Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells

Summary

Deriving enteric nervous system (ENS) lineages from human pluripotent stem cells (hPSC) provides a scalable source of cells to study ENS development and disease, and to use in regenerative medicine. Here, a detailed in vitro protocol to derive enteric neurons from hPSCs using chemically defined culture conditions is presented.

Abstract

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls bowel motility, enzyme secretion, and nutrient absorption and interacts with the immune system and the gut microbiome. Consequently, developmental and acquired defects of the ENS are responsible for many human diseases and may contribute to symptoms of Parkinson's disease. Limitations in animal model systems and access to primary tissue pose significant experimental challenges in studies of the human ENS. Here, a detailed protocol is presented for effective in vitro derivation of the ENS lineages from human pluripotent stem cells, hPSC, using defined culture conditions. Our protocol begins with directed differentiation of hPSCs to enteric neural crest cells within 15 days and yields diverse subtypes of functional enteric neurons within 30 days. This platform provides a scalable resource for developmental studies, disease modeling, drug discovery, and regenerative applications.

Introduction

The enteric nervous system (ENS) is the largest component of the peripheral nervous system. The ENS contains more than 400 million neurons that are located within the GI tract and control nearly all functions of the gut¹. Molecular understanding of the ENS development and function and its defects in enteric neuropathies requires access to a reliable and authentic source of enteric neurons. Access to human primary tissue is limited, and animal models fail to recapitulate key disease phenotypes in many enteric neuropathies. Human pluripotent stem cell (hPSC) technology has proven exceedingly beneficial in providing an unlimited source of desired cell types, especially those that are difficult to isolate from primary sources^2,3,4,5,6,7. Here, we provide details of a stepwise and robust in vitro method to obtain ENS cultures from hPSCs. These scalable hPSC-derived cultures open avenues for developmental studies, disease modeling, and high-throughput drug screening and can provide transplantable cells for regenerative medicine.

Enteric neuron lineages are derived from neural crest (NC) cells following the ENS developmental path during embryogenesis. In embryos, NC cells emerge along the margins of the folding neural plate. They proliferate, migrate and give rise to many different cell types including sensory neurons, Schwann cells, melanocytes, craniofacial skeleton and enteric neurons and glia^8,9,10. The cell fate decision depends on the distinct region along the anterior-posterior axis that the NC cells emerge from, i.e., cranial NC, vagal NC, trunk NC and sacral NC. The ENS develops from vagal and sacral NC cells with the former dominating the population of the enteric neurons owing to the extensive migration along the length of the bowel and colonizing the gut¹¹.

Derivation of NC cells from PSCs commonly involves a combination of dual SMAD inhibition and WNT pathway activation^12,13. Until recently, all NC induction protocols involved serum and other animal product additives in the culture conditions. Chemically undefined media not only lower the reproducibility of NC induction but also challenge mechanistic developmental studies. To overcome these challenges, Barber et al¹⁴ developed an NC induction protocol using chemically defined culture conditions and was hence advantageous over alternative methods that rely on serum-replacement factors (e.g., KSR)^13,14. This was obtained by basal media replacements and optimizing a protocol originally presented by Fattahi et al to derive enteric NC cells from hPSCs. This improved system is the basis of the hPSC differentiation protocol that is presented here. It begins with induction of enteric neural crest (ENC) cells in a 15-day period by precise modulation of BMP, FGF, WNT and TGFβ signaling in addition to retinoic acid (RA). We then derive the ENS lineages by treating cells with glial cell line-derived neurotrophic factor (GDNF). All media compositions are chemically defined and yield robust enteric neuronal cultures within 30-40 days.

In addition to the monolayer cell culture system described here, alternative NC induction approaches have been developed which use free-floating embryoid-bodies^15,16. Migratory cells in these cultures have been shown to express NC markers with a subset representing the vagal NC. Co-cultures with primary gut tissue have been used to enrich enteric NC precursors in these cultures. Media compositions in these studies contain a combination of different factors such as nerve growth factor, NT3 and brain-derived neurotrophic factor. At this stage, it is not fully clear how these factors might affect the enteric neuron precursor commitment identities. For an efficient comparison of the enteric NC induction in the monolayer and the embryoid-body-based cultures more data is required. Given the different culture layouts in these strategies, the use of each method should be considered and optimized according to specific application in mind.

The protocol presented here is reproducible and has been successfully tested by us and others using different hPSC (induced and embryonic) lines^8,14,16.

Protocol

1. Media preparation

NOTE: Concentrations mentioned throughout the protocol are final concentrations of the media components. Prepare all media under sterile conditions in a laminar flow hood, and store at 4 °C in the dark. Use within 2 weeks.

1. hPSC maintenance medium: Mix feeder-free hPSC maintenance medium supplement (20 µL/mL) with its base medium.
2. Medium A: Add bone morphogenetic protein 4 (BMP4; 1 ng/mL), SB431542 (10 μM), and CHIR99021 (600 nM) to differentiation base medium.
3. Medium B: Add SB431542 (10 μM), and CHIR99021 (1.5 μM) to differentiation base medium.
4. Medium C: Add SB431542 (10 μM), CHIR99021 (1.5 μM) and RA (1 μM) to differentiation base medium.
5. Neural crest complete (NCC) Medium: Add FGF2 (10 ng/mL), CHIR99021 (3 μM), N2 supplement (10 μL/mL), B27 supplement (20 μL/mL), L-glutamine supplement (10 μL/mL), and MEM NEAAs (10 μL/mL) to neurobasal medium.
6. ENC Medium: Add GDNF (10 ng/mL), ascorbic acid (100 μM), N2 supplement (10 μL/mL), B27 supplement (20 μL/mL), L-glutamine supplement (10 μL/mL), and MEM NEAAs (10 μL/mL) to neurobasal medium.
7. Ethylenediaminetetraacetic acid (EDTA) 1x: Dilute 500 mM EDTA (1000x) to a final concentration of 500 μM in PBS.
   NOTE: Use Ca²⁺ and Mg²⁺ free PBS throughout this protocol unless otherwise stated.

2. Coating of culture plates

1. Basement membrane matrix coated plates: Quickly thaw, and dilute a 500 μL frozen aliquot of basement membrane matrix in 50 mL of chilled DMEM:F12 by vigorously pipetting using a 50 mL serological pipette. Coat the wells with the diluted basement membrane matrix solution (100 μL per cm² of well surface area) and incubate them overnight at 37 °C. Aspirate the coating medium before use. To prevent gelatinization of basement membrane matrix, perform this step as quickly as possible and use cold DMEM:F12 for dissolving the frozen aliquot.
   NOTE: Basement membrane matrix temperature should be kept cold during thawing (on ice) and aliquoting (tubes on ice) to prevent coagulation.
2. PO/FN/laminin coated plates: Prepare a 15 μg/mL polyornithine (PO) solution in PBS by diluting a 1000x stock. Coat wells with 100 μL of PO/PBS solution per cm² of well surface area and incubate the plates overnight at 37 °C. The following day, aspirate PO/PBS and coat the wells at 100 μL per cm² of well surface area with freshly made FN/laminin/PBS solution containing 2 μg/mL of fibronectin (FN) and 2 μg/mL of laminin. Plates can be used after a minimum of 2 h incubation at 37 °C. Aspirate FN/laminin/PBS before use.

3. Maintenance of hPSC culture

NOTE: All cell incubation steps are at 5% CO₂ and at 37 °C in a humidified incubator.

1. Aspirate hPSC medium from the hPSC culture and add fresh medium (200 μL per cm² of well surface area) every other day until colonies are ~80% confluent.
2. To passage, aspirate hPSC medium and wash cells with 100 μL of PBS per cm² of well surface area.
   NOTE: It is important to use Ca²⁺ and Mg²⁺ free PBS to wash the cells.
3. Add 500 μM EDTA (100 μL per cm² of well surface area). Replace the lid of the plate. Watch through an inverted microscope and monitor the cells for detachment of colony edges (2-4 min).
4. Use a 5 or 10 mL serological pipette to transfer the same volume of hPSC medium into each well and mechanically harvest the cells by pipetting up and down a few times.
   NOTE: Optimal EDTA incubation time depends on the iPSC line. Avoid vigorous pipetting for detaching colonies as it can lead to excessive colony dissociation and cell death. When passaging to start a differentiation plate, incubate cells with EDTA for 1-2 min longer.
5. Transfer the cell suspension into a 15 mL conical tube. Spin the cells (2 min, 290 x g, 20-25 °C) and carefully discard the supernatant.
6. Resuspend the cells in an appropriate volume of hPSC medium and transfer to wells of a new basement membrane matrix coated plate.
   NOTE: For regular hPSC maintenance use a 1:18 - 1:24 passaging ratio (1:18 means transferring one third of the cells dissociated from a single well of a 6-well plate into a full new plate). To start a differentiation plate, follow a 5:6 passaging ratio.
7. Incubate at 5% CO₂ and 37 °C.

