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Lentiviral Barcode Labeling and Transplantation of Fetal Liver Hematopoietic Stem and Progenitor Cells
胎儿肝脏造血干细胞和祖细胞的慢病毒条形码标记和移植   

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Abstract

Cellular barcoding enables the dissection of clonal dynamics in heterogeneous cell populations through single cell lineage tracing. The labeling of hematopoietic stem and progenitor cells (HSPCs) with unique and heritable DNA barcodes, makes it possible to resolve donor cell heterogeneity in terms of differentiation potential and lineage bias at the single cell level, through subsequent transplantation and high-throughput sequencing. Furthermore, cellular barcoding allows for bona fide hematopoietic stem cells (HSCs) to be defined based on functional rather than immunophenotypic parameters.

This protocol describes the work flow of lentiviral cellular barcoding, tracking 14.5 days post coitum (d.p.c.) fetal liver (FL) Lineage-Sca+cKit+ (LSK) HSPCs following long-term reconstitution (Figure 1) (Kristiansen et al., 2016), but can be adapted to the cell type or time frame of choice.


Figure 1. Summary of experimental workflow (Naik et al., 2013)

Keywords: Cellular barcoding(细胞条形码), Lentiviral transduction(慢病毒转导), Fetal liver(胎儿肝脏), Hematopoietic stem and progenitor cells(造血干细胞和祖细胞), Single cell lineage tracing(单细胞谱系追踪), Transplantation(移植)

Background

The cellular barcoding technique was initially established to resolve single cell dynamics upon transplantation of hematopoietic cells in vivo and has in recent years contributed significantly to our appreciation of the functional heterogeneity within blood cell populations in a transplantation setting (Schepers et al., 2008; Gerrits et al., 2010; Lu et al., 2011; Naik et al., 2013; Verovskaya et al., 2013; Kristiansen et al., 2016). The generation and characterization of lentiviral barcode libraries, the importance of library complexity as well as the associated analytical challenges have been carefully reviewed (Bystrykh et al., 2014; Naik et al., 2014; Bystrykh and Belderbosv, 2016) and need to be considered before starting this protocol to ensure proper experimental design. The current protocol pertains our adaptation of the technology as seen in our recent article (Kristiansen et al., 2016), to trace the long-term reconstitution capacity of FL derived HSPCs.

Materials and Reagents

  1. 96-well non-treated plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 260836 )
  2. Tissue paper (KCWW, Kimberly-Clark, catalog number: 75512 )
  3. Autoclaved 1.5 ml Eppendorf tubes (Fisher Scientific, FisherbrandTM, catalog number: 05-408-129 )
  4. 50 ml tubes (Corning, Falcon®, catalog number: 352070 )
  5. 1 ml pipette tips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2179-05-HR )
  6. Cell strainers, 40 µm (Corning, catalog number: 431750 )
  7. 60 mm Petri-dishes (Corning, catalog number: 430166 )
  8. Cup filters, 50 µm (BD, BD Biosciences, catalog number: 340630 )
  9. 1 ml syringe with 29 gauge ½ inch needle (Terumo Medical, catalog number: BS-N1H2913 )
  10. FACS tubes (Corning, Falcon®, catalog number: 352058 )
  11. Mice: Males and females of appropriate experimental genotypes for timed pregnancies
    Recipient mice of the appropriate experimental genotype
    One mouse of the appropriate experimental genotype for support bone marrow cells
    Note: This protocol was optimized using C57BL/6 mice. An example of animal usage is shown for a litter of 6 pups (Figure 2). If desired, donor derived cells can be distinguished from support and recipient derived cells using congenic mouse strains. However, this is not necessary since barcoded donor cells can be distinguished based on lentiviral GFP expression.
    Optional: We recommend 2 recipients for each biological replicate containing HSPCs pooled from 3 pups (Figure 2), see discussion (Notes).
  12. Virus: Concentrated lentiviral supernatants containing barcode library
  13. Retronectin (Takara Bio, Clontech, catalog number: T100A )
  14. Sterile PBS (GE Healthcare, catalog number: SH30028.02 )
  15. Antibodies
    Ter119 biotin (Biolegend, catalog number: 116204 )
    Ter119 PE-Cy5 (Biolegend, catalog number: 116210 )
    CD3 PE-Cy5 (Biolegend, catalog number: 100310 )
    Gr1 PE-Cy5 (Biolegend, catalog number: 108410 )
    B220 PE-Cy5 (Biolegend, catalog number: 103210 )
    Sca1 PE-Cy7 (Biolegend, catalog number: 108114 )
    c-kit APC (Biolegend, catalog number: 105812 )
  16. StemSpanTM SFEM (SFEM) (STEMCELL Technologies, catalog number: 09650 )
  17. Penicillin/Streptomycin (P/S) solution (Nordic Biolabs, catalog number: SV30010 )
  18. Cytokines
    SCF (PreproTech, catalog number: 250-03 )
    FLT3 (PreproTech, catalog number: 300-19 )
    IL3 (PreproTech, catalog number: 213-13 )
    IL6 (PreproTech, catalog number: 216-16 )
    IL7 (PreproTech, catalog number: 217-17 )
  19. Trypan blue (Sigma-Aldrich, catalog number: T6146 )
  20. Anti-Biotin MicroBeads (Miltenyi Biotec, catalog number: 130-090-485 )
  21. 7AAD (Sigma-Aldrich, catalog number: A9400 )
  22. 10% bovine serum albumin (BSA) in Iscove’s MDM (STEMCELL Technologies, catalog number: 09300 )
  23. Ciprofloxacin, 250 mg/tablet (Teva Pharmaceutical Industries, catalog number: 01 16 40 )
  24. Agencourt AMPure XP beads (Beckman Coulter, catalog number: A63880 )
  25. Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, catalog number: Q32854 )
  26. High Fidelity Taq polymerase (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11304011 )
  27. High Sensitivity DNA Bioanalyzer Kit (Agilent Technologies, catalog number: 5067-4626 )
  28. BSA ≥ 98% (Sigma-Aldrich, catalog number: A9647 )
  29. EDTA (Sigma-Aldrich, catalog number: E5134 )
  30. HBSS without MgCl2 (Thermo Fisher Scientific, GibcoTM, catalog number: 14175053 )
  31. Sodium chloride (NaCl)
  32. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 74255 )
  33. Proteinase K
  34. Staining buffer (see Recipes)
  35. Lysis buffer (see Recipes)

Equipment

  1. Dissection tools: fine tipped tweezers and scissors for FL dissection
  2. Pestle and mortar
  3. Pipette  
  4. Centrifuge with temperature control (for 50 and 15 ml tubes) (Sigma Laborzentrifugen, model: 3-18K , catalog number: 10290)
  5. Centrifuge with temperature control (for Eppendorf tubes) (Beckman Coulter, model: Microfuge® 20R centrifuge , catalog number: B31607)
  6. MACS columns (Miltenyi Biotec, catalog number: 130-042-401 )
  7. MACS separator magnet (Miltenyi Biotec, model: QuadroMACSTM separator , catalog number: 130-090-976)
  8. Small animal heat lamp
  9. Cell culture room and equipment certified for lentivirus work
  10. Agilent 2100 Bioanalyzer Instrument (Agilent Technologies, model: 2100 Bioanalyzer Instrument )
  11. Qubit Fluorometer
  12. DynaMag 96 well plate magnet (Thermo Fisher Scientific, catalog number: 12331D )
  13. BD FACS Aria III (BD, model: FACS Aria III) or comparable sorter
  14. Deep sequencing facility and reagents (e.g., Illumina MiSeq system [Illumina, model: MiSeq system ] with MiSeq Reagent Kit v3 150-cycle or the Ion PGM System [Thermo Fisher Scientific, model: Ion PGM System ] with Ion 314 Chip Kit v2)

Procedure

  1. Lentiviral barcode library
    The generation of barcode libraries has been previously reviewed (Bystrykh et al., 2014). The complexity and distribution of barcodes in the virus library impact the risk of labelling of two cells with the same barcode and therefore poses a constraint on the number of single cell fates that can be faithfully traced. Thus, these properties need to be empirically determined for each new barcode library and virus prep (Lu et al., 2011; Naik et al., 2014; Bystrykh and Belderbos, 2016). As a rule of thumb the number of cells traced per recipient should not exceed 10% of the library diversity (Naik et al., 2014). Furthermore, the virus library needs to be titrated using the donor cell type of interest prior to experiment as transduction efficiency can vary greatly from cell type to cell type. In our recent study, a previously characterized barcode library was used (Lu et al., 2011).

