In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Table 1. Media recipes for all cell types included in the protocol. Media should be filtered after mixing all components with either a sterile vacuum filtration system for big volumes or syringe and 0.2 um syringe filters. Conversion media (with factors) must be prepared fresh every week.

1. Direct conversion of adult human fibroblasts to neuronal progenitor cells

NOTE: A schematic timeline of this protocol can be reviewed in Meyer (2014)¹⁹.

1. Use primary human skin fibroblasts that can be obtained from cell banks or skin biopsies³¹. Culture cells at 37 °C in a 5% CO₂ tissue culture incubator. Passage cells for at least 1-2 passages post thawing prior to use in the experiment. Once the cells are ready, coat a well of a 6-well plate with human fibronectin (1:200 diluted in PBS) at room temperature for 15-20 min.
2. When the cells are ready to be plated, remove the fibronectin from the dish and immediately add cell solution or 2 mL of fibroblast media (Table 1) – do not let the dish dry out prior to adding media. Depending on how fast the cells grow, plate 150,000 (fast growing) – 200,000 (slow growing) fibroblasts in two wells of a human fibronectin coated 6-well plate each.
   NOTE: The cells should be around 70% confluent for viral transduction to be able to keep them in the same plate for several more days after transduction. It can be beneficial to seed at two different densities in order to have a choice the next day for conversion.
3. The day after seeding, check the fibroblasts under the microscope and verify cell density (between 70-85%) and even seeding throughout the well for optimal results.
4. Transduce one well with all four retroviral vectors (Oct3/4, Klf4, Sox2 and c-Myc) – use a multiplicity of infection (MOI) of 10 for fast growing or 15 for slow growing fibroblasts (transduction efficiency should exceed 70% or more positive cells for each vector). Add regular fibroblast medium to viral mix to a total volume of 1 mL per well and incubate overnight at 37 °C in a 5% CO₂ tissue culture incubator. Leave the second well untreated and return cells to tissue culture incubator.
   NOTE: Retroviral vectors can be purchased by a vendor or made in-house, protocols are available online³².
5. The day after transduction, wash cells 1x with PBS and add 1 mL of fresh fibroblast medium.
6. The next day, change medium to conversion medium. Wash cells 1x with PBS to get rid of the residual fibroblast medium, and then add 1 mL of conversion medium. Change the media of the untreated well to conversion media as well.
   NOTE: Some morphological changes can be observed even by changing the media on the fibroblasts that did not receive retroviral vector treatment, thus having an untreated well (optional) will be helpful when determining effects of the viral vectors. Cells should start changing morphology 2-4 days after switching to conversion media.
7. Observe cells under the microscope and watch for changes in morphology (Figure 1). Cell media should be replaced daily throughout the conversion process (1-2 mL of conversion media, Table 1) and for subsequent iNPC culture. Change medium by carefully removing 70% of medium from the well while making sure that the cells remain covered in the remaining 30% of media.
   NOTE: Media with growth factors in it needs to be consumed within 1 week of preparation.
   1. Continue to observe cells and change media daily (1-2 mL of conversion media).
   2. Some cell lines start forming loose elevated round formations growing in ball like structures or loose neuronal spheres (Supplemental Figure 1 and ^19,33). Take care to not detach these cells. If the balls detach, they can be collected and re-plated in a new fibronectin coated well of a 6-well plate.
      NOTE: If they are expanded on their own, the cells will have reduced diversity compared to the initial plate and may represent a distinctly different cell line. We recommend combining these cells with the rest of the converted cells at the next round of splitting.
8. After about 5-6 days in conversion media (up to 12 days for difficult cell lines) or when cells become very dense, passage them 1:2 or maximum 1:3.
   ​NOTE: It is very important to keep the cells at a high density throughout the conversion process.

