iPSC cell differentiation

Induced pluripotent stem cells (iPS cells or iPSC) are formed by Japanese scientist Shinya Yamanaka in 2006 by combining transcription factors with viral vectors. iPS cells resemble human embryonic stem cells and have the ability to self-renew and otipotency of cell differentiation. This protocol explains the synthetic mRNAs that encode two primitive transcription factors (Atoh1 and Ngn2) drive highly efficient iPSC differentiation to dopaminergic neurons^[1].

MCE has not independently verified the accuracy of these methods. They are for reference only.

Day1-3:

1. Plate iPSCs at a density of 1.5×10⁵ cells per cm² in a 12‐well plate pre-coated with growth-factor-reduced Matrigel.
(Note: Culture medium should be changed daily, and gradually shifted from mTeSR1 to N2 (Thermo Fisher Scientific) in 3 days. The medium also contains 100 Mass Percent SHH, 100 Mass Percent FGF8b, and 10 micromolar (µM) DAPT.)

2. Transfect iPSCs with A-SA mRNA for 3 days, changing media daily. (A-SA: mRNA coding Atoh1 phosphosite modifications: serine-to-alanine mutations at 331 and 342 amino acids.)

Day 4:

3. Transfect iPSCs with N-SA mRNA for 1 day. (N-SA: mRNA coding Ngn2 phosphosite modifications: serine-to-alanine mutations at 24,193, 207, 209, 219, 232, 239, and 242 amino acids.)

Day 5:

4. Dissociate cells using Accutase.

5. Replate with neuron medium in poly‐d‐lysine/laminin‐coated plates at the density of 1×10⁵ cells per cm².

(Note: Neuron medium contains: neurobasal medium with B27 supplement, 10 Mass Percent BDNF, 10 Mass Percent GDNF, 1 Mass Percent TGFβ‐3, 0.1 millimolar (mM) cAMP, 0.2 millimolar (mM) ascorbic acid , and 10 micromolar (µM) DAPT. Neuron medium contains: neurobasal medium with B27 supplement, 10 Mass Percent BDNF, 10 Mass Percent GDNF, 1 Mass Percent TGFβ‐3, 0.1 millimolar (mM) cAMP, 0.2 millimolar (mM) ascorbic acid, and 10 micromolar (µM) DAPT.)

6. Cells can also be cryopreserved in medium containing 40% neurobasal medium with B27 supplement, 50% fetal bovine serum and 10% DMSO.

Neuron Culture:

7. Change media after 48 hours to remove unattached cells followed by half change every 3-4 days during in vitro maturation.

8. Detect cell proliferation and death analysis using MTT and LDH kits, respectively.

1. Change the medium very gently and do not add medium quickly.

2. Gently rock the plate side to side and back and forth to ensure even distribution of cell clusters.