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Small Molecule-Based Retinal Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells
基于小分子的人胚胎干细胞和诱导多能干细胞的视网膜分化   

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Cell Stem Cell
Mar 2017

Abstract

Retinal degeneration leads to loss of light-sensing photoreceptors eventually resulting in vision impairment and impose a heavy burden on both patients and the society. Currently available treatment options are very limited and mainly palliative. Ever since the discovery of human pluripotent stem cell technologies, cell replacement therapy has become a promising therapeutic strategy for these patients and may help restore visual function. Reproducibly generating enriched retinal cells including retinal progenitors and differentiated retinal neurons such as photoreceptors using human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells in a dish is an essential first step for developing stem cell-based therapies. In addition, this will provide a reliable and sufficient supply of human retinal cells for studying the mechanisms of diseases. Here we describe a small molecule-based retinal induction protocol that has been used to generate retinal progenitors and differentiated retinal neurons including photoreceptors from several human ES and iPS cell lines. The retinal cells generated by this protocol can survive and functionally integrate into normal and diseased mouse retinas for several months following subretinal transplantation.

Keywords: Human (人), ES Cells (ES细胞), iPS Cells (iPS细胞), Retina (视网膜), Differentiation (分化)

Background

A number of groups around the world are developing methodologies to generate specific cell types from human pluripotent stem cells. These cells will likely play a critical role in the future of regenerative medicine as a source of replacement cells. These newly generated human cells will be very useful in developing better and more accurate human disease models that can then be used for discovery of novel drugs with better efficacy and safety profiles.

Our work focuses on retinal degenerative diseases such as macular degeneration and retinitis pigmentosa which affect millions of people worldwide. Death of light-sensing photoreceptors in the retina is commonly associated with those diseases and results in severe impairment or total loss of vision. There are no effective medical treatments available to cure those diseases.

Under specific conditions, human ES and iPS cells can be specifically used to generate retinal progenitor cells, and consequentially differentiate into specialized retinal neuronal subtypes (retinal ganglion cells, amacrine cells, bipolar cells, horizontal cells, and photoreceptors) (Osakada et al., 2008 and 2009; Hirami et al., 2009; Lamba et al., 2006, 2009 and 2010; Meyer et al., 2009; Hambright et al., 2012; Zhong et al., 2014). Establishment of effective and chemically-defined protocols to generate retinal progenitors as well as differentiated retinal cell types including photoreceptors from human ES cells and iPS cells is a critical step for developing cell replacement therapies for patients with a variety of incurable retinal degenerative diseases.

Here, we report a detailed small molecule-based retinal induction protocol that has been used to generate retinal cells in vitro and the derived retinal cells were used as donor cells in the transplantation studies carried out by Dr. Lamba’s research group. The derived retinal progenitors and retinal photoreceptors were tested in multiple host mouse lines with and without retinal degeneration conditions and showed the ability to survive and functionally integrate into the host mouse retina following transplantation (Zhu et al., 2017 and 2018).

Materials and Reagents

  1. 6-well plate (DNase and RNase free, treated) (Greiner Bio One International, catalog number: 657160 )
  2. Cryogenic vials (Corning, catalog number: 430659 )
  3. 2 ml serological pipette (VWR, catalog number: 76093-882 )
  4. 1,000 µl micropipette (VWR, catalog number: 89079-974 )
  5. 15 ml conical tube (VWR, catalog number: 490001-623 )
  6. Micropipette tip (VWR, catalog number: 490000-468 )
  7. Cell scraper (Corning, catalog number: 3008 )
  8. Sterile Disposable Filter System with PES Membrane (0.22 µm pore size) (Thermo Fisher Scientific, catalog number: 566-0020 )
  9. Cover glasses (VWR, catalog number: 89015-725 )
  10. 70% ethanol (Sigma-Aldrich, catalog number: 459836 )
  11. Matrigel (BD, BD Biosciences, catalog number: 354234 )
  12. DMEM/F-12 basal medium (GE Healthcare, catalog number: SH30023.02 )
  13. Essential 8 medium (Thermo Fisher Scientific, catalog number: A2858301 )
  14. Penicillin Streptomycin Amphotericin B (Lonza, catalog number: 17-745E )
  15. Lyophilized Y27632 (Stemgent, catalog number: 04-0012 )
  16. 1x Dulbecco’s phosphate buffered saline (DPBS) without calcium and magnesium (Thermo Fisher Scientific, catalog number: 14190144 )
  17. 0.1% EDTA (Diluted 1x EDTA in DPBS at 1:1,000) (Corning, catalog number: 46-034-CI )
  18. Sodium Pyruvate (Corning, catalog number: 25-000-Cl )
  19. HEPES Buffer (Corning, catalog number: 25-060-CI )
  20. Sodium Bicarbonate (Corning, catalog number: 25-035-CI )
  21. Non-essential Amino Acid (Corning, catalog number: 25-025-CI )
  22. N1 Supplement (Sigma-Aldrich, catalog number: N6530 )
  23. Fetal Bovine Serum (FBS) (Atlanta Biologicals, catalog number: S11150 )
  24. KnockOutTM Serum Replacer (KSR) (Thermo Fisher Scientific, catalog number: 10828028 )
  25. IWR1-endo (Stemgent, catalog number: 04-0010 )
  26. LDN 193189 (Stemgent, catalog number: 04-0074 )
  27. Insulin-like growth factor 1 (IGF1) (R&D Systems, catalog number: 291-G1 )
  28. TrypLE Express dissociation reagent (Thermo Fisher Scientific, catalog number: 12604021 )
  29. 1x HBSS (Corning, catalog number: 21-022-CM )
  30. Dimethyl Sulfoxide (DMSO) (Fisher Scientific, catalog number: BP231-1 )
  31. Poly-D-lysine (Sigma-Aldrich, catalog number: P6407 )
  32. 4% Paraformaldehyde solution (Electron Microscopy Sciences, catalog number: 157-4 )
  33. 10% Normal Donkey Serum Blocking Solution (Jackson Immuno Research Laboratories, catalog number: 017-000-001 )
  34. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  35. Anti-fading Fluoromount G mounting medium (Electron Microscopy Sciences, catalog number: 17984-25 )
  36. 4',6-diamidino-2-phenylindole (DAPI, Enzo Life Scienes, catalog number: ENZ-52404 )
  37. Monoclonal mouse anti PAX6 antibody (DHSB, catalog number: PAX6 )
  38. Polyclonal goat anti LHX2 antibody (Santa Cruz Biotechnology, catalog number: sc-19344 or sc-517243 )
  39. Biotinylated polyclonal goat anti human OTX2 antibody (R&D Systems, catalog number: BAF1979 )
  40. Polyclonal Rabbit anti CRX antibody (GeneTex, catalog number: GTX124188 )
  41. Anti-Recoverin Antibody (EMD Millipore, catalog number: AB5585 )
  42. Freezing Medium (NSC + 0.5% FBS Medium + 10% DMSO)
  43. Essential 8 human ESC/iPSC culture medium (see Recipes) 
  44. Rock inhibitor Y-27632 (10 mM, 1,000x, Stemgent, catalog number: 04-0012 ) (see Recipes)
  45. Neuronal Stem Cell Culture Medium (NSC) (see Recipes)
  46. NSC + 0.5% FBS Medium (see Recipes) 
  47. ISLI + KSR Retinal Induction Medium (see Recipes)

