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Dec 2018
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Direct Reprogramming of Mouse Embryonic Fibroblasts to Conventional Type 1 Dendritic Cells by Enforced Expression of Transcription Factors
转录因子强化表达直接将小鼠胚胎成纤维细胞重编程为常规1型树突状细胞   

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Abstract

Ectopic expression of transcription factor combinations has been recently demonstrated to reprogram differentiated somatic cells towards the dendritic cell (DC) lineage without reversion to a multipotent state. DCs have the ability to induce potent and long-lasting adaptive immune responses. In particular, conventional type 1 DCs (cDC1s) excel on antigen cross-presentation, a critical step for inducing CD8+ T cell cytotoxic responses. The rarity of naturally occurring cDC1s and lack of in vitro methodologies for the generation of pure cDC1 populations strongly hinders the study of cDC1 lineage specification and function. Here, we describe a protocol for the generation of induced DCs (iDCs) by lentiviral-mediated expression of the transcription factors PU.1, IRF8 and BATF3 in mouse embryonic fibroblasts. iDCs acquire DC morphology, cDC1 phenotype and transcriptional signatures within 9 days. iDCs generated with this protocol acquire functional ability to respond to inflammatory stimuli, engulf dead cells, process and cross-present antigens to CD8+ T cells. DC reprogramming provides a simple and tractable system to generate high numbers of cDC1-like cells for high content screening, opening new avenues to better understand cDC1 specification and function. In the future, faithful induction of cDC1 fate in fibroblasts may lead to the generation of patient-specific DCs for vaccination.

Keywords: Cell reprogramming (细胞重编程), Transcription factor (转录因子), Cell fate conversion (细胞命运转换), Dendritic cell (树突状细胞), Antigen presenting cell (抗原呈递细胞), Immunity (免疫), Mouse embryonic fibroblast (小鼠胚胎成纤维细胞), Immunotherapy (免疫疗法)

Background

Dendritic cells (DCs) are professional antigen presenting cells specialized in the recognition, processing and presentation of antigens to T cells, playing a pivotal role in the induction of adaptive immune responses and immunological memory (Merad et al., 2013). DCs can be divided into 4 main subsets: plasmacytoid DCs (pDC), producers of large amounts of type 1 interferons, monocyte-derived DCs, derived from circulating monocytes, and conventional DCs (cDC), a functionally heterogeneous subset further subdivided in conventional DCs type 1 (cDC1) and type 2 (cDC2). While cDC1s excel on priming cytotoxic CD8+ T cell responses by cross-presenting exogenous antigens on major histocompatibility complex (MHC) class I, cDC2s are specialized in the presentation of extracellular antigens to CD4+ T cells via MHC class II. This functional specialization results from stage-specific interplay between different transcription factors (TFs) that specify DC subsets. For instance, it has been shown that conditional deletion of E26 transformation-specific (ETS) family TF PU.1 in hematopoietic progenitors impacts the specification of the whole DC lineage (Carotta et al., 2010). Interferon regulatory factor 8 (IRF8) knock-out mice lack pDC and cDC1 subsets, and basic leucine zipper ATF-Like transcription factor 3 (BATF3) deficient mice have impaired cDC1 specification (Schiavoni et al., 2002; Hildner et al., 2008; Carotta et al., 2010).

Growing evidence supports the crucial role of cDC1s in initiating immune responses in the context of cancer (Bottcher and Reis, 2018), and their in vivo rarity has propelled attempts to generate cDC1 in vitro (Perez and De Palma, 2019; Wculek et al., 2019). Several strategies have been explored to generate bona fide DCs from blood monocytes or bone marrow (BM) precursors in the presence of different hematopoietic cytokines (Inaba et al., 1992; Lu et al., 1995; Lutz et al., 1999; Son et al., 2002; Naik et al., 2005; Poulin et al., 2010; Proietto et al., 2012; Balan et al., 2014; Mayer et al., 2014; Lee et al., 2015; Balan et al., 2018; Kirkling et al., 2018). However, limiting numbers of BM progenitors, together with the generation of mixed populations of DC subsets with conflicting functions, strongly limits the use of these approaches to elucidate DC development and harness cDC1s for therapy (Yona and Mildner, 2018).

Cell reprogramming offers an exciting opportunity to overcome these challenges. Through enforced expression of TFs, it is possible to reprogram somatic cells into induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006; Takahashi et al., 2007). Protocols to differentiate DCs from iPSCs have been described (Choi et al., 2009; Senju et al., 2009 and 2011; Horton et al., 2019). However, these rely on complex and long culture conditions that give rise to mixed populations of distinct DC subsets. Alternatively, somatic cells can be reprogrammed directly into other specialized cell types without transiting through pluripotency or multipotency. Mouse and human fibroblasts were directly reprogrammed into several cell types, such as neurons, cardiomyocytes and hematopoietic stem and progenitor cells, using TFs specifying target-cell identity (Pereira et al., 2012 and 2013, Xu et al., 2015).

Here, we describe a direct cell reprogramming approach to generate cDC1-like cells (induced DCs, or iDCs) by enforced expression of the TFs PU.1, IRF8 and BATF3 in mouse embryonic fibroblasts (MEFs) (Rosa et al., 2018). MEF-derived iDCs acquire DC morphology, cDC1 phenotype and transcriptional program characteristic of natural cDC1s. Functionally, iDCs acquire the ability to respond to toll-like receptor 3 (TLR3) and TLR4 stimuli, engulf protein and dead cells and, importantly, cross-present antigens to CD8+ T cells. Thus, the protocol described here enables the fast generation (within 9 days) of cDC1-like cells in vitro using a controlled and adaptable direct reprogramming approach suitable for screening studies aiming to better understand cDC1 developmental specification and functional maturation. This approach, coupled with pharmacological inhibition, gene knock-out or knock-down, will enable the discovery of regulators of DC specification acting individually or in combination (Pires et al., 2019). Employing high-content screening methodologies based on recent CRISPR-Cas9 technologies in MEFs paves the way for defining novel critical regulators of cDC1 fate and how cross-presentation ability is established de novo in multiple cell types. In the future, DC reprogramming will serve as a platform to develop immunotherapies for cancer and infectious disease.

Materials and Reagents

  1. 100 mm tissue culture plates (Corning, catalog number: 430167 )
  2. 150 mm tissue culture plates (Corning, catalog number: 430599 )
  3. 1.5 ml microtubes (Sarstedt, catalog number: 72.690.001 )
  4. 15 ml centrifuge tubes (Sarstedt, catalog number: 62.547.205 )
  5. 50 ml centrifuge tubes (Sarstedt, catalog number: 62.547.205 )
  6. Polypropylene round-bottom FACS tubes (Falcon, catalog number: 352063 )
  7. 150 ml bottle top vacuum filters, 0.45 μm low-protein binding (Corning, catalog number: 430625 )
  8. Amicon ultra-15 centrifugal filter units (Merck Millipore, catalog number: UFC903096 )
  9. Wild type mice (C57BL/6) or double homozygous Clec9aCre/Cre RosatdTomato/tdTomato (Clec9a-tdT) mice (Rosa et al., 2018)
  10. Human embryonic kidney (HEK) 293T cell line (ATCC, catalog number: CRL-3216 )
  11. Sterile 40 µm cell strainers (Corning, catalog number: 431750 )
  12. Gelatin from porcine skin type A (Sigma-Aldrich, catalog number: G1890 )
  13. Polyethylenimine (PEI) transfection reagent (Sigma-Aldrich, catalog number: 408727 )
  14. Hexadimethrine bromide (Polybrene) (Sigma-Aldrich, catalog number: H9268 )
  15. Doxycycline hyclate (DOX) (Sigma-Aldrich, catalog number: D9891 )
  16. Trypsin-EDTA (0.25%), phenol red (Thermo Fisher Scientific, catalog number: 25200-056 )
  17. TrypLE Express (1x) no phenol red (Thermo Fisher Scientific, catalog number: 12604-021 )
  18. HyClone Phosphate Buffered Saline solution (PBS) (GE Healthcare, catalog number: SH30256.01 )
  19. HyClone high-glucose Dulbecco's Modified Eagles Medium (DMEM) (GE Healthcare, catalog number: SH30243.01 )
  20. Opti-MEM reduced serum medium (Thermo Fisher Scientific, catalog number: 31985070 )
  21. HyClone 10,000 U/ml Penicillin 10,000 μg/ml Streptomycin Solution (Pen/Strep) (GE Healthcare, catalog number: SV30010 )
  22. HyClone Fetal Bovine Serum (FBS) (GE Healthcare, catalog number: SV30160.03 )
  23. Rat serum (GeneTex, catalog number: GTX73226 )
  24. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  25. Plasmid pMD2.G (Addgene, catalog number: 12259 )
  26. Plasmid psPAX2 (Addgene, catalog number: 12260 )
  27. Plasmid FUW-M2rtTA (Addgene, catalog number: 20342 )
  28. Plasmid pFUW-TetO-PIB containing a tri-cistronic cassette encoding PU.1, IRF8 and BATF3 (PIB) reprogramming factors (Rosa et al., 2018). Cell Reprogramming in Hematopoiesis and Immunity Lab, Division of Molecular Medicine and Gene Therapy, Lund University, Sweden.
  29. 4’,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: D9542 )
  30. Hoechst staining solution (Sigma-Aldrich, catalog number: H6024 )
  31. APC conjugated anti-mouse CD45 monoclonal antibody (Biolegend, catalog number: 103112 )
  32. PE-Cy7 conjugated anti-mouse MHC-II monoclonal antibody (Biolegend, catalog number: 107630 )
  33. DMEM Complete medium (see Recipes)
  34. Dissociation solution (see Recipes)
  35. 0.1% Gelatin solution (see Recipes)
  36. Wash solution (see Recipes)
  37. Staining solution (see Recipes)
  38. Freezing solution (see Recipes)

