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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

Streszczenie

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

Wprowadzenie

In both mice and humans, roughly half of all cortical inhibitory interneurons (CIns) originate within a transient subcortical structure known as the medial ganglionic eminence (MGE), where the neuroepithelial progenitors of CIns and other neuronal and glial subgroups express the transcription factor Nkx2.11,2. CIn subgroups or subtypes are defined by intersecting morphological, neurochemical, electrophysiological, and connectivity characteristics3,4. The MGE-derived CIns can be grouped into mostly non-overlapping subgroups based on their expression of either PV or SST, the expression of which correlates with particular electrophysiological and connectivity tendencies5. Dysfunction of interneurons, especially those in the PV subgroup, has been implicated in multiple neuropsychiatric disorders and diseases6,7. The overall goal of this method is to produce stem cell-derived mitotic progenitors and migratory precursors enriched for either PV or SST CIn fate for studying cortical interneuron biology and for use in cell-based therapies.

We have developed a scalable, highly efficient method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mESCs. Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line8, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via FACS and subsequently used in a number of downstream applications. By manipulating a number of signaling pathways, duration of culture, and mode of neurogenesis, we can obtain millions of fluorescently labeled interneuron precursors suitable for a host of downstream applications.

Although several other methods exist for generating MGE-like progenitors from mESCs9,10,11,12,13,14, our method, which relies on the Wnt antagonist XAV-939, is particularly efficient at generating Foxg1/Nkx2.1 co-expressing telencephalic progenitors. In addition, the ability to select for interneuron progenitors or their post-mitotic Lhx6-expressing progeny via our dual reporter system, greatly enhances the capacity to generate distinct progenitors and their progeny.

Protokół

NOTE: The dual reporter mESC line described in this protocol is available upon request (sande@mail.med.upenn.edu).

1. Media Preparation

NOTE: Warm all media to 37 °C before use in cell culture.

  1. Mouse Embryonic Fibroblast (MEF) media (for preparing 500 mL)
    1. Add 50 mL fetal bovine serum (FBS) to 449 mL of Dulbecco's Modified Eagle's Medium (DMEM), and filter through a 500 mL 0.22 µm pore filter unit.
    2. Add 1 mL antimicrobial agent (50 mg/mL) after filtration. Store the media at 4 °C for up to 1 month or aliquot and store at ≤ -20 °C.
  2. N2 media (for preparing 500 mL)
    1. Add 5 mL L-alanine-L-glutamine (100x) and 500 µL 2-Mercaptoethanol (55 mM) to 489.5 mL DMEM: Nutrient Mixture F-12 (DMEM/F-12), and filter through a 500 mL 0.22 µm pore filter unit.
    2. Add 1 mL antimicrobial agent (50 mg/mL) and 5 mL N2 supplement-B after filtration. Store the media at 4 °C in the dark for up to 1 month.
  3. Serum-free growth medium (KSR) (for preparing 500 mL)
    1. Add 75 mL serum-free medium supplement, 5 mL L-glutamine (100x), 5 mL MEM Non-Essential Amino Acids (MEM-NEAA) (100x), and 500 µL 2-Mercaptoethanol (55 mM) to 413.5 mL non-glutamine containing DMEM, and filter through a 500 mL 0.22 µm pore filter unit.
    2. Add 1 mL antimicrobial agent (50 mg/mL) after filtration. Store the media at 4 °C for up to 1 month.
  4. Mouse embryonic stem cell media (for preparing 500 mL)
    1. Add 75 mL stem cell grade FBS, 5 mL MEM-NEAA (100x), 5 mL L-glutamine (100x), and 500 µL 2-Mercaptoethanol (55 mM) to 413.5 mL non-glutamine containing DMEM, and filter through a 500 mL 0.22 µm pore filter unit.
    2. Add 1 mL antimicrobial agent (50 mg/mL) after filtration. Store the media at 4 °C in the dark for up to 1 month or aliquot and store at ≤ -20 °C.

