Derivation of a Human Brain Organoid with Microglia Development

Summary

We present a protocol to generate a human brain organoid with resident microglia by incorporating Induced pluripotent stem cell (iPSC)-derived hematopoietic progenitor cells (HPCs) into organoid development.

Abstract

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

Introduction

Microglia are residential immune cells in the brain, playing critical roles in both brain development and homeostasis^1,2. The activation of microglia results in the production of proinflammatory factors, elevated phagocytosis, and reactive oxidative stress, which removes the invading pathogens and compromised cells. However, overactivation or prolonged activation of microglia may, on the other hand, cause neurodegeneration as a mechanism of pathogenesis in many neurological disorders, including Parkinson's disease^3,4. It is important that microglia....

Protocol

The original blood samples from healthy adult donors were collected at the Transfusion Medicine Blood Bank of the NIH, and signed informed consent forms were obtained in accordance with the NIH Institutional Review Board.

1. Producing hematopoietic progenitor cells (HPCs) from human iPSCs

NOTE: Human iPSC cells 510 and 507 were used to produce the representative results. The methods of generation and maintenance of the iPSCs can be found in a previous.......

Discussion

Here, a detailed protocol for making 3D neural organoids containing innate microglia from EBs derived from mixed iPSCs and iPSC-differentiated HPCs is presented. It is a relatively short and easy approach involving only cell culture techniques and equipment generally available in most laboratories.

The most critical factor for the success of this protocol is the quality of HPC differentiation. We adopted the published method¹⁷ using a commercial kit to differentiate HPC.......

Materials

Name	Company	Catalog Number	Comments
12 well cell culture plates	Corning	#3512
24 well cell culture plate	SARSTEDT	#83.3922
Accutase	Thermo	A1110501
Aggrewell 400 plate	Stemcell technologies	#34411	Referred to as microwell culture plate
Alexa Fluor 488 goat anti-mouse antibody	Life techniologies	A11001	1:400 dilution
Alexa Fluor 594 goat anti-rabbit antibody	Life techniologies	A11012	1:400 dilution
Allegra X-30R Centrifuge with rotor S6069	Beckman Couler
Anti- Adherence  Rinsing solution	Stem Cell Technologies	#07010
anti-CD34 antibody	Stem Cell Technologies	#60013	1:100 dilution
anti-Human CD43 antibody	Stem Cell Technologies	#60085	1:100 dilution
anti-IBA1 rabbbit antibody	Fujifilm	019-19741	2.5 µg/mL
anti-TREM2  rat pAb	RD Systems	mab17291	2.5 µg/mL
Antibiotic-antimycotic	Gibco	15240-062	1x
B27 supplement	Life technologies	17504-044	1x
bFGF	Peprotech	100-18B	20 ng/mL
CD200	Novoprotein	C311	100 ng/mL
CryoTube vials	Thermo	#368632
CX3CL1	Peprotech	300-31	100 ng/mL
DAPI	Sigma	D9542	1 µg/mL
DMEM/F12	Life technologies	12400-024	1x
DMSO	Sigma	D2650
DPBS	Gibco	#4190136	1x
E8 Flex medium kit	Thermo	A2858501
EDTA	Mediatech	46-034-Cl	0.5 mM
EGF	Peprotech	AF-100-15	20 ng/mL
EVOS FL Auto Microscope	Thermo		Fluorescence microscope
FastStart Universal SYBR Green PCR master mix	Roche	#4913850001
Glutamax	Gibco	#35050079
Goat serum	Sigma	G9023	4%
IL-34	Peprotech	200-34	100 ng/mL
ImageXpress Micro Confocal	Molecular Devices
Knockout DMEM/F12	Gibco	#10829018
M-CSF	Peprotech	300-25	25 ng/mL
Matrigel	Corning	#354277	Basement membrane matrix (BMM)
Mouse anti-βIII-tubulin antibody	Promega	G712A	1:1000 dilution
Mr. Frosty container	Thermo	5100-0001
N2 supplement	Life technologies	17502-048	1x
Paraformadehyde	Sigma	P6148	4%
PSC Neural Induction Medium	Gibco	A1647801
Rock inhibitor Y27632	Stemcell technologies	#72304	1 mM stock
RT LTS 1000 ul pipette tips	RAININ	#30389218	for transferring organoids
STEMdiff Cerebral Organoid Kit	Stem Cell Technologies	#08570
STEMdiff Hematopoietic Kit	StemCell Technologies	#5310	Referred to as hematopoietic Kit
StemPro Neural Supplement	Gibco	A1050801	Referred to as neural supplement
TGF-β1	Peprotech	100-21	50 ng/mL
Total RNA Purification Plus Kit	Norgen	#48400
TritonX-100	Sigma	T9284	0.10%
Visikol Histo-Starter Kit	Visikol	HSK-1	Contains organoid clearing solution HISTO-M, washing buffer
Zeiss LSM 510-META Confocal Microscope	Zeiss