miltenyi Biotec, catalog number: 130-094-013 )
  30. Recombinant human vascular endothelial growth factor (VEGF) (PeproTech, catalog number: 100-20 )
  31. 1x StemPro-34 SFM (Thermo Fisher Scientific, catalog number: 10639011 )
  32. Penicillin-streptomycin 10,000 U/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  33. L-glutamine 200 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  34. Hyper-interleukin 6 (IL-6) (kindly provided by S. Rose John, Institute of Biochemistry, Medical Faculty, Christian-Albrechts-University, Kiel, Germany, [Fischer et al., 1997], see also Notes)
  35. α-Minimal essential medium (α-MEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 12571063 )
  36. β-mercaptoethanol (50 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 31350010 )
  37. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10500064 )
  38. Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11875093 )
  39. Collagenase IV solution (1 mg/ml) (see Recipes)
  40. 0.1% gelatin solution (see Recipes)
  41. 1-Thioglycerol stock solution (100 mM) (see Recipes)
  42. L-ascorbic acid stock solution (50 mg/ml) (see Recipes)
  43. 0.1% BSA solution (see Recipes)
  44. BMP4 stock solution (25 µg/ml) (see Recipes)
  45. bFGF stock solution (100 µg/ml) (see Recipes)
  46. FLT3L stock solution (25 µg/ml) (see Recipes)
  47. GM-CSF stock solution (100 µg/ml) (see Recipes)
  48. IGF1 stock solution (40 µg/ml) (see Recipes)
  49. IL-3 stock solution (150 µg/ml) (see Recipes)
  50. IL-4 stock solution (20 µg/ml) (see Recipes)
  51. SCF stock solution (100 µg/ml) (see Recipes)
  52. TPO stock solution (20 µg/ml) (see Recipes)
  53. VEGF stock solution (100 µg/ml) (see Recipes)
  54. Hematopoietic progenitor basal differentiation medium (see Recipes)
  55. Induction medium d0 (see Recipes)
  56. Induction medium d1 (see Recipes)
  57. Induction medium d2 (see Recipes)
  58. Induction medium d4 (see Recipes)
  59. Induction medium d6 (see Recipes)
  60. OP9 culture medium (see Recipes)
  61. DC differentiation basal medium (see Recipes)
  62. DC differentiation FSG4 medium (see Recipes)
  63. DC differentiation FSG medium (see Recipes)

Equipment

  1. 1,000 µl pipette (Gilson, catalog number: F123602 )
  2. 200 µl pipette (Gilson, catalog number: F123601 )
  3. 20 µl pipette (Gilson, catalog number: F123600 )
  4. 10 µl pipette (Gilson, catalog number: F144802 )
  5. Pipetboy (INTEGRA Biosciences, catalog number: 155000 )
  6. Water bath (JULABO, model: SW22 )
  7. Inverted light microscope (Leica Microsystems, model: Leica DM IL LED )
  8. Autoclave
  9. Centrifuge (Thermo Fisher Scientific, model: HeraeusTM Multifuge 3 L )
  10. Flow hood (Heraeus)
  11. Automatic CO2 incubator with nitrogen supply and O2 sensor (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM 240i , catalog number: 51026331)
  12. Vacuum pump (INTEGRA Biosciences, catalog number: 158300 )
  13. Standard fridge
  14. Standard non-defrosting freezer

Procedure

  1. The differentiation of iPS cells (or ES cells) into hematopoietic progenitors is summarized in Figure 1.


    Figure 1. Schematic representation of the differentiation protocol of iPS cells (or ES cells) into hematopoietic progenitors. Differentiation time and corresponding differentiation steps and cytokine compositions (in colored boxes) are shown.

    1. To differentiate iPS cells (or ES cells) into hematopoietic progenitors, prewarm required amounts of collagenase IV solution (1 mg/ml, 37 °C, see Recipes) and KO-DMEM (room temperature, RT) before starting the experiment. Prepare induction medium d0 (see Recipes) and prewarm at RT.
    2. Harvest iPS cells (or ES cells) when they reached 70-80% confluence. Therefore, aspirate medium and add collagenase IV solution to the cells. The volume depends on the culture dish used. Make sure to completely cover the cells with collagenase IV solution (e.g., 1 ml per well of a 6-well plate, 4 ml per 10 cm dish).
    3. Incubate cells with collagenase IV at 37 °C in a normoxic incubator with 5% CO2 for 40-60 min. Check if colony edges start to roll up. Only if colony edges have rolled up from all sites and colonies are only loosely adherent to the plate proceed with step A4. If necessary, incubate 10-15 min longer but do not exceed 80 min collagenase IV treatment because this compromises cell viability.
    4. Dilute collagenase IV with KO-DMEM in a ratio 1/3, meaning add 2 ml KO-DMEM for each ml collagenase IV solution. Use a 10 ml pipette to rinse off colonies, resuspend and collect them in 50 ml Falcon tubes.
    5. If iPS cells (or ES cells) were cultured on mouse embryonic fibroblasts (MEF), separate iPS cell (or ES cell) clusters and single MEF by gravity for 10 min at RT. Large iPS cell (or ES cell) clusters will settle to the bottom, while single MEF will remain in suspension. Aspirate 80% of the supernatant to remove MEF. If iPS cells (or ES cells) were cultured feeder-free, directly continue with step A6.
    6. Collect iPS cell (or ES cell) clusters by centrifugation at 212 x g for 4 min. If not stated otherwise, all centrifugation steps are performed at RT. Aspirate supernatant and resuspend pellet in 1 ml induction medium d0. Pipette up and down with 1 ml pipette to break up clusters. Clusters should contain 50-100 cells (Figure 2). Therefore, transfer a drop of the cell suspension onto a microbiology grade Petri dish and check cluster size by microscopy.


      Figure 2. From iPS cells to hematopoietic progenitors. Representative phase contrast images of iPS cells during differentiation into hematopoietic progenitors. Undifferentiated iPS cell colonies on day 0 and small EB with some cell debris on day 2 and 6. Scale bars = 500 µm. Hemato-endothelial patches and hematopoietic progenitors on day 8, 10 and 14. Scale bars = 200 µm.

