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Jun 2019

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In vitro Differentiation of Human iPSC-derived Retinal Pigment Epithelium Cells (iPSC-RPE)
人诱导多功能干细胞来源的视网膜色素上皮细胞的体外分化   

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Abstract

Induced Pluripotent Stem Cells (iPSCs) serve as an excellent model system for studying the molecular underpinnings of tissue development. Human iPSC-derived retinal pigment epithelium (iPSC-RPE) cells have fetal-like molecular profiles. Hence, biobanks like iPSCORE, which contain iPSCs generated from hundreds of individuals, are an invaluable resource for examining how common genetic variants exert their effects during RPE development resulting in individuals having different propensities to develop Age-related Macular Degeneration (AMD) as adults. Here, we present an optimized, cost-effective and highly reproducible protocol for derivation of human iPSC-RPE cells using small molecules under serum-free condition and for their quality control using flow cytometry and immunofluorescence. While most previous protocols have required laborious manual selection to enrich for iPSC-RPE cells, our protocol uses whole culture passaging and yields a large number of iPSC-RPE cells with high purity (88-98.1% ZO-1 and MiTF double positive cells). The simplicity and robustness of this protocol would enable its adaption for high-throughput applications involving the generation of iPSC-RPE samples from hundreds of individuals.

Keywords: Human induced pluripotent stem (hiPSC) (人诱导多功能干细胞), Retinal pigment epithelium (RPE) (视网膜色素上皮细胞), Human induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) (人诱导多功能干细胞来源的视网膜色素上皮细胞), Age-related macular degeneration (AMD) (老年性黄斑变性), Differentiation (分化), Genetic studies (遗传研究), Small molecules (小分子), Genetic variant (遗传变异)

Background

Age-related macular degeneration (AMD) is a leading cause of vision loss in developed countries affecting 11 million individuals in the United States and about 170 million worldwide (Pennington and DeAngelis, 2016). Moreover, considering age as a main factor, in our current aging society, the incidence of AMD is estimated to increase to 198 million in 2020 and 288 million by 2040 and to 22 million in the United States alone by 2050 (Wong et al., 2014, Pennington and DeAngelis, 2016). Current therapeutic strategies, although effective, are expensive and limited to delaying the speed of disease progression, and AMD still eventually leads to a complete loss of vision (Al-Zamil and Yassin, 2017; Mitchell et al., 2018). At the time of preparation of this article, there are seven active clinical trials aimed to evaluate the effectiveness and optimize the conditions of the transplantation of the human iPSC-RPEs or human embryonic stem cell-derived retinal pigment epithelium (ESC-RPE) cells (NIH-ClinicalTrials.gov). Thus, the development of a robust and cost-effective method for generating large amounts of high quality iPSC-RPEs is imperative for the advancement of future therapeutic treatments of AMD and potentially other eye diseases in humans as well as domestic animals (Sparrow et al., 2010).

We have previously demonstrated the utility of employing iPSC-RPE cells to identify and study genetic variants playing a role in the development of AMD (Smith et al., 2019). In this study, we derived iPSC-RPE cells from six individuals (3 European Americans, 2 East Asian Americans, and 1 African American) and showed that they have morphological and molecular characteristics similar to those of naïve RPE cells. We showed that iPSC-RPE gene expression profiles are highly similar to that of human fetal RPE, and that their ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) peaks are enriched for relevant transcription factor motifs. We performed fine mapping of AMD risk loci integrating the molecular data from iPSC-RPE cells that resulted in the prioritization of four variants, including a potential regulatory SNP (rs943080) near VEGFA and a coding SNP (rs34882957, pP167S) in the C9 gene (Smith et al., 2019). Our findings illustrate that iPSC-RPE cells are an excellent model system to study the molecular functions of genetic variation associated with AMD.

The initial protocols for deriving iPSC-RPE cells developed over a decade ago involved spontaneous differentiation and were highly inefficient requiring the manual separation of pigmented iPSC-RPE patches from non-differentiated cells (Klimanskaya et al., 2004; Vugle et al., 2008). Subsequently, induced directed differentiation protocols resulted in higher yielded and quality of iPSC-RPE cells but required numerous animal-derived components and thus had relatively low reproducibility or were very time consuming (Aoki et al., 2006; Osakada et al., 2008; Buchholz et al., 2009; Idelson et al., 2009; Reichman et al., 2014; Hazim et al., 2017). The introduction of small molecules into iPSC-RPE differentiation protocols greatly simplified the procedure and resulted in higher reproducibility (Osakada et al., 2009; Maruotti et al., 2013; Maruotti et al., 2015). Previous studies have optimized differentiation protocols to derive iPSC-RPEs from a limited number of iPSC or ESC lines and in most cases utilized small format culture vessels. Here, we present an optimized protocol for deriving iPSC-RPEs from multiple human iPSC lines in large sized culture flasks. It is similar to the protocol of Maruotti et al. (2015), but initiates differentiation when iPSCs reach 80% confluency, which we found to be optimal for all tested iPSCORE lines (Panopoulos et al., 2017). Additionally, we modified the length of time of exposure to small molecules at two steps, which resulted in further increase of yield and purity of iPSC-RPEs (88-98.1% ZO-1 and MiTF double positive cells). Our optimized protocol allowed us to derive iPSC-RPEs from six iPSC lines generated from ethnically diverse individuals under identical culturing conditions without the requirement of any individualized optimization steps.

Materials and Reagents

  1. iPSC Cell Culture
    1. 6-well plates (Corning, catalog number: 3506)
    2. Syringe filter 0.2 μm (VWR, catalog number: 28145-501)
    3. Soft-Ject® 3-Part Disposable Syringe, Air-Tite-3 ml (VWR, catalog number: 89215-234)
    4. 5 ml Borosilicate serological pipettes (Fisher Scientific, catalog number: 1367827E)
    5. 5 ml Serological pipettes (Bio Pioneer, catalog number: GEX0050-S01)
    6. 10 ml Serological pipettes (Bio Pioneer, catalog number: GEX0100-S01)
    7. P20 pipette tips sterile with filter
    8. P1000 pipette tips sterile with filter
    9. 15 ml conical tubes (Bio Pioneer, catalog number: CNT-15R)
    10. iPSC cells
    11. 70% ethanol
    12. UltraPureTM DNase/RNase-Free Distilled Water (Thermo Fisher Scientific, catalog number: 10977023)
    13. Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, catalog number: 354230)
    14. mTeSRTM1 (Stem Cell Technologies, catalog number: 85850)
    15. DMEM/F-12 medium (Thermo Fisher Scientific, catalog number: 11330057)
    16. Dispase II (Thermo Fisher Scientific, catalog number: 17105041)
    17. Matrigel solution (Matrigel) (see Recipes: Table 1)
    18. 10 mM ROCK inhibitor, Y-27632 dihydrochloride solution (ROCK Inhibitor) (see Recipes: Table 2)
    19. 10x Dispase (see Recipes: Table 3)
    20. mTeSRTM1 complete medium (mTeSR) (see Recipes: Table 4)

  2. Monolayer plating
    1. 100 mm tissue culture dishes (Corning, catalog number: 430167)
    2. Automated cell counter slides (Bio-Rad Laboratories, catalog number: 1450019) or a hemocytometer (Hausser Scientific, catalog number: 1483) or equivalent
    3. 5 ml Serological pipettes (Bio Pioneer, catalog number: GEX0050-S01)
    4. 10 ml Serological pipettes (Bio Pioneer, catalog number: GEX0100-S01
    5. P20 pipette tips sterile with filter
    6. P200 pipette tips sterile with filter
    7. P1000 pipette tips sterile with filter
    8. 15 ml conical tubes (Bio Pioneer, catalog number: CNT-15R)
    9. 50 ml conical tubes (Bio Pioneer catalog number: CNT-50R)
    10. 70% ethanol
    11. Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix (Matrigel) (Corning, catalog number: 354230)
    12. mTeSRTM1 (Stem Cell Technologies, catalog number: 85850)
    13. DMEM/F-12 medium (Thermo Fisher Scientific, catalog number: 11330-057)
    14. Accutase (Innovative Cell Technologies, Inc., catalog number: AT 104)
    15. Trypan Blue Solution, 0.4% (Thermo Fisher Scientific, catalog number: 15250061)
    16. ROCK inhibitor, Y-27632 dihydrochloride (Selleck hem, catalog number: S1049)
    17. iPSC cell culture
    18. Matrigel solution (see Recipes: Table 1)
    19. 10 mM ROCK inhibitor, Y-27632 dihydrochloride solution (see Recipes: Table 2)
    20. mTeSRTM1 complete medium (see Recipes: Table 4)

  3. iPSC-RPE differentiation
    1. 100 mm tissue culture dishes (Corning, catalog number: 430167)
    2. (Optional) T150 tissue culture flasks, vented (Sigma, catalog number: Z707929)
      Note: At the time of preparation of this manuscript Z707929 was no longer available. The same flasks are available under the catalog number Z707511-36EA (Sigma, catalog number Z707511-36EA).
    3. 70 μm strainers (Fisher Scientific, catalog number: 431751)
    4. Automated cell counter slides (Bio-Rad Laboratories, catalog number: 1450019) or a hemocytometer (Hausser Scientific, catalog number: 1483) or equivalent
    5. 10 ml Serological pipettes (Bio Pioneer, catalog number: GEX0100-S01)
    6. 25 ml Serological pipettes (Bio Pioneer catalog number: GEX250-S01)
    7. 50 ml Serological pipettes (Bio Pioneer, catalog number: GEX500-S01)
    8. P20 pipette tips sterile with filter
    9. P200 pipette tips sterile with filter
    10. P1000 pipette tips sterile with filter
    11. Cell scraper (VWR International, catalog number: 179707)
    12. 15 ml conical tubes (Bio Pioneer, catalog number: CNT-15R)
    13. 50 ml conical tubes (Bio Pioneer, catalog number: CNT-50R)
    14. Nalgene Cryogenic vials (Thermo Fisher Scientific, catalog number: 5000-1020)
    15. iPSCs monolayer
    16. 70% ethanol
    17. Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix (Matrigel) (Corning, catalog number: 354230)
    18. DMEM/F-12 medium (Thermo Fisher Scientific, catalog number: 11330057)
    19. DMEM medium (Thermo Fisher Scientific, catalog number: 11965092)
    20. Ham’s F12 Nutrient Mix (Thermo Fisher Scientific, catalog number: 11765054)
    21. 1x Dulbecco’s phosphate buffered saline (DPBS) without calcium and magnesium (Thermo Fisher Scientific, catalog number: 14190250)
    22. B27 Supplement (50x), serum free (Thermo Fisher Scientific, catalog number: 17504044)
    23. KnockOutTM Serum Replacement (KOSR) (Thermo Fisher Scientific, catalog number: 10828028)
    24. L-Glutamine 200 mM (Thermo Fisher Scientific, catalog number: 25030081)
    25. MEM Non-Essential Amino Acids Solution 100x (Thermo Fisher Scientific, catalog number: 11140050)
    26. Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher Scientific, catalog number: 15140122)
    27. β-Mercaptoethanol (Thermo Fisher Scientific, catalog number: 21985023)
    28. Accutase (Innovative Cell Technologies, Inc., catalog number: AT 104)
    29. Nicotinamide (Sigma, catalog number: N3376)
    30. Chetomin (Sigma, catalog number: C9623)
    31. Trypan Blue Solution, 0.4% (Thermo Fisher Scientific, catalog number: 15250061)
    32. Dimethyl Sulfoxide (DMSO) (Sigma, catalog number: D2650-100ML)
    33. Liquid nitrogen
    34. RPE DM medium (see Recipes: Table 5)
    35. RPE medium (see Recipes: Table 6)
    36. 2x iPSC-RPE freezing medium (see Recipes: Table 7)
    37. 1 mM Chetomin solution (see Recipes: Table 8)
    38. 1 M Nicotinamide (100x) solution (see Recipes: Table 9)

  4. Flow cytometry
    1. 96-well round bottom assay plates (Genesee Scientific, catalog number: 25-224)
    2. CorningTM FalconTM Test Tube with Cell Strainer Snap Cap (Fisher Scientific, catalog number: 352235)
    3. CorningTM CostarTM Sterile Disposable Reagent Reservoirs (Fisher Scientific, catalog number: 4870)
    4. 5 ml Serological pipettes (Bio Pioneer, catalog number: GEX0050-S01)
    5. 10 ml Serological pipettes (Bio Pioneer, catalog number: GEX0100-S01)
    6. P20 pipette tips sterile with filter
    7. P200 pipette tips sterile with filter
    8. P1000 pipette tips sterile with filter
    9. P20 pipette tips without filter
    10. (Optional) P200 pipette tips without filter
    11. Fixation/Permeabilization Solution Kit with BD GolgiStopTM (BD Biosciences, catalog number: 554715)
    12. 1x Dulbecco’s phosphate buffered saline (DPBS) without calcium and magnesium (Thermo Fisher Scientific, catalog number: 14190250)
    13. Bovine Serum Albumin (BSA) (Sigma, catalog number: A2153-100G)
    14. (Optional) NaN3 (Sigma, catalog number: S2002-5G)
    15. 37% Formaldehyde (Sigma, catalog number: F-1635-500ML)
    16. Rabbit polyclonal anti-ZO-1 antibody (abcam, catalog number: ab59720)
      Note: During preparation of this manuscript the antibody ab59720 was no longer available. A potential replacement: abcam, catalog number: ab221547.
    17. Mouse monoclonal anti-MiTF antibody (abcam, catalog number: ab12039)
    18. Recombinant Rabbit IgG, monoclonal [EPR25A]–Isotype Control (abcam, catalog number: ab172730)
    19. Mouse IgG1, kappa monoclonal [15-6E10A7]–Isotype Control (abcam, catalog number: ab170190)
    20. Donkey-anti-Rabbit Alexa FluorTM 647 conjugated antibody (abcam, catalog number: ab150075)
    21. Goat-anti-Mouse Alexa FluorTM 488 conjugated antibody (Thermo Scientific, catalog number: A-11001)
    22. FACS Buffer (see Recipes: Table 10)
    23. FACS-FIX Buffer (see Recipes: Table 11)
    Note: For antibody working concentration, see Recipes: Table 15.

