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Jan 2021

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Differentiation of Human Induced Pluripotent Stem Cell into Macrophages
人诱导多能干细胞向巨噬细胞的分化   

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Abstract

As a model to interrogate human macrophage biology, macrophages differentiated from human induced pluripotent stem cells (hiPSCs) transcend other existing models by circumventing the variability seen in human monocyte-derived macrophages, whilst epitomizing macrophage phenotypic and functional characteristics over those offered by macrophage-like cell lines (Mukherjee et al., 2018). Furthermore, hiPSCs are amenable to genetic manipulation, unlike human monocyte-derived macrophages (MDMs) (van Wilgenburg et al., 2013; Lopez-Yrigoyen et al., 2020), proposing boundless opportunities for specific disease modelling.


We outline an effective and efficient protocol that delivers a continual production of hiPSC-derived-macrophages (iMACs), exhibiting human macrophage surface and intracellular markers, together with functional activity.


The protocol describes the resuscitation, culture, and differentiation of hiPSC into mature terminal macrophages, via the initial and intermediate steps of expansion of hiPSCs, formation into embryoid bodies (EBs), and generation of hematopoietic myeloid precursors.


We offer a simplified, scalable, and adaptable technique that advances upon other protocols, utilizing feeder-free conditions and reduced growth factors, to produce high yields of consistent iMACs over a period of several months, economically.

Keywords: Macrophages (巨噬细胞), Human induced pluripotent stem cells (人诱导多能干细胞), Embryoid Bodies (胚状体), Feeder-free (采用无饲养层), Differentiation (分化)

Background

Macrophages occupy multiple tissues and orchestrate both innate and adaptive immune responses. Research into macrophage function and characterization has been impeded by multiple limitations suffered by model systems, such as human primary monocyte-derived macrophages (MDMs) and leukaemia-derived human macrophage-like cell lines (Hale et al., 2015; Alasoo et al., 2015; Mukherjee et al., 2018). The large volumes of blood required from donors presents ethical and logistical challenges, only exacerbated by the need for repeated venesection owing to the high variability observed within and between donors (van Wilgenburg et al., 2013). Human myeloid cell lines, such as THP-1, demonstrate karyotypical abnormalities and do not entirely represent human macrophages phenotypically or functionally (van Wilgenburg et al., 2013; Lopez-Yrigoyen et al., 2020, Baldassarre, et al., 2021).


Furthermore, human macrophages are typically resistant to genetic manipulation, hampering disease-specific modelling (Hale et al., 2015). Technology that employs human induced pluripotent stem cells (hiPSCs), boasting a self-renewing source of cells capable of differentiation into three germ layers, that are amenable to genetic manipulation, poses multiple attractive qualities as a model system over others (Alasoo et al., 2015; Lachmann et al., 2015; Alasoo et al., 2018; Lee et al., 2018, Nenasheva et al., 2020, Mukhopadhyay et al., 2020).


This protocol improves upon predecessors by the utilisation of feeder-free culture conditions with reduced requirements for growth factors, whilst still generating high yields of consistent terminally differentiated macrophages, which share phenotypical and functional characteristics to primary human MDMs. Millions of hiPSC-derived-macrophages (iMACs) can be conveniently harvested every 5–7 days from generated embryoid bodies (EBs) maintained in culture for several months. The protocol is easy to follow, cost-effective, and can be scaled to the user’s requirements, creating reproducible results for repeated experiments over time.

Materials and Reagents

  1. Human induced Pluripotent Stem cells culture and expansion

    1. Human induced Pluripotent Stem Cells used in the development of this protocol:

      1. NL9—a fibroblast-derived hiPSC line from a healthy individual, which is widely available (Baghbaderani et al., 2016), obtained from the National Heart, Lung & Blood Institute (NHLBI) iPSC core (please see the Acknowledgments section for more information on the source of the NL9 hiPSC line)

      2. KOLF_2—a skin tissue derived hiPSC line from a healthy individual, generated at the Sanger institute, as part of the HipSci initiative

        (https://www.hipsci.org/lines/#/lines/HPSI0114i-kolf_2)

    2. Complete Essential 8 Basal Medium (Thermo Fisher, catalog number: A1517001)

    3. Vitronectin (rhVTN-N) (Gibco, catalog number: A14700) 500 µg/mL

    4. Dulbecco's phosphate-buffered saline (DPBS) without Ca2+/Mg2+ 500 mL (Thermo Fisher, catalog number: 14190144) (Storage conditions: 15–30°C. Shelf life: 36 months from date of manufacture)

    5. ROCK inhibitor (ROCKi) (Y-27632 dihydrochloride) (Sigma, catalog number: Y0503-1MG)

    6. 10 cm or 6 well tissue culture treated plates (Corning, catalog number: 430167 or 3516)

    7. 0.22 µm filter steri-cups (Merck Millipore, catalog number: 15780319)

    8. 15 mL and 50 mL Falcon tubes (Falcon, catalog numbers: 352097 and 352098)


  2. Embryoid body generation in a feeder-free system

    1. 0.5 M ultrapure Ethylenediaminetetraacetic acid (EDTA) (Invitrogen, catalog number: 15575020), 100 mL

    2. Cryopreservation/freezing medium: KnockOut serum replacement (KSR) (Gibco, catalog number: 10828028), 500 mL

    3. Dimethyl sulfoxide (DMSO) (Sigma, catalog number: D2438), 50 mL

    4. Dulbecco's phosphate-buffered saline (DPBS) with Ca2+/Mg2+, 500 mL (Thermo Fisher, catalog number: 14040091) (Storage conditions: 2–8°C. Shelf life: 36 months from date of manufacture)

    5. Bovine serum albumin (BSA) low endotoxin tissue culture-grade (Sigma, catalog number: A9543-5G)

    6. Recombinant Human BMP-4 Protein 10 µg (R&D Systems, catalog number: 314-BP-010)


  3. Embryoid Body Transfer, Culture and Macrophage Differentiation

    1. Sterile water for embryo transfer (Sigma, catalog number: W1503)

    2. Gelatin from porcine skin (Sigma, catalog number: G1890)

    3. X-VIVO 15 Serum-free Hematopoietic Cell Medium 500 mL (Lonza, catalog number: LZBE02-060F)

    4. L-Glutamine (200 mM), 100 mL (Gibco, catalog number: 25030181)

    5. Penicillin-Streptomycin (10,000 U/mL), 100 mL (Gibco, catalog number: 15140122)

    6. 2-Mercaptoethanol (Sigma, catalog number: M3148)

    7. Recombinant human M-CSF (Peprotech, catalog number: 300-25)

    8. Recombinant human IL-3 (Peprotech, catalog number: 200-03)

    9. RPMI 1640 Medium 500 mL (Sigma, catalog number: R0883)

    10. Ultra-low adherent U bottom 96-well plates (Costar, catalog number: 7007)

    11. 10 cm tissue culture dishes or 6-well plates

    12. 70-100 µm cell strainers (Falcon)

    13. Complete Essential 8 Basal Medium (see Recipes)

    14. Vitronectin 1 mL vial (see Recipes)

    15. ROCK inhibitor (ROCKi) (see Recipes)

    16. 0.5 mM Ethylenediaminetetraacetic acid (EDTA) solution (see Recipes)

