Protocol for neurogenin-2-mediated induction of human stem cell-derived neural progenitor cells

Summary Human pluripotent stem cell-derived neural progenitor cells (NPCs) are an essential tool for the study of brain development and developmental disorders such as autism. Here, we present a protocol to generate NPCs rapidly and reproducibly from human stem cells using dual-SMAD inhibition coupled with a brief pulse of mouse neurogenin-2 (Ngn2) overexpression. We detail the 48-h induction scheme deployed to produce these cells—termed stem cell-derived Ngn2-accelerated progenitor cells—followed by steps for expansion, purification, banking, and quality assessment. For complete details on the use and execution of this protocol, please refer to Wells et al.1

Sterile filter (0.22 mm) and store at 4 C. 100 mg/mL human bFGF: Add 500 mL of 0.1% BSA in D-PBS to 50 mg tube of bFGF.
Aliquot into 25 mL and store at À20 C until needed.100 mg/mL human EGF: Add 1 mL of sterile water to 100 mg tube of EGF.
Aliquot into 25 mL and store at À20 C until needed.
Accutase: Thaw 500 mL bottle of accutase for 12-18 h at 4 C Aliquot into 10 mL and store at À20 C until needed.

Reagent
Final concentration Amount mTeSR-Plus base N/A 400 mL mTeSR-Plus supplement 13 100 mL (Continued on next page) Alternatives: This protocol uses mTeSR-Plus, but e8 or standard mTesR could be used as alternatives.

Final concentration Amount
Normocin 5003 1 mL Total 500 mL mTeSR-Plus media should be protected from light and stored at 4 C for no more than 2 days.SNaP base media should be protected from light and stored at 4 C for no more than 2 months.Cell culture plates need to be coated with a basement matrix prior to plating cells.Failure to perform this step properly will prevent cells from sticking to the plate and growing.

Final concentration Amount
1. Thaw a tube (5 mL) of LDEV-Free Geltrex for 4-6 h at 4 C until the entire solution is fluid.Once thawed, Geltrex can be stored at 4 C for up to 2 weeks.
Alternatives: This protocol uses Geltrex, but Matrigel could be used in its place.
2. Dilute Geltrex in cold MEM at a ratio of 1:100 to make Geltrex coating solution.This coating solution is stable at 4 C for up to one week.
CRITICAL: Geltrex solidifies in 5 min when kept above 15 C. Therefore, keep working vials on ice when in use to prevent premature gelling.
3. Add the Geltrex coating solution to each well of the cell culture-grade plates at 20 C-22 C according to Table 1. 4. Rotate the plates briefly to ensure equal distribution and full coating of the bottom of each well, and incubate for at least 30 min in a 37 C/5% CO 2 incubator.5.If using immediately, aspirate the Geltrex coating solution after 30 min and quickly replace with culture media.If not using immediately, the Geltrex-coated plates can be left in the incubator for up to 3 days.

