Differentiation of Induced Pluripotent Stem Cells into Neural Progenitor Cells

Abstract

Source: Phillips, A. W., et al. Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays. J. Vis. Exp. (2017).

The video demonstrates a technique for differentiating human induced pluripotent stem cells (hiPSCs) into neural progenitor cells (NPCs). Initially, the hiPSCs are cultured on a feeder layer of mouse embryonic fibroblasts (MEFs). Subsequently, the cells are detached and transferred to a biopolymer-coated plate to allow the MEFs to adhere to and separate from the hiPSCs. The suspended hiPSCs are then transferred to a V-bottom plate and centrifuged to form aggregates. Finally, the addition of a differentiation medium induces the differentiation of hiPSCs within the aggregate into NPCs.

Protocol

1. Generation of Neural Progenitor Cells

1. Maintain hiPSCs derived from fibroblasts and peripheral blood mononuclear cells (PBMCs) in 6-well plates on a γ-irradiated mouse embryonic feeder (MEF) cell layer in human iPSC medium supplemented with small-molecules.
   NOTE: Daily maintenance is a modified version of previously reported procedures.
   1. Plate 6 x 10⁵ MEFs onto each well of a 6-well tissue culture grade plate in 300 μL/well recommended medium (following manufacturer's protocol).
   2. After 48 h, replace MEFs medium with 300 μL/well hiPSC medium for 1 h before adding hiPSCs.
   3. Quickly thaw hiPSCs in a 37 °C water bath and slowly add 1 mL hiPSC medium dropwise. Transfer the hiPSCs and medium to a 15 mL conical tube. Add medium to bring the total volume to 5 mL. Centrifuge for 4 min at 129 x g, aspirate the supernatant, gently resuspend the pellet in 1 mL hiPSC medium with Rho-associated protein kinase (ROCK) inhibitor at 1:1,000 concentration.
   4. Plate the cell suspension (typically seeded at 300,000 cells/well) onto the MEF cell layer and incubate at 37 °C, 5% CO₂, 95% humidity.
   5. Monitor iPSC culture closely using a light microscope. After 48 h, remove cultures that have uneven edges, have grown to become cystic-like or appear yellowish-brown under the light microscope to minimize spontaneous differentiation.
      NOTE: After 7 - 10 days, hiPSC colonies should form. Colonies should be round, with clearly defined borders and uniform cell densities, and devoid of loosely packed non-uniform cells.
   6. Manually select round hiPSC colonies to clear the culture of spontaneously differentiated cells. Expand the colonies by placing them on fresh MEF layers. Expand 1:3 every 7 days when cultures near confluency and freeze.
2. After expansion and freezing cells (for backup), grow hiPSC colonies on plates until they reach 50 - 75% confluence.
3. Seven-days post plating, use mild enzymatic treatment (5-10 min with 300 μL of enzyme solution) and gentle trituration (2-4 times with a 1,000 µL pipet) to harvest and prepare hiPSCs for neural precursor cell differentiation.
4. Centrifuge the cell suspension at 129 x g for 4 min, aspirate the supernatant, and resuspend the pellet in 5 mL human iPSC medium. Transfer the cell suspension to a 0.1% gelatin coated 5 cm cell culture dish for 1 h at 37 °C to eliminate MEFs and to maximize the hiPSC yield. Transfer the medium (with non-adherent cells) to another 5 cm gelatin coated dish.
5. Transfer the non-adherent cells to a 15 mL centrifuge tube using a transfer pipet. Gently rinse the plates with 3 mL medium and add it to the 15 mL tube.
6. Centrifuge the cell suspension at 129 x g for 4 min, aspirate the supernatant, and gently resuspend the pellet in medium. Determine the cell count using an automated cell counter. Plate 9,000 cells/well in a 96-well low-adhesion V-bottom plate.
7. Centrifuge the plate at 163 x g for 3 min. Incubate the plate at 37 °C, 95% humidity and 5% CO₂. Using a pipette, change 50% medium every other day by removing half of the medium and replacing it with fresh chemically-defined differentiating medium 1 (DM1; Figure 1; Table of Materials) for 14 days.

Results

Figure 1: Schematic Outlining the Early Steps in Preparing SFEBs. After iPSCs have been harvested using an enzymatic dissociation, they are plated in 96-well plates and centrifuged at 163 x g for 3 min to induce quick aggregation. After 14 days in vitro (DIV) cell aggr...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SFEB Neuronal Differentiation Cell culture Media. Reagents. Components
STEMdiff Neural Induction Medium (hiPSC Media)	STEMCELL Technologies	0-5835	250ml
PluriQ ES-DMEM Medium (MEF Media)	GlobalStem	GSM-2001
DM1 Media Components
D-MEM/F-12 (1X), Glutamax liquid, 1:1	Invitrogen	10565018	385ml
Knockout Serum Replacement	Invitrogen	10828028	20% 100ml
Pen/Strep	Invitrogen	15140122	5ml
Glutamax 200mM	Invitrogen	35050061	5ml
MEM Non-Essential Amino Acids Solution 10 mM (100X), liquid	Invitrogen	11140050	5ml
2-Mercaptoethanol (1,000X), liquid	Invitrogen	21985023	900ul
Small Molecules
Y27632 (ROCKi)	Stemgent	04-0012-10	10uM
Components/Materials
Mouse Embryonic Fibroblasts	GlobalStem	GSC-6301G
96 well V bottom w/Lids	Evergreen	222-8031-01V
StemPro Accutase Cell Dissociation Reagent	ThermoFisher	A1110501
6 well flat bottom	Falcon	353046