Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders

Patient-derived or genomically modiﬁed human induced pluripotent stem cells (iPSCs) offer the opportu-nity to study neurodevelopmental and neurodegenerative disorders. Overexpression of certain neurogenic transcription factors (TFs) in iPSCs can induce efﬁcient differentiation into homogeneous populations of the disease-relevant neuronal cell types. Here we provide protocols for genomic manipulations of iPSCs by CRISPR/Cas9. We also introduce two methods, based on lentiviral delivery and the piggyBac transposon system, to stably integrate neurogenic TFs into human iPSCs. Furthermore, we describe the TF-mediated neuronal differentiation and maturation in combination with astrocyte cocultures.


Introduction
Induced pluripotent stem cells (iPSCs) enable studying neurodevelopmental and neurodegenerative diseases such as autism spectrum disorders including fragile X syndrome and Rett syndrome, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, or spinal muscular atrophy [1]. Human iPSC lines are generated by reprogramming of fibroblasts, hair, or blood samples [2], which are either directly donated by patients with a disease-relevant phenotype and a known genotype or disease-causing mutations can be introduced into the genome of the iPSCs by genomic modifications such as CRISPR/Cas9 [3]. To study the effect of the mutations on the cellular level, iPSCs can be differentiated into the disease-relevant neuronal subtypes. Conventional differentiation protocols rely on the addition of specific soluble growth factors and compounds to the culturing media. These factors trigger intracellular signaling pathways affecting transcription factors (TFs), which in turn induce neuronal differentiation by changing gene expression levels and triggering gene regulatory networks. However, these protocols can be very delicate and time-consuming, lasting from several weeks to months, and yield a heterogeneous mixture of different neuronal subtypes at different developmental stages and glia cells. The forced expression of certain neurogenic TFs in human iPSCs shortcuts neuronal differentiation resulting in rapid neurogenesis that yields highly homogeneous populations of neurons [4][5][6][7]. Here we describe the culturing of a robust inducible-neuronal iPSC line as well as different methods to introduce neurogenic TFs and genomic modifications into human iPSCs and how to differentiate those iPSCs into mature neurons.
Neurogenic TFs under the control of a doxycycline-inducible promoter can be stably integrated in the genome of iPSCs either by lentiviral delivery [8] or via the piggyBac transposon system [9]. While lentiviruses have a high efficiency in delivering transgenes, the preparation of viral particles is laborious, timeconsuming and requires biosafety level 2. In contrast, the piggyBac transposon system offers a nonviral alternative to efficiently cut and paste transgenes into the genome. The production of plasmids is faster and cheaper and the piggyBac system requires only standard laboratory biosafety levels. For genome editing of human iPSCs with great precision, the CRISPR/Cas9 technology is the method of choice since it is easy-to-use, efficient, and cost-effective. Genomically modified iPSCs can be differentiated into neurons by doxycycline-induced overexpression of TFs and maturation is achieved by astrocyte coculture.
9. 4 M NaCl solution. Store at 4 C. 10. PBS pH 7.2 without calcium and magnesium. Store at room temperature.
12. Antibiotic: If you would like to select the cells for the integrated lentiviral construct, use the appropriate antibiotic (such as blasticidin or puromycin). Store aliquots at À20 C, after thawing store at 4 C, protected from light.
15. TaqMan ® PCR master mix, such as TaqMan ® Universal PCR Master Mix (Thermo Fisher Scientific). Store at 4 C. 16. TaqMan ® primer and probes for WPRE and albumin detection (see Table 1). Dilute in ddH 2 O to a concentration of 10 μM and store at À20 C.
Store at À20 C.
3. 1Â PBS with calcium and magnesium. Store at 4 C.
4. Doxycycline solution: dissolve 10 mg doxycycline hyclate powder in 20 ml PBS (0.5 mg/ml ¼ 1000Â), sterile-filter (0.22 μm). Store aliquots at À20 C; after thawing store at 4 C, protected from light. Put the tubes in a freezing container and store at À80 C for at least 2 h. Subsequently, store in liquid nitrogen.

Nucleofection of iPSCs
1. In order to electroporate piggyBac and transposase vectors into iPSCs in suspension, use the X-Unit of the 4D-Nucleofector™ System in combination with the P3 Primary Cell 4D-Nucleo-fector™ X Kit according to the manufacturer's guidelines.
2. First of all, prepare the DNA, the Nucleofector™ solution and the cell culture plates. For a nucleofection reaction in 100 μl cuvettes, mix 10 μg piggyBac vector and 2.5 μg transposase vector in less than 10 μl volume (maximum 10% of the final sample volume) in a 1.5 ml tube. In a separate tube, mix 82 μl Nucleofector™ solution with 18 μl supplement per nucleofection reaction and bring to room temperature. Prepare Matrigel-coated cell culture plates with the desired volume of mTeSR™1 medium with ROCKi and prewarm in the incubator (see Note 11).
