The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Summary

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.

Abstract

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.

Introduction

Human pluripotent stem cells (PSCs), including both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have the capacity to generate every cell type in the body, including neurons^1-3. Directed differentiation of various neuronal subtypes from human PSCs holds the key for the application of these cells in regenerative medicine. The generation of functional neuronal subtypes during development is a complex process involving the induction of neural lineage, the specification of regional progenitors along the rostro-caudal axis, and the differentiation of post-mitotic neuron types from the regional progenitors^4,5. Beginning in 2001, several systems have been established to generate neural lineage from hESCs, which have provided a platform for the subsequent generation of neuronal subtypes^6,7. Based upon developmental principles, several neuron types such as spinal motor neurons^8-12, midbrain dopaminergic neurons^13-15, and neural retinal cells^16,17 have been efficiently specified from human PSCs. This was done by applying critical morphogens which are important for the specification of these neuron types during in vivo development. Other protocols have also been developed to promote the differentiation of hESCs into neurons using either additional factors^18-20 such as small molecules or by co-culturing with other cell types to help promote differentiation²¹.

The human neocortex is highly developed and contains many cell types, including glutamatergic neurons which play an important role in learning, memory, and cognitive function^22,23. The first step in generating glutamatergic neurons in culture is to specify telencephalic progenitor cells. Yoshiki Sasai's group first reported the directed differentiation of telencephalic precursors from mouse ESCs (mESCs) using a serum-free suspension culture in the presence of DKK1 (which inhibits Wnt signaling) as well as LeftyA (which inhibits nodal signaling)²⁴. Subsequently, several groups including ours have also reported the specification of telencephalic precursors from human PSCs in serum free medium ^25-27. The generation of telencephalic precursors from human PSCs does not require the use of exogenous morphogens and the efficiency in generating these precursors is much higher than that from mESCs ^26,27. Here, a chemically defined system for neural induction which was well established by Zhang's group⁷ has been described. Without the addition of exogenous caudalizing factors, this protocol efficiently generates telencephalic precursors from human PSCs²⁷. These progenitors can then be differentiated into dorsal or ventral progenitors by regulating the signaling of Wnt and sonic hedgehog (SHH).The dorsal progenitors can further differentiate into glutamatergic neurons efficiently²⁷. In addition, this protocol also works well for the generation of glutamatergic neurons from human iPSCs²⁸, which allows for the generation of patient-specific neurons that can be utilized to explore the mechanism of action as well as potential therapies for a large array of diseases. Moreover, our system also provides a platform to explore the development and specification of diverse neuronal types in the telencephalon.

Protocol

1. Generation of Human Pluripotent Stem Cell Aggregates (D1-D4)

1. Human pluripotent stem cells are cultured on mouse embryonic fibroblast (MEF) feeders in the presence of hESC medium supplemented with basic fibroblast growth factor (bFGF, 4 ng/ml). After 5-7 days in culture, when the colonies are big but still undifferentiated, they are ready for the next step.
2. The enzyme solution should first be prepared. In a 50 ml tube, dissolve the dispase (or collagenase) at a 1 mg/ml concentration into DMEM/F12 medium. Since these solutions mix best when warmed, place the tube containing the medium and enzyme into a 37 °C water bath for 10-15 min and then sterilize it using a Steriflip filter.
3. Aspirate off the medium from the cells, rinse them with DMEM/F12, and aspirate this off as well. Add 1 ml of dispase to each well and place in the incubator for 3-5 min for hESCs (up to 10 min for iPSCs). Look at the cells under the microscope; when the edges curl up slightly/look a bit darker, the cells are ready for the next step. It is best to check the cells after 3 min and put them back in if necessary. When using this protocol for the first time, it is best to start with only one plate of cells at a time as it is important to remove the cells from the dispase as quickly as possible.
4. Aspirate off the dispase from the cells. Gently (the cells are very vulnerable at this stage and will come off of the plate relatively easily) add 1.5 ml of DMEM/F12 medium to each well in order to rinse off the dispase. Aspirate off the DMEM/F12 and add 1.5 ml of hESC medium to each well.
5. To detach and break up the cells, place the tip of a 10 ml glass pipette towards the bottom right hand corner of a well (touching the cells) and move the pipette up and down (vertically) - the upward stoke should be in close proximity to the proceeding downward stroke. Once the other side of the well is reached, repeat in the horizontal direction.
6. Once all of the cells are in suspension, place them in 15 ml tubes and centrifuge them at 200 x g for 2 min. There should be a pellet of cells at the bottom of the tube. Aspirate off the medium from above the pellet.
7. The size of the cell culture flask that should be utilized to keep the aggregates in depends upon the original quantity of cells. For 6 wells (1 plate) of hESCs, one non-tissue culture treated T75 flask with 50 ml of hESC medium should be utilized. Transfer the cells from step 1.6 to the flask and incubate at 37 °C.
8. In order to get rid of the cell debris, the cell medium and flask should be replaced approximately 12 hr later. To do this, place all of the medium/cells into a 50 ml tube, spin the cells down (200 x g for 2 min), and aspirate off the old medium. Next, add 50 ml of fresh hESC medium to the cells, and place into a new T75 flask. The cells can then be fed daily following this same procedure (until day 5).