  5. Immunofluorescence
    1. Millicell EZ SLIDE 8-well glass slides (Millipore, catalog number: PEZGS0816)
    2. Cover glass slides (Fisherbrand, catalog number: 12-545-F)
    3. 5 ml Serological pipettes (Bio Pioneer, catalog number: GEX0050-S01)
    4. 10 ml Serological pipettes (Bio Pioneer, catalog number: GEX0100-S01)
    5. P20 pipette tips sterile with filter
    6. P200 pipette tips sterile with filter
    7. 1x Dulbecco’s phosphate buffered saline (DPBS) without calcium and magnesium (Thermo Fisher Scientific, catalog number: 14190250)
    8. Bovine Serum Albumin (BSA) (Sigma, catalog number: A2153-100G)
    9. Paraformaldehyde (PFA)
    10. Tween® 20 (Sigma, catalog number: P9416-100ML)
    11. Triton X-100 (Manufacturer, catalog number: X-100-500ML)
    12. Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix (Matrigel) (Corning, catalog number: 354230)
    13. Rabbit polyclonal anti-ZO-1 antibody (abcam, catalog number: ab59720)
    14. Mouse monoclonal anti-MiTF antibody (abcam, catalog number: ab12039)
    15. Mouse monoclonal anti-Bestrophin 1 antibody (Novus Biologicals, catalog number: NB300-164SS)
    16. Recombinant Rabbit IgG, monoclonal [EPR25A]–Isotype Control (abcam, catalog number: ab172730)
    17. Mouse IgG1, kappa monoclonal [15-6E10A7]–Isotype Control (abcam, catalog number: ab170190)
    18. Donkey-anti-Rabbit Alexa FluorTM 647 conjugated antibody (abcam, catalog number: ab150075)
    19. Goat-anti-Mouse Alexa FluorTM 488 conjugated antibody (Thermo Scientific, catalog number: A-11001)
    20. ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technologies, catalog number: 8961)
    21. IF Wash Buffer (see Recipes: Table 12)
    22. IF Perm Buffer (see Recipes: Table 13)
    23. IF Staining Buffer (see Recipes: Table 14)
    Note: For antibody working concentration, see Recipes: Table 15.

Equipment

  1. iPSC Cell Culture
    1. Biosafety cabinet (Labconco, model: Logic+)
    2. Incubator with humidity and gas control set to maintain 37 °C and 95% humidity in an atmosphere of 5% CO2 in air (Panasonic, model: MCO-170AICUVH-PA)
    3. Water bath (Thermo Scientific, model: Precision)
    4. Tissue culture centrifuge with rotors for 15 ml conical tubes and 50 ml conical tubes (Thermo Scientific, model: Legend RT+)
    5. Phase contrast inverted microscope (objectives: x4, x10, x20) (Olympus, model: CKX41SF)
    6. (Optional) Phase contrast inverted microscope with camera (objectives: x4, x10, x20) (Thermo Scientific, model: EVOS XL Core)
    7. Microscope Object marker (Nikon, model MBW10020)
    8. Pipette aid
    9. P20 Micropipette
    10. Freezer -20 °C
    11. Refrigerator 2-8 °C

  2. Monolayer plating
    1. Biosafety cabinet (Labconco, model: Logic+)
    2. Incubator with humidity and gas control set to maintain 37 °C and 95% humidity in an atmosphere of 5% CO2 in air (Panasonic, model: MCO-170AICUVH-PA)
    3. Tissue culture centrifuge with rotors for 15 ml conical tubes and 50 ml conical tubes (Thermo Scientific, model: Legend RT+)
    4. Phase contrast inverted microscope (objectives: x4, x10, x20) (Olympus, model: CKX41SF)
    5. Phase contrast inverted microscope with camera (objectives: x4, x10, x20) (Thermo Scientific, model: EVOS XL Core)–Optional
    6. Pipette aid
    7. P20 Micropipette
    8. P200 Micropipette
    9. P1000 Micropipette
    10. Automated cell counter (Bio-Rad, model: TC20) or a hemocytometer (Hausser Scientific, catalog number: 1483) or equivalent
    11. Freezer -20 °C
    12. Refrigerator 2-8 °C

  3. iPSC-RPE differentiation and cryopreservation
    1. Biosafety cabinet (Labconco, model: Logic+)
    2. Incubator with humidity and gas control set to maintain 37 °C and 95% humidity in an atmosphere of 5% CO2 in air (Panasonic, model: MCO-170AICUVH-PA)
    3. Tissue culture centrifuge with rotors for 15 ml conical tubes and 50 ml conical tubes (Thermo Scientific, model: Legend RT+)
    4. Phase contrast inverted microscope (objectives: x4, x10, x20) (Olympus, model: CKX41SF)
    5. (Optional) Phase contrast inverted microscope with camera (objectives: x4, x10, x20) (Thermo Scientific, model: EVOS XL Core)
    6. Pipette aid
    7. P20 Micropipette
    8. P200 Micropipette
    9. P1000 Micropipette
    10. Automated cell counter (Bio-Rad, model: TC20) or a hemocytometer (Hausser Scientific, catalog number: 1483) or equivalent.
    11. Mr. Frosty freezing container (Corning, model: CoolCell® FTS30)
    12. Refrigerator 2-8 °C
    13. Freezer -20 °C
    14. Freezer -80 °C
    15. Liquid nitrogen vapor tank

  4. Flow cytometry
    1. Pipette aid
    2. P20 Micropipette
    3. P200 Micropipette
    4. P1000 Micropipette
    5. P200 Multichannel micropipette
    6. Refrigerator 2-8 °C
    7. Freezer -20 °C
    8. Flow cytometer (BD Biosciences, model: FACSCanto II) or equivalent

  5. Immunofluorescence
    1. Pipette aid
    2. P20 Micropipette
    3. P200 Micropipette 
    4. P1000 Micropipette
    5. Refrigerator 2-8 °C
    6. Freezer -20 °C
    7. Confocal laser scanning fluorescence microscope (Olympus, FluoView1000)

Software

  1. FlowJo (Version 10) (FlowJo, LLC, https://www.flowjo.com/)
  2. FlowView ASW V03.01.03.03 or V4.2a (Olympus Life Science, https://www.olympus-lifescience.com/en/support/downloads/)

Procedure

  1. iPSC cell culture
    1. Thaw iPSC cells
      1. Prepare 12 ml of mTeSR containing 10 μM ROCK Inhibitor.
      2. Transfer 9 ml of mTeSR containing 10 μM ROCK Inhibitor into a sterile conical tube labeled with the name of the line.
      3. Remove vial of cryopreserved cells from liquid nitrogen tank. Keep vial on dry ice.
      4. Place and shake gently in a 37 °C water bath until a pea-sized ice crystal remains (around 2 min).
      5. Wipe off excess water from the vial, spray with 70% ethanol before placing in the hood.
      6. Remove thawed cells from the vial and add gently into 9 ml mTeSR containing 10 μM ROCK Inhibitor in a conical tube. Wash the vial with 1-2 ml of mTeSR containing 10 μM ROCK Inhibitor. Collect all cells in the same conical tube.
      7. Centrifuge cells for 5 min at 53 x g (500 RPM in a Sorvall 75006445 rotor with 75006441 K buckets) at room temperature.
      8. Aspirate supernatant, and gently resuspend cell pellet in 2 ml of mTeSR containing 10 μM ROCK Inhibitor (1 cryovial is thawed into 1 well of 6-well plate).
      9. Label a Matrigel plate with name of line, clone and passage number. Aspirate DMEM/F-12 from Matrigel-coated plate. Add +1 to the passage number after thawing.
        Note: Do not add +1 to the passage number if the passage number was increased during cryopreservation of iPSCs.
      10. Plate cells resuspended in 2 ml into one well of a Matrigel-coated 6-well plate (final volume 2 ml/well).
      11. 24 h after plating, observe cells. Wash cells gently with DMEM/F-12 (2 ml/well) to remove cell debris and feed using fresh mTeSR medium without ROCK Inhibitor (2 ml/well).
      12. Daily, observe the iPSCs, remove the differentiated cells, and change the medium with fresh mTeSR (2 ml/well).
        Note: It is critical to maintain iPSC culture differentiation free.
      13. Cells should reach 80-90% of confluency and be ready for passage in about 5 days.
    2. iPSC passaging using Dispase
      1. Prepare 1x (2 mg/ml) Dispase solution by adding 9 ml DMEM/F-12 to 1 ml of 10x Dispase (20 mg/ml).
      2. Allow 1x Dispase solution to come to room temperature.
        Note: 1x Dispase solution can be stored at 4 °C for maximum 2 weeks.
      3. Mark any areas of differentiation on the well to be split using the Microscope Object marker.
      4. Aspirate spent media. Aspirate marked areas of differentiation, if any, by gently tapping a Pasteur pipette within the marked circle. Wash with 2 ml of DMEM/F-12 per well.
      5. Add 1 ml of 1x Dispase in each well to be split. Incubate at 37 °C for 5 min.
      6. Check morphology of colonies after 5 min.
        When edges of the colonies are slightly curled up, cells are ready to be passaged. If edges of colonies are not curled up, incubate cells at 37 °C for another 1-2 min. Do not incubate with Dispase for longer than 8 min.
      7. Aspirate Dispase from all wells.
      8. Rinse the wells gently 3 times with DMEM/F12 (2 ml/well).
      9. Add 1 ml of mTeSR media to each well to be passaged.
      10. Use a glass serological pipette to detach colonies. Hold the pipette at a 90° angle to the surface of the plate. Scrape across the surface of the 6-well plate in the motion outlined in Figure 1 (start from top left side of the well and zig-zag tightly down to bottom-right side, then turn plate clockwise or counterclockwise and scrape again). Scrape until at least 90% of the colonies are detached from the well.


        Figure 1. Pattern of movement of a glass serological pipette during the iPSCs passaging with Dispase. After scraping the well in one direction, turn the plate clockwise or counterclockwise by 90° and scrape remaining iPSC colonies again. About 90% cells should be detached from the well. Please refer to the section on iPSC passaging using Dispase for details.

      11. Wash plate with the volume of mTeSR required to bring cells up to the final volume needed to seed a new Matrigel-coated vessel. Calculate the final volume considering 2 ml per each well to be seeded with passaged cells. For example, if cells are to be passaged 1 to 3 the final volume will be 6 ml, therefore the volume of mTeSR used to wash the plate is 5 ml.
      12. Seed cells on a new Matrigel plate plating 1 ml per well and then add 1 ml more. Plate cells dropwise across the entire surface of the well to ensure uniform plating.
      13. Observe seeded cells under microscope to ensure even plating.
      14. Place in a 37 °C incubator. Shake the plate in T-shape to homogenously distribute the colonies pieces in the well.
      15. Twenty-four hours after plating gently, wash cells with DMEM/F-12 before adding fresh mTeSR medium.
        Note: For a healthy and efficient iPSC culture, it is critical to plate cells uniformly. Plate cells uniformly across the entire surface of the well and, when plating multiple well,s plate cells uniformly across all wells.

  2. Monolayer plating
    Note: After thawing an iPSC line, passage cells with Dispase at least once before plating monolayer.
    1. Remove 6-well plates from the incubator. When iPSC cells are at around 80% confluency (cells are ready for a passage), iPSC cells are ready for Monolayer. Mark all differentiated cells, which need to be removed.
    2. Aspirate the spent medium. Remove all marked differentiated cells and wash cells with DMEM/F-12 (2 ml/well).
    3. Aspirate DMEM/F-12 and add 1 ml of room temperature Accutase to well of a 6-well plate. Incubate cell for 8 min at 37 °C.
    4. After 8 min of incubation, add 1 ml per well of mTeSR containing 5 μM ROCK inhibitor and re-suspend cells as single cells without scraping plate surface, using a P1000 pipette. Pipette cells 10-12 times, turn the dish by 180° (upside down) and pipette 5 more times. Collect cells from all wells in a 50 ml conical tube. You should not see any cell clumps.
    5. Wash all wells twice with 5 ml of mTeSR containing 5 μM ROCK Inhibitor. Collect all cells in the same conical tube.
    6. Centrifuge the cells at 53 x g (500 RPM in a Sorvall 75006445 rotor with 75006441 K buckets) for 7 min at room temperature. Aspirate the supernatant and resuspend cells in 10 ml of mTeSR containing ROCK Inhibitor.
    7. Mix the pooled cell suspension by inverting 20 times or more if necessary. Perform the live cell count using 0.4% Trypan Blue Solution.
      Note: iPSC cell viability should be not lower than 80%.
    8. Prepare required number of cells. Optimal cell number will vary depending on the scale of differentiation. iPSC-RPE differentiation protocol requires 2.5 x 104 live cells per cm2 (2 x 106 per one 100 mm dish). For one 100 mm dish, prepare in a 15 ml conical tube 11 ml of cell suspension containing 2.2 x 106 cells. Mix cell suspension very well by inverting the tube 20 times.
    9. Add 10 ml of cell suspension per 100 mm dish dropwise using a 10 ml pipette.
      e (T-shape).
    Note: It is critical to plate cells uniformly on the entire surface of the plate. To help distribute the cells uniformly plate one dish at the time and shake the newly plated dish in a cross shape (T-shape).
    1. Place plates in the incubator without stacking the plates. Incubate the cells until next morning, at 37 °C, 5% CO2.
    2. Next day change medium for fresh mTeSR without ROCK inhibitor (10 ml/100 mm dish). Monolayer for iPSC-RPE cell differentiation requires culturing cells for about 4-5 days until the monolayer reaches 80% confluency. Change medium with fresh mTeSR daily.