    17. Cryopreservation/freezing Medium (see Recipes)

    18. Bovine serum slbumin (BSA) (see Recipes)

    19. Recombinant Human BMP-4 Protein (see Recipes)

    20. Sterile water for embryo transfer (see Recipes)

    21. Gelatin from porcine skin (see Recipes)

    22. EB-Myeloid precursor base medium (see Recipes)

    23. Recombinant Human M-CSF (see Recipes)

    24. Recombinant Human IL-3 (see Recipes)

    25. Macrophage Differentiation base medium (see Recipes)


    Flow Cytometry Antibodies used in this protocol

    1. CD14 (BV605, Biolegend, catalogue number: 301834)

    2. CD16 (APC, eBioscience, catalogue number: 17-0168-42)

    3. CD80 (BV711, Biolegend, catalogue number: 305236)

    4. CD86 (PE-Dazzle594, Biolegend, catalogue number: 374217)

    5. CD206 (PerCP/Cyanine 5.5, Biolegend, Catelogue number: 321122)

    6. CD204 (PE/Cyanine7 Biolegend, catalogue number: 371908)


    Primer sequences used in this protocol

    1. TATA-Binding Protein (TBP) (F: GGGAAGGGGCATTATTTG, R: CCAGATAGCAGCACGGTA).

    2. CD68 (F: GGAAATGCCACGGTTCATCCA, R: TGGGGTTCAGTACAGAGATGC)

    3. CSFR1 (F: TCCAACATGCCGGCAACTA, R: GCTCAAGTTCAAGTAGGCACTCTCT)

    4. CD163 (F: TTTGTCAACTTGAGTCCCTTCAC, R: TCCCGCTACACTTGTTTTCAC)

    5. SOX2 F: GCTACAGCATGATGCAGGACCA, R: TCTGCGAGCTGGTCATGGAGTT)

    6. NANOG (F: CTCCAACATCCTGAACCTCAGC, R: CGTCACACCATTGCTATTCTTCG)

    7. OCT4 (F: CCTGAAGCAGAAGAGGATCACC, R: AAAGCGGCAGATGGTCGTTTGG)

Equipment

  1. Class 2 microbiological safety hood or cabinet using an aseptic technique

  2. 37°C incubator with 5% CO2

  3. Centrifuge

  4. Phase-contrast microscope (4×, 10×, 40× magnification)

  5. Water bath set at 37°C

  6. -80°C storage

  7. Liquid Nitrogen storage

  8. Cell freezing containers (“Mr Frosty”)

  9. Pipette controller and selection of different volume stripettes (5 mL, 10 mL), which usually have large-bore orifices

  10. Pipettes (P1000, P200, P20, P2) and corresponding sterile tips.

  11. ParafilmTM

Procedure

General Considerations:

  1. All cell culture, media preparation, tissue culture vessels, and other laboratory work is to be conducted inside a Class 2 microbiological hood, using aseptic technique.

  2. The hood should be cleaned thoroughly before use with 3% of Distel (or equivalent disinfectant) and 70% ethanol.

  3. Prepare all media, solutions, and reagents at room temperature (RT). See the Recipes section for more detailed information on media preparation.


  1. Human induced Pluripotent Stem cell culture and expansion

  1. Prepare all cell maintenance base media and reagents, as described in the Materials and Reagents or Recipes sections.

  2. Prepare vitronectin plates. Vitronectin is a glycoprotein used to coat the surface of cell culture plates, to facilitate hiPSC attachment and spreading. Thaw one 60 μL aliquot to RT and dilute into 6 mL DPBS without Ca2+/Mg2+. If using a 6-well plate, add 1–1.5 mL per well; alternatively, add 6 mL to one 10 cm dish. Ensure the vitronectin covers the entire surface, and incubate in a hood for 1–2 h at RT. Vitronectin plates can be sealed with ParafilmTM and stored at 4°C for 5 days. Allow to warm to RT for 1–2 h prior to use.

  3. Prepare the E8 basal medium by adding the thawed supplement and allowing the media to warm to RT. We recommend that thawed hiPSCs are cultured and expanded in E8 basal media, and that a master stock of hiPSC lines is frozen and stored early on.

  4. Set up a 15 mL Falcon with 9 mL of E8 media plus 0.312 µM ROCKi for one frozen vial of 1 mL of hiPSC, i.e., 1 µL of ROCKi into a final volume to 10 mL. Although ROCKi allows attachment and recovery of hiPSC, whilst preventing apoptosis & spontaneous differentiation, it must be removed from the media after 24 h, to prevent interference with colony expansion.

  5. Partially thaw the frozen hiPSCs quickly until just defrosted, with some ice remaining in the vial, using the water bath at 37°C (thawing should be quick, to avoid prolonged contact with DMSO; however, do not warm the entire vial to 37°C). Add 1 mL of E8 in a drop-wise fashion to the vial and gently transfer the cells to the 15 mL Falcon, using a wide-bore pipette tip or stripette. Invert the tube to mix and dilute out the DMSO.

    Note: The exact cell number or density in a cryovial is usually not known nor determined at the time of freezing, to avoid creating a single cell suspension, and to improve recovery after thawing for subsequent culture. Between 6–10 cryovials are generated from one previously 70–80% confluent 10 cm plate of hiPSCs.

  6. Spin at 290 × g and 20°C for 3 min. Discard the supernatant.

  7. Aspirate the diluted vitronectin from the plate surface. One thawed cryovial should give a small 5–20 µL cell pellet in a 15 mL Falcon after centrifugation. Resuspend the pellet gently in the desired volume of E8 medium supplemented with 3.12 µM ROCKi (1 µL: 1,000 µL), avoiding excessive pipetting.

    Note: ROCKi has been previously used in earlier adapted protocols at 10 µM, but we have titrated this down further, and have seen effective hiPSC attachment and recovery at a lower concentration of 3.12 µM.

  8. Plate 2 mL of cell suspension per well of a 6-well plate or 8 mL per 10 cm dish; aim to use minimum volumes of media to enhance adherence. One frozen vial of hiPSC (equivalent to 1 well of a 6-well plate, or one-tenth of a 10 cm dish at 70–80% confluence), can be split into 2–3 wells of a 6-well plate or if the hiPSC line demonstrates good recovery, then cells can be seeded less densely straight into a 10 cm dish. Recovery is described as good when multiple small colonies have attached to the vitronectin plate 24 h after seeding (see Figure 1), and colony expansion occurs in the following days (see Figure 2).

  9. Place the plate into a humidified 37°C incubator with 5% CO2. Ensure the plate is initially gently agitated to distribute the colonies evenly over the vitronectin.

  10. Check for attachment under a phase-contrast microscope after 24 h (see Figure 1).

  11. Change the medium every 24 h, remove the unattached colonies, dead cells, and spent media with a stripette or aspirator. If there are a lot of dead cells or debris, then wash the plate with DBPS before replacing the E8 media. Replace with fresh E8 basal media gently, using a stripette against the side wall of the vessel (ROCKi supplementation is not required after the first 24 h). hiPSCs are difficult to maintain in culture and are prone to differentiation, media cytokines, and factors that maintain pluripotency, necessitating daily media change.

  12. Continue to change media daily until the cells reach 70–80% confluency and ready for passaging. Do not allow the clusters/colonies to fuse together (see Figures 3, 4, and 5), which helps to avoid spontaneous differentiation. Once thawed hiPSCs are established, subsequent steps, culture systems, and further differentiation should take place after 1–2 passages.



Figure 1. Day 1 hiPSC attachment and growing in small closely neighbouring colonies on vitronectin.

Images taken using an Olympus CKX41 Inverted Microscope with 10× objective.



Figure 2. Example of heterogenous hiPSC colonies growing and expanding on vitronectin.

Image taken using EVOSTM XL Core imaging system, with a 4× objective.



Figure 3. A 70–80% confluent plate of hiPSCs, growing in colonies that are almost touching one another, on the screen of an EVOSTM XL Core imaging system, imaged with a 4× objective.



Figure 4. Close-up image of the 70–80% confluent plate, where multiple large colonies are visible to the naked eye.



Figure 5. hiPSC at 70–80% confluency prior to passaging. Images obtained with a Olympus CKX41 Inverted Microscope, equipped with a 20× objective.


  1. Passaging and freezing of hiPSC

  1. Prepare vitronectin plates as before.

  2. Remove the exhausted media from the plate with hiPSC at 70–80% confluency and wash the plate/wells twice with DPBS without Ca2+/Mg2+ (approximately 6 mL for 10 cm dish).

  3. Remove the DPBS and add 6 mL of 0.5 mM PBS-EDTA solution, allowing full coverage of the plate surface.

  4. Observe the cells under a phase-contrast microscope every 2 min for up to 4 min, to monitor cell morphology changes and detachment (bright halos and edge enhancement to the colonies) but do not to allow full detachment and separation of cells into single cells.

  5. Gently remove and discard the PBS-EDTA with a stripette by tilting the plate, but do not to dislodge the colonies.

  6. Using a stripette add 8 mL of E8 media to the 10 cm plate aiming to detach and wash the colonies in small clumps and clusters into suspension, by repeated aspiration and flushing of the media through the stripette up to four times with the same 8 mL volume. Collect the media and cells into a new 50 mL Falcon. Repeat this step up to twice more to maximise colony recovery, while avoiding excessive agitation and pipetting, which will result in single cells.