2. Culturing mESCs

  1. Plate mitotically inactive MEF feeder cells in MEF media onto tissue culture treated plates at 3-4 x 104 cells/cm2. Allow at least 12 h for them to settle in a cell culture incubator at 37 °C with ≥ 95% relative humidity and 5% CO2 before plating the mESCs. If not used immediately, replace the MEF media every 3 days. Dispose of MEFs if not used within 7 days of plating.
  2. Add mESCs (density range from 1-4 x 104 cells/cm2) to MEF plates in mESC media containing Mouse Leukemia Inhibitory Factor (mLIF) (1,000 U/mL). Incubate the cells at 37 °C with ≥ 95% relative humidity and 5% CO2.
  3. Passage mESCs using trypsin-EDTA (0.05% trypsin) (1:5-1:10) once the dish becomes ~ 70-80% confluent or if the colonies are beginning to touch. This typically occurs every 2 days, but can take up to 4 or 5 days depending on the initial plating density.
  4. Maintain the mESCs on a MEF feeder layer in mESC medium to keep pluripotency.
  5. Before starting differentiation, passage the cells at least once onto gelatin coated plates without MEFs to dilute out the MEFs.
    1. Prepare gelatin coated plates by adding 0.1% gelatin in phosphate buffered saline (PBS) with Ca2+/Mg2+ to a tissue culture treated dish and leaving at 37 °C for at least 1 h. Plate 1.5-2 x 106 cells/10 cm plate (2.7-3.6 x 104 cells/cm2) and allow the cells to expand for 2 days before beginning differentiation.

3. Differentiating mESCs Toward MGE-like Telencephalic Progenitors

  1. Differentiation day (DD) 0 = "float cells"
    1. After the mESCs have grown on gelatin coated plates for 2 days, aspirate the media and wash the cells once with PBS without Ca2+/Mg2+. Add enough trypsin-EDTA (0.05% trypsin) to cover the surface of the plate (typically 4 mL trypsin-EDTA for one 10 cm tissue culture dish), and place the cells back into the incubator at 37 °C with ≥95% relative humidity and 5% CO2 for 4 min.
    2. After 4 min, quench the trypsin-EDTA using 2 times the volume with mESC media. Transfer the cells to an appropriately sized centrifuge tube and centrifuge the cells at 200 x g for 5 min. After 5 min, remove the tube and aspirate the media without disturbing the pellet. Resuspend the pellet in 1 mL KSR:N2 media (1:1) containing the BMP inhibitor LDN-193189 (250 nM) and the Wnt inhibitor XAV-939 (10 µM).
    3. Measure the cell concentration using a hemocytometer or automated cell counter. Start growing cells as embryoid bodies (EBs) by adding 75,000 cells/mL in KSR:N2 media (1:1) containing LDN-193189 (250 nM) and XAV-939 (10 µM) in non-adherent tissue culture dishes. Incubate the cells at 37 °C with ≥ 95% relative humidity and 5% CO2.
  2. On DD1, prepare for cell "landing" by coating tissue culture treated dishes with poly-L-lysine (10 µg/mL in PBS with Ca2+/Mg2+) overnight (O/N) at 37 °C with ≥ 95% relative humidity or for at least 1 h.
  3. On DD2, aspirate the poly-L-lysine and coat the plates with laminin (10 µg/mL in PBS with Ca2+/Mg2+) O/N at 37 °C with ≥ 95% relative humidity.
    NOTE: While O/N is optimal, as little as 2 h may be sufficient. If plates are not used by the next day, aspirate laminin, replace with PBS without Ca2+/Mg2+, and store at 4 °C for up to 2 weeks.
  4. DD3 ("land cells")
    1. Before beginning EB dissociation, aspirate the laminin and allow the plates to completely dry in a tissue culture hood. Do not use plates that appear shiny or visibly wet. Transfer the EBs with media into a 15 mL tube and centrifuge for 3-4 min at 15 x g or until the EBs have pelleted.
  5. Aspirate the media and add 3 mL of cell detachment solution containing DNase (2 U/mL) and incubate at 37 °C with ≥ 95% relative humidity and 5% CO2 for 15 min. Gently flick the tube every 3 min to aid in EB dissociation.
  6. Once the EBs are no longer visible or 15 min have elapsed, add 6 mL KSR:N2 (1:1) containing DNase (1 U/mL) and centrifuge for 5 min at 200 x g.
  7. Plate the cells in KSR:N2 (1:1) containing LDN-193189 (250 nM), XAV-939 (10 µM), and the ROCK inhibitor Y-27632 (10 µM) at 4.5-5 x 104 cells/cm2.