    7. If cluster size is appropriate, pass cell suspension through a 70 µm cell strainer to remove remaining larger clusters. Fill up flow-through to the final volume with induction medium d0 and transfer to microbiology grade Petri dishes to allow embryoid bodies (EB) to form. We usually use 10 ml induction medium d0 for two 6-well plates with iPS cells (or ES cells).
    8. Place cells in a 37 °C, 5% CO2 incubator and set O2 regulator of the automatic incubator to 5%. Thus, O2 levels will slowly decrease to 5%.
    9. On day 1 of differentiation (24 h after EB formation), many cells (approx. 40-50%) will have died. Prepare induction medium d1 (see Recipes) and prewarm at RT. Dilute old medium (induction medium d0) with fresh medium (induction medium d1) in a ratio of 1/2. Calculate volume of bFGF in induction medium d1 according to total volume (old medium + fresh medium).
      Example: 10 ml induction medium d0 + 5 ml induction medium d1 medium containing 1.5 µl bFGF and 1.6 µl BMP4. Final bFGF concentration should be 10 ng/ml. Continue culture under hypoxic conditions (5% O2, 5% CO2).
    10. On day 2 of differentiation, there will be a lot of cell debris visible (Figure 2). Collect EB in 50 ml Falcon tubes. Despite the use of microbiology grade Petri dishes, some EB loosely adhere to the dish. Rinse loosely adherent EB off the dishes twice with 1x PBS (prewarmed to RT) and add to EB suspension. Centrifuge EB at 136 x g for 4 min. Aspirate supernatant, gently resuspend pellet (if necessary with 1 ml pipette to break up aggregated EB) in induction medium d2 (prewarmed to RT, see Recipes) and transfer to new microbiology grade Petri dishes. Use the same volume as on day 0 (see step A7). Continue culture under hypoxic conditions (5% O2, 5% CO2).
    11. On day 4 of differentiation, proceed as in step A10. Finally, resuspend EB in induction medium d4 (prewarmed to RT, see Recipes) and transfer to new microbiology grade Petri dishes. Use the same volume as on day 0 (see step A7). Continue culture under hypoxic conditions (5% O2, 5% CO2).
    12. On day 6 of differentiation, proceed as in step A10. Finally, resuspend EB in induction medium d6 (prewarmed to RT, see Recipes) and transfer to tissue culture plastic (TCP) dishes coated with 0.1% gelatin (see Recipes). Use the same volume as on day 0 (see step A7). Continue culture under hypoxic conditions (5% O2, 5% CO2).
    13. On day 8 of differentiation, most EB are adherent on TCP. Collect medium and centrifuge at 136 x g for 4 min. Aspirate supernatant, resuspend in induction medium d6 (prewarmed to RT) and transfer carefully back to the TCP dish. Use the same volume as on day 0 (see step A7). From now on, avoid detaching EB from the dish. Transfer cells to 37 °C, 5% CO2 and normoxic incubator.
    14. On day 10 and 12 of differentiation, medium change is performed as in step A13.
    15. Depending on the iPS cell (or ES cell) clone used hematopoietic progenitors will start to bud off from adherent or loosely adherent EB between day 6-14 (iPS cells) or day 21-28 (ES cells) of differentiation, respectively (Figure 2, Video 1). iPS cell (or ES cell) derived hematopoietic progenitors are CD43+ (Figure 3) and used for DC differentiation between day 10-14 (iPS cells) and day 24-28 (ES cells).

      Video 1. Emergence of iPS cell (or ES cell) derived hematopoietic progenitors. Representative video of iPS cell derived EB on day 6 of differentiation.


      Figure 3. iPS cells develop via hemogenic endothelium into hematopoietic progenitors. Representative flow cytometry data on day 6 and 10 of differentiation. Upper row shows gating for CD34+ CD31+ hemogenic endothelium population. Center row shows gating for CD43+ CD31+ hematopoietic progenitors. Bottom row shows gating for mature CD43+ CD45+ hematopoietic progenitors.

  2. The differentiation of iPS cell (or ES cell) derived hematopoietic progenitors into DC subsets is summarized in Figure 4.


    Figure 4. Differentiation protocol of iPS cell (or ES cell) derived hematopoietic progenitors into DC subsets. CD43+ hematopoietic progenitors are co-cultured on OP9 stromal cells with FSG4 and FSG cytokines to generate cDC1 and cDC2 (FSG4), or cDC1 and pDC (FSG), respectively.

    1. To differentiate iPS cell (or ES cell) derived hematopoietic progenitors into DC, seed irradiated OP9 stromal cells (30 Gray) resuspended in OP9 culture medium (see Recipes) in a density of 1.2 x 104 cells/cm2 onto 0.1% gelatin coated TCP dishes one day prior to DC differentiation. We prefer using 6-well plates because later during DC differentiation we can use the same cells for different treatments and experiments.
    2. Collect hematopoietic progenitors from step A15 by gentle pipetting and pass cell suspension through a 40 µm cell strainer in order to remove remaining EB. Wash plates twice with 1x PBS (prewarmed to RT) and pass this suspension through the same cell strainer.
    3. Centrifuge flow-through at 416 x g for 4 min. Aspirate supernatant and wash pellet in defined volume of 1x PBS (prewarmed to RT, e.g., 5 ml). Count cells (e.g., with a Neubauer chamber and trypan blue as live/dead cell marker).
    4. Centrifuge cell suspension at 416 x g for 4 min. Aspirate supernatant and resuspend cells in DC differentiation basal medium (prewarmed to RT, see Recipes) in a density of 1 x 106-1.5 x 106 cells/ml. Split cell suspension in half in two separate Falcon tubes labeled FSG4 and FSG, respectively.
    5. Add 100 ng/ml FLT3L, 20 ng/ml SCF, 20 ng/ml GM-CSF and 20 ng/ml IL-4 final concentrations in one Falcon tube (FSG4). Add 100 ng/ml FLT3L, 20 ng/ml SCF and 10 ng/ml GM-CSF final concentrations in the other Falcon tube (FSG).
    6. Aspirate OP9 culture medium from OP9 stromal cells and distribute FSG4 and FSG cell suspension onto the OP9 stromal cells. For example, 6 ml of FSG4 and FSG suspension can be distributed in 3 wells of a 6-well plate (2 ml per well), respectively.
    7. Culture cells at 37 °C and 5% CO2.
    8. Perform a partial medium change every second day. Prepare fresh medium: take half of the volume of basal DC differentiation medium as in step B4 but add double the amount of cytokines. Carefully tilt the plate and slowly aspirate the supernatant. Cells are loosely adherent to the feeder layer and those in suspension will settle down to the bottom if the medium is removed slowly. Avoid disturbing the cells e.g., by rinsing them off the feeder layer. Remove approx. 50% of the old medium (e.g., 1 ml per 6-well). Add 50% of freshly prepared medium (see above, prewarmed to RT). Thereby cytokine concentrations in the total volume will be the same as given in step B5. 
    9. DC have acquired DC morphology by DC differentiation day (dd) 4 (Figure 5) and can be used for analyses between dd4-dd7. To collect DC, carefully rinse them off the feeder layer. Wash plates twice with 1x PBS (prewarmed to RT). Avoid detaching OP9 stromal cells. Collect cells in a 50 ml Falcon tube on ice and proceed to required analyses and experiments, respectively.