  2. Timed pregnancy set-up
    Day -14
    1. In the afternoon, add two female mice into each cage containing one male mouse (Figure 2).
    2. The following morning, separate females from male and check for vaginal plugs.
      Note: Detection of a vaginal plug at this time point is counted as 0.5 d.p.c. The presence of a vaginal plug is not a guarantee of the female being pregnant. Likewise, the lack of a vaginal plug does not mean the female is not pregnant. To increase chances of fertilization, more than one cage of timed pregnancy should be set up. After E12 females can be palpated for abdominal distention, bilateral bulging and weight increase to confirm pregnancy. However if the female is pregnant with low number of embryos it can be difficult to distinguish.

  3. Transduction and cell sorting preparations
    Day 0
    1. Transduction preparations
      1. Dilute Retronectin with sterile PBS to 40 μg/ml.
      2. Coat one well per biological replicate by adding 100 μl of the diluted Retronectin from step C1a to a non-culture treated 96-well plate.
        Note: If more than one biological replicate is desired, a litter of 6 embryos could be split up into 2 equal biological replicates to be transduced in 2 separate wells (Figure 2).
        Optional: Transduced LSKs from each biological replicate can later be divided into two equal parts and transplanted into two recipients to facilitate data interpretation (see Notes) (Figure 2).
      3. Coat one additional well for an untransduction control.
      4. Store the plate at 4 °C overnight.


        Figure 2. Example of experimental animal usage based on estimated number of pups and cell yield

    2. Tools
      Autoclave dissection tools, pestle and mortar, tissue paper, Eppendorf tubes.
    3. Cell sorting and culturing preparations
      Optional: The following steps can be performed on Day 1.
      1. Prepare LSK antibody cocktail for FACS (store at 4 °C in the dark until use).
        Notes:
        1. The fluorochromes listed here are suggestions and can be substituted with other colors.
        2. Lineage antibodies include: CD3 PE-Cy5, Gr1 PE-Cy5, Ter119 PE-Cy5, B220 PE-Cy5.
        Antibody

        fluorochrome

        Dilution

        Final concentration
        (μg/100 μl)
        c-Kit 
        APC
        1:200
        0.1
        Sca1 
        PE-Cy7
        1:200
        0.1
        Lineage Antibodies
        PE-Cy5
        1:400 each
        0.05 each

      2. Prepare antibody dilutions for single stains and appropriate staining controls.
      3. Prepare cytokine mix in SFEM containing 1% P/S (store at 4 °C until use).
        Cytokine
        Final concentration (ng/ml)
        SCF
        50
        IL6
        50
        FLT3
        50
        IL3
        10
        IL7
        10

  4. Cell sorting and transduction
    Note: All dissections should be performed in appropriate laboratory areas and cell preparations in sterile biosafety cabinets.
    Day 1
    1. Harvesting of total BM support cells
      1. In the morning, euthanize mouse for support cells (e.g., cervical dislocation or CO2 asphyxiation) (Figure 2).
      2. Dissect hind leg bones (femur, tibia and iliac crest) and collect in 50 ml tube with cold (4 °C) staining buffer (see Recipes). Crush bones using pestle and mortar.
      3. Make single cell suspension by gently pipetting up and down with a 1 ml pipette tip.
      4. Filter through a 40 µm cell strainer and adjust volume to 10 ml with staining buffer.
      5. Store at 4 °C until intravenous injections on Day 2.
    2. Harvesting of 14.5 d.p.c. FL donor cells
      1. Euthanize the pregnant female (e.g., cervical dislocation or CO2 asphyxiation) in the morning.
      2. Lift skin over the peritoneal cavity and make a small incision with scissors.
      3. Pull out the uterine horns and release the uterus by cutting the cervix.
      4. Dissect the embryos from the uterine membranes in a 60 mm Petri-dish filled with 5 ml cold (4 °C) staining buffer.
      5. Place an embryo on autoclaved tissue paper and use fine tipped tweezers to isolate the FL.
      6. Transfer the FL to a new 60 mm Petri-dish with 1 ml with cold staining buffer per FL and repeat for all embryos, combining only the FLs intended for the same biological replicate into the same Petri-dish.
      7. Remove any attached connective tissue and intestine prior to dissociating by gentle pipetting using a 1 ml pipette tip.
      8. Filter the resulting single cell suspension through a 50 µm cup filter and repeat for biological replicates.
      9. Count the cells and centrifuge cells at 350 x g for 5 min at 4 °C.
        Note: Expect approximately 40 x 106/FL including red blood cells, although some variation in cell number can be expected (depending on loss of cells during dissection and size of the embryo). 
      10. Remove the supernatant and prepare for Ter119 depletion.
    3. Ter119 depletion using MACS
      Ter119 is expressed by mature erythrocytes and erythroid progenitors which are the most abundant cells in E14.5 FL. Depletion of these cells enrich for the LSK population and shortens sorting time.
      1. Resuspend the cell pellet to a volume of 50 μl staining buffer/107 cells and add Ter119 biotin antibody to a final concentration of 0.5 μg/ml.
      2. Incubate at 4 °C for 25 min.
      3. Add 10 ml cold staining buffer and centrifuge at 350 x g for 5 min at 4 °C, remove supernatant.
      4. Resuspend the pellet to a volume of 40 μl staining buffer/107 cells and add 10 μl of anti-Biotin MicroBeads/107 cells.
      5. Incubate at 4 °C for 15 min.
      6. Add 10 ml staining buffer and centrifuge at 350 x g for 5 min at 4 °C, remove supernatant.
      7. Resuspend cell pellet in 500 μl staining buffer.
      8. Put MACS columns in MACS separator magnet, placing a 50 µm cup filter on top of each column.
      9. Equilibrate the columns by running 3 ml buffer through each and discard the liquid flow through.
      10. Place labeled collection tubes under each column and add the 500 μl samples through the filter onto the column.
      11. Rinse the original sample containing tube with an additional 500 μl staining buffer and add onto the column through the same filter.
      12. Add 3 x 3 ml buffer onto each column to rinse off unbound cells.
        Note: For each round of 3 ml rinse, wait until the existing buffer has almost entirely run through before adding the next 3 ml.
      13. Count the cells in the 10 ml flow-through.
      14. Set aside approximately 200K cells for FACS staining controls.
      15. Centrifuge the remaining cells at 350 x g for 5 min at 4 °C and remove supernatant, the cell pellet should be white. 
    4. Cell staining and sorting
      Our workflow using the BD FACS Aria IIu or III is briefly described below.
      1. Resuspend the cell pellets in the LSK antibody cocktail (step C3a) at a volume of 100 μl/107 Ter119 depleted cells.
      2. Stain the sample and appropriate staining controls in the dark at 4 °C for 30 min.
      3. For each sample prepare one Eppendorf tube containing 100 μl cold staining buffer for post-sort purity check and one containing 500 μl SFEM to collect the sorted cells.
      4. Rinse stained cells with staining buffer, centrifuge at 350 x g for 5 min at 4 °C and remove supernatant.
      5. Resuspend samples in staining buffer containing 7AAD to a final concentration of 1 μg/ml 7AAD.
        Note: Volume of buffer to resuspend cells in for sorting depends on the cell number and sort settings (e.g., 7 x 106 cells can be resuspended in 700 μl when sorted on BD FACS Aria III at approximately 4,000 events/sec, flow rate < 4, purity mask 0-32-0, using a 70 μm nozzle).
      6. Set-up staining compensation matrix using single stained controls.
      7. Set LSK gates as shown in Figure 3.