  3. iPSC-RPE differentiation
    Refer to Figure 2 for a schematic representation of the differentiation protocol.


    Figure 2. Schematic representation of the iPSC-RPE differentiation protocol. Nicotinamide (NIC), Chetomin (CTM). iPSC monolayer is cultured until the cells reach 80% confluency. iPSC-RPE differentiation is initiated and driven using small molecules (NIC and CTM). iPSC-RPEs continue to differentiate and expand for a total of 12 weeks. Whole culture passages promote expansion of iPSC-RPE cells. Adapted from Smith et al., 2019.

    1. Day 0 (D0)–When iPSC monolayer reaches about 80% confluency initiate the iPSC-RPE differentiation by replacing mTeSR medium with RPE DM medium (see Recipes: Table 5) (24 ml/100 mm dish)
    2. D1–24 h after initiation of the differentiation replace spent RPE DM medium with fresh RPE DM medium supplemented with 10 mM Nicotinamide (see Recipes: Table 9) and 50 nM Chetomin (see Recipes: Table 8) (24 ml/100 mm dish).
    3. Daily change spent medium with fresh RPE DM medium supplemented with 10 mM Nicotinamide (NIC) and 50 nM Chetomin (CTM) (24 ml/10 cm dish).
      Note: Due to very high cell number, high proliferative rate and metabolic activity of iPSC-RPE cells to obtain a healthy and robust iPSC culture it is strongly recommended to maintain the schedule of 24 h media change throughout the entire differentiation.
    4. On D14 change spent medium with fresh RPE DM medium supplemented with 10 mM Nicotinamide (24 ml/100 mm dish).
    5. Daily change spent medium with fresh RPE DM medium supplemented with 10 mM Nicotinamide (24 ml/100 mm dish).
      Note: First cells start to acquire melanin pigmentation and the characteristic polygonal shape after 2-3 weeks.
    6. On D28 passage the cells–first passage.
      1. (Optional) Take images of the cells–(Figure 3-D28-left panel).


        Figure 3. Images of the iPSC-RPE cells. Bright-field image of iPSC-RPE at Day 28 (D28) of the differentiation iPSC-RPE cells appear as small clusters of polygonal pigmented cells which expand over time (left); T150 flasks containing iPSC-RPE cells (iPSCORE 87_1) at Day 84 (D84) (middle left); Bright-field image of iPSC-RPE sample (iPSCORE_42_1) at Day 84 illustrating a highly organized monolayer with strong melanin pigmentation (middle right) and characteristic polygonal shape (right). Adapted from Smith et al., 2019.

      2. Aspirate spent medium and wash cells with PBS (10 ml/100 mm dish).
      3. Aspirate PBS and add 5 ml of room temperature Accutase to a 100 mm dish. Incubate cells for 12 min at 37 °C.
      4. After 12 min of incubation add 5 ml per dish of RPE DM medium, re-suspend cells in Accutase as single cells without scraping the surface of the plate, using a P1000 pipette. If cells are difficult to remove from a dish, use a cell scraper.
      5. Collect cells in a 50 ml conical tube.
      6. Wash plate three additional times, each with 5 ml of RPE DM medium. Collect all cells in the same conical tube.
      7. Centrifuge cells for 8 min at 136 x g (800 RPE in a Sorvall 75006445 rotor with 75006441 K buckets) at room temperature.
      8. After centrifugation, aspirate the supernatant and resuspend the cells in 10 ml of RPE medium (see Recipes: Table 6).
      9. Gently pass all cells through a 70 μm strainer. Add medium for a total of 20 ml of final volume passing it through the strainer.
      10. Plate cells on two fresh 100 mm dishes which were coated overnight with Matrigel.
      11. Using a 10 ml pipette add dropwise 10 ml of cell suspension per 100 mm dish.
        Note: It is critical to plate cells uniformly on the entire surface of the plates. To help distribute the cells uniformly plate one dish at the time and shake the newly plated dish in a cross shape (T-shape).
        Optional: Instead of using two 100 mm dishes plate cells on one T150 flask.
      12.  Place plates in the incubator. Incubate the cells until next day, at 37 °C, 5% CO2 without stacking the plates.
    7. Twenty-four hours after split, change medium for fresh RPE medium (10 ml/100 mm dish). If using T150 flask use 25 ml/T150 flask.
    8. Change medium daily for fresh RPE medium (10 ml/100 mm dish) for 4 weeks. If using T150 flask use 25 ml/T150 flask.
    9. On D56 passage the cells–second passage.
      Perform the passage following Steps C6a-C6g.
      Note: If cells were plated on a T150 flask, incubate cells with 10 ml of Accutase per flask and use a cell scraper to recover all the cells. To collect all the cells, wash flask three times, each time with 10 ml of medium.
      1. After centrifugation, aspirate the supernatant and resuspend the cells in 20 ml of RPE medium.
      2. Gently pass all cells through a 70 μm strainer. Add medium for a total of 50 ml of final volume passing at least 20 ml of it through the strainer.
      3. Plate cells onto two fresh T150 flasks which were coated overnight with Matrigel.
      4. Add 25 ml of cell suspension per each T150 flask dropwise using a 10 ml pipette.
        Note: It is critical to plate cells uniformly on the entire surface of the plates which were coated overnight with Matrigel. To help distribute the cells uniformly plate one flask at the time and shake the newly plated flasks in a cross shape (T-shape). 
      5. Place flasks in the incubator without stacking. Incubate the cells until next morning, at 37 °C, 5% CO2.
    10. Twenty-four hours after split, change medium with fresh RPE medium (25 ml/T150 flask).
    11. Change medium daily with fresh RPE medium (25 ml/T150 flask) for 4 weeks.
    12. On D84 collect all cells.
      1. (Optional) Take images of the cells. Refer to Figure 3 for an example of expected iPSC-RPE yield, pigmentation (Figure 3–panel middle left and middle right) and cell morphology (Figure 3–panel middle right and right).
      2. Aspirate spent medium and wash cells with PBS (20 ml/T150 flask).
      3. Aspirate PBS and add 10 ml of room temperature Accutase to each flask. Incubate for 12 min at 37 °C.
      4. After 12 min of incubation, add 10 ml per well of RPE medium re-suspend cells. Use cell scraper to remove all cells.
      5. Collect cells in a 50 ml conical tube.
      6. Wash plate three additional times with 10 ml of RPE medium. Collect all cells in the same conical tube.
      7. Centrifuge cells for 8 min at 136 x g (800 RPE in a Sorvall 75006445 rotor with 75006441 K buckets) at room temperature.
      8. After centrifugation, aspirate the supernatant and resuspend the cells in 20 ml of RPE medium.
      9. Gently pass all cells through a 70 μm strainer. Add medium for a total of 40 ml of final volume passing at least 20 ml of it through the strainer.
      10. Mix the cell suspension by inverting 20-30 times. Perform the live cell count using 0.4% Trypan Blue Solution.
      Notes:
      1. When performing the live cell count of iPSC-RPE using automated cell counter, cell viability may be inaccurately scored as high due to the high melanin concentration in the cells. Depending on the experimental needs, iPSC-RPE cells can be cryopreserved for future experiments, fixed for flow cytometry analysis and/or plated for immunofluorescence analysis.
      2. Cells start to acquire melanin pigmentation and the characteristic polygonal shape after 2-3 weeks in culture and about 1 week after each passage. The first signs of the cells starting to acquire the pigmentation is slightly grayish (“dirty”) hue of the color of the medium. When cells are strongly pigmented the medium also acquires dark color. Cell suspension of concentrated iPSC-RPE at D84 appears black, similarly to the pelleted iPSC-RPE-cells which are also black (Figure 4).


      Figure 4. Images of the iPSC-RPE cells at D84. iPSC-RPE cells (iPSCORE_29_1) collected prior to the centrifugation (left) and after centrifugation (right).

  4. Cryopreservation of iPSC-RPE
    1. Prepare 2x iPSC-RPE freezing medium by preparing 20% DMSO solution in FBS. Prepare 0.25 ml of 2x iPSC-RPE freezing medium per each cryovial intended to be cryopreserved. Freeze cells at a final density of 1.2 x 107/ml (depending on the downstream experiments the volume and the concentration of the cryopreserved iPSC-RPE cells in a single cryovial can be modified).
      Optional: If a serum free conditions are required, prepare the 2x iPSC-RPE freezing medium using KOSR instead of FBS.
    2. Prepare and print the labels for cryovials. Prepare n + 2 number of labels (n = number of cryovials to be cryopreserved). Prepare and affix the labels on all cryovials to be frozen, use one label for the Mr. Frosty and one label for record keeping (i.e., lab book).
    3. After the live cell count (Step C12j), determine how many cells should be cryopreserved and transfer desired number of cells into a new 15 ml or 50 ml conical tube.
    4. Centrifuge cells for 5-8 min at 136 x g (800 RPM in a Sorvall 75006445 rotor with 75006441 K buckets) at room temperature (adjust the time of centrifugation depending on the volume of cells).
    5. After centrifugation, aspirate the supernatant and resuspend the cells in 0.25 ml of FBS (or KOSR) per each cryovial to be frozen at the concentration of 2.4 x 107/ml (i.e., for 10 cryopreserved vials resuspend 6 x 107 cells in 2.5 ml of FBS or KOSR).
    6. Open all pre-labeled cryovials and add 0.25 ml of cell suspension to each cryovial.
    7. Add 0.25 ml of 2x iPSC-RPE freezing medium to each cryovial containing the iPSC-RPE cell suspension.
    8. Close all cryovials and gently invert them 5-6 times to mix cell suspension and 2x iPSC-RPE freezing medium. Transfer cryovials to Mr. Frosty freezing container.
    9. Immediately transfer Mr. Frosty into a -80 °C freezer. When freezing large number of cryovials (i.e., multiple Mr. Frosties) prepare individual batches, with each batch containing only the number of cryovials that will fit into one Mr. Frosty.
    10. After 24-48 h, transfer the cells into a liquid nitrogen vapor tank. Update accordingly the records (i.e., box maps).

    Here, we provide detailed protocols for flow cytometry (FC) and immunofluorescence (IF) which can be applied to perform quantitative (FC) and qualitative (FC and IF) quality control of derived iPSC-RPE cells.

