Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

Summary

Here we report a protocol to develop a three-dimensional (3-D) system from human induced-pluripotent stem cells (hiPSCs) called the serum free embryoid body (SFEB). This 3-D model can be used like an organotypic slice culture to model human cortical development and for the physiological interrogation of developing neural circuits.

Abstract

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not represent the underlying cytoarchitectural and functional complexity of the affected individuals carrying suspected disease variants. Conventional 2-D models remain incomplete representations of in vivo-like structures and do not adequately capture the complexity of the brain. Thus, there is an emerging need for more 3-D hiPSC-based models that can better recapitulate the cellular interactions and functions seen in an in vivo system.

Here we report a protocol to develop a 3-D system from undifferentiated hiPSCs based on the serum free embryoid body (SFEB). This 3-D model mirrors aspects of a developing ventralized neocortex and allows for studies into functions integral to living neural cells and intact tissue such as migration, connectivity, communication, and maturation. Specifically, we demonstrate that the SFEBs using our protocol can be interrogated using physiologically relevant and high-content cell based assays such as calcium imaging, and multi-electrode array (MEA) recordings without cryosectioning. In the case of MEA recordings, we demonstrate that SFEBs increase both spike activity and network-level bursting activity during long-term culturing. This SFEB protocol provides a robust and scalable system for the study of developing network formation in a 3-D model that captures aspects of early cortical development.

Introduction

We have previously reported a 3-D model system, generated from patient-derived human induced pluripotent stem cells (hiPSCs) that recapitulates some aspects of early cortical network development¹. This 3-D model, a serum-free embryoid body (SFEB), improves on previous simple aggregation hiPSC models^2,3. A growing body of work is revealing that 3-D structures like our SFEBs, approximate aspects of neural development commonly observed in vivo and at an earlier time point than observed in 2-dimensional (2-D)/monolayer hiPSC models^4,5. Initial studies have been focused on the self-organizing complexity of 3-D bodies without demonstrating their physiological complexity².

The protocol described here has been used on undifferentiated hiPSCs derived from fibroblasts and peripheral blood mononuclear cells (PBMCs). These cells are maintained on γ-irradiated mouse embryonic feeders (MEFs). These hiPSC colonies are manually cleaned of spontaneously differentiated cells, enzymatically harvested, and resuspended in medium containing Rho-Kinase inhibitor Y-27632 (ROCKi). Undifferentiated hiPSCs are subjected to dissociation and centrifugation before being transferred to 96-well low adhesion V-bottom plates. After plating, neural induction is initiated using dual SMAD inhibition (SB431542 and LDN193189 along with dickkopf 1 (DKK-1)) to drive an anterior-frontal forebrain neuronal-fate lineage⁶. After 14 days, the SFEBs are transferred to cell culture inserts in a 6-well plate. Once transferred, the round SFEBs begin to spread and thin, while maintaining local network connections as is often observed in hippocampal organotypic slice culture preparations using similar cell culture inserts^1,7.

The use of an SFEB based 3-D platform in this format is amenable to the efficient production of cortical networks that may be interrogated using cell based physiological assays such as calcium imaging or electrophysiological assays such as single cell recordings or multi-electrode array (MEA)¹. Although 3-D systems bear the markers of early cortical development, other studies have shown that these 3-D bodies may require longer incubation times to allow for the inherently slower pace of human tissue development⁸. This SFEB protocol successfully generates 3-D SFEBs from undifferentiated hiPSCs that captures aspects of the early development of the cortex.