        Figure 3. FACS plots for LSK HSPC gating during cell sorting. Fluorescence minus one (FMO) controls for Sca1 and c-Kit are shown to illustrate the stringent gating that we draw to maximize sort purity (reanalysis plot). The expected HSC content within the FL LSK gate varies from 4-10% in our experience.

      8. Take appropriate precautions to ensure sorting purity of > 95%.
      9. Sort LSK cells for all samples into prepared collection tubes containing 500 μl cold SFEM.
        Note: Sort at least 11,000 LSKs for each planned recipient to compensate for cell loss in steps E9-E10. It is possible to obtain enough LSKs from 3 FLs for 2 recipients if technical replicates are desired (see Notes).
      10. Record the cell number yields from the sort and store tubes on ice.
    5. Transduction
      This step should be performed in an appropriate cell culture hood using procedures approved for lentiviral transduction.
      Note: HSPCs from fetal liver and adult bone marrow differ in transduction efficiency with less virus required to achieve the same transduction efficiency in fetal HSPCs. Before starting the protocol titrate your lentiviral barcoding library specifically for the population of choice, determining the amount of virus needed to achieve a transduction efficiency that ensures that the number of transduced cells per recipient does not exceed 10% of the complexity of the lentiviral barcode library. For an estimated barcode complexity of 80K (Lu et al., 2011), we aimed for a transduction efficiency of 15-30% meaning that 1,500-3,000 cells out of the 10K cells injected per recipient mouse were barcoded (Figure 4). This routinely yielded hundreds of cell fates traced in each recipient out of which around 60 clones demonstrated functional characteristics of active HSCs and minimal instances in which the same barcode transduced more than one cell (Kristiansen et al., 2016) (see Notes).


      Figure 4. HSC frequency within the FL LSK population. (Left) representative FACS plots for FL HSCs defined by LSK CD48-CD150+. (Right) %frequency of HSCs within LSK compiled from 6+3 individual embryos from 2 litters in 2 separate experiments. 

      1. Remove Retronectin from the wells.
      2. Immediately add 200 μl 2% BSA (Note: BSA in Iscove’s MDM diluted in PBS.) to each of the coated wells.
      3. Incubate the plate for 30 min at room temperature.
      4. Set aside approximately 300 LSK cells in a separate tube for an untransduced control.
      5. Centrifuge tubes containing LSK cells at 350 x g for 5 min at 4 °C and remove supernatant.
        Note: When working with low numbers of sorted cells, every precaution to minimize cell loss must be taken to get reproducible results. Therefore, omit further counting and filtering steps and always leave an appropriate volume when removing supernatant following centrifugation so as not to disturb the cell pellet.
      6. Resuspend cell pellet to a final volume of 150 μl cytokine mix.
      7. Remove 2% BSA from the coated wells and plate the sorted cells from each biological replicate into a single well. Also plate the LSK cells set aside from step D5d into the well designated for the untransduced control (step C1c).
      8. Add the previously optimized amount of lentivirus to each well except for the untransduced control well.
      9. Gently rock the plate to disperse the virus.
      10. Incubate in humidified CO2 incubator at 37 °C and 5% CO2 overnight.
    6. Irradiation of recipient mice
      1. Irradiate recipients with 2 x 450 cGy with 3 h between the doses. Transplantation should be performed within 24 h of the first dose.
        Note: Split dose irradiation is more gentle on the mice while still achieving the desired effects of lethal myeloablation. Recipient mice do not need treatment with antibiotics prior to irradiation.
      2. Add Ciprofloxacin to a final concentration of 125 mg/L in the drinking water for 14 days. 

  5. Intravenous injections
    The LSK population is heterogeneous containing stem and progenitor cells that survive and proliferate differently in culture. To minimize cell loss we omit additional cell counting steps and treat the donor cell number on Day 2 as equivalent to the number of sorted cells from Day 1. 
    Note: The following steps must strictly adhere to biosafety protocols for lentiviral handling and approved animal use protocols.
    Day 2
    1. In the morning, filter support cells from Day 1 through a 40 µm cell strainer as significant aggregation may have taken place over night. Count the cells.
      Note: The cell count includes red blood cells.
    2. Centrifuge support cells at 350 x g for 5 min at 4 °C and resuspend in cold sterile PBS to a concentration of 200K/100 μl.
    3. Collect each transduced LSK cell sample in autoclaved Eppendorf tubes containing 500 μl cold PBS.
      Note: To minimize cell loss associated with filtering, donor cells are collected directly from the well for transplantation.
    4. Rinse each well with at least 2 rounds of 200 μl PBS to recover all cells and combine in corresponding Eppendorf tubes.
      Note: Check that all cells have been harvested from the wells using a microscope.
    5. Transfer a small volume of each sample back to their original well for transduction efficiency readout on Day 3. Add cytokine mix to a final volume of 150 μl and incubate the cells at 37 °C in 5% CO2 overnight.
    6. Add an additional 500 μl cold PBS to each sample tube and centrifuge at 350 x g for 5 min at 4 °C.
    7. Dispose of lentiviral supernatant according to appropriate biosafety protocols.
    8. Add 200K support cells (100 μl from step E2)/10K sorted and transduced FL LSKs and add 1 ml cold PBS.
    9. Centrifuge samples at 350 x g for 5 min and discard supernatant according to appropriate biosafety protocols.
    10. Resuspend the pellet in 275 μl cold PBS/11,000 sorted and transduced FL LSKs (step D4i).
    11. Inject 250 μl (the equivalent of 10,000 sorted and transduced LSKs) of sample into the tail-vein of each recipient using a 29 gauge ½ inch 1 ml needle.
      Note: We routinely warm up the recipient mice for 5 min under a heat lamp to facilitate intravenous injections.

  6. Transduction efficiency readout
    Day 3
    1. Collect the transduced cells that were set aside on Day 2 for transduction efficiency readout (step E5) and the untransduced control into individual FACS tubes.
    2. Centrifuge samples at 350 x g for 5 min at 4 °C and remove supernatant.
    3. Resuspend each sample in 150 μl staining buffer containing 7AAD.
    4. Analyze by FACS the percentage GFP+ events among of live (7AAD-) cells (Figure 5).


      Figure 5. Example of GFP transduction efficiency readout on Day 3. 7AAD- singlet LSK cells from untransduced control (left) and transduced sample (right) are shown.

  7. Peripheral blood analysis
    4-8 weeks post transplantation
    We recommend checking initial engraftment of GFP+ barcoded cells in the peripheral blood. This is done by tail vein bleeding of recipient mice and assessment the percentage GFP+ cells e.g., among granulocytes and B cells using FACS.
    Note: We frequently observe a higher GFP% in the peripheral blood compared to the transduction efficiency readout on Day 3 with FL HSPCs. This is likely due to a preferential transduction of highly proliferating HSPCs that engraft better.