      Figure 5. iPS derived DC acquire DC morphology during differentiation. Representative phase contrast images of iPS cell derived DC (yellow arrow) on irradiated OP9 stromal cells (white arrow) during differentiation. Scale bars = 25 µm.

Data analysis

iPS cell (or ES cell) derived DC subsets can be analyzed as described in our recent publication (Sontag et al., 2017).

Notes

Instead of hyper-IL-6 we also tested recombinant human IL-6 (Miltenyi Biotec, catalog number: 130-095-352) in a final concentration of 100 ng/ml and obtained similar results.

Recipes

  1. Collagenase IV solution (1 mg/ml)
    1,000 mg collagenase IV
    1,000 ml KO-DMEM
    Note: Sterile filter with 0.2 µm top bottle filter. Aliquot (e.g., 50 ml) and store at -20 °C.
  2. 0.1% gelatin solution
    0.5 g gelatin
    500 ml distilled water
    Note: Autoclave at 121 °C for 30 min. Gelatin will solubilize during autoclaving. Store at RT. For coating, cover surface of tissue culture dish with gelatin solution and incubate at 37 °C for at least 15 min. Aspirate gelatin solution prior to plating cells. Use immediately without additional washing or drying.
  3. 1-Thioglycerol stock solution (100 mM)
    87 µl 1-thioglycerol
    10 ml distilled and sterile water
    Note: 1-Thioglycerol has a high viscosity. Pipette slowly in order to dispense 1-thioglycerol accurately. Aliquot (e.g., 1 ml) and store at -20 °C.
  4. L-ascorbic acid stock solution (50 mg/ml)
    100 mg L-ascorbic acid
    2 ml distilled and sterile water
    Note: Aliquot (e.g., 250 µl) and store at -20 °C. Protect from light.
  5. 0.1% BSA solution
    0.1 g BSA
    100 ml 1x PBS
    Note: Store at 4 °C.
  6. BMP4 stock solution (25 µg/ml)
    10 µg BMP4
    400 µl 0.1% BSA solution
  7. bFGF stock solution (100 µg/ml)
    50 µg bFGF
    500 µl 0.1% BSA solution
  8. FLT3L stock solution (25 µg/ml)
    100 µg FLT3L
    4 ml 0.1% BSA solution
  9. GM-CSF stock solution (100 µg/ml)
    20 µg GM-CSF
    200 µl 0.1% BSA solution
  10. IGF1 stock solution (40 µg/ml)
    100 µg IGF1
    2.5 ml 0.1% BSA solution
  11. IL-3 stock solution (150 µg/ml)
    25 µg IL-3
    167 µl 0.1% BSA solution
  12. IL-4 stock solution (20 µg/ml)
    10 µg IL-4
    500 µl 0.1% BSA solution
  13. SCF stock solution (100 µg/ml)
    10 µg SCF
    100 µl 0.1% BSA solution
  14. TPO stock solution (20 µg/ml)
    100 µg TPO
    5 ml 0.1% BSA solution
  15. VEGF stock solution (100 µg/ml)
    10 µg VEGF
    100 µl 0.1% BSA solution

    Note: All cytokines (BMP4, bFGF, FLT3L, GM-CSF, IGF1, IL-3, IL-4, SCF, TPO and VEGF) are stored in aliquots (e.g., 10-50 µl) at -20 °C in a standard non-defrosting freezer. After thawing, we keep them at 4 °C and use them for four weeks.

  16. Hematopoietic progenitor basal differentiation medium
    237 ml 1x StemPro-34 SFM
    6.5 ml StemPro-34 supplements (provided with 1x StemPro-34 SFM)
    2.5 ml penicillin-streptomycin (final concentration 100 U/ml penicillin, 100 µg/ml streptomycin)
    2.5 ml L-glutamine (final concentration 2 mM)
    1.0 ml 1-thioglycerol stock solution (final concentration 0.4 mM)
    250 µl L-ascorbic acid stock solution (final concentration 50 µg/ml)
  17. Induction medium d0
    20 ml hematopoietic progenitor basal differentiation medium
    6.7 µl BMP4 stock solution (final concentration 8 ng/ml)
  18. Induction medium d1
    20 ml hematopoietic progenitor basal differentiation medium
    6.7 µl BMP4 stock solution (final concentration 8 ng/ml)
    4.0 µl bFGF stock solution (final concentration 10 ng/ml, see Procedure step A9)
  19. Induction medium d2
    20 ml hematopoietic progenitor basal differentiation medium
    6.7 µl BMP4 stock solution (final concentration 8 ng/ml)
    2.0 µl bFGF stock solution (final concentration 10 ng/ml)
    2.0 µl VEGF stock solution (final concentration 10 ng/ml)
    20 µl hyper-IL-6 (final concentration 10 ng/ml)
    12.5 µl IGF1 stock solution (final concentration 25 ng/ml)
    20 µl SCF stock solution (final concentration 100 ng/ml)
  20. Induction medium d4
    20 ml hematopoietic progenitor basal differentiation medium
    2.0 µl bFGF stock solution (final concentration 10 ng/ml)
    2.0 µl VEGF stock solution (final concentration 10 ng/ml)
    20 µl hyper-IL-6 (final concentration 10 ng/ml)
    12.5 µl IGF1 stock solution (final concentration 25 ng/ml)
    20 µl SCF stock solution (final concentration 100 ng/ml)
    8.0 µl FLT3L stock solution (final concentration 10 ng/ml)
    20 µl TPO stock solution (final concentration 20 ng/ml)
    4.0 µl IL-3 stock solution (final concentration 30 ng/ml)
  21. Induction medium d6
    20 ml hematopoietic progenitor basal differentiation medium
    20 µl SCF stock solution (final concentration 100 ng/ml)
    8.0 µl FLT3L stock solution (final concentration 10 ng/ml)
    20 µl TPO stock solution (final concentration 20 ng/ml)
    4.0 µl IL-3 stock solution (final concentration 30 ng/ml)
  22. OP9 culture medium
    195 ml α-MEM
    50 ml FBS (final concentration 20%)
    2.5 ml penicillin-streptomycin (final concentration 100 U/ml penicillin, 100 µg/ml streptomycin)
    2.5 ml L-glutamine (final concentration 2 mM)
  23. DC differentiation basal medium
    220 ml RPMI 1640
    25 ml FBS (final concentration 10%)
    2.5 ml penicillin-streptomycin (final concentration 100 U/ml penicillin, 100 µg/ml streptomycin)
    2.5 ml L-glutamine (final concentration 2 mM)
    500 µl β-mercaptoethanol (final concentration 0.1 mM)
  24. DC differentiation FSG4 medium
    20 ml DC differentiation basal medium
    80 µl FLT3L stock concentration (final concentration 100 ng/ml)
    40 µl SCF stock concentration (final concentration 20 ng/ml)
    4.0 µl GM-CSF stock solution (final concentration 20 ng/ml)
    20 µl IL-4 stock concentration (final concentration 20 ng/ml)
  25. DC differentiation FSG medium
    20 ml DC differentiation basal medium
    80 µl FLT3L stock concentration (final concentration 100 ng/ml)
    40 µl SCF stock concentration (final concentration 20 ng/ml)
    2.0 µl GM-CSF stock solution (final concentration 20 ng/ml)

Acknowledgments

This work was supported in part by the Ministry for Innovation, Science and Research of German Federal State of North Rhine-Westphalia, Duesseldorf, Germany (S. S. and M. Z.) and by a research grant of the Interdisciplinary Center for Clinical Research (IZKF) Aachen, Germany (M. Z.). We thank Marion Kennedy and Gordon Keller for their advice on hematopoietic progenitor differentiation.