2. Induction of Neuroepithelial Cells (D5-D17)

1. Transfer the cells/medium to a 50 ml tube, and centrifuge the cells for 2 min at 200 x g. Next, aspirate off the supernatant, and transfer the cells to a new (non-tissue culture treated) T75 flask with 50 ml of neural induction medium (NIM).
2. Once the cells are in NIM, half of the medium should be replaced every other day. The aggregates should become large enough to where they will sink to the bottom of a 50 ml conical tube on their own after a few minutes (no centrifugation required). After 2-3 days in NIM (D7-D8 total), the cells should be ready to plate. Laminin or fetal bovine serum (FBS) can be utilized to help the cells attach to the plates.
3. The number of tissue culture treated 6 well plates that are needed depends upon the density of the cells as each aggregate should be able to grow on the plate for several more days without touching any of the others. On average, the cells from one T75 flask will yield about 4-6 6 well plates worth of cells.
4. 1. If using laminin, dilute it in NIM to 10-20 μg/ml to coat the 6-well plate (~0.5 ml/well). After coating for 2-3 hr, plate cell aggregates onto 6-well plates (~20-30 aggregates per well). After the aggregates have attached, add 1.5 ml of NIM.
   2. If using FBS, prepare a mixture of 90% NIM and 10% FBS (1.5 ml/well). Add 1.5 ml of medium with the cells to each well, gently shake the plate back and forth to spread out the aggregates, and allow the cells to attach O/N in an incubator. Be sure to change the medium (2 ml/well of NIM) the following morning to remove the FBS.
5. After a day or two of being plated, each aggregate will spread out to form a monolayer. Within a couple of days, the centers of most of these cells will morphologically appear similar to columnar cells (elongated). During this period, change the NIM every other day. Some PSC lines (especially iPSC), may form columnar cells less efficiently. If this happens, the addition of BMP and TGF inhibitors (Dorsomorphin²⁹, SB431542¹⁹ at 2 μM) during stage 2 can be helpful.
6. Around days 14-17, the columnar cells will appear more compact. Sometimes these cells form ridges or rings with visible lumen inside. Besides the columnar morphology, cells at this stage express neuroepithelial markers, such as PAX6 and SOX1^12,26.
7. Before the isolation can begin, one must first examine the cells to determine if there are any non-neuroepithelial cells present. An objective marker can be utilized to indicate the non-neuroepithelial colonies. These can then be eliminated by scraping them off with a pipette tip.