The potential of SFEBs to model network aberrations in neurological disorders is a strength of this system. The hiPSCs derived from patient tissue can be grown into cells of the nervous system that are subject to assays relating to cell biology as well as concomitant gene expression. Human iPSCs are being used to ascertain the genetic profile of large groups of individuals with varying neurological disorders with complex etiologies such as autism spectrum disorder (ASD), schizophrenia⁹, Rett Syndrome¹⁰, and Alzheimer's disease^11,12. Until recently, iPSC models were typically monolayer preparations that, while proficient in evaluating molecular interactions, were inadequate in deciphering the complex cellular interactions seen in vivo. Animal models have been the default substitute for recreating the whole-organ platform. These animal models are plagued by poor translation of findings and have limited ability to replicate human genetic profiles identified by large genetic screening studies. Thus, the development of 3-D systems from iPSCs adds a needed layer of complexity in human disease modeling^13,14. The next step for 3D hiPSC platforms is to accommodate the large scale requirements of high throughput screening using cell based assays¹⁵.

Protocol

1. Generation of Neural Progenitor Cells

1. Maintain hiPSCs derived from fibroblasts and PBMCs in 6-well plates on a γ-irradiated mouse embryonic feeder (MEF) cell layer in human iPSC medium supplemented with small-molecules (see the Materials Table).
   NOTE: Daily maintenance is a modified version of previously reported procedures^1,16.
   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 re-suspend the pellet in 1 mL hiPSC medium with 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^1,16.
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.

2. Serum-Free Embryoid Body (SFEB) Induction (Figure 2)

1. Place 40 µm cell culture inserts into 6-well plates and add 1 mL DM1 medium at least 1 h prior to the addition of aggregates.
2. On day 14, transfer the aggregates with 20 µL medium using a 200 µL wide mouth tip onto 40 µm cell culture inserts and change the medium to DM2.
   NOTE: Cell culture inserts are specialized inserts that sit slightly off the bottom of the well and consist of a polytetrafluoroethylene membrane suspended across a plastic frame. This membrane is biocompatible and can efficiently sustain nutrient and oxygen transport to the SFEBs, which are placed on top. They are commonly used with organotypic hippocampal slice cultures taken from mouse or rat^7,17,18.
   1. Transfer 4-6 aggregates to one cell culture insert and allow adequate space between the aggregates. Remove as much excess solution as possible using a fine pipette tip (e.g. 200 μL tip).
      NOTE: It is acceptable to briefly disturb the SFEB as they will move on the cell culture insert as the solution in removed from around the SFEBs.
   2. Maintain the cultures in 1 mL of DM2 at 37 °C, 95% humidity and 5% CO₂. Using a pipette, change 75% medium every other day by removing three quarters of the medium and replacing with three quarters fresh medium. For example, remove 750 μL of medium and add 750 μL of fresh medium.
3. After 14 days of culture, switch to DM3 medium with 75% medium change every other day and for another 16 days.
4. To maintain the SFEBs on cell culture inserts beyond 30 days, change the medium every other day for 60, 90 and 120 days.
   NOTE: The SFEBs will grow to about 1,000 μm in diameter and are typically 100-150 μm thick¹.