References

  1. Bellin, M., Marchetto, M. C., Gage, F. H. and Mummery, C. L. (2012). Induced pluripotent stem cells: the new patient? Nat Rev Mol Cell Biol 13(11): 713-726.
  2. Belz, G. T. and Nutt, S. L. (2012). Transcriptional programming of the dendritic cell network. Nat Rev Immunol 12(2): 101-113.
  3. Choi, K. D., Vodyanik, M. and Slukvin, II (2011). Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells. Nat Protoc 6(3): 296-313.
  4. Fischer, M., Goldschmitt, J., Peschel, C., Brakenhoff, J. P., Kallen, K. J., Wollmer, A., Grotzinger, J. and Rose-John, S. (1997). A bioactive designer cytokine for human hematopoietic progenitor cell expansion. Nat Biotechnol 15(2): 142-145.
  5. Gilliet, M., Boonstra, A., Paturel, C., Antonenko, S., Xu, X. L., Trinchieri, G., O'Garra, A. and Liu, Y. J. (2002). The development of murine plasmacytoid dendritic cell precursors is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor. J Exp Med 195(7): 953-958.
  6. Grskovic, M., Javaherian, A., Strulovici, B. and Daley, G. Q. (2011). Induced pluripotent stem cells--opportunities for disease modelling and drug discovery. Nat Rev Drug Discov 10(12): 915-929.
  7. Joffre, O. P., Segura, E., Savina, A. and Amigorena, S. (2012). Cross-presentation by dendritic cells. Nat Rev Immunol 12(8): 557-569.
  8. Jongbloed, S. L., Kassianos, A. J., McDonald, K. J., Clark, G. J., Ju, X., Angel, C. E., Chen, C. J., Dunbar, P. R., Wadley, R. B., Jeet, V., Vulink, A. J., Hart, D. N. and Radford, K. J. (2010). Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens. J Exp Med 207(6): 1247-1260.
  9. Kennedy, M., Awong, G., Sturgeon, C. M., Ditadi, A., LaMotte-Mohs, R., Zuniga-Pflucker, J. C. and Keller, G. (2012). T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures. Cell Rep 2(6): 1722-1735.
  10. Lee, J., Breton, G., Oliveira, T. Y., Zhou, Y. J., Aljoufi, A., Puhr, S., Cameron, M. J., Sekaly, R. P., Nussenzweig, M. C. and Liu, K. (2015). Restricted dendritic cell and monocyte progenitors in human cord blood and bone marrow. J Exp Med 212(3): 385-399.
  11. Li, Y., Liu, M. and Yang, S. T. (2014). Dendritic cells derived from pluripotent stem cells: Potential of large scale production. World J Stem Cells 6(1): 1-10.
  12. Lundberg, K., Albrekt, A. S., Nelissen, I., Santegoets, S., de Gruijl, T. D., Gibbs, S. and Lindstedt, M. (2013). Transcriptional profiling of human dendritic cell populations and models--unique profiles of in vitro dendritic cells and implications on functionality and applicability. PLoS One 8(1): e52875.
  13. Merad, M., Sathe, P., Helft, J., Miller, J. and Mortha, A. (2013). The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu Rev Immunol 31: 563-604.
  14. Poulin, L. F., Salio, M., Griessinger, E., Anjos-Afonso, F., Craciun, L., Chen, J. L., Keller, A. M., Joffre, O., Zelenay, S., Nye, E., Le Moine, A., Faure, F., Donckier, V., Sancho, D., Cerundolo, V., Bonnet, D. and Reis e Sousa, C. (2010). Characterization of human DNGR-1+ BDCA3+ leukocytes as putative equivalents of mouse CD8α+ dendritic cells. J Exp Med 207(6): 1261-1271.
  15. Robinton, D. A. and Daley, G. Q. (2012). The promise of induced pluripotent stem cells in research and therapy. Nature 481: 295-305.
  16. Rossi, R., Hale, C., Goulding, D., Andrews, R., Abdellah, Z., Fairchild, P. J. and Dougan, G. (2012). Interaction of Salmonella typhimurium with dendritic cells derived from pluripotent embryonic stem cells. PLoS One 7(12): e52232.
  17. Schlitzer, A. and Ginhoux, F. (2014). Organization of the mouse and human DC network. Curr Opin Immunol 26: 90-99.
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  20. Senju, S., Suemori, H., Zembutsu, H., Uemura, Y., Hirata, S., Fukuma, D., Matsuyoshi, H., Shimomura, M., Haruta, M., Fukushima, S., Matsunaga, Y., Katagiri, T., Nakamura, Y., Furuya, M., Nakatsuji, N. and Nishimura, Y. (2007). Genetically manipulated human embryonic stem cell-derived dendritic cells with immune regulatory function. Stem Cells 25(11): 2720-2729.
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简介