  5. Flow cytometry
    1. After the live cell count (Step C12j), determine how many cells should be fixed for flow cytometry analysis and transfer desired number of cells into a 15 ml conical tube. Use at least 2-5 x 106 cells.
    2. Centrifuge cells for 5 min at 136 x g (800 RPM in a Sorvall 75006445 rotor with 75006441 K buckets) at room temperature.
    3. After centrifugation, aspirate the supernatant and resuspend the cells in 10 ml of PBS.
      Optional: If the volume of cell suspension used for flow cytometry in smaller than 0.5 ml then add directly to the cells 14 ml of PBS and centrifuge mix of cells and PBS for 8 min at 136 x g (800 rpm in a Sorvall 75006445 rotor with 75006441 K buckets) at room temperature.
    4. Fix and permeabilize iPSC-RPE cells using the Fixation/Permeabilization Solution Kit with BD GolgiStopTM following manufacturer recommendations. After the last centrifugation, aspirate the supernatant and resuspend the cells in the 1x BD Perm/WashTM Buffer at the concentration of 1 x 107/ml. For each flow cytometry staining use 2.5 x 105 cells.
      Note: Staining of 2.5 x 105 cells allows for an efficient cells and reagent usage, however it is also possible to use 1 x 106 cells per staining maintaining the same antibodies dilution ratios.
    5. Transfer 25 μl of fixed and permeabilized cells into 5 wells of a 96-well round bottom assay plate. In order to limit usage of the antibodies and cells when staining multiple lines, mix equal number of cells from each line and transfer 25 μl of the cell mix into four control wells [Recombinant Rabbit IgG, monoclonal class control antibody (Rb-IgG), Mouse IgG1, kappa monoclonal (M-IgG1), anti ZO-1 antibody (ZO1) and anti MiTF antibody (MiTF)].
    6. Following Table 15 add appropriate concentrations of the antibodies in each well. Using a multichannel pipette set for 20 μl mix cells and antibodies gently by pipetting up and down 20 times.
    7. Incubate cells with primary antibodies for 1 h at room temperature.
    8. After 1 h, add 150 μl of FACS buffer (see Recipes: Table 10).
    9. Centrifuge plate at 863 x g (2,000 RPM in a Sorvall 75006445 rotor with 75006441 K buckets) for 8-10 s counting from when the speed reaches 863 x g (2,000 rpm in a Sorvall 75006445 rotor with 75006441 K buckets) at room temperature.
      In detail:
      1. Set the centrifuge for 863 x g (2,000 RPM), 1 min, room temperature); start the centrifuge and wait until the speed reaches 863 x g (2,000 rpm).
      2. Count to 8-10 s and stop the centrifuge.) The pellet after the centrifugation should be clearly visible especially when using 1 x 106 cells.
    10. After centrifugation, gently aspirate the supernatant, being very careful not to aspirate any cells. If using vacuum to aspirate cells, use a P20 tip without a filter (or a P200 + P20 tips without filters). Leave about 20 μl of liquid in each well to avoid aspirating the cells.
    11. Using a multichannel pipette add 200 μl of FACS buffer and mix cells gently 5-6 times.
    12. Centrifuge plate like in Step E9.
    13. Repeat Steps E10-E12 to wash the cells one more time.
    14. Resuspend cells in 50 μl of 1x BD Perm/WashTM Buffer.
    15. Following Table 15 add appropriate concentrations of the antibodies in each well. Using a multichannel pipette set for 40 μl mix cells and antibodies gently by pipetting up and down 20 times.
    16. Incubate cells with secondary antibodies for 45 min at room temperature in darkness.
    17. After 45 min repeat Steps E8-E13.
    18. After the last centrifugation aspirate the supernatant and resuspend the cells in 200 μl of FACS-FIX Buffer (see Recipes: Table 11). Using a multichannel pipette resuspend the cells by pipetting 5-6 times.
    19. Using a P1000 pipette transfer each sample, one at a time, into a CorningTM FalconTM Test Tubes with Cell Strainer Snap Cap passing the cells through the strainer in a cap.
    20. With an additional 250 μl wash each well and transfer to the appropriate tube passing the cells through the strainer in a cap. Depending on the number of cells used for staining dilute the cells to an appropriate concentration to avoid clogging the flow cytometer.
    21. Place all the tubes in an appropriate rack and wrap them in an aluminum foil to protect from light.
    22. Proceed with acquisition using a flow cytometer FACS Canto II (or alternative flow cytometer).
    23. Perform the flow cytometry analysis using FlowJo software V 10.4. Refer to the Figure 5 for an example of iPSC-RPE flow cytometry staining results.


      Figure 5. Flow-cytometry analysis of iPSC-RPE (iPSCORE_42_1) at Day 84 showing high co-staining of Zonula Occludens 1 (ZO-1) and Microphthalmia-associated Transcription Factor (MiTF). Adapted from Smith et al., 2019.

  6. Immunofluorescence
    1. Coat Millicell EZ SLIDE 8-well glass slides overnight with Matrigel.
    2. Plate fresh or cryopreserved iPSC-RPE cells on the Matrigel coated Millicell EZ SLIDE 8-well glass slides. Plate at least 6 wells per line at the density of 1-1.5 x 106/cm2.
    3. Culture cells for 10 days until they reach full confluency, re-acquire polygonal shape and pigmentation.
    4. Aspirate the medium and wash cells twice with PBS. Aspirate the PBS.
    5. Fix cells with 4% PFA for 10 min at room temperature.
    6. Remove the PFA solution and wash cells twice with freshly prepared IF Wash Buffer (see Recipes: Table 12). Aspirate the IF Wash Buffer.
    7. Saturate and permeabilize the cells using IF Perm Buffer (see Recipes: Table 13). Incubate the cells for 20 min at room temperature.
    8. In the last 5 min of the saturation and permeabilization prepare the primary antibody solutions in IF Staining Buffer (see Recipes: Table 14 and Table 15) for the appropriate concentrations of the antibodies. Store antibodies solutions on ice until use.
    9. After saturation and permeabilization aspirate all the buffer and add antibodies solutions to the appropriate wells.
    10. Incubate cells with the antibodies solution overnight at 4 °C.
    11. Next day (morning) prepare the secondary antibody solutions in IF staining Buffer. Refer to the Table 15 for the appropriate concentrations of the antibodies. Keep antibodies solutions on ice until use, protected from light.
    12. Aspirate the primary antibodies solutions and wash cells three times with PBS. After last wash aspirate all PBS.
    13. Immediately add the secondary antibodies solutions to the appropriate wells. Incubate cells for 1 h at room temperature in darkness.
    14. Aspirate the secondary antibodies solutions and wash cells three times with PBS. After last wash aspirate all PBS.
    15. Detach the walls of the Millicell EZ SLIDE 8-well glass slides.
    16. Add ProLong Gold Antifade Reagent with DAPI following manufacturer’s recommendations and gently mount the cover glass slide avoiding bubbles. Use a pencil rubber to gently remove any bubbles. Store the slide(s) at room temperature for several hours (best until next day) in darkness to allow proper mounting.
    17. Acquire images using an appropriate immunofluorescence microscope (best is a confocal laser scanning fluorescence microscope). Refer to Figure 6 for an example of iPSC-RPE immunofluorescence staining.


      Figure 6. Immunofluorescence analysis of Bestrofin 1 (BEST1) (iPSCORE_29_1), ZO-1 (iPSCORE_29_1), and MiTF (iPSCORE_42_1). ZO-1 appears as sharp cell membrane staining; BEST1 membrane staining appears “fuzzier” compared to ZO1 staining; MiTF nuclear staining. Adapted from Smith et al., 2019.

Recipes

  1. Cell culture reagents and media preparation

    Table 1. Preparation of Matrigel solution


    Table 2. Preparation of 1. 10 mM ROCK inhibitor, Y-27632 dihydrochloride


    Table 3. Preparation of 10x Dispase Solution


    Table 4. Preparation of mTeSRTM1 complete medium


    Table 5. Preparation of RPE DM medium (following Maruotti et al., 2015)


    Table 6. Preparation of RPE medium (following Maruotti et al., 2015)


    Table 7. Preparation of 2x iPSC-RPE freezing medium (for 5 ml)


    Table 8. Preparation of 1 mM Chetomin solution


    Table 9. Preparation of 1M Nicotinamide (100x) solution


  2. Buffer preparation

    Table 10. Preparation of FACS Buffer


    Table 11. Preparation of FACS-FIX Buffer


    Table 12. Preparation of IF Wash Buffer


    Table 13. Preparation of IF Perm Buffer


    Table 14. Preparation of IF Staining Buffer


  3. Antibodies

    Table 15. Antibodies concentrations

Acknowledgments

This work was supported in part by a CIRM grant GC1R-06673-B and NIH grants HG008118, HL107442, DK105541, DK112155, and EY021237. This protocol was adapted from previous work (Smith, D'Antonio-Chronowska et al., 2019).

Competing interests

Authors declare no competing interests.

Ethics

iPSC lines generated from individuals of different ethnicities (3 European Americans, 2 East Asian Americans, and 1 African American) were obtained from iPSCORE (Panopoulos et al., 2017). Donors were all females ranging from 21 to 62 years of age at the time of donation. The recruitment of these individuals was approved by the Institutional Review Boards of the University of California, San Diego, and The Salk Institute (project no. 110776ZF).

References

  1. Al-Zamil, W. M. and Yassin, S. A. (2017). Recent developments in age-related macular degeneration: a review. Clin Interv Aging 12: 1313-1330. 
  2. Aoki, H., Hara, A., Nakagawa, S., Motohashi, T., Hirano, M., Takahashi, Y. and Kunisada, T. (2006). Embryonic stem cells that differentiate into RPE cell precursors in vitro develop into RPE cell monolayers in vivo. Exp Eye Res 82(2): 265-274.
  3. Buchholz, D. E., Hikita, S. T., Rowland, T. J., Friedrich, A. M., Hinman, C. R., Johnson, L. V. and Clegg, D. O. (2009). Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells. Stem Cells 27(10): 2427-2434. 
  4. Hazim, R. A., Karumbayaram, S., Jiang, M., Dimashkie, A., Lopes, V. S., Li, D., Burgess, B. L., Vijayaraj, P., Alva-Ornelas, J. A., Zack, J. A., Kohn, D. B., Gomperts, B. N., Pyle, A. D., Lowry, W. E. and Williams, D. S. (2017). Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization. Stem Cell Res Ther 8(1): 217. 
  5. Idelson, M., Alper, R., Obolensky, A., Ben-Shushan, E., Hemo, I., Yachimovich-Cohen, N., Khaner, H., Smith, Y., Wiser, O., Gropp, M., Cohen, M. A., Even-Ram, S., Berman-Zaken, Y., Matzrafi, L., Rechavi, G., Banin, E. and Reubinoff, B. (2009). Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. Cell Stem Cell 5(4): 396-408. 
  6. Klimanskaya, I., Hipp, J., Rezai, K. A., West, M., Atala, A. and Lanza, R. (2004). Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. Cloning Stem Cells 6(3): 217-245. 
  7. Maruotti, J., Sripathi, S. R., Bharti, K., Fuller, J., Wahlin, K. J., Ranganathan, V., Sluch, V. M., Berlinicke, C. A., Davis, J., Kim, C., Zhao, L., Wan, J., Qian, J., Corneo, B., Temple, S., Dubey, R., Olenyuk, B. Z., Bhutto, I., Lutty, G. A. and Zack, D. J. (2015). Small-molecule-directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells. Proc Natl Acad Sci U S A 112(35): 10950-10955.
  8. Maruotti, J., Wahlin, K., Gorrell, D., Bhutto, I., Lutty, G. and Zack, D. J. (2013). A simple and scalable process for the differentiation of retinal pigment epithelium from human pluripotent stem cells. Stem Cells Transl Med 2(5): 341-354. 
  9. Mitchell, P., Liew, G., Gopinath, B. and Wong, T. Y. (2018). Age-related macular degeneration. Lancet 392(10153): 1147-1159. 
  10. Osakada, F., Ikeda, H., Mandai, M., Wataya, T., Watanabe, K., Yoshimura, N., Akaike, A., Sasai, Y. and Takahashi, M. (2008). Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nat Biotechnol 26(2): 215-224.
  11. Osakada, F., Jin, Z. B., Hirami, Y., Ikeda, H., Danjyo, T., Watanabe, K., Sasai, Y. and Takahashi, M. (2009). In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction. J Cell Sci 122(Pt 17): 3169-3179. 
  12. Panopoulos, A. D., D'Antonio, M., Benaglio, P., Williams, R., Hashem, S. I., Schuldt, B. M., DeBoever, C., Arias, A. D., Garcia, M., Nelson, B. C., Harismendy, O., Jakubosky, D. A., Donovan, M. K. R., Greenwald, W. W., Farnam, K., Cook, M., Borja, V., Miller, C. A., Grinstein, J. D., Drees, F., Okubo, J., Diffenderfer, K. E., Hishida, Y., Modesto, V., Dargitz, C. T., Feiring, R., Zhao, C., Aguirre, A., McGarry, T. J., Matsui, H., Li, H., Reyna, J., Rao, F., O'Connor, D. T., Yeo, G. W., Evans, S. M., Chi, N. C., Jepsen, K., Nariai, N., Muller, F. J., Goldstein, L. S. B., Izpisua Belmonte, J. C., Adler, E., Loring, J. F., Berggren, W. T., D'Antonio-Chronowska, A., Smith, E. N. and Frazer, K. A. (2017). iPSCORE: A resource of 222 iPSC lines enabling functional characterization of genetic variation across a variety of cell types. Stem Cell Reports 8(4): 1086-1100. 
  13. Pennington, K. L. and DeAngelis, M. M. (2016). Epidemiology of age-related macular degeneration (AMD): associations with cardiovascular disease phenotypes and lipid factors. Eye Vis (Lond) 3: 34.
  14. Reichman, S., Terray, A., Slembrouck, A., Nanteau, C., Orieux, G., Habeler, W., Nandrot, E. F., Sahel, J. A., Monville, C. and Goureau, O. (2014). From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium. Proc Natl Acad Sci U S A 111(23): 8518-8523.
  15. Smith, E. N., D'Antonio-Chronowska, A., Greenwald, W. W., Borja, V., Aguiar, L. R., Pogue, R., Matsui, H., Benaglio, P., Borooah, S., D'Antonio, M., Ayyagari, R. and Frazer, K. A. (2019). Human iPSC-derived retinal pigment epithelium: a model system for prioritizing and functionally characterizing causal variants at AMD risk loci. Stem Cell Reports 12(6): 1342-1353. 
  16. Sparrow, J. R., Hicks, D. and Hamel, C. P. (2010). The retinal pigment epithelium in health and disease. Curr Mol Med 10(9): 802-823. 
  17. Vugler, A., Carr, A. J., Lawrence, J., Chen, L. L., Burrell, K., Wright, A., Lundh, P., Semo, M., Ahmado, A., Gias, C., da Cruz, L., Moore, H., Andrews, P., Walsh, J. and Coffey, P. (2008). Elucidating the phenomenon of HESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation. Exp Neurol 214(2): 347-361.
  18. Wong, W. L., Su, X., Li, X., Cheung, C. M., Klein, R., Cheng, C. Y. and Wong, T. Y. (2014). Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Glob Health 2(2): e106-116.