Equipment

  1. Vertical laminar flow hood certified for Level II handling of biological materials 
  2. Water bath
  3. Incubator with humidity and gas control to maintain 37 °C and 95% humidity in an atmosphere of 5% CO2 in air
    Note: The incubator needs to be able to adjust the O2 level, as required for Step A4i.
  4. Low-speed centrifuge with a swinging bucket rotor (e.g., Beckman Coulter, model: GS-6 ) with an adaptor for plate holders 
  5. Pipette-aid with appropriate serological pipettes 
  6. Hemocytometer 
  7. Micropipette with appropriate tips 
  8. Mr. Frosty freezing container at room temperature (Thermo Fisher Scientific, catalog number: 5100-0001 )
  9. -150 °C freezer or liquid nitrogen (LN2) vapor tank 
  10. -80 °C freezer 
  11. -20 °C freezer
  12. Refrigerator (2-8 °C)
  13. Inverted microscope

Procedure

  1. Thawing and passaging human ES and iPS cells
    1. Sterilization before handling the cells
      Expose the Biological Safety Cabinet to UV light for 1 h prior use, spray 70% ethanol and wipe the working area inside the cabinet, the tools and supplies that will be used inside the cabinet.
    2. Preparation of Matrigel-coated plates
      1. Take 1 aliquot of frozen Matrigel (110 µl) (BD Biosciences) and thaw on ice.
        Note: The Matrigel aliquots are prepared ahead of time and stored in -80 °C freezer.
      2. Dissolve 110 µl Matrigel in 12 ml chilled (4 °C) DMEM/F-12 basal medium (GE Healthcare), pipette up and down to mix the Matrigel and medium very well.
      3. Add 2 ml of the mixture of Matrigel and medium to each well of a 6-well plate (Greiner Bio One, DNase and RNase free, treated), and let the plate sit in an incubator (37 °C) for 2-12 h before seeding the cells into the plate.
    3. Preparation of Essential 8 human ESC/iPSC culture medium (see Recipe 1)
    4. Thawing and seeding cells
      In general, 1 well of the cells in a 6-well plate that is at 60-70% of confluence is prepared to be frozen into 2 cryovials. One vial of cryopreserved cells can be successfully recovered into 1-2 wells of a Matrigel-coated 6-well plate.
      Note: The number of wells plated may need to be adjusted depending on how many cell aggregates were cryopreserved. Typically, twice as many cell aggregates will need to be plated per well upon thawing to account for lower post-thaw viability than during routine passaging.
      1. Label the Matrigel-coated plate, warm Essential 8 culture medium (with supplement of Rock Inhibitor [Recipe 2]) to room temperature (~25 °C), and prepare all other cell culture supplies before starting the protocol to ensure that the thawing procedure is done as quickly as possible.
      2. Quickly thaw the cells in a 37 °C water bath by gently shaking the cryovial continuously until only a small frozen cell pellet remains.
      3. Remove the cryovial from the water bath and wipe it with 70% ethanol.
      4. Use a 2 ml serological pipette to transfer the contents of the cryovial to a 15 ml conical tube.
        Note: Using a 2 ml serological pipette instead of a 1 ml micropipette will minimize breakage of cell aggregates.
      5. Add 5-7 ml of warm Essential 8 medium (supplied with Rock Inhibitor) dropwise to the 15 ml tube, gently mixing as the medium is added.
      6. Centrifuge cells at 270 x g for 3 min at room temperature.
      7. Aspirate the medium, leaving the cell pellet intact, gently resuspend the cell pellet in 1 ml of Essential 8 medium (with Rock Inhibitor) using a 2 ml serological pipette, maintain the cells as aggregates.
      8. Transfer 0.5 ml of the cell mixture onto a well of a Matrigel-coated 6-well plate containing Essential 8 with Rock Inhibitor (i.e., two wells can be plated from each cryovial).
      9. Place the plate in a 37 °C hypoxia-capable incubator (5% CO2, 5% O2), move the plate in several quick, short, back-and-forth and side-to-side motions to evenly distribute the cell aggregates. Do not disturb the plate for 24 h.
        Note: Uneven distribution of aggregates may result in increased differentiation of human ES and iPS cells (Figure 2).
      10. Perform daily medium changes using Essential 8 medium and visually assess cultures to monitor the growth until the next passaging time. Check for undifferentiated colonies that are ready to be passaged (dense-centered) approximately 6-7 days after thawing (Figure 1 and Figure 2A).
        Note: If only a few undifferentiated colonies are observed after thawing, it may be necessary to select only these colonies for passaging and replate them in the same size well (i.e., without splitting) on a newly-coated plate.


      Figure 1. Undifferentiated iPSC colonies in Essential 8 media (scale bar = 400 μm)

    5. Passaging human ES and iPS cells
      Human ES and iPS cells maintained in Essential 8 medium can be passaged using several different methods. The non-enzymatic EDTA dissociation method is described below: 
      1. Clean off any flat or differentiating cells (Figure 2B) with a 1,000 µl pipette tip under a microscope in a sterilized working area.


      Figure 2. Undifferentiated and differentiating human ESC colonies in culture. A. An undifferentiated human ESC colony. B. A differentiating human ESC colony with whorled appearance that needs to be cleaned up prior to subculture. (scale bars = 400 µm)

      1. Rinse cells with 2 ml of DPBS once or twice.
      2. Add 1 ml of 0.1% EDTA dissociation reagent per well on a 6-well plate, incubate at room temperature for 3-5 min.
      3. Check under a microscope for signs of dissociation.
      4. Aspirate EDTA dissociation reagent without disturbing the cells.
      5. Add 1 ml of Essential 8 medium (with Rock Inhibitor supplement).
      6. Use a cell scraper to lift the cells from the bottom of the well, triturate with a 1,000 µl pipette gently to break down into cell clusters containing 20-40 cells.
      7. Add 2 ml of Essential 8 medium (with Rock Inhibitor added) to a new well.
      8. Add cells from original well to the new well to reach 20% of confluence. Usually 1:5 ratio for a confluent plate.
        Note: If there are too few cells, cells don't grow as well; if there are too many cells, cells grow too quickly and should be split soon. Small clumps are ideal, if cells remain as big chunks, gently triturate the large size cell aggregates with a 1,000 µl pipette for a few times to break them down.
      9. Place the plate back into a hypoxia 37 °C incubator (5% CO2 and 5% O2) and evenly distribute cells by shaking plate front to back and side to side as above.
      10. Perform daily medium changes, 2 ml Essential 8 medium per well, and visually assess cultures to monitor growth until the next passaging time.