3. Determining SFEB Composition

1. Detach the SFEBs from the insert by gently pipetting medium using a 200 µL wide mouth pipette tip. To transfer the SFEBs, use a wide mouth pipette tip to gently suction the bodies into the pipet tip. After loading SFEB into the pipet tip, gently transfer to 12-well plates and wash with 300 μL phosphate buffered saline (PBS) per well.
   NOTE: SFEBs will be suspended in solution in the 12-well plates. This is important to allow full penetration of the fixative, the blocking solution, and antibodies. If using multiple SFEBs for staining, up to 10 SFEBs can be added per well of a 12 well plate. This will conserve solutions and antibodies.
2. Fix SFEBs with 300 μL per well 4% paraformaldehyde at room temperature for 30-45 min. Wash fixed SFEBs twice with 300 μL PBS, 3-5 min each wash.
   Caution: Wear appropriate personal protective equipment when handling paraformaldehyde.
   NOTE: Probe for immunoreactivity to markers to determine maturity, cell type etc. For marking neurons in this study chicken-Tuj1 was used at 1:500, for additional markers please see Table 2 and references^1,16.
3. Permeabilize and block with 0.1% Triton-X100 in PBS and 10% normal donkey serum (blocking solution; 300 μL per well) for 1 h at room temperature.
4. Remove the blocking solution and treat SFEBs with 300 μL primary antibodies in blocking solution. Incubate at 4 °C overnight. Wash SFEBs thrice (3-5 min per wash) in 300 μL PBS containing 0.1% Triton-X.
5. Prepare secondary antibodies 1:1000 in blocking solution. Incubate SFEBs for 1 h at room temperature with secondary antibodies. Wash SFEBs with 300 μL PBS, 3 times for 3-5 min each.
   NOTE: At this stage SFEBs can be prepared for imaging.
6. To image, pipette SFEBs onto a glass slide using a wide-bore pipette. Remove excess solution around the SFEB using gentle suction.
   NOTE: For similar staining conditions, multiple SFEBs can be placed on the same glass slide.
7. Add a drop of mounting medium on the SFEBs, place a glass coverslip, and gently press to ensure that there are no air bubbles. Allow the mounting medium to harden and proceed to imaging and quantification (step 3.8).
   NOTE: After the mounting medium hardens, the SFEBs are ready for imaging.
8. Perform cell quantification by taking multiple regions of interest (ROI) across the SFEB and using a z-stack combined with a maximal projection image through each ROI on a standard confocal microscope as described in references^1,16.
   NOTE: Whole SFEBs can be quantified using image-based tiling (see references^1,16 for details). Since the SFEBs thin to approximately 100 μm, there is minimal scattering of light in the tissue and thus there is no need for 2-photon microscopy. Earlier uses of this protocol with fibroblast-derived iPSCs produced a SFEB fate map that revealed complex and diverse structure with subpopulations of interneurons with transcriptional identities that resembled CGE and anterior forebrain fates^1,16.

4. Recording Neuronal activity in SFEBs Using MEAs

1. Prepare 12-well MEA plates as per manufacturer's instructions.
2. Wash the MEA plates 3 times for 5 min with sterile H₂O under aseptic conditions to clean. Wash for 5 min with 75% ethanol and then with 100% ethanol to sterilize the plate.
3. Bake the plate inverted in an oven for 4-5 h at 50 °C to complete the sterilization process.
4. Add 500 µL 0.2% polyethyleneimine solution (PEI) to each well and incubate 1 h at room temperature. Aspirate PEI and wash the wells 4x with sterile distilled H₂O. Air dry in the hood overnight.
5. Prepare a 20 µL/mL solution of laminin in L15 medium and add 10 µL laminin to the center of each well.
   NOTE: Do not coat the surrounding reference and grounding electrodes.
6. Add sterile dH₂O to the surrounding reservoirs to prevent medium or laminin evaporation. Incubate the plate for 1 h at 37 °C.
7. Add SFEBs (see sections 1 and 2) to the laminin coated MEA plates by gently suctioning them into a wide mouth pipette tip and transfer them. Add 200 µL DM2 medium to each well and incubate overnight at 37 °C, 95% humidity and 5% CO₂.
8. Add an additional 200 µL medium after 24 h and allow it to recover for 30 min at 37 °C, 95% humidity, 5% CO₂ before reading the plate.
9. Place the MEA plate in the plate reader (preheated to 37 °C) to record neuronal activity. Record activity for 10 min with the associated MEA recording software. See reference¹⁹ for details.
10. Generate raw data-continuous streams and raster plots of neuronal activity using statistical analysis software¹⁹.
    NOTE: MEA recordings are taken 7 days post SFEB plating. For this study, recordings were taken for 10 min to detect network burst activity, continuous trace, and raster plots. SFEBs can be maintained long-term on MEA plates and can be recorded over longer periods of time using environmentally controlled conditions (Figure 6).

Results

SFEBs grown using our technique yielded tissue with morphological characteristics that resemble an early developing cortical subventricular zone replete with extensive Tuj1-positive neurons, as well as neural progenitors (Figure 3A). Numerous developing cortical rosettes were observed in the outer layers and inner layers of the SFEB (Figure 3B). The outer edge of the SFEB resembles a developing cortical plate containing postmitot...