3. Switch on the X-Unit of the 4D-Nucleofector™ System and choose the cell-type specific program for the human embryonic stem cell line H9, the cuvette size, P3 primary solution and the pulse CB-156 or CB-150 (see Note 12).
4. Dissociate the cells to be nucleofected using TrypLE, centrifuge (400 Â g, 4 min) and resuspend in mTeSR™1 with ROCKi. Determine the cell number, transfer 800,000 cells for each nucleofection into a 1.5 ml tube and centrifuge (400 Â g, 4 min). Aspirate the supernatant and resuspend the cells in 100 μl room temperature Nucleofector™ solution with supplement, mix with the DNA and transfer into an electroporation cuvette and close the lid. Avoid air bubbles while pipetting. Gently tap the cuvette to make sure that the sample covers the bottom.
5. Quickly put the cuvette(s) into the Nucleofector™ and press the start button to apply the pulse CB-156 or CB-150. Immediately after, carefully remove the samples, add mTeSR™1 with ROCKi into the cuvette, mix by gently pipetting up and down 6. The next day, wash the cells with 1Â PBS w/o Ca 2+ and Mg 2+ and change the medium to mTeSR™1 w/o ROCKi. Change the medium every day until next passaging (see Fig. 2b). Starting 48 h after nucleofection, select the cells with an integrated construct with the appropriate antibiotic (see Note 14). In order to determine the number of the integrated piggyBac constructs, use the piggyBac copy number kit from System Biosciences (see Note 15). To prepare genomic DNA, seed the cells in a 12-well plate (see Note 16). When confluent, wash once with 1Â PBS w/o Ca 2+ and Mg 2+ and add 250 μl lysis buffer to each well. Freeze the cells at À80 C and thaw the plate at room temperature to ensure complete cellular lysis. Detach the cells by pipetting up and down, transfer the lysates to 1.5 ml tubes and heat them at 95 C for 2 min. Centrifuge at 17,000 Â g for 2 min and transfer the supernatant to a new 1.5 ml tube. The lysates should be placed on ice if used immediately or stored at À20 C. 9. Calculate the copy number as follows [11]: , divide the ΔΔC t by 2 as there are two copies of the UCR1 sequence per genome.

Lentivirus Production and Transduction
1. For the production and transduction of lentiviruses, titration and copy number determination, we follow the protocol from the Trono lab [8].
2. One day prior to transfection, seed 8,000,000,293T/17 cells in a 10 cm culture dish. The next day, replace the culture medium with 4 ml fresh DMEM with 10% FBS. The cells are transfected using 45 μg of polyethylenimine (PEI) combined with 15 μg DNA containing the plasmid of interest (see Fig. 3a, b), the viral packaging (psPAX2) plasmid, and the viral envelope (pMD2G) plasmid in a 4:2:1 ratio.    . 3c). 9. Plot the standard curve using the software of your qPCR machine or manually using other software such as Microsoft Excel, and calculate the quantity of albumin and WPRE for each sample using the equation of the standard curve.
10. Calculate the copy number for each sample as follows: Copy number ¼ (quantity mean of WPRE sequence/quantity mean of Alb sequence) Â 2.
11. Calculate the viral titer with the following formula: Titer (viral genome/ml) ¼ (number of target cells counted at day 1 Â number of copies per cell of the sample)/volume of supernatant (ml). 2. Seed the iPSCs at a density of 30,000-50,000 cells per cm 2 in mTeSR™1 medium with ROCKi supplemented with 0.5 μg/ ml doxycycline. On the next day, wash the cells with 1Â PBS w/o Ca 2+ and Mg 2+ and change the medium to mTeSR™1 w/o ROCKi supplemented with 0.5 μg/ml doxycycline. Change the medium daily until day 4 (Fig. 4).
3. When culturing the neurons for longer time periods, it is recommended to change the stem cell medium (mTeSR™1) to maturation medium (BrainPhys™ with supplements).
Change half of the medium on day 5 of differentiation to BrainPhys™ medium with supplements. Repeat changing half of the medium 2 days later. After those two adaptation medium changes, it is sufficient to change half of the medium once per week. Volume loss due to evaporation should be compensated with ddH 2 O.

Coculturing with Astrocytes
1. In order to increase the maturation of neurons for electrophysiological measurements, coculturing with astrocytes is highly recommended [4,15]. We adapted the protocol from Kaech and Banker [16] to our cell culture.
2. Rat primary cortical astrocytes are cultured in astrocyte medium at 37 C and 5% CO 2 according to the manufacturer's instructions. For passaging, aspirate the culture medium and store it in a Falcon tube as a washing solution (see Note 26).