3. Generation of Telencephalic Progenitors (D17-D24)

1. The centers of the colonies contain the neuroepithelial cells, and should be isolated from the rest of the colony. This can be done using a micropipette and a sterile 1 ml filtered pipette tip. When a small amount of pressure is applied to the center portion of the colony, it generally lifts off quite readily. Be cautious to not let the outer portion of the colony detach as well. If the centers are being stubborn and will not come off on their own, they can be removed by excising them using a needle under a microscope in a sterile hood. Transfer the center portions of the colonies to a 15 ml tube and spin at 200 x g for 2 min.
2. The medium should be aspirated off of the cells. Two plates worth of cells can then be transferred to a non-tissue culture treated T25 flask containing 10 ml of NIM with the addition of B27 (without vitamin A, 1:50). Use 5-10 ml of medium per one plate of cells.
3. When the cells are viewed the following day, they should have a sphere like appearance. It is necessary to transfer these cells into a new flask (in the presence of NIM and B27, 1:50) as it is often the peripheral cells which sneak in and attach to the bottom of the flask.
4. The neurospheres should then be cultured in suspension for one week using NIM supplemented with B27 (1:50). In order to promote cell endurance and proliferation, cyclic AMP (cAMP, 1 μM) as well as insulin growth factor (IGF1, 10 ng/ml) can be added into the medium during suspension culture. At this point, the neurospheres contain telencephalic progenitors (FOXG1⁺) and are ready to be plated onto coverslips for terminal differentiation.

4. Further Differentiation into the Telencephalic Neurons (D25+)

1. 1. Prepare the poly-ornithine/laminin coated coverslips (in 24-well plates) for plating the cells. Coverslips are first cleaned, and are then coated with ploy-ornithine (at 0.1 mg/ml, 37 °C, O/N, and stored in the freezer).
      Poly-ornithine coating:
      1. Sterilize the coverslips: Place the coverslips in a beaker containing nitric acid and gently shake in the fume hood for one hour. Pour out the nitric acid, rinse several times with DI water, and leave the coverslips under running DI water for at least 15 min. Place the coverslips in 95% ethanol and either shake for 30 min (if using immediately) or store in ethanol at RT.
      2. In a sterile hood, pour out the contents of the 50 ml tube containing sterilized coverslips into the lid of a 6-well plate. Using sterilized forceps, pick up one coverslip at a time and set upright in a single well of a 24-well plate. Repeat so that each well has a coverslip.
      3. After they dry completely, tap the plates until coverslips have fallen flat in each well. Next add 100 μl poly-ornithine (0.1 mg/ml in sterile dH₂O) to each coverslip and incubate the plates O/N at 37 °C.
      4. The next day, remove plates from the incubator and allow them to cool to RT. Aspirate poly-ornithine off of each coverslip (from the edge of the coverslip) making sure not to touch or scratch the coverslips in the process. Allow the coverslips to dry for approximately 30 min before washing.
      5. Add 1 ml sterile dH₂O to each well, let sit for 10 min, and aspirate off the water from each well. Repeat this wash step two more times. After the plates dry completely, (leave covers off in the hood) place the lids on, cover with aluminum foil, and store at -20 °C.
   2. 1. To coat the coverslips with laminin, add 50 μl of a mixture of neural differentiation medium and laminin (final concentration of 20 μg/ml) to each poly-ornithine treated coverslip being careful to only have the liquid touching the coverslip itself (and not spilling off). Incubate them at 37 °C for 1-2 hr before preparing the cells for attachment.
2. Collect and centrifuge the neurospheres at 200 x g for 2 min in a 15 ml tube. Aspirate off the supernatant.
3. 1. Rinse the cells with DMEM-F12 and centrifuge the cells again. If the neurospheres are too large to attach properly, they can be dissociated with Accutase at this step.
   2. If using Accutase: After washing the cells with DMEM-F12, the neurospheres are dissociated to small clusters by adding Accutase (~2 ml) and incubating the cells at 37 °C for 3 min. Next, add the same amount of trypsin inhibitor (~2 ml) and centrifuge the cells at 200 x g for 3 min. The cells should then be re-suspended in NDM and broken using a P200 pipette. The small clusters can then be plated onto pre-coated coverslips.
4. Aspirate off the majority of the medium and place the cells onto a 6 cm Petri dish in a few drops of the medium so that selecting them becomes easier. Add about 4-5 neurospheres to each pre-coated coverslip. There is no need to aspirate off the laminin first.
5. Let the neurospheres attach for at least 2-4 hr. Once they have attached well, more medium (0.5 ml/coverslip) should be added. It should consist of neural differentiation medium supplemented with B27 (1:50), cAMP (1 μM), and neurotrophic factors (BDNF, GDNF, and IGF, all at 10 ng/ml). At this point half of the medium can be changed every other day and these cells can be maintained for several months.