诱导的多能干细胞(iPS细胞)是工程干细胞,其表现出与胚胎干细胞(ES细胞,Takahashi和Yamanaka,2016)非常相似的性质。 iPS细胞和ES细胞都具有非凡的自我更新能力,可以分化成我们身体的所有细胞类型,包括造血干细胞/祖细胞和源自其的树突状细胞(DC)。这使得iPS细胞特别适用于研究疾病,药物发现和再生治疗的分子机制(Grskovic等人,2011; Bellin等人,2012; Robinton和Daley,2012)。
  DC是免疫系统的主要抗原呈递细胞,因此它们是调节和引导免疫应答的关键参与者(Merad等人,2013)。 DC巡逻外周和界面组织(例如,肺,肠和皮肤)以检测入侵的病原体,并且在激活时,它们迁移到淋巴结以激活和引发淋巴细胞。
  DC包含具有功能专门子集的表型异质家族(Schlitzer和Ginhoux,2014)。通常,经典DC(cDC)和浆细胞样DC(pDC)是分别表现出典型的和等离子体细胞样的DC形态。 cDC识别许多病原体并在激活后分泌促炎细胞因子,而pDC专门检测细胞内病原体并分泌I型干扰素(Merad等,2013; Schlitzer和Ginhoux,2014)。在被称为CD141 Clec9a + cDC1和CD1c + CD14的人类系统中,cDC进一步分为交叉呈递cDC1和常规cDC2-cDC2。人类pDC的特征在于CD303 CD304 (Jongbloed等人,2010; Joffre等人, 2012; Swiecki和Colonna,2015)
为了调查人类DC的子集规范和功能,我们建立了一种从人类iPS细胞(或ES细胞)体外产生cDC1,cDC2和pDC 的方案(Sontag等人, ,2017)。因此,我们将iPS细胞(或ES细胞)通过中胚层承诺和血细胞内皮规格分化到CD43 + CD31 + 造血祖细胞。随后,将它们分别接种到具有FLT3L,SCF,GM-CSF和IL-4或FLT3L,SCF和GM-CSF的灭活的OP9基质细胞上,分别指定cDC1和cDC2或cDC1和pDC。
【背景】DC及其发育主要在小鼠中进行研究(Belz和Nutt,2012; Merad等人,2013; Schlitzer和Ginhoux,2014)。由于它们在非淋巴组织中的稀少性和对人淋巴组织的限制性获取,所以在人类中研究DC是具有挑战性的(Jongbloed等人,2010; Villadangos和Shortman,2010)。然而,理解DC子集规范中的发育途径,起源和机制对于治疗应用(例如抗肿瘤剂)在体外产生自体DC亚群是很重要的。具有无限自我更新和分化潜能的iPS细胞(和ES细胞)提高了希望,可以产生和修饰DC(例如,,加载患者和疾病特异性抗原),数量和质量>体外研究DC发展和功能,并改善DC免疫治疗(Sontag等人,2017)。
几个组从具有常规粒细胞巨噬细胞集落刺激因子(GM-CSF)/白细胞介素4(IL-4)方案的iPS细胞(和ES细胞)中产生单核细胞衍生的DC,但是这种DC代表炎性DC并且不是子集特异性(Senju等人,2007; Tseng等人,2009; Choi等人,2011; Senju等人, ,2011; Belz和Nutt,2012; Rossi等人,2012; Yanagimachi等人,2013; Li等人, em>,2014)。从小鼠研究中,已知fms样酪氨酸激酶配体(FLT3L)是DC发育的关键细胞因子,并且FLT3L和GM-CSF信号的组合在体内指定DC亚群(Gilliet et al。,2002; Schmid等人,2010)。最近,人类cDC1,cDC2和pDC由MST基质细胞上的FLT3L,干细胞因子(SCF)和GM-CSF的脐带血(CB)产生(Lee等人,2015)。另外,Poulin等人(2010)报道了在无饲养者环境中使用FLT3L,SCF,GM-CSF和IL-4从CB产生cDC1(Poulin等人, ,2010)。相比之下,Silk等人(2012)描述了cDC1在无饲养细胞的GM-CSF / IL-4系统中的分化(Silk等人,2012) 。然而,最近的基因组广泛的转录分析研究突出了DC发展期间微环境线索的影响,表明饲养者支持是重要的(Lundberg等人,2013)。因此,我们使用FLT3L,SCF,GM-CSF和IL-4(简称FSG4)和FLT3L,SCF和GM-CSF(简称FSG)与OP9基质细胞组合,从iPS中产生cDC1,cDC2和pDC细胞(或ES细胞)衍生的造血祖细胞。
这里我们描述两步法:首先,人类iPS细胞(或ES细胞)被分化成造血祖细胞(改编自Kennedy等人,2012)。其次,这些造血干细胞/祖细胞进一步分化成cDC1,cDC2和pDC(Sontag等人,2017)。

关键字:iPS细胞, ES细胞, 造血, 造血分化, 人树突状细胞, 树突状细胞分化

材料和试剂

  1. 6孔组织培养板(TPP Techno Plastic Products,目录号:92006)
  2. 10厘米微生物学培养皿(SARSTEDT,目录号:82.1473.001)
  3. 50ml Falcon管(Corning,Falcon ®,目录号:352070)
  4. 70μm细胞过滤器(Greiner Bio One International,目录号:542070)
  5. 40μm细胞过滤器(Greiner Bio One International,目录号:542040)
  6. 顶级过滤器(TPP Techno Plastic Products,目录号:99505)
  7. 5ml血清移液管(Corning,Falcon ®,目录号:357543)
  8. 10ml血清移液管(Corning,Falcon ®,目录号:357551)
  9. 25 ml血清移液管(康宁,Falcon ®,目录号:357525)
  10. 移液瓶头10μl(STARLAB INTERNATIONAL,目录号:S1180-3810)
  11. 移液瓶提示20μl(STARLAB INTERNATIONAL,目录号:S1120-1810)
  12. 移液瓶提示200μl(STARLAB INTERNATIONAL,目录号:S1120-8810)
  13. 移液瓶提示1000μl(STARLAB INTERNATIONAL,目录号:S1126-7810)
  14. 胶原酶IV(Thermo Fisher Scientific,Gibco TM,目录号:17104019)
  15. Knockout-Dulbecco的改良Eagle培养基(KO-DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:10829018)
  16. 明胶(Sigma-Aldrich,目录号:G1890)
  17. 1-Thiogylcerol(Sigma-Aldrich,目录号:M1753)
  18. L-抗坏血酸(Sigma-Aldrich,目录号:A4403)
  19. 牛血清白蛋白(BSA)低内毒素(PAA,目录号:K31-011)
  20. 1x Dulbecco的磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190094)
  21. 人类骨形态发生蛋白4(BMP4)研究级(Miltenyi Biotec,目录号:130-110-921)
  22. 重组人碱性成纤维细胞生长因子(bFGF)(PeproTech,目录号:100-18B)
  23. 重组人FLT3L(PeproTech,目录号:300-19)
  24. 重组人GM-CSF(PeproTech,目录号:300-03)
  25. 重组人类胰岛素样生长因子1(IGF1)(PeproTech,目录号:100-11)
  26. 人白细胞介素3(IL-3)研究级(Miltenyi Biotec,目录号:130-094-193)
  27. 人IL-4研究级(Miltenyi Biotec,目录号:130-095-373)
  28. 人类SCF研究级(Miltenyi Biotec,目录号:130-096-693)
  29. 人血小板生成素(TPO)研究级(Miltenyi Biotec,目录号:130-094-013)
  30. 重组人血管内皮生长因子(VEGF)(PeproTech,目录号:100-20)
  31. 1x StemPro-34 SFM(Thermo Fisher Scientific,目录号:10639011)
  32. 青霉素 - 链霉素10,000U / ml(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  33. L-谷氨酰胺200mM(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  34. 超细胞白介素6(IL-6)(由S.Rose John,德国基尔,阿尔布雷希奇大学医学院生物化学研究所提供,[Fischer等人,1997] ,另见备注)
  35. α-最小必需培养基(α-MEM)(Thermo Fisher Scientific,Gibco TM,目录号:12571063)
  36. β-巯基乙醇(50mM)(Thermo Fisher Scientific,Gibco TM,目录号:31350010)
  37. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10500064)
  38. 罗斯韦尔公园纪念研究所(RPMI)1640培养基(Thermo Fisher Scientific,Gibco TM,目录号:11875093)
  39. 胶原酶IV溶液(1mg / ml)(参见食谱)
  40. 0.1%明胶溶液(见配方)
  41. 1-硫代甘油储备溶液(100mM)(参见食谱)
  42. L-抗坏血酸储备溶液(50mg / ml)(参见食谱)
  43. 0.1%BSA溶液(见配方)
  44. BMP4储备溶液(25μg/ ml)(参见食谱)
  45. bFGF储备溶液(100μg/ ml)(参见食谱)
  46. FLT3L储备溶液(25μg/ ml)(见配方)
  47. GM-CSF储备溶液(100μg/ ml)(见配方)
  48. IGF1储备溶液(40μg/ ml)(参见食谱)
  49. IL-3储备溶液(150μg/ ml)(参见食谱)
  50. IL-4储备溶液(20μg/ ml)(参见食谱)
  51. SCF储备液(100μg/ ml)(见食谱)
  52. TPO储备溶液(20μg/ ml)(参见食谱)
  53. VEGF储备溶液(100μg/ ml)(参见食谱)
  54. 造血祖细胞基础分化培养基(见配方)
  55. 感应介质d0(见配方)
  56. 感应介质d1(见配方)
  57. 感应介质d2(参见食谱)
  58. 感应介质d4(见配方)
  59. 感应介质d6(参见食谱)
  60. OP9培养基(参见食谱)
  61. DC分化基础培养基(见食谱)
  62. DC分化FSG4培养基(参见食谱)
  63. DC分化FSG培养基(参见食谱)

