Jun 28, 2023

Public workspaceiNIL/iNIP transcription factor-induced motor neuron differentiation v2 (Mazzoni lab)

This protocol is a draft, published without a DOI.
  • Dylan E. Iannitelli1,
  • Hoa (Emma) Nguyen2,1,
  • Albert Tan1,
  • Esteban O. Mazzoni1
  • 1Department of Cell Biology, NYU Grossman School of Medicine, New York, NY 10016, USA;
  • 2Department of Biology, New York University, New York, NY 10003, USA
Emma Nguyen
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Protocol Citation: Dylan E. Iannitelli, Hoa (Emma) Nguyen, Albert Tan, Esteban O. Mazzoni 2023. iNIL/iNIP transcription factor-induced motor neuron differentiation v2 (Mazzoni lab). protocols.io https://protocols.io/view/inil-inip-transcription-factor-induced-motor-neuro-cwfuxbnw
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: June 28, 2023
Last Modified: June 28, 2023
Protocol Integer ID: 84180
Keywords: Differentiation, Motor Neurons, Cranial motor neurons, Spinal motor neurons, Transcription factor, iPSC, Stem cell
Funders Acknowledgement:
CZI New roles of the proteasome in preventing neurodegeneration
Grant ID: 2020-222014 (5022)
CZI Chromatin encoding of repeat expansion in neurodegeneration
Grant ID: CP2-0000000013
NIH A comparative inter-neuronal and inter-species platform to understand neuronal differential sensitivity to neurodegeneration
Grant ID: R21 AG067174
Abstract
Protocol overview (Day 0 - Day 28)
Attachments
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iNIL iNIP motor neur...
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Guidelines


Expected result
hNIL differentiation (Day 8-28)


hNIP differentiation (Day 8-28)





Materials


Before start
Preparing base human motor neuron media (hMNM)
  • 1X Neurobasal Media 500mL
  • 50X B27 10mL
  • 100X N2 5mL
  • 100X L-Glutamine 5mL
Thawing and maintaining human iPSCs prior to differentiation
Thawing and maintaining human iPSCs prior to differentiation
Preparing Matrigel


Thaw an aliquot of 500 μL of Matrigel TemperatureOn ice in the Temperature4 °C fridge for a few hours

Dilute aliquot 1:100 in cold Neurobasal Media

Coat plates overnight with diluted Matrigel at Temperature37 °C

Note
Make sure the entire surface of the plate is covered.


Thaw vial of cells from liquid nitrogen by placing the vial in a Temperature37 °C bath until partially thawed
Note
Avoid letting the cells thaw completely.


Add Amount1 mL of pre-warmed E8 + Y-27632 (10 μM) media into the partially thawed vial and transfer cells to a 15 mL tube

Add an additional Amount1-2 mL of pre-warmed E8 + Y-27632 (10 μM) media into the 15 mL tube

Spin at Centrifigation900 rpm for 4 minutes
While waiting for the spin to complete, prepare plate
Aspirate Matrigel from coated plate

Note
!! DO NOT LET THE PLATE DRY OUT

Wash plate with 1X PBS
Aspirate 1X PBS, and add the appropriate amount of pre-warmed E8 + Y-27632 (10 μM) media
At the end of spin, aspirate media and resuspend cell pellet in 1 mL of E8 Media + Y-27632 (10 μM)
Seed directly into E8 Media + Y-27632 (10 μM)
Change media the next day to E8 with no Y-27632 (Rock inhibitor)

Note
Removing Rock inhibitor will result in a change in morphology of iPSCs from "spiky" looking cells to smoother cells.

Change media with E8 every other day until iPSCs are near confluent and ready for re-replating and differentiation
Day 0
Day 0
Coat plates with Matrigel as described above
Aspirate media and gently wash iPSCs with 1X DPBS
Harvest iNIL/iNIP iPSCs

Aspirate 1X PBS and dissociate iNIL/iNIP iPSCs by adding Accutase
Neutralize Accutase with E8 Media + Y-27632 (10 μM) 2 times the amount of Accutase added (eg. If 2 mL of Accutase was used, resuspend in 4 mL of E8 Media + Y-27632 (10 μM))
Transfer resuspended cells to a 15 mL tube
Spin at Centrifigation900 rpm for 4 minutes

Aspirate media and resuspend cell pellet in Amount3-5 mL of E8 Media + Y-27632 (10 μM)

Count cells
Seed directly into hMN media + Y-27632 (10 μM) + dox (3 μM)
  • Seed at 8,000 cells/cm2 (Example: 600,000 cells/10cm dish)
Note
Optional: Add antibiotic (ie. Penstrep) to hMN media to avoid contamination

Day 2
Day 2
Aspirate all media
Replenish with hMN media + Y-27632 (10 μM) + dox (3 μM), adding Retinoic Acid (RA)
  • iSpMNs: 1.0 μM RA
  • iCrMNs: 0.01 μM RA
Day 4
Day 4
Aspirate all media
Replenish hMN media + dox (3 μM) + RA (iSpMN 1.0 μM/iCrMN 0.01 μM), removing Y-27632
Day 6
Day 6
Carefully aspirate half of the total volume of media per well/dish
Replenish hMN media + dox (3 μM) + RA (iSpMN 1.0 μM/iCrMN 0.01 μM) + Adarotene (1 μM)
Day 8
Day 8
Carefully aspirate half of the total volume of media per well/dish
Replenish hMN media + dox (3 μM) + RA (iSpMN 1.0 μM/iCrMN 0.01 μM) + Adarotene (1 μM) + supplements:
  • BDNF (10 ng/mL)
  • GDNF (10 ng/mL)
  • CNTF (10 ng/mL)
  • L-ascorbic acid (200 ng/mL)
Day 10 + (for a healthy culture, change media every 4-5 days)
Day 10 + (for a healthy culture, change media every 4-5 days)
Carefully aspirate half of the total volume of media per well/dish
Replenish hMN media + dox (3 μM) + RA (iSpMN 1.0 μM/iCrMN 0.01 μM) + Adarotene (1 μM), + supplements:
  • BDNF (10 ng/mL)
  • GDNF (10 ng/mL) 
  • CNTF (10 ng/mL)
  • L-ascorbic acid (200 ng/mL)
Optional: adding maturation cocktail (GENtonik)
  • GSK (1 μM)
  • EPZ (1 μM)
  • NMDA (1 μM)
  • BayK (1 μM)