Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells

Published: May 17, 2024

doi:

Homa Majd², Mikayla N. Richter², Ryan M. Samuel², Ali Kalantari², Jonathan T. Ramirez², Faranak Fattahi^2,3

¹Department of Cellular and Molecular Pharmacology,University of California, San Francisco, ²Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research,University of California, San Francisco, ³Program in Craniofacial Biology,University of California, San Francisco

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 sources²^,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 glia⁸^,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 activation¹²^,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-bodies¹⁵^,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) lines⁸^,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. hPSC maintenance medium: Mix feeder-free hPSC maintenance medium supplement (20 µL/mL) with its base medium. Medium A: Add bone morphogenetic protein 4 (BMP4; 1 ng/mL), SB431542 (10 μM), and CHIR99021…

Representative 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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

The work was supported by grants from UCSF Program for Breakthrough Biomedical Research and Sandler Foundation, March of Dimes grant no. 1-FY18-394 and 1DP2NS116769-01, the NIH Director's New Innovator Award (DP2NS116769) to F.F. and the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK121169) to F.F., H.M. is supported by Larry L. Hillblom Foundation postdoctoral fellowship, NIH T32-DK007418 fellowship and UCSF Program for Breakthrough Biomedical Research independent postdoctoral fellowship.

Materials

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

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