Equipment

  1. Sterile dissecting scissors (Sigma-Aldrich, catalog number: S3146 )
  2. Sterile stainless steel forceps (Sigma-Aldrich, catalog number: Z168777 )
  3. Freezing container Nalgene Mr. Frosty (Sigma-Aldrich, catalog number: C1562 )
  4. Hemacytometer cell counting chamber (Marienfeld Superior, catalog number: 13444890 )
  5. 4-16K Refrigerated centrifuge (Sigma)
  6. FACSCanto II flow cytometry analyzer (8-color, blue/red/violet) (BD Biosciences)
  7. BD FACSAria III cell sorter (BD Biosciences)
  8. Heracell 150i CO2 incubator (Thermo Scientific)
  9. ScanLaf Mars class 2 laminar flow hood (LaboGene)
  10. IX70 inverted tissue culture microscope (Olympus)
  11. Celldiscoverer 7 inverted microscope (Zeiss)

Software

  1. GraphPad PRISM 6, https://www.graphpad.com/
  2. FlowJo V10, https://www.flowjo.com/
  3. ZEN 3.1 (blue edition), https://www.zeiss.com/
  4. Adobe Photoshop CS6, https://www.adobe.com/se

Procedure

A schematic representation of the direct reprogramming protocol described herein can be found in Figure 1.


Figure 1. Schematic representation of the protocol for direct reprogramming of mouse embryonic fibroblasts to dendritic cells. Mouse embryonic fibroblasts (MEFs) were isolated from C57BL/6 wild type 13.5 days-old embryos. Embryos were dissected to remove the head (grey), liver and heart (red) and dissociated to a single-cell suspension. MEFs were plated and allowed to grow to confluency (Passage 0, P0). Cells were dissociated and purified by fluorescence-activated cell sorting (FACS) to remove contaminant hematopoietic cells (CD45+ or MHC-II+) and expanded until passage 3 to 4. If a reporter was used such as the double homozygous Clec9aCre/Cre RosatdTomato/tdTomato, tdTomato+ cells were removed as well. 250,000 MEFs were plated per 100 mm gelatin-coated Petri dish and transduced twice with lentiviral particles encoding PU.1, IRF8 and BATF3 (PIB) reprogramming factors (FUW-TetO-PIB), and M2rtTA (FUW-M2rtTA). Doxycycline (DOX) was added to the culture media (Day 0, d0) to induce expression of PU.1, IRF8 and BATF3 and reprogramming was followed by flow cytometry at reprogramming Day 5 (d5), 7 (d7), 8 (d8) and 9 (d9). Emergent induced dendritic cells (iDCs) may be purified by FACS and characterized by mRNA-sequencing (mRNA-seq), reverse transcriptase quantitative real-time PCR (RT-qPCR), immunofluorescence, scanning electron microscopy (SEM) and functional assays at reprogramming day 9.


  1. Isolation of mouse embryonic fibroblasts
    Note: MEFs can be purchased from vendors such as Sigma and ScienCell. In this case, thaw and expand fibroblasts, purify (Step A26), and use them in reprogramming experiments until passage 3 (P3) or P4. Alternatively, MEFs can be isolated from pregnant mice as described in the protocol below.
    1. Set up timed matings for wild-type (C57BL/6) or double homozygous Clec9aCre/Cre RosatdTomato/tdTomato (Clec9a-tdT) mice (Rosa et al., 2018).
      Note: If using cells isolated from Clec9a-tdT reporter mice, tracking of induction of DC fate during reprogramming will be facilitated by the activation of tdTomato expression.
    2. The following morning, check for vaginal plugs. If these are detected, isolate plugged females in individual cages and consider this time-point as Embryonic day (E) 0.5.
      Note: Plugs can be difficult to detect. As an alternative set up weekly matings keeping two females and one male overnight and removing the male the following day.
    3. Check isolated plugged females at E13.5 to confirm pregnancy before MEF isolation.
    4. Sacrifice pregnant females via CO2 inhalation followed by cervical dislocation.
    5. Disinfect the abdomen of pregnant female with 70% ethanol. Dissect the abdominal cavity with sterile scissors and forceps and expose the abdominal wall by pulling the skin with both hands.
    6. Using new scissors and forceps, cut the peritoneum and expose the uterine horns containing the embryo sacs. Isolate the uterus with scissors and transfer it to a 50 ml centrifuge tube with 30 ml of ice-cold PBS.
      Note: Uterus can be kept on ice for several hours but PBS supplemented with 10% FBS should be used instead.
    7. Inside a cell culture hood, wash uterus twice with sterile chilled PBS and transfer it to a 100 mm dish with 5 ml of sterile chilled PBS.
      Note: Typically, 6-8 embryos are recovered per pregnant female.
    8. With sterile scissors and forceps, make an incision along the uterine horns to isolate the sacs carrying the embryos.
      Note: Don’t perform deep cuts in order to avoid piercing the embryos.
    9. Using forceps, separate embryos from placentas and gently cut-open each individual sac.
    10. Transfer the isolated embryos to a new 100 mm dish containing 5 ml of chilled PBS and leave on ice while completing the remaining dissections.
    11. Using forceps, remove embryo heads. If needed, this tissue can be used for embryo genotyping.
    12. Position the forceps underneath the red-colour visceral organs (fetal liver and heart) and remove them in order to avoid extensive contamination of MEF cultures with hematopoietic cells.
    13. Transfer the dissected embryos to a new 100 mm dish without PBS. Using scissors cut the remaining embryonic tissue into small pieces. Add 1.5 ml of dissociation solution (see Recipes) per embryo, and transfer the tissue to a 50 ml conical tube. Rinse the plate with additional 1.5 ml of dissociation solution per embryo and incubate at 37 °C for 20 min.
    14. Add additional 3 ml of dissociation solution per embryo, pipette up and down to help tissue dissociation and incubate at 37 °C for additional 20 min.
    15. Add 6 ml of DMEM complete medium per embryo to inactivate trypsin and pipette up and down a few times to help with tissue dissociation.
    16. Incubate for 5 min at room temperature (20-25 °C) without agitation to deposit cell debris. Filter the supernatant through a 40 μm sterile cell strainer into a new 50 ml conical tube.
    17. Centrifuge at 350 x g for 5 min, discard supernatant and resuspend the cell pellet in 10 ml of fresh complete medium.
    18. Count cell number and adjust cell concentration to 106 cells per ml of DMEM complete medium.
      Note: On average, 107 cells are obtained per embryo. Alternatively, the cell counting step can be skipped and one embryo equivalent of cells plated per 100 mm dish.
    19. Pre-coat a 100 mm tissue culture dish with 5 ml of 0.1% gelatin solution and incubate for 15 min at 37 °C.
    20. Aspirate gelatin solution, transfer 10 ml of cell suspension (107 cells per dish, or one embryo equivalent) to the pre-coated 100 mm tissue culture dishes and incubate at 37 °C with 5% CO2 for 24 h.
    21. Next day, change culture media to remove floating cells and check cell density under an inverted tissue culture microscope.
      Note: It is expected to have some cell death due to trypsin digestion but the plates should be at least 60-70% confluent. If less cells are recovered, consider reducing the dissociation incubation times to 15 min. At this point, MEFs will have small size and 107 cells can be plated per dish. After passages, MEFs will increase the size and become flatter, and a reduced number of cells should be plated per dish.
    22. When cells become confluent (consider this passage 0 or P0), remove medium, wash once with PBS, and dissociate with 3 ml of TrypLE Express per plate by incubating at 37 °C with 5% CO2 for 5 to 10 min. After cell detaching, add 7 ml of DMEM complete medium and resuspend cells by pipetting up and down a few times. Transfer cell suspension to a 15 ml canonical tube.
      Note: MEFs P0 can be frozen at this time in freezing solution. It is recommended to resuspend 1 confluent plate of MEFs per ml of freezing solution. 1 ml of cell suspension should be transferred to a cryovial and frozen overnight at -80 °C using a Mr. Frosty freezing container. For long-term storage, cryovials should be transferred to -150 °C ultra low freezer or liquid nitrogen.
    23. Count cells using a hemocytometer and centrifuge cells at 350 x g for 5 min.
    24. Discard supernatant, resuspend the cell pellet in 100 μl of staining solution (see Recipes) per 106 cells and incubate for 30 min at 4 °C.
    25. Wash cells in 10 ml of ice-cold wash buffer and centrifuge at 350 x g for 5 min.
    26. Discard the supernatant and resuspend the cell pellet in 300 μl of wash buffer per 106 cells.
    27. Purify CD45-MHC-II- MEFs or, in case of using a reporter such as Clec9a-tdT MEFs, exclude tdTomato+ cells as well, by Fluorescence-activated cell sorting (FACS) to remove rare contaminant hematopoietic cells.
      Note: During FACS purification, we recommend using 100 micron nozzle and collecting purified MEFs in polypropylene round-bottom FACS tubes coated with 500 μl of FBS supplemented with Pen/Strep diluted 1:100. Typically, contaminant hematopoietic cells represent less than 1% of MEFs before purification (Rosa et al., 2018), but can be a confounding population during reprogramming experiments. After FACS sorting the MEF population should be > 99% CD45-MHC-II-.
    28. After FACS purification, transfer purified MEFs to a 50 ml canonical tube with 10 ml of DMEM complete medium and centrifuge at 350 x g for 5 min.
    29. Discard the supernatant and resuspend the cell pellet in fresh complete medium. Count cell number and adjust cell concentration to 106 cells per ml of DMEM complete medium.
    30. Transfer cell suspension to gelatin pre-coated 100 mm tissue culture dishes with 9 ml of DMEM complete medium (106 cells per dish, final volume 10 ml) and incubate at 37 °C with 5% CO2 for 24 h. Consider this as passage 1.
    31. Next day, replace culture media to remove floating dead cells and check cell density.
      Note: Typically, 50-60% cell confluency is achieved at this step.
    32. When confluent, split cells to new gelatin pre-coated 100 mm tissue culture dishes at 1:4 dilution. Consider this Passage 2. For reprogramming experiments use MEFs from passages 3-4 to avoid cellular senescence.
    33. If required, purified MEFs can be frozen from confluent plates. Prepare 1 vial per confluent plate in 1 ml of freezing solution (as described in Step A22).

