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

从人多能干细胞衍生肠道神经元

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.

作者聚焦：来自人多能干细胞的人肠道神经元的稳健培养

肠神经系统谱系与人多能干细胞的分化

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.

肠神经嵴球体和人多能干细胞神经元诱导的分化用于肠神经系统研究

首先，对经过神经嵴诱导的人多能干细胞进行 12 天的培养。轻轻地从细胞中吸出培养基 C。每平方厘米孔表面积添加 100 微升 PBS 以洗涤细胞，然后去除 PBS。向细胞中加入等体积的细胞分离溶液，并在 37 摄氏度的 5% 二氧化碳补充下孵育 30 分钟。向板中加入等体积的 NCC 培养基。然后，使用血清移液管收集细胞悬液并将其转移到 15 毫升锥形管中。在 20 至 25 摄氏度之间以 290G 离心细胞 2 分钟。小心丢弃上清液，不要干扰细胞沉淀。使用 10 毫升血清移液器将 12 毫升 NCC 培养基移液到细胞沉淀中。机械上下移液以产生悬浮液。然后，将细胞悬液转移到超低附着板上。第 14 天，轻轻旋转细胞培养板，在每个孔的中心收集小球体。使用 P1000 微量移液器从每个孔的圆周缓慢吸出用过的培养基。用原始体积的 NCC 培养基重新喂养细胞。第 15 天，小心去除培养基。将 PBS 添加到孔中以洗涤球体。然后，去除尽可能多的 PBS，确保球状体不受干扰。接下来，每平方厘米孔表面积添加 100 微升细胞分离溶液。将细胞在溶液中在 37 摄氏度的 5% 二氧化碳下孵育 30 分钟。使用 10 毫升血清移液管，向每个孔中加入等体积的 ENC 培养基。机械移液溶液以破坏任何剩余的球体。然后将细胞转移到 50 毫升锥形管中。像以前一样离心后弃去上清液。然后，将细胞重悬于 5 至 10 mL ENC 培养基中。使用台盼蓝和血细胞计数器计数活细胞的浓度。现在，添加足够体积的 ENC 培养基，以每平方厘米表面积接种约 300， 000 至 400， 000 个活细胞，密度为每毫升约 100 万个细胞。然后，从孔中吸出溶液。接下来，将细胞添加到带有 PO/FN 层粘连蛋白包被孔的板中。每隔一天用每平方厘米孔表面积 200 微升 ENC 培养基喂养细胞，直至第 30 至 40 天。神经嵴诱导后观察到具有光滑表面的高质量自由漂浮球体。球体表达神经嵴标志物 SOX10。在肠道神经元诱导期间，在 25 至 30 天之间观察到神经突。

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 ...

科研

神经科学

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.

从人类多能干细胞形成神经嵴用于肠道神经系统研究

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.