  2. Induction of neuro-retinal fate
    1. Medium preparation
      1. Preparation of Neuronal Stem Cell Culture Medium (NSC) (500 ml) (see Recipe 3).
      2. Preparation of NSC medium with 0.5% FBS (100 ml) (see Recipe 4).
      3. Preparation of ISLI + KSR Retinal Induction Medium (50 ml) (see Recipe 5).
    2. Retinal induction in human ES and iPS cells
      When the cells in wells reach 60-70% confluence, start treating cells with ISLI + KSR retinal induction medium (2 ml per well of a 6-well plate), daily medium change for 5-6 days. Once the cells are in ISLI + KSR medium, culture the cells in a normoxic 37 °C incubator with 5% CO2 level.
      Procedure of splitting differentiating cells (Days 6-7)
      1. Change 2 ml fresh ISLI + KSR retinal induction medium for cells at least 1 h before splitting.
      2. At the time of splitting, aspirate the medium.
      3. Add 1 ml TrypLE Express dissociation reagent to the well; incubate 6-8 min at room temperature until the cells loose contact with each other and the plate.
      4. Scrape the cells gently from the bottom of the well and transfer the cell suspension into a 15 ml centrifuge tube. Take care not to make single cell suspension.
      5. Rinse the well with 2 ml 1x HBSS once to collect the remaining cells from the well.
      6. Centrifuge at 270 x g for 3 min.
      7. Aspirate the supernatant gently without disturbing the cell pellet.
      8. Add 3-4 ml 1x HBSS to resuspend the cell pellet, centrifuge again at 270 x g for 3 min.
      9. Aspirate 1x HBSS gently without disturbing the cell pellet.
      10. Resuspend the cell pellet in fresh ISLI + KSR retinal induction medium. The splitting ratio is 1:3.
      11. Evenly distribute the cells as above by shaking the plate and return to the incubator under normoxic conditions.
      12. Next day, check the cell survival by looking the percentage of cells attached to the bottom of the plate, dead cells do not attach and float in culture medium. If there is too much cell death, rinse cells with 2 ml 1x HBSS once or twice to clean up the dead cells.
      13. Wean cells into NSC culture medium supplemented with 0.5% FBS gradually by adding 1 ml ISLI + KSR medium and 1 ml NSC + 0.5% FBS medium on Day 1 post-split; 0.5 ml ILSI + KSR medium + 1.5 ml NSC + 0.5% FBS medium on Day 2; from Day 3 and onward, the differentiating cells will be cultured in NSC + 0.5% FBS medium to let them undergo further differentiation. When the cells reach confluence, the cells need to be split into new Matrigel-coated plates with the TrypLE dissociation method. This is usually done every 7 days with a split ratio of 1:3 to 1:5 depending on the cell line.
        Note: Rock Inhibitor can be added to the NSC medium at the step to help cell survive after dissociation if the cells are old or stressed.

  3. Isolation of Neuroretinal Rosettes (Days 18-21)
    The differentiating cells are mixed populations that are composed of retinal progenitor cell population and retinal pigmented epithelial progenitor cells (RPE) (Figure 3A) as they both arise from the same optic vesicle. The retinal stem/progenitors form clusters (neuroretinal rosettes) and can be manually separated and expanded (Figure 3B).


    Figure 3. Differentiated Early Retinal Rosettes in Culture. A. Retinal Rosettes prior to picking and sorting at 3 weeks; B. Purified neuro-retinal cultures following picking and replating. Scale bars = 100 μm.

    Procedure of manually separation of the retinal stem/progenitor cells and retinal pigmented epithelial cells
    1. Sterilize the bench surface and any areas on and around the microscope and tools that need to be in direct contact with the cells with 70% ethanol.
    2. Change to fresh NSC + 0.5% FBS medium for the cells before picking.
    3. Under a microscope, gently scrape the areas that have densely-packed neuronal cells with a sterile micropipette tip. If too large, scrape regions containing 100-200 cell clusters.
    4. After most of neuronal areas are lifted, collect the medium that contains the floating neuronal rosettes.
    5. Transfer them into a new Matrigel-coated well with a 1,000 µl pipette to let the cells attach and grow in NSC medium in the incubator (37 °C, 5% CO2).

  4. Retinal Stem/Progenitor cell expansion
    The manually purified retinal stem/progenitors need to be cultured in NSC + 0.5% FBS medium for several months to allow the cells undergo further differentiation to give rise to all types of differentiated retinal neurons (Figure 4). The dissociation method is described below:
    TrypLE Dissociation Method of Differentiating Retinal Cells
    1. Aspirate the old medium.
    2. Rinse cells with 1-2 ml 1x HBSS (or DPBS) once or twice.
    3. Aspirate 1x HBSS.
    4. Add 1 ml of TrypLE Express dissociation reagent to the cells, incubate for 6-8 min at room temperature, and check under the microscope for signs of dissociation (The cell-cell connections loosen up and empty spaces show up among densely packed cells). Do not wait until the cells are lifted (Figure 4).


      Figure 4. TrypLE dissociation of differentiating retinal cells for expansion. A. Morphology of retinal cells before dissociation. B. Morphology of retinal cells after 6 min of TrypLE dissociation. Scale bars = 400 µm.

    5. Scrape cells using a cell scraper.
    6. Transfer cells into a 15 ml centrifuge tube.
    7. Spin at 270 x g for 3 min.
    8. Aspirate the supernatant and add 2 ml 1x HBSS and resuspend the cells.
    9. Spin at 270 x g for 3 min.
    10. Aspirate the supernatant and add NSC + 0.5% FBS medium to resuspend the cells (2 ml per well), usually the splitting ratio is 1:3.
      Note: Rock Inhibitor can be added to the medium at the step to help cell survive after dissociation if the cells appear stressed.
    11. Add 2 ml of cells in NSC + 0.5% FBS medium into the wells.
    12. Put the plate into an incubator (37 °C, 5% CO2) and shake left to right and back and forth to evenly distribute the cells.
    13. Cells will be cultured in NSC + 0.5% FBS medium.
    14. Change media every 2-3 days as needed.
    At 1 months of induced retinal differentiation, seed cells onto Poly-D-lysine (Sigma-Aldrich) and Matrigel-coated cover glasses and followed by immunocytochemistry analysis to check the retinal differentiation status. At this stage, most of cells are retinal progenitors by their expression of retinal stem/progenitor cell markers PAX6, and LHX2. After characterization, cells can be cryopreserved.

  5. Retinal Stem/Progenitor cell cryopreservation
    Freezing Neuro-retinal Stem Cells
    1. Rinse cells with 1-2 ml of 1x HBSS twice.
    2. Add 1 ml of TrypLE per well on a 6-well plate and incubate for 6-8 min.
    3. During this time, start labeling cryovials with date, cell line, info, etc.
    4. Check under a microscope for signs of dissociation as in Step D4.
    5. Scrape cells using a cell scraper.
    6. Transfer everything into a 15 ml tube.
    7. Spin for 3 min at 270 x g.
    8. Dispose of the supernatant.
    9. Add 2 ml of 1x HBSS and resuspend the cells.
    10. Spin for 3 min at 270 x g.
    11. Dispose of the supernatant.
    12. Add the calculated amount of freezing medium to resuspend the cells.
    13. Add 1 ml of cells and freezing medium to each cryovial.
    14. Immediately place all cryovials into a freezing chamber.
    15. Transfer chamber into a -80 °C freezer.
    16. Next day, remove vials and place into an appropriate box in the -80 °C freezer.
    17. Transfer vials to liquid nitrogen next day.