  7. Spin cells at 290 × g and 20°C for 3 min. Discard the supernatant.

  8. Resuspend the colonies in fresh E8 base media supplemented with 3.12 mM ROCKi at the desired splitting ratio (1:5–1:30, depending on the growth rate of the hiPSC line).

  9. Aspirate the vitronectin solution from pre-prepared plates. Transfer and seed the split colonies into a new vitronectin plate/well.

  10. Cryopreservation/freezing of hiPSC:

    To freeze hiPSC cells, freshly prepare freezing medium (10% DMSO in KSR) at RT. After harvesting the hiPSCs (Steps B2–B7), resuspend the cell pellet in 10% DMSO-KSR and transfer 1 mL of cell suspension per cryovial. It is important that the cells spend minimal time in presence of DMSO at RT. Cryovials should be placed immediately into a Mr. Frosty or appropriate freezing container and into the -80°C freezer and then into liquid nitrogen after 24 h, for long term storage.

    Note: The volume of freezing medium to prepare will depend on the number of cryovials to be stored. We advise 1 mL of freezing media per cryovial and 2 cryovials per well of a 6-well plate and 6–10 mL or 6–10 cryovials per 10 cm dish at 70–80% confluency.


  1. Embryoid Body formation utilizing a feeder-free system

  1. After harvesting the hiPSC (as described in Steps B2–B7), add 1 mL of fresh E8 media and pipette the hiPSCs up and down more than usual (3–4 pipetting strokes), to break up clumps and create a single-cell suspension.

  2. Using a haemocytometer, count the cells in 10 µL of cell suspension, after adding 10 µL of Trypan blue (dilution factor = 2), and determine cell density:


    (Number of cells in 3 squares / 3) × dilution factor × 10,000 = Number of cells/mL


  3. Make individual, homogenous, and equally-sized EBs using an ultra-low adherent U bottom 96-well plate (1× EB per well). With the knowledge of the total cell number harvested above, both the desired EB size (cell number) and the overall total number of the individual EBs to be made, can be calculated. For example, if 3 × 106 hiPSCs are harvested = 100× individual EBs of 3 × 104 size can be made.


    Note: The size of EB may need to be determined for each hiPSC line. However, in our hands, EB sizes of between 1 × 104–5 × 104 cells generate the highest yields of myeloid precursors for both the hiPSC lines used in the generation of this protocol (EBs up to 1 × 105 cells can also work well).

  4. Each individual EB is made up in 100 µL of E8 media supplemented with 50 ng/mL recombinant BMP-4 and 3.12 µM ROCKi. Depending on the desired EB size, create a cell suspension of 1 × 105–10 × 105 cells per mL.

    For the example calculated above, a cell suspension of 3 × 105 cell/mL should be created to generate individual EBs of 3 × 104 size in 100 µL of supplemented E8 per well of ultra-low adherent U bottom 96 well plate.

  5. Transfer 100 µL of hiPSC cell suspension into each well of an ultra-low adherent U bottom 96-well plate.

  6. Spin the plate at 800 rpm for 1 min and transfer to a humidified 37°C incubator with 5% CO2.

  7. Leave these EBs undisturbed for 48 h and, on day 2 of culture, remove 50 µL of spent media and carefully replace with 50 µL of E8 media supplemented with 50 ng/mL BMP-4 only, aiming to not disturb the forming EB. Repeat for day 3 (see Figure 6). ROCKi can interfere with late EB and germ layer formation.

  8. It is unusual to find more than one EB per well (see Figures 7 and 8); however, providing the appearance of each EB within one well is similar to that seen in Figure 6, with a clear rim and 3D structure, then all EBs within the well can be transferred. Whenever multiple small clumps or a broken and shattered EB appearance is found, the well and cellular material should be discarded.

  9. On day 4, prepare gelatin-coated 10 cm dishes. Add 6 mL of 0.1% gelatine solution to a 10 cm dish and incubate in a hood for 1–2 h, allowing the gelatin to coat the entire surface and warm to RT (plates can be pre-prepared and stored at 4°C for 5 days).

  10. Remove the gelatin solution from the plate prior to EB transfer.

  11. Using wide bore P1000 filter tips (alternatively, P1000 filter tips can be cut higher up their length with a scalpel, to create a wide bore orifice), carefully aspirate the individual EBs which are easily visible, and transfer them to the gelatin-coated plate (Figure 9). The number of EBs per cm2 may need to be determined per hiPSC line, nevertheless, in our experience, up to 35× EBs per 10 cm dish provides a high yield of myeloid precursors.



    Figure 6. Embryoid Bodies should have this appearance after 24 h in culture.

    This is an EB in one well of a 96-well ultra-low adherent plate, on day 4 prior to transfer into a gelatin plate. EB size is 3 × 104 hiPSCs. Images taken using EVOSTM XL Core imaging system with a 4× objective.



    Figure 7. Single individual EBs in the centre of each well of a 96-well ultra-low adherent plate are visible to the naked eye.



    Figure 8. Single EBs at close range and labelled with arrows.



    Figure 9. Single EB aspirated into wide bore pipette for transfer into gelatin-coated plate below.


  12. Once transferred, gently tilt the plate and remove the spent EB media from the plate, without removing EBs.

  13. Slowly add 10–12 mL of fresh EB-Myeloid precursor media supplemented with 50 ng/mL M-CSF, which is critical for myeloid differentiation, and 25 ng/mL IL-3, which is critical for hematopoietic specification and proliferation.

  14. Carefully move these plates to a humidified 37°C incubator with 5% CO2. And leave undisturbed for 1 week, to allow EBs to attach to the gelatin (see Figure 10).

  15. At day 7, carefully remove spent EB media with a large bore stripette to avoid damaging any unattached EBs. Take care not to dislodge any attached EBs and pass the media through a 70–100 µm strainer over a 50 mL Falcon tube, to catch and retain all the unattached EBs that are in suspension.

  16. Return the EBs in the strainer back into the original plate, by inverting the strainer over the 10 cm dish and gently adding fresh EB media supplemented with 50 ng/mL M-CSF and 25 ng/mL IL-3 warmed to RT over the inverted strainer. Continue to take care not to dislodge the attached EBs in the plate.

  17. Once the EB media has been exchanged, and EBs returned to their dish, spin the 50 mL Falcon at 290 × g and 20°C for 3 min. At week 3, this tube will contain the harvested myeloid precursors in suspension. Discard supernatant.

  18. Add 1 mL of macrophage differentiation media to the cell pellet and observe the appearance of the cells under a microscope, counting them with a haemocytometer (see Step C1).

  19. EB media should continue to be exchanged as described in Steps C14–C17 each week. Depending on the hiPSC line, myeloid precursors should be harvested from weeks 3–4 of EB culture.

  20. EBs can be maintained in culture for several months. In our hands, myeloid precursor yields begin to fall from 12–16 weeks.



    Figure 10. Embryoid Body attachment: 1 week post transfer onto the gelatin plate.

    Images taken using EVOSTM XL Core imaging system, with a 4× objective.


  1. Macrophage differentiation

  1. Harvest the myeloid precursors as described in Step C14–C17. Following counting, resuspend the harvested myeloid precursors in the desired volume of macrophage differentiation media supplemented with high concentration 100 ng/mL M-CSF.

  2. Seed the cells into the desired culture plates, ideally 0.5 × 106–1 × 106 in approximately 8–10 mL of macrophage differentiation media per 10 cm dish, or alternatively 1.5 × 105 in 3 mL per well of a 6-well plate, or 1.5 × 104 in 150 µL per well on a 96-well plate (see Figure 11a).

  3. Transfer the cells to a humidified 37°C incubator with 5% CO2 and leave undisturbed for 5–7 days (see Figure 11b).

  4. Cells can then be lifted with 0.5 mM EDTA solution, with or without the help of a cell scraper. The 0.5 mM EDTA solution should be left covering the cells for 10 min. Cells should be recounted and phenotypically characterized by flow cytometry or qPCR, or functionally thereafter (see Figures 12 and 13).

    Note: The number of macrophages should be similar to the number of myeloid precursors seeded onto the plate, but this number can vary between cell lines and also be dependent on EB size.

  5. Cells left in culture for 10–14 days post harvest tend to become fibroblast-like (Figure 11c).



Figures 11. Myeloid precursor apperance.
 (a) Myeloid precursors following harvest (b) differentiated attached macrophages 7 days later. Images taken using EVOSTM XL Core imaging system with a 20× objective. (c) Fibroblast-like cells at 14 days post harvest. Image taken using Olympus CKX41 Inverted Microscope with a 20× objective.