  2. Lentiviral Production
    Note: Perform lentiviral production in a biosafety level 2 laboratory in a dedicated laminar flow hood and place all viral contaminated waste (tubes, tips, plates) in an appropriate residue container for biohazardous materials.
    1. Expand HEK 293T cells in 150 mm plates with 20 ml of DMEM complete medium. When confluent, remove the medium, wash once with PBS, and dissociate with 6 ml of TrypLE Express by incubating at 37 °C with 5% CO2 for 5 to 10 min.
      Note: HEK 293T cells are semi-adherent so removal of medium and washing with PBS needs to be performed slowly to avoid cell detachment.
    2. Add 6 ml of DMEM complete medium and resuspend cells by pipetting up and down a few times. Transfer cells into 50 ml canonical tubes and centrifuge at 350 x g for 5 min. Remove supernatant and split cells to new 150 mm tissue culture dishes at 1:6 dilution 48 h prior transfection.
    3. In 15 ml tubes, add 10 μg of pFUW-TetO-PIB (inducible lentiviral construct with the tri-cistronic cassette encoding PU.1, IRF8 and BATF3 reprogramming factors) or FUW-M2rtTA, for each 150 mm dish to be transfected. To both tubes add 7.5 μg of packaging psPAX2 and 2.5 μg of envelope pMD2.G plasmids. Adjust the final volume to 2 ml by adding Opti-MEM. Add 60 μl of Polyethylenimine (PEI) transfection reagent (1 mg/ml), vortex thoroughly and incubate transfection mix for 30 min at room temperature to allow the formation of DNA-lipid complexes.
      Note: The lentiviral transfer plasmid pFUW-TetO drives expression of PU.1, IRF8 and BATF3 under the control of a doxycycline-responsive promoter. The FUW-M2rtTA lentiviral plasmid drives expression of the reverse tetracycline transactivator M2rtTA under the control of a constitutively active human ubiquitin C promoter. In this system, when both vectors are co-expressed in the presence of doxycycline, M2rtTA protein bridges doxycycline to the doxycycline-inducible promoter, inducing the expression of PU.1, IRF8 and BATF3. The inducible system is recommended for DC reprogramming experiments because it allows timed induction of transgene expression.
    4. Change HEK 293T cell culture media for 10 ml DMEM without FBS and Pen/Strep. Add transfection mix dropwise to the cells and incubate at 37 °C with 5% CO2 for 6 h.
    5. Replace culture media of transfected HEK 293T cells with 20 ml of DMEM complete medium and incubate cells at 37 °C with 5% CO2 for 24 h.
    6. On the following day, replace HEK 293T cell culture media with 12 ml of fresh DMEM complete medium and incubate cells at 37 °C with 5% CO2.
    7. Harvest viral supernatants 36 h, 48 h and 60 h after transfection into 50 ml polypropylene tubes, store viral supernatants at 4 °C and replace culture media with 12 ml of fresh DMEM complete medium between harvests.
      Note: In total, 36 ml of viral supernatant are harvested per 150 mm plate. Viral particles are not stable at 37 °C but last 1-2 weeks at 4 °C. By harvesting at three time points viral titers are maximized.
    8. Filter harvested supernatants containing viral particles through a 150 ml bottle top 0.45 μm low-protein binding PES filter system.
      Note: Low-protein binding filters should be used to minimize the loss of viral particles by filter adsorption.
    9. Pipette 12 ml of filtered viral supernatant into an Amicon ultra-15 centrifugal filter unit, centrifuge at 2,000 x g at 4 °C for 30 min and discard flow-through liquid.
    10. Add additional 12 ml of filtered viral supernatant to the concentrated viral volume in the filter unit, mix the tube content and centrifuge at 2,000 x g at 4 °C for an additional 30 min. Discard the flow-through.
    11. Add the remaining filtered viral supernatant to the concentrated viral solution left in the filter, mix the contents by inverting the tube and centrifuge once again at 2,000 x g at 4 °C for 20 to 40 min. The final centrifugation time should be adjusted to obtain 600-800 μl of the total volume of concentrated viruses in the filter.
      Note: If the final volume is larger than 800 μl, mix the concentrate and centrifuge again at 2,000 x g at 4 °C for an additional 10 to 20 min.
    12. Prepare aliquots in microtubes on ice (50-200 μl depending on the size of future reprogramming experiments) of each type of concentrated lentiviruses and store at -80 °C for long-term storage (1-2 years) or at 4 °C for short-term storage (1-2 weeks). Concentrated or non-concentrated lentiviruses can also be used fresh.
      Note: Freezing concentrated viruses at -80 °C once leads to a reduction of approximately 10-20% of viral titer. Do not freeze and thaw repeatedly as this results in reduced titer.

  3. Dendritic Cell Reprogramming
    1. Pre-coat 100 mm tissue culture dishes with 5 ml of 0.1% gelatin solution per plate and incubate for at least 15 min at 37 °C with 5% CO2.
    2. Thaw MEF vials at 37 °C. As soon as the cell solution is thawed, add cells dropwise into 10 ml of DMEM complete medium pre-warmed at 37 °C and mix carefully.
    3. Centrifuge at 350 x g for 5 min. Aspirate supernatant and resuspend the cell pellet in 10 ml of DMEM complete medium per vial.
    4. Aspirate gelatin solution of pre-incubated 100 mm plates. Dispense 10 ml of MEF suspension per gelatin pre-coated 100 mm tissue culture dish and incubate overnight at 37 °C with 5% CO2. If required, expand fibroblasts to increase starting cell numbers until passage 3-4 is reached.
      Note: Typically, 2 to 5 x 106 MEFs can be recovered from a confluent 100 mm plate, depending on the passage number.
    5. To start DC reprogramming experiments, plate 250,000 MEFs per gelatin pre-coated 100 mm plate in 10 ml of DMEM complete medium and incubate overnight at 37 °C with 5% CO2.
      Note: Depending on the required number of iDCs or purpose of the experiment, the reprogramming protocol can be scaled up by starting with a large number of 100 mm plates (i.e., 50 to 100 plates to purify iDCs for functional assays) or scaled down to 6-well plates (i.e., to test the influence of different culture conditions in DC reprogramming efficiency), adjusting cell number and viral volume according to the surface area.
    6. On the afternoon of the following day, perform the first lentiviral transduction by replacing medium with 10 ml of DMEM complete medium supplemented with polybrene (8 μg/ml) and 30 to 60 μl of a 1:1 mix of FUW-TetO-PIB and FUW-M2rtTA concentrated lentiviruses. Incubate overnight at 37 °C with 5% CO2.
      Note: For DC reprograming experiments it is recommended to define the optimal volume of lentiviral mix for efficient reprogramming without compromising cell viability (Figure 2). Depending on the aim of the experiment, this may be important to maximize the number of reprogrammed cells obtained in the end of the protocol. MEFs with higher passage number may require higher volumes of viruses.


