Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Summary

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

Abstract

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26−28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Introduction

Supplemental oxygen is often required to treat newborn infants with respiratory distress¹. However, hyperoxia therapy in infants has adverse long-term effects. Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD)². BPD is a major complication of hyperoxia treatment that can occur in spite of early surfactant therapy, optimal ventilation procedures, and increased use of noninvasive positive pressure ventilation in premature infants. While many treatment strategies have been reported for BPD³, no known therapy can reduce this complication.

Corticosteroid use is one potential treatment to prevent BPD, because pulmonary inflammation plays a crucial role in its pathogenesis. However, systemic corticosteroid therapy is not usually recommended for preterm infants due to long-term adverse effects^4,5.

Mesenchymal stromal cells (MSCs) have pluripotent characteristics and can differentiate into various cell types, including bone, cartilage, adipose tissue, muscle, and tendons⁶. MSCs have immunomodulatory, anti-inflammatory, and regenerative effects⁷, and animal studies show the therapeutic benefits of MSCs and their secreted components in hyperoxia-induced lung injury in rodents^8,9. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and corticosteroid therapy might be a potential treatment strategy to treat newborns with respiratory disorders. Preclinical studies have used intratracheