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 are included in the relevant models for studying human neurological disorders. In recent years, human stem cells have been used to develop 3D organoids as in vitro models as an alternative to animal models and human subject studies⁵. Ideally, human organoids constitute multiple cell types and tissue structures similar to the corresponding human organs, better representing human physiology and pathogenesis than animal models but without the ethical concerns involved in studies of human individuals directly. They may represent the future of human disease modeling for the study of pathogenesis and drug development and for the guidance of individualized therapies⁶. As an example, 3D human brain organoids derived from human induced pluripotent stem cells (iPSCs) have prevailed in the field of neuroscience research, modeling neural infectious diseases including ZIKA, SARS-CoV-2⁷, and neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease^8,9. However, conventional 3D neural organoids using dual-SMAD inhibition to induce neuronal differentiation¹⁰ produce brain organoids lacking microglia, as they are derived from progenitors recruited from the blood instead of the neuroectoderm lineage that neurons are from^11,12. Without the presence of microglia, the organoids are inadequate to model CNS infections, inflammation, and the associated neurodegeneration.

To address this critical issue, attempts have been made to incorporate differentiated microglia into the brain organoids¹³ or inducing microglial differentiation within organoids from the beginning using alternative approaches instead of dual-SMAD inhibition¹³. However, by incorporating differentiated microglia into the brain organoids, the early interactions between neuronal and microglial development are missed. This could be important in CNS development or the pathogenesis of neuroinfectious disorders targeting infant brain development, such as in ZIKA virus infection¹⁴. On the other hand, differentiating innate microglia within iPSC-derived brain organoids without intermittent stages involves a prolonged process and has higher variability within the end products¹⁵. In this reported protocol, we incorporated the iPSC-derived hematopoietic progenitor cells (HPCs) into the iPSCs to make embryoid bodies (EBs), which were further differentiated into 3D organoids including both neurons and microglia.

Our protocol provides an easy approach that can be adopted to study the human central nervous system involving early neuron-microglial interactions and the pathogenesis of neural infectious disorders and neuroinflammation involving microglial activation.

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 publication¹⁶.

1. Day 0: Coat a 12-well cell plate by adding 500 μL/well of ice-cold Matrigel (basement membrane matrix [BMM]) solution diluted in DMEM/F12 medium and incubate it for at least 30 min at room temperature (RT).
2. Remove the coating supernatant completely and replace it with 1 mL of E8 Flex medium per well.
3. Check the iPSC culture in a 6 well plate under the microscope to confirm the iPSCs are of high quality and without signs of differentiation. Choose a middle-sized colony by marking it with a marker pen underneath the bottom of the plate.
4. Remove the medium from the iPSC culture and add 500 μL of ethylenediaminetetraacetic acid (EDTA) iPSC dissociation buffer (0.5 mM EDTA, 0.45 g/L NaCl in DPBS) to the well.
5. Observe under a microscope for signs of cell dissociation when three to four rows of cells from the edges of the colony start to shrink, with empty space appearing between cells. This usually takes 1-3 min.
6. Discard the EDTA solution completely. Dislodge the marked iPSC colony by pipetting 1 mL of E8 Flex medium forcefully and directly onto it. Ensure that the colony detaches completely into cell patches containing 20-50 cells each after 1-3 times of pipetting.
7. Collect the supernatant containing cell patches and add it to the first well of the coated wells in the 12-well plate.
8. Mix the well by pipetting one or two times and transfer 1 mL of the cells into the second well that contains 1 mL of medium.
9. Repeat step 1.8 twice to make serial diluted iPSC cultures in a total of four wells.
10. Incubate the plate in a 37 °C, 5% CO2 incubator.
11. After 24 h, on day 1, check the colonies under the microscope and count the numbers of colonies by going through all the fields in a well continuously. Choose one well that contains 10-20 iPSC colonies.
12. Replace the medium with 1 mL of medium A from the hematopoietic Kit. Put the plate back into the incubator for 48 h.
13. On day 3, remove 500 μL of spent medium and add 500 μL of fresh medium A.
14. On day 4,observe under the microscope and notice significant cell growth and differentiation from the iPSC colonies. Replace the medium with 1 mL of medium B.
15. Monitor the cell differentiation under the microscope and do half-medium change with 500 μL of Medium B every other day. The HPC-like cells appear on days 6-7.
16. On day 10, observe under the microscope bright single round cells with the morphology of normal HPCs floating in the medium or loosely attached to the single bottom layer of flat cells, with some loose cell aggregates.
    NOTE: The differentiated HPCs are ready for collection for characterization or further experiment.
17. Collect all the differentiated HPCs by pipetting up and down three times using a 1 mL pipette tip to break the cell aggregates and detach the HPCs from the plate surface.
18. Add the cells into a 15 mL tube and centrifuge the cells at 300 x g for 5 min. Remove the supernatant and resuspend the cell pellet into 1 mL of medium B.
19. Count the cells and adjust the concentration to 1 million cells/mL. At this stage, more than 1 million of HPCs are generated. When checked with flow cytometry, ensure that the purity is more than 85% of cells with CD34+/CD43+. Ensure no significant dead cells (more than 5%) are noticed.

