Generating Brain Endothelial Cells from Induced Pluripotent Stem Cells

Abstract

Source: Endres, L. M., et al., Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells. J. Vis. Exp. (2020)

This video demonstrates a protocol to induce the differentiation of induced pluripotent stem cells into brain endothelial cells.

Protocol

1. Preparation of materials required for iPSC culture and BEC differentiation.

1. Matrix coating of tissue culture (TC) plastic for IMR90-4 iPSC culture
   1. Aliquot basement membrane matrix gel (e.g., Matrigel) into 2.5 mg aliquots and store at -20 °C.
      NOTE: Work quickly when handling the matrix gel and aliquot on ice, as it forms a gel above 4 °C and cannot be aliquoted once it has solidified.
   2. For coating TC plastic, quickly add one aliquot of matrix gel to 30 mL of Dulbecco's Modified Eagle Medium (DMEM)/F12 medium in a 50 mL conical tube. Add 1 mL of the medium onto the frozen matrix ge....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Accutase (1x)	Sigma	A6964
All-trans retinoic acid (RA)	Sigma	R2625
b-Mercaptoethanol	Merck (Sigma-Aldrich)	805740
Cell culture plates and flasks	Sarstedt
Centrifuge (Heraeus Megafuge 1.0R)	Thermo Scientific
Class II biosafety cabinet	Nuaire	NU-437-400E
Carbon dioxide incubator (DHD Autoflow Carbon dioxide Air-Jacketed Incubator)	Nuaire
Collagen IV	Sigma	C5533
Costar Transwell polyester filters (12- or 24-well)	Corning	3460, 3470
Fibronectin	Sigma	F1141
Hemacytometer (Neubauer)	A. Hartenstein	ZK06
Human basic fibroblast growth factor (bFGF)	PeproTech	100-18B
Human Endothelial Serum Free Medium (hESFM)	Gibco	11111-044
Inverted microscope (Wilovert)	Hund (Will Wetzlar)
iPS(IMR90)-4 cells	WiCell
Knockout serum replacement (KOSR)	Gibco	10828-028
L-glutamine (GlutaMAX)	Invitrogen	35050-038
Matrigel Matrix	Corning	354230
Nonessential amino acids (NEAA)	Gibco	11140-035
StemFlex basal medium + 50x StemFlex supplement	Gibco	A3349401	Stem-cell maintenance medium
Swinging Bucket Rotor (Heraeus #2704)	Thermo Scientific