      Figure 2. Optimization of lentiviral mix volume for efficient dendritic cell direct reprogramming. Clec9a-tdT mouse embryonic fibroblasts (MEFs) were transduced with increasing volumes (30 to 60 μl) of concentrated pooled lentiviral particles encoding PU.1, IRF8 and BATF3 (PIB) transcription factors (FUW-TetO-PIB), together with M2rtTA (FUW-M2rtTA), at 1:1 ratio. Reprogrammed cells were analyzed at day 9 by flow cytometry to define an optimal volume of transduction for dendritic cell reprogramming. The percentage of tdTomato+ cells (A) indicates reprogramming efficiency and the percentage of DAPI-negative cells (B) indicates cell viability. Non-transduced Clec9a-tdT MEFs were used as negative control. In the example, 45 μl of pooled viral particles induces the maximum reprogramming efficiency without compromising cell viability.

    7. After 16 h of incubation, replace media with fresh DMEM complete medium. Incubate for 6-8 h at 37 °C with 5% CO2 to allow cells to recover.
    8. Perform second lentiviral transduction as described in Step C6. Incubate overnight at 37 °C with 5% CO2.
      Note: The transduction mix (DMEM complete with FUW-TetO-PIB, FUW-M2rtTA and polybrene) can be prepared for both transductions and kept at 4 °C.
    9. After 16 h of incubation, replace media with fresh DMEM complete medium supplemented with DOX (1 μg/ml).
      Note: FUW-TetO-PIB and FUW-M2rtTA are a DOX inducible lentiviral system. Thus, adding DMEM complete medium supplemented with DOX will initiate expression of PIB reprogramming factors. Consider this as reprogramming Day 0.
    10. Replace culture media with DMEM complete medium supplemented with DOX every 2-3 days for the duration of the DC reprogramming cultures (9 days).
      Note: It is recommended to follow cell cultures during reprogramming under an inverted microscope. Reprogramming-associated cell death is expected in the first days after DOX supplementation, which depends on MEF passage and viral volume used. Cells will become progressively more confluent at later stages of reprogramming due to the proliferation of non-transduced and non-reprogrammed MEFs. When using Clec9a-tdT MEFs, tdTomato+ cells start being detected approximately 30 h after DOX supplementation (Rosa et al., 2018). iDCs remain adherent throughout the reprogramming process.
    11. The emergence of iDCs during DC reprogramming can be analyzed at different time points. For this purpose, remove the medium, wash once with PBS, and dissociate with 3 ml of TrypLE Express per plate by incubating at 37 °C with 5% CO2 for 5 to 10 min.
    12. After cell detaching, add 7 ml of DMEM complete medium and resuspend cells by pipetting up and down a few times. Transfer cell suspension to a 15 ml canonical tube and centrifuge at 350 x g for 5 min.
    13. Discard the supernatant and resuspend 1 plate equivalent cell pellet in 200 μl of wash buffer for downstream analysis by flow cytometry or FACS sorting for microscopy, mRNA-sequencing or RT-qPCR analysis.

Data analysis

DC reprogramming can be evaluated at different time points between Day 0 and Day 9 (Figure 1). The emergence of CD45+MHC-II+ indicates iDC reprogramming efficiency and can be detected by flow cytometry at reprogramming Day 0, 5, 7, 8 and 9 (Figures 3A-3B). If using a reporter such as the Clec9a-tdT, the emergence of tdTomato+MHC-II+ iDCs can be followed by flow cytometry at the same time-points (Figure 3C). CD45, MHC-II or tdTomato are not detected in transduced MEFs at Day 0 or in M2rtTA-transduced MEFs at Day 9 (Figures 3B-3C). At Day 9, iDCs can be analyzed by microscopy (Figures 3D-3E). For more detailed morphology analysis, tdTomato+ cells can be purified by FACS and analyzed by scanning electron microscopy (SEM) (Figure 3F).


Figure 3. Flow cytometry and microscopy analysis of induced dendritic cells generated by direct reprogramming of mouse embryonic fibroblasts. A. Gating strategy to analyze the emergence of induced dendritic cells (iDCs) by flow cytometry during reprogramming. Population was selected with FSC and SSC, followed by exclusion of doublets and dead cells (DAPI+). B. The emergence of CD45+MHC-II+ iDCs was quantified by flow cytometry 5 days (d5), 7 days (d7), 8 days (d8) and 9 days (d9) after induction of PU.1, IRF8 and BATF3 (PIB) in wild type (C57BL/6) mouse embryonic fibroblasts (MEFs). Transduced MEFs at Day 0 and M2rtTA-transduced MEFs at Day 9 were included as controls. CD45+MHC-II+ gating strategy was performed based on single-cell stainings for CD45 and MHC-II 9 days after induction of PIB in wild type MEFs. C. Flow cytometry analysis of tdTomato+MHC-II+ cells at d5, d7, d8 and d9 after induction of M2rtTA and PIB in Clec9a-tdT MEFs. Transduced MEFs at Day 0 and M2rtTA-transduced MEFs at Day 9 were included as controls. D. Immunofluorescence for tdTomato and Hoechst (blue) highlighting the emergence and frequency of tdTomato+ cells. M2rtTA-transduced MEFs at Day 9 were included as control. Scale bar = 500 μm. E. Bright-field micrographs of C57BL/6 MEFs 9 days after induction of M2rtTA or M2rtTA and PIB. Scale bars = 100 μm. F. Scanning electron microscopy analysis of a tdTomato+ cell 9 days after induction of M2rtTA and PIB in Clec9a-tdT MEFs. M2rtTA-transduced MEFs at Day 9 were included as control. Scale bars = 10 μm.

Additionally, mRNA-sequencing of purified tdTomato+CD45+MHC-II+ iDCs at reprogramming Day 9 (iDCs Day 9) and splenic MHC-II+CD11c+CD8a+ cDC1s can be performed to further validate successful reprogramming (Rosa et al., 2018). High expression levels of genes encoding the reprogramming factors (Spi1 [encoding PU.1], Irf8 and Batf3) are detected in iDCs Day 9 and cDC1s (Figure 4A). The fibroblast-specific genes Actg2 and Col9a2 are down regulated in iDCs Day 9 and not expressed in cDC1s (Figure 4B). Expression of the DC genes Xcr1, Clec9a, Cd207, Naaa, Ucp2, Ifi205, Cd74, Ciita, H2-aa and H2-dmb1 is not detected in MEFs, is activated in iDCs Day 9 and expressed in splenic cDC1s (Figure 4C). This set of markers can be used to assess successful reprogramming either by mRNA-sequencing or RT-qPCR. Further functional evaluation of iDCs can be performed after FACS purification including: secretion of inflammatory cytokines, dead cell engulfment, antigen export to cytosol and antigen cross-presentation to OT-I CD8+ T cells (Rosa et al., 2018).


Figure 4. Gene signature to assess successful dendritic cell reprogramming. A. Genes encoding the reprogramming factors PU.1, IRF8 and BATF3 (PIB) are expressed in induced dendritic cells (iDCs) at reprogramming Day 9 (tdTomato+CD45+MHC-II+) and in conventional dendritic cells type 1 (cDC1s, MHC-II+CD11c+CD8a+). B. Fibroblast-associated genes Actg2 and Col9a2 are down regulated in iDCs at reprogramming Day 9 and are not expressed in cDC1s. C. Dendritic cell genes Xcr1, Clec9a, Cd207, Naaa, Ucp2, Ifi205, CD74, Ciita, H2-aa and H2-dmb1 are expressed in iDCs at reprogramming Day 9 and in cDC1s. After induction of PIB in mouse embryonic fibroblasts (MEF), iDCs (tdTomato+CD45+MHC-II+) and splenic cDC1s (CD11c+MHC-II+CD8α+) were FACS sorted and expression of genes quantified by mRNA-sequencing (Rosa et al., 2018; GSE103618). Gene expression is displayed in bar plots as mean of gene counts ± standard deviation. As an alternative, RT-qPCR can be used to quantify the expression of this gene set and assess successful reprogramming.

Recipes

  1. DMEM Complete medium
    DMEM supplemented with 10% (v/v) heat-inactivated FBS and 1:100 dilution of 10,000 U/ml Penicillin, 10,000 μg/ml Streptomycin stock solution
  2. Dissociation solution
    Dilute 0.25% trypsin-EDTA solution in the same volume of PBS (dilution 1:2) for a final concentration of 0.125% trypsin-EDTA
  3. 0.1% Gelatin solution
    Dissolve 0.1 g of gelatin in 100 ml of MilliQ-grade water and autoclave
  4. Wash solution
    PBS supplemented with 2.5% FBS
  5. Staining solution
    Wash solution supplemented with rat serum (1/100), anti-mouse CD45 monoclonal antibody (0.25 µg per 100 μl) and anti-mouse MHC-II monoclonal antibody (0.25 µg per 100 μl)
  6. Freezing solution
    Heat-inactivated FBS supplemented with 10% DMSO

Acknowledgments

This project was co-funded by Cancerfonden (CAN 2017/745), the Swedish research council (2018-02042), Crafoord Foundation (20190561), NovoNordisk Fonden (0056527) and FCT (CENTRO-01-0145-FEDER-030013). The Knut and Alice Wallenberg foundation, the Medical Faculty at Lund University and Region Skåne are acknowledged for generous financial support. F.F.R and C.F.P. and F.F.R. are supported by FCT doctoral (SFRH/BD/130845/2017) and postdoctoral (SFRH/BPD/121445/2016) fellowships, respectively. This protocol was adapted from Rosa et al., 2018.