4. SST-enriching Protocol: "DD12 GFP High Sonic Hedgehog (SHH)" (continuation from step 3.7)

  1. On DD5, change media with KSR:N2 (1:1) containing FGF-2 (10 ng/mL), IGF-1 (20 ng/mL), and SSH (50 ng/mL).
  2. Prepare tissue culture plates for re-plating on day 8 (step 4.4) using the instructions outlined in steps 3.2-3.3.
  3. On DD7, change media with KSR:N2 (1:1) containing FGF-2 (10 ng/mL), IGF-1 (20 ng/mL), and SHH (50 ng/mL).
  4. On DD8, re-plate the cells.
    NOTE: While this step is not essential, re-plating the cells can reduce early-differentiated, unwanted cell types. Re-plating also breaks up balls of cells that make downstream immunohistochemical analyses difficult, and increases the efficiency of FACS at later time points.
    1. To re-plate cells, detach the cells from the plate with trypsin-EDTA (0.05% trypsin) for 5 min at 37 °C with ≥95% relative humidity and 5% CO2. Quench the trypsin-EDTA using 2 times the volume with KSR:N2, and centrifuge at 200 x g for 5 min. Filter the cells through a 40 µm filter tube to remove any clumps. Re-plate the cells at 250,000 cells/cm2 in N2/KSR (1:1) containing FGF-2 (10 ng/mL), IGF-1 (20 ng/mL), and Y-27632 (10 µM) with SHH (50 ng/mL).
      NOTE: If instead, the decision is made not to re-plate the cells on DD8, change the media with KSR:N2 containing SHH (50 ng/mL) every 2 days from DD7 to DD12 and continue to add IGF-1 (20 ng/mL) and FGF-2 (10 ng/mL) until DD9.
  5. On DD10, change the media with KSR:N2 containing SHH (50 ng/mL).
  6. On DD12, to obtain cells that are enriched for SST subtypes, use FACS to isolate Lhx6::GFP-only expressing cells.
    1. To detach the cells, use a non-trypsin containing cell-dissociation reagent rather than trypsin-EDTA. Wash cells once with PBS without Ca2+/Mg2+. Add pre-warmed non-trypsin containing cell-dissociation reagent containing DNase (2 U/mL) to the cells. Place them in the incubator at 37 °C with ≥95% relative humidity and 5% CO2 for 10-30 min or until the cells have detached. Gently tap the plates every 5 min to aid the dissociation.
      NOTE: For DD11-12 cultures, only 10-15 min may be necessary. For DD15-16 cultures, 30 min or more may be required for complete dissociation.
    2. Once the cells have completely lifted off the plate, proceed to FACS isolation using standard protocols or use the cells in other downstream analyses.

5. PV-enriching Protocol: "DD11/DD17 mCherry + aPKCi" (continuation from step 3.7)

  1. On DD5, change the media with KSR:N2 (1:1) containing FGF-2 (10 ng/mL) and IGF-1 (20 ng/mL).
  2. Prepare tissue culture plates for re-plating on DD8 (step 4.4) using the instructions outlined in steps 3.2-3.3.
  3. On DD7, change the media with KSR:N2 (1:1) containing FGF-2 (10 ng/mL) and IGF-1 (20 ng/mL).
  4. On DD8, re-plate the cells as described in step 4.4 with the following exceptions: when re-plating the cells, replace SHH with smoothened agonist (SAG; 30 nM) and add atypical PKC inhibitor (aPKCi; 2 µM). Taken together, the re-plating media is now N2/KSR (1:1) containing FGF-2 (10 ng/mL), IGF-1 (20 ng/mL), Y-27632 (10 µM), SAG (30 nM), and aPKCi (2 µM).
  5. On DD10, change the media with KSR:N2 containing aPKCi (2 µM).
  6. DD11
    NOTE: At this point, Nkx2.1::mCherry+ cells are enriched to become PV CIns.
    1. Detach the cells from the plate for FACS using the same protocol outlined in step 4.6. If the decision is made to collect Nkx2.1::mCherry+ cells at a later differentiation day, disregard this step.
  7. On DD12, change the media with KSR:N2 containing aPKCi (2 µM). On DD14, change the media with KSR:N2 containing aPKCi (2 µM). On DD16, change the media with KSR:N2 containing aPKCi (2 µM). On DD17, collect the Nkx2.1::mCherry+ cells using the same protocol outlined in step 4.6.

6. PV-enriching Protocol: "DD17 mCherry Low SHH" (continuation from step 3.7)

  1. On DD5, change the media with KSR:N2 (1:1) containing FGF-2 (10 ng/mL) and IGF-1 (20 ng/mL). On DD7, change the media with KSR:N2 (1:1) containing FGF-2 (10 ng/mL) and IGF-1 (20 ng/mL).
  2. On DD9, change the media with KSR:N2 (1:1). From this point onward, no additional growth factors are necessary.
    1. Continue to change the media every 2 days until harvesting the cells on DD17.