Data analysis


Figure 12. Representative flow cytometry plots demonstrating positivity for macrophage markers CD14 (BV605), CD16 (APC), CD80 (BV711), CD86 (PE-Dazzle594), CD206 (PerCP/Cyanine 5.5), and CD204 (PE/Cyanine7) in differentiated iMACS. (Black = Unstained iMACs, Red = iMACs).


Figure 13. Representative qPCR data demonstrating macrophage characterization of iMACs. Relative expression is normalized to TATA-Bing Protein (TBP).

There is increased expression in pan-macrophage markers: CD68, CSFR1, and CD163 in iMACs over hiPSCs, and increased expression of pluripotent markers SOX2, NANOG, and OCT4 are seen in hiPSCs compared to iMACs.

Recipes

All media preparation and laboratory work to be conducted inside a Class 2 microbiological hood using aseptic techniques. Prepare all media, solutions and reagents at RT.


  1. Complete Essential 8 Basal Medium

    Made up of two components:

    490 mL of media (store at 2–8°C and protect from light) with 10 mL of supplement (50×) (store at -5 to -20°C. Shelf life: 12 months).

    To reconstitute, thaw supplement to RT and add to 490 mL of media.

    Alternatively, aliquot supplement into smaller volumes for longer storage at -20 to -80°C and thaw as required.

    Swirl or invert to mix.

    Store reconstituted base media at 2–8°C, shelf-life 2 weeks.

    Allow to warm to RT before use.

  2. Vitronectin: 1 mL vial (store at -80°C. Shelf-life: 24 months)

    Divide into 60 µL sterile aliquots.

    To coat a plate: Dilute 60 µL into 6 mL of DPBS without Ca2+/Mg2+, or use at a concentration 2.5–10 µg/mL.

    Invert to mix.

    Ensure parafilm is applied to seal the edges of the plate, for any pre-prepared plates prior to storage.

  3. ROCK inhibitor (ROCKi) (store at 2–8°C)

    Prepare a 3.12 mM working concentration stock, i.e., reconstitute 1 mg with 1 mL of sterile ultrapure water.

    Divide into 10 µL aliquots (store at -20°C).

    For use at 0.312 µM or 3.12 µM final concentrations.

    Once thawed, stock ROCKi can be stored at 2–8°C for 7 days.

  4. 0.5 mM Ethylenediaminetetraacetic acid (EDTA) solution (store at RT)

    Dilute 50 µL in 50 mL of DPBS without Ca2+/Mg2+ for a 0.5 mM working concentration, store at RT, and use on the day of preparation.

  5. Cryopreservation/freezing Medium (Store at -5 to -20°C and protect from light. Shelf-life 18 months)

    Aliquot into small volumes.

    Add 1 mL of DMSO to 9 mL of thawed KnockOut serum replacement (KSR), to make a final concentration of 10% DMSO for freezing.

    Use on the day of preparation and for long term storage of hiPSC in liquid nitrogen.

  6. Bovine serum slbumin (BSA) (store at 2–8°C)

    Add 100 mg BSA to 100 mL of DPBS with Ca2+/Mg2+, mix, and leave in 37°C water bath until the BSA has dissolved.

    Filter-sterilise (store at 2–8°C, shelf-life 4 months).

  7. Recombinant Human BMP-4 Protein

    (Store at -20 to -70°C. Shelf life 12 months)

    Reconstitute to 50 µg/mL working concentration stock (10 µg in 200 µL of 4 mM HCl and 0.1% BSA).

    Aliquot into small volumes.

    For use at 50 ng/mL final concentration (store at -20 to -80°C).

  8. Sterile water for embryo transfer (store at RT)

  9. Gelatin from porcine skin (store at RT)

    Make 0.1% gelatin solution: add 500 mg gelatin to 500 mL of water, heat to 56°C to dissolve, and filter-sterilise (Ssore at 4°C for up to 4 months).

  10. EB-Myeloid precursor base medium

    To 500mL of X-VIVO 15 Serum-free Hematopoietic Cell Medium, add:

    2 mM (5 mL) L-Glutamine (store at -5 to -20°C. Shelf life 24 months),

    100 IU/ mL (5 mL) Penicillin-Streptomycin (store at -5 to -20°C. Shelf life 12 months)

    0.1 mM (3.5 µL) 2-Mercaptoethanol (store at RT)

    Filter-sterilise (store at 2–8°C).

  11. Recombinant Human M-CSF

    Reconstitution: add 500 µL of 0.1% BSA solution to 0.5 mg lyophilised rhM-CSF, to achieve a 1 mg/mL stock concentration (store in 5 µL aliquots at -20 to -80°C. Shelf life 12 months).

    Thaw to add to fresh media prior to use (for use at 50ng/mL final concentration).

    Avoid repeated free-thaw cycles.

  12. Recombinant Human IL-3

    Reconstitution: add 1 mL of 0.1% BSA solution to 0.5 mg lyophilised rhIL-3, to achieve a 0.5 mg/mL stock concentration (store in 5 µl aliquots at -20 to -80°C. Shelf life 12 months).

    Thaw to add to fresh media prior to use (for use at 25 ng/mL final concentration).

    Avoid repeated free-thaw cycles.

  13. Macrophage Differentiation base medium

    To 500mL of RPMI 1640 Medium add:

    10% (50 mL) of foetal bovine serum

    2 mM (5 mL) L-Glutamine

    Filter-sterilise (store at 2–8°C).

Acknowledgments

Funding for research provided by Medical Research Council, Kidney Research UK, National Institute for Health Research, The Royal Society, and The Wellcome Trust.

The NL9 hiPSC used in the development of this protocol were generously donated by Professor Claudia Kemper and the National Heart, Lung and Blood Institute (NHLBI) iPSC core (https://www.nhlbi.nih.gov/science/ipsc-core/research), Bathesda, Maryland, USA.

Thanks also go to Professor Anthony Dorling at King’s College London, for the patient guidance, supervision and enduring encouragement.

This protocol is derived and adapted from previously published protocols (van Wilgenburg et al., 2013; Mukherjee et al., 2018; Lopez- Yrigoyen et al., 2020).

Competing interests

There are no financial or non-financial competing interests to declare.

References

  1. Alasoo, K., Martinez, F. O., Hale, C., Gordon, S., Powrie, F., Dougan, G., Mukhopadhyay, S. and Gaffney, D. J. (2015). Transcriptional profiling of macrophages derived from monocytes and iPS cells identifies a conserved response to LPS and novel alternative transcription. Sci Rep 5: 12524.
  2. Alasoo, K., Rodrigues, J., Mukhopadhyay, S., Knights, A. J., Mann, A. L., Kundu, K., Consortium, H., Hale, C., Dougan, G. and Gaffney, D. J. (2018). Shared genetic effects on chromatin and gene expression indicate a role for enhancer priming in immune response. Nat Genet 50(3): 424-431.
  3. Baldassarre, M., Solano-Collado, V., Balci, A., Colamarino, R. A., Dambuza, I. M., Reid, D. M., Wilson, H. M., Brown, G. D., Mukhopadhyay, S., Dougan, G. et al. (2021). The Rab32/BLOC-3-dependent pathway mediates host defense against different pathogens in human macrophages. Sci Adv 7(3): eabb1795.
  4. Lopez-Yrigoyen, M., May, A., Ventura, T., Taylor, H., Fidanza, A., Cassetta, L., Pollard, J. W. and Forrester, L. M. (2020). Production and Characterization of Human Macrophages from Pluripotent Stem Cells. J Vis Exp(158). DOI: 10.3791/61038-v.
  5. Mukherjee, C., Hale, C. and Mukhopadhyay, S. (2018). A Simple Multistep Protocol for Differentiating Human Induced Pluripotent Stem Cells into Functional Macrophages. Methods Mol Biol 1784: 13-28.
  6. Mukhopadhyay, S., Heinz, E., Porreca, I., Alasoo, K., Yeung, A., Yang, H. T., Schwerd, T., Forbester, J. L., Hale, C., Agu, C. A., et al. (2020). Loss of IL-10 signaling in macrophages limits bacterial killing driven by prostaglandin E2. J Exp Med 217(2): e20180649.
  7. van Wilgenburg, B., Browne, C., Vowles, J. and Cowley, S. A. (2013). Efficient, long term production of monocyte-derived macrophages from human pluripotent stem cells under partly-defined and fully-defined conditions. PLoS One 8(8): e71098.
  8. Lee, C. Z. W., Kozaki, T. and Ginhoux, F. (2018). Studying tissue macrophages in vitro: are iPSC-derived cells the answer? Nat Rev Immunol 18(11): 716-725.
  9. Hale, C., Yeung, A., Goulding, D., Pickard, D., Alasoo, K., Powrie, F., Dougan, G. and Mukhopadhyay, S. (2015). Induced pluripotent stem cell derived macrophages as a cellular system to study salmonella and other pathogens. PLoS One 10(5): e0124307.
  10. Lachmann, N., Ackermann, M., Frenzel, E., Liebhaber, S., Brennig, S., Happle, C., Hoffmann, D., Klimenkova, O., Luttge, D., Buchegger, T., et al. (2015). Large-scale hematopoietic differentiation of human induced pluripotent stem cells provides granulocytes or macrophages for cell replacement therapies. Stem Cell Reports 4(2): 282-296.
  11. Nenasheva, T., Gerasimova, T., Serdyuk, Y., Grigor'eva, E., Kosmiadi, G., Nikolaev, A., Dashinimaev, E. and Lyadova, I. (2020). Macrophages Derived From Human Induced Pluripotent Stem Cells Are Low-Activated "Naive-Like" Cells Capable of Restricting Mycobacteria Growth. Front Immunol 11: 1016.
  12. Baghbaderani, B. A., Syama, A., Sivapatham, R., Pei, Y., Mukherjee, O., Fellner, T., Zeng, X. and Rao, M. S. (2016). Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications.Stem Cell Rev Rep 12(4): 394-420.

