Feb 19, 2020
Version 2
iNeuron pre-differentiation & differentiation protocol V.2
- Merissa Chen1,
- Nina Draeger1,
- Martin Kampmann1,
- Kun Leng1,
- Emmy Li1,
- Connor Ludwig1,
- Gregory Mohl1,
- Avi Samelson1,
- Sydney Sattler1,
- Ruilin Tian1
- 1University of California, San Francisco
Protocol Citation: Merissa Chen, Nina Draeger, Martin Kampmann, Kun Leng, Emmy Li, Connor Ludwig, Gregory Mohl, Avi Samelson, Sydney Sattler, Ruilin Tian 2020. iNeuron pre-differentiation & differentiation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bcrjiv4n
Manuscript citation:
Tian et al (2019). CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons. Neuron pii: S0896-6273(19)30640-3. [Epub ahead of print] PubMed PMID: 31422865.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 19, 2020
Last Modified: February 19, 2020
Protocol Integer ID: 33291
Keywords: iNeuron, iPSCs, differentiation, CRISPR interference, CRISPRi, CROP-seq, Perturb-Seq, essential genes, functional genomics, high-content microscopy, neuron, single-cell RNA sequencing, stem cell
Abstract
This protocol describes thedifferentiation of iPSCs with stably integrated doxycycline-inducible Ngn2 (such as i3Ns).
Attachments
Materials
MATERIALS
Glutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
DPBS no calcium no magnesiumGibco - Thermo Fisher ScientificCatalog #14190250
KnockOut™ DMEMThermo FisherCatalog #10829018
MEM Non-Essential Amino Acids Solution (100X)Thermo FisherCatalog #11140050
DMEM/F-12Thermo FisherCatalog #11320033
B-27™ Supplement (50X), minus vitamin AThermo FisherCatalog #12587010
KnockOut™ DMEM/F-12Thermo FisherCatalog #12660012
N-2 Supplement (100X)Thermo FisherCatalog #17502048
Laminin Mouse Protein, NaturalThermo FisherCatalog #23017015
StemPro™ Accutase™ Cell Dissociation ReagentThermo FisherCatalog #A1110501
Essential 8™ MediumThermo FisherCatalog #A1517001
Rock inhibitor Y-27632 dihydrochlorideTocrisCatalog #125410
Neurobasal™-A MediumThermo ScientificCatalog #10888022
BrainPhys™ Neuronal MediumSTEMCELL Technologies Inc.Catalog #05790
Recombinant Human/Murine/Rat BDNFpeprotechCatalog #450-02
Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane MatrixCorningCatalog #356231
Recombinant Human NT-3peprotechCatalog #450-03
Doxycycline hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #D3447
Culture iPSCs
Culture iPSCs
Protocol
NAME
WTc11 iPSC Culture and Maintenance
CREATED BY
Martin Kampmann
Thaw the frozen StemFlex Supplement 10X at Room temperature for ~02:00:00 or overnight at 2 °C to 8 °C .
Note
IMPORTANT! Do not thaw the frozen supplement at 37°C.
Mix the thawed supplement by gently inverting 3−5 times.
Aseptically transfer 50 mL of StemFlex Supplement 10X to the bottle of StemFlexTM Basal Medium (450 mL fill).
Gently invert the bottle several times to obtain 500 mL of homogenous complete medium.
Note
Following reconstitution, complete StemFlexTM Medium can be stored at 2°C to 8°C for up to 2 weeks or aliquoted and stored at -5 °C to -20 °C for up to 6 months. Alternatively, usage size aliquots of the supplement can be made and frozen at -5 °C to -20 °C for up to 6 months. Avoid multiple freeze-thaw cycles.
Feed the PSCs the day after seeding followed by every-other-day thereafter.
Note
If the cells are to be left without feeding for two days (for example, over a weekend), then double the feed volume (i.e., 4 mL added per well of 6-well plate).
iPSCs should be split when cells are ~ 80% confluent.
Thaw Matrigel on ice and dilute in pre-chilled Knockout DMEM for a final volume of 100 Mass Percent .
