Cell differentiation

Directed neuronal differentiation of human induced pluripotent stem cells (ipscs) or neural progenitors using transcription factors can achieve rapid and highly reproducible differentiation of mature and functional neurons. Exogenous expression of the transcription factor Neurogenin-2(NGN2) has been widely used to induce the generation of different neuronal cells, which have been used in neurodevelopmental studies, disease modeling, drug screening and neuronal replacement therapy. This protocol describes the differentiation of iPSCs with stably integrated doxycycline-inducible Ngn2 (such as i3Ns)^[1].

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

• Methods

1. Culture iPSCs

1.1. Culture iPS cells with ROCK inhibitor only when plating or when colonies constitute fewer than about 20 cells.

1.2. Generally, change the media every other day, unless cells are more than 50% confluent, change StemFlex every day.

1.3. Passage cells when 60-75% confluent. Should never be over 90% confluent.

2. Days -3-0: Pre-differentiation.

2.1. Coat plate with matrigel diluted in Knockout DMEM. Coat for at least 30 minutes (or O/N). Coated plates can last in the 37℃ incubator for 14 days.

2.2. Aspirate media from iPSCs and wash with DPBS.

2.3. Add accutase and incubate at 37℃ for 3 minutes; if necessary, incubate for an additional time up to 7 minutes.

2.4. Use gentle agitation to release cells, and collect them in an Eppendorf tube with DPBS.

2.5. Spin cells at 200 x g for 5 minutes, resuspend in N2 pre-diff media.

2.6. Count cells and add desired amount to an Eppendorf tube, spin, resuspend in N2 pre-differentiation media, and plate onto Matrigel-coated cell culture vessels for the pre-differentiation.

2.7. Predifferentiated medium was half-changed daily during day 0-3 of predifferentiation, or full change was performed on day 1 or day 2.

2.8. Going into differentiation, it is best if cells are in single-cell suspension. Optional: use a cell strainer.

2.9. Pre-differentiated cells can be frozen in 10% DMSO + N2/B27 Differentiation media (Can also freeze in pre-diff media) .

3. Day 0: Releasing and Plating Pre-Differentiated neurons.

3.1. Aspirate media and wash with DPBS.

3.2. Add accurate and incubate at 37℃ for 3 minutes; if necessary, incubate for an additional time up to 7 minutes.

3.3. Use gentle agitation to release cells, and collect them in an Eppendorf tube or conical with DPBS.

3.4. Spin cells at 200 x g for 5 minutes.

3.5. Carefully aspirate DPBS/accutase solution and resuspend in Classic N2/B27 Differentiation Media

3.6. Count cells, dilute them to appropriate density, and plate them onto PDL-coated cell culture vessels.

3.7. Leftover cells can be frozen down in pre-differentiation media + 10% DMSO.

4. Day 0+: Neuronal differentiation.

4.1. Full media change on day 3 post-plating, once debris is observed.

4.2. Pipet at the wall of the plate. Do not touch the bottom of the plate.

4.3. Change Classic N2/B27 Differentiation media at minimum once every week without dox or rock inhibitor.

4.4. Closely monitor. Wait for a week at minimum before using it for the experiment. Preferably, use after 2 weeks.

1. Keep reagents at 4℃ 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. Your cells must be single cells.