简介

诱导多能干细胞(iPSC)是研究组织发育的分子基础的出色模型系统。人iPSC衍生的视网膜色素上皮(iPSC-RPE)细胞具有胎儿样的分子特征。因此,像iPSCORE这样的生物库包含数百个个体产生的iPSC,是检查RPE发育过程中常见遗传变异体如何发挥作用的宝贵资源,导致个体具有不同的成年年龄相关性黄斑变性(AMD)倾向。在这里,我们提出了一种优化的,具有成本效益的和高度可重复的方案,用于在无血清条件下使用小分子衍生人iPSC-RPE细胞,并使用流式细胞仪和免疫荧光进行质量控制。尽管大多数以前的方案需要费力的手动选择才能富集iPSC-RPE细胞,但我们的方案使用了整个培养传代过程,并产生了大量高纯度的iPSC-RPE细胞(88-98.1%ZO-1和MiTF双阳性细胞) 。该协议的简单性和鲁棒性使其能够适应涉及数百个人的iPSC-RPE样本生成的高通量应用。

【背景】与年龄相关的黄斑变性(AMD)是发达国家视力丧失的主要原因,影响了美国的1100万人和全世界的约1.7亿(Pennington和DeAngelis,2016)。此外,考虑到年龄是主要因素,在我们当前的老龄化社会中,AMD的发病率估计将在2020年增加到1.98亿,到2040年将增加到2.88亿,到2050年仅在美国就将增加到2200万(Wong et等,2014年,Pennington和DeAngelis,2016年)。当前的治疗策略虽然有效,但价格昂贵且仅限于延缓疾病进展速度,AMD最终仍会导致视力完全丧失(Al-Zamil和Yassin,2017年; Mitchell等人。 ,2018)。在撰写本文时,有七项正在进行的临床试验旨在评估人iPSC-RPEs或人胚胎干细胞源性视网膜色素上皮细胞(ESC-RPE)的有效性和优化移植条件( NIH-ClinicalTrials.gov)。因此,开发一种强大且具有成本效益的方法来生成大量高质量的iPSC-RPE,对于人类以及家畜中AMD和其他潜在眼病的未来治疗方法的发展势在必行(Sparrow等。 ,2010)。



我们之前已经证明了使用iPSC-RPE细胞来鉴定和研究在AMD的发展中发挥作用的遗传变异的实用性(Smith et al。,2019)。在这项研究中,我们从6个个体(3个欧洲裔美国人,2个东亚裔美国人和1个非裔美国人)中衍生了iPSC-RPE细胞,并显示了它们的形态和分子特征与单纯RPE细胞相似。我们显示iPSC-RPE基因表达谱与人类胎儿RPE高度相似,并且其ATAC-seq(使用测序法测定转座酶可及的染色质)峰富含相关的转录因子基序。我们对整合了来自iPSC-RPE细胞的分子数据的AMD风险基因座进行了精细定位,从而确定了四个变体的优先级,包括在 VEGFA 附近的潜在调控SNP(rs943080)和一个编码SNP(rs34882957, C9 基因中的pP167S)(史密斯等人,2019年)。我们的发现表明,iPSC-RPE细胞是研究与AMD相关的遗传变异的分子功能的优秀模型系统。



十年前开发的用于衍生iPSC-RPE细胞的初始方案涉及自发分化,并且效率极低,需要从未分化的细胞中手动分离色素性iPSC-RPE斑块(Klimanskaya等,2004)。 ; Vugle等,2008)。随后,诱导的定向分化方案导致iPSC-RPE细胞的产量和质量更高,但需要大量动物来源的成分,因此具有相对较低的可重复性或非常耗时(Aoki等,2006;大阪田等人,2008年;布赫霍尔兹等人,2009年;爱德森等人,2009年;赖希曼等人。 / em>,2014年; Hazim 等人,2017年)。将小分子引入iPSC-RPE分化方案极大地简化了程序,并提高了重现性(Osakada等,2009; Maruotti等,2013; Maruotti et al。,2015)。先前的研究已经对分化方案进行了优化,以从有限数量的iPSC或ESC品系中衍生出iPSC-RPE,并且在大多数情况下使用的是小型培养皿。在这里,我们提出了从大型培养瓶中的多个人iPSC品系中提取iPSC-RPEs的优化方案。它类似于Maruotti等人的协议。(2015),但当iPSC达到80%融合时开始分化,我们发现这对于所有测试的iPSCORE品系都是最佳的(Panopoulos等人,2017)。此外,我们在两个步骤中修改了暴露于小分子的时间长度,从而进一步提高了iPSC-RPEs(88-98.1%ZO-1和MiTF双阳性细胞)的产量和纯度。我们的优化方案使我们能够在相同的培养条件下,从六种iPSC系衍生出iPSC-RPE,它们来自不同种族的个体,无需任何个性化的优化步骤。

关键字:人诱导多功能干细胞, 视网膜色素上皮细胞, 人诱导多功能干细胞来源的视网膜色素上皮细胞, 老年性黄斑变性, 分化, 遗传研究, 小分子, 遗传变异

材料和试剂

  1. iPSC细胞培养
    1. 6孔板(Corning,目录号:3506)
    2. 针头过滤器0.2μm(VWR,目录号:28145-501)
    3. Soft-Ject ®三部分式一次性注射器,Air-Tite-3 ml(VWR,目录号:89215-234)
    4. 5 ml硼硅酸盐血清移液管(Fisher Scientific,目录号:1367827E)
    5. 5 ml血清移液器(生物先锋,目录号:GEX0050-S01
    6. 10 ml血清移液管(Bio Pioneer,目录号:GEX0100-S01)
    7. P20移液器吸头,无菌,带过滤器
    8. P1000移液器吸头,无菌,带过滤器
    9. 15 ml锥形管(Bio Pioneer,目录号:CNT-15R)
    10. iPSC细胞
    11. 70%乙醇
    12. UltraPure TM 不含DNase / RNase的蒸馏水(Thermo Fisher Scientific,目录号:10977023)
    13. 康宁® Matrigel ®生长因子降低(GFR)地下室膜基质(Corning,目录号:354230)
    14. mTeSR TM 1(干细胞技术公司,目录号:85850)
    15. DMEM / F-12培养基(Thermo Fisher Scientific,目录号:11330057)
    16. Dispase II(Thermo Fisher Scientific,目录号:17105041)
    17. 基质胶溶液(Matrigel)(请参阅配方:表1)
    18. 10 mM ROCK抑制剂,Y-27632二盐酸盐溶液(ROCK抑制剂)(请参阅配方:表2)
    19. 10倍分配(请参阅食谱:表3)
    20. mTeSR TM 1完全培养基(mTeSR)(请参阅食谱:表4)

  2. 单层电镀
    1. 100 mm组织培养皿(Corning,目录号:430167)
    2. 自动细胞计数片(Bio-Rad Laboratories,目录号:1450019)或血细胞计数器(Hausser Scientific,目录号:1483)或同等功能。
    3. 5 ml血清移液管(Bio Pioneer,目录号:GEX0050-S01)
    4. 10 ml血清移液器(生物先锋,目录号:GEX0100-S01
    5. P20移液器吸头,无菌,带过滤器
    6. P200移液器吸头无菌,带过滤器
    7. P1000移液器吸头,无菌,带过滤器
    8. 15 ml锥形管(Bio Pioneer,目录号:CNT-15R)
    9. 50 ml锥形管(Bio Pioneer目录号:CNT-50R)
    10. 70%乙醇
    11. 康宁®基质胶®生长因子降低(GFR)的地下膜基质(Matrigel)(Corning,目录号:354230)
    12. mTeSR TM 1(干细胞技术公司,目录号:85850)
    13. DMEM / F-12培养基(Thermo Fisher Scientific,目录号:11330-057)
    14. Accutase(Innovative Cell Technologies,Inc.,目录号:AT 104)
    15. 台盼蓝溶液,0.4%(Thermo Fisher Scientific,目录号:15250061)
    16. ROCK抑制剂,Y-27632二盐酸盐(塞勒克下摆,目录号:S1049)
    17. iPSC细胞培养
    18. 基质胶溶液(请参见配方:表1)
    19. 10 mM ROCK抑制剂,Y-27632二盐酸盐溶液(请参见配方:表2)
    20. mTeSR TM 1完全培养基(请参见配方:表4)

  3. iPSC-RPE分化
    1. 100 mm组织培养皿(Corning,目录号:430167)
    2. (可选)排气的T150组织培养瓶(西格玛,目录号:Z707929)
      注意:在编写本手稿时,Z707929已不再可用。相同的烧瓶的商品目录号为Z707511-36EA(Sigma,商品目录号为Z707511-36EA)。
    3. 70μm过滤器(Fisher Scientific,目录号:431751)
    4. 自动细胞计数片(Bio-Rad Laboratories,目录号:1450019)或血细胞计数器(Hausser Scientific,目录号:1483)或同等产品
    5. 10 ml血清移液管(Bio Pioneer,目录号:GEX0100-S01)
    6. 25 ml血清移液管(Bio Pioneer目录号:GEX250-S01)
    7. 50 ml血清移液管(Bio Pioneer,目录号:GEX500-S01)
    8. P20移液器吸头,无菌,带过滤器
    9. P200移液器吸头无菌,带过滤器
    10. P1000移液器吸头,无菌,带过滤器
    11. 细胞刮板机(VWR International,目录号179707)
    12. 15 ml锥形管(Bio Pioneer,目录号:CNT-15R)
    13. 50 ml锥形管(Bio Pioneer,目录号:CNT-50R)
    14. Nalgene低温瓶(Thermo Fisher Scientific,目录号:5000-1020)
    15. iPSC单层
    16. 70%乙醇
    17. 康宁®基质胶®生长因子降低(GFR)的地下膜基质(Matrigel)(Corning,目录号:354230)
    18. DMEM / F-12培养基(Thermo Fisher Scientific,目录号:11330057)
    19. DMEM介质(Thermo Fisher Scientific,目录号:11965092)
    20. 火腿F12营养混合物(Thermo Fisher Scientific,目录号:11765054)
    21. 1个不含钙和镁的Dulbecco磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,目录号:14190250)
    22. B27补充剂(50x),无血清(Thermo Fisher Scientific,目录号:17504044)
    23. KnockOut TM 血清置换(KOSR)(Thermo Fisher Scientific,目录号:10828028)
    24. L-谷氨酰胺200 mM(Thermo Fisher Scientific,目录号:25030081)
    25. MEM非必需氨基酸溶液100x(Thermo Fisher Scientific,目录号:11140050)
    26. 青霉素-链霉素(10,000 U / ml)(Thermo Fisher Scientific,目录号:15140122)
    27. β-巯基乙醇(Thermo Fisher Scientific,目录号:21985023)
    28. Accutase(Innovative Cell Technologies,Inc.,目录号:AT 104)
    29. 烟酰胺(Sigma,目录号:N3376)
    30. Chetomin(Sigma,目录号:C9623)
    31. 台盼蓝溶液,0.4%(Thermo Fisher Scientific,目录号:15250061)
    32. 二甲基亚砜(DMSO)(Sigma,目录号:D2650-100ML)
    33. 液氮
    34. RPE DM介质(请参阅配方:表5)
    35. RPE介质(请参阅配方:表6)
    36. 2x iPSC-RPE冷冻培养基(请参阅配方:表7)
    37. 1 mM Chetomin溶液(请参见配方:表8)
    38. 1 M烟酰胺(100x)溶液(请参见配方:表9)

  4. 流式细胞仪
    1. 96孔圆底测定板(Genesee Scientific,目录号:25-224)
    2. 带有细胞过滤器卡扣帽的康宁 TM Falcon TM 试管(Fisher Scientific,目录号:352235)
    3. 康宁 TM CostarT M 无菌一次性试剂容器(Fisher Scientific,目录号:4870)
    4. 5 ml血清移液管(Bio Pioneer,目录号:GEX0050-S01)
    5. 10 ml血清移液管(Bio Pioneer,目录号:GEX0100-S01)
    6. P20移液器吸头,无菌,带过滤器
    7. P200移液器吸头无菌,带过滤器
    8. P1000移液器吸头,无菌,带过滤器
    9. P20移液器吸头,无过滤器
    10. (可选)不带过滤器的P200移液器吸头
    11. 带有BD GolgiStop TM 的固定/通透溶液套件(BD Biosciences,目录号:554715)
    12. 1个不含钙和镁的Dulbecco磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,目录号:14190250)
    13. 牛血清白蛋白(BSA)(Sigma,目录号:A2153-100G)
    14. (可选)NaN3(Sigma,目录号:S2002-5G)
    15. 37%甲醛(Sigma,目录号:F-1635-500ML)
    16. 兔抗ZO-1多克隆抗体(abcam,目录号:ab59720)
      注:在编写此手稿期间,抗体ab59720不再可用。可能的替代产品:abcam,目录号:ab221547。
    17. 小鼠单克隆抗MiTF抗体(abcam,目录号:ab12039)
    18. 重组兔IgG,单克隆[EPR25A]-同型对照(abcam,目录号:ab172730)
    19. 小鼠IgG1,κ单克隆抗体[15-6E10A7]-同型对照(abcam,目录号:ab170190)
    20. 驴抗兔Alexa Fluor TM 647共轭抗体(abcam,目录号:ab150075)
    21. 山羊抗小鼠Alexa Fluor TM 488偶联抗体(Thermo Scientific,目录号:A-11001)
    22. FACS缓冲液(请参阅配方:表10)
    23. FACS-FIX缓冲区(请参阅配方:表11)
    注意:有关抗体的工作浓度,请参见配方:表15。