Protocol
Note: Do not let the Geltrex-coated plate dry if leaving in the incubator for a prolonged period of time or after aspirating the coating solution.
Thaw and expansion of human pluripotent stem cells (hPSCs) These steps describe how to thaw and culture hPSCs in 6-well plates in preparation for lentiviral transduction of doxycycline-inducible Ngn2 expression vectors.This feeder-free protocol works for both human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs).
6. Thaw hPSCs.a. Prepare two Geltrex-coated wells of a 6-well plate.b.Label a 15 mL conical tube with the hPSC donor identifier.c.Remove the cryogenic vial of hPSCs from the liquid nitrogen storage.
Note: If liquid nitrogen tank is more than a 2 min walk from the biosafety cabinet, or if you are thawing multiple lines at the same time, we recommend temporarily placing the cryovials on dry ice to prevent premature thawing.
d. Quickly thaw cells by gently shaking the vial in a 37 C water bath until only a small frozen pellet of cells is visible.This should take 1-2 min depending on the cell suspension volume.e. Make sure the label on the vial is ethanol resistant.
i. Wipe down the vial with 70% ethanol before transferring into the sterile biosafety cabinet.
ii. Open the vial and transfer the cell suspension to 15 mL conical tube.f.Slowly add 5 mL mTeSR-Plus media dropwise to cell suspension.Gently swirl the tube to ensure complete resuspension and dilution of freezing media.g.Centrifuge cells for 5 min at 200 x g to pellet.h.Prepare 5 mL mTeSR-Plus with 10 mM Y-27632 Rock Inhibitor (RI).i. Aspirate the media from the 15 mL conical tube so that only the cell pellet remains.CRITICAL: Do not aspirate the cell pellet at this step.If you are concerned about using a vacuum aspirator, use a manual pipet-aid instead.j.Add 1 mL of mTeSR-Plus with RI to the cell pellet and gently resuspend without introducing bubbles.k.Aspirate the Geltrex coating solution from the 6-well plate and immediately add 2 mL of mTeSR-Plus with RI to each well.l.Add 1 million cells dropwise to one well ($100,000 cells/cm 2 ) and 250,000 cells dropwise to another ($25,000 cells/cm 2 ).i.If the total number of cells is unknown, instead add 750 mL of cell suspension dropwise to one well and 250 mL to dropwise to the other well.ii Gently rock the plate in a T-shape to ensure even distribution of the cells.
Note: Plating at multiple densities ensures that at least one well will not be overly dense or sparse during the first 2 days post-thawing.It is best not to passage hPSCs within 2 days of thawing.m.Incubate cells for 12-18 h at 37 C/5% CO 2 .7. Maintain hPSCs.
a.The day after plating, cells should be fed daily with 2 mL mTeSR-Plus (without RI).
Note: mTeSR-Plus allows for a less stringent feeding schedule in which cells can be fed every 48 h.Nevertheless, we feed cells daily during the week and allow for one 48-h feed on weekends.
b.When cells reach 80%-90% confluency, they should be prepared for passage and expansion.

Note:
The amount of time it takes to reach this level of confluency depends on the cell line and the initial plating density and survival of the frozen hPSCs.Typically, hPSCs require 3-7 days to reach $80%-90% confluency from a thaw.Note: At the 5-min mark, briefly remove cells from the incubator and rotate the plate.You should see cells lifting off the plate.
d.At the 10 min mark, dissociate cells by pipetting up and down 5-10 times.If the cells do not go into suspension, put the plate back into the incubator for an additional 5 min and repeat the dissociation.e. Collect the cells into a 15 mL conical tube containing 2 mL mTeSR-Plus so that the ratio of mTeSR-Plus to accutase is at least 2:1.f.Centrifuge tube for 5 min at 200 x g to pellet cells.g.Prepare 5 mL mTeSR-Plus with 10 mM Y-27632 Rock Inhibitor (RI).h.Aspirate the media from the 15 mL conical tube so that only the cell pellet remains.i. Add 1 mL of mTeSR-Plus with RI to the cell pellet and gently pipette mix to resuspend.j.Aspirate the Geltrex coating solution from the 6-well plate and immediately add 2 mL of mTeSR-Plus with RI to each well.k.Add 100 mL of cell suspension dropwise to one well (1:10 passage ratio) and 50 mL dropwise to the other well (1:20 passage ratio).Gently move the plate in a T-shaped motion to ensure even distribution of the cells, pausing between changing directions.l.Incubate cells for 12-18 h at 37 C/5% CO 2 .m. Feed the cells on the next day by aspirating the spent media and replacing it with mTeSR-Plus media without RI.Cells will be ready for the next passage when they reach 80%-85% confluency 3-5 days later depending on the growth rate of the hPSC line.
CRITICAL: Stem cells should undergo at least one passage prior to transduction to maximize post-transduction survival.One confluent well of a 6-well should be more than enough to generate 1 million cells for lentiviral transduction.Here, we introduce doxycycline-inducible mouse Ngn2 (and optionally, GFP) into the genome of human stem cells via lentiviral integration.This protocol has not been optimized for human NGN2.