Competing interests

Fábio F. Rosa, Cristiana F. Pires and Carlos-Filipe Pereira have filed a Patent Cooperation Treaty (PCT) protecting the intellectual property described here on 2018-04-05.

Ethics

This protocol was performed according to Lund University's research ethics committee guidelines and should be done in accordance with individual institutional guidelines. Procedures involving animal experimentation were approved by the Swedish Ethical Review board (5.8.18-19343/2017).

References

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简介

[摘要] 转录因子组合的异位表达最近被证明可以将分化的体细胞重编程为树突状细胞(DC)谱系,而不会回复到多能状态。DC具有诱导有效和持久的适应性免疫应答的能力。在特定的常规类型1的DC(cDC1s)练成抗原交叉呈递,用于诱导CD8的关键步骤+ T细胞的细胞毒性应答。天然存在的cDC1的稀有性和缺乏用于生成纯cDC1群体的体外方法论,严重阻碍了对cDC1谱系规格和功能的研究。在这里,我们描述了用于生成感应DC(iDC)的协议 慢病毒介导的转录因子PU.1,IRF8和BATF3在小鼠胚胎成纤维细胞中的表达。iDC 在9天内获得DC形态,cDC1表型和转录特征。使用此协议生成的iDC 具有对炎症刺激,吞噬死细胞,将抗原加工并交叉呈递给CD8 + T细胞的功能。DC重新编程提供了一个简单易处理的系统,可以生成大量的cDC1类细胞用于高内涵筛选,从而开辟了新途径,可以更好地了解cDC1的规格和功能。将来,在成纤维细胞中忠实诱导cDC1命运可能会导致产生患者特定的疫苗接种DC。

[背景技术树突状细胞(DC)是专业的抗原呈递细胞,专门用于识别,加工和呈递T细胞抗原,在诱导适应性免疫应答和免疫记忆中起关键作用(Me rad 等,2013)。DC可以分为4个主要子集:浆细胞样DC(pDC ),大量1型干扰素的产生者,循环单核细胞衍生的单核细胞衍生DC 和常规DC(cDC ),即功能上异质的子集,可以进一步细分为常规DC类型1(cDC1)和类型2(cDC2)。尽管cDC1s 通过交叉呈递主要组织相容性复合体(MHC)I类上的外源抗原而擅长引发细胞毒性CD8 + T细胞反应,但cDC2s专门通过MHC II类将细胞外抗原呈递给CD4 + T细胞。此功能专业化是由指定DC子集的不同转录因子(TF)之间的特定阶段相互作用所导致的。例如,它已经表明,转化特异性E26(ETS)家族TF的条件删除在造血PU.1 祖细胞的影响š 整个DC谱系的说明书(Carotta 等人,2010)。干扰素调节因子8(IRF8)剔除小鼠缺乏pDC 和cDC1亚型,碱性亮氨酸拉链ATF样转录因子3(BATF3)缺陷型小鼠的cDC1指标受损(Schiavoni 等,2002;Hildner 等,2008)。 ;Carotta 等,2010)。

越来越多的证据支持cDC1在癌症背景下引发免疫反应中的关键作用(Bottcher 和Reis,2018),并且它们的体内稀有性推动了体外生成cDC1的尝试(Perez和De Palma,2019; Wculek 等人。,2019)。已经探索了几种在存在不同造血细胞因子的情况下从血液单核细胞或骨髓(BM)前体产生真正DC的策略(Inaba 等,1992; Lu 等,1995; Lutz 等,1999; Son 等人,2002 ; Naik 等人,2005 ; Poulin 等人,2010 ; Proietto 等人,2012 ; Balan 等人,2014 ; Mayer 等人,2014 ; Lee 等人,2015 ; Balan 等人等人,2018 ; Kirkling 等人,2018 )。然而,有限数量的BM祖细胞以及具有冲突功能的DC亚群的混合种群的产生,强烈地限制了这些方法的使用,以阐明DC的发展和利用cDC1进行治疗(Yona 和Mildner ,2018)。

单元重编程为克服这些挑战提供了令人兴奋的机会。通过强制表达TF,可以将体细胞重编程为诱导性多能干细胞(iPSC )(Takahashi and Yamanaka,2006; Takahashi et al。,2007)。已经描述了区分DC与iPSC的方案(Choi 等,2009; Senju 等,2009和2011; Horton 等,2019)。但是,这些依赖于复杂且长期的培养条件,这会导致不同DC子集的混合种群。或者,可以将体细胞直接重编程为其他专门的细胞类型,而无需通过多能性或多能性转移。使用指定靶细胞身份的TF将小鼠和人的成纤维细胞直接重编程为几种细胞类型,例如神经元,心肌细胞以及造血干细胞和祖细胞(Pereira 等人,2012和2013; Xu 等人,2015)。

在这里,我们描述了通过在小鼠胚胎成纤维细胞(MEF)中强制表达TFs PU.1,IRF8和BATF3 来生成cDC1样细胞(诱导的DC或iDC )的直接细胞重编程方法(Rosa 等人,2018) 。MEF衍生的iDC 具有天然cDC1的DC形态,cDC1表型和转录程序特征。在功能上,iDC 具有对Toll样受体3(TLR3)和TLR4刺激,吞噬蛋白和死细胞做出反应的能力,重要的是,抗原可以交叉呈递给CD8 + T细胞。因此,此处描述的协议能够使用受控且可调节的直接重编程方法在体外快速生成cDC1样细胞(9天之内),该方法适用于筛选研究,目的是更好地了解cDC1的开发规格和功能成熟度。这种方法,加上药理学抑制,基因敲除- 出或敲- 向下,将使单独或组合作用DC规格的调节剂的发现(皮雷斯等人,2019)。采用高- 内容根据近期MEF中CRISPR-Cas9技术筛选方法铺平了道路,定义新的关键REGUL方式CDC1命运,以及如何建立交叉呈递能力的ators 从头在多种细胞类型。将来,DC重编程将用作开发针对癌症和传染病的免疫疗法的平台。

关键字:细胞重编程, 转录因子, 细胞命运转换, 树突状细胞, 抗原呈递细胞, 免疫, 小鼠胚胎成纤维细胞, 免疫疗法

材料和试剂


 


100 mm组织培养板(Corning,目录号:430167 )
150 mm组织培养板(Corning,目录号:430599 )
1.5 ml微管(Sarstedt,目录号:72.690.001 )
15 ml离心管(Sarstedt ,目录号:62.547.205 )
50 ml离心管(Sarstedt ,目录号:62.547.205 )
聚丙烯圆底FACS管(Falcon,目录号:352063 )
150毫升瓶顶部真空过滤器,0.45 微米的低蛋白结合(康宁,目录号:430625 )
Amicon ultra-15离心过滤器单元(Merck Millipore,目录号:UFC903096 )
野生型小鼠(C57BL / 6)或双重纯合的Clec9a Cre / Cre Rosa tdTomato / tdTomato (Clec9a-tdT)小鼠(Rosa 等,2018)
人胚肾(HEK)293T细胞系(ATCC,目录号:CRL-3216 )
40 µm无菌细胞过滤器(Corning,目录号:431750 )
猪皮肤A型明胶(Sigma-Aldrich,目录号:G1890 )
聚乙烯绝缘体升亚胺(PEI)转染试剂(Sigma-Aldrich公司,目录号:408727 )
海美溴铵(聚凝胺)(Sigma-Aldrich公司,目录号:H9268 )
盐酸强力霉素(DOX)(Sigma-Aldrich,目录号:D9891 )
胰蛋白酶-EDTA(0.25%),酚红(Thermo Fisher Scientific,目录号:25200-056 )
TrypLE Express(1x)无酚红(Thermo Fisher Scientific,目录号:12604-021 )
HyClone 磷酸盐缓冲盐溶液(PBS)(GE Healthcare,目录号:SH30256.01 )
HyClone 高葡萄糖Dulbecco改良的Eagles培养基(DMEM)(GE Healthcare,目录号:SH30243.01 )
Opti -MEM还原血清培养基(Thermo Fisher Scientific,目录号:31985070 )
HyClone公司万U /米升青霉素万微克/米升链霉素溶液(笔/ 链霉素)(GE Healthcare公司,目录号:SV30010 )
HyClone 胎牛血清(FBS)(GE Healthcare,目录号:SV30160.03 )
大鼠血清(GeneTex ,目录号:GTX73226 )
二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418 )
质粒pMD2.G(Addgene ,目录号:12259 )
质粒psPAX2(Addgene ,目录号:12260 )
质粒FUW-M2rtTA(Addgene ,目录号:20342 )
包含编码PU.1,IRF8和BATF3(PIB)重编程因子的三顺反子盒的质粒pFUW - TetO -PIB (Rosa 等,2018)。瑞典隆德大学分子医学与基因治疗学系造血与免疫实验室的细胞重编程。
4',6-二mid基-2-苯基吲哚(DAPI)(Sigma-Aldrich,目录号:D9542 )
Hoechst染色液(Sigma-Aldrich,目录号:H6024 )
APC共轭抗小鼠CD45单克隆抗体(Biolegend ,目录号:103112 )
PE-Cy7共轭抗小鼠MHC-II单克隆抗体(Biolegend ,目录号:107630 )
DMEM完全培养基(请参阅食谱)
解离解决方案(请参阅食谱)
0.1%明胶溶液(请参阅食谱)
洗涤液(请参阅食谱)
染色溶液(请参阅食谱)
冻结解决方案(请参阅食谱)
 