2. Developing embryoid bodies from mixed iPSCs and HPCs

1. On the same day of collecting the HPCs, make sure an iPSC culture also reaches 80% confluence in one well in a 6-well plate (or two wells in a 12-well plate).
2. Treat one well of the microwell culture plate for EB formation with 500 µL of anti-adherence rinsing solution by adding it to minimize bubbles.
3. Spin the plate at 2000 x g for 5 min in a swinging bucket rotor fitted with plate holders to remove possible bubbles.
4. Remove the rinsing solution completely by pipetting and wash the wells twice with 1 mL of DPBS and then 1 mL of DMEM/F12 without generating bubbles.
5. Dissociate the iPSCs from the plate by treating the cells with 1 mL of Accutase solution.
6. Observe under the microscope until the cells show signs of separation but are still attached to the bottom. This usually happens within 1-3 min of Accutase treatment. Do not over-digest the iPSCs.
7. Remove the Accutase solution completely without disturbing the cells and add1 mL of DMEM/F12 medium to the wells. Further, dissociate the cells into single cells by pipetting up and down a couple of times using a 1 mL tip.
8. Collect the cells into a 15 mL tube and fill it to 5 mL with DMEM/F12 medium. Spin the cells at 300 x g for 5 min.
9. Remove the supernatant by pipetting and resuspend the cells in 1 mL of E8 Flex medium. Count the cells and adjust the cell concentration to 1 million cells per mL in the E8 Flex medium.
10. Mix iPSCs with HPCs in a ratio of 2:1 by adding 1 million of iPSCs (1 mL) and a half million of HPCs (500 µL) together. Add all the mixed cells into the previously treated well of the microwell culture plate.
11. Add 1 µL of Rock inhibitor Y27632 stock solution (1 mM) per 1 mL medium into the supernatant. Shake the plate from side to side a few times to distribute the cells evenly.
12. Spin the microwell culture plate at 300 x g for 5 min in a swinging bucket rotor fitted with plate holders. Observe under the microscope to see if the cells have settled into the microwells. (Supplementary Figure 1A).
13. Without disturbing the cells, put the plate in a 37 °C, 5% CO₂ cell incubator.
14. On day 2, 48 h later, observe the cells under the microscope for the formation of EBs. When the EBs are clearly formed, continue to the next step (Supplementary Figure 1B).
15. Treat a 24-well cell culture plate with anti-adherence rinsing buffer (500 µL) for 15 min to make a low attachment plate.
16. Remove the rinsing buffer completely and wash the wells twice with 1 mL of DPBS, then add 1 mL of E8 Flex medium into the wells.
17. Resuspend the EBs in the microwell culture plate by pipetting them with a 1 mL wide orifice pipette tip for a few times.
18. For each well of the low adherence treated 24 well plates, collect and add 100 µL of medium containing EBs. This will result in 10-20 EBs per well. Add E8 Flex medium to the old well containing the leftover EBs as backups.
19. Incubate the plate in a 37 °C, 5% CO₂ cell incubator for 48 h.