简介

从人类多能干细胞衍生的人类巨噬细胞(抽象的多能干细胞)模型,同时,与巨噬细胞样细胞系相比,巨噬细胞的表型和功能特征具有代表性(Mukherjee等人,2018年)。此外,与人类单核细胞源性巨噬细胞(MDMs)不同,hiPSCs易受基因操纵(van Wilgenburg等人,2013年;Lopez-Yrigoyen等人,2020年),为特定疾病建模提供了无限的机会。

我们概述了一个有效且高效的方案,该方案持续产生hiPSC衍生的巨噬细胞(IMAC),展示人类巨噬细胞表面和细胞内标记物,以及功能活性。

该方案描述了hiPSC的复苏、培养和分化为成熟终末巨噬细胞的过程,包括hiPSC扩增、胚状体(EBs)形成和造血髓样前体生成的初始和中间步骤。

我们提供了一种简化的、可扩展的、适应性强的技术,它在其他协议的基础上取得了进步,利用无饲养条件和减少的生长因子,在几个月的时间内经济地生产出高产量的一致iMac。


[背景]巨噬细胞占据多个组织,协调先天性和适应性免疫反应。对巨噬细胞功能和特性的研究受到了模型系统的多种限制,例如人类原代单核细胞源性巨噬细胞(MDM)和白血病源性人类巨噬细胞样细胞系(Hale等人,2015;Alasoo等人,2015;Mukherjee等人,2018)。捐献者需要大量血液,这带来了道德和后勤方面的挑战,但由于捐献者内部和捐献者之间的高度变异性,需要重复剖腹产的情况进一步加剧了这一挑战(van Wilgenburg等人,2013年)。人类髓样细胞系,如THP-1,表现出核型异常,并不完全代表人类巨噬细胞


表型或功能(van Wilgenburg等人,2013年;Lopez-Yrigoyen等人,2020年;Baldassarre等人,2021年)。

)此外,人类巨噬细胞通常对基因操纵具有抵抗力,阻碍了疾病特异性建模(Hale等人,2015)。采用人类诱导多能干细胞(HIPSC)的技术,该技术拥有自我更新的细胞来源,能够分化为三个胚层,并且易于基因操纵,与其他系统相比,作为一个模型系统,具有多种吸引人的品质(阿拉苏等人,2015年;拉赫曼等人,2015年;阿拉苏等人,2018年;李等人,2018年;内纳舍瓦等人,2020年;穆霍帕德海耶等人,2020年)。

该方案通过利用无饲养层培养条件,减少对生长因子的需求,同时仍能产生高产量的一致终末分化巨噬细胞,与原代人类MDM具有相同的表型和功能特征,从而改进了前人的研究。每5-7天,可以方便地从培养数月的胚状体(EBs)中获取数百万个hiPSC衍生的巨噬细胞(IMAC)。该方案易于遵循,具有成本效益,并且可以根据用户的要求进行缩放,随着时间的推移,为重复实验创建可重复的结果。

关键字:巨噬细胞, 人诱导多能干细胞, 胚状体, 采用无饲养层, 分化





材料和试剂
A.人类诱导的多能干细胞培养和扩增
1.用于制定本方案的人类诱导多能干细胞:


a、 NL9-一种来自健康个体的成纤维细胞衍生的hiPSC系,可广泛获得(Baghbaderani等人,2016),从国家心肺血液研究所(NHLBI)iPSC核心获得(有关NL9 hiPSC系来源的更多信息,请参阅确认部分)
b、 KOLF_2——一种来自健康个体的皮肤组织衍生hiPSC细胞系,由桑格研究所产生,作为HipSci倡议的一部分
(https://www.hipsci.org/lines/#/lines/HPSI0114i-科尔夫2号)


2.完整的Essential 8基础培养基(赛默飞世尔,目录号:A1517001)


3.Vitron(Vtron-4700)产品目录号


4.Dulbecco磷酸盐缓冲盐水(DPBS),不含Ca2+/Mg2+500 mL(赛默飞世尔,目录号:14190144)(储存条件:15-30°C。保质期:自生产之日起36个月)


5.岩石抑制剂(ROCKi)(Y-27632二盐酸盐)(西格玛,目录号:Y0503-1MG)


6.10 cm或6孔组织培养处理板(康宁,目录号:430167或3516)


7.0.22 µm过滤器steri杯(默克密理博,目录号:1578019)


8.15 mL和50 mL猎鹰管(猎鹰,目录号:352097和352098)


 


 


B.在无饲养系统中产生胚状体
1.0.5 M超纯乙二胺四乙酸(EDTA)(Invitrogen,目录号:




15575020),100毫升


2.冷冻保存/冷冻培养基:敲除血清置换(KSR)(Gibco,目录号:10828028),500毫升


3.二甲基亚砜(DMSO)(西格玛,目录号:D2438),50毫升


4.Dulbecco磷酸盐缓冲盐水(DPBS),含Ca2+/Mg2+,500 mL(赛默飞世尔,目录号:14040091)(储存条件:2-8°C。保质期:自生产之日起36个月)


5.牛血清白蛋白(BSA)低内毒素组织培养级(西格玛,目录号:A9543-5G)


6.重组人BMP-4蛋白10µg(研发系统,目录号:314-BP-010)


 


 


C.胚状体转移、培养和巨噬细胞分化
1.胚胎移植用无菌水(Sigma,目录号:W1503)


2.猪皮明胶(西格玛,目录号:G1890)


3.X-VIVO 15无血清造血细胞培养基500 mL(Lonza,目录号:LZBE02060F)


4.L-谷氨酰胺(200mM),100mL(Gibco,目录号:25030181)


5.青霉素链霉素(10000 U/mL),100 mL(Gibco,目录号:15140122)


6.2-巯基乙醇(西格玛,目录号:M3148)


7.重组人M-CSF(Peprotech,目录号:300-25)


8.重组人IL-3(Peprotech,目录号:200-03)


9.RPMI 1640中等浓度500毫升(西格玛,目录号:R0883)


10.超低粘性U型底部96孔板(Costar,目录号:7007)


11.10 cm组织培养皿或6孔板


12.70-100 µm细胞过滤器(猎鹰)


13.完整的8种基本培养基(见配方)


14.卵黄连蛋白1毫升小瓶(见配方)


15.岩石抑制剂(ROCKi)(见配方)


16.0.5 mM乙二胺四乙酸(EDTA)溶液(见配方)


17.冷冻保存/冷冻介质(见配方)


18.牛血清白蛋白(BSA)(见配方)