  8. Barcode extraction
    Clonal dynamics of long-term HSCs stabilize after approximately 12 weeks post transplantation (Verovskaya et al., 2013). Therefore, we recommend reading out barcode distribution no earlier than 16 weeks post transplantation.
    16-20 weeks post transplantation
    1. Cell sorting and lysis
      1. Cell populations of interest based on the specific questions posed are FACS sorted. Cells are sorted into 500 μl staining buffer in non-stick tubes as described for step D4 and can be stored at -80 °C as cell pellets initially.
        Note: Granulocytes have a short turnover time of a few days and is therefore a good representation of recent HSC activity. Lymphocytes on the other hand can survive for weeks and even months and will therefore contain barcodes derived from both long and short term progenitors at the time of barcode labelling.
      2. Prepare cell lysis buffer (see Recipes).
      3. Cell pellets are lysed for genomic DNA in 100 μl cell lysis buffer/sample (2 h at 56 °C + 10 min at 95 °C to inactivate the proteinase K) within a week of sorting and can subsequently be stored at -20 °C.
        Note: With a smaller number of cells it is recommended to sort directly into lysis buffer for immediate lysis. Cell numbers are a limiting factor to getting a good sequencing coverage of the population.
    2. Library preparation and barcode deep sequencing
      1. Genomic DNA is purified using Ampure XP beads according to manufacturer’s recommendations.
      2. Measure genomic DNA concentration using a high sensitivity method e.g., the Qubit Fluorometer and the Qubit dsDNA HS Assay Kit.
      3. Up to 100 ng DNA from each sample is divided equally into two technical replicate PCR reactions (50 ng each) using the appropriate primers and a high fidelity DNA Taq polymerase (e.g., Q5 Taq DNA polymerase, NEB) for amplification of the barcode fragment (Figure 1). The amplified fragments are purified using Ampure XP beads according to manufacturer’s recommendations.
        Optional: Depending on the design of PCR primers and the choice of sequencing platform, a second step of PCR amplification can be performed to add multiplexing adapters.
      4. Amplicons are quantified using the Bioanalyzer HS DNA Analysis Kit and according to manufacturers’ instructions. A product of the correct size without contamination of primer dimers is critical for successful sequencing. The presence of primer dimers can be removed with another round of Ampure XP bead purification according to manufacturer’s recommendations.
      5. Pool equivalent amounts of amplified and indexed barcode libraries for sequencing–the required concentration of product depends on the sequencing platform of choice.
        Note: The extent of multiplexing should be informed by the read capacity of the sequencing platform of choice and the expected complexity of your barcoded cells.
      6. Sequencing is performed by experienced technical personnel according to manufacturer’s instructions.
        Note: We have thus far had good success using the Illumina MiSeq system (MiSeq Reagent Kit v3 150-cycle, Illumina) and the Ion PGM (Ion 314 Chip Kit v2, Thermo Scientific) platforms.

Data analysis

The initial steps of cellular barcode analysis aims at generating a list of reliable barcodes for each sorted sample coupled with the barcode read frequency reflecting the abundance of each clone within the population. The most crucial part of this analysis is filtering out barcodes introduced by e.g., sequencing errors, which would otherwise bias the results. The filtering steps are customized for the barcode library of choice and experimental design. For example, if a reference library exists for the barcode library of choice, the sequenced barcodes can be mapped back to the reference and the filtering step can be streamlined. Here, we briefly provide an example for the analysis of randomly generated barcode libraries lacking a known reference (Figure 1; Kristiansen et al., 2016). For in depth recommendations, we have provided a number relevant references.

  1. Barcodes are isolated by the identification of flanking sequences using custom R scripts and the R package ShortReads.
  2. Correct for sequencing errors by subjecting each barcode list to e.g., the Starcode algorithm (Zorita et al., 2015). Based on Levenshtein distances, daughter barcodes generated by sequencing errors are identified and removed while their read frequencies are merged with that of the parent barcode.
  3. The cut-off for low frequency barcodes is individually defined for each sorted population based on the total read number retrieved and the cell number sampled such that barcodes that fall below the expected read representation of a single cell are excluded from further analyses.
  4. The barcode lists obtained from the two technical PCR replicates of each population are assessed for correlation of read frequencies with a criterion of r > 0.85 as previously described (Naik et al., 2014). Only barcodes that pass all filtering criteria in both technical replicates are considered for further analysis.
    Note: These stringent filtering criteria produce a conservative barcode list for downstream analyses. However, it is important to note that these filtering steps may exclude true barcodes of very low representation (Naik et al., 2014) (Figure 1).
  5. Barcode lists can be further analyzed for overlap, read frequency distributions etc. using custom scripts.

Notes

  1. For kinship analysis of cells of different lineages, qualitative barcode overlap is a critical readout. To avoid false positive barcode overlap readout, it is particularly important to control that the same barcode does not enter more than one cell. This can be done by comparing the filtered barcode lists from two technical replicate recipients (Figure 2) that received LSK cells transduced in the same well (Naik et al., 2014). Since the short virus incubation time is not likely to permit HSC division events, any overlap between the two recipients can be considered as noise. The current protocol is designed to accommodate two technical replicate recipients per biological replicate (Figure 2) by simply sorting (step D4i) and plating double the number of LSK cells per Retronectin coated well. In choosing populations for barcode content readout, we also recommend including two populations with expected barcode overlap as a positive control. These controls will allow for proper validation of the signal versus noise ratio in your experimental set up (Kristiansen et al., 2016).
  2. If the experimental set up is sound, and the appropriate controls and barcode filtering steps are applied, reproducibility between individual experiments should be high. However, donor HSPCs of different ages vary significantly in their propensity to become transduced and will yield age specific barcode distributions.

Recipes

  1. Staining buffer
    0.5% BSA
    Note: Use BSA ≥ 98%.
    2 mM EDTA
    in HBSS without MgCl2
  2. Lysis buffer
    0.1 M Tris-HCl pH 8.5
    0.5 mM EDTA pH 8
    0.2 M NaCl
    0.2% SDS
    0.1 mg/ml proteinase K
    Note: To be added to lysis buffer immediately before use.

Acknowledgments

This protocol was originally described in and adapted from Kristiansen et al. (2016). This work was supported by grants from the Swedish Cancer Foundation, the Swedish Research Council, StemTherapy, and The Knut and Alice Wallenberg Foundation.

References

  1. Bystrykh, L. V. and Belderbos, M. E. (2016). Clonal analysis of cells with cellular barcoding: When numbers and sizes matter. Methods Mol Biol 1516: 57-89.
  2. Bystrykh, L. V., de Haan, G. and Verovskaya, E. (2014). Barcoded vector libraries and retroviral or lentiviral barcoding of hematopoietic stem cells. Methods Mol Biol 1185: 345-360.
  3. Gerrits, A., Dykstra, B., Kalmykowa, O. J., Klauke, K., Verovskaya, E., Broekhuis, M. J., de Haan, G. and Bystrykh, L. V. (2010). Cellular barcoding tool for clonal analysis in the hematopoietic system. Blood 115(13): 2610-2618.
  4. Kristiansen, T. A., Jaensson Gyllenback, E., Zriwil, A., Bjorklund, T., Daniel, J. A., Sitnicka, E., Soneji, S., Bryder, D. and Yuan, J. (2016). Cellular barcoding links B-1a B cell potential to a fetal hematopoietic stem cell state at the single-cell level. Immunity 45(2): 346-357.
  5. Lu, R., Neff, N. F., Quake, S. R. and Weissman, I. L. (2011). Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding. Nat Biotechnol 29(10): 928-933.
  6. Naik, S. H., Perie, L., Swart, E., Gerlach, C., van Rooij, N., de Boer, R. J. and Schumacher, T. N. (2013). Diverse and heritable lineage imprinting of early haematopoietic progenitors. Nature 496(7444): 229-232.
  7. Naik, S. H., Schumacher, T. N. and Perie, L. (2014). Cellular barcoding: a technical appraisal. Exp Hematol 42(8): 598-608.
  8. Schepers, K., Swart, E., van Heijst, J. W., Gerlach, C., Castrucci, M., Sie, D., Heimerikx, M., Velds, A., Kerkhoven, R. M., Arens, R. and Schumacher, T. N. (2008). Dissecting T cell lineage relationships by cellular barcoding. J Exp Med 205(10): 2309-2318.
  9. Verovskaya, E., Broekhuis, M. J., Zwart, E., Ritsema, M., van Os, R., de Haan, G. and Bystrykh, L. V. (2013). Heterogeneity of young and aged murine hematopoietic stem cells revealed by quantitative clonal analysis using cellular barcoding. Blood 122(4): 523-532.
  10. Zorita, E., Cusco, P. and Filion, G. J. (2015). Starcode: sequence clustering based on all-pairs search. Bioinformatics 31(12): 1913-1919.

