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&#39;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.

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 &#176;C in the dark. Use within 2 weeks.
<ol>
	<li>hPSC maintenance medium: Mix feeder-free hPSC maintenance medium supplement (20 &#181;L/mL) with its base medium.</li>
	<li>Medium A: Add bone morphogenetic protein 4 (BMP4; 1 ng/mL), SB431542 (10 &#956;M), and CHIR99021 (600 nM) to differentiation base medium.</li>
	<li>Medium B: Add SB431542 (10 &#956;M), and CHIR99021 (1.5 &#956;M) to differentiation base medium.</li>
	<li>Medium C: Add SB431542 (10 &#956;M), CHIR99021 (1.5 &#956;M) and RA (1 &#956;M) to differentiation base medium.</li>
	<li>Neural crest complete (NCC) Medium: Add FGF2 (10 ng/mL), CHIR99021 (3 &#956;M), N2 supplement (10 &#956;L/mL), B27 supplement (20 &#956;L/mL), L-glutamine supplement (10 &#956;L/mL), and MEM NEAAs (10 &#956;L/mL) to neurobasal medium.</li>
	<li>ENC Medium: Add GDNF (10 ng/mL), ascorbic acid (100 &#956;M), N2 supplement (10 &#956;L/mL), B27 supplement (20 &#956;L/mL), L-glutamine supplement (10 &#956;L/mL), and MEM NEAAs (10 &#956;L/mL) to neurobasal medium.</li>
	<li>Ethylenediaminetetraacetic acid (EDTA) 1x: Dilute 500 mM EDTA (1000x) to a final concentration of 500 &#956;M in PBS. 
	NOTE: Use Ca2+ and Mg2+ free PBS throughout this protocol unless otherwise stated.</li>
</ol>
2. Coating of culture plates
<ol>
	<li>Basement membrane matrix coated plates: Quickly thaw, and dilute a 500 &#956;L 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 &#956;L per cm2 of well surface area) and incubate them overnight at 37 &#176;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.</li>
	<li>PO/FN/laminin coated plates: Prepare a 15 &#956;g/mL polyornithine (PO) solution in PBS by diluting a 1000x stock. Coat wells with 100 &#956;L of PO/PBS solution per cm2 of well surface area and incubate the plates overnight at 37 &#176;C. The following day, aspirate PO/PBS and coat the wells at 100 &#956;L per cm2 of well surface area with freshly made FN/laminin/PBS solution containing 2 &#956;g/mL of fibronectin (FN) and 2 &#956;g/mL of laminin. Plates can be used after a minimum of 2 h incubation at 37 &#176;C. Aspirate FN/laminin/PBS before use.</li>
</ol>
3. Maintenance of hPSC culture
NOTE: All cell incubation steps are at 5% CO2 and at 37 &#176;C in a humidified incubator.
<ol>
	<li>Aspirate hPSC medium from the hPSC culture and add fresh medium (200 &#956;L per cm2 of well surface area) every other day until colonies are ~80% confluent.</li>
	<li>To passage, aspirate hPSC medium and wash cells with 100 &#956;L of PBS per cm2 of well surface area. 
	NOTE: It is important to use Ca2+ and Mg2+ free PBS to wash the cells.</li>
	<li>Add 500 &#956;M EDTA (100 &#956;L 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).</li>
	<li>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.</li>
	<li>Transfer the cell suspension into a 15 mL conical tube. Spin the cells (2 min, 290 x g, 20-25 &#176;C) and carefully discard the supernatant.</li>
	<li>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.</li>
	<li>Incubate at 5% CO2 and 37 &#176;C.</li>
</ol>
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.
<ol>
	<li>On day 0, replace hPSC maintenance medium with Medium A containing 1 ng/mL BMP4, 10 &#956;M SB431542, 600 nM CHIR99021 (200 &#956;L of medium per cm2 of well surface area).</li>
	<li>On day 2, cells should be 100% confluent. Gently aspirate spent Medium A and feed cells with Medium B containing 10 &#956;M SB431542, 1.5 &#956;M CHIR99021 (200 &#956;L 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.</li>
	<li>On day 4, feed cells again with Medium B (200 &#956;L of medium per cm2 of well surface area).</li>
	<li>On day 6, replace medium B with Medium C containing 10 &#956;M SB431542, 1.5 &#956;M CHIR99021 and 1 &#956;M retinoic acid (400 &#956;L of medium per cm2 of well surface area).</li>
	<li>On days 8 and 10, feed as day 6.</li>
</ol>
5. ENC spheroid formation
NOTE: This step is schematically represented in Figure 1B.
<ol>
	<li>On day 12, gently aspirate Medium C and wash cells with 100 &#956;L 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 &#176;C.</li>
	<li>Dilute cell detachment solution by adding the same volume of NCC medium containing 10 ng/mL FGF2, 3 &#956;M CHIR99021, 10 &#956;L/mL N2 supplement, 20 &#956;L/mL B27 supplement, 10 &#956;L/mL L-glutamine supplement, and 10 &#956;L/mL MEM NEAA. Using a serological pipette harvest the single cell suspension and add it to a 15 mL conical tube.</li>
	<li>Spin the cells (2 min, 290 x g, 20-25 &#176;C) and carefully discard the supernatant.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Feed the cells with the original volume of NCC medium.</li>
</ol>
6. EN induction
NOTE: This step is schematically represented in Figure 1B.
<ol>
	<li>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.</li>
	<li>Add cell detachment solution (100 &#956;L per cm2 of well surface area) and dissociate the spheroids into single cells by incubating for 30 min at 5% CO2 and 37 &#176;C.</li>
	<li>Using a 10 mL serological pipette, add an equal volume of ENC medium (containing 10 ng/mL GDNF, 100 &#956;M ascorbic acid, 10 &#956;L/mL N2 supplement, 20 &#956;L/mL B27 supplement, 10 &#956;L/mL L-glutamine supplement, and 10 &#956;L/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.</li>
	<li>Spin the cells (2 min, 290 x g, 20-25 &#176;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.</li>
	<li>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.</li>
	<li>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 &#956;M Y-27632 ROCK inhibitor. Plate cells in the desired plate format. Replace the media with fresh ENC after a couple of hours.</li>
	<li>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.</li>
	<li>Feed cells every other day with ENC medium (200 &#956;L 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 &#956;L 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 &#181;g/mL) and laminin (2 &#181;g/mL) once a week.</li>
</ol>

