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

We use this protocol to make neural progenitor cells from patient skin fibroblasts and study neurological and neurodegenerative diseases. We feel like this protocol has a lot of advantages over classic IPS technology, including the speed, but also getting in certain cases a more severe disease phenotype. We study neurological and neurodegenerative diseases and using skin samples allows us really to evaluate the diversity of the patients as well.

My lab is particularly interested in the role of astrocytes in diseases and we use the astrocytes that we make from the NPCs to test medications and also to study disease mechanisms, but also in co-cultures with human and mouse neurons. We hope this protocol is going to be informative and please reach out if you have any questions. Thank you.

Use primary human skin fibroblasts that can be obtained from cell banks or skin biopsies. Culture cells at 37 degrees Celsius in a 5%CO2 tissue culture incubator. Passage cells for at least one to two passages post thawing prior to use in the experiment.

Once the cells are ready, coat a well of a six-well plate with human fibronectin at a 1:200 concentration diluted in PBS at room temperature for 15 to 20 minutes. Prepare fibroblast media as shown in table one. When the cells are ready to be plated, remove the fibronectin from the dish and immediately add cell solution or two mils of fibroblast media.

Do not let the dish dry out prior to adding media. Depending on how fast the cells grow, plate 150, 000 to 200, 000 fibroblasts in two wells of a human fibronectin-coated six-well plate. Culture cells at 37 degrees Celsius in a 5%CO2 tissue culture incubator.

The day after seeding, check the fibroblasts under the microscope and verify that cell density is between 70 to 85%Transduce one well with all four retroviral vectors:Oct3/4, Klf4, Sox2, and c-Myc. Use a multiplicity of infection of 10 for fast-growing or 15 for slow-growing fibroblasts. Transduction efficiency must exceed 70%or more positive cells for each vector.

Add regular fibroblast medium to viral mix for a total volume of one mil per well. Leave the second well untreated and return cells to the tissue culture incubator to incubate overnight. The day after transduction, wash cells one time with PBS and add one mil fresh fibroblast media.

Culture cells at 37 degrees Celsius in a 5%CO2 tissue culture incubator. The next day, prepare conversion media as shown in table one and wash cells one time with PBS to get rid of the residual fibroblast media, then add one mil of conversion medium. Change the media of the untreated well to conversion media as well.

Observe cells under the microscope and watch for changes in morphology. Cell media should be replaced daily throughout the conversion process with one to two mils of conversion media and for subsequent iNPC culture. Change media by carefully removing 70%media from the well while making sure that the cells remain covered in the remaining 30%of media.

Continue to observe the cells and change media daily with one to two mils of conversion media. After about five to six days in conversion media or when cells become very dense, passage them one to two or maximum one to three. Warm up Accutase and coat an appropriate number of wells in a six-well plate with one mil total fibronectin diluted 1:200 in PBS for 15 to 20 minutes at room temperature.

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 aspirating.

Add 500 microliters Accutase and incubate two to three minutes at 37 degrees Celsius in a tissue culture incubator. Check under the microscope to verify that most cells have detached. If cells have come off, add two mils of fresh conversion media and gently pipette up and down two to three times to dissociate clumps.

Collect the cells in a 15 mil tube and add three mils media to further dilute the Accutase. Centrifuge for four minutes at 200 G at room temperature. Remove supernatant from the cell pellet and resuspend the cells in one mil of fresh medium.

Gently pipette up and down two to three times to resolve cell clumps. Remove fibronectin from coated six wells and add one mil fresh media to each well immediately. For a split ratio of 1:2, add 500 microliters of the cell suspension in one well and the other 500 in a second well.

Distribute the cells by gently shaking the six well in north-south east-west direction and put in 37 degree tissue culture incubator. Observe the cells under the microscope the next day and change media with one to two mils. Change media every day until cells are ready to be split again.

Prepare NPC media containing FGF at increased concentration without EGF or heparin as seen in table one. At this new split, seed one well of cells in conversion media and the other well in NPC media. Keep changing media of iNPCs daily with one to two mils of NPC media.

Prepare astrocyte media according to table one. To make astrocytes from fresh iNPCs, seed iNPCs directly in 10 mils of astrocyte media in 10 centimeter fibronectin-coated plates so that they are around 10%confluency the following day. Culture cells at 37 degrees Celsius in a 5%CO2 tissue culture incubator.

Change medium with 10 mils of astrocyte media three days after plating. Keep the cells differentiating for five to seven days. To seed for experimentation, split the astrocytes using trypsin or Accutase as described in step 2 through 2.6.

Recommended seeding densities are included in table two. Instructions on how to portion iNPCs to generate induced astrocytes are located in table three. To make astrocytes from frozen iNPC stocks, remove portion stock file from liquid nitrogen storage tank and quickly thaw at 37 degrees Celsius.

As soon as cells are defrosted, pipette cell solution in a 15 mil tube containing five mils of astrocyte media. Centrifuge for four minutes at 200 G room temperature. Remove supernatant and resuspend in one mil fresh astrocyte medium.

Add nine mils fresh astrocyte medium to a fibronectin-coated 10 centimeter dish and add one mil cell solution, gently distributing cells in north-south east-west motion. Culture cells at 37 degrees Celsius in a 5%CO2 tissue culture incubator. This protocol allows for the rapid and easy generation of iNPCs directly from human skin fibroblasts using retroviruses containing the Yamanaka factors.

This method allows bypassing the stem cell state and the need for clonal selection, thereby avoiding clonal variation. Important steps to keep in mind while cells are undergoing the conversion process are the fibroblast seeding density, media pH, and keeping the cells at an optimal confluency. Examples of optimal confluency and morphology changes during the conversion process can be found in figure one.

After the conversion process is complete, the NPCs will show strongly reduced expression or no expression of fibroblast markers and morphology and express NPC-specific cell markers. Moreover, they can also be used for generating different cell lines like induced neurons, oligodendrocytes, and astrocytes. In our experience, induced astrocytes in particular are very valuable for drug and disease mechanism testing as they can be generated in pure populations at reproducible large numbers.

Induced astrocytes can be differentiated from iNPCs by taking a small aliquot of cells during splitting or a previously frozen iNPC portion and directly plating in astrocyte media. Important considerations during this process are maintaining the iNPCs'initial seeding density low as a high seeding density has been shown to hinder the differentiation process, and paying additional attention to the media pH as acidification can activate even healthy astrocytes. In summary, the direct conversion protocol is a very quick method to be able to make neural progenitor cells from patient skin samples.

This allows to study a larger number of cell samples at the same time and really look at diversity in the disease context. We feel this is a very strong assay that you can use to make neural progenitor cells, neurons, but also astrocytes that can then be used either in co-culture or by themselves for drug testing or mechanistic studies as well.