  6. Thawing Retinal Stem/Progenitor cells
    One vial of cryopreserved retinal stem/progenitor cells can be successfully thawed into 1 well of a Matrigel-coated 6-well plate. 
    1. Label the Matrigel-coated plate, warm NSC + 0.5% FBS medium (with supplement of Rock Inhibitor) to room temperature (~25 °C) and prepare all other cell culture supplies before starting the protocol to ensure that the thawing procedure is done as quickly as possible.
    2. Quickly thaw the cells in a 37 °C water bath by gently shaking the cryovial continuously until only a small frozen cell pellet remains.
    3. Remove the cryovial from the water bath and wipe it with 70% ethanol or isopropanol to sterilize.
    4. Use a 1 ml micropipette to transfer the contents of the cryovial to a 15 ml conical tube.
    5. Add 7 ml of 1x HBSS dropwise to the 15 ml tube, gently mixing as the medium is added.
    6. Centrifuge at 270 x g for 3 min.
    7. Aspirate the supernatant and add 2 ml of warm NSC medium (supplied with Rock Inhibitor) to the 15 ml tube to resuspend the cells.
    8. Transfer the cell mixture onto an empty well of a Matrigel-coated 6-well plate.
    9. Place the plate in a 37 °C normoxic incubator (5% CO2), move the plate in several quick, short, back-and-forth and side-to-side motions to distribute the cell aggregates. Do not disturb the plate for 24 h.
    10. Change media next day. If excessive cell death occurs, rinse cells with 1x HBSS before medium change.
    11. Perform medium changes every 2-3 days as needed using NSC medium and visually assess cultures to monitor the growth until the next passaging time.
    At 2 and 3 months of induced retinal differentiation, seed cells onto Poly-D-lysine (Sigma-Aldrich) and Matrigel-coated cover glasses and followed by immunocytochemistry analysis to check the photoreceptor differentiation status (Figures 5 and 6).


    Figure 5. Confluent Neuro-retinal culture following three months of culture (scale bar = 400 μm)


    Figure 6. Immunocytochemistry analysis of the retinal cells generated from human iPSCs at 6 weeks of retinal differentiation. Cells expressed retinal stem cell markers PAX6 and LHX2 as well as photoreceptor markers, OTX2 and RCVRN (scale bars = 10 µm).

  7. Immunocytochemistry protocol
    1. Aspirate the old culture media. 
    2. Rinse cells in 1x HBSS 2 times gently without lifting the cells.
    3. Fix cells in 4% Paraformaldehyde solution (Electron Microscopy Sciences) for 30 min at room temperature.
    4. Rinse cells in 1x PBS for 3 times, 5 min for each rinse.
    5. Block the cells in 10% Normal Donkey Serum Blocking Solution (Jackson Immuno Research Laboratories, the blocking solution is prepared by diluting the stock in 1x PBS that contains 0.5% Triton X-100) for 1 h at room temperature.
    6. Add the primary antibodies with pre-determined concentrations (Table 1) (prepared in serum blocking solution) to the cells and incubate overnight in a cool room in a humidified chamber.

      Table 1. Primary Antibodies for retinal analysis


    7. Next day, rinse off the non-binding primary antibodies with 1x PBS, 3 times, 5 min for each rinse.
    8. Add the secondary Antibodies and incubate at room temperature for 1 h in the dark in a humidified chamber.
    9. Rinse off the non-binding secondary antibodies with 1x PBS 3 times, 5 min for each rinse.
    10. DAPI (working concentration is 0.5 µg/ml, prepared in PBS) stains the nuclei of cells for 3-5 min.
    11. Mount the cells on cover glasses onto histology slides with anti-fading Fluoromount G mounting medium (Electron Microscopy Sciences).
    Note: The samples are ready for image collection on a fluorescence-capable microscope.

Data analysis

Data analysis for the differentiated retinal cells is carried out by t-test. Total cells expressing a given antibody as well as total cells in a field (marked by DAPI) were counted from 3-5 fields and at least 3 independent experiments. The results were compared to the marker expression in either undifferentiated cells or alternate differentiation media. Detailed analysis of induced retinal cell differentiation of human ES cells and iPS cells via this protocol can be found in a recently published open access paper by the lab (Zhu et al., 2018).

Notes

While the protocol works reliably in multiple cell lines tested by our lab. There is some inherent variability due to different culture conditions experienced by various undifferentiated cells or methods of derivation. Authors also advise all labs to routinely test lines for mycoplasma contamination using commercial kits as they significantly affect differentiation capacity.

Recipes

  1. Essential 8 human ESC/iPSC culture medium

    For newly thawed cells, use Essential 8 medium with supplementation of Rock inhibitor (Y-27632, 10 µM) to promote cell survival.
  2. Rock inhibitor (Y-27632) stock (10 mM, 1,000x)
    Dissolve 2 mg lyophilized Y27632 (Stemgent) in 625 µl double distilled water to make 10 mM mother stock and make 10 µl aliquots and store in -20 °C freezer
  3. Neuronal Stem Cell Culture Medium (NSC) (500 ml)

  4. NSC medium with 0.5% FBS (100 ml)

  5. ISLI + KSR Retinal Induction Medium (50 ml)
    Note: In “ISLI + KSR”, “I” stands for IWR1, “S” stands for SB431542, “L” stands for LDN193189, the last “I” stands for IGF1, and KSR stands for KnockOutTM Serum Replacer.

Acknowledgments

We are grateful to Dr. Xianmin Zeng for the human iPS cell lines (NCL-1, NCL1-GFP). The human ES line was purchased from WiCell. The work was supported through funds from NEI grant EY025779, Buck Institute funds. This protocol is based on our previous work (Zhu et al., 2018).

References

  1. Hambright, D., Park, K. Y., Brooks, M., McKay, R., Swaroop, A. and Nasonkin, I. O. (2012). Long-term survival and differentiation of retinal neurons derived from human embryonic stem cell lines in un-immunosuppressed mouse retina. Mol Vis 18: 920-936.
  2. Hirami, Y., Osakada, F., Takahashi, K., Okita, K., Yamanaka, S., Ikeda, H., Yoshimura, N. and Takahashi, M. (2009). Generation of retinal cells from mouse and human induced pluripotent stem cells. Neurosci Lett 458(3): 126-131.
  3. Lamba, D. A., Karl, M. O., Ware, C. B. and Reh, T. A. (2006). Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc Natl Acad Sci U S A 103(34): 12769-12774.
  4. Lamba, D. A., Gust, J. and Reh, T. A. (2009). Transplantation of human embryonic stem cell-derived photoreceptors restores some visual function in Crx-deficient mice. Cell Stem Cell 4(1): 73-79.
  5. Lamba, D. A., McUsic, A., Hirata, R. K., Wang, P. R., Russell, D. and Reh, T. A. (2010). Generation, purification and transplantation of photoreceptors derived from human induced pluripotent stem cells. PLoS One 5(1): e8763.
  6. Meyer, J. S., Shearer, R. L., Capowski, E. E., Wright, L. S., Wallace, K. A., McMillan, E. L., Zhang, S. C. and Gamm, D. M. (2009). Modeling early retinal development with human embryonic and induced pluripotent stem cells. Proc Natl Acad Sci U S A 106(39): 16698-16703.
  7. 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.
  8. 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.
  9. Zhong, X., Gutierrez, C., Xue, T., Hampton, C., Vergara, M. N., Cao, L. H., Peters, A., Park, T. S., Zambidis, E. T., Meyer, J. S., Gamm, D. M., Yau, K. W. and Canto-Soler, M. V. (2014). Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs. Nat Commun 5: 4047.
  10. Zhu, J., Cifuentes, H., Reynolds, J. and Lamba, D. A. (2017). Immunosuppression via loss of IL2rγ enhances long-term functional integration of hESC-derived photoreceptors in the mouse retina. Cell Stem Cell 20(3): 374-384 e375.
  11. Zhu, J., Reynolds, J., Garcia, T., Cifuentes, H., Chew, S., Zeng, X. and Lamba, D. A. (2018). Generation of transplantable retinal photoreceptors from a current good manufacture practice-manufactured human induced pluripotent stem cell line. Stem Cells Transl Med.