Lentiviral transduction of hPSCs
CRITICAL: Safe handling practices and institutional approvals are required for lentiviral transductions.Note: We typically purchase lentivirus from Alstem Inc that produces titers between 108 and 10 9 .This protocol is therefore optimized for high-titer viruses.Lower titers may require additional optimization steps.
Note: Co-transduction with a tetO-GFP lentivirus is not required but is recommended for test inductions; this enables real-time visualization of cells that were properly transduced and estimations of transduction efficiency.
b. Add the appropriate volume of lentiviruses to cell suspension of 1 million cells in a 1 mL mTesR-Plus to reach a multiplicity of infection (MOI) of 2 for each lentivirus.Pipette mix 5-10 times.
Note: To calculate the volume in microliters of lentivirus needed for MOI = 2 in 1 million cells: Divide 1 million by the viral titer (provided by the commercial lentiviral producer), multiply by 1000 to convert to mL, and multiply that number by 2 (because MOI of 2 is desired).For example, if the viral titer is 1 3 10 9 , then 2 mL should be added to the suspension of 1 million cells [(1 million / 1 3 10 9 ) * 1000 * 2)].This calculation will need to be repeated for each lentivirus, and the appropriate volume of each lentivirus will need to be added to the cell suspension.
Note: Lentiviral titers decrease with each freeze-thaw cycle.We recommend making 10 mL aliquots of high-titer virus upon receipt, or purchasing pre-aliquoted lentivirus.
c. Aspirate the Geltrex coating solution from the 12-well plate and immediately add 500 mL of mTeSR-Plus with RI and protamine sulfate to each well.d.Add 500 mL of cell suspension dropwise to each well.Gently rock the plate in a T-shape to ensure even distribution of the cells.

Note:
The relatively low plating volume (1 mL) ensures adequate concentrations of the lentiviruses.
e. Centrifuge the plate for 1 h at 1,000 3 g at 25 C to increase lentiviral contact with the cells (''spinoculation'').

Note:
We have observed no differences in viability or transduction efficiency when this centrifugation step is performed at 20 C-22 C.
f. Incubate cells for 12-18 h at 37 C/5% CO 2 .g.The next day, add 1 mL mTeSR-Plus media (but no RI) to each well.Incubate for 12-18 h at 37 C/5% CO 2 .h.The next day, remove virus-containing media and dispense into a 15 mL conical tube containing 5 mL of 10% bleach.Feed the cells with 2 mL mTeSR-Plus.
Note: Cell death is typical within 24-48 h post-transduction.In some cases, this death is concentrated in the center of the well.
i. Once transduced cells reach 80%-85% confluency (which typically takes 3-5 days depending on the growth rate of the hPSC line and its ability to tolerate lentiviral infection), they can be passaged for freezing, transduction efficiency tests, and/or induction.11.Banking transduced hPSCs.
a. Use Accutase to dissociate the cells, then count the cell suspension.b.Take 1 million cells and resuspend in 500 mL of Cryostor-10.c. Immediately transfer the cell suspension to a labeled 2 mL cryovial.d.Place the vial in a Mr. Frosty Freezing container filled with 100%.Place the container in a À80 C freezer.e.The next day, move the vial into a liquid nitrogen tank for long-term storage.
Pause point: Transduced hPSCs can be stored indefinitely in liquid nitrogen.
a. Use Accutase to dissociate the cells, then count the cell suspension.b.Take 75,000 cells and resuspend in 300 mL of mTeSR-Plus with 2 mg/mL Doxycycline.c. Plate 100 mL of cell suspension per well of a Geltrex-coated 96-well plate with clear bottoms that are suitable for epifluorescence imaging (3 wells total).d.The next day, image the cells using an epifluorescence microscope.e.If the GFP lentivirus was included, transduction efficiency can be calculated by estimating the percentage of GFP-positive cells in the culture.f.If the GFP lentivirus was not included, transduction efficiency can be calculated by estimating the percentage of cells that show noticeable morphological changes (Figure 1B).
CRITICAL: Transduction efficiencies >30% are required for successful NPC induction in the subsequent steps of this protocol.Many factors contribute to differential efficiencies, including the cell line, vendor source of the lentivirus, and the temporal proximity to the lentivirus shelf life.For the SW7388-1 hPSC line, we typically achieve between 75%-95% transduction efficiency.We have observed a broader range of efficiencies across the >100 hPSC lines we have transduced ($25%-100%).