Results

Here, a protocol to differentiate human PSCs into telencephalic glutamatergic neurons through several critical steps: the formation of PSC aggregates, the induction of neuroepithelial cells, the generation of telencephalic progenitors, and the terminal differentiation of these progenitors into telencephalic neurons (Figure 1) has been described. This system is robust and efficient in the generation of telencephalic progenitors and glutamatergic neurons. As an example (Figure 2), without ...

Discussion

There are several critical steps during the neural differentiation process. It is important to ensure that the human PSCs are pluripotent because otherwise the cells may already be biased towards becoming a non-neuronal lineage. This can be confirmed by staining the human PSCs with antibodies against pluripotency markers such as Oct4, Sox2, Nanog, and Tra-1-60 ^1-3. If the human PSCs do not attach very well after passaging them, ROCK inhibitor (Y27632) can be added to help. For those having difficulty with keep...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's modified eagle medium with F12 nutrient mixture (DMEM/F12)	Gibco	11330-032
Knockout Serum Replacer	Gibco	10828-028
L-glutamine (200 mM)	Gibco	25030
Non Essential Amino Acids	Gibco	1140-050
2-Mercapt–thanol (14.3 M)	Sigma	M-7522
Neurobasal medium	Gibco	21103-049
N2	Gibco	17502-048
B27	Gibco	12587-010
Heparin	Sigma	H3149
Poly-L-ornithine hydrobromide (polyornithine)	Sigma	116K5103
Laminin (human)	Sigma	L-6274
Laminin (mouse)	Invitrogen	23017-015
FBS	Gemini	100-106
Bovine serum albumin (BSA)	Sigma	A-7906
Dispase	Gibco	17105-041
Collagenase	Invitrogen	17104-019
Accutase	Innovative Cell Technologies	AT104
ROCK Inhibitor	Stemgent	04-0012
SB431542	Stemgent	04-0010
Dorsomorphin	Stemgent	04-0024
Fibroblast growth factor 2 (FGF2, bFGF)	Invitrogen	13256-029
Trypsin inhibitor	Gibco	17075
0.1% gelatin	Millipore	ES-006-B
Foxg1 antibody	Dr. Y. Sasai
Hoxb4 antibody (1:50)	Developmental Studies Hybridoma Bank	I12
Pax6 antibody (1:5000)	Developmental Studies Hybridoma Bank	PAX6
Nkx2.1 antibody (1:200)	Chemicon	MAB5460
Tbr1 antibody (1:2000)	Chemicon	AB9616
vGLUT1 antibody (1:100)	Synaptic Systems	135302
Brain derived neurotrophic factor (BDNF)	PrepoTech Inc.	450-02
Glial derived neurotrophic factor (GDNF)	PrepoTech Inc.	450-10
Insulin growth factor 1 (IGF1)	PrepoTech Inc.	100-11
Cyclic AMP (cAMP)	Sigma	D-0260
Sonic hedgehog (SHH)	R&D	1845-SH
50 ml tubes	Becton Dickinson (BD)	352098
15 ml tubes	BD	352097
6 well plates	BD	353046
24 well plates	BD	353047
T25 flasks (untreated)	BD	353009
T75 flasks (untreated)	BD	353133
Coverslips	Chemiglass Life Sciences	1760-012
6 cm Petri dishes	BD	353004
9'' glass pipetes	Fisher	13-678-20D
Steriflip filters (0.22 μM)	Millipore	SCGP00525
Stericup filters 1,000 ml (0.22 μM)	Millipore	SCGPU10RE
Phase contrast microscope (Observer A1)	Zeiss	R2625
Carbon dioxide incubator (Hera Cell 150)	Thermo Electron Corporation
Biosafety hood (Sterilgard III Advance)	The Baker Company
Centrifuge (5702 R)	Eppendorf