19.重组人BMP-4蛋白(见配方)


20.用于胚胎移植的无菌水(见配方)


21.猪皮制成的明胶(见配方)


22.EB髓样前体基础培养基(见配方)


23.重组人M-CSF(见配方)


24.重组人IL-3(见配方)


25.巨噬细胞分化基础培养基(见配方)


 


本方案中使用的流式细胞仪抗体




1.CD14(BV605,Biolegend,目录号:301834)


2.CD16(APC,eBioscience,目录号:17-0168-42)


3.CD80(BV711,Biolegend,目录号:305236)


4.CD86(PE-Dazzle594,Biolegend,目录号:374217)


5.CD206(PerCP/Cynine 5.5,Biolegend,分类号:321122)


6.CD204(PE/Cyanine7 Biolegend,目录号:371908)


 


本协议中使用的引物序列


1.TATA结合蛋白(TBP)(F:GGGAGGCATATTTG,R:CCAGAGCAGGTA)。


2.CD68(F:GGAAATGCCAGGTTCATCCA,R:TGGGGTTCAGATACAGAGATGC)


3.CSFR1(F:TCCAACATGCCGGCAACTA,R:GCTCAAGTCAAGTCTCT)


4.CD163(F:TTTGTCaactTGAGTCTCTC,R:TCCGCTACTCTTTTTCAC)


5.SOX2 F:GCTACAGCATGATCAGACCA,R:TCTGCGAGCGGTCATGGAGTT)


6.NANOG(F:CTCCACATCTGACTGACTCACG,R:CGTCACACATGCTATTCTCG)


7.十月四日(F:CCTGAGAGAGAGATCACC,R:AAAGCGAGAGATGGTCGTTTGG)


 


 


设备


 


 


1.使用无菌技术的2级微生物安全罩或柜


2.37°C培养箱,含5%二氧化碳


3.离心机


4.相衬显微镜(4倍、10倍、40倍放大)


5.水浴温度设置为37°C


6.-80℃储存


7.液氮储存


8.细胞冷冻容器(“Frosty先生”)


9.移液管控制器和不同容量条带(5毫升、10毫升)的选择,这些条带通常有大口径孔


10.移液管(P1000、P200、P20、P2)和相应的无菌尖端。


11.ParafilmTM


 


 


程序


 


 


一般考虑:


1.所有细胞培养、培养基制备、组织培养容器和其他实验室工作均应使用无菌技术在2级微生物罩内进行。


2.使用前,应使用3%的Distel(或同等消毒剂)和70%的乙醇彻底清洁发动机罩。


3.在室温(RT)下准备所有介质、溶液和试剂。有关详细信息,请参见“食谱”部分




更多关于媒体准备的详细信息。


 


 


A.人类诱导的多能干细胞培养和扩增
1.按照“材料和试剂”或“配方”部分中的说明,准备所有细胞维护基础培养基和试剂。


2.准备卵黄连蛋白板。卵黄连蛋白是一种糖蛋白,用于覆盖细胞培养板表面,促进hiPSC附着和扩散。将一份60μL小份解冻至RT,并稀释至不含Ca2+/Mg2+的6 mL DPBS中。如果使用6孔板,每孔添加1-1.5毫升;或者,在一个10厘米的盘子中加入6毫升。确保卵黄连蛋白覆盖整个表面,并在室温下在罩中培养1-2小时。卵黄连蛋白板可使用ParafilmTM密封,并在4°C下储存5天。Allow to warm to RT for 1–2 h prior to use.


3. 通过添加解冻的补充剂并让培养基升温至 RT 来制备 E8 基础培养基。我们建议在 E8 基础培养基中培养和扩增解冻的 hiPSC,并尽早冷冻和储存大量的 hiPSC 系。




4. 用 9 mL E8 培养基加 0.312 µM ROCKi 设置 15 mL Falcon,将一瓶冷冻的 1 mL hiPSC,即 1 µL ROCKi 放入最终体积为 10 mL。虽然 ROCKi 允许 hiPSC 的附着和恢复,同时防止细胞凋亡和自发分化,但必须在 24 小时后将其从培养基中取出,以防止干扰集落扩展。




5. 使用 37°C 的水浴快速部分解冻冷冻的 hiPSC,直到刚刚解冻,小瓶中残留一些冰块(解冻应该快速,以避免与 DMSO 长时间接触;但是,不要将整个小瓶加热至37℃)。将 1 mL 的 E8 以逐滴方式添加到小瓶中,然后使用大口径移液器尖端或条纹轻轻地将细胞转移到 15 mL Falcon。倒置管子以混合和稀释 DMSO。




注意:冷冻管中的确切细胞数量或密度通常在冷冻时是未知的,也无法确定,以避免产生单细胞悬液,并提高解冻后的恢复率,以便后续培养。从一个先前 70-80% 汇合的 10 厘米 hiPSC 板产生 6-10 个冷冻管。




6. 在 290 × g 和 20°C 下旋转 3 分钟。弃去上清液。




7.从板表面吸出稀释的玻连蛋白。一个解冻的冷冻管在离心后应在 15 mL Falcon 中提供一个小的 5–20 µL 细胞沉淀。将颗粒轻轻重悬在所需体积的 E8 培养基中,辅以 3.12 μM ROCKi(1 μL:1,000 μL),避免过度移液。




注意:ROCKi 以前曾在 10 µM 的早期改编协议中使用过,但我们有




进一步滴定,并在 3.12 µM 的较低浓度下看到有效的 hiPSC 附着和恢复。




8. 6 孔板每孔 2 mL 细胞悬液或每 10 cm 培养皿 8 mL;旨在使用最少的媒体量来提高依从性。一瓶冷冻的 hiPSC(相当于 6 孔板的 1 个孔,或 70-80% 汇合度时 10 cm 培养皿的十分之一),可以分成 6 孔板的 2-3 个孔,或者如果hiPSC 系表现出良好的恢复,然后细胞可以








直接播种到 10 厘米的培养皿中。当多个小菌落在播种后 24 小时附着在玻连蛋白板上时,恢复被描述为良好(参见图 1),并且在接下来的几天内发生菌落扩张(参见图 2)。




9. 将培养板置于 37°C 加湿 5% CO2 的培养箱中。确保板最初轻轻搅动,使菌落均匀分布在玻连蛋白上。




10. 24 小时后在相差显微镜下检查附件(见图 1)。




11.每 24 小时更换一次培养基,用吸管或吸管去除未附着的菌落、死细胞和用过的培养基。如果有很多死细胞或碎片,则在更换 E8 介质之前用 DBPS 清洗板。轻轻更换新鲜的 E8 基础培养基,在容器侧壁上使用条纹(前 24 小时后不需要补充 ROCKi)。 hiPSCs 难以在培养中维持,并且容易分化、培养基细胞因子和维持多能性的因素,需要每天更换培养基。




12.继续每天更换培养基,直到细胞达到 70–80% 融合并准备好




传代。不要让簇/菌落融合在一起(参见图 3、4 和 5),这有助于避免自发分化。一旦建立解冻的 hiPSC,后续步骤、培养系统和进一步分化应在 1-2 次传代后进行。












图 1. 第 1 天 hiPSC 附着并在玻连蛋白上紧密相邻的小菌落中生长。




使用带有 10 倍物镜的 Olympus CKX41 倒置显微镜拍摄的图像。








 












图 2. 在玻连蛋白上生长和扩展的异源 hiPSC 菌落示例。




使用带有 4 倍物镜的 EVOSTM XL Core 成像系统拍摄的图像。




 












图 3. 在 EVOSTM XL Core 成像系统的屏幕上,用 4 倍物镜成像的 70–80% hiPSCs 汇合板,在几乎相互接触的菌落中生长。








 












图 4. 70–80% 汇合板的特写图像,其中多个大菌落肉眼可见。












图 5. 传代前 70–80% 融合的 hiPSC。使用配备 20 倍物镜的 Olympus CKX41 倒置显微镜获得的图像。




 




B.hiPSC的传代和冷冻
1. 像以前一样准备玻连蛋白板。




2. 从具有 70–80% 融合度的 hiPSC 板中取出用尽的培养基,并用不含 Ca2+/Mg2+ 的 DPBS 清洗板/孔两次(10 cm 培养皿约 6 mL)。