3. 3D neural organoid induction, proliferation, and maturation

1. On day 2 after EB formation, thaw aliquots of BMM on ice for at least 30 min.
2. Use a pre-chilled 20 µL tip, and add 15 µL ice-cold BMM drop wisely on top of the medium to coat the EBs. This makes a film of BMM that EBs will attach and grow onto.
3. Put the plate in a 37 °C, 5% CO₂ cell incubator.
4. On day 4, after the EB formation, remove 500 µL of medium carefully without discarding EBs and repeat BMM coating by following step 3.2.
5. Immediately start neural induction by adding 500 µL of PSC Neural Induction Medium on top of cells.
6. On days 6 and 8, perform half medium change by carefully removing 500 µL of medium without touching cells and adding 500 µL of neural induction medium.
7. On days 10, 12, and 14, perform half medium change with neural stem cell (NSC) medium: Knockout DMEM/F12 + 1x Glutamax supplement + 1x Neural supplement + bFGF + EGF.
8. On day 15, transfer the whole well of spheres with medium to a new 12-well plate treated with anti-adherence rinsing solution as previously described. Add 500 µL of neuronal maturation medium (DMEM/F12 + 1x N2 supplement + 1x B27 supplement + 1x antibiotics-antimycotic) on top of the culture.
9. Perform half-medium change with neuronal maturation medium every other day.
10. During the stage of maturation, if there are signs of nutrition depletion, such as color change of medium, move the organoid to 6 well plates, which hold up to 3 mL of medium per well.
11. Optionally, 17 days after the EB formation, supplement the maturation medium with cytokines (IL-34, M-CSF, TGF-β1, CD200, CX3CL1) for 6 days to facilitate microglial maturation.
12. On day 23, collect the resulting neural organoids for characterization or further experiments.

4. Clearance and immunostaining of 3D neural organoids

1. Fix up to 10 organoids by immersion in 1 mL of 4% paraformaldehyde (PFA) at 4 °C for 24 h.
2. Transfer the organoids per well to a 96-well clear bottom plate, and wash with 200 µL of DPBS 2 times for 1 h each time with gentle shaking.
3. Permeabilize organoids by washing them once in 50% methanol in DPBS, 80% methanol in double distilled (dd) water, and 100% dry methanol for 10 min each, at 4 °C with gentle shaking.
4. Wash the organoids serially in 20% DMSO/methanol, 80% methanol in dd water, 50% methanol in DPBS, in 100% DPBS, then in DPBS with 0.2% Triton X-100, for 10 min each at 4 °C with gentle shaking. Make an effort to remove the residual buffer completely after each washing.
5. Incubate in permeabilizing buffer (DPBS with 0.2% Triton X100, 0.3 M glycine, and 20% DMSO) with gentle shaking for 40 min at RT.
6. Block in blocking buffer (DPBS with 0.2% Triton X100, 6% Goat serum, and 10% DMSO) for 1 h at 37 °C with gentle shaking on a shaker at 80 rpm.
7. Incubate with antibodies in antibody dilution buffer (IBA1 1:100, TREM2 1:100, βIII-tubulin, 1:1000, 100 µL /well in DPBS with 0.2% Tween 20, 100 µg/mL Heparin, 3% Goat serum and 5% DMSO) at 37 °C for 2 h or in cold room with gentle shaking at 80 rpm for 3 days.
8. Wash with DPBS with 0.2% Tween 20, 100 µg/mL Heparin 5 times, 10 min each at RT with gentle shaking on a shaker at 80 rpm.
9. Incubate with secondary antibody (1:200 goat anti-mouse Alexa488) at 37 °C for 1 h followed by RT overnight with gentle shaking on a shaker at 80 rpm.
10. For nuclear contrast staining, perform DAPI staining together with antibody incubation or in a washing buffer.
11. Wash the organoids 10 times in washing buffer, 10 min each time at 37 °C, with gentle shaking.
12. Observe under a microscope. If the background is still high, keep washing it 5 times.
13. Discard the supernatant as much as possible. Add 200 µL of organoid clearing solution and incubate it for 5 min before observation and take images using a confocal microscope.

Results

Our protocol follows a scheme to differentiate HPCs from iPSCs and then mix the HPCs with iPSCs to make EBs, followed by neural induction, differentiation, and maturation (Figure 1). High quality of HPC differentiation is critical for the success of EB formation and later organoid differentiation. A serial dilution culture technique is used to produce the appropriate numbers and size of iPSC colonies to start the HPC differentiation (Figu...

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