2. Conversion and iNPC splitting procedure

1. Warm up cell detachment solution (e.g., Accutase) and coat an appropriate number of wells of a 6-well with fibronectin (1:200 in PBS, 1 mL of coating volume) for 15-20 min at room temperature. Fibronectin coating can be longer without negative impact, but care should be taken to not shorten the coating time.
2. Wash cells carefully with PBS without detaching any cells (use the wall of the well to apply PBS gently to the cells). Carefully remove PBS with pipettes or by aspiration.
3. Add 0.5 mL of cell detachment solution and incubate 2-3 min at 37 °C in a tissue culture incubator. Check under the microscope to verify that most cells have detached. If all cells have come off, add 2 mL of fresh conversion media and gently pipette up and down 2-3 times to dissociate clumps (if required).
4. Collect the cells in a 15 mL tube and add additional 3 mL of media to further dilute the cell detachment solution. Wash the well with the extra media first to ensure all cells have been collected.
5. Centrifuge for 4 min at 200 x g at room temperature.
   NOTE: Converting cells or iNPCs are extremely sensitive to the presence of cell detachment solution in the media. It is absolutely necessary to remove the residual enzyme by centrifugation and withdrawal of supernatant.
6. Remove supernatant from cell pellet and resuspend the cells in 1 mL of fresh medium. Gently pipette up and down 2-3 times to resolve cell clumps. Remove fibronectin from coated 6-wells and add 1 mL of fresh media to each well immediately (do not let fibronectin dry). For a split ratio of 1:2, add 0.5 mL of the cell suspension in one well and the other 0.5 mL in a second well.
   1. Culture cells at 37 °C in a tissue culture incubator.
7. Distribute the cells by gently shaking the 6-well in north-south-east-west direction (not circular) and put in tissue culture incubator.
8. Observe the cells under the microscope the next day and change media (1-2 mL). Change media every day until the cells are ready to be split again.
   NOTE: At this point, the cell proliferation should start to accelerate, and cells should be ready for a second split within 2-3 days (4 days for very slow growing lines).
   1. At this new split, seed one well of cells in conversion media and the other well in NPC media containing only FGF at increased concentration without EGF/heparin (Table 1).
      NOTE: Some cell lines reach a stagnant state in conversion media after about 10 days and switching to NPC media might help with cell growth. In addition, NPCs have a more specific, smaller shape, when grown in NPC media¹⁹. At this point, the iNPCs are ready for differentiation and further use.
9. Keep changing media (1-2 mL) of the iNPCs daily.
   1. Expand NPCs into 10 cm dishes by combining two or three confluent wells of a 6-well. The media volume for 10 cm dishes is typically 12-15 mL.
   2. At the next split, further expand these cells to three or four 10 cm dishes (12-15 mL of media) for generations of large numbers of cell stocks. 10 cm dishes are usually split at 1:3 or 1:4 every 3-4 days depending on proliferation rate.

3. Generating induced astrocytes from NPCs

1. To make astrocytes from fresh iNPCs, seed iNPCs (in 10 cm fibronectin coated plates) directly in 10 mL astrocyte medium (Table 1) so that they are around 10% confluency the following day. Culture cells at 37 °C in a 5% CO₂ tissue culture incubator.
   NOTE: The recommended seeding density is usually 50 µL of the cell resuspension of a 10 cm dish of NPCs, provided they are diluted to a final volume based on their split ratio (e.g., 3 mL for a 1:3 split, 4 mL for a 1:4 split, etc.).
2. Change medium (10 mL astrocyte media) of the iAs three days after plating. Keep the cells differentiating for 5-7 days.
   NOTE: iAs do not tolerate multiple passages well, therefore it is better to make fresh astrocytes every time you split the NPCs.
3. To seed for experimentation, split the astrocytes using trypsin or cell detachment solution as described in steps 2.1-2.7. Recommended seeding densities are included in Table 2.

Table 2. Recommended seeding density of iAs per plate area. Typical number of iAs seeded to generate a monolayer on the most common tissue culture plates.

4. Preparing and defrosting portions for astrocyte differentiation from frozen NPC stocks (alternative to step 3)

NOTE: As an alternative to maintaining iNPCs in culture, astrocytes can also be produced directly from a frozen stock. To do so, the iNPCs are frozen in smaller portions. Table 3 shows suggested freezing and thawing volumes for portions to be defrosted into a 10 cm dish containing 10 mL of Astrocyte media.

Table 3. Instructions on how to portion iNPC to generate iAs. Proportion of iNPC suspension and freezing media to generate portions. Note that the final DMSO concentration is 10% when cell suspension is mixed with freezing media.

1. To make astrocytes from frozen iNPC stocks, remove portion stock vial from liquid nitrogen storage tank and quickly thaw at 37 °C. As soon as cells are defrosted, pipette cell solution in a 15 mL tube containing 5 mL astrocyte media.
2. Centrifuge for 4 min at 200 x g and room temperature. Remove the supernatant and resuspend in 1 mL of fresh astrocyte medium.
3. Add 9 mL of fresh astrocyte medium to a fibronectin coated 10 cm dish and add 1 mL of cell solution. Gently distributing cells in north-south-east-west motion. Culture cells at 37 °C in a 5% CO₂ tissue culture incubator.