  5. 免疫荧光
    1. Millicell EZ SLIDE 8孔玻璃载玻片(Millipore,目录号:PEZGS0816)
    2. 盖玻片(Fisherbrand,目录号:12-545-F)
    3. 5 ml血清移液管(Bio Pioneer,目录号:GEX0050-S01)
    4. 10 ml血清移液管(Bio Pioneer,目录号:GEX0100-S01)
    5. P20移液器吸头,无菌,带过滤器
    6. P200移液器吸头无菌,带过滤器
    7. 1个不含钙和镁的Dulbecco磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,目录号:14190250)
    8. 牛血清白蛋白(BSA)(Sigma,目录号:A2153-100G)
    9. 多聚甲醛(PFA)
    10. Tween ® 20(Sigma,目录号:P9416-100ML)
    11. 海卫一X-100(制造商,目录号:X-100-500ML)
    12. 康宁®基质胶®生长因子降低(GFR)的地下膜基质(Matrigel)(Corning,目录号:354230)
    13. 兔多克隆抗ZO-1抗体(abcam,目录号:ab59720)
    14. 小鼠单克隆抗MiTF抗体(abcam,目录号:ab12039)
    15. 小鼠单克隆抗Bestrophin 1抗体(Novus Biologicals,目录号:NB300-164SS)
    16. 重组兔IgG,单克隆[EPR25A]-同型对照(abcam,目录号:ab172730)
    17. 小鼠IgG1,κ单克隆抗体[15-6E10A7]-同型对照(abcam,目录号:ab170190)
    18. 驴抗兔Alexa Fluor TM 647共轭抗体(abcam,目录号:ab150075)
    19. 山羊抗小鼠Alexa Fluor TM 488偶联抗体(Thermo Scientific,目录号:A-11001)
    20. 具有DAPI的ProLong金抗褪色试剂(Cell Signaling Technologies,目录号:8961)
    21. 中频洗涤缓冲液(参见配方:表12)
    22. IF烫发缓冲液(请参阅配方:表13)
    23. 中频染色缓冲液(见配方:表14)
    注意:有关抗体的工作浓度,请参见配方:表15。

设备

  1. iPSC细胞培养
    1. 生物安全柜(Labconco,型号:Logic +)
    2. 具有湿度和气体控制功能的培养箱可在空气中5%CO 2 的气氛中维持37°C和95%湿度(Panasonic,型号:MCO-170AICUVH-PA)
    3. 水浴(Thermo Scientific,型号:Precision)
    4. 带转子的组织培养离心机,用于15 ml锥形管和50 ml锥形管(Thermo Scientific,型号:Legend RT +)
    5. 相衬倒置显微镜(物镜:x4,x10,x20)(奥林巴斯,型号:CKX41SF)
    6. (可选)带相机的相衬倒置显微镜(物镜:x4,x10,x20)(Thermo Scientific,型号:EVOS XL Core)
    7. 显微镜物标(Nikon,MBW10020型)
    8. 移液器辅助
    9. P20微量移液器
    10. 冷冻室-20°C
    11. 冰箱2-8°C

  2. 单层电镀
    1. 生物安全柜(Labconco,型号:Logic +)
    2. 具有湿度和气体控制功能的培养箱可在空气中5%CO 2 的气氛中维持37°C和95%湿度(Panasonic,型号:MCO-170AICUVH-PA)
    3. 带转子的组织培养离心机,用于15 ml锥形管和50 ml锥形管(Thermo Scientific,型号:Legend RT +)
    4. 相衬倒置显微镜(物镜:x4,x10,x20)(奥林巴斯,型号:CKX41SF)
    5. 带相机的相衬倒置显微镜(物镜:x4,x10,x20)(Thermo Scientific,型号:EVOS XL Core)–可选
    6. 移液器辅助
    7. P20微量移液器
    8. P200微量移液器
    9. P1000微量移液器
    10. 自动细胞计数器(Bio-Rad,型号:TC20)或血细胞计数器(Hausser Scientific,目录号:1483)或同等产品
    11. 冷冻室-20°C
    12. 冰箱2-8°C

  3. iPSC-RPE的分化和冷冻保存
    1. 生物安全柜(Labconco,型号:Logic +)
    2. 具有湿度和气体控制功能的培养箱可在空气中5%CO 2 的气氛中维持37°C和95%湿度(Panasonic,型号:MCO-170AICUVH-PA)
    3. 带转子的组织培养离心机,用于15 ml锥形管和50 ml锥形管(Thermo Scientific,型号:Legend RT +)
    4. 相衬倒置显微镜(物镜:x4,x10,x20)(奥林巴斯,型号:CKX41SF)
    5. (可选)带相机的相衬倒置显微镜(物镜:x4,x10,x20)(Thermo Scientific,型号:EVOS XL Core)
    6. 移液器辅助
    7. P20微量移液器
    8. P200微量移液器
    9. P1000微量移液器
    10. 自动细胞计数器(Bio-Rad,型号:TC20)或血细胞计数器(Hausser Scientific,目录号:1483)或同等功能。
    11. Frosty先生冷冻容器(Corning,型号:CoolCell ® FTS30)
    12. 冰箱2-8°C
    13. 冷冻室-20°C
    14. 冷冻室-80°C
    15. 液氮蒸气罐

  4. 流式细胞仪
    1. 移液器辅助
    2. P20微量移液器
    3. P200微量移液器
    4. P1000微量移液器
    5. P200多通道微量移液器
    6. 冰箱2-8°C
    7. 冷冻室-20°C
    8. 流式细胞仪(BD Biosciences,型号:FACSCanto II)或同等

  5. 免疫荧光
    1. 移液器辅助
    2. P20微量移液器
    3. P200微量移液器
    4. P1000微量移液器
    5. 冰箱2-8°C
    6. 冷冻室-20°C
    7. 共聚焦激光扫描荧光显微镜(Olympus,FluoView1000)

软件

  1. FlowJo(版本10)(FlowJo,LLC, https://www.flowjo.com/ )
  2. FlowView ASW V03.01.03.03或V4.2a(奥林巴斯生命科学, https:// www.olympus-lifescience.com/en/support/downloads/ )

程序

  1. iPSC细胞培养
    1. 解冻iPSC细胞
      1. 准备含有10μMROCK抑制剂的12 ml mTeSR。
      2. 将9 ml含10μMROCK抑制剂的mTeSR转移到标有管线名称的无菌锥形管中。
      3. 从液氮罐中取出冷冻保存的小瓶。将小瓶放在干冰上。
      4. 将其放在37°C水浴中轻轻摇动,直到剩下豌豆大小的冰晶(约2分钟)。
      5. 从小瓶中擦去多余的水,然后再将70%的乙醇喷入引擎盖。
      6. 从小瓶中取出融化的细胞,并在锥形管中轻轻加入9 ml含10μMROCK抑制剂的mTeSR中。用1-2 ml含10μMROCK抑制剂的mTeSR洗涤小瓶。将所有细胞收集在同一锥形管中。
      7. 在室温下以53 x g (在带有75006441 K桶的Sorvall 75006445转子中以500 RPM)离心细胞5分钟。
      8. 吸出上清液,然后将细胞沉淀轻轻重悬于2 ml含10μMROCK抑制剂的mTeSR中(将1个冷冻小管融化到6孔板的1个孔中)。
      9. 用行名称,克隆名称和传代号标记Matrigel板。从Matrigel涂层板上吸出DMEM / F-12。解冻后,在段落编号上加上+1。
        注意:如果在iPSC的低温保存过程中增加了传代次数,则不要在传代次数上加上+1。
      10. 将平板细胞重悬于2 ml溶液中,涂在Matrigel包被的6孔板的一个孔中(最终体积2 ml /孔)。
      11. 接种后24小时,观察细胞。用DMEM / F-12(2 ml /孔)轻轻洗涤细胞以去除细胞碎片,并使用不含ROCK抑制剂(2 ml /孔)的新鲜mTeSR培养基进料。
      12. 每天观察iPSC,去除分化的细胞,并用新鲜的mTeSR(2 ml /孔)更换培养基。
        注意:保持无iPSC文化分化的关键。
      13. 细胞应达到融合度的80-90%,并准备在5天内通过。
    2. 使用Dispase进行iPSC传递
      1. 通过将9 ml DMEM / F-12添加到1 ml 10x Dispase(20 mg / ml)中,制备1x(2 mg / ml)Dispase溶液。
      2. 让1x Dispase溶液恢复到室温。
        注意:1x Dispase溶液可在4°C下最多保存2周。
      3. 使用显微镜对象标记在要拆分的孔上标记任何分化区域。
      4. 吸出用过的媒体。轻轻敲打标记圆圈内的巴斯德吸管,吸出标记的分化区域(如有)。每孔用2 ml DMEM / F-12洗涤。
      5. 在每个孔中加入1 ml 1x Dispase进行拆分。在37°C下孵育5分钟。
      6. 5分钟后检查菌落的形态。
        当菌落的边缘稍微卷曲时,细胞就可以传代了。如果菌落边缘未卷曲,则将细胞在37°C下再孵育1-2分钟。不要与Dispase一起温育超过8分钟。
      7. 从所有孔中吸出Dispase。
      8. 用DMEM / F12(2 ml /孔)轻轻冲洗孔3次。
      9. 向每个孔中加入1 ml mTeSR培养基进行传代。
      10. 使用玻璃血清移液管分离菌落。将移液器与平板表面成90°角。按照图1所示的动作刮擦6孔板的表面(从孔的左上侧开始,然后用锯齿紧紧地向下弯曲到右下角,然后顺时针或逆时针旋转板并再次刮擦)。刮擦直到至少有90%的菌落从孔中脱离。


        图1.在用Dispase传递iPSC期间,玻璃血清移液器的移动方式。沿一个方向刮擦孔后,将板顺时针或逆时针旋转90°,然后再次刮擦剩余的iPSC菌落。应从孔中分离出约90%的细胞。有关详细信息,请参阅有关使用Dispase进行iPSC传递的部分。

      11. 用使细胞升至播种新的Matrigel涂层容器所需的最终体积的mTeSR体积洗涤板。考虑到每个孔中要接种传代细胞的每个孔2 ml,计算最终体积。例如,如果要将细胞传代1-3次,则最终体积将为6 ml,因此用于洗涤板的mTeSR体积为5 ml。
      12. 在新的Matrigel板上接种种子细胞,每孔1 ml,然后再添加1 ml。平板细胞在孔的整个表面上滴落,以确保均匀铺板。
      13. 在显微镜下观察接种的细胞,以确保接种均匀。
      14. 放入37°C的培养箱中。将平板摇成T形,以将菌落块均匀分布在孔中。
      15. 轻轻铺板后二十四小时,在添加新鲜的mTeSR培养基之前,用DMEM / F-12洗涤细胞。
        注意:对于健康高效的iPSC培养,至关重要的是均匀铺板细胞。平板细胞均匀地分布在孔的整个表面上,当镀多个孔时,平板细胞均匀地分布在所有孔中。

  2. 单层电镀
    注意:解冻iPSC线后,在铺单层之前至少用Dispase传代细胞一次。
    1. 从培养箱中取出6孔板。当iPSC细胞达到约80%汇合度(细胞已准备好传代)时,iPSC细胞已准备好用于单层。标记所有需要去除的分化细胞。
    2. 吸出用过的培养基。除去所有标记的分化细胞,并用DMEM / F-12(2 ml /孔)洗涤细胞。
    3. 吸出DMEM / F-12,然后在6孔板的孔中加入1 ml室温Accutase。在37°C下孵育细胞8分钟。
    4. 孵育8分钟后,每孔含5μMROCK抑制剂的mTeSR加入1 ml,并使用P1000移液器将细胞重悬为单细胞,而不刮板表面。用移液器吸移10-12次,将培养皿旋转180°(倒置),再移吸5次。在50 ml锥形管中收集所有孔中的细胞。您应该看不到任何细胞团块。
    5. 用5 ml含5μMROCK抑制剂的mTeSR将所有孔洗涤两次。将所有细胞收集在同一锥形管中。
    6. 在室温下以53 x g (在带有75006441441 K桶的Sorvall 75006445转子中以500 RPM)离心细胞7分钟。吸出上清液并将细胞重悬于10 ml含ROCK抑制剂的mTeSR中。
    7. 如有必要,通过颠倒20次或更多次来混合合并的细胞悬液。使用0.4%的台盼蓝溶液进行活细胞计数。
      注意:iPSC细胞生存力应不低于80%。
    8. 准备所需数量的单元格。最佳细胞数将取决于分化程度。iPSC-RPE分化方案需要每cm 2 2.5 x 10 4 活细胞(每100毫米培养皿2 x 10 6 )。对于一个100毫米的培养皿,在15毫升的锥形管中准备11毫升的细胞悬浮液,其中含有2.2 x 10 6个细胞。通过颠倒试管20次,将细胞悬液充分混合。
    9. 使用10毫升移液器向每100毫米培养皿中滴加10毫升细胞悬液。
      e(T形)。
    注意:在板的整个表面上均匀铺板细胞至关重要。为帮助一次分配细胞均匀地镀上一个皿,然后将新镀的皿摇成十字形(T形)。
    1. 将板放在培养箱中,不要堆叠板。将细胞孵育到第二天早上,在37°C,5%CO 2 处。
    2. 第二天更换不含ROCK抑制剂的新鲜mTeSR培养基(10 ml / 100 mm培养皿)。用于iPSC-RPE细胞分化的单层细胞需要培养约4-5天,直到单层细胞达到80%融合为止。每天更换含有新鲜mTeSR的培养基。

  3. iPSC-RPE的区别
    请参阅图2,以了解区分方案的示意图。


    图2。iPSC-RPE分化方案的示意图。烟酰胺(NIC),Chemomin(CTM)。培养iPSC单层直到细胞达到80%融合为止。iPSC-RPE分化是使用小分子(NIC和CTM)启动和驱动的。iPSC-RPE持续分化和扩展了总共12周。整个培养传代会促进iPSC-RPE细胞的扩增。改编自Smith et al。,2019.