Timing: 4 days
These steps describe how to induce stem cells into neural progenitors through the overexpression of Ngn2.The resulting cells, termed Stem cell-derived Ngn2-accelerated Progenitors (SNaPs), resemble early dorsal telencephalic NPCs at protein, transcriptional, and functional level. 1 CRITICAL: If starting from a thawed vial of transduced hPCS, passage at least one time prior to the start of the induction protocol.
a.The next day, aspirate media and feed with 2 mL SNaP induction complete media per well.b.Twenty-four hours later, aspirate media and feed with 2 mL SNaP induction selection media per well.
Note: At this stage (24 h post-induction), cells should have noticeable morphological changes (Figure 1B).If dox-inducible GFP-expressing lentivirus was included in the transduction, bright green cells should be present.The percentage of cells expressing GFP should be at least 30% to ensure the successful production of an established SNaP line.Note: There may be non-GFP-positive stem cell colonies that remain; these should be eliminated during the following additional selection step.c.Aspirate media from transduced cells and add 500 mL of Accutase to each well.Incubate for 10 min in 37 C/5% CO 2 incubator.d.After 10 min, dissociate cells by pipetting the cell suspension up and down 5-10 times while applying gentle force to the bottom of the well.If the cells do not go into suspension, put back into the incubator for an additional 5 min and repeat the dissociation.e. Move cell suspension to a labeled 15 mL conical tube containing 3 mL SNaP base media and centrifuge for 5 min at 200 3 g to pellet.f.Prepare 4 mL SNaP complete media with 10 mM RI and 5 mg/mL puromycin.g.Aspirate the media from the conical tube so that only the pellet remains.h.Add 1 mL SNaP complete media with RI and puromycin to the cell pellet and gently resuspend.i. Count the cells using a Countess III (or any other counting device).j.Aspirate the Geltrex coating solution from the 24-well plate and immediately add 1 mL SNaP complete media with RI and puromycin to each well.k.Plate 240,000 cells per well (120,000 cells/cm 2 ) by adding the appropriate volume of cell suspension dropwise.Gently rock the plate in a T-shape to ensure even distribution of the cells.l.Incubate for 12-16 h at 37 C/5% CO 2 .m.No more than 12-16 h later, aspirate the media and feed the cells with 1 mL SNaP complete media.
CRITICAL: Do not leave the cells in puromycin-containing media for more than 16 h after passaging.The puromycin resistance cassette is driven by the presence of doxycycline in the media.Doxycycline is removed from our media at 48 h post-induction, as prolonged Ngn2 overexpression forces the cells out of the proliferative state.Therefore, the additional puromycin selection step that takes place after 48-h post-induction relies on the residual protein expression of the puromycin expression cassette.Selection beyond 16 h post-passage will result in excessive death.Failure to include this additional selection step could result in the presence and expansion of stem cell colonies in the SNaP culture.

Timing: 1-2 weeks
The induction phase of the SNaP protocol produces cells that resemble dorsal telencephalic NPCs.However, like many hPSC-to-NPC induction protocols, the cells' morphologies sometimes do not resemble NPCs after the first post-induction passage.The goal of the next set of procedures is to establish a stable line of purified SNaPs that can be maintained for up to 10-15 passages.
16. SNaP expansion.a. Continue to feed the cells daily with 1 mL SNaP complete media for one week.
Note: In the first 1-2 days post-passaging, it is common to observe cell death, post-mitotic neurons, and flattened cells that do not resemble NPCs.These contaminating cell types, as well as the abnormal morphology of NPCs, tend to resolve themselves over the course of 4-7 days depending on the cell line and the initial transduction efficiency.