Rinse the cells once with 1Â PBS w/o Ca 2+ and Mg 2+ . Add prewarmed Accutase and incubate the cells at 37 C until all of them are detached (usually 5 min are sufficient). Stepwise add the cell culture medium stored in the first step to flush cells and collect all cells to a prerinsed 15 ml Falcon tube. Centrifuge at 400 Â g for 5 min. Aspirate the supernatant and resuspend the pellet in prewarmed astrocyte growth medium. Count the cells using Trypan Blue and seed the appropriate amount in uncoated tissue-culture treated dishes at a seeding density of approximately 5000 cells per cm 2 . Change the growth medium every 3-4 days.
3. For the coculture with neurons, prepare astrocytes to be~80% confluent at day 4 of neuronal differentiation. One day before the reseeding of neurons, wash the astrocytes three times with 1Â PBS w/o Ca 2+ and Mg 2+ and add BrainPhys™ medium with minimal supplements.
4. Thoroughly clean the coverslips in a big glass petri dish. First, rinse the coverslips in ddH 2 O for 2 h and then shake in 50 ml 8. After 2 h, place the coverslips with the differentiated iPSCs upside down into culture wells containing 80% confluent rat astrocytes. Every 7 days, exchange 50% of the BrainPhys™ medium and compensate the volume loss due to evaporation with ddH 2 O (see Note 30).
1. Aliquot the Matrigel according to the protocol and the dilution factor provided with it (varies for each bottle of Matrigel). We prepare aliquots for dilution in 12 ml coating medium. Briefly, thaw the Matrigel on ice in the cold room or the fridge and prepare a box with dry ice to precool 1.5 ml tubes. Quickly distribute the Matrigel solution into the tubes and store at À20 C.
2. The piggyBac vector backbone can be obtained from Addgene (to be submitted, containing the EGFP gene under the control of the doxycycline-inducible promoter). For cloning of transcription factors, the EGFP can be excised using NheI and XhoI, the transcription factor cDNA can be amplified by PCR and introduced into the piggyBac vector using Gibson Assembly cloning [17].
3. There are two different lentiviral vector systems that can be used: the pLV system that consists of two constructs, one expressing the rtTA transactivator from the constitutively active EF1α promoter and the other one expressing the transgene under the control of the doxycycline-inducible TRE promoter [4] (Addgene plasmids #61472 and #61471, respectively) or the pLIX403 system that expresses the rtTA transactivator and the transgene under the TRE promoter on a single construct (Addgene plasmid #41395). The pLV plasmids that are referred to in this protocol do not contain any selection markers. If selection for the integrated constructs is required, it should be cloned into the plasmids before the production of lentiviral particles.
4. For better attachment of the neurons, freshly add 1 μl of a 1 mg/ml laminin solution per 1 ml supplemented BrainPhys™ medium to a final concentration of 1 μg/ml.

5.
For the coculture with astrocytes, we use BrainPhys™ medium with a minimal supplementation since we found that astrocytes do not grow well in the presence of cAMP. Addition of BDNF and GDNF was neither found to enhance maturation nor affect astrocytes but might be beneficial depending on experiment design.
6. Use 1 ml of diluted Matrigel solution per well of a 6-well plate, 0.5 ml per well of a 12-well plate and 0.25 ml per well of a 24-well plate.
7. Use 2 ml mTeSR™1 medium per well of a 6-well plate, 1 ml per well of a 12-well plate and 0.5 ml per well of a 24-well plate. If you would like to avoid feeding the cells on the weekend, add at least the 1.5-fold amount of medium on Friday.
8. The optimal cell density depends on the growth rate of your iPSC line. For our cells, we seed 15,000-25,000 cells/cm 2 for maintenance of stem cells, and 30,000-50,000 cells/cm 2 for differentiation experiments. 9. Check iPSCs in 4-week intervals for mycoplasma contamination using the Universal Mycoplasma Detection Kit (ATCC ® 30-1012K™) according to the manufacturer's instructions. 10. The optimal density for freezing depends on your iPSC line. For our cells, a density of 500,000-1,000,000 cells/cryotube in 0.5-1 ml mFreSR™ works well.
11. Cells of one 100 μl nucleofection reaction can be seeded to one well of a 6-well plate or distributed to multiple wells of a 12-or 24-well plate.
12. The pulse CB-156 is recommended if higher transfection efficiency is favored at expenses of a lower survival rate, whereas the pulse CB-150 results in higher viability with lower transfection efficiency.
13. Leaving the cells in Nucleofector™ solution for extended periods of time may lead to reduced transfection efficiency and viability so it is important to work as quickly as possible. If you face problems such as low transfection efficiency due to very big plasmids etc. you can try to incubate the cells after nucleofection in the Nucleofector™ solution at room temperature for approximately 10 min.