设备

  1. 1,000μl移液器(Gilson,目录号:F123602)
  2. 200μl移液器(Gilson,目录号:F123601)
  3. 20μl移液器(Gilson,目录号:F123600)
  4. 10μl移液器(Gilson,目录号:F144802)
  5. Pipetboy(INTEGRA Biosciences,目录号:155000)
  6. 水浴(JULABO,型号:SW22)
  7. 倒置光学显微镜(Leica Microsystems,型号:Leica DM IL LED)
  8. 高压灭菌器
  9. 离心机(Thermo Fisher Scientific,型号:Heraeus TM Multifuge 3 L)
  10. 流动罩(Heraeus)
  11. 具有氮供应和O 2传感器的自动CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus sup> 240i,目录号:51026331)
  12. 真空泵(INTEGRA Biosciences,目录号:158300)
  13. 标准冰箱
  14. 标准非除霜冷冻机

程序

  1. iPS细胞(或ES细胞)分化成造血祖细胞总结在图1中

    图1. iPS细胞(或ES细胞)分化为造血祖细胞的分化方案的示意图。 显示分化时间和相应的分化步骤和细胞因子组成(彩色盒子)。

    1. 为了将iPS细胞(或ES细胞)分化成造血祖细胞,在开始实验之前,预热所需量的胶原酶IV溶液(1mg / ml,37℃,参见食谱)和KO-DMEM(室温,RT)。准备诱导培养基d0(参见食谱)并在室温下预热。
    2. 收获iPS细胞(或ES细胞)达到70-80%汇合时。因此,吸出培养基并将胶原酶IV溶液加入细胞。体积取决于所用的培养皿。确保使用胶原酶IV溶液(例如,每孔1ml,6孔板,每10cm皿4ml)完全覆盖细胞。
    3. 在含有5%CO 2的正常培养箱中,在37℃下将细胞与胶原酶IV孵育40-60分钟。检查菌落边缘是否开始卷起。只有当菌落边缘从所有部位卷起,并且菌落才松散地粘附在板上,继续步骤A4。如有必要,孵育10-15分钟,但不要超过80分钟的胶原酶IV治疗,因为这会影响细胞活力。
    4. 用KO-DMEM稀释胶原酶IV,比例为1/3,意味着每ml胶原酶IV溶液加入2 ml KO-DMEM。使用10毫升移液管清洗菌落,重新悬浮并收集在50ml Falcon管中。
    5. 如果将iPS细胞(或ES细胞)在小鼠胚胎成纤维细胞(MEF)上培养,则在室温下通过重力将独立的iPS细胞(或ES细胞)簇和单个MEF通过重力培养10分钟。大的iPS细胞(或ES细胞)簇将沉降到底部,而单个MEF将保持悬浮状态。吸出80%的上清液去除MEF。如果将iPS细胞(或ES细胞)培养为无饲养细胞,则直接继续步骤A6。
    6. 通过在212×g下离心4分钟收集iPS细胞(或ES细胞)簇。如果没有另外说明,所有的离心步骤都是在RT进行的。吸出上清并将沉淀重悬于1ml诱导培养基d0中。用1毫升移液管上下移动以分裂群集。群集应包含50-100个细胞(图2)。因此,将一滴细胞悬浮液转移到微生物学级培养皿上,并通过显微镜检查簇大小。


      图2.从iPS细胞到造血祖细胞。在分化成造血祖细胞期间,iPS细胞的代表性的对比图像。未分化的iPS细胞集落在第0天,小EB和一些细胞碎片在第2和6天。比例尺=500μm。血液内皮斑块和造血祖细胞在第8,10和14天。比例尺=200μm。