设备


 


无菌解剖剪刀(Sigma-Aldrich,目录号:S3146 )
无菌不锈钢钳(Sigma-Aldrich,目录号:Z168777 )
冷冻容器Nalgene Frosty先生(Sigma-Aldrich,目录号:C1562 )
血细胞计数器细胞计数室(Marienfeld Superior,目录号:13444890 )
4-16K冷冻离心机(Sigma )
FACSCanto II流式细胞仪分析仪(8色,蓝色/红色/紫色)(BD Biosciences )
BD FACSAria III细胞分选仪(BD Biosciences )
Heracell 150i CO 2 培养箱(Thermo Scientific )
ScanLaf Mars 2级层流罩(LaboGene )
IX70倒置组织培养显微镜(奥林巴斯)
Celldiscoverer 7倒置显微镜(Zeiss ) 
 


软件


 


1. GraphPad PRISM 6,https://www.graphpad.com/                                                                                                                                                                                                               


2. FlowJo V10,https: //www.flowjo.com/      


3. ZEN 3.1(蓝色版本),https://www.zeiss.com/      


4. Adobe Photoshop CS6,https: //www.adobe.com/cn      


 


程序


 


本文所述的直接重编程协议的示意图可在图1中找到。


 






图1.将小鼠胚胎成纤维细胞直接重编程为树突状细胞的方案示意图。从C57BL / 6野生型13.5天大的胚胎中分离出小鼠胚胎成纤维细胞(MEF)。胚胎解剖,以除去所述头部(灰色),肝脏和心脏(红色)和离解为单个- 细胞悬浮液。将MEF铺板并使其生长至汇合(第0代,P0)。将细胞解离并通过荧光激活细胞分选术(FACS)进行纯化,以去除造血细胞(CD45 + 或MHC-II + ),并扩增至3到4代。如果使用了报道基因,如双重纯合子Clec9a Cre / Cre Rosa tdTomato / tdTomato 和tdTomato + 细胞也被移除。250000分的MEF每100mm明胶包被铺板的Petri 培养皿,并用慢病毒颗粒的编码转导的PU.1两次,IRF8和BATF3(PIB)重编程因子(FUW- TETO -PIB),和M2rtTA(FUW-M2rtTA)。将强力霉素(DOX)添加到培养基中(第0 天,d0)以诱导PU.1,IRF8和BATF3的表达,并在重编程第5 天(d5),第7天(d7),第8天(d8)进行流式细胞术重编程)和9(d9)。射诱导的树突状细胞(iDC中)可以通过FACS进行纯化,并通过mRNA的测序(mRNA- SEQ ),逆转录酶定量实时- 时间PCR(RT-qPCR的),免疫荧光,扫描电子显微镜(SEM)和在重编程功能测定第9天


 


小鼠胚胎成纤维细胞的分离
注意:MEF可以从Sigma和ScienCell 等供应商处购买。在这种情况下,解冻并扩增成纤维细胞,纯化(步骤A26),并将其用于重编程实验中,直到第3代(P3)或P4。备选地,可以如以下方案中所述从怀孕小鼠中分离MEF 。


为野生型(C57BL / 6)或双重纯合子Clec9a Cre / Cre Rosa tdTomato / tdTomato (Clec9a-tdT)小鼠设置定时交配(Rosa 等,2018)。
注意:如果使用从Clec9a-tdT报告基因小鼠中分离的细胞,则tdTomato 表达的激活将有助于在重编程过程中追踪DC命运。


在第二天早晨,检查阴道塞。如果检测到这些,将隔离的雌性个体隔离在单独的笼子中,并将该时间点视为胚胎日(E)0.5。
注意:插头可能很难检测到。作为替代方案,每周交配一次,让两名雌性和一名雄性过夜,然后第二天将其移出。


在隔离MEF之前,请在E13.5处检查隔离的雌性插头,以确认怀孕。
通过吸入CO 2 牺牲孕妇,然后进行颈脱位。
用70%的乙醇消毒孕妇的腹部。用无菌剪刀和镊子解剖腹腔,用双手拉皮肤揭露腹壁。
用新的剪刀和镊子切开腹膜,露出包含胚囊的子宫角。隔离用剪刀子宫并将其转移到用30毫升冰的50毫升离心管中- Ç 老PBS。
注意:子宫可在冰上放置数小时,但应使用补充有10%FBS的PBS。


在细胞培养罩内,用无菌冰冷的PBS清洗子宫两次,然后将其转移至装有5 ml无菌冰冷的PBS的100 mm皿中。
注意:通常,每位怀孕的女性可回收6-8个胚胎。


用无菌剪刀和镊子在子宫角上切开一个切口,以隔离携带胚胎的囊。
注意:请勿进行深切,以免刺破胚胎。


使用镊子,从胎盘中分离出胚胎,并轻轻切开每个单独的囊。
将分离的胚胎转移到一个新的100 mm培养皿中,该培养皿中含有5 ml的冷PBS,并在完成剩余解剖的同时置于冰上。
用镊子取出胚胎头。如果需要,该组织可用于胚胎基因分型。
定位红-下方钳子颜色内脏器官(胎肝和心脏)和为了避免MEF培养物与造血细胞的广泛污染除去它们。
将解剖的胚胎转移到没有PBS的新的100毫米培养皿中。用剪刀将剩余的胚胎组织切成小块。每个胚胎添加1.5 ml解离溶液(请参阅“ 食谱” ),并将组织转移至50 ml锥形管中。每个胚胎用另外的1.5 ml解离溶液冲洗平板,并在37孵育  °C 20分钟。
每个胚胎添加额外的3 ml解离溶液,上下吸管以帮助组织解离,并在37 °C 下再孵育20分钟。
每个胚胎添加6 ml DMEM完全培养基以使胰蛋白酶失活,并上下吸移几次以帮助组织解离。
孵育在室温下(20 5分钟- 25 ℃下)不搅拌以存款细胞碎片。通过40μm 无菌细胞过滤器将上清液过滤到新的50 ml锥形管中。
以350 x g 离心5分钟,弃去上清液,将细胞沉淀重悬于10 ml新鲜完全培养基中。
计数细胞数并将细胞浓度调整为每毫升DMEM完全培养基10 6个细胞。
注意:每个胚胎平均可获得10 7个细胞。或者,可以跳过细胞计数步骤,每100毫米培养皿接种一个当量的胚胎细胞。


用5 ml的0.1%明胶溶液预涂100 mm的组织培养皿,并在37 °C 下孵育15分钟。   
吸出明胶溶液,将10 ml细胞悬液(每培养皿10 7个细胞,或一个胚胎当量)转移至预涂的100 mm组织培养皿中,并在37 °C 和5%CO 2 下孵育24小时。
第二天,更换培养基以除去漂浮细胞,并在倒置的组织培养显微镜下检查细胞密度。
注意:由于胰蛋白酶的消化,预计会导致一些细胞死亡,但平板应至少融合60-70 %。如果回收的细胞较少,请考虑将解离孵育时间缩短至15分钟。此时,MEF的尺寸很小,每个培养皿可铺10 7个细胞。后的通道,将MEF将增加的大小和变得平坦,并应每皿被镀覆的细胞的数量减少。


当细胞汇合时(考虑该传代0或P0),除去培养基,用PBS洗涤一次,并通过在37 °C 和5%CO 2中孵育5至10分钟,与每板3 ml TrypLE Express 分离。细胞分离后,添加7 ml DMEM完全培养基,并通过上下移液几次重悬细胞。将细胞悬浮液转移到15 ml的标准管中。
注意:此时可以在冷冻溶液中冷冻MEF P0。据推荐,以悬浮每毫升冷冻溶液的MEF中的1个汇合的平板。1ml细胞悬浮液应转移到一个离心管在-80和冷冻过夜℃下使用雾先生冷冻容器。对于长期储存,冷冻管应转移至-150 ℃的超低冰箱或液氮。


使用血细胞计数器计数细胞并以350 x g 离心5分钟。
弃去上清液,将细胞沉淀重悬于每10 6个细胞100μl 的染色溶液中(请参见食谱),并在4 °C 下孵育30分钟。
在10 ml冰冷的洗涤缓冲液中洗涤细胞,并以350 x g 离心5分钟。
丢弃的上清液和重悬的细胞沉淀在300 微升每10个洗涤缓冲液6 细胞。
纯化CD45 - MHC-II - MEF,或者在使用报告子(例如Clec9a-tdT MEF)的情况下,也通过荧光激活细胞分选(FACS)去除罕见的造血细胞,排除tdTomato + 细胞。
注意:在FACS纯化,我们建议使用100微米喷嘴和在涂布有500聚丙烯圆底FACS管收集纯化的MEF 微升FBS的补充有笔/ 链霉素稀释1:100。通常情况下,污染的造血细胞在纯化前仅占不到1%的MEF(Rosa等人,2018),但在重编程实验期间可能是混杂的种群。FACS分选后的MEF人口应> 99%CD45 - MHC-II - 。