简介

视网膜变性导致光感受器丧失,最终导致视力损害,并给患者和社会带来沉重的负担。目前可用的治疗方案非常有限,主要是姑息治疗。自从人类多能干细胞技术的发现以来,细胞替代疗法已成为这些患者的有希望的治疗策略,并可能有助于恢复视觉功能。使用人类胚胎干(ES)细胞和诱导多能干(iPS)细胞在培养皿中重现性地产生包括视网膜祖细胞和分化的视网膜神经元(例如光感受器)的富集视网膜细胞是开发基于干细胞的治疗的重要的第一步。此外,这将为研究疾病机制提供可靠和充足的人类视网膜细胞供应。在这里,我们描述了一种小分子视网膜诱导协议,已被用于生成视网膜祖细胞和分化的视网膜神经元,包括来自几个人ES和iPS细胞系的光感受器。通过该方案产生的视网膜细胞可以在视网膜下移植后的几个月内存活并且功能性地整合到正常和患病的小鼠视网膜中。

【背景】世界各地的一些团体正在开发用于从人多能干细胞产生特定细胞类型的方法。这些细胞可能在再生医学的未来作为替代细胞的来源中发挥关键作用。这些新产生的人类细胞在开发更好和更准确的人类疾病模型中非常有用,然后可用于发现具有更好功效和安全性的新药。

我们的工作重点是影响全球数百万人的视网膜退行性疾病,如黄斑变性和视网膜色素变性。视网膜中光感受器的死亡通常与这些疾病相关,并导致严重损伤或全部视力丧失。没有有效的药物治疗可以治愈这些疾病。

在特定条件下,人类ES和iPS细胞可以专门用于产生视网膜祖细胞,并且随后分化成特化的视网膜神经元亚型(视网膜神经节细胞,无长突细胞,双极细胞,水平细胞和光感受器)(Osakada等人,2008和2009; Hirami等人,2009; Lamba等人,2006,2009和2010; Meyer等人, ,2009; Hambright et al。,2012; Zhong et al。,2014)。建立有效的和化学定义的方案来产生视网膜祖细胞以及分化的视网膜细胞类型,包括来自人类ES细胞和iPS细胞的光感受器,对于开发具有各种不可治愈的视网膜退行性疾病的患者的细胞替代疗法是关键的一步。

在这里,我们报告了一种详细的小分子视网膜诱导方案,该方案已被用于在体外产生视网膜细胞,并且衍生的视网膜细胞被用作由Lamba博士进行的移植研究中的供体细胞研究小组。衍生的视网膜祖细胞和视网膜光感受器在具有和不具有视网膜变性条件的多个宿主小鼠系中进行测试,并显示在移植后存活和功能性整合到宿主小鼠视网膜中的能力(Zhu等人,2017年和2018年)。

关键字:人, ES细胞, iPS细胞, 视网膜, 分化

材料和试剂

  1. 6孔板(不含DNA酶和RNase,处理)(Greiner Bio One International,目录号:657160)
  2. 低温瓶(Corning,目录号:430659)
  3. 2毫升血清移液管(VWR,目录号:76093-882)
  4. 1,000μl微量吸管(VWR,目录号:89079-974)

  5. 15毫升锥形管(VWR,目录号:490001-623)
  6. 微管吸头(VWR,目录号:490000-468)
  7. 细胞刮刀(康宁,目录号:3008)
  8. 带PES膜(0.22μm孔径)的无菌一次性过滤系统(Thermo Fisher Scientific,目录号:566-0020)
  9. 盖玻片(VWR,目录号:89015-725)
  10. 70%乙醇(Sigma-Aldrich,目录号:459836)
  11. Matrigel(BD,BD Biosciences,目录号:354234)
  12. DMEM / F-12基础培养基(GE Healthcare,目录号:SH30023.02)
  13. Essential 8培养基(Thermo Fisher Scientific,目录号:A2858301)
  14. 青霉素链霉素两性霉素B(Lonza,目录号:17-745E)
  15. 冻干的Y27632(Stemgent,目录号:04-0012)
  16. 1x不含钙和镁的Dulbecco's磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,目录号:14190144)
  17. 0.1%EDTA(在1:1000的DPBS中稀释1倍EDTA)(Corning,目录号:46-034-CI)
  18. 丙酮酸钠(Corning,目录号:25-000-Cl)
  19. HEPES缓冲液(康宁,目录号:25-060-CI)
  20. 碳酸氢钠(康宁,目录号:25-035-CI)
  21. 非必需氨基酸(Corning,目录号:25-025-CI)
  22. N1补充物(Sigma-Aldrich,目录号:N6530)
  23. 胎牛血清(FBS)(Atlanta Biologicals,目录号:S11150)
  24. KnockOut TM血清替代品(KSR)(Thermo Fisher Scientific,目录号:10828028)
  25. IWR1-endo(Stemgent,目录号:04-0010)
  26. LDN 193189(Stemgent,目录号:04-0074)
  27. 胰岛素样生长因子1(IGF1)(R& D Systems,目录号:291-G1)
  28. TrypLE Express解离试剂(Thermo Fisher Scientific,目录号:12604021)
  29. 1x HBSS(Corning,目录号:21-022-CM)
  30. 二甲基亚砜(DMSO)(Fisher Scientific,目录号:BP231-1)
  31. 聚-D-赖氨酸(Sigma-Aldrich,目录号:P6407)
  32. 4%多聚甲醛溶液(电子显微镜科学,目录号:157-4)
  33. 10%正常的驴血清封闭液(Jackson Immuno Research Laboratories,目录号:017-000-001)
  34. Triton X-100(Sigma-Aldrich,目录号:T8787)

  35. 防褪色Fluoromount G固定介质(电子显微镜科学,目录号:17984-25)
  36. 4',6-二脒基-2-苯基吲哚(DAPI,Enzo Life Scienes,目录号:ENZ-52404)
  37. 单克隆小鼠抗PAX6抗体(DHSB,目录号:PAX6)
  38. 多克隆山羊抗LHX2抗体(Santa Cruz Biotechnology,目录号:sc-19344或sc-517243)
  39. 生物素化多克隆山羊抗人OTX2抗体(R& D Systems,目录号:BAF1979)
  40. 多克隆兔抗CRX抗体(GeneTex,目录号:GTX124188)
  41. 抗Recoverin抗体(EMD Millipore,目录号:AB5585)
  42. 冷冻培养基(NSC + 0.5%FBS培养基+ 10%DMSO)
  43. Essential 8人类ESC / iPSC培养基(见食谱) 
  44. 岩石抑制剂Y-27632(10mM,1,000x,Stemgent,目录号:04-0012)(见食谱)
  45. 神经元干细胞培养基(NSC)(见食谱)
  46. NSC + 0.5%FBS培养基(见食谱) 
  47. ISLI + KSR视网膜诱导培养基(见食谱)