    7. 如果聚类尺寸合适,请将细胞悬浮液通过70μm的细胞过滤器,以除去剩余的较大簇。用感应培养基d0填充到最终体积,并转移到微生物学级培养皿中以形成胚状体(EB)。我们通常使用10 ml的诱导培养基d0作为两个具有iPS细胞(或ES细胞)的6孔板
    8. 将细胞置于37℃,5%CO 2培养箱中,将自动培养箱的O 2调节物置于5%。因此,O 2级别将缓慢降低至5%。
    9. 在分化的第1天(EB形成后24小时),许多细胞(约40-50%)将死亡。准备感应介质d1(参见食谱)并在室温预热。用新鲜培养基(感应培养基d1)以1/2的比例稀释老培养基(诱导培养基d0)。根据总体积(老培养基+新鲜培养基)计算诱导培养基中bFGF的体积。
      实施例:10ml诱导培养基d0 + 5ml含有1.5μlbFGF和1.6μlBMP4的诱导培养基d1培养基。最终的bFGF浓度应为10ng / ml。继续在缺氧条件下培养(5%O 2 ,5%CO 2
    10. 在分化的第2天,会有很多细胞碎片可见(图2)。收集EB在50ml Falcon管中。尽管使用微生物学级培养皿,但有些EB松散地贴在菜上。用1x PBS(预热至RT)将松散粘附的EB漂洗两次,并加入EB悬浮液中。离心EB在136 x g处理4分钟。吸入上清液,在诱导培养基d2(预热至RT,参见食谱)中轻轻重悬沉淀(如果需要使用1ml移液管分解聚集的EB)并转移到新的微生物学培养皿中。使用与第0天相同的音量(参见步骤A7)。继续在缺氧条件下培养(5%O 2,5%CO 2)。
    11. 在分化的第4天,如步骤A10那样进行。最后,将EB置于诱导培养基d4(预热至RT,参见食谱)中并转移到新的微生物学培养皿中。使用与第0天相同的音量(参见步骤A7)。继续在缺氧条件下培养(5%O 2,5%CO 2)。
    12. 在分化的第6天,如步骤A10那样进行。最后,将EB重新悬浮在诱导培养基d6(预热至RT,参见食谱)中并转移至涂有0.1%明胶的组织培养塑料(TCP)培养皿(参见食谱)。使用与第0天相同的音量(参见步骤A7)。继续在缺氧条件下培养(5%O 2,5%CO 2)。
    13. 在分化的第8天,大多数EB在TCP上是粘附的。收集介质并以136 x g离心4分钟。将吸出的上清液重新悬浮于诱导培养基d6(预热至RT)中,并小心地转移到TCP培养皿中。使用与第0天相同的音量(参见步骤A7)。从现在开始,避免将EB从菜中分离出来。将细胞转移至37℃,5%CO 2和常氧培养箱。
    14. 在分化的第10天和第12天,如步骤A13那样进行中等变化。
    15. 根据iPS细胞(或ES细胞)克隆,使用的造血祖细胞将分别从第6-14天(iPS细胞)或第21-28天(ES细胞)分化的贴壁或松散粘附的EB发芽(图2 ,视频1)。 iPS细胞(或ES细胞)衍生的造血祖细胞是CD43 +(图3),并用于第10-14天(iPS细胞)和第24-28天(ES细胞)之间的DC分化。 />
      视频1


      图3. iPS细胞通过血液内皮发育成造血祖细胞。在分化的第6和10天有代表性的流式细胞术数据。上行显示CD34 + CD31 + 血液内皮细胞群体的门控。中央排显示CD43 + CD31 + 造血祖细胞的门控。下图显示成熟CD43 + CD45 + 造血祖细胞的门控。

  2. 将iPS细胞(或ES细胞)衍生的造血祖细胞分化为DC亚型,如图4所示。


    图4.将iPS细胞(或ES细胞)衍生的造血祖细胞分化为DC亚型的分化方案 CD43 + 造血祖细胞在OP9基质细胞上与FSG4和FSG共培养细胞因子分别产生cDC1和cDC2(FSG4),或cDC1和pDC(FSG)。

    1. 为了将iPS细胞(或ES细胞)衍生的造血祖细胞分化为DC,将种子照射的OP9基质细胞(30Gy)以1.2×10 4个细胞/孔重悬浮于OP9培养基(参见食谱)在DC分化前一天,在0.1%明胶包被的TCP培养皿上。我们更喜欢使用6孔板,因为在DC分化过程中,我们可以使用相同的细胞进行不同的处理和实验。
    2. 通过轻轻移液从步骤A15收集造血祖细胞,并将细胞悬液通过40μm细胞过滤器,以除去剩余的EB。用1x PBS(预热至RT)洗涤板两次,并将该悬浮液通过相同的细胞过滤器。
    3. 离心机以416×g流通4分钟。吸出上清液,并在规定体积的1x PBS(预热至RT,例如5ml)中洗涤沉淀。使用Neubauer室计数细胞(例如,,并将台盼蓝作为活/死细胞标记)。
    4. 离心细胞悬浮液在416×g下静置4分钟。吸出上清液并将细胞重悬于DC分化基础培养基(预热至RT,参见Recipes)中,密度为1×10 6细胞/ ml〜1.5×10 6细胞/ ml。分别在两个独立的Falcon管中分离细胞悬浮液,分别标记为FSG4和FSG。
    5. 在一个Falcon管(FSG4)中加入100ng / ml FLT3L,20ng / ml SCF,20ng / ml GM-CSF和20ng / ml IL-4终浓度。在其他Falcon管(FSG)中加入100ng / ml FLT3L,20ng / ml SCF和10ng / ml GM-CSF终浓度。
    6. 从OP9基质细胞吸出OP9培养基,并将FSG4和FSG细胞悬浮液分散到OP9基质细胞上。例如,可以将6ml的FSG4和FSG悬浮液分别分布在6孔板(2ml /孔)的3个孔中。
    7. 培养细胞在37℃和5%CO 2
    8. 每隔一天进行一次介质更换。准备新鲜培养基:如步骤B4中所取的基础DC分化培养基体积的一半,但加入细胞因子的量的两倍。小心地倾斜板,慢慢吸出上清液。细胞松散地粘附到饲养层上,如果缓慢移除培养基,那些悬浮液将会沉降到底部。避免干扰细胞例如,通过将其从供料层上冲洗掉。删除约50%的旧培养基(例如,,每6孔1ml)。加入50%新鲜制备的培养基(见上文,预热至RT)。因此,总体积中的细胞因子浓度将与步骤B5中给出的相同。 
    9. DC通过DC分化天(dd)4(图5)获得了DC形态,可用于dd4-dd7之间的分析。要收集DC,请小心地将其从饲料层中冲洗干净。用1x PBS(预热至RT)洗涤板两次。避免分离OP9基质细胞。在冰上将细胞收集在50ml Falcon管中,分别进行必要的分析和实验

      图5. iPS衍生的DC在分化期间获得DC形态。 在分化期间照射的OP9基质细胞(白色箭头)上的iPS细胞衍生的DC(黄色箭头)的代表性的对比图像。刻度棒=25μm。

数据分析

iPS细胞(或ES细胞)衍生的DC亚群可以如我们最近的出版物(Sontag等人,2017)所述进行分析。

笔记

而不是超IL-6,我们还测试了终浓度为100ng / ml的重组人IL-6(Miltenyi Biotec,目录号:130-095-352),并获得了类似的结果。