4. Neural crest induction

NOTE: This step is schematically represented in Figure 1A. Begin NC differentiation when cells are about 80% confluent. This will be achieved within 1-2 days when cells are passaged at a 5:6 ratio (see above). Start NC differentiation on day 0 with pluripotent and fully undifferentiated hPSC cells. For a high efficiency, the colony borders should have minimal differentiating cells. Passage and plate hPSC for differentiation when colonies are large, or the center of the colony starts to thicken/darken when monitored using an inverted microscope. Paying attention to the confluency and morphology tends to be more reliable than the number of plated cells as this might vary depending on the cell line and can vary from 50,000-200,000 per cm².

1. On day 0, replace hPSC maintenance medium with Medium A containing 1 ng/mL BMP4, 10 μM SB431542, 600 nM CHIR99021 (200 μL of medium per cm² of well surface area).
2. On day 2, cells should be 100% confluent. Gently aspirate spent Medium A and feed cells with Medium B containing 10 μM SB431542, 1.5 μM CHIR99021 (200 μL of medium per cm² of well surface area).
   NOTE: As cultures grow during the NC induction, cells may detach from the underlying monolayer. Avoid excess cell loss by gently removing old media and adding fresh media to the side of the well.
3. On day 4, feed cells again with Medium B (200 μL of medium per cm² of well surface area).
4. On day 6, replace medium B with Medium C containing 10 μM SB431542, 1.5 μM CHIR99021 and 1 μM retinoic acid (400 μL of medium per cm² of well surface area).
5. On days 8 and 10, feed as day 6.

5. ENC spheroid formation

NOTE: This step is schematically represented in Figure 1B.

1. On day 12, gently aspirate Medium C and wash cells with 100 μL of PBS per cm² of well surface area. Add the same volume of cell detachment solution and incubate for 30 min at 5% CO₂ and 37 °C.
2. Dilute cell detachment solution by adding the same volume of NCC medium containing 10 ng/mL FGF2, 3 μM CHIR99021, 10 μL/mL N2 supplement, 20 μL/mL B27 supplement, 10 μL/mL L-glutamine supplement, and 10 μL/mL MEM NEAA. Using a serological pipette harvest the single cell suspension and add it to a 15 mL conical tube.
3. Spin the cells (2 min, 290 x g, 20-25 °C) and carefully discard the supernatant.
4. Using a 10 mL serological pipette, add 12 mL (for a full 6-well plate) of NCC medium and resuspend the cells completely by mechanically pipetting up and down 1-2 times. Transfer the cell suspension to an ultra-low attachment plate.
   NOTE: Approximately 10 cm² of ENC monolayer cells are resuspended in 2 mL of NCC medium and transferred to one well of an ultra-low attachment plate. This equals to a full 6-well NC induction plate transferred into a full 6-well ultra-low attachment plate.
5. On day 14, gently swirl the plates until small spheroids are gathered in the center of each well. Using a P1000 micropipette and in a slow circular motion, aspirate the spent NCC medium around the circumference of each well. Try to avoid removing the free-floating spheroids.
6. Feed the cells with the original volume of NCC medium.

6. EN induction

NOTE: This step is schematically represented in Figure 1B.