3. 除去 DPBS,加入 6 mL 0.5 mM PBS-EDTA 溶液,使板面完全覆盖。




4.在相差显微镜下观察细胞
每 2 分钟最多 4 分钟,以监测细胞形态变化和脱离(明亮的光晕和集落边缘增强),但不要让细胞完全脱离和分离成单个细胞。




5. 倾斜平板轻轻取出并丢弃 PBS-EDTA,但不要去除菌落。








6. 使用条带将 8 mL E8 培养基添加到 10 cm 板中,目的是分离和洗涤小团块和簇中的菌落,使其悬浮,使用相同的 8 通过条带反复抽吸和冲洗培养基多达四次毫升体积。将培养基和细胞收集到新的 50 mL Falcon 中。重复此步骤最多两次,以最大限度地恢复菌落,同时避免过度搅拌和移液,这将导致单细胞。




7. 在 290 × g 和 20°C 下旋转细胞 3 分钟。弃去上清液。




8. 将菌落重悬在补充有 3.12 mM ROCKi 的新鲜 E8 基础培养基中,并以所需的分裂比例(1:5–1:30,取决于 hiPSC 系的生长速率)。




9.从预先准备好的板中吸出玻连蛋白溶液。将分裂的菌落转移并播种到新的玻连蛋白板/孔中。




10.hiPSC的冷冻保存/冷冻:




要冷冻 hiPSC 细胞,请在 RT 处新鲜制备冷冻培养基(KSR 中的 10% DMSO)。收获 hiPSC(步骤 B2-B7)后,将细胞颗粒重新悬浮在 10% DMSO-KSR 中,并在每个冷冻管中转移 1 mL 的细胞悬浮液。重要的是,细胞在 RT 存在 DMSO 时花费最少的时间。冷冻管应立即放入 Mr. Frosty 或适当的冷冻容器中,然后放入 -80°C 冰箱中,然后在 24 小时后放入液氮中,以便长期储存。注意:准备冷冻培养基的体积取决于要储存的冷冻管的数量。我们建议在 70-80% 的汇合度下,每个冷冻管 1 mL 冷冻培养基和 6 孔板每孔 2 个冷冻管和每 10 cm 培养皿 6-10 mL 或 6-10 个冷冻管。




 




C.利用无饲养系统形成胚体
1. 收获 hiPSC 后(如步骤 B2-B7 中所述),加入 1 mL 新鲜 E8 培养基并比平时更多地上下移取 hiPSC(3-4 次移液),以打散团块并创建一个细胞悬液。




2.使用血细胞计数器,加入10 µL台盼蓝(稀释因子=2)后,在10 µL细胞悬液中计数细胞,测定细胞密度:




 




(3 个方格中的细胞数 / 3)× 稀释倍数 × 10,000 = 细胞数/mL




 




 




3. 使用超低粘附性 U 型底 96 孔板(每孔 1 个 EB)制备单个、均质且大小相同的 EB。有了上面收获的总细胞数的知识,可以计算所需的 EB 大小(细胞数)和要制造的单个 EB 的总数。例如,如果收获 3 × 106 hiPSC = 100 × 可以制作 3 × 104 大小的单个 EB。




 




注意:可能需要为每个 hiPSC 线确定 EB 的大小。然而,在我们手中,1 × 104–5 × 104 细胞之间的 EB 大小为该协议生成中使用的两个 hiPSC 系产生最高产量的骨髓前体(高达 1 × 105 细胞的 EB 也可以很好地工作) .








4. 每个单独的 EB 由 100 µL 的 E8 培养基组成,辅以 50 ng/mL 重组 BMP-4 和 3.12 µM ROCKi。根据所需的 EB 大小,创建每毫升 1 × 105–10 × 105 个细胞的细胞悬浮液。




对于上面计算的示例,应创建 3 × 105 细胞/mL 的细胞悬浮液,以在超低粘附 U 底 96 孔板的每孔 100 μL 补充 E8 中生成 3 × 104 大小的单个 EB。




5. 将 100 µL hiPSC 细胞悬液转移到超低贴壁 U 型底 96 孔板的每个孔中。




6. 将板以 800 rpm 转速旋转 1 分钟,然后转移到 5% CO2 的 37°C 加湿培养箱中。




7. 让这些 EB 不受干扰 48 小时,在培养的第 2 天,去除 50 µL 用过的培养基,小心地更换为 50 µL 的 E8 培养基,仅补充 50 ng/mL BMP-4,目的是不干扰正在形成的 EB .重复第 3 天(参见图 6)。 ROCKi 可以干扰晚期 EB 和胚层的形成。




8.每口井发现一个以上的EB是不常见的(见图7和图8);但是,如果一个井中每个 EB 的外观与图 6 中看到的相似,具有清晰的边缘和 3D 结构,则可以转移井内的所有 EB。每当发现多个小团块或破碎和破碎的 EB 外观时,应丢弃孔和细胞材料。




9.第 4 天,准备涂有明胶的 10 厘米盘子。在 10 cm 培养皿中加入 6 mL 0.1% 明胶溶液,在通风橱中孵育 1-2 小时,使明胶覆盖整个表面并加热至室温(板可预先制备并在 4°C 下储存5天)。




10. 在 EB 转移之前从板上除去明胶溶液。




11.使用宽口径 P1000 滤芯吸头(或者,可以用手术刀将 P1000 滤芯吸头向上切割,以形成大口径或 
Měi 2 fēnzhōng zuìd
ifice), 小心吸出容易看到的单个 EB, 并将它们转移到明胶涂层板上 (图 9)。每 cm2 的 EB 数量可能需要确定每个 hiPSC 系,然而,根据我们的经验,每 10 cm 培养皿高达 35× EB 可提供高产量的骨髓前体。








 












图 6. 胚体在培养 24 小时后应具有这种外观。




这是 96 孔超低粘附板的一个孔中的 EB,在转移到明胶板之前的第 4 天。 EB 大小为 3 × 104 hiPSC。使用带有 4 倍物镜的 EVOSTM XL Core 成像系统拍摄的图像。












图 7. 肉眼可见 96 孔超低粘附板的每个孔中心的单个 EB。








 












图 8. 近距离的单个 EB 并用箭头标记。




 












图 9. 将单个 EB 吸入大口径移液器中以转移到下方的明胶涂层板中。




 




12. 转移后,轻轻倾斜板并从板中取出用过的 EB 培养基,不要去除 EB。




13.慢慢加入 10–12 mL 新鲜的 EB-骨髓前体培养基,辅以 50 ng/mL M-




CSF,对骨髓分化至关重要,25 ng/mL IL-3,对造血规范和增殖至关重要。








14. 小心地将这些板移至 37°C 加湿 5% CO2 的培养箱中。静置 1 周,让 EB 附着在明胶上(见图 10)。




15.在第 7 天,小心地取出带有大孔条纹的用过的 EB 培养基,以避免损坏任何未连接的 EB。注意不要移动任何附着的 EB,并将介质通过 50 mL Falcon 管上的 70-100 μm 过滤器,以捕获并保留所有未连接的悬浮 EB。




16. 将过滤器中的 EB 放回原盘中,将过滤器倒置在 10 cm 培养皿上,轻轻加入补充有 50 ng/mL M-CSF 和 25 ng/mL IL-3 的新鲜 EB 培养基,加热至 RT倒置的过滤器。继续注意不要将附加的 EB 移出板中。




17. 更换 EB 培养基并将 EB 放回培养皿后,将 50 mL Falcon 在 290 × g 和 20°C 下旋转 3 分钟。在第 3 周,该管将包含悬浮的收获的骨髓前体。弃去上清液。




18. 向细胞沉淀中加入 1 mL 巨噬细胞分化培养基,在显微镜下观察细胞外观,用血细胞计数器计数(见步骤 C1)。




19.EB 培养基应继续按照步骤 C14-C17 中的说明每周更换。根据 hiPSC 线,应从 EB 培养的第 3-4 周收获骨髓前体。




20.EBs 可以在培养物中维持数月。在我们手中,骨髓前体的产量从 12-16 周开始下降。












图 10. 胚体附着:转移到明胶板上 1 周后。




使用带有 4 倍物镜的 EVOSTM XL Core 成像系统拍摄的图像。




 




 




D.巨噬细胞分化
1.如步骤 C14–C17 中所述收获骨髓前体。计数后,将收获的骨髓前体重新悬浮在所需体积的巨噬细胞分化培养基中,并辅以高浓度 100 ng/mL M-CSF。