食谱

  1. 胶原酶IV溶液(1mg / ml)
    1,000毫克胶原酶IV
    1000 ml KO-DMEM
    注意:无菌过滤器带有0.2μm顶部瓶子过滤器。等分试样(如50ml)并储存于-20℃。
  2. 0.1%明胶溶液
    0.5克明胶
    500毫升蒸馏水
    注意:在121°C高压灭菌30分钟。明胶会在高压灭菌过程中溶解。在室内存放对于涂层,用明胶溶液覆盖组织培养皿的表面,并在37℃下孵育至少15分钟。吸出明胶溶液前电镀细胞。立即使用,无需额外洗涤或干燥。
  3. 1-硫代甘油储备液(100mM)
    87μl1-硫甘油
    10毫升蒸馏水和无菌水 注意:1-硫代甘油具有高粘度。缓慢移液以准确分配1-硫代甘油。等分试样(如1毫升)并储存于-20℃。
  4. L-抗坏血酸储备溶液(50 mg / ml)
    100毫克L-抗坏血酸
    2 ml蒸馏水和无菌水 注意:等分试样(如250μl)并保存在-20°C。防光。
  5. 0.1%BSA溶液
    0.1克BSA
    100 ml 1x PBS
    注意:请于4°C储存。
  6. BMP4储备溶液(25μg/ ml)
    10μgBMP4
    400μl0.1%BSA溶液
  7. bFGF储备液(100μg/ ml)
    50μgbFGF
    500μl0.1%BSA溶液
  8. FLT3L储备溶液(25μg/ ml)
    100μgFLT3L
    4 ml 0.1%BSA溶液
  9. GM-CSF储备溶液(100μg/ ml)
    20μgGM-CSF
    200μl0.1%BSA溶液
  10. IGF1储备溶液(40μg/ ml)
    100μgIGF1
    2.5 ml 0.1%BSA溶液
  11. IL-3储备溶液(150μg/ ml)
    25μgIL-3
    167μl0.1%BSA溶液
  12. IL-4储备溶液(20μg/ ml)
    10μgIL-4
    500μl0.1%BSA溶液
  13. SCF储备溶液(100μg/ ml)
    10μgSCF
    100μl0.1%BSA溶液
  14. TPO储备溶液(20μg/ ml)
    100μgTPO
    5 ml 0.1%BSA溶液
  15. VEGF储备溶液(100μg/ ml)
    10μgVEGF
    100μl0.1%BSA溶液

    注意:所有细胞因子(BMP4,bFGF,FLT3L,GM-CSF,IGF1,IL-3,IL-4,SCF,TPO和VEGF)以等分试样储存(例如,10-50μl) C在标准的非除霜冷冻室。解冻后,我们将其保存在4°C,并使用它们四周。

  16. 造血祖细胞基础分化培养基
    237 ml 1x StemPro-34 SFM
    6.5 ml StemPro-34补充剂(附带1个StemPro-34 SFM)
    2.5 ml青霉素 - 链霉素(终浓度100 U / ml青霉素,100μg/ ml链霉素)
    2.5ml L-谷氨酰胺(终浓度2mM)
    1.0ml 1-硫代甘油储备溶液(终浓度0.4mM)
    250μlL-抗坏血酸储备溶液(终浓度50μg/ ml)
  17. 感应介质d0
    20毫升造血祖细胞基础分化培养基
    6.7μlBMP4储备液(终浓度8 ng / ml)
  18. 感应介质d1
    20毫升造血祖细胞基础分化培养基
    6.7μlBMP4储备液(终浓度8 ng / ml)
    4.0μlbFGF储备溶液(终浓度10ng / ml,参见方法步骤A9)
  19. 感应介质d2
    20毫升造血祖细胞基础分化培养基
    6.7μlBMP4储备液(终浓度8 ng / ml)
    2.0μlbFGF储备液(终浓度10ng / ml)
    2.0μlVEGF储备溶液(终浓度10ng / ml)
    20μl超IL-6(终浓度10 ng / ml)
    12.5μlIGF1储备溶液(终浓度25ng / ml)
    20μlSCF储备溶液(终浓度100 ng / ml)
  20. 感应介质d4
    20毫升造血祖细胞基础分化培养基
    2.0μlbFGF储备液(终浓度10ng / ml)
    2.0μlVEGF储备溶液(终浓度10ng / ml)
    20μl超IL-6(终浓度10 ng / ml)
    12.5μlIGF1储备溶液(终浓度25ng / ml)
    20μlSCF储备溶液(终浓度100 ng / ml)
    8.0μlFLT3L储备溶液(终浓度10 ng / ml)
    20μlTPO储备溶液(终浓度20ng / ml)
    4.0μlIL-3储备溶液(终浓度30 ng / ml)
  21. 感应介质d6
    20毫升造血祖细胞基础分化培养基
    20μlSCF储备溶液(终浓度100 ng / ml)
    8.0μlFLT3L储备溶液(终浓度10 ng / ml)
    20μlTPO储备溶液(终浓度20ng / ml)
    4.0μlIL-3储备溶液(终浓度30 ng / ml)
  22. OP9培养基
    195mlα-MEM
    50ml FBS(终浓度20%)
    2.5 ml青霉素 - 链霉素(终浓度100 U / ml青霉素,100μg/ ml链霉素)
    2.5ml L-谷氨酰胺(终浓度2mM)
  23. DC分化基础培养基
    220ml RPMI 1640
    25毫升FBS(终浓度10%)
    2.5 ml青霉素 - 链霉素(终浓度100 U / ml青霉素,100μg/ ml链霉素)
    2.5ml L-谷氨酰胺(终浓度2mM)
    500μlβ-巯基乙醇(终浓度0.1mM)
  24. DC分化FSG4培养基
    20 ml DC分化基础培养基 80μlFLT3L储备液浓度(终浓度100ng / ml)
    40μlSCF原料浓度(终浓度20ng / ml)
    4.0μlGM-CSF储备溶液(终浓度20ng / ml)
    20μlIL-4储备液浓度(终浓度20 ng / ml)
  25. DC分化FSG培养基
    20 ml DC分化基础培养基 80μlFLT3L储备液浓度(终浓度100ng / ml)
    40μlSCF原料浓度(终浓度20ng / ml)
    2.0μlGM-CSF储备溶液(终浓度20ng / ml)

致谢

这项工作部分得到了德国联邦莱茵 - 威斯特法伦州德国杜塞尔多夫(SS和MZ)的创新科学与研究部的支持,并得到了亚琛临床研究跨学科中心(IZKF)的研究资助,德国(MZ)。我们感谢Marion Kennedy和Gordon Keller对造血祖细胞分化的建议。

参考

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sontag, S., Förster, M., Seré, K. and Zenke, M. (2017). Differentiation of Human Induced Pluripotent Stem Cells (iPS Cells) and Embryonic Stem Cells (ES Cells) into Dendritic Cell (DC) Subsets. Bio-protocol 7(15): e2419. DOI: 10.21769/BioProtoc.2419.
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