Derivation of Enteric Neurons from Human Pluripotent Stem Cells

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The authors have nothing to disclose.

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 (&#62; day 55)13,14,19,22. These neurons and glia are induced by stepwise differentiation of hPSCs into vagal and ente...

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.

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

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.

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...

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

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.

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 ...

differentiation-enteric-nervous-system-lineages-from-human

Research

JoVE Journal

Neuroscience

1.2K Views.  University of California, San Francisco. Hier bieden we een methode voor het genereren van menselijke enterische neuronen uit menselijke pluripotente stamcellen onder volledig gedefinieerde omstandigheden, en we gebruiken deze cellen om de ontwikkeling en functie van het menselijk enterisch zenuwstelsel te begrijpen. Dus ik denk dat de ontwikkeling, fysiologie en pathofysiologie van het menselijk enterisch zenuwstelsel een grote uitdaging is geweest, en dat komt door de zeldzaamheid van de cellen van het enterische zenuwstelsel en d...

Video: Auteur in de schijnwerpers: robuuste cultuur van menselijke darmneuronen uit menselijke pluripotente stamcellen

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Developmental Biology

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing gastrointestinal (GI) tract. Regional specification in the intestine is driven by multiple developmental pathways, including the bone morphogenetic protein (BMP) pathway. Based on normal regional specification, a method to generate human colonic organoids (HCOs) from human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hES) and induced pluripotent stem cells (iPSCs), was developed. A three-day induction of BMP signaling sufficiently patterns mid/hindgut tube cultures into special AT-rich sequence-binding protein 2 (SATB2)-expressing HCOs containing all of the main epithelial cell types present in human colon as well as co-developing mesenchymal cells. Omission of BMP (or addition of the BMP inhibitor NOGGIN) during this critical patterning period resulted in the formation of human intestinal organoids (HIOs). HIOs and HCOs morphologically and molecularly resemble human developing small intestine and colon, respectively. Despite the utility of HIOs and HCOs for studying human intestinal development, the generation of HIOs and HCOs is challenging. This paper presents methods for generating, maintaining, and characterizing HIOs and HCOs. In addition, the critical steps in the protocol and troubleshooting recommendations are provided.

Here, detailed methods for generating, maintaining, and characterizing human pluripotent stem cell-derived small intestinal and colonic organoids are described. These methods are designed to improve reproducibility, expand scalability, and decrease the working time required for plating and passaging of organoids.

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing ...

Innervation of Human Intestinal Organoids

The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques for generating human intestinal organoids (HIOs) resembling human small intestine have been previously reported. HIOs contain epithelium and mesenchyme but lack other critical components of functional intestine such as the enteric nervous system (ENS), immune cells, vasculature, and microbiome. Two independent research groups have published distinct methods to innervate HIOs with an ENS. Here we discuss a unique method of incorporating the ENS into an HIO-derived bioengineered small intestine, which utilizes components of these prior reports to optimize progenitor cell identity as well as developmental timing.
Human pluripotent stem cells (hPSCs) are differentiated to independently generate HIOs and enteric neural crest cells (ENCCs) by temporal regulation of differentiation markers over a period of several days per published protocols. Once HIOs reach the mid-hindgut spheroid stage (approximately day 8), day 15-21 ENCC spheroids are dissociated, co-cultured with HIOs, and suspended within clear three-dimensional (3D) basement membrane matrix droplets. HIO + ENCC co-cultures are maintained in vitro for 28-40 days before transplantation into &#62;9-week-old immunodeficient mice for further development and maturation. Transplanted HIOs (tHIOs) with ENS can be harvested 4-20 weeks later. This method integrates elements from two previously published techniques by utilizing ENCCs generated from hPSCs and co-culturing them with HIOs at an early stage of development to maximize exposure to early developmental cues that likely contribute to the formation of a more mature intestinal morphology.

Human intestinal organoids must be innervated to better recapitulate the structure and function of the native human intestine. Here, we present one method for incorporating an enteric nervous system into these constructs.

The complexity of intestinal cytoarchitecture and function poses significant challenges for the creation of the bioengineered small intestine. Techniques ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells

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