Transnasal Transplantation of Induced Pluripotent Stem Cell-Derived Human Microglial Cells into the Mouse Brain

Abstract

Source: Parajuli, B., et.al. Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route. J. Vis. Exp. (2022).

This video demonstrates the trans-nasal transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) cells into a mouse brain. Hyaluronidase is applied to the nostrils of a microglial-depleted mouse to enhance permeability. iPSMG suspension is administered intranasally, and the mouse is allowed to recover. Growth factor-rich media is administered regularly to support iPSMG survival in the brain tissue.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of cell medium, transplantation medium, and anesthesia mixture

1. Prepare the cell medium by adding 10% fetal bovine serum and 0.1% penicillin/streptomycin in Dulbecco's modified Eagle medium (DMEM).
2. Prepare the transplantation medium by adding human colony stimulating factor 1 or hCSF1 (250 ng/mL) and human transforming growth factor beta 1 or hTGF-β1 (100 ng/mL) to the cell medium.
3. Prepare the anesthesia mixture for intraperitoneal injection by mixing 0.45 mL of medetomidine hydrochloride, 1.2 mL of midazolam, and 1.5 mL of butorphanol tartrate in 11.8 mL of normal saline.

2. Preparation of induced pluripotent stem cell-derived human microglia (iPSMG) cells

NOTE: Frozen iPSMG (Table of Materials) was kept at -80 °C until use.

1. Thaw the frozen cells quickly in a 37 °C water bath. Swirl the samples until all visible ice has melted.
2. Add the thawed induced pluripotent stem cell-derived human microglia (iPSMG) to the culture media warmed to 37 °C. Add 1 mL of thawed cells containing medium (1 × 10⁶ cells) to 10 mL of the culture media.
3. Centrifuge the cells at 300 x g for 5 min to obtain a cell pellet.
4. After centrifugation, remove all supernatant without disturbing the cell pellet. Complete removal of supernatant is desirable to reduce dilution of the cytokine concentration in the transplantation medium.
5. Add the transplantation medium to obtain a cell concentration of 1 x 10⁵ cells/µL.
6. Place the iPSMG on ice and immediately proceed to transplantation.​
   NOTE: Preparation of iPSMG for transplantation is performed on a clean bench to avoid contamination.

3. Preparation of the mouse for transnasal transplantation (Tsn)

1. Feed the wild-type male mice (C57BL/6J, 8 weeks old) with a PLX5622-containing diet for 7 days.
2. At the end of the 7^th day, cease the PLX diet and feed the mice with a normal diet till the end of study.
   NOTE: PLX5622 containing diet is prepared by adding 1.2 g of PLX5622 in 1 kg of AIN-93G (Table of Materials).

4. Transnasal transplantation of cells

1. 24 h after the cessation of PLX feeding, weigh the mice and anesthetize them using an intraperitoneal injection of the anesthesia mixture (0.2 mL/20 g).
2. After the mice are completely anesthetized as assessed by unresponsiveness to pedal withdrawal reflex (firm toe pinch), administer 2.5 µL of hyaluronidase in phosphate buffered saline (PBS) (100 U/mL), 1 h before transnasal transplantation of iPSMG to each nostril twice using a 10 µL pipette tip to increase the permeability of the nasal mucosa.
3. After the application of hyaluronidase, place the mice in the supine position.
4. Repeat step 4.2 10 min before the transnasal transplantation of iPSMG.
5. Apply 2.5 µL of cell suspension into one nostril of the mouse using a 10 µL pipette tip.
6. Place the mouse in the supine position for 5 min before administration of the cell suspension to the other nostril.
7. Repeat steps 4.5 and 4.6 four times, applying a total volume of 20 µL per animal.
8. Place the mouse in the supine position on a 37 °C heat pad until recovery from anesthesia.
9. At 48 h after the cessation of PLX feeding, repeat steps 4.1-4.7 on the same mice once again.
   ​NOTE: It should be noted that iPSMG cells are placed on ice during the transplantation.

5. Application of cytokines

1. Anesthetize the mice by an intraperitoneal injection of anesthesia mixture (0.2 mL/20 g).
2. Apply 2.5 µL of the transplantation medium into one nostril of the mouse using a 10 µL pipette tip.
3. Place the mouse in the supine position for 5 min before administering the transplantation medium to the other nostril.
4. Repeat steps 5.2 and 5.3 four times, applying a total volume of 20 µL per animal.
   NOTE: It should be noted that transnasal administration of transplantation medium (human cytokines) is required every 12 h for the viability of transplanted iPSMG till the end of the study.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium (DMEM)	Thermo Fisher Scientific	10566
AIN 93G	Oriental Yeast Co
Fetal bovine serum	GE Healthcare Life Sciences	SH30070.03
Frozen iPSMG	Shionogi & Co., Ltd		Laboratory for Drug Discovery and Disease Research
Human colony stimulating factor 1 (hCSF1)	PeproTech	300-25
Hyaluronidase	Sigma-Aldrich	H-3506
Medetomidine hydrochloride	Meiji Seika	VETLI5
Midazolam	Astellas	18005A2
Paraformaldehyde	Wako Pure Chemical Industries	162-16065
Penicillin/streptomycin	Thermo Fisher Scientific	15140-122
Pipette	Eppendorf	3120000011
Pipette tip	Eppendorf	30076028
PLX5622	Amadis Chemical	A930097
Transforming growth factor-β1 (Tgf-b1)	PeproTech	100-21