FACS纯化后,将纯化的MEF转移至含有10 ml DMEM完全培养基的50 ml标准管中,并以350 x g 离心5分钟。
丢弃的上清液和重悬的细胞在新鲜的完全培养基沉淀。计数细胞数并将细胞浓度调整为每毫升DMEM完全培养基10 6个细胞。
将细胞悬浮液转移至装有9 ml DMEM完全培养基(每个培养皿10 6个细胞,最终体积10 ml)的明胶预包被的100 mm组织培养皿中,并在5%CO 2 于37 °C 孵育24小时。将此视为第一段。
第二天,更换培养基以去除漂浮的死细胞并检查细胞密度。
注意:通常,此步骤可达到50-60%的细胞融合度。


汇合后,将细胞以1:4的比例稀释至新的明胶预涂100毫米组织培养皿中。考虑该通道2。对于重新编程实验,请使用3-4通道中的MEF,以避免细胞衰老。
如果需要,可以从融合板上冷冻纯化的MEF。每个融合板在1 ml冷冻溶液中准备1小瓶(如步骤A22中所述)。
 


慢病毒生产
注意:在专门的层流通风橱中在生物安全性2级实验室中进行慢病毒生产,并将所有被病毒污染的废物(管子,尖端,板)放在适当的生物危害材料残留容器中。


用20 ml DMEM完全培养基在150 mm平板中扩增HEK 293T细胞。当汇合时,除去该介质,用PBS,并用6ml的解离洗一次的TrypLE 快车通过在37温育℃下用5%CO 2 为5至10分钟。
注意:HEK 293T细胞为半贴壁细胞,因此需要缓慢除去培养基并用PBS洗涤,以避免细胞脱离。


加入6 ml DMEM完全培养基,并通过上下移液几次重悬细胞。将细胞转移到50 ml标准管中,并以350 x g 离心5分钟。转染前48小时,以1:6的稀释度去除上清液并将细胞分裂至新的150 mm组织培养皿中。
在15ml管中,加入10 微克的pFUW - TETO -PIB(可诱导慢病毒构建与三顺反子盒编码PU.1,IRF8和BATF3重编程因子)或FUW-M2rtTA,对于被转染以150毫米的菜。到两个管添加7.5 微克包装psPAX2和2.5 微克信封pMD2.G质粒。通过添加Opti -MEM 将最终体积调节至2 ml 。加入60 微升的聚乙烯绝缘体升亚胺(PEI)转染试剂(1毫克/毫升),涡旋并彻底在室温下30分钟温育转染混合物,以允许所述DNA-脂质复合物的形成。
注意:慢病毒转移质粒pFUW- TetO在强力霉素反应性启动子的控制下驱动PU.1,IRF8和BATF3的表达。FUW-M2rtTA慢病毒质粒在组成型活性人泛素C启动子的控制下驱动逆四环素反式激活因子M2rtTA的表达。在该系统中,当两种载体在强力霉素存在下共表达时,M2rtTA蛋白将强力霉素桥接至强力霉素诱导型启动子,从而诱导PU.1,IRF8和BATF3的表达。推荐将诱导型系统用于DC重编程实验,因为它可以定时诱导转基因表达。


将不含FBS和Pen / Strep的10 ml DMEM更换为HEK 293T细胞培养基。将转染混合物滴加到细胞中,并在37 °C 和5%CO 2中孵育6小时。
用20 ml DMEM完全培养基替换转染的HEK 293T细胞的培养基,并将细胞在37 °C 和5%CO 2 下孵育24小时。
第二天,用12 ml新鲜的DMEM完全培养基替换HEK 293T细胞培养基,并在5%CO 2 下于37 °C 孵育细胞。
转染到50 ml 聚丙烯管中后36 h,48 h和60 h收获病毒上清液,将病毒上清液保存在4 °C,并在收获之间用12 ml新鲜DMEM完全培养基代替培养基。
注意:每150毫米平板总共可收获36毫升病毒上清液。病毒颗粒在37 °C下不稳定,但在4 °C下可持续1-2周。通过在三个时间点收获,病毒滴度达到最大。


通过含有病毒颗粒过滤器收获上清液150毫升瓶顶0.45 微米的低蛋白结合过滤器PES系统。
注:低蛋白结合过滤器应被用于最小化的病毒颗粒通过过滤器损失吸附。


吸取12 ml过滤的病毒上清液到Amicon ultra-15离心过滤器中,在4 °C下以2,000 x g 离心30分钟,并丢弃流通液。
添加额外12毫升过滤病毒上清液,以在过滤器单元中的浓缩的病毒量,混合管的内容和离心机中以2000 X 克在4 ℃下进行的30分钟。丢弃流通物。
将剩余的过滤后的病毒上清液添加到留在过滤器中的浓缩病毒溶液中,通过颠倒试管来混合内容物,并在4 x 2,000 x g 下再次离心20至40分钟。最后一次离心时间应被调整以获得600-800 微升的所述的在过滤浓缩的病毒总体积。
注意:如果最终体积大于800 微升,在2再次混合所述浓缩物和离心机,在4 000×g离心℃下进行一个附加的10至20分钟。


在制备等分试样的微管在冰(50-200 微升取决于未来重新编程实验大小)每种类型的浓缩慢病毒和商店中的在-80 ℃下长期储存(1-2岁)或在4 ℃下进行短期储存(1-2周)。浓缩或非浓缩慢病毒也可以新鲜使用。
注意:在-80 °C 冷冻浓缩的病毒一次可导致病毒滴度降低约10-20%。不要反复冷冻和融化,因为这样会降低滴度。


 


树突状细胞重编程
每块板用5 ml 0.1%明胶溶液预涂100 mm组织培养皿,并在5%CO 2 下于37 °C 孵育至少15分钟。
在37 °C下解冻MEF小瓶。细胞溶液解冻后,将细胞滴加到10 ml在37 °C 预热的DMEM完全培养基中,小心混合。
以350 xg离心5分钟。吸去上清液和重悬的细胞沉淀在10ml每小瓶DMEM完全培养基中。
吸取预先孵育的100 mm平板的明胶溶液。每个预先涂明胶的100 mm组织培养皿分配10 ml MEF悬浮液,并在5%CO 2 下于37°C 孵育过夜。如果需要,扩增成纤维细胞以增加起始细胞数,直到达到传代3-4。
注意:通常,可以从汇合的100 mm平板上回收2至5 x 10 6个MEF,具体取决于通过次数。


要开始DC重新编程实验,在10 ml DMEM完全培养基中,将每个明胶预涂100 mm平板接种250,000个MEF,并在5%CO 2 下于37°C 孵育过夜。
注意:根据所需的iDC 数量或实验目的,可通过从大量100 mm平板开始(例如,纯化50至100个平板以纯化iDC 以进行功能测定)来扩大重编程方案,或缩小至6孔板(即,测试的不同培养条件在DC重编程效率的影响),调整细胞数和根据病毒体积的表面积。


在接下来的一天的下午,通过用10ml DMEM完全培养基中补充有更换介质执行第一慢病毒转导的聚凝胺(8 微克/毫升)和30至60 微升FUW-的1混合:1的TETO -PIB和FUW-M2rtTA浓缩慢病毒。在5%CO 2 和37°C下孵育过夜。
注意:对于DC重编程实验,建议定义慢病毒混合物的最佳体积,以进行有效的重编程而不损害细胞活力(图2 )。取决于实验的目的,这对于最大化协议末尾获得的重编程细胞的数量可能很重要。具有较高通过次数的MEF可能需要更多数量的病毒。


 






图2.优化慢病毒混合物体积以有效地进行树突状细胞直接重编程。CLEC9A-TDT 小鼠胚胎成纤维细胞(MEF中)与转分别增加体积(30〜60 微升编码PU.1,IRF8和BATF3(PIB)转录因子(FUW-浓缩合并的慢病毒颗粒的)TETO 与M2rtTA -PIB),一起( FUW-M2rtTA),比例为1:1。重编程的细胞以进行分析d AY 通过流式细胞术9以限定转导树突细胞重编程的最佳的体积。tdTomato + 细胞的百分比(A)表示重新编程效率,DAPI阴性细胞的百分比(B)表示细胞活力。未转导的Clec9a-tdT MEF用作阴性对照。在该例子中,45 微升合并病毒颗粒的诱导而不会损害细胞活力的最大重编程效率。


 


孵育16小时后,用新鲜的DMEM完全培养基替换培养基。在5%CO 2 下于37°C孵育6-8小时,以使细胞恢复。
如步骤C6 所述进行第二次慢病毒转导。在5%CO 2 和37°C下孵育过夜。
注意:转导混合物(DMEM完全与FUW- TETO -PIB,FUW-M2rtTA和聚凝胺)可以用于转导二者来制备,并保持在4℃。


孵育16小时后,用新鲜的DMEM完全培养基补充DOX(1μg / ml)替换培养基。
注意:FUW - TetO -PIB和FUW-M2rtTA是DOX诱导的慢病毒系统。因此,添加补充有DOX的DMEM完全培养基将启动PIB重编程因子的表达。将其视为第0 天的重新编程。


在DC重编程培养期间(9天),每2-3天用补充有DOX的DMEM完全培养基替换培养基。
注意:建议在倒置显微镜下进行重编程期间遵循细胞培养。重新编程相关的细胞死亡,预计在DOX补充后的第一天,这取决于小号在MEF通道,并用于病毒体积。由于未转导的和未重编程的MEF 的增殖,细胞在重编程的后期将逐渐变得更加融合。当使用Clec9a-tdT MEF时,在添加DOX约30小时后开始检测到tdTomato +细胞(Rosa等人,2018)。在重新编程的整个过程中,iDC 始终保持不变。


所述Ë 的合并的iDC DC重新编程期间可在不同的时间点进行分析。为了这个目的,除去了介质,用PBS洗一次,并解离用3ml 的TrypLE 每板快速通过在37温育C° 与5%CO 2 为5至10分钟。
细胞分离后,添加7 ml DMEM完全培养基,并通过上下移液几次重悬细胞。将细胞悬浮液转移到15 ml的标准管中,并以350 x g的速度离心5分钟。
弃去上清液,并在200μl 洗涤缓冲液中重悬1板当量细胞沉淀,以通过流式细胞仪或FACS进行下游分析,以便进行显微镜检查,mRNA测序或RT-qPCR分析。
 


数据分析


 


可以在第0 天和第9 天之间的不同时间点评估DC重新编程(图1)。所述Ë CD45的合并+ MHC-II + 表示的iDC 重编程效率,并且可以通过流式细胞术在重新编程来检测日0,5,7,8和9(图3A-3B)。如果使用记者如CLEC9A-TDT,所述的出现tdTomato + MHC-II + 的iDC 可以在同一时间点(图3C)之后通过流式细胞术。在第0 天未在转导的MEF中或在第9 天在M2rtTA转导的MEF中检测到CD45,MHC-II或tdTomato (图3B-3C)。在日9,将iDC 可以通过显微术(图进行分析小号3D-3E )。对于更详细的形态分析,可以通过FACS纯化tdTomato + 细胞,并通过扫描电子显微镜(SEM)进行分析(图3F)。


 






图3. 通过直接重编程小鼠胚胎成纤维细胞产生的诱导树突状细胞的流式细胞仪和显微镜分析。A.门控策略进行分析的诱导的树突状细胞(出苗的iDC 重新编程期间)通过流式细胞术。使用FSC和SSC选择种群,然后排除双峰和死细胞(DAPI + )。B. 该Ë CD45的合并+ MHC-II + 的iDC 用流式细胞仪5天(D5),7天(D7),8定量天(D8)和9天(D9)PU.1,IRF8和诱导后野生型(C57BL / 6)小鼠胚胎成纤维细胞(MEF)中的BATF3(PIB)。包括在第0天的转导MEF和在第9 天的M2rtTA转导的MEF 作为对照。CD45 + MHC-II + 的门控策略是基于单执行- 细胞染色为CD45和MHC-II在野生型MEF中PIB的诱导后9天。C. 在Clec9a-tdT MEF中诱导M2rtTA和PIB后,在d5,d7,d8和d9 时tdTomato + MHC-II + 细胞的流式细胞术分析。包括在第0天的转导MEF和在第9 天的M2rtTA转导的MEF 作为对照。D. tdTomato 和Hoechst的免疫荧光(蓝色)突出了tdTomato + 细胞的出现和频率。在第9 天将M2rtTA转导的MEF 包括在内作为对照。比例尺= 500 微米。E.诱导M2rtTA或M2rtTA和PIB后9天的C57BL / 6MEF的明场显微照片。比例尺= 100μm 。F. 在Clec9a-tdT MEF中引入M2rtTA和PIB 后9天,对tdTomato + 细胞进行扫描电子显微镜分析。在第9 天将M2rtTA转导的MEF 包括在内作为对照。比例尺= 10μm 。


 


  此外,可以在重编程第9 天(iDC 第9 天)和脾MHC-II + CD11c + CD8a + cDC1s进行纯化的tdTomato + CD45 + MHC-II + iDC的mRNA测序,以进一步验证成功的重编程(Rosa 等, 2018)。在iDC 第9天和cDC1s 中检测到编码重编程因子的基因(Spi1 [ 编码PU.1 ] ,Irf8 和Batf3 )的高表达水平(图4A)。成纤维细胞特异性基因Actg2 和Col9a2 在iDC 第9 天被下调,而在cDC1中不表达(图4B)。在MEF中未检测到DC基因Xcr1 ,Clec9a ,Cd207 ,Naaa ,Ucp2 ,Ifi205 ,Cd74 ,Ciita ,H2-aa 和H2-dmb1的表达,在第9 天的iDC中被激活并在脾脏cDC1中表达(图4C)。这套标记可用于通过mRNA测序或RT-qPCR评估成功的重编程。可以在FACS纯化后对iDC 进行进一步的功能评估,包括:炎性细胞因子的分泌,死细胞吞噬,抗原输出到胞质溶胶和抗原交叉呈递到OT-1 CD8 + T细胞(Rosa 等,2018)。


 






图4. 评估成功的树突状细胞重编程的基因标记。编码重编程因子PU.1 A.基因,IRF8和BATF3(PIB)在诱导的树突状细胞(表达的iDC 在重新编程)日9(tdTomato + CD45 + MHC-II + ),并在常规树突细胞1型(cDC1s, MHC-II + CD11c + CD8a + )。B.成纤维细胞相关基因Actg2 和Col9a2 在重新编程的第9 天在iDC 中被下调,而在cDC1中不表达。C.树突状细胞基因Xcr1 ,Clec9a ,Cd207 ,Naaa ,Ucp2 ,Ifi205 ,CD74 ,Ciita ,H2-aa 和H2-dmb1 在重编程第9 天的iDC 和cDC1 中表达。在小鼠胚胎成纤维细胞(MEF)中诱导PIB后,将iDC (tdTomato + CD45 + MHC-II + )和脾cDC1s(CD11c + MHC-II + CD8α + )进行FACS分选,并通过mRNA测序定量基因的表达(Rosa 等人,2018 ; GSE103618)。基因表达在条形图中显示为基因计数的平均值± 标准偏差。作为替代,RT-qPCR可用于量化该基因集的表达并评估成功的重编程。






菜谱


 


DMEM完全培养基
DMEM补充有10%(v / v)热灭活的FBS和10,000 U / m l 青霉素,10,000μg / m l 链霉素储备溶液的1:100稀释液


解离溶液
在相同体积的PBS中稀释0.25%胰蛋白酶-EDTA溶液(稀释度为1:2),最终浓度为0.125%胰蛋白酶-EDTA


0.1%明胶溶液
将0.1g明胶的在100毫升的MilliQ -grade水和高压釜


洗涤液
PBS补充2.5%FBS


染色液
洗涤溶液补充有大鼠血清(1/100),抗小鼠CD45单克隆抗体(每100 0.25微克微升)和抗小鼠MHC-II单克隆抗体(每100 0.25微克微升)


冷冻液
热灭活的FBS辅以10%DMSO


                                         


致谢


 


该项目由Cancerfonden (CAN 2017/745),瑞典研究委员会(2018-02042),Crafoord Foundation(20190561),NovoNordisk Fonden (0056527)和FCT(CENTRO-01-0145-FEDER-030013)共同资助。Knut和Alice Wallenberg基金会,隆德大学的医学系和斯科讷地区得到了慷慨的财政支持。FFR和CFP和FFR分别由FCT博士(SFRH / BD / 130845/2017)和博士后(SFRH / BPD / 121445/2016)奖学金提供支持。该协议改编自Rosa 等。,2018。


 


利益争夺


 


FábioF . Rosa,Cristiana F.Pires 和Carlos-Filipe Pereira已于2018-04-05提交了一项专利合作条约(PCT),以保护此处所述的知识产权。


 


伦理


 


该协议是根据隆德大学研究伦理委员会的指导方针执行的,应根据个人机构指导方针进行。涉及动物实验的程序已获得瑞典伦理审查委员会(5.8.18-19343 / 2017)的批准。


 


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Rosa, F. F., Pires, C. F., Zimmermannova, O. and Pereira, C. (2020). Direct Reprogramming of Mouse Embryonic Fibroblasts to Conventional Type 1 Dendritic Cells by Enforced Expression of Transcription Factors. Bio-protocol 10(10): e3619. DOI: 10.21769/BioProtoc.3619.
  2. Rosa, F. F., Pires, C. F., Kurochkin, I., Ferreira, A. G., Gomes, A. M., Palma, L. G., Shaiv, K., Solanas, L., Azenha, C., Papatsenko, D., Schulz, O., Reis e Sousa, C. and Pereira, C. F. (2018). Direct reprogramming of fibroblasts into antigen-presenting dendritic cells. Sci Immunol 3(30).
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