Protocol
b.When confluency reaches 95%-100% and the cell morphology becomes more uniform within the well, passage and count the cells.
Note: Quality control assays (see below) can be performed at this stage of culture development.The SNaP line is considered stable when QC metrics are passed.
c. Resuspend the cells in 1 mL SNaP complete media with 10 mM RI. d.Plate the cells on Geltrex-coated plates in the desired cell culture format at 120,000 cells/cm 2 by adding the appropriate volume of cell suspension dropwise.Gently rock the plate in a T-shape to ensure even distribution of the cells.e. Incubate cells for 12-18 h at 37 C/5% CO 2 .f.The next day, feed cells with SNaP complete media.g.At this stage, cells should be fed daily and passaged every 7 days.17. SNaP banking.
a. Use Accutase to dissociate the cells, then count the cell suspension.b.Take 1-2 M cells and resuspend in 500 mL of Cryostor-10.c. Immediately transfer the cell suspension to a labeled 2 mL cryovial.d.Place the vial in a Mr. Frosty Freezing container filled with 100% isopropanol.Place the container in a À80 C freezer.e.The next day, move the vial into a liquid nitrogen tank for long-term storage.
Pause point: SNaPs can be stored indefinitely in liquid nitrogen.
a. Prepare two Geltrex-coated wells of a 24-well plate.b.Label a 15 mL conical tube with the donor identifier.c.Remove the cryogenic vial of SNaPs from the liquid nitrogen storage.
Note: If liquid nitrogen tank is more than a 2 min walk from the biosafety cabinet, or if you are thawing multiple lines at the same time, we recommend temporarily placing the cryovials on dry ice to prevent premature thawing.
d. Quickly thaw cells by gently shaking the vial in a 37 C water bath until only a small frozen pellet of cells is visible.This should take 1-2 min depending on the cell suspension volume.e. Make sure the label on the vial is ethanol resistant.
i. Wipe down the vial with 70% ethanol before transferring into the sterile biosafety cabinet.
ii. Open the vial and transfer the cell suspension to 15 mL conical tube.f.Slowly add 5 mL SNaP base media dropwise to cell suspension.Gently swirl the tube to ensure complete resuspension and dilution of freezing media.g.Centrifuge cells for 5 min at 200 3 g to pellet.h.Prepare 5 mL SNaP complete media with 10 mM Y-27632 Rock Inhibitor (RI) i. Aspirate the media from the 15 mL conical tube so that only the cell pellet remains.j.Add 1 mL of SNaP complete media with RI to the cell pellet and gently resuspend without introducing bubbles.k.Aspirate the Geltrex coating solution from the 24-well plate and immediately add 500 mL of SNaP complete media with RI to each well.l.Add 500,000 cells dropwise to one well ($250,000 cells/cm 2 ) and 250,000 cells dropwise to another ($125,000 cells/cm 2 ).i.If the total number of cells is unknown, instead add 750 mL of cell suspension dropwise to one well and 250 mL to dropwise to the other well.ii.Gently rock the plate in a T-shape to ensure even distribution of the cells.
Note: Plating at multiple densities ensures that at least one well will not be overly dense or sparse during the first 2 days post-thawing.It is best not to passage SNaPs within 2 days of thawing.m.Incubate cells for 12-18 h at 37 C/5% CO 2 .

SNaP quality control assays
Timing: 1-3 weeks NPCs express canonical protein markers and have the ability to differentiate into neurons and glia.We leverage these molecular and cellular proteins to test the purity of our SNaP cultures and validate the functionality of these NPC models.

