differentiation of enteric neural crest spheroid and neuronal induction from human pluripotent stem cells for enteric nervous system research

Derivation of Enteric Neurons from Human Pluripotent Stem Cells

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 TGF&#946; 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.

Author Spotlight: Robust Culture of Human Enteric Neurons from Human Pluripotent Stem Cells

Diferenciação de linhagens do sistema nervoso entérico de células-tronco pluripotentes humanas

Diferenciação da Crista Neural Entérica Esferóide e Indução Neuronal de Células-Tronco Pluripotentes Humanas para Pesquisa do Sistema Nervoso Entérico

Para começar, faça culturas de 12 dias de células-tronco pluripotentes humanas que foram submetidas à indução da crista neural. Aspire suavemente o meio C das células. Adicione 100 microlitros de PBS por centímetro quadrado de área de superfície do poço para lavar as células e, em seguida, remova o PBS.Adicione um volume igual de solução de descolamento celular às células e incube por 30 minutos sob suplementação de dióxido de carbono a 5% a 37 graus Celsius. Adicione um volume igual de meio NCC na placa. Em seguida, use uma pipeta sorológica para colher a suspensão celular e transfira-a para um tubo cônico de 15 mililitros.Gire as células a 290G por dois minutos entre 20 a 25 graus Celsius. Descarte cuidadosamente o sobrenadante sem perturbar o pellet celular. Use uma pipeta sorológica de 10 mililitros para pipetar 12 mililitros de meio NCC para o pellet celular.Pipete mecanicamente a suspensão para cima e para baixo para criar uma suspensão. Em seguida, transfira a suspensão da célula para uma placa de fixação ultrabaixa. No dia 14, gire suavemente as placas de cultura de células para coletar pequenos esferoides no centro de cada poço.Use uma micropipeta P1000 para aspirar lentamente o meio gasto da circunferência de cada poço. Realimente as células com o volume original do meio NCC. No dia 15, remova cuidadosamente o meio.Adicione PBS aos poços para lavar os esferóides. Em seguida, remova o máximo de PBS possível, garantindo que os esferóides não sejam perturbados. Em seguida, adicione 100 microlitros de solução de descolamento de células por centímetro quadrado de área de superfície do poço.Incube as células na solução por 30 minutos sob 5% de dióxido de carbono a 37 graus Celsius. Com uma pipeta sorológica de 10 mililitros, adicione um volume igual de meio ENC a cada poço. Pipetar mecanicamente a solução para quebrar os esferóides restantes.Em seguida, transfira as células para um tubo cônico de 50 mililitros. Rejeitar o sobrenadante após centrifugação como antes. Em seguida, ressuspenda as células em 5 a 10 mililitros de meio ENC.Conte a concentração de células viáveis usando azul de tripano e hemocitômetro. Agora, adicione volume suficiente de meio ENC para colocar cerca de 300.000 a 400.000 células viáveis por centímetro quadrado de área de superfície e uma densidade de cerca de 1 milhão de células por mililitro. Em seguida, aspire as soluções dos poços.Em seguida, adicione as células a uma placa com poços revestidos de laminina PO / FN. Alimente as células em dias alternados com 200 microlitros de meio ENC por centímetro quadrado de área de superfície do poço até os dias 30 a 40. Esferas flutuantes livres de alta qualidade com superfícies lisas foram observadas após a indução da crista neural.Os esferoides expressaram o marcador de crista neural SOX10. Neurites foram observados entre os dias 25 a 30 durante a indução do neurônio entérico.

Research

JoVE Journal

Neuroscience

Differentiation of Enteric Neural Crest Spheroid and Neuronal Induction from Human Pluripotent Stem Cells for Enteric Nervous System Research

Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls ...

Neurociência

Formation of Neural Crests from Human Pluripotent Stem Cells for Enteric Nervous System Research

Formação de cristas neurais a partir de células-tronco pluripotentes humanas para pesquisa do sistema nervoso entérico