简介

细胞条形码可以通过单细胞谱系追踪来分离异种细胞群体中的克隆动力学。通过独特和可遗传的DNA条形码对造血干细胞和祖细胞(HSPC)进行标记,可以通过随后的移植和高通量测序,在单细胞水平上分化供体细胞异质性,分化潜力和谱系偏倚。此外,细胞条形码可以根据功能而不是免疫表型参数定义真正的造血干细胞(HSC)。
&nbsp;该协议描述了慢病毒细胞条形码的工作流程,追踪1450天后(dpc)胎肝(FL)Lineage-Sca+ cKit + (LSK)HSPC经过长期重建(图1)(Kristiansen等人,2016),但可以适应于选择的细胞类型或时间框架。


图1.实验工作流程摘要(Naik 等人,2013)

最初建立了细胞条形码技术来解决在体内移植造血细胞后的单细胞动力学,并且近年来在移植中对血细胞群体中功能​​异质性的认识有显着贡献(Schepers ,2008; Gerrits等人,2010; Lu等人,2011; Naik等人 ,2013; Verovskaya等人,2013; Kristiansen等人,2016)。已经仔细审查了慢病毒条形码图书馆的生成和表征,图书馆复杂性的重要性以及相关的分析挑战(Bystrykh等人,2014; Naik等人, ,2014年; Bystrykh和Belderbosv,2016),并且在启动本协议之前需要考虑以确保适当的实验设计。目前的方案与我们最近文章(Kristiansen等人,2016)中所看到的技术适应有关,以追踪FL衍生HSPC的长期重组能力。

关键字:细胞条形码, 慢病毒转导, 胎儿肝脏, 造血干细胞和祖细胞, 单细胞谱系追踪, 移植

材料和试剂

  1. 96孔未处理的板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:260836)
  2. 组织纸(KCWW,Kimberly-Clark,目录号:75512)
  3. 高压灭菌的1.5ml Eppendorf管(Fisher Scientific,Fisherbrand TM ,目录号:05-408-129)
  4. 50ml管(Corning,Falcon ®,目录号:352070)
  5. 1 ml移液管吸头(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:2179-05-HR)
  6. 细胞过滤器,40μm(Corning,目录号:431750)
  7. 60毫米培养皿(康宁,目录号:430166)
  8. 杯式过滤器,50μm(BD,BD Biosciences,目录号:340630)
  9. 1 ml注射器,配有29½寸针头(Terumo Medical,目录号:BS-N1H2913)
  10. FACS管(Corning,Falcon ®,目录号:352058)
  11. 小鼠:用于定时怀孕的适当实验基因型的男性和女性
    适当实验基因型的受体小鼠
    支持骨髓细胞的适当实验基因型的一只小鼠
    注意:本协议是使用C57BL/6小鼠进行了优化的。对于6只幼仔的小狗,显示了动物使用的一个例子(图2)。如果需要,可以使用同源小鼠品系将供体来源的细胞与载体和受体来源的细胞区分开来。然而,这不是必需的,因为可以基于慢病毒GFP表达来区分条形码供体细胞。
    可选:我们建议从3只幼仔中收集含有HSPCs的每个生物复制品的2个接收者(见图2),参见讨论(注)。
  12. 病毒:含有条形码文库的浓缩慢病毒上清液
  13. Retronectin(Takara Bio,Clontech,目录号:T100A)
  14. 无菌PBS(GE Healthcare,目录号:SH30028.02)
  15. 抗体
    Ter119生物素(Biolegend,目录号:116204)
    Ter119 PE-Cy5(Biolegend,目录号:116210)
    CD3 PE-Cy5(Biolegend,目录号:100310)
    Gr1 PE-Cy5(Biolegend,目录号:108410)
    B220 PE-Cy5(Biolegend,目录号:103210)
    Sca1 PE-Cy7(Biolegend,目录号:108114)
    c-kit APC(Biolegend,目录号:105812)
  16. StemSpan TM SFEM(SFEM)(STEMCELL Technologies,目录号:09650)
  17. 青霉素/链霉素(P/S)溶液(Nordic Biolabs,目录号:SV30010)
  18. 细胞因子
    SCF(PreproTech,目录号:250-03)
    FLT3(PreproTech,目录号:300-19)
    IL3(PreproTech,目录号:213-13)
    IL6(PreproTech,目录号:216-16)
    IL7(PreproTech,目录号:217-17)
  19. 台盼蓝(Sigma-Aldrich,目录号:T6146)
  20. 抗生物素MicroBeads(Miltenyi Biotec,目录号:130-090-485)
  21. 7AAD(Sigma-Aldrich,目录号:A9400)
  22. Iscove的MDM中的10%牛血清白蛋白(BSA)(STEMCELL Technologies,目录号:09300)
  23. 环丙沙星,250mg /片(Teva Pharmaceutical Industries,目录号:011640)
  24. Agencourt AMPure XP珠(Beckman Coulter,目录号:A63880)
  25. Qubit dsDNA HS测定试剂盒(Thermo Fisher Scientific,目录号:Q32854)
  26. 高保真度Taq 聚合酶(Thermo Fisher Scientific,Invitrogen TM,目录号:11304011)
  27. 高灵敏度DNA生物分析仪(Agilent Technologies,目录号:5067-4626)
  28. BSA≥98%(Sigma-Aldrich,目录号:A9647)
  29. EDTA(Sigma-Aldrich,目录号:E5134)
  30. 不含MgCl 2的HBSS(Thermo Fisher Scientific,Gibco TM,目录号:14175053)
  31. 氯化钠(NaCl)
  32. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:74255)
  33. 蛋白酶K
  34. 染色缓冲液(见配方)
  35. 裂解缓冲液(见配方)

设备

  1. 解剖工具:精细镊子和剪刀用于FL解剖
  2. 杵和砂浆
  3. 移液器
  4. 离心机,温度控制(50和15 ml管)(Sigma Laborzentrifugen,型号:3-18K,目录号:10290)
  5. 离心机温度控制(用于Eppendorf管)(Beckman Coulter,型号:Microfuge 20R离心机,目录号:B31607)
  6. MACS列(Miltenyi Biotec,目录号:130-042-401)
  7. MACS分离器磁体(Miltenyi Biotec,型号:QuadroMACS TM分离器,目录号:130-090-976)
  8. 小动物热灯
  9. 细胞培养室和设备认证为慢病毒工作
  10. Agilent 2100生物分析仪仪器(Agilent Technologies,型号:2100 Bioanalyzer Instrument)
  11. Qubit荧光计
  12. DynaMag 96孔板磁体(Thermo Fisher Scientific,目录号:12331D)
  13. BD FACS Aria III(BD,型号:FACS Aria III)或类似的分选机
  14. 使用MiSeq Reagent Kit v3 150循环或Ion PGM系统[Thermo Fisher Scientific,型号:Ion PGM系统]的深度测序设备和试剂(例如,Illumina MiSeq系统[Illumina,型号:MiSeq系统] ]与Ion 314芯片组v2)