Marker protein expression.
a. Use Accutase to dissociate the cells, then count the cell suspension.b.Passage 1-2 SNaPs are cultured in 100 mL per well of SNaP complete media with RI at 120,000 cells/cm 2 on Geltrex-coated 96-well plates with clear bottoms that are suitable for epifluorescence imaging.c.The next day, aspirate the media and feed the cells with SNaP complete media without RI.d.The next day, fix and stain cells with antibodies directed against NESTIN, PAX6, SOX1, and OCT4 (see immunocytochemistry protocol below).20.Differentiation assay.
a. Passage 1-2 SNaPs are cultured at 10,000 cells/cm 2 in Geltrex-coated 6-well plates in SNaP base media with RI. b.After one week, use Accutase to dissociate the cells, then count the cell suspension and plate cells at 10,000 cells/cm 2 in Geltrex-coated clear-bottomed 96-well plates in SNaP base media without RI.c.After one additional week (i.e., after 2 weeks of spontaneous differentiation), fix and stain cells with antibodies directed against HuCD, CD44, and S100ß.21.Immunocytochemistry protocol.
a. Aspirate media and gently wash SNaPs with 150 mL 13 PBS with Ca 2+ and Mg 2+ .
CRITICAL: The cells will detach from the plate if PBS without Ca 2+ and Mg 2+ is used.
b. Immediately remove 13 PBS and add 50 mL of 4% paraformaldehyde.Incubate at 20 C-22 C for 10 min.c.Remove the 4% paraformaldehyde and wash cells three times with 150 mL 13 PBS.
CRITICAL: SNaPs tend to lift off the plate if washed too harshly.We recommend leaving a small volume of PBS (10-20 mL) in the wells during each wash.
Pause point: Fixed cells can be stored at 4 C protected from light for 1-2 weeks before permeabilization.d.After the final wash, add 50 mL of 0.1% Triton for 15 min to permeabilize cells.e. Remove 0.1% Triton and add 50 mL of blocking solution (10% normal donkey serum diluted in 13 PBS) for 1 h at 20 C-22 C.This incubation step should be performed on a plate rocker or shaker.f.Prepare the primary antibody in the blocking solution as detailed in Table 2. Remove the blocking solution and add 30-50 mL of primary antibody solution per well.g.Incubate for 12-18 h by shaking gently at 4 C. h.The next day, remove the plate from 4 C and allow to equilibrate to 20 C-22 C. i. Remove the primary antibody solution and wash three times with 13 PBS.j.Prepare the secondary antibody at 1:1000 in blocking solution.(Related to Step 10).Some hPSC cell lines are resistant to lentiviral infection, and investigators will have difficulty achieving adequate transduction efficiencies.We have also observed differences in transduction efficiencies across stem cell maintenance media.

Potential solution
Repeat transduction at a higher MOI ($3).If not using mTeSR/mTeSR-Plus, switch to this maintenance media as this has been effective across a range of stem cell lines.

Problem 3
Low SNaP survival after the 48 h post-induction passage.
(Related to Step 15L-m).This scenario is often the result of failing to remove the puromycin selection media from the culture within 12-18 h after the first passage.The timing of this step is critical.(Related to Steps 15-16).We have on occasion noticed uneven distribution of SNaPs on cell culture surfaces in which large segments of the plate are missing cells.Typically, we observe these gaps on just one side and/or the center of the wells.In almost all cases, this was the result of media being added too forcefully or directly on top of the cells rather than along the side of the well (N.B. the absence of cells on the left side of the well was common to right-handed feeders; absence on the right side of the well was common to left-handed feeders).SNaPs appear to be most sensitive to feed force in the first couple of days after the 48 h post-induction passage.

Potential solution
Always feed cells by adding media along the side of the well, rather than directly from above.For especially sensitive cell lines, leave 10% of the media volume in the well prior to feeding with fresh media to reduce cell detachment.

Problem 5
Abnormal morphology in the first week after induction.
(Related to Step 16).In the first 24 h after the 48-h SNaP passage, cells may appear spindle-like (Figure 5A), which is expected and is the result of ROCK pathway inhibition.During the next few days, some SNaP cultures may appear sparse, flat, and/or neuronal in morphology (Figure 5B).This scenario tends to resolve itself with daily feeds no later than 5-7 days after this passage.If the culture is not uniform in morphology within 10 days and/or if the culture does not meet R 75% NPC markerpositivity threshold, we recommend repeating the induction for this line.We have observed a small number of lines that cannot be made into SNaPs; the most common cause of these induction failures is low transduction efficiency.Therefore, if the line fails a second time, we advise taking steps to increase transduction efficiency prior to repeating induction attempts.