1. On day 15, apply the same technique used on day 14 to gently remove the medium and wash the spheroids with PBS. Remove as much PBS as possible avoiding the spheroids.
2. Add cell detachment solution (100 μL per cm² of well surface area) and dissociate the spheroids into single cells by incubating for 30 min at 5% CO₂ and 37 °C.
3. Using a 10 mL serological pipette, add an equal volume of ENC medium (containing 10 ng/mL GDNF, 100 μM ascorbic acid, 10 μL/mL N2 supplement, 20 μL/mL B27 supplement, 10 μL/mL L-glutamine supplement, and 10 μL/mL MEM NEAA in neurobasal medium) to each well and break the remaining spheroids by 2-3 rounds of mechanical pipetting. Transfer the cells to a 50 mL conical tube.
   NOTE: Avoid excessive sheer stress and cell death that can occur from forced pipetting or use of pipettes with smaller tip opening.
4. Spin the cells (2 min, 290 x g, 20-25 °C) and carefully discard the supernatant. Using a 10 mL serological pipette, add ~ 5-10 mL of ENC medium and resuspend the cells completely by pipetting up and down 1-2 times.
5. Count the concentration of viable cells using trypan blue and a cell counting method such as a hemocytometer.
   CAUTION: Trypan blue is a suspected carcinogen. Handle with care, dispose appropriately.
6. Add enough volume of ENC medium aiming to plate approximately 300,000-400,000 viable cells per cm² of surface area at a density of about ~ 1,000,000 cells per mL. Aspirate FN/laminin/PBS solution from wells of the plate.
   NOTE: Choose the plate format according to the assays that are planned for the EN cultures as this stage marks the final re-plating step of the protocol. Enteric neuron progenitors can be frozen on day 15. After dissociating the spheres with cell detachment solution, resuspend the progenitors at approximately 5 x 10⁶ cells/mL in freezing medium such as 10% DMSO or Stem-cellbanker. When needed, thaw in warm ENC media with 5 μM Y-27632 ROCK inhibitor. Plate cells in the desired plate format. Replace the media with fresh ENC after a couple of hours.
7. Gently add the cells into the PO/FN/laminin coated wells. Avoid bubbles getting trapped under the cell suspension preventing cells attachment to the PO/FN/laminin coating, especially when using plates with smaller well size such as 384-well plates. This could lead to cell death.
8. Feed cells every other day with ENC medium (200 μL per cm² of well surface area) until day 30-40, after which reduce the feeding frequency (to once or twice a week) but double the feeding volume (to 400 μL of medium per cm² of well surface area).
   NOTE: This is important as excessive removing and adding media could facilitate detachment of neuronal culture from the surface of the wells. To prospectively prevent detachment especially for longer-term cultures, supplement ENC with FN (2 µg/mL) and laminin (2 µg/mL) once a week.

Results

This protocol provides a method to derive enteric neural crest and enteric neurons from hPSCs using chemically defined culture conditions (Figure 1A-B). Generating high-quality neurons depends on an efficient enteric neural crest induction step. This can be visually assessed by checking the morphology of the free-floating spheres that should look round with smooth surfaces with a size of approximately 0.1 - 0.4 mm as seen in Figure 1C. These sph...

Discussion

The differentiation protocol described here provides a robust in vitro method to obtain enteric neurons from hPSCs within 30-40 days (Figure 1E) and enteric glia expressing glial fibrillary acidic protein, GFAP, and SOX10 in older cultures (> day 55)^13,14,19,22. These neurons and glia are induced by stepwise differentiation of hPSCs into vagal and ente...

Materials

Name	Company	Catalog Number	Comments
Ascorbic acid	Sigma-Aldrich	A5960
B27 supplement (serum free, minus vitamin A)	Gibco	12587-010
Basement membrane matrix, Geltrex	Gibco	A14133-2
BMP4	R&D systems	314-BP
Cell culture centrifuge	Eppendorf, model no. 5810R	02262501
Cell detachment solution, Accutase	Stemcell Technologies	07920
CHIR99021	Tocris	4423
Conical tubes	USA scientific	1475-0511, 1500-1211
Differentiation base medium, Essential 6	Life Technologies	A1516401
DMEM/F-12 no glutamine	Life Technologies	21331020
EDTA	Corning	MT-46034CI
Feeder-free hPSC maintenance medium, Essential 8 Flex Medium Kit	Life Technologies	A2858501
FGF2	R&D systems	233-FB/CF
Fibronectin	Corning	356008
GDNF	Peprotech	450-10
Hemocytometer	Hausser Scientific	1475
Human pluripotent stem cells, H9 ESC	WiCell	RRID: CVCL_1240
Incubator with controlled humidity, temperature and CO2	Thermo Fisher Scientific	Herralcell 150i
Inverted microscope	Thermo Fisher Scientific	EVOS FL
Laminar flow hood	Thermo Fisher Scientific	1300 series class II, type A2
Laminin	Cultrex	3400-010
L-glutamine supplement, Glutagro	Corning	25-015-CI
MEM NEAAs	Corning	25-025-CI
Multiwell plates, Falcon	BD	353934, 353075
N-2 Supplement	CTS	A1370701
Neurobasal Medium	Life Technologies	21103049
PBS (Ca and Mg free)	Life Technologies	10010023
Pipette filler	Eppendorf	Z768715-1EA
Pipette tips	USA scientific	1111-2830
Pipettes	Fisherbrand	13-678-11E, 13-678-11F
PO	Sigma-Aldrich	P3655
polymer coverslip bottom imaging plates, ibidi	ibidi	81156
RA	Sigma-Aldrich	R2625
SB431542	R&D systems	1614
Trypan blue stain, 0.4%	Thermo Fisher Scientific	15250-061
Ultra-low attachment plates	Fisher Scientific	07-200-601
Y-27632 dihydrochloride	R&D systems	1254