    1. 第0天(D0)–当iPSC单层达到约80%融合度时,通过用RPE DM培养基替换mTeSR培养基来启动iPSC-RPE分化(请参见食谱:表5)(24 ml / 100 mm皿)
    2. 分化开始后的D1–24小时,将用过的RPE DM培养基替换为新鲜的RPE DM培养基,其中补充了10 mM烟酰胺(见配方:表9)和50 nM壳聚糖(见配方:表8)(24 ml / 100 mm皿) 。
    3. 每日更换用过的培养基,用新鲜的RPE DM培养基补充10 mM烟酰胺(NIC)和50 nM Chetomin(CTM)(24 ml / 10 cm皿)。
      注意:由于iPSC-RPE细胞具有很高的细胞数量,高的增殖速率和代谢活性,因此可以获得健康,稳定的iPSC培养物,因此强烈建议在整个分化过程中保持24 h培养基更换的时间表。 / em>
    4. 在D14上,更换用过的新鲜RPE DM培养基补充10 mM烟酰胺(24 ml / 100 mm皿)的用过的培养基。
    5. 每日更换用过的培养基,以及新鲜的RPE DM培养基和10 mM烟酰胺(24 ml / 100 mm皿)。
      注意:2-3周后,第一批细胞开始出现黑色素沉淀和特征性的多边形形状。
    6. 在D28传代细胞–第一代。
      1. (可选)拍摄细胞图像–(图3-D28左面板)。


        图3. iPSC-RPE细胞的图像。 iPSC-RPE细胞在分化的第28天(D28)的明场图像表现为小的多角形色素细胞簇,它们在剩下的时间); 在第84天(D84),装有iPSC-RPE细胞(iPSCORE 87_1)的T150烧瓶(左中);第84天的iPSC-RPE样品(iPSCORE_42_1)的明场图像显示了高度组织化的单层膜,具有强烈的黑色素色素沉着(右中)和特征多边形形状(右)。改编自Smith et al。,2019.

      2. 吸出用过的培养基并用PBS(10 ml / 100 mm皿)洗涤细胞
      3. 吸出PBS,将5 ml室温Accutase加入100 mm皿中。在37°C下孵育细胞12分钟。
      4. 孵育12分钟后,每碟RPE DM培养基添加5 ml,使用P1000移液器将细胞重新悬浮在Accutase中作为单细胞,而不会刮擦板表面。如果难以从培养皿中取出细胞,请使用细胞刮刀。
      5. 将细胞收集在50 ml锥形管中。
      6. 再用3 ml RPE DM培养基洗涤板3次。将所有细胞收集在同一锥形管中。
      7. 在室温下,以136 x g (800 RPE在带有75006441 K桶的Sorvall 75006445转子中离心800分钟)离心细胞。
      8. 离心后,吸出上清液,并将细胞重悬于10 ml RPE培养基中(请参见配方:表6)。
      9. 轻轻将所有细胞通过70μm过滤器。添加培养基,使最终体积总计20毫升通过过滤器。
      10. 将细胞接种在两个新鲜的100毫米培养皿上,并用Matrigel包被过夜。
      11. 使用10毫升移液器,每100毫米培养皿中滴加10毫升细胞悬液。
        注意:在板的整个表面上均匀铺板细胞至关重要。为帮助一次将细胞均匀分布在一个培养皿中,并以十字形(T形)摇晃新培养的培养皿。
        可选:代替在一个T150烧瓶上使用两个100 mm皿式培养皿。
      12.  将板放入培养箱中。将细胞在37°C,5%CO 2 中孵育至第二天,而无需将板堆叠。
    7. 分割后二十四小时,将培养基换为新鲜的RPE培养基(10 ml / 100 mm皿)。如果使用T150烧瓶,请使用25 ml / T150烧瓶。
    8. 每天更换培养基,换上新鲜的RPE培养基(10毫升/ 100毫米培养皿),持续4周。如果使用T150烧瓶,请使用25 ml / T150烧瓶。
    9. 在D56传代中,细胞第二次传代。
      按照步骤C6a-C6g执行段落。
      注意:如果将细胞接种在T150烧瓶上,则将每个烧瓶用10 ml的Accutase孵育细胞,并使用刮板回收所有细胞。为了收集所有细胞,将烧瓶洗涤3次,每次用10 ml培养基洗涤。
      1. 离心后,吸出上清液,将细胞重悬于20 ml RPE培养基中。
      2. 轻轻将所有细胞通过70μm过滤器。添加培养基,使最终体积总计为50 ml,使至少20 ml的培养基通过滤网。
      3. 将细胞接种到两个新鲜的T150烧瓶中,并用Matrigel包被过夜。
      4. 使用10 ml移液管向每个T150烧瓶中逐滴添加25 ml细胞悬液。
        注意:至关重要的是,将细胞均匀地铺板在用Matrigel包被过夜的板的整个表面上。为帮助一次将细胞均匀地分布在一个烧瓶中,并以十字形(T形)摇动新接种的烧瓶。
      5. 将烧瓶放在培养箱中,不要堆叠。将细胞孵育到第二天早上,在37°C,5%CO 2 处。
    10. 分流后二十四小时,更换新鲜RPE培养基(25 ml / T150烧瓶)。
    11. 每天更换新鲜的RPE培养基(25 ml / T150烧瓶),持续4周。
    12. 在D84上,收集所有细胞。
      1. (可选)拍摄单元格的图像。请参阅图3,以了解iPSC-RPE预期产量,色素沉着(图3 –面板中左和中右)和细胞形态(图3 –面板中右和右)的示例。
      2. 吸出用过的培养基,并用PBS(20 ml / T150烧瓶)洗涤细胞。
      3. 吸出PBS,向每个烧瓶中加入10 ml室温Accutase。在37°C下孵育12分钟。
      4. 孵育12分钟后,每孔加入10 ml RPE培养基重悬细胞。使用细胞刮板除去所有细胞。
      5. 将细胞收集在50 ml锥形管中。
      6. 再用10 ml RPE培养基洗涤板3次。将所有细胞收集在同一锥形管中。
      7. 在室温下,以136 x g (800 RPE在带有75006441 K桶的Sorvall 75006445转子中离心800分钟)离心细胞。
      8. 离心后,吸出上清液,将细胞重悬于20 ml RPE培养基中。
      9. 轻轻将所有细胞通过70μm过滤器。添加培养基,使最终体积总计为40 ml,使至少20 ml的培养基通过滤网。
      10. 颠倒20-30次,混合细胞悬液。使用0.4%台盼蓝溶液进行活细胞计数。
      注意:
      1. 使用自动细胞计数器对iPSC-RPE进行活细胞计数时,由于细胞中黑色素浓度高,可能无法准确地将细胞生存力评分为高。根据实验需要,可以将iPSC-RPE细胞冷冻保存用于将来的实验,固定用于流式细胞术分析和/或铺板用于免疫荧光分析。
      2. 培养2-3周后和每次传代后约1周,细胞开始获得黑色素色素沉着和特征性多边形形状。细胞开始获得色素沉着的最初迹象是培养基颜色略带灰色(“脏”)。当细胞色素沉着时,培养基也会获得深色。浓缩的iPSC-RPE细胞在D84处的细胞悬浮液呈黑色,类似于沉淀的iPSC-RPE细胞也呈黑色(图4)。


      图4. D84处iPSC-RPE细胞的图像。在离心前(左)和离心后(右)收集的iPSC-RPE细胞(iPSCORE_29_1)。

  4. iPSC-RPE的冷冻保存
    1. 通过在FBS中制备20%DMSO溶液来制备2x iPSC-RPE冷冻培养基。每个要冷冻保存的冷冻管,准备0.25 ml 2x iPSC-RPE冷冻培养基。冷冻细胞的最终密度为1.2 x 10 7 / ml(取决于下游实验,可以修改单个冷冻管中冷冻保存的iPSC-RPE细胞的体积和浓度)。
      可选:如果需要无血清条件,请使用KOSR代替FBS制备2x iPSC-RPE冷冻培养基。
    2. 准备并打印冷冻管的标签。准备n + 2个标签数(n =要冷冻保存的冷冻管数量)。准备并粘贴所有要冷冻的冷冻管的标签,为Frosty先生使用一个标签,并为保存记录使用一个标签( ie ,实验手册)。
    3. 活细胞计数后(步骤C12j),确定应冷冻保存多少个细胞,并将所需数量的细胞转移到新的15 ml或50 ml锥形管中。
    4. 在室温下以136 x g (在带有75006441 K桶的Sorvall 75006445转子中以800 RPM)离心细胞5-8分钟(根据细胞体积调整离心时间)。
    5. 离心后,吸出上清液,将细胞悬浮于每个冷冻小管的0.25 ml FBS(或KOSR)中,以2.4 x 10 7 / ml( ie ,对于10个冷冻保存的小瓶,将6 x 10 7 细胞重悬于2.5 ml FBS或KOSR中)。
    6. 打开所有预先标记的冷冻管,并向每个冷冻管中添加0.25 ml细胞悬液。
    7. 将0.25 ml的2x iPSC-RPE冷冻培养基添加到每个含有iPSC-RPE细胞悬液的冷冻管中。
    8. 关闭所有冷冻小管,轻轻颠倒5-6次以混合细胞悬液和2x iPSC-RPE冷冻培养基。将冷冻管转移到Frosty先生的冷冻容器中。
    9. 立即将Frosty先生转移到-80°C的冰箱中。冻结大量冷冻管时( ie ,多个Frosty先生),请准备单独的批次,每批次仅包含可放入一个Frosty先生的冷冻管数量。
    10. 24-48小时后,将细胞转移到液氮蒸气罐中。相应地更新记录( ie ,箱形图)。

    在这里,我们提供了流式细胞仪(FC)和免疫荧光(IF)的详细协议,可用于对衍生的iPSC-RPE细胞进行定量(FC)和定性(FC和IF)质量控制。

  5. 流式细胞仪
    1. 活细胞计数后(步骤C12j),确定应固定多少细胞以进行流式细胞术分析,并将所需数量的细胞转移到15 ml锥形管中。至少使用2-5 x 10 6 电池。
    2. 在室温下以136 x g (在带有75006441 K桶的Sorvall 75006445转子中以800 RPM)离心细胞5分钟。
    3. 离心后,吸出上清液,将细胞重悬于10 ml PBS中。
      可选:如果用于流式细胞术的细胞悬液体积小于0.5 ml,则直接向细胞中加入14 ml PBS,并以136 xg(在Sorvall 75006445中为800 rpm)离心细胞和PBS的混合物8分钟带有75006441 K桶的转子)。
    4. 根据制造商的建议,使用带有BD GolgiStop TM 的Fixation / Permeabilization Solution Kit固定和透化iPSC-RPE细胞。最后一次离心后,吸出上清液,将细胞以1 x 10 7 / ml的浓度重悬于1x BD Perm / Wash TM 缓冲液中。对于每种流式细胞仪染色,请使用2.5 x 10 5 细胞。
      注意:染色2.5 x 105个细胞可以有效利用细胞和试剂,但是每次染色也可以使用1 x 106个细胞,以保持相同的抗体稀释比。
    5. 将25μl固定和透化的细胞转移到96孔圆底测定板的5孔中。为了限制对多条线染色时抗体和细胞的使用,请从每条线中混合相等数量的细胞,并将25μl细胞混合物转移至四个对照孔中[重组兔IgG,单克隆类别对照抗体(Rb-IgG),小鼠IgG1,κ单克隆抗体(M-IgG1),抗ZO-1抗体(ZO1)和抗MiTF抗体(MiTF)]。
    6. 在表15之后,在每个孔中添加适当浓度的抗体。使用用于20μl的多通道移液器,通过上下移液20次,轻轻地混合细胞和抗体。
    7. 在室温下将细胞与一抗孵育1小时。
    8. 1小时后,添加150μlFACS缓冲液(请参见配方:表10)。
    9. 从863 xg (转速为863 xg (2,000 rpm时,转速为863)室温下带有75006441 K铲斗的Sorvall 75006445转子)。
      详细:
      1. 将离心机设置为863 x g (2,000 RPM),1分钟,室温); 启动离心机,直到速度达到863 x g (2,000 rpm)。
      2. 计数到8-10 s并停止离心。)离心后的沉淀应清晰可见,尤其是使用1 x 10 6 细胞时。
    10. 离心后,轻轻吸出上清液,非常小心不要吸出任何细胞。如果使用真空抽吸细胞,请使用不带过滤器的P20吸头(或不带过滤器的P200 + P20吸头)。在每个孔中保留约20μl液体,以避免抽吸细胞。
    11. 使用多通道移液器添加200μlFACS缓冲液,轻轻混合细胞5-6次。
    12. 像步骤E9一样离心板。
    13. 重复步骤E10-E12,再次洗涤细胞。
    14. 将细胞重悬于50μl1x BD Perm / Wash TM 缓冲液中。
    15. 在表15之后,在每个孔中添加适当浓度的抗体。使用用于40μl的多通道移液器,通过上下移液20次,轻轻地混合细胞和抗体。
    16. 在黑暗中于室温下将细胞与二抗孵育45分钟。
    17. 45分钟后,重复步骤E8-E13。
    18. 最后一次离心后,吸出上清液,将细胞重悬于200μlFACS-FIX缓冲液中(参见配方:表11)。使用多通道移液器通过移液5-6次来重悬细胞。
    19. 使用P1000移液器一次将每个样品转移到带有Cell Strainer Snap Cap的Corning TM Falcon TM 试管中,使细胞穿过盖中的过滤器。
    20. 用另外的250μl洗涤每个孔,并转移到适当的试管中,使细胞穿过瓶盖中的过滤器。根据用于染色的细胞数量,将细胞稀释至适当的浓度,以免堵塞流式细胞仪。
    21. 将所有管子放在适当的架子上,并用铝箔纸包裹以防光照。
    22. 使用流式细胞仪FACS Canto II(或其他流式细胞仪)进行采集。
    23. 使用FlowJo软件V 10.4执行流式细胞仪分析。有关iPSC-RPE流式细胞仪染色结果的示例,请参见图5。