程序

  1. 慢病毒条形码库
    之前已经检查过条形码图书馆的生成(Bystrykh等人,2014)。条形码在病毒库中的复杂性和分布影响了使用相同条形码标记两个细胞的风险,因此对可以忠实追踪的单细胞命运数量构成了限制。因此,这些性质需要根据每个新的条形码库和病毒准备(Lu et al。,2011; Naik等人,2014年; Bystrykh和Belderbos, 2016)。根据经验,每个接收者追踪的细胞数不应超过图书馆多样性的10%(Naik等人,2014)。此外,在实验之前,需要使用感兴趣的供体细胞类型对病毒库进行滴定,因为转导效率可以从细胞类型到细胞类型变化很大。在我们最近的研究中,使用了先前描述的条形码库(Lu等人,2011)。

  2. 定时怀孕设置
    一天-14
    1. 下午,将两只雌性小鼠加入每只含有一只雄鼠的笼子(图2)
    2. 第二天早晨,女性与男性分离,检查阴道栓塞。
      注意:在这个时间点检测阴道塞计数为0.5 d.p.c.阴道塞的存在不是怀孕女性的保证。同样地,缺少阴道塞并不意味着女性没有怀孕。为了增加受精的机会,应建立一个以上定期怀孕的笼子。 E12女性可以触诊腹部膨胀,双侧鼓胀和体重增加以确认怀孕。然而,如果女性怀孕的胚胎数量很少,可能难以区分。

  3. 转导和细胞分选制剂
    第0天
    1. 转导制剂
      1. 用无菌PBS稀释Retronectin至40μg/ml。
      2. 通过将100μl稀释的Retronectin从步骤C1a加入到非培养处理的96孔板中,每个生物复制物涂一个孔。
        注意:如果需要多于一个生物复制物,可以将6只胚胎的胚胎分成2个相等的生物重复,以在2个单独的孔中转导(图2)。
        可选:每个生物复制体的转导的LSK可以稍后分为两个相等的部分,并移植到两个收件人中以便于数据解释(见注释)(图2)。
      3. 涂一个另外一个井,以进行转移控制。
      4. 将板在4℃下过夜。


        图2.基于估计的幼崽数量和细胞产量的实验动物使用实例

    2. 工具
      高压灭菌工具,杵和砂浆,薄纸,Eppendorf管。
    3. 细胞分选和培养制剂
      可选:可以在第1天执行以下步骤。
      1. 准备用于FACS的LSK抗体混合物(在暗处存储4℃,直到使用) 注意:
        1. 这里列出的荧光染料是建议,可以用其他颜色代替。
        2. 谱系抗体包括:CD3 PE-Cy5,Gr1 PE-Cy5,Ter119PE-Cy5,B220PE-Cy5。//em>
        抗体

        荧光染料

        稀释

        最终集中
        (μg/100μl)
        c-Kit 
        APC
        1:200
        0.1
        Sca1 
        PE-Cy7
        1:200
        0.1
        谱系抗体
        PE-Cy5
        1:400每个
        0.05每个

      2. 为单一染色剂和适当的染色对照制备抗体稀释液
      3. 在含有1%P/S的SFEM中制备细胞因子混合物(在4℃下储存直至使用)
        细胞因子
        终浓度(ng/ml)
        SCF
        50
        IL6
        50
        FLT3
        50
        IL3
        10
        IL7
        10

  4. 细胞分选和转导
    注意:所有解剖应在适当的实验室进行,并在无菌生物安全柜中进行细胞制备。
    第1天
    1. 收获总BM支持细胞
      1. 早晨,安乐死小鼠支持细胞(例如,宫颈脱位或CO 2窒息)(图2)。
      2. 解剖后腿骨(股骨,胫骨和髂嵴),并用冷(4℃)染色缓冲液收集在50ml管中(参见食谱)。使用杵和砂浆粉碎骨头。
      3. 通过用1毫升移液器吸头上下轻轻移液来制备单细胞悬浮液。
      4. 通过40μm细胞过滤器过滤,并用染色缓冲液调节体积至10ml。
      5. 储存于4°C,直至注射第2天。
    2. 收获14.5 d.p. FL供体细胞
      1. 在早晨安乐死怀孕的女性(例如,,颈椎脱位或CO <2>窒息)。
      2. 将皮肤提起腹膜,并用剪刀做一个小切口。
      3. 拔出子宫角并切开子宫释放子宫。
      4. 在装有5ml冷(4℃)染色缓冲液的60mm培养皿中从子宫膜解剖胚胎。
      5. 将胚胎放置在高压消毒的薄纸上,并使用精细的镊子隔离FL。
      6. 将FL转移到一个新的60毫米培养皿,1毫升,每个FL的冷染色缓冲液,并重复所有的胚胎,仅将同一生物复制品的FL组合成相同的培养皿。
      7. 通过轻轻移液使用1 ml移液管吸头解离之前,取出任何附着的结缔组织和肠
      8. 通过50μm杯过滤器过滤所得的单细胞悬浮液,并重复进行生物学重复
      9. 计数细胞,并在4℃以350×g离心细胞5分钟。
        注意:预期大约40 x 10 6 /FL包括红细胞,尽管可以预期细胞数量的一些变化(取决于解剖期间细胞的损失和胚胎的大小)。 ;
      10. 去除上清液并准备Ter119消耗。
    3. Ter119耗尽使用MACS
      Ter119由成熟的红细胞和红细胞祖细胞表达,它是E14.5 FL中最丰富的细胞。这些细胞的消耗丰富了LSK群体,缩短了排序时间。
      1. 将细胞沉淀重悬于50μl染色缓冲液/10μg/ml细胞的体积中,并加入终浓度为0.5μg/ml的Ter119生物素抗体。
      2. 在4℃下孵育25分钟。
      3. 加入10ml冷染色缓冲液,并在4℃下以350×g离心5分钟,除去上清液。
      4. 将沉淀重悬于40μl染色缓冲液/10μg/ml细胞的体积中,加入10μl抗生物素MicroBeads /10μg/ml细胞。
      5. 在4℃下孵育15分钟。
      6. 加入10 ml染色缓冲液,并在4℃下以350 x g离心5 min,除去上清液。
      7. 将细胞沉淀重悬于500μl染色缓冲液中
      8. 将MACS柱放入MACS分离器磁体中,在每列顶部放置一个50微米的杯式过滤器
      9. 通过运行3ml缓冲液平衡柱,并丢弃液体流过。
      10. 将标记的收集管放在每列下方,并将500μl样品通过过滤器添加到色谱柱上
      11. 用另外的500μl染色缓冲液冲洗含有原始样品的管,并通过相同的过滤器加入柱中。
      12. 在每个柱上加入3×3ml缓冲液以冲洗未结合的细胞。
        注意:对于每一轮3毫升冲洗液,等到现有的缓冲液几乎完全通过,然后再加入下一个3 ml。
      13. 计数细胞在10毫升流通。
      14. 放置约200K细胞用于FACS染色对照。
      15. 在4℃下以350×g离心剩余的细胞5分钟,除去上清液,细胞沉淀物应为白色。 
    4. 细胞染色和分选
      以下简要描述我们使用BD FACS Aria IIu或III的工作流程。
      1. 将细胞沉淀物重悬于LSK抗体混合物(步骤C3a)中,体积为100μl/10μg/孔的Ter119耗尽细胞。
      2. 将样品和适当的染色对照在4℃下在黑暗中染色30分钟
      3. 对于每个样品,准备一个含有100μl冷染色缓冲液的Eppendorf管,用于后排序纯度检查,一个含有500μlSFEM以收集分选的细胞。
      4. 用染色缓冲液冲洗染色的细胞,在4℃下以350×g离心5分钟,除去上清液。
      5. 将样品重悬于含有7AAD的染色缓冲液中,终浓度为1μg/ml 7AAD。
        注意:重新悬浮细胞进行排序的缓冲液体积取决于细胞数量和排序设置(例如,当在BD FACS Aria III上分选时,可将7×10 6细胞重新悬浮在700μl中大约4,000事件/秒,流速<4,纯度掩模0-32-0,使用70μm喷嘴)。
      6. 使用单染色控制设置染色补偿矩阵
      7. 设置LSK门,如图3所示

