Transplantation of Stem Cell-Derived Neuronal Progenitors into Human Cortical Slices

Abstract

Source: Palma-Tortosa, S., et.al. Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation. J. Vis. Exp. (2022)

This video demonstrates the transplantation of stem cell-derived neuronal progenitor cells into human cortical tissue slices. The procedure involves isolating and resuspending progenitor cells in a basement membrane matrix, followed by their injection into a cortical slice placed on a transwell assembly. After incubation to allow matrix solidification and neuronal differentiation, the transplanted tissue is prepared for further analysis.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Proliferation and differentiation of lt-NES cells

NOTE: human induced pluripotent stem cell-derived long-term self-renewing neuroepithelial-like stem (lt-NES) cells are generated and transduced with a lentiviral vector carrying green fluorescent protein (GFP) under a constitutive promoter (GFP-lt-NES cells). Vials containing 3 x 10⁶ cells are stored at −150 °C until use.

1. Preparation of the stock solutions and media

1. Dilute 100 µg of basic fibroblast growth factor (bFGF) in 10 mL of phosphate-buffered saline (PBS)-0.1% bovine serum albumin (BSA) to have a stock concentration of 10 µg/mL. Prepare 100 µL aliquots.

2. Dilute 100 µg of epidermal growth factor (EGF) in 10 mL of PBS-0.1% BSA to have a stock concentration of 10 µg/mL. Prepare 100 µL aliquots.

3. Aliquot 50x B27 supplement in a volume of 100 µL per aliquot.

4. Dilute 10 mg of poly-L-ornithine in 100 mL of deionized water to have a stock concentration of 100 µg/mL. Make 1 mL aliquots.

5. Prepare 30 µL aliquots of 1.20 mg/mL mouse laminin.

6. Dilute 10 mL of trypsin-ethylenediaminetetraacetic acid (EDTA) (0.25%) in 90 mL of PBS to a stock concentration of 0.025%. Prepare 1 mL aliquots.

7. Dilute 0.025 g of trypsin inhibitor in 50 mL of PBS to a stock concentration of 0.5 mg/mL. Prepare 1 mL aliquots.

8. Dilute 10 µg of Wnt3a (Wingless-type MMTV integration site family member 3A) in 1 mL of PBS-0.1% BSA to have a stock concentration of 10 µg/mL. Prepare BMP4 (bone morphogenetic protein 4) in the same way. Aliquot into a volume of 100 µL.

9. Dilute 1 mg of cyclopamine in 2.5 mL of dimethyl sulfoxide (DMSO) to a final concentration of 400 µg/mL, and make 100 µL aliquots.

10. Prepare basic medium (Table 1) and differentiation defined medium (DDM, Table 2), and store at 4 °C for up to 2 weeks.

2. Coating of culture dishes

1. To pre-coat the dishes to culture the GFP-It-NES cells during proliferation or differentiation, dilute poly-L-ornithine 1:100 in deionized water and add 5 mL of the solution to a T25 flask. Incubate overnight at room temperature (RT).

2. Wash the poly-L-ornithine coated plates 1x with deionized water and 1x with PBS.

3. For GFP-It-NES cells in proliferation, dilute mouse laminin at 1:500 in PBS and incubate for at least 2 h at 37 °C. For GFP-It-NES cells in differentiation, dilute mouse laminin at 1:100 in PBS and incubate for at least 2 h at 37 °C.

3. Proliferation of the GFP-lt-NES cells

1. Warm 5 mL (for washing) and 5 mL (for seeding) of basic medium in two different 15 mL tubes.

2. Rapidly thaw one vial of GFP-lt-NES cells at 37 °C, transfer them to the washing tube, and centrifuge at 300 x g for 5 min.

3. Aspirate the medium carefully without touching the pellet and resuspend the cells in 1 mL of prewarmed basic medium. Transfer the cell suspension to the seeding tube containing basic medium supplemented with proliferation factors: EGF (10 ng/ mL), bFGF (10 ng/mL), and B27 (10 ng/mL). Seed the cells on a poly-L-ornithine/laminin-coated T25 flask.

4. Feed the cells with proliferation factors every day. Replace the medium if it turns yellow. Passage the cells every third or fourth day (1 day after they reach 100% confluency).

4. Splitting the GFP-lt-NES cells for proliferation and differentiation

1. Pre-warm 5 mL of basic medium per flask (to collect the cells), plus the total volume needed to reseed them.
NOTE: The passage is done at a 1:3 dilution to keep the cells in proliferation, requiring 15 mL of basic medium, and a 1:6 dilution to start differentiation, requiring 30 mL of basic medium.

2. Remove the medium from the cell culture flask by aspiration and add 500 µL of pre-warmed 0.025% trypsin. Incubate for 5-10 min at RT. The detachment of the cells can be confirmed under a standard light microscope at 10x magnification.

3. Add an equal volume of trypsin inhibitor (0.5 mg/ mL final concentration) followed by 5 mL of warm basic medium. Detach and collect the cells by gently pipetting up and down. Transfer the cells to a 15 mL tube and centrifuge for 5 min at 300 x g.

4. To keep the cells in proliferation, replate at a 1:3 dilution in fresh basic medium supplemented with proliferation factors, and repeat step 1.3.4.

5. Cortical differentiation of the GFP-lt-NES cells

1. On day 0, resuspend the cells (to be used for differentiation) in 30 mL of basic medium supplemented with proliferation factors, and plate them in six differentiation-coated T25 flasks (split 1:6).

