Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Summary

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

Abstract

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Introduction

The blood-brain barrier (BBB), and the meningeal blood-CSF barrier (mBCSFB) are extremely tight cellular barriers that separate the circulation from the central nervous system (CNS) and are primarily comprised of highly specialized brain endothelial cells (BECs)^1,2. Together, BECs maintain proper brain homeostasis by regulating nutrients and waste products in and out of the brain, while excluding many toxins, drugs, and pathogens^1,2. Bacterial meningitis occurs when blood-borne bacteria are able to interact with, and penetrate the barrier formed by BEC....

Protocol

NOTE: All media / reagent preparation, stem-cell maintenance, and differentiation steps are adapted from Stebbins et al.²².

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 matri....

Results

The protocol described here is adapted from Stebbins et al. and highlights the process to differentiate iPSCs into brain-like endothelial cells that possess BBB properties, and how to utilize this model for infection studies using iPSC-BECs with Nm^19,22. The iPSC-BECs, when differentiated properly, exhibit tight barrier properties measured by TEER that are often greater than 2000 Ω·cm², and express endothelial markers such as VE-cadherin and CD31 (PECA.......

Discussion

Modeling BECs and the BBB has had challenges, as primary and immortalized human BECs, in vitro, tend to lack robust barrier phenotypes. The advent of human stem cell technologies has allowed for the generation of iPSC derived BEC-like cells that retain expected hallmark BBB phenotypes such as endothelial markers, tight junction expression, barrier properties, response to other CNS cell types, and functional efflux transporters^12,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Accutase (1x)	Sigma	A6964	Enzymatic cell dissociation reagent
Acetic acid	Sigma	A6283
All-trans retinoic acid (RA)	Sigma	R2625
Anti-CD31 (PECAM-1)	Thermo Scientific (Labvision)	RB-10333
Anti-Claudin-5	Invitrogen	4C3C2
Anti-Glut-1	Thermo Scientific (Labvision)	SPM498 (MA5-11315)
Anti-Occludin	Invitrogen	33-1500
Anti-VE-cadherin	Santa Cruz	sc-52751
Anti-ZO-1	Invitrogen	33-9100
Bacto Proteose Peptone	BD	211684
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
CO2 Incubator (DHD Autoflow CO2 Air-Jacketed Incubator)	Nuaire
Collagen IV	Sigma	C5533
Columbia ager + 5 % sheep blood	Biomerieux	43049
Costar Transwell polyester filters (12- or 24-well)	Corning	3460, 3470
D(+)-Glucose	Merck (Sigma-Aldrich)	G8270
DAPI	Invitrogen	D1306
DMEM/F12	Gibco	31330-038
DMSO	ROTH	A994.1
Dulbecco's phosphate-buffered saline (DPBS)	Gibco	21600-069
Epithelial Volt-Ohm Meter (Millicell ERS-2) with STX electrode	Merck (Millipore)	MERS00002
Fe(NO3)3	ROTH	5632.1
Fibronectin	Sigma	F1141
Fluoresence microscope (Eclipse Ti)	Nikon
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
Kellogg's supplement			To prepare 110 ml of Kellogg's supplement, prepare 100 ml of 4 g/ml glucose, 0.1 g/ml glutamine, and 0.2 mg/ml thiamine pyrophosphate and 10 ml of 5 mg/ml Fe(NO3)3 and combine the solutions. Filter sterilize and store aliquoted at -20 °C.
Knockout serum replacement (KOSR)	Gibco	10828-028
L-glutamine (GlutaMAX)	Invitrogen	35050-038
LunaScript RT SuperMix Kit	NEB	E3010L	cDNA synthesis kit
Matrigel Matrix	Corning	354230
Methanol	ROTH	4627.5
MgCl2	ROTH	KK36.1
Micropipettes (Research Plus)	Eppendorf
NaHCO3	ROTH	6329
Nonessential amino acids (NEAA)	Gibco	11140-035
NucleoSpin RNA isolation kit	Machery-Nagel	740955	RNA isolation kit
Pipette boy (Accu-Jet Pro)	Brand
Platelet poor plasma-derived serum, bovine (PDS)	Fisher	50-443-029
PowerUp SYBR Green Master Mix	Applied Biosystems	A25742	qPCR master mix
qPCR film (MicroAmp Optical Adhesive Film)	Applied Biosystems	4211971
qPCR plates (MicroAmp Fast 96-well)	Applied Biosystems	4346907
ROCK inhibitor, Y27632 dihydrochloride	Tocris	1254
RT-PCR thermo cycler (StepOnePlus)	Applied Biosystems	4376600
Serological pipettes	Sarstedt
StemFlex basal medium + 50x StemFlex supplement	Gibco	A3349401	Stem-cell maintenance medium
Swinging Bucket Rotor (Heraeus #2704)	Thermo Scientific
Thiamine pyrophosphate	Sigma	C8754-5G
Trypan Blue Solution, 0.4%	Gibco	15250061
Versene	Gibco	15040-033	Non-enzymatic cell dissociation reagent (EDTA)