      图5.第84天对iPSC-RPE(iPSCORE_42_1)的流式细胞术分析,显示Zonula Occludens 1(ZO-1)和小眼症相关转录因子(MiTF)的高共染色。史密斯等人,2019年。

  6. 免疫荧光
    1. 用Matrigel将Millicell EZ SLIDE 8孔玻璃载玻片涂过夜。
    2. 在Matrigel包被的Millicell EZ SLIDE 8孔玻璃载玻片上将新鲜或冷冻保存的iPSC-RPE细胞平板接种。每行以1-1.5 x 10 6 / cm 2 的密度接种至少6孔。
    3. 培养细胞10天,直到它们完全融合,重新获得多边形形状和色素沉着。
    4. 吸出培养基并用PBS洗涤细胞两次。吸出PBS。
    5. 在室温下用4%PFA固定细胞10分钟。
    6. 除去PFA溶液,并用新鲜制备的IF洗涤缓冲液洗涤细胞两次(请参见配方:表12)。吸出IF Wash Buffer。
    7. 使用IF Perm Buffer浸透和渗透细胞(请参见配方:表13)。在室温下孵育细胞20分钟。
    8. 在饱和和通透性的最后5分钟内,在IF染色缓冲液中准备一级抗体溶液(请参见配方:表14和表15)以获取适当浓度的抗体。将抗体溶液储存在冰上直至使用。
    9. 饱和和透化后,吸出所有缓冲液,并将抗体溶液添加到适当的孔中。
    10. 将细胞与抗体溶液在4°C下孵育过夜。
    11. 第二天(早上)在IF染色缓冲液中准备二抗溶液。有关抗体的适当浓度,请参见表15 。将抗体溶液在冰上保存,直至使用,并避光。
    12. 吸出一级抗体溶液,并用PBS洗涤细胞3次。最后清洗后,吸出所有PBS。
    13. 立即将二抗溶液添加到适当的孔中。在黑暗中于室温下孵育细胞1小时。
    14. 吸出二抗溶液并用PBS洗涤细胞三遍。最后清洗后,吸出所有PBS。
    15. 卸下Millicell EZ SLIDE 8孔玻璃载玻片的壁。
    16. 遵循制造商的建议,将ProLong金抗褪色剂与DAPI添加在一起,并轻轻地安装盖玻片,以免产生气泡。用铅笔橡皮轻轻地去除任何气泡。在黑暗中将载玻片在室温下存放数小时(最好直到第二天),以确保正确安装。
    17. 使用适当的免疫荧光显微镜(最好是共聚焦激光扫描荧光显微镜)获取图像。有关iPSC-RPE免疫荧光染色的示例,请参见图6。


      图6. Bestrofin 1(BEST1)(iPSCORE_29_1),ZO-1(iPSCORE_29_1)和MiTF(iPSCORE_42_1)的免疫荧光分析。 ZO-1表现为清晰的细胞膜染色。与ZO1染色相比,BEST1膜染色显得“模糊”。MiTF核染色。改编自Smith et al。,2019。

菜谱

  1. 细胞培养试剂和培养基制备

    表1.制备基质胶溶液


    表2。1 . 10 mM ROCK抑制剂Y-27632二盐酸盐的制备


    表3. 10倍分散液的制备


    表4. mTeSRTM1完全培养基的制备


    表5. RPE DM培养基的制备(继Maruotti等,2015年之后)


    表6. RPE培养基的制备(紧随Maruotti等人,,2015年)


    表7.制备2x iPSC-RPE冷冻培养基(5毫升)


    表8.制备1 mM Chetomin溶液


    表9. 1M烟酰胺(100x)溶液的制备


  2. 缓冲液制备

    表10. FACS缓冲液的制备


    表11. FACS-FIX缓冲液的制备


    表12.中频洗涤缓冲液的制备


    表13.中频烫发缓冲液的制备


    表14.中频染色缓冲液的制备


  3. 抗体

    表15.抗体浓度

致谢

这项工作得到了CIRM赠款GC1R-06673-B和NIH赠款HG008118,HL107442,DK105541,DK112155和EY021237的部分支持。该协议改编自以前的工作(Smith,D'Antonio-Chronowska et al。,2019)。

利益争夺

作者宣称没有利益冲突。

伦理

从不同种族的个体(3个欧洲裔美国人,2个东亚裔美国人和1个非裔美国人)产生的iPSC品系是从iPSCORE获得的(Panopoulos et al。,2017)。捐赠时,捐赠者均为21至62岁的女性。招募这些人得到了加利福尼亚大学圣地亚哥分校的机构审查委员会和The Salk Institute的批准(项目号110776ZF)。

参考文献

  1. WM的Al-Zamil和SA的Yassin(2017)。与年龄有关的黄斑变性的最新发展:回顾。 Clin Interv Aging 12:1313-1330。
  2. 青木H.,原野A.,中川S.,本桥T.,平野M.,高桥Y.和国立贞(2006)。在体外 分化为RPE细胞前体的胚胎干细胞发展成为RPE细胞单层体内。 Exp Eye Res 82(2):265-274。
  3. Buchholz,DE,Hikita,ST,Rowland,TJ,Friedrich,AM,Hinman,CR,Johnson,LV和Clegg,DO(2009)。从诱导性多能干细胞中衍生出功能性视网膜色素上皮。 干细胞 27(10):2427-2434。
  4. 哈兹姆(Hazim,RA),卡伦巴亚拉姆(Karumbayaram),S。,姜(M.),迪马什基(Dimashkie),答。洛普斯(Lopes),VS,李,D。伯吉斯,BL,维杰亚拉杰(P. ,Gomperts,BN,Pyle,AD,Lowry,WE和Williams,DS(2017)。 RPE细胞与无整合iPS细胞的区别及其细胞生物学特性。 Stem Cell Res Ther 8(1):217. 
  5. Idelson,M.,Alper,R.,Obolensky,A.,Ben-Shushan,E.,Hemo,I.,Yachimovich-Cohen,N.,Khaner,H.,Smith,Y.,Wiser,O.,Gropp ,M.,Cohen,MA,Even-Ram,S.,Berman-Zaken,Y.,Matzrafi,L.,Rechavi,G.,Banin,E。和Reubinoff,B。(2009)。指导人类胚胎干细胞分化为功能性视网膜色素上皮细胞。 细胞干细胞 5(4):396-408。
  6. Klimanskaya,I.,Hipp,J.,Rezai,KA,West,M.,Atala,A. and Lanza,R.(2004)。使用转录组学从人类胚胎干细胞中提取和比较视网膜色素上皮的情况。 克隆干细胞 6(3):217-245。
  7. Maruotti,J.,Sripathi,SR,Bharti,K.,Fuller,J.,Wahlin,KJ,Ranganathan,V.,Sluch,VM,Berlinicke,CA,Davis,J.,Kim,C.,Zhao,L. ,Wan,J.,Qian,J.,Corneo,B.,Temple,S.,Dubey,R.,Olenyuk,BZ,Bhutto,I.,Lutty,GA和Zack,DJ(2015)。由小分子定向的人多能干细胞有效产生视网膜色素上皮。 Proc Natl Acad Sci US A 112(35):10950-10955。
  8. Maruotti,J.,Wahlin,K.,Gorrell,D.,Bhutto,I.,Lutty,G.和Zack,DJ(2013)。从人多能干细胞分化视网膜色素上皮的简单且可扩展的过程。 Stem Cells Transl Med 2(5):341-354。
  9. Mitchell,P.,Liew,G.,Gopinath,B.和Wong,TY(2018)。与年龄有关的黄斑变性。 柳叶刀 392(10153 ):1147-1159。 
  10. F.Osakada,F.,Ikeda,H.,Mandai,M.,Wataya,T.,Watanabe,K.,Yoshimura,N.,Akaike,A.Sasai,Y. and Takahashi(2008)。努力从小鼠,猴和人类胚胎干细胞中产生杆状和锥状光感受器。 Nat Biotechnol 26(2):215-224。
  11. F.Osakada,F.Jin,ZB,Hirami,Y.,Ikeda,H.,Danjyo,T.,Watanabe,K.,Sasai,Y. and Takahashi(2009)。 体外视网膜细胞与人多能干细胞的分化, J Cell Sci 122(Pt 17):3169-3179。
  12. Panopoulos,AD,D'Antonio,M.,Benaglio,P.,Williams,R.,Hashem,SI,Schuldt,BM,DeBoever,C.,Arias,AD,Garcia,M.,Nelson,BC,Harismendy,O 。,Jakubosky,DA,Donovan,MKR,Greenwald,WW,Farnam,K.,Cook,M.,Borja,V.,Miller,CA,Grinstein,JD,Drees,F.,Okubo,J.,Diffenderfer,KE ,Hishida,Y.,Modesto,V.,Dargitz,CT,Feiring,R.,Zhao,C.,Aguirre,A.,McGarry,TJ,Matsui,H.,Li,H.,Reyna,J.,Rao ,F.,O'Connor,DT,Yeo,GW,Evans,SM,Chi,NC,Jepsen,K.,Nariai,N.,Muller,FJ,Goldstein,LSB,Izpisua Belmonte,JC,Adler,E., Loring,JF,Berggren,WT,D'Antonio-Chronowska,A.,Smith,EN和Frazer,KA(2017)。 iPSCORE:222种iPSC品系的资源,可对多种细胞类型的遗传变异进行功能表征。 干细胞报告 8(4):1086-1100。
  13. KL Pennington和MM DeAngelis(2016)。与年龄有关的黄斑变性(AMD)的流行病学:与心血管疾病表型和脂质因子的关联。 / a> Eye Vis(Lond) 3:34。
  14. Reichman,S.,Terray,A.,Slembrouck,A.,Nanteau,C.,Orieux,G.,Habeler,W.,Nandrot,EF,Sahel,JA,Monville,C.和Goureau,O.(2014) 。从融合的人iPS细胞到自形成的神经视网膜和视网膜色素上皮细胞。 Proc Natl Acad Sci US A 111(23):8518-8523。
  15. 史密斯(Smith),EN,D'Antonio-Chronowska,A.,Greenwald,WW,Borja,V.,Aguiar,LR,Pogue,R.,Matsui,H.,Benaglio,P.,Booooah,S.,D'Antonio, M.,Ayyagari,R.和弗雷泽,KA(2019)。人类iPSC衍生的视网膜色素上皮细胞:一种模型系统,用于对因AMD引起的风险变异进行优先排序和功能表征 干细胞报告 12(6):1342-1353。
  16. Sparrow,JR,Hicks,D.和Hamel,CP(2010)。健康和疾病中的视网膜色素上皮。 Curr Mol Med 10(9):802-823。
  17. Vugler,A.,Carr,AJ,Lawrence,J.,Chen,LL,Burrell,K.,Wright,A.,Lundh,P.,Semo,M.,Ahmado,A.,Gias,C.,da Cruz ,L.,Moore,H.,Andrews,P.,Walsh,J。和Coffey,P。(2008)。阐明HESC衍生的RPE现象:细胞起源,扩张和视网膜移植的解剖学。 Exp Neurol 214(2):347-361。
  18. Wong,WL,Su,X.,Li,X.,Cheung,CM,Klein,R.,Cheng,CY and Wong,TY(2014)。 2020年和2040年与年龄相关的黄斑变性和疾病负担预测的全球患病率:系统回顾和荟萃分析。 柳叶刀球的健康状况 2(2):e106-116。
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引用:D’Antonio-Chronowska, A., D’Antonio, M. and Frazer, K. A. (2019). In vitro Differentiation of Human iPSC-derived Retinal Pigment Epithelium Cells (iPSC-RPE). Bio-protocol 9(24): e3469. DOI: 10.21769/BioProtoc.3469.
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