Coat desired wells/plates with diluted Matrigel and incubate at 37 °C for 00:30:00 -01:00:00 using the following table for volumes to add per well:
| Per: | 96-well | 24-well | 12-well | 6-well | 10-cm dish | 15-cm dish | |
| Volume to add: | 40 μL | 200 μL | 0.5 mL | 1 mL | 5 mL | 10 mL |
Note
Matrigel may be re-used during this time only to coat additional plates. Original plates should
have PBS or media to prevent the matrix from drying out. Matrigel coated plates must be used within 14 days of coating.
Tilt cell-containing plate towards you and aspirate existing media.
Wash wells once with ample PBS (about 2x amount of media).
Add accutase to well(s) using the following table for volumes per well and incubate at 37 °C for 00:03:00 ; add another 01:00:00 -00:02:00 if cells have not mostly lifted/dissociated.
| Per: | 96-well | 24-well | 12-well | 6-well | 10-cm dish | 15-cm dish | |
| Volume to add: | 20 μL | 100 μL | 250 μL | 0.5 mL | 2 mL | 4 mL |
Add ample PBS to accutase-containing well(s) to dilute accutase using the following table for volumes per well:
| Per: | 96-well | 24-well | 12-well | 6-well | 10-cm dish | 15-cm dish | |
| Volume to add: | 200 μL | 1 mL | 2.5 mL | 5 mL | 10 mL | 20 mL |
Pipette up and down gently to mechanically release remaining cells, collect, and add to appropriately-sized conical tubes.
Spin cells at 200 x g for 00:05:00 at Room temperature .
Carefully aspirate supernatant from pelleted conicals.
Add appropriate volume of StemFlex + Rock inhibitor at 10 micromolar (µM) to conicals according to pellet size for counting.
Note
Rock inhibitor should only be used when cells are individualized or in small colonies (typically the first two days after passaging); the presence of Ri at higher densities results in cell stress/death, and in general, Rock inhibitor greatly reduces proliferation.
Note
For first time use of Rock inhibitor, it is suggested to aliquot at 10mM [1000x], diluting in DPBS, and use on cells at concentration 10 Mass Percent .
Triturate to resuspend cells in StemFlex + Rock inhibitor and remove 10μL and add this volume to a 1.5mL Eppendorf tube.
Note
Be careful to minimize contact of pipette with the side of the conical wall.
Count cells and calculate desired number of cells to seed, and dilute this volume with additional StemFlex + Rock inhibitor to plate using the following table for volume to add per well:
| Per: | 96-well | 24-well | 12-well | 6-well | 10-cm dish | 15-cm dish | |
| Volume to add: | 50-100 μL | 250-500 μL | 0.75-1 mL | 1.5-2 mL | 8-12 mL | 15-25 mL |
Note
For general passaging where exact cell number seeded is not important, adding resuspended cells at 1:100, 1:50, and 1:20 the final well volume typically provides near-confluency in 7 days, 5 days, and 3 days, respectively.
iPSCs can be frozen in StemFlex + 10% DMSO.
Days -3-0: Pre-differentiation
Days -3-0: Pre-differentiation
Note
Pre-Differentiation and Differentiation Notes:
1. Keep reagents at 4 °C for a maximum of 4 weeks, If needing long term storage, don’t freeze-thaw more than 3 times.
2. Sometimes pre-differentiated cells take a long time to come off of the plate on Day 0. This is usually okay. It is important that your cells are single cells.
3. Feeding schedule and volumes:
Make N2 Pre-differentiation Media:
| Component | Stock Concentration | Final Concentration | Dilution Factor | For 100 mL (mL) | For 50 mL (units in cell) | |
| Konckout DMEM/F12 | 1X | 1X | 1 | 100 | 50mL | |
| NEAA | 100X | 1X | 100 | 1 | 500uL | |
| N2 Supplement | 100X | 1X | 100 | 1 | 500uL | |
| NT-3 | 10ug/mL | 10ng/mL | 1000 | 0.1 | 50uL | |
| BDNF | 10ug/mL | 10ng/mL | 1000 | 0.1 | 50uL | |
| Mouse Laminin* | 1.0mg/mL | 1ug/mL | 1000 | 0.1 | 50uL | |
| ROCK Inhibitor** | 10mM | 10uM | 1000 | 0.1 | 50uL | |
| Doxycycline*** | 2mg/mL | 2ug/mL | 1000 | 0.1 | 50uL |
*Mouse Laminin comes at variable concentrations, so make sure the final concentration is correct in the media you are making!