设备


  1. 垂直层流罩认证适用于生物材料的二级处理 
  2. 水浴
  3. 培养箱中的湿度和气体控制在空气中5%CO 2气氛中保持37°C和95%湿度
    注意:孵化器需要能够根据步骤A4i的要求调整O 2 级别。 br />
  4. 低速离心机带有摆动式转子(,例如,Beckman Coulter,型号:GS-6),带有一个用于固定板的转接器
  5. 用适当的血清移液器移液助剂 
  6. 血细胞计数器 
  7. 带适当提示的微量移液器 
  8. 先生在室温下冷冻冷冻容器(Thermo Fisher Scientific,目录号:5100-0001)
  9. -150°C冰箱或液氮(LN2)蒸气罐
  10. -80°C冰柜 
  11. -20°C冷冻机
  12. 冰箱(2-8°C)
  13. 倒置显微镜

程序

  1. 解冻并传代人ES和iPS细胞
    1. 处理细胞之前进行灭菌
      使用前将生物安全柜暴露于紫外线灯下1小时,喷洒70%乙醇并擦拭橱柜内的工作区域,橱柜内使用的工具和耗材。
    2. 制备Matrigel包被的平板
      1. 取1等分的冷冻基质胶(110μl)(BD Biosciences)并在冰上解冻。
        注意:Matrigel等分试样需要提前准备,并储存在-80°C冰箱中。

      2. 在12 ml冷冻(4°C)DMEM / F-12基础培养基(GE Healthcare)中溶解110μlMatrigel,上下移动以混合Matrigel和培养基。
      3. 向6孔板(Greiner Bio One,DNase和RNase free,处理)的每个孔中加入2ml Matrigel和培养基的混合物,并将板置于培养箱(37℃)中2-12小时然后将细胞接种到平板中。
    3. Essential 8人ESC / iPSC培养基的制备(见配方1)
    4. 解冻和播种细胞
      一般而言,准备6孔板中60%70%汇合处的1孔细胞冷冻成2个冷冻管。一瓶冷冻保存的细胞可成功回收到Matrigel涂层6孔板的1-2孔中。
      注意:根据冷冻保存多少个细胞聚集体,可能需要调整孔的数量。通常,解冻后每孔需要铺板两倍的细胞聚集体,以解释比常规传代期间更低的解冻后活力。
      1. 将标记Matrigel包被的平板,温热的Essential 8培养基(补充Rock抑制剂[配方2])至室温(〜25°C),并在开始方案前制备所有其他细胞培养物,以确保解冻程序尽快完成。
      2. 在37℃水浴中快速融化细胞,轻轻摇动冷冻管,直到只剩下一小块冷冻细胞团。

      3. 除去水浴中的冷冻管并用70%乙醇擦拭。
      4. 使用2毫升血清移液管将冷冻管内容物转移到15毫升锥形管中。
        注意:使用2毫升血清移液器代替1毫升微量移液器可以最大限度地减少细胞聚集体的破损。
      5. 将5-7毫升温热的Essential8培养基(含有抑制剂)滴加到15毫升试管中,在加入培养基时轻轻混合。

      6. 在室温下将细胞在270×g离心3分钟。
      7. 吸出培养基,使细胞沉淀完好,用2毫升血清移液管轻轻地将细胞沉淀物重悬于1毫升Essential 8培养基(含有抑制剂)中,保持细胞为聚集体。
      8. 将0.5ml细胞混合物转移到含有岩石抑制剂必需品8的基质胶包被的6孔板的孔中(即,每个冷冻管可以铺两个孔)。
      9. 将培养板置于37℃缺氧培养箱(5%CO 2,5%O 2)中,移动培养板几个快速,短暂,背部和第四次和左右移动以均匀分布细胞聚集体。
        请勿打扰24小时 注意:聚集体的不均匀分布可能导致人ES和iPS细胞的分化增加(图2)。
      10. 使用Essential 8培养基进行每日培养基更换,并视觉评估培养物以监测直到下一次传代时间的增长。检查解冻后约6-7天准备传代的未分化集落(密集居中)(图1和图2A)。
        注意:如果在解冻后只观察到少数未分化的菌落,则可能需要仅选择这些菌落进行传代,并在新涂布的平板上以相同大小重新铺平它们(即不分裂)。 />

      图1. Essential 8培养基中未分化的iPSC菌落(比例尺= 400μm)

    5. 传递人类ES和iPS细胞
      在Essential 8培养基中维持的人ES和iPS细胞可以使用几种不同的方法进行传代。非酶促EDTA分解方法如下所述: 

      1. 用无菌工作区的显微镜下的1,000μl移液器吸头清除任何扁平或分化的细胞(图2B)。


      图2.培养物中未分化和分化的人ESC集落A.未分化的人ESC集落。 B.具有轮状外观的区分性人类ESC集落需要在继代培养之前清理。 (比例尺= 400μm)

      1. 用2毫升DPBS冲洗细胞一次或两次。

      2. 在6孔板上每孔加入1ml 0.1%EDTA解离试剂,在室温下孵育3-5分钟。

      3. 在显微镜下检查分离迹象。
      4. 吸入EDTA解离试剂而不干扰细胞。

      5. 添加1毫升Essential 8培养基(含防石剂补充剂)。
      6. 使用细胞刮刀从孔底部提起细胞,用1,000微升移液管轻轻研磨,分解成含有20-40细胞的细胞团。

      7. 添加2毫升Essential 8培养基(添加了抑制剂)
      8. 将原始孔中的细胞添加到新孔中以达到20%的汇合。通常为汇合板的1:5比例。
        注意:如果细胞数量太少,细胞就不会生长;如果细胞太多,细胞生长过快,应该很快分裂。小块是理想的,如果细胞仍然是大块,用1,000μl移液管轻轻研磨大尺寸细胞聚集体几次,以分解它们。
      9. 将板放回到37℃的低氧培养箱(5%CO 2和5%O 2)中,并通过前后摇晃板和左右摇晃均匀分布细胞如上所述。
      10. 每天进行培养基更换,每孔2毫升Essential8培养基,目视评估培养物以监测生长,直到下一次传代时间。

  2. 诱导神经 - 视网膜的命运
    1. 中等准备
      1. 神经元干细胞培养基(NSC)(500ml)的制备(参见配方3)。
      2. 制备含有0.5%FBS(100ml)的NSC培养基(参见配方4)。
      3. ISLI + KSR视网膜诱导培养基(50ml)的制备(参见配方5)。
    2. 人类ES和iPS细胞中的视网膜诱导
      当孔中细胞达到60-70%汇合时,开始用ISLI + KSR视网膜诱导培养基处理细胞(每孔6孔板2ml),每日培养基更换5-6天。一旦细胞处于ISLI + KSR培养基中,在具有5%CO 2水平的常氧37℃培养箱中培养细胞。
      分化分化细胞的过程(第6-7天)

      1. 在分离前至少1小时更换2毫升新鲜ISLI + KSR视网膜诱导培养基
      2. 在分裂时,吸入介质。
      3. 向孔中加入1 ml TrypLE Express解离试剂;
        在室温下孵育6-8分钟,直到细胞彼此间松动接触
      4. 从孔底部轻轻刮去细胞并将细胞悬液转移到15ml离心管中。注意不要让单细胞悬液。

