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

Dérivation de neurones entériques à partir de cellules souches pluripotentes humaines

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.

Pleins feux sur l’auteur : Culture robuste de neurones entériques humains à partir de cellules souches pluripotentes humaines

Différenciation des lignées du système nerveux entérique à partir de cellules souches pluripotentes humaines

Here we provide a method for generation of human enteric neurons from human pluripotent stem cells under fully defined conditions, and we use these cells in order to understand human enteric nervous system development and function. So I think human enteric nervous system development, physiology and pathophysiology has been a great challenge, and it's because of the rarity of the enteric nervous system cells and the transient nature of the neural crest progenitors, and difficulty in access to tissue and isolation of the tissue. This is a robust platform for generating authentic and functional human enteric neurons from HPSCs, and by overcoming longstanding technical challenges in tissue access, it provides scalable cultures of enteric nervous system components for disease modeling, drug discovery, and studying ENS developments and function.

Différenciation de la crête neurale entérique sphéroïde et induction neuronale à partir de cellules souches pluripotentes humaines pour la recherche sur le système nerveux entérique

Pour commencer, prenez des cultures de 12 jours de cellules souches pluripotentes humaines qui ont été soumises à l’induction de la crête neurale. Aspirez doucement le milieu C des cellules. Ajoutez 100 microlitres de PBS par centimètre carré de surface de puits pour laver les cellules, puis retirez le PBS.Ajoutez un volume égal de solution de détachement cellulaire aux cellules et incubez pendant 30 minutes sous une supplémentation en dioxyde de carbone de 5 % à 37 degrés Celsius. Ajouter un volume égal de milieu NCC dans la plaque. Ensuite, à l’aide d’une pipette sérologique, prélevez la suspension cellulaire et transférez-la dans un tube conique de 15 millilitres.Faites tourner les cellules à 290 G pendant deux minutes entre 20 et 25 degrés Celsius. Jetez soigneusement le surnageant sans déranger la pastille cellulaire. À l’aide d’une pipette sérologique de 10 millilitres, pipeter 12 millilitres de milieu NCC sur la pastille de cellule.Pipetez mécaniquement la suspension de haut en bas pour créer une suspension. Ensuite, transférez la suspension cellulaire sur une plaque de fixation ultra basse. Le 14e jour, faites tourner doucement les plaques de culture cellulaire pour recueillir de petits sphéroïdes au centre de chaque puits.À l’aide d’une micro-pipette P1000, aspirez lentement le milieu usé de la circonférence de chaque puits. Alimentez à nouveau les cellules avec le volume d’origine de milieu NCC. Le 15e jour, retirez délicatement le milieu.Ajoutez du PBS dans les puits pour laver les sphéroïdes. Ensuite, retirez autant de PBS que possible, en vous assurant que les sphéroïdes ne sont pas dérangés. Ensuite, ajoutez 100 microlitres de solution de détachement cellulaire par centimètre carré de surface de puits.Incuber les cellules dans la solution pendant 30 minutes à 5% de dioxyde de carbone à 37 degrés Celsius. À l’aide d’une pipette sérologique de 10 millilitres, ajoutez un volume égal de milieu ENC dans chaque puits. Pipetez mécaniquement la solution pour casser les sphéroïdes restants.Transférez ensuite les cellules dans un tube conique de 50 millilitres. Jetez le surnageant après la centrifugation comme précédemment. Ensuite, mettez les cellules en suspension dans 5 à 10 millilitres de milieu ENC.Comptez la concentration de cellules viables à l’aide du bleu de trypan et de l’hémocytomètre. Maintenant, ajoutez suffisamment de volume de milieu ENC pour plaquer environ 300 000 à 400 000 cellules viables par centimètre carré de surface, et une densité d’environ 1 million de cellules par millilitre. Ensuite, aspirez les solutions des puits.Ensuite, ajoutez les cellules à une plaque avec des puits recouverts de laminine PO/FN. Nourrissez les cellules tous les deux jours avec 200 microlitres de milieu ENC par centimètre carré de surface de puits jusqu’aux jours 30 à 40. Des sphères flottantes de haute qualité avec des surfaces lisses ont été observées après l’induction de la crête neurale.Les sphéroïdes exprimaient le marqueur de crête neurale SOX10. Des neurites ont été observées entre les jours 25 et 30 lors de l’induction des neurones entériques.

differentiation-enteric-neural-crest-spheroid-neuronal-induction-from

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 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.

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.

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

Recherche

Neurosciences

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

To induce neural crest formation, replace the maintenance medium of the human pluripotent stem cell culture with 200 microliters of medium A per square centimeter of the well-surface. Incubate the culture under 5%carbon dioxide supplementation at 37 degrees Celsius for two days. Gently aspirate the medium from the well-containing 100%confluence cells then pipette about 200 microliters of medium B per square centimeter of the well-surface area.On day four, feed the cells with 200 microliters of medium B per square centimeter of the well-surface. Incubate the cells for two more days, then replace the medium B with 400 microliters per square centimeter of medium C.

Formation de crêtes neurales à partir de cellules souches pluripotentes humaines pour la recherche sur le système nerveux entérique

To begin, take 12-day cultures of human pluripotent stem cells that have been subjected to neural crest induction. Gently aspirate medium C from the cells. Add 100 microliters of PBS per square centimeter of well surface area to wash the cells, and then remove the PBS.Add an equal volume of cell detachment solution to the cells and incubate for 30 minutes under 5%carbon dioxide supplementation at 37 degrees Celsius. Add an equal volume of NCC medium into the plate. Then, use a serological pipette to harvest the cell suspension and transfer it to a 15-milliliter conical tube.Spin the cells at 290G for two minutes between 20 to 25 degrees Celsius. Carefully discard the supernatant without disturbing the cell pellet. Use a 10-milliliter serological pipette to pipette 12 milliliters of NCC medium to the cell pellet.Mechanically pipette the suspension up and down to create a suspension. Then, transfer the cell suspension to an ultra low attachment plate. On day 14, gently swirl the cell culture plates to gather small spheroids at the center of each well.Use a P1000 micro pipette to slowly aspirate the spent medium from the circumference of each well. Refeed the cells with the original volume of NCC medium. On day 15, carefully remove the medium.Add PBS to the wells to wash the spheroids. Then, remove as much PBS as possible, ensuring spheroids are not disturbed. Next, add 100 microliters of cell detachment solution per square centimeter of well surface area.Incubate the cells in the solution for 30 minutes under 5%carbon dioxide at 37 degrees Celsius. With a 10-milliliter serological pipette, add an equal volume of ENC medium to each well. Mechanically pipette the solution to break any remaining spheroids.Then transfer the cells to a 50-milliliter conical tube. Discard the supernatant after centrifuging as before. Then, resuspend the cells in 5 to 10 milliliters of ENC medium.Count the concentration of viable cells using trypan blue and hemocytometer. Now, add enough volume of ENC medium to plate about 300, 000 to 400, 000 viable cells per square centimeter of surface area, and a density of about 1 million cells per milliliter. Then, aspirate the solutions from the wells.Next, add the cells to a plate with PO/FN laminin coated wells. Feed cells every other day with 200 microliters of ENC medium per square centimeter of well surface area until days 30 to 40. High quality free floating spheres with smooth surfaces were observed after neural crest induction.The spheroids expressed the neural crest marker SOX10. Neurites were observed between days 25 to 30 during the enteric neuron induction.