2. 将细胞接种到所需的培养板中,理想情况下为 0.5 × 106-1 × 106,大约 8-10








每 10 cm 培养皿中加入 mL 巨噬细胞分化培养基,或者 6 孔板每孔 3 mL 中加入 1.5 × 105,或者在 96 孔板上每孔加入 1.5 × 104 × 150 µL(参见图 11a)。




3.将细胞转移到 37°C 加湿 5% CO2 的培养箱中,静置 5–7




天(见图 11b)。




4. 然后可以用 0.5 mM EDTA 溶液提起细胞,有或没有细胞刮刀的帮助。 0.5 mM EDTA 溶液应覆盖细胞 10 分钟。应重新计数细胞并通过流式细胞术或 qPCR 或随后的功能表征细胞表型(参见图 12 和 13)。




注:巨噬细胞数量应与骨髓前体细胞数量相近




接种到板上,但这个数字可能因细胞系而异,也取决于 EB 大小。




5. 收获后培养 10-14 天的细胞往往会变成成纤维细胞样(图 11c)。




 












图 11. 骨髓前体外观 (a) 收获后的骨髓前体 (b) 7 天后分化的附着巨噬细胞。使用带有 20 倍物镜的 EVOSTM XL Core 成像系统拍摄的图像。 (c) 收获后 14 天的成纤维细胞样细胞。使用带有 20 倍物镜的 Olympus CKX41 倒置显微镜拍摄的图像。




 




数据分析








 












 




图 12. 显示巨噬细胞标志物 CD14 (BV605)、CD16 (APC)、CD80 (BV711)、CD86 (PE-Dazzle594)、CD206 阳性的代表性流式细胞仪图




(PerCP/Cyanine 5.5) 和 CD204 (PE/Cyanine7) 在分化的 iMACS 中。 (黑色 = 未染色的 iMAC,红色 = iMAC)。




 












 




图 13. 展示 iMAC 巨噬细胞特征的代表性 qPCR 数据。相对表达是常态


化为 TATA-Bing 蛋白 (TBP)。




与 iMACs 相比,iMACs 中泛巨噬细胞标志物的表达增加:CD68、CSFR1 和 CD163,并且与 iMACs 相比,hiPSCs 中的多能标志物 SOX2、NANOG 和 OCT4 的表达增加。








食谱




 




 




所有培养基制备和实验室工作都将使用无菌技术在 2 级微生物罩内进行。在 RT 准备所有介质、溶液和试剂。




 




1.Complete Essential 8 基础培养基由两部分组成:




490 mL 培养基(储存于 2–8°C 并避光)和 10 mL 补充剂 (50×)(储存于 - 5 至 -20°C。保质期:12 个月)。




要重组,解冻补充 RT 并添加到 490 mL 的媒体。




或者,将补充剂分装到较小的体积中,以便在 -20 至 -80°C 下长时间储存,并根据需要解冻。




旋转或倒置混合。




将重构的基础培养基储存在 2–8°C,保质期 2 周。允许在使用前升温至 RT。




2.Vitronectin:1 mL 小瓶(-80°C 保存。保质期:24 个月)分成 60 µL 无菌等分试样。




涂板:将 60 µL 稀释到 6 mL 不含 Ca2+/Mg2+ 的 DPBS 中,或以 2.5–10 µg/mL 的浓度使用。




反转混合。




确保在储存前使用封口膜密封板的边缘,用于任何预先准备好的板。




3.ROCK抑制剂(ROCKi)(储存在2-8°C)




准备 3.12 mM 工作浓度库存,即用 1 mL 无菌超纯水重组 1 mg。




分成 10 µL 等份(-20°C 储存)。




用于 0.312 µM 或 3.12 µM 最终浓度。




解冻后,ROCKi 原液可在 2–8°C 下保存 7 天。




4.0.5 mM 乙二胺四乙酸 (EDTA) 溶液(室温储存)




在 50 mL 的 DPBS 中稀释 50 μL,不含 Ca2+/Mg2+,工作浓度为 0.5 mM,储存在 RT,并在制备当天使用。




5. 冷冻保存/冷冻培养基(-5 至 -20°C 保存,避光。保质期 18 个月) 分装成小体积。




将 1 mL 的 DMSO 添加到 9 mL 的解冻敲除血清替代物 (KSR) 中,使最终浓度为 10% 的 DMSO 进行冷冻。




在制备当天使用,并在液氮中长期储存 hiPSC。




6.牛血清白蛋白(BSA)(储存在2-8°C)




将 100 mg BSA 添加到 100 mL 含有 Ca2+/Mg2+ 的 DPBS 中,混合,然后置于 37°C 水浴中直至 BSA 溶解。




过滤除菌(储存在 2–8°C,保质期 4 个月)。








7.重组人BMP-4蛋白(-20至-70°C保存。保质期12个月)




重组为 50 µg/mL 工作浓度库存(10 µg 在 200 µL 的 4 mM HCl 和 0.1% BSA 中)。分装成小体积。




最终浓度为 50 ng/mL(储存在 -20 至 -80°C)。




8.胚胎移植用无菌水(室温储存)




9.来自猪皮的明胶(储存于室温)




配制 0.1% 明胶溶液:将 500 mg 明胶加入 500 mL 水中,加热至 56°C 溶解,过滤除菌(4°C 酸痛长达 4 个月)。




10.EB-骨髓前体基础培养基:




向 500mL X-VIVO 15 无血清造血细胞培养基中添加: 2 mM (5 mL) L-谷氨酰胺(储存于 -5 至 -20°C。保质期 24 个月),




100 IU/mL (5 mL) 青霉素-链霉素(-5 至 -20°C 储存。保质期 12 个月)




0.1 mM (3.5 µL) 2-巯基乙醇(室温储存) 过滤除菌(2–8°C 储存)。




11.重组人M-CSF




复溶:将 500 µL 0.1% BSA 溶液添加到 0.5 mg 冻干 rhM-CSF 中,以达到 1 mg/mL 储备浓度(在 -20 至 -80°C 下以 5 µL 等分试样储存。保质期 12 个月)。




使用前解冻添加到新鲜培养基中(以 50ng/mL 最终浓度使用)。避免重复的解冻循环。




12.重组人IL-3




复溶:将 1 mL 的 0.1% BSA 溶液添加到 0.5 mg 冻干的 rhIL-3 中,以达到 0.5 mg/mL 的储备浓度(在 -20 至 -80°C 下以 5 µl 等分试样储存。保质期 12 个月)。




在使用前解冻以添加到新鲜培养基中(用于 25 ng/mL 最终浓度)。避免重复的解冻循环。




13.巨噬细胞分化基础培养基向500mL RPMI 1640培养基中加入:10%(50mL)胎牛血清




2 mM (5 mL) L-谷氨酰胺过滤灭菌(储存在 2–8°C)。




 




致谢




 




 




由医学研究委员会、英国肾脏研究中心、国家健康研究所、皇家学会和威康信托基金提供的研究资金。




用于开发该协议的 NL9 hiPSC 由 Claudia Kemper 教授和国家心肺血液研究所 (NHLBI) iPSC 核心 (https://www.nhlbi.nih.gov/science/ipsc-core/) 慷慨捐赠研究),巴塞斯达,马里兰州,美国。




还要感谢伦敦国王学院的 Anthony Dorling 教授的耐心指导,








监督和持久的鼓励。




该协议源自先前发布的协议(van Wilgenburg 等人,2013 年;Mukherjee 等人,2018 年;Lopez-Yrigoyen 等人,2020 年)。




 




利益争夺




 




 




没有需要申报的财务或非财务竞争利益。




 




参考文献



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Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Douthwaite, H., Arteagabeitia, A. B. and Mukhopadhyay, S. (2022). Differentiation of Human Induced Pluripotent Stem Cell into Macrophages. Bio-protocol 12(6): e4361. DOI: 10.21769/BioProtoc.4361.
  2. Baldassarre, M., Solano-Collado, V., Balci, A., Colamarino, R. A., Dambuza, I. M., Reid, D. M., Wilson, H. M., Brown, G. D., Mukhopadhyay, S., Dougan, G. et al. (2021). The Rab32/BLOC-3-dependent pathway mediates host defense against different pathogens in human macrophages. Sci Adv 7(3): eabb1795.
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