      5. 用2 ml 1x HBSS冲洗孔一次,以收集剩余细胞。

      6. 在270×g离心3分钟。

      7. 。轻轻吸出上清液,不会干扰细胞沉淀。
      8. 加入3-4毫升1×HBSS以重悬细胞沉淀,再以270×g离心3分钟。

      9. 。轻轻吸取1x HBSS,不要干扰细胞团。
      10. 在新鲜的ISLI + KSR视网膜诱导培养基中重悬细胞沉淀。分光比为1:3。
      11. 通过摇动平板并在含氧量正常的条件下返回培养箱,均匀地分配上述细胞。
      12. 第二天,通过查看附着在平板底部的细胞的百分比来检查细胞存活,死细胞不附着并漂浮在培养基中。如果细胞死亡太多,用2 ml 1x HBSS冲洗细胞一次或两次以清除死细胞。
      13. 通过在分裂后第1天加入1ml ISLI + KSR培养基和1ml NSC + 0.5%FBS培养基,将细胞逐渐加入补充有0.5%FBS的NSC培养基中;第2天0.5ml ILSI + KSR培养基+ 1.5ml NSC + 0.5%FBS培养基;从第3天开始,分化细胞将在NSC + 0.5%FBS培养基中培养以使它们进一步分化。当细胞达到汇合时,需要用TrypLE解离方法将细胞分裂成新的Matrigel包被的平板。这通常每7天进行一次,拆分比率为1:3至1:5,具体取决于细胞系。
        注意:如果细胞老化或受到压力,Rock Inhibitor可以添加到NSC培养基中,以帮助细胞在解离后存活。

  3. 神经视网膜玫瑰花结的分离(第18-21天)
    分化细胞是由视网膜祖细胞群和视网膜色素上皮祖细胞(RPE)组成的混合群体(图3A),因为它们都来自相同的视泡囊。视网膜干/祖细胞形成簇(神经视网膜玫瑰花结),可以手动分离和扩展(图3B)。


    图3.文化中差异化的早期视网膜玫瑰花结A.视网膜玫瑰花结在3周采摘和分选之前; B.采摘和重新接种后纯化的神经视网膜培养物。比例尺= 100μm。

    手动分离视网膜干/祖细胞和视网膜色素上皮细胞的程序
    1. 消毒台面以及显微镜上和周围的任何区域以及需要用70%乙醇直接接触细胞的工具。

    2. 选择新的NSC + 0.5%FBS培养基
    3. 在显微镜下,用无菌微量移液器尖端轻轻刮去密集的神经元细胞区域。如果太大,则刮去含有100-200个细胞簇的区域。
    4. 在大部分神经元区域被解除后,收集含有浮动神经元玫瑰花结的介质。
    5. 使用1,000μl移液管将它们转移至新的Matrigel包被的孔中,使细胞附着并在培养箱中的NSC培养基(37°C,5%CO 2)中生长。

  4. 视网膜干/祖细胞扩张
    手动纯化的视网膜干/祖细胞需要在NSC + 0.5%FBS培养基中培养数月,以使细胞进一步分化以产生所有类型的分化的视网膜神经元(图4)。解离方法如下所述:
    TrypLE解离法分化视网膜细胞
    1. 吸引旧媒体。
    2. 用1-2ml 1x HBSS(或DPBS)冲洗细胞一次或两次。
    3. 吸取1x HBSS。
    4. 向细胞中加入1ml TrypLE Express解离试剂,在室温下孵育6-8分钟,并在显微镜下检查解离的迹象(细胞 - 细胞连接松动并在密集细胞中出现空白空间)。不要等到细胞被提起(图4)。


      图4. TrypLE分化的视网膜分化细胞扩增。A.解离前视网膜细胞的形态学。 B.TripLE解离6分钟后视网膜细胞的形态学。比例尺= 400微米。

    5. 使用细胞刮刀刮细胞。
    6. 将细胞转移到15 ml离心管中。

    7. 以270 x g旋转3分钟
    8. 吸出上清液并加入2 ml 1x HBSS并重悬细胞。

    9. 以270 x g旋转3分钟
    10. 吸出上清液并加入NSC + 0.5%FBS培养基重悬细胞(每孔2ml),通常分离比例为1:3。
      注意:如果细胞出现压力,可以在步骤中添加岩石抑制剂以帮助细胞存活。

    11. 在NSC + 0.5%FBS培养基中加入2ml细胞到孔中。
    12. 将培养板放入培养箱(37°C,5%CO 2)中并左右摇晃,来回均匀分布细胞。
    13. 细胞将在NSC + 0.5%FBS培养基中培养。
    14. 根据需要每2-3天更换媒体。
    在诱导视网膜分化1个月后,将种子细胞加到聚-D-赖氨酸(Sigma-Aldrich)和Matrigel包被的盖玻片上,然后进行免疫细胞化学分析以检查视网膜分化状态。在这个阶段,大多数细胞是视网膜祖细胞通过其视网膜干/祖细胞标志物PAX6和LHX2的表达。表征后,细胞可以冷冻保存。

  5. 视网膜干/祖细胞冷冻保存
    冻结神经视网膜干细胞
    1. 用1-2ml 1x HBSS冲洗细胞两次。

    2. 每孔加入1 ml TrypLE于6孔板中孵育6-8分钟
    3. 在此期间,开始用日期,细胞系,信息,等标记冷冻管。

    4. 在步骤D4中检查显微镜下是否有分离迹象
    5. 使用细胞刮刀刮细胞。
    6. 将所有东西都转移到15毫升的管子里。

    7. 旋转3分钟,270克x克
    8. 处理上清液。
    9. 加入2毫升1x HBSS并重新悬浮细胞。

    10. 旋转3分钟,270克x克
    11. 处理上清液。
    12. 添加计算量的冷冻介质以重新悬浮细胞。

    13. 加入1 ml细胞和冷冻培养基到每个冷冻管。
    14. 立即将所有冷冻管放入冷冻室。
    15. 将室转移到-80°C的冷冻箱中。
    16. 第二天,取出小瓶,放入-80°C冷冻箱的适当盒子中。
    17. 第二天将小瓶转移到液氮中。

  6. 解冻视网膜干/祖细胞
    一瓶冷冻保存的视网膜干细胞/祖细胞可以成功地解冻成1孔的Matrigel包被的6孔板。 
    1. 标记Matrigel包被的平板,将NSC + 0.5%FBS培养基(补充岩石抑制剂)温至室温(〜25°C),并在开始方案前制备所有其他细胞培养物,以确保解冻过程完成为尽可能迅速。
    2. 在37℃水浴中快速融化细胞,轻轻摇动冷冻管,直到只剩下一小块冷冻细胞团。
    3. 从水浴中取出冷冻管并用70%乙醇或异丙醇擦拭以消毒。
    4. 使用1毫升微量移液管将冷冻管的内容物转移到15毫升的锥形管中。

    5. 添加7毫升1x HBSS至15毫升试管中,轻轻混合,并加入培养基。

    6. 在270×g离心3分钟。
    7. 吸出上清液,并添加2毫升温暖的NSC培养基(由Rock Inhibitor提供)到15毫升管中以重悬细胞。
    8. 将细胞混合物转移至Matrigel包被的6孔板的空孔中。
    9. 将板置于37℃常氧培养箱(5%CO 2)中,以几个快速,短暂,前后和左右移动的方式移动板以分配细胞聚集体。
      请勿打扰24小时
    10. 第二天更换媒体。如果发生过度的细胞死亡,请在培养基更换前用1x HBSS冲洗细胞。
    11. 根据需要使用NSC培养基每2-3天进行一次培养基更换,并视觉评估培养物以监测直至下一次传代时间的生长。
    在2和3个月诱导的视网膜分化后,将种子细胞加到聚-D-赖氨酸(Sigma-Aldrich)和Matrigel包被的盖玻片上,然后进行免疫细胞化学分析以检查光感受器分化状态(图5和6) >