Potential solution
Increase lentiviral MOI (Step 10) and use mTeSR/mTeSR-Plus to increase transduction efficiency.

Problem 6
Contaminating cell types in passage 2 SNaP cultures.SNaP cultures should reach high purity by the end of the first passage, at which point the percentage of cells that remain pluripotent (OCT4 + ) should be less than 0.1%.If, however, this threshold is not met, it is common to observe contaminating cell types in the culture in passages 2 and beyond.

Potential solution
Repeat the induction with a prolonged puromycin selection phase during the 48-h post-induction passage (e.g., 18 h instead of 12 h) (Step 15L-m).

Problem 7
Contaminating cell types in passage 3+ SNaP cultures.
(Related to Step 16).SNaPs should remain proliferative and multipotent for at least 10 passages.Beyond 10 passages, it is common to see low levels of spontaneous differentiation to neural crest, radial glia, neurons, and astrocytes.This differentiation may increase with each passage beyond passage 10.However, we have at times observed SNaP cultures that, despite passing QC and not containing detectable numbers of pluripotent stem cells, show contaminating cells starting at passage 3 (Figure 5C).In almost each case, we were able to attribute this to the inappropriate use of expired EGF/bFGF stocks or repeated feeding of cells with SNaP complete media that was not fresh (i.e., used within 48 h of preparation).

8.
Passage hPSCs.a. Thaw a frozen aliquot of Accutase in a 37 C water bath to reach 20 C-22 C. b.Prepare two Geltrex-coated wells of a 6-well plate.c.Aspirate media from cells and add 750 mL of Accutase to each well.Incubate for 10 min at 37 C/5% CO 2 .

9.
Passage hPSCs for transduction.a. Thaw a frozen aliquot of Accutase in a 37 C water bath to reach 20 C-22 C. b.Prepare two Geltrex-coated wells of a 12-well plate for Ngn2 lentiviral transduction.c.Prepare 5 mL of mTeSR-Plus with 10 mM RI and 8 mg/mL protamine sulfate.d.Incubate hPSCs in accutase, dissociate, pellet, and resuspend in 1 mL mTeSR-Plus with RI and protamine.e. Count cells using an automated Countess III device (or any other cell counter device) i. Add 10 mL of Trypan Blue and 10 mL of cell suspension to a 0.6 mL Eppendorf tube and pipette mix.ii.Add 10 mL of cell suspension + Trypan Blue to one side of a Countess slide.iii.Place slide in a Countess III automated cell counter, and record the concentration of live cells.10. hPSC lentiviral transduction.a. Remove vials of high-titer TetO-Ngn2-Puromycin and FUdeltaGW-rtTA lentiviruses from the À80 C and place on dry ice.When ready, remove from ice and briefly thaw at 20 C-22 C.
c. Twenty-four hours later (48 h post-induction), dead cells from the puromycin selection should be visible and floating in the media (Figure 1B, top).i.If GFP lentivirus was used, a large percentage of remaining cells should be green (Figure 1B, bottom).

Figure 1 .
Figure 1.SNaP induction and representative images (A) Schematic describing SNaP induction, selection, and expansion protocol.(B) Bright-field (top) and epifluorescence GFP images (bottom) of cells during the 48 h induction phase.Day 0 stem cells show spindle-like morphology because of the presence of RI.Scale bar = 100 mm.

Figure 3 .
Figure 3. Expansion of SNaP cultures (A-C) Bright-field images of Passage 2 SNaPs (A) the day after passage in the presence of RI, (B) 48 h after passage (24 h after removal of RI), and (C) 5 days post-passage.Top row images captured using a 43 objective (scale bar = 250 mm) and bottom row images captured using a 103 objective (Scale bar = 100 mm).
SNaP induction media should be protected from light and stored at 4 C for no more than 2 days.SNaP induction complete media should be protected from light and stored at 4 C for no more than 2 days.SNaP induction selection media should be protected from light and stored at 4 C for no more than 2 days.

Table 1 .
SNaP complete media with RI and puromycin should be protected from light and stored at 4 C for no more than 2 days.Media and reagent volumes for different plate formats