Discussion

The protocol described here provides the conditions for differentiating a hiPSC source into a 3-D structure that recapitulates an early developmental stage of the frontal cortex. This procedure yields structures that can be interrogated for electrophysiology while also being amenable to microscopy. The final morphology of the SFEB resembles that of organotypic brain slice cultures and allows high quality detailed confocal imaging. This protocol can successfully generate SFEBs from both fibroblast and peripheral-blood mon...

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	250 mL
PluriQ ES-DMEM Medium (MEF Media)	GlobalStem	GSM-2001
Name	Company	Catalog Number	Comments
DM1 Media Components
D-MEM/F-12 (1x), Glutamax liquid, 1:1	Invitrogen	10565018	385 mL
Knockout Serum Replacement	Invitrogen	10828028	20% 100 mL
Pen/Strep	Invitrogen	15140122	5 mL
Glutamax 200 mM	Invitrogen	35050061	5 mL
MEM Non-Essential Amino Acids Solution 10 mM (100x), liquid	Invitrogen	11140050	5 mL
2-Mercaptoethanol (1,000x), liquid	Invitrogen	21985023	900 μL
Name	Company	Catalog Number	Comments
DM2 Media Components
D-MEM/F-12 (1x), Glutamax liquid, 1:1	Invitrogen	10565018	500 mL
Glutamax 200 mM	Invitrogen	35050061	5 mL
Pen/Strep	Invitrogen	15140122	5 mL
N-2 Supplement (100x), liquid	Invitrogen	17502048	10 mL
Name	Company	Catalog Number	Comments
DM3 Media Components
NEUROBASAL Medium (1x), liquid	Invitrogen	21103049	500 mL
B-27 Supplement Minus Vitamin A (50x), liquid	Invitrogen	12587010	10 mL
Glutamax 200 mM	Invitrogen	35050061	5 mL
Pen/Strep	Invitrogen	15140122	5 mL
Name	Company	Catalog Number	Comments
Small Molecules
Thiazovivin	Stemgent	04-0017	2 μM
SB431542	Stemgent	04-0010-10	1:1,000 (10 μM)
Dorsomorphin	Stemgent	04-0024	1 μM
LDN-193189	Stemgent	04-0074-10	250 nM
Y27632 (ROCKi)	Stemgent	04-0012-10	10 μM
Name	Company	Catalog Number	Comments
Recombinant Protiens
DKK-1	Peprotech	120-30	200 ng/mL
Name	Company	Catalog Number	Comments
Components/Materials
Cell Culture inserts 0.4 μM, 30mm Diameter	Millicell	PICM0RG50
Mouse Embryonic Fibroblasts	GlobalStem	GSC-6301G
96 well V bottom w/Lids	Evergreen	222-8031-01V
StemPro Accutase Cell Dissociation Reagent	ThermoFisher	A1110501
TritonX-100	ThermoFisher	85111
Phosphate Buffered Saline (PBS)	ThermoFisher	10010023	500 mL
Normal Donkey Serum	Jackson Labs	017-000-121
Leibovitz's L-15 Medium	ThermoFisher	11415114	500 mL
DRAQ5 (Nuclear Marker)	ThermoFisher	65-0880-96
MEA Plates	Axion Biosystems	M768-GL1-30Pt200
6 well flat bottom	Falcon	353046
Name	Company	Catalog Number	Comments
Antibody
Nestin	Millipore	MAB5326
Brn-2	Protein tech	14596-1-AP
VGLUT1	Synaptic Systems	135 303
Pax6	abcam	ab5790
Calretinin	abcam	ab702
Calbindin	abcam	ab11426
CoupTFII	R&D Systems	PPH714700
Nkx 2.1	abcam	ab12650
Tuj1	abcam	ab41489
Reelin	Millipore	MAB5364
Tbr1	Millipore	MAB2261
NFH	Dako	M0762