    图5.培养三个月后的融合神经视网膜培养(比例尺= 400μm)


    图6.视网膜分化6周时由人iPSC产生的视网膜细胞的免疫细胞化学分析细胞表达视网膜干细胞标记物PAX6和LHX2以及光感受器标记物OTX2和RCVRN(比例尺= 10 μm)。

  7. 免疫细胞化学方案
    1. 吸引旧的文化媒体

    2. 。轻轻冲洗1次HBSS中的细胞2次,而不用提起细胞。
    3. 在室温下将细胞固定在4%多聚甲醛溶液(电子显微镜科学)中30分钟。
    4. 用1x PBS冲洗细胞3次,每次冲洗5分钟。
    5. 在室温下封闭10%正常驴血清封闭溶液(Jackson Immuno Research Laboratories,封闭溶液通过在含有0.5%Triton X-100的1x PBS中稀释该溶液制备)1小时来封闭细胞。
    6. 将具有预定浓度的初级抗体(表1)(在血清封闭溶液中制备)添加至细胞并在潮湿室中的冷室中孵育过夜。

      表1.用于视网膜分析的一级抗体


    7. 第二天,用1x PBS冲洗掉未结合的一抗,每次冲洗3次,每次5分钟。
    8. 加入第二抗体,并在室温下在潮湿的室内黑暗中孵育1小时。

    9. 用1x PBS冲洗3次未结合的二抗,每次冲洗5分钟。
    10. DAPI(工作浓度为0.5μg/ ml,用PBS制备)染色细胞核3-5分钟。

    11. 使用防褪色Fluoromount G固定介质(电子显微镜科学)将盖玻片上的细胞安装到组织学幻灯片上。
    注意:样品已准备好在具有荧光功能的显微镜上进行图像采集。

数据分析

分化的视网膜细胞的数据分析通过 t - 测试进行。从3-5个田地和至少3个独立实验计数表达给定抗体的总细胞以及田间总细胞(由DAPI标记)。将结果与未分化细胞或交替分化培养基中的标志物表达进行比较。通过该方案对人ES细胞和iPS细胞诱导的视网膜细胞分化的详细分析可以在最近公开的实验室开放获取论文(Zhu等人,2018)中找到。

笔记

虽然协议在我们实验室测试的多个细胞系中可靠工作。由于各种未分化细胞或衍生方法的不同培养条件,存在一些固有的变异性。作者还建议所有实验室使用商业试剂盒定期检测支原体污染的细胞系,因为它们会显着影响分化能力。

食谱

  1. Essential 8人类ESC / iPSC培养基



    对于新解冻的细胞,使用含有Rock抑制剂(Y-27632,10μM)的Essential8培养基以促进细胞存活。
  2. Rock抑制剂(Y-27632)储备(10 mM,1,000x)
    将2 mg冻干的Y27632(Stemgent)溶于625μl双蒸水中制成10 mM母液,并制成10μl等分试样并储存在-20°C冰箱中。

  3. 神经元干细胞培养基(NSC)(500毫升)



  4. 含0.5%FBS(100ml)的NSC培养基



  5. ISLI + KSR视网膜诱导培养基(50毫升)
    注意:在“ISLI + KSR”中,“I”代表IWR1,“S”代表SB431542,“L”代表LDN193189,最后的“I”代表IGF1,KSR代表KnockOut TM 血清替代品。


致谢

我们感谢曾宪民博士为人类iPS细胞系(NCL-1,NCL1-GFP)。人类ES系购自WiCell。这项工作得到了NEI基金EY025779,Buck学院基金的资助。该协议基于我们以前的工作(Zhu等人,2018年)。

参考

  1. Hambright,D.,Park,K.Y.,Brooks,M.,McKay,R.,Swaroop,A。和Nasonkin,I。O.(2012)。 未免疫抑制小鼠中源自人胚胎干细胞系的视网膜神经元的长期存活和分化视网膜。 Mol Vis 18:920-936。
  2. Hirami,Y.,Osakada,F.,Takahashi,K.,Okita,K.,Yamanaka,S.,Ikeda,H.,Yoshimura,N。和Takahashi,M。(2009)。 从小鼠和人类诱导的多能干细胞产生视网膜细胞 Neurosci Lett 458(3):126-131。
  3. Lamba,D.A.,Karl,M.O.,Ware,C.B。和Reh,T.A。(2006)。 从人类胚胎干细胞高效生成视网膜祖细胞 Proc Natl美国科学院院报103(34):12769-12774。
  4. Lamba,D.A.,Gust,J。和Reh,T.A。(2009)。 人类胚胎干细胞衍生的光感受器的移植恢复了Crx缺陷小鼠的一些视觉功能。 a> Cell Stem Cell 4(1):73-79。
  5. Lamba,D.A.,McUsic,A.,Hirata,R.K。,Wang,P.R。,Russell,D。和Reh,T.A。(2010)。 衍生自人类诱导多能干细胞的光感受器的产生,纯化和移植 PLoS One 5(1):e8763。
  6. Meyer,J.S.,Shearer,R.L.,Capowski,E.E。,Wright,L.S.,Wallace,K.A.,McMillan,E.L.,Zhang,S.C。和Gamm,D.M。(2009)。 用人胚胎和诱导多能干细胞建模早期视网膜发育 Proc美国国家科学院院士106(39):16698-16703。
  7. Osakada,F.,Ikeda,H.,Mandai,M.,Wataya,T.,Watanabe,K.,Yoshimura,N.,Akaike,A.,Sasai,Y。和Takahashi,M.(2008)。 向小鼠,猴子和人类胚胎干细胞产生棒状和锥状感光细胞。 Nat Biotechnol 26(2):215-224。
  8. Osakada,F.,Jin,Z.B.,Hirami,Y.,Ikeda,H.,Danjyo,T.,Watanabe,K.,Sasai,Y.和Takahashi,M。(2009)。 人体多能干细胞视网膜细胞的体外分化,分子诱导。 J Cell Sci 122(Pt 17):3169-3179。
  9. Zhong,X.,Gutierrez,C.,Xue,T.,Hampton,C.,Vergara,MN,Cao,LH,Peters,A.,Park,TS,Zambidis,ET,Meyer,JS,Gamm,DM,Yau ,KW和Canto-Soler,MV(2014)。 用来自人类iPSCs的功能性光感受器产生三维视网膜组织 Nat Commun 5:4047.
  10. Zhu,J.,Cifuentes,H.,Reynolds,J。和Lamba,D.A。(2017)。 通过丧失IL2rγ引起的免疫抑制增强了hESC衍生的光感受器在小鼠视网膜中的长期功能整合。 细胞干细胞 20(3):374-384 e375。
  11. Zhu,J.,Reynolds,J.,Garcia,T.,Cifuentes,H.,Chew,S.,Zeng,X。和Lamba,D.A。(2018)。 从目前良好的生产实践中制造的可移植的视网膜光感受器 - 制造的人诱导多能干细胞系。 / a> 干细胞转移。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhu, J. and Lamba, D. A. (2018). Small Molecule-Based Retinal Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells. Bio-protocol 8(12): e2882. DOI: 10.21769/BioProtoc.2882.
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