**Only add during plating of iPSCs on Day -3 and omit for any further media changes
***Add immediately before use
Coat plate with matrigel diluted in Knockout DMEM. Coat for at least 00:30:00 (or O/N). Coated plates can last in the 37 °C incubator for 14 days.
Aspirate media from iPSCs and wash with DPBS.
Add Accutase and incubate at 37 °C for 00:03:00 ; if necessary, incubate for additional time up to 00:07:00 .
Use gentle agitation to release cells, and collect in an Eppendorf tube or conical with DPBS.
Spin cells at 200 x g for 00:05:00 resuspend in N2 pre-diff media.
Count cells and add desired amount to an Eppendorf tube or conical, spin, resuspend in N2 pre-differentiation media, and plate onto Matrigel-coated cell culture vessels for the pre-differentiation. Reference seeding density chart for plating
Perform ½ pre-differentiation media change every day (w/dox, no RI) throughout days 0-3 pre-differentiation period or full media change on day -1 or day -2. Be consistent.
Going into differentiation, it is best if cells are in single-cell suspension. Optional: use cell strainer.
Pre-differentiated cells can be frozen in 10%DMSO + N2/B27 Differentiation media (**can also freeze in pre-diff media).
Day 0: Releasing and Plating Pre-Differentiated iNeurons
Day 0: Releasing and Plating Pre-Differentiated iNeurons
Make N2/B27 Differentiation Media:
| Component | Stock Concentration | Final Concentration | Dilution Factor | For 100mL (mL) | For 50 mL (units in cell) | |
| DMEM/F12* | 1X | 0.5X | 2 | 50 | 25mL | |
| Neurobasal-A | 1X | 0.5X | 2 | 50 | 25mL | |
| NEAA | 100X | 1X | 100 | 1 | 500uL | |
| GlutaMAX | 100X | 0.5X | 200 | 0.5 | 250uL | |
| N2 Supplement | 100X | 0.5X | 200 | 0.5 | 250uL | |
| B27-VA Supplement | 50X | 0.5X | 100 | 1 | 500uL | |
| NT-3 | 10ug/mL | 10ng/mL | 1000 | 0.1 | 50uL | |
| BDNF | 10ug/mL | 10ng/mL | 1000 | 0.1 | 50uL | |
| Mouse Laminin** | 1.0mg/mL | 1ug/mL | 1000 | 0.1 | 50uL | |
| Doxycycline*** | 2mg/mL | 2ug/mL | 1000 | 0.1 | 50uL |
*DMEM/F12 with either bicarbonate or HEPES buffer is okay, but we have had qualitatively better neurons with HEPES buffer when grown and differentiated side-by-side. HEPES is essential for imaging, as media will change color after ~30 minutes if using bicarbonate buffered media.
**Mouse Laminin comes at variable concentrations, so make sure the final concentration is correct in the media you are making!
***Add immediately before use and only on Day 0
Aspirate media and wash with DPBS.
Add Accutase and incubate at 37 °C for 00:03:00 ; if necessary, incubate for additional time up to00:07:00 .
Use gentle agitation to release cells, and collect in an Eppendorf tube or conical with DPBS.
Spin cells at 200 x g for 00:05:00 .
Carefully aspirate DPBS/accutase solution and resuspend in Classic N2/B27 Differentiation Media.
Count cells, dilute to appropriate density, and plate onto PDL-coated cell culture vessels.
Leftover cells can be frozen down in pre-differentiation media + 10% DMSO.
Day 0+: Neuronal differentiation
Day 0+: Neuronal differentiation
Full media change on day 3 post-plating, once debris is observed.
Pipet at the wall of the plate. DO NOT TOUCH BOTTOM OF PLATE! Be extra careful with 96 well plates.
Change Classic N2/B27 Differentiation media at minimum once every week without dox or rock inhibitor.
Closely monitor. Wait for a week at minimum before using for experiment. Preferably, use after 2 weeks.