Wyniki

The protocol described in this paper is a modified version of our published protocols15,16,17 and has been optimized for use with our Nkx2.1::mCherry:Lhx6::GFP dual-reporter mESC line. By adding the Wnt inhibitor XAV-939 from DD0-5, combined with re-plating at DD8, we achieve robust Nkx2.1 induction, wherein upwards of 50% of all DAPI+ nuclei in culture are also Nkx2.1 expressing (

Dyskusje

While this method is highly effective at patterning J1-derived mESCs (SCRC-1010), we have experienced variable success with other mESC lines and clonal isolates. For instance, Foxg1::venus mESCs (EB3-derived; Danjo et al.13) respond poorly to this protocol and Foxg1 induction by DD12 is typically on the order of 1-2%. For reasons that we do not fully understand, another Nkx2.1::mCherry:Lhx6::GFP dual reporter clone (termed JQ59) that was isolated simultaneously as the line described in th...

Ujawnienia

The authors have nothing to disclose.

Podziękowania

We are grateful to Qing Xu for developing the Nkx2.1::mCherry:Lhx6:GFP dual reporter mESC line as well as Jennifer Tyson, Asif Maroof, and Tim Petros for their early work on helping to develop this protocol. We also thank the CHOP flow cytometry core for technical assistance. This work was supported by an NIH R01 MH066912 (SA) and F30 MH105045-02 (DT).

Materiały

NameCompanyCatalog NumberComments
Bottle-top vacuum filter systemCorningCLS430769
Test Tube with Cell Strainer Snap CapThermoFisherCorning 352235
Mouse embryonic fibroblasts (CF-1 MEF IRR 7M)MTI-GlobalstemGSC-6101G1 vial of 7M MEFs is sufficient for four 10-cm TC plates. References: 29,35
FBSAtlanta BiologicalsS11150H
PrimocinInvivogenAnt-pm-2Also known as antimicrobial agent. Do not filter with base media -- add after filtration. References: 9,11,36,37
N2 supplement-BStemcell Technologies7156Do not filter with base media -- add after filtration
Glutamax (100x)ThermoFisher35050061Also known as L-alanine-L-glutamine. References: 9,11,38,39
KnockOut Serum Replacement (KSR)ThermoFisher10828028Also known as serum-free medium supplement. References: 9,11
L-glutamine (100x)ThermoFisher25030081
MEM-NEAA (100x)ThermoFisher11140050
2-MercaptoethanolThermoFisher21985023
KnockOut DMEMThermoFisher10829018Also known as non-glutamine containing DMEM. References: 9,11
Hyclone FBSVWR82013-578Also known as stem cell grade FBS. References: 9,11
Tissue culture treated dish (10cm)BD Falcon353003
Non-adherent sterile petri dish (10cm)VWR25384-342
Leukemia inhibitory factor (mLIF)ChemiconESG1107Do not freeze, store at 4'C. References: 9,11
DMEM/F12ThermoFisher11330032
0.1% Gelatin SolutionATCCATCC PCS-999-027
LamininSigmaL2020
Poly-L-lysineSigmaP6282
Trypsin-EDTA (0.05%)ThermoFisher25300054
AccutaseThermoFisherA1110501Also known as non-trypsin containing cell dissociation reagent. References: 9,11
RQ1 RNase-Free DNasePromegaM610A
LDN-193189Stemgent04-0074Resuspend in DMSO and store at -80'C in single use aliquots
XAV939Stemgent04-0046Resuspend in DMSO and store at -80'C in single use aliquots
rhFGF-2R&D Systems233-FBResuspend in PBS with 0.1% BSA and store at -80'C in single use aliquots
rhIGF-2R&D Systems291-G1Resuspend in PBS with 0.1% BSA and store at -80'C in single use aliquots
ROCK inhibitor (Y-27632)Tocris1254Resuspend in DMSO and store at -80'C in single use aliquots
Smoothened agonist (SAG)Millipore566660-1MGResuspend in H20 and store at -80'C in single use aliquots
rm Sonic Hedgehog/SHHR&D Systems464-SH-025Resuspend in PBS with 0.1% BSA and store at -80'C in single use aliquots
PKCζ Pseudosubstrate Inhibitor, MyristoylatedEMD Millipore539624Resuspend in H20 and store at -80'C in single use aliquots

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