14. The concentration of antibiotic optimal for selection depends on the specific iPSC line of choice and should be determined with a killing curve. We use a final concentration of 20 μg/ml for blasticidin, 3 μg/ml for puromycin, and 250 μg/ml for hygromycin.
15. Alternatively, the copy number can be determined as described for the lentiviral transduction (see Subheading 3.3) by performing a TaqMan ® -based qPCR on genomic DNA. Use the albumin gene for normalization and a gene specific for the piggyBac construct for counting the integration events. We recommend using primers and probes for the antibiotic resistance gene, if not otherwise present in the genome of your iPSC line. It is important to have both genes present on the same plasmid used for the standard curve since the preparation of the serial dilutions is prone to small variations. 16. Before performing the copy number determination, the cells must be passaged at least once to avoid the interference of nonintegrated piggyBac plasmids with the qPCR.
17. The optimal settings of the qPCR protocol may vary with the qPCR machine and the SYBR ® Green or TaqMan ® mix used. 18. From this step on, the cells are producing viral particles and should be handled at biosafety level 2. All viral particles that are collected are also biosafety level 2.
19. If your centrifuge is not able to run at 7000 Â g, the centrifugation step can be carried out at lower g for a longer period of time (e.g., at 5000 Â g for 30 min).
20. We usually use one aliquot of viral particles to transduce one well of a 6-or 12-well plate. In order to optimize the viral transduction, it is recommended to determine the viral titer. Therefore, transduce cells with different volumes of the viruscontaining supernatant and perform a qPCR on genomic DNA counting the number of integrated copies per cell.
21. Directly after transfection, the iPSCs are biosafety level 2 and should be handled as such, after the medium change the next day, they are back at biosafety level 1.
23. Avoid placing the PAM sequence into your sgRNA-expressing vector and the potential donor construct. It will be cut once sgRNA and Cas9 are expressed. The vectors from the Zhang lab can be ordered in different versions, that is, with GFP or puromycin expression. If positive cells should be sorted with flow cytometry, GFP is optimal. When expanding of single cells and subsequent picking of monoclonal colonies is preferred, use puromycin with the version V2 on Addgene, which is corrected from a previous version. The success rate of this cloning strategy is usually very high.
24. One or two guanines can be added for more efficiency of the U6 promoter if the designed sgRNA is not starting with it. They have to be added to the bottom oligo as reverse complement in addition to the sgRNA sequence as well.
25. The T7 endonuclease I assay is performed as follows: Transfect the sgRNA-and Cas9-expressing constructs into a test cell line (e.g., 293T/17, see Subheadings 2.3 and 3.3). Isolate the DNA using a DNA extraction kit, such as the DNeasy® Blood and Tissue Kit (Qiagen). Amplify the locus using flanking primers tested for specificity in advance. Purify the reaction using a PCR Purification kit (Qiagen). Elute in 30 μl. Mix 200 ng purified PCR product, 2 μl NEBuffer™ 2 and water to a total volume of 19 μl. Hybridize the PCR product in a thermocycler by heating to 95 C and ramp down to 85 C with À2 C/s, then to 25 C with 0.1 C/s and hold at 4 C. Add 1 μl T7 endonuclease I to the reaction and incubate at 37 C for 20 min. Run on a 2% agarose gel (30 min, 90 V) to see if one or more bands appear. If two or three bands are visible, the sgRNA works fine. The T7 endonuclease cuts at wobbles that appear with reannealing of nonfitting DNA [13] strands. This happens if the Cas9 cuts parts of the DNA of the population of cells used as the test cell line.
26. Rat Primary Cortical Astrocytes stick to the plastic used in cell culture dishes and centrifuge tubes. Prior to use, rinse all material that will come in contact with the cells with medium to prevent cells from sticking to the plastic.
27. Since the cleaning of the coverslips is very time-consuming, it can also be done in 1 day. Briefly, rinse the coverslips two times in ddH 2 O, then add 50 ml 1 M HCl and shake for 1 h. Rinse three times with ddH 2 O by shaking for 2 min, and then rinse once more with ddH 2 O by shaking for 1 h. Shake three times in 100% ethanol for 2 min and one time for 1 h. Sterilize the coverslips at 225 C for 2-3 h. Successful cleaning will be accompanied by an even spread of coating solution across the whole surface of the coverslip. If problems with adhesion occur, go back to the long protocol.
28. The purpose of the spacers is to allow growth of the induced neurons in close proximity to the astrocyte feeder layer but without physical contact.
29. The coverslips can have any size depending on the requirements of the experiment. We routinely use 12 mm coverslips equipped with three paraffin feet in a 24-well plate. It is recommended to add additional volume of medium to the well to completely cover the coverslips in order to avoid floating.
30. We add approximately 50 μl ddH 2 O per week for a 24-well plate to compensate for volume loss due to evaporation. Store a test plate full of H 2 O in the incubator and weigh in weekly intervals to check for evaporation.