2. On day 1, change half of the medium to DDM, and add proliferation factors at half of their concentration.

3. On day 2, change the medium completely to DDM supplemented with differentiation factors: BMP4 (10 ng/mL), Wnt3a (10 ng/mL), and cyclopamine (400 ng/mL).

4. On day 4, add the differentiation factors alone. Replace the medium if it turns yellow.

5. On day 6, change the medium to DDM supplemented with BMP4 and Wnt3a. The cyclopamine is removed at this step.

6. On day 7, detach the cells as stated in step 1.4.2 and step 1.4.3.

2. Transplantation of the GFP-lt-NES cells into organotypic hACtx slices

NOTE: The adult human cortical (hACtx) tissue should be cultured for 1 week prior to cell transplantation. To facilitate the transplantation procedure, it is necessary to remove 2 mL of the hACtx medium from the top of the insert to prevent the tissue from floating.

1. Resuspend the cortically primed GFP-lt-NES cells (from step 1.5.6) in cold pure basement membrane matrix (see the Table of Materials) at a concentration of 1 x 105 cells/µL and transfer the solution to a smaller sterile tube.

NOTE: During the transplantation procedure, all the materials (pipette tips, tubes, capillary, etc.) should be pre-cooled to avoid gel solidification. Thaw the basement membrane matrix gel on ice for 30 min before its use.

2. Collect the cell suspension into a cold glass capillary connected to a rubber teat for suction. Inject the cell suspension as small drops (approx. 1 µL each) by stabbing the semi-dry tissue slice at various sites.

3. Incubate at 37 °C for 30 min for the gel to solidify. Transfer the plate from the incubator back to the hood and carefully add 2 mL of hACtx medium to the top of the insert to completely submerge the tissue.

4. Replace the culture medium with fresh hACtx medium once per week.

Results

Table 1: Composition of proliferation medium of lt-NES cells (basic medium).

Basic Medium	Stock concentration	Final concentration	Per 100 mL
DMEM/F12 with L-Glutamine	1x		98.7 mL
N-2 supplement	100x	1:100	1 mL
Glucose	45%	3.5 mL/L	350 µL

...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MANTAINANCE AND CULTURE OF HUMAN NEOCORTICAL TISSUE
6-well plate	ThermoFisher Scientific	140675
Alvetex scaffold 6 well insert	Reinnervate Ltd	AVP004-96
B27 Supplement (50x)	ThermoFisher Scientific	17504001
BrainPhys without Phenol Red	StemCell technologies	#05791	Referenced as neuronal medium in the text
Filter units 250 mL or 500 mL	Corning Sigma	CLS431096/97
Forceps	Any suitable
Gentamicin (50 mg/mL)	ThermoFisher Scientific	15750037
Glutamax Supplement (100x)	ThermoFisher Scientific	35050061	Referenced as L-glutamine in the text
Rubber teat + Glass pipette	Any suitable
GENERATION OF lt-NES cells
2-Mercaptoethanol 50 mM	ThermoFisher Scientific	31350010
Animal Free Recombinant EGF	Peprotech	AF-100-15
B27 Suplemment (50x)	Thermo Fisher Scientific	17504001
bFGF	Peprotech	AF-100-18B
Bovine Albumin Fraction V (7.5% solution)	ThermoFisher Scientific	15260037
Cyclopamine, V. calcifornicum	Calbiochem	# 239803
D (+) Glucose solution (45%)	Sigma	G8769
Dimethyl sulfoxide (DMSO)	Sigma Aldrich	D2438-10mL
DMEM/F12	ThermoFisher Scientific	11320074
Dulbecco's Phosphate Buffer Saline (DPBS)	Thermo Fisher Scientific	14190-144	Without calcium and magnesium
Laminin Mouse Protein, Natural	Thermo Fisher Scientific	23017015
MEM Non-essential aminoacids solutions (100x)	ThermoFisher Scientific	11140050
N-2 Supplement (100 x)	ThermoFisher Scientific	17502001
Poly-L-Ornithine	Merk	P3655
Recombinant Human BMP-4 Protein	R&D Systems	314-BP-010
Recombinant Human Wnt-3a Protein	R&D Systems	5036-WN
Sodium Pyruvate (100 mM)	ThermoFisher Scientific	11360070
Soybean Trypsin Inhibitor, powder	Thermo Fisher Scientific	17075029
Sterile deionized water	MilliQ		MilliQ filter system
Trypsin EDTA (0.25%)	Sigma	T4049-500ML
EQUIPMENT FOR CELL CULTURE
Adjustable volume pipettes 10, 100, 200, 1000 µL	Eppendorf	Various
Basement membrane matrix ESC-qualified (Matrigel)	Corning	CLS354277-1EA
Centrifuge	Hettich Centrifugen	Rotina 420R	5% CO2, 37 °C
Incubator	ThermoForma Steri-Cult CO2	HEPA Class100
Stem cell cutting tool 0.190-0.210 mm	Vitrolife	14601
Sterile tubes	Sarstedt	Various
Sterile Disposable Glass Pasteur Pipettes 150 mm	VWR	612-1701
Sterile pipette tips 0.1-1000 µL	Biotix VWR	Various
Sterile Serological Pipettes 5, 10, 25, 50 mL	Costar	Various
T25 flasks Nunc	ThermoFisher Scientific	156367