        图3.细胞分选期间LSK HSPC门控的FACS图。 Sca1和c-Kit的荧光减少一个(FMO)对照显示了我们绘制的严格门控,以最大化排序纯度(再分析图)。 FL LSK门口的预期HSC内容在我们的经验中从4-10%不等。

      8. 采取适当的预防措施,确保分选纯度> 95%。
      9. 将所有样品的LSK细胞分选成含有500μl冷SFEM的收集管。
        注意:为每个计划的收件人排序至少11,000个LSK,以补偿步骤E9-E10中的细胞损失。如果需要技术重复,可以从2个收件人处获得3 FLF的足够的LSK(见注释)。
      10. 记录来自排序和储存管的细胞数量。
    5. 转导
      该步骤应在适用于慢病毒转导的程序的适当的细胞培养罩中进行。
      注意:来自胎肝和成骨骨髓的HSPC在转导效率上差异很小,在胎儿HSPCs中达到相同的转导效率所需的病毒较少。在启动协议之前,您的慢病毒条形码库专门针对所选择的群体进行滴定,确定实现转导效率所需的病毒数量,以确保每个接受者的转导细胞数量不超过慢病毒条形码库复杂度的10% 。对于80K的估计条形码复杂度(Lu等人,2011),我们的目标是转导效率为15-30%,意味着每个受体小鼠注射的10K细胞中的1,500-3,000个细胞被条形码化(图4)。这常常产生了在每个接受者中追踪的数百个细胞命运,其中大约60个克隆显示了活性HSC的功能特征和相同条形码转导多于一个细胞的最小情况(Kristiansen等,2016)(参见附注)。/em>


      图4. FL LSK群体内的HSC频率(左)由LSK CD48 - CD150 + 定义的FL HSC的代表性FACS图。 (右)在两个单独的实验中,从2升的6 + 3个别胚胎编制的LSK内的HSC的%频率。 

      1. 从孔中除去Retronectin。
      2. 立即向每个涂覆的孔中加入200μl2%BSA(注意:在Iscove的在PBS中稀释的MDM中的BSA)。
      3. 在室温下孵育30分钟。
      4. 在单独的管中放置约300个LSK细胞用于未转导的对照。
      5. 离心管在350℃下含有LSK细胞,在4℃下5分钟,并除去上清液。
        注意:使用低数量的分选细胞时,必须采取预防措施以尽量减少细胞损失,以获得可重复的结果。因此,省略进一步的计数和过滤步骤,并且在离心后除去上清液时始终保持适当的体积,以免扰乱细胞沉淀。
      6. 将细胞沉淀重新悬浮至最终体积为150μl的细胞因子混合物。
      7. 从涂覆的孔中除去2%BSA,并将来自每个生物复制体的分选细胞平板化成单个孔。还将从步骤D5d放置的LSK细胞置于未转染对照的指定的孔中(步骤C1c)。
      8. 将以前优化的慢病毒量添加到每个孔中,除了未转导的对照孔外。
      9. 轻轻摇动板块分散病毒。
      10. 在加湿的CO 2培养箱中于37℃和5%CO 2孵育过夜。
    6. 受体小鼠的照射
      1. 在剂量之间3小时,辐射2 x 450 cGy的接受者。移植应在第一次给药的24 h内进行 注意:分裂剂量照射对小鼠更温和,同时仍然达到预期的致死性骨髓消除作用。接受者小鼠在照射之前不需要用抗生素治疗。
      2. 在饮用水中加入环丙沙星至终浓度为125 mg/L,持续14天。

  5. 静脉注射
    LSK群体是多种含有茎和祖细胞,其在培养中存活和增殖不同。为了最大限度地减少细胞损失,我们省略了额外的细胞计数步骤,并将第2天的供体细胞数量与第1天的分选细胞数相当。
    注意:以下步骤必须严格遵守慢病毒处理和批准的动物使用协议的生物安全协议。
    第2天
    1. 早上,从第1天到40μm细胞过滤器的过滤器支持细胞可能会在晚上发生显着的聚集。计数细胞。
      注意:细胞计数包括红细胞。
    2. 在4℃下以350μg离心支持细胞5分钟,并重悬于冷的无菌PBS中至浓度为200K /100μl。
    3. 在含有500μl冷PBS的高压灭菌的Eppendorf管中收集每个转导的LSK细胞样品 注意:为了最小化与过滤相关的细胞损失,供体细胞直接从孔中收集用于移植。
    4. 用至少2轮200μlPBS冲洗每个孔,以回收所有细胞,并在相应的Eppendorf管中结合。
      注意:使用显微镜检查所有细胞是否已从孔中采集。
    5. 将每个样品的小体积转移到原始孔中,以在第3天转导效率读数。将细胞因子混合物添加至最终体积为150μl,并将细胞在37℃下孵育5%CO 2一夜之间
    6. 向每个样品管中加入另外500μl冷PBS,并在4℃下以350 x g离心5分钟。
    7. 根据适当的生物安全方案处理慢病毒上清液。
    8. 加入200K支持细胞(100μl来自步骤E2)/10K分选和转导的FL LSK并加入1ml冷PBS。
    9. 以350 x g离心样品5分钟,并根据适当的生物安全方案弃去上清液。
    10. 将沉淀重悬于275μl冷PBS/11,000分选转导的FL LSK中(步骤D4i)。
    11. 使用29磅1/2英寸1毫升的针头,将样品的250μL(相当于10000个分选和转导的LSK)注入每个受体的尾静脉。
      注意:我们通常在热灯下加热受体小鼠5分钟,以促进静脉注射。

  6. 转导效率读数
    第3天
    1. 收集在第2天放置的转导细胞,用于转导效率读数(步骤E5)和未转染的控制到各个FACS管中。
    2. 在4℃下以350×g离心样品5分钟,除去上清液
    3. 将每个样品重悬于含有7AAD的150μl染色缓冲液中
    4. 通过FACS分析活体(7AAD - )细胞之间的GFP + 事件百分比(图5)。



      图5显示了在第3天时GFP转导效率读数的实施例。显示了来自未转导对照(左)和转导样品(右)的7AAD-单链LSK细胞。

  7. 外周血分析
    移植后4-8周
    我们建议检查外周血中GFP + 条形码细胞的初始植入。这是通过受体小鼠的尾静脉出血进行的,并且使用FACS评估粒细胞和B细胞之间的GFP + 细胞百分比。
    注意:与FL HSPCs在第3天的转导效率读数相比,我们经常观察到外周血中的GFP%较高。这可能是由于优先转运高度增殖的HSPC,其更好地植入
  8. 条码抽取
    长期HSCs的克隆动力学在移植后约12周后稳定(Verovskaya等人,2013)。因此,我们建议在移植后16周内阅读条形码分发。
    移植后16-20周
    1. 细胞分选和裂解
      1. 基于提出的具体问题的感兴趣的细胞群体是FACS分类的。如步骤D4所述,将细胞分类到不粘管中的500μl染色缓冲液中,并且可以最初以-80℃作为细胞沉淀储存。
        注意:粒细胞几周的周转时间短,因此是近期HSC活动的良好表现。另一方面,淋巴细胞可以存活数周甚至数月,因此在条形码标签时将含有来源于长期和短期祖细胞的条形码。
      2. 准备细胞裂解缓冲液(参见食谱)。
      3. 将细胞沉淀物在100μl细胞裂解缓冲液/样品(在56℃下2小时,95℃下10分钟灭活蛋白酶K)的基因组DNA中裂解,并在随后的-20℃保存。
        注意:使用较少数量的细胞时,建议直接将其分解为裂解缓冲液以立即溶解。细胞数量是获得群体良好测序覆盖率的限制因素
    2. 图书馆准备和条码深度测序
      1. 根据制造商的建议,使用Ampure XP珠纯化基因组DNA。
      2. 使用高灵敏度方法(例如,Qubit荧光计和Qubit dsDNA HS测定试剂盒)测量基因组DNA浓度。
      3. 使用适当的引物和高保真DNA Taq 聚合酶(例如)将来自每个样品的多达100ng DNA平均分配到两个技术重复PCR反应(每个50ng)中。 ,Q5 DNA聚合酶,NEB)扩增条形码片段(图1)。根据制造商的建议,使用Ampure XP珠纯化扩增的片段。
        可选:根据PCR引物的设计和测序平台的选择,可以进行PCR扩增的第二步,以添加多重适配器。
      4. 使用Bioanalyzer HS DNA分析试剂盒并根据制造商的说明书对Amplions进行定量。正确大小的产品,不污染引物二聚体对成功测序至关重要。根据制造商的建议,可以用另一轮Ampure XP珠粒纯化除去引物二聚体的存在。
      5. 用于测序的池等效量的扩增和索引条形码库 - 所需的产品浓度取决于所选择的测序平台。
        注意:多路复用的程度应该通过选择的测序平台的读取容量和您的条形码单元的预期复杂度来通知。
      6. 经验丰富的技术人员根据制造商的说明进行排序。
        注意:我们迄今为止使用Illumina MiSeq系统(MiSeq Reagent Kit v3 150周期,Illumina)和Ion PGM(Ion 314 Chip Kit v2,Thermo Scientific)平台取得了很好的成功。

数据分析

细胞条形码分析的初始步骤旨在为每个排序样本生成可靠条形码列表,并与条形码读取频率相结合,反映群体内每个克隆的丰度。该分析中最重要的部分是过滤出由引入的条形码,例如,排序错误,否则会偏离结果。过滤步骤是为选择条件库和实验设计定制的。例如,如果选择的条形码库存在参考库,则可将顺序条形码映射回参考,并且过滤步骤可以被简化。在这里,我们简要提供一个例子,分析随机生成的缺少已知参考文献的条形码库(图1; Kristiansen等人,2016)。对于深入的建议,我们提供了一些相关的参考。

  1. 通过使用定制R脚本和R包ShortReads来识别侧翼序列来隔离条形码。
  2. 通过使每个条形码列表例如,Starcode算法(Zorita等人,2015)来纠正排序错误。基于Levenshtein距离,识别和删除由排序错误产生的子条形码,同时读取频率与父条形码的合并。
  3. 基于所检索的总读取次数和采样的单元数量,针对每个排序的总体单独定义低频条形码的截止值,使得低于单个单元的预期读取表示的条形码被排除在进一步分析之外。
  4. 评估从每个群体的两个技术性PCR重复获得的条形码列表中的读取频率与标准的相关性。如前所述(Naik等人,2014)。只有通过技术重复的所有过滤标准的条形码才能进一步分析。
    注意:这些严格的过滤条件为下游分析产生了保守的条形码列表。然而,重要的是要注意,这些过滤步骤可以排除非常低的表示的真实条形码(Naik等人,2014)(图1)

  5. 可以使用自定义脚本进一步分析条形码列表的重叠,读取频率分布。

笔记

  1. 对于不同谱系的细胞的亲缘关系分析,定性条码重叠是关键读数。为了避免错误的正条形码重叠读出,特别重要的是控制相同的条形码不会进入多个单元格。这可以通过比较接收在同一孔中转导的LSK细胞的两个技术重复接收者(图2)的过滤条形码列表来完成(Naik等人,2014)。由于短病毒孵化时间不太可能允许HSC分裂事件,所以两个接收者之间的任何重叠可以被认为是噪声。目前的协议被设计为通过简单地分选(步骤D4i)并且每个每个Retronectin包被的孔的数量的两倍来容纳每个生物复制物(图2)来容纳两个技术重复接收者。在选择条形码读取的人群时,我们还建议将两个预期条形码重叠的人群作为阳性对照。这些控制将允许正确验证您的实验设置中的信号与噪声比(Kristiansen等人,2016)。
  2. 如果实验设置正确,并且应用了适当的控制和条形码滤波步骤,则各个实验之间的重复性应该很高。然而,不同年龄的供体HSPCs在转化的倾向上有很大差异,并将产生年龄特异性条形码分布。

食谱

  1. 染色缓冲液
    0.5%BSA
    注意:使用BSA≥98%。
    2 mM EDTA
    在没有MgCl 2的HBSS中,
  2. 裂解缓冲液
    0.1M Tris-HCl pH 8.5
    0.5 mM EDTA pH 8
    0.2 M NaCl
    0.2%SDS
    0.1 mg/ml蛋白酶K
    注意:使用前立即加入裂解缓冲液。

致谢

该协议最初在Kristiansen等人描述和改编。 (2016)。这项工作得到瑞典癌症基金会,瑞典研究委员会,StemTherapy以及The Knut和Alice Wallenberg基金会的资助。

参考文献

  1. Bystrykh,LV和Belderbos,ME(2016)。克隆使用细胞条形码分析细胞:当数字和大小重要时。 Methods Mol Biol 1516:57-89。
  2. Bystrykh,LV,de Haan,G.and Verovskaya,E.(2014)。< 造血干细胞的条形码载体文库和逆转录病毒或慢病毒条形码。方法Mol Biol 1185:345-360。
  3. Gerrits,A.,Dykstra,B.,Kalmykowa,OJ,Klauke,K.,Verovskaya,E.,Broekhuis,MJ,de Haan,G.and Bystrykh,LV(2010)。< a class = insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/20093403"target ="_ blank">用于造血系统中克隆分析的细胞条形码工具。 em> 115(13):2610-2618。
  4. Kristiansen,TA,Jaensson Gyllenback,E.,Zriwil,A.,Bjorklund,T.,Daniel,JA,Sitnicka,E.,Soneji,S.,Bryder,D.and Yuan,J.(2016)一个class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27533015"target ="_ blank">细胞条形码链接到胎儿造血干细胞的B-1a B细胞电位状态为单细胞水平。免疫力 45(2):346-357。
  5. Lu,R.,Neff,NF,Quake,SR和Weissman,IL(2011)。使用高通量测序结合病毒基因条形码来跟踪单个造血干细胞体内。 Nat Biotechnol 29(10 ):928-933。
  6. Naik,SH,Perie,L.,Swart,E.,Gerlach,C.,van Rooij,N.,de Boer,RJ和Schumacher,TN(2013)。  早期造血祖细胞的多样性和遗传谱系印记 自然 496(7444 ):229-232。
  7. Naik,SH,Schumacher,TN和Perie,L。(2014)。  细胞条形码:技术鉴定。 Exp Hematol 42(8):598-608。
  8. Schepers,K.,Swart,E.,van Heijst,JW,Gerlach,C.,Castrucci,M.,Sie,D.,Heimerikx,M.,Velds,A.,Kerkhoven,RM,Arens,R.and Schumacher ,TN(2008)。通过以下方式解剖T细胞谱系关系细胞条形码。 J Exp Med 205(10):2309-2318。
  9. Verovskaya,E.,Broekhuis,MJ,Zwart,E.,Ritsema,M.,van Os,R.,de Haan,G.and Bystrykh,LV(2013)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/23719303"target ="_ blank">通过使用细胞条形码的定量克隆分析揭示了年轻和老年鼠型造血干细胞的异质性。 > Blood 122(4):523-532。
  10. Zorita,E.,Cusco,P.和Filion,GJ(2015)。  星号:基于全对搜索的序列聚类。生物信息学31/12(12):1913-1919。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kristiansen, T. A., Doyle, A. and Yuan, J. (2017). Lentiviral Barcode Labeling and Transplantation of Fetal Liver Hematopoietic Stem and Progenitor Cells. Bio-protocol 7(8): e2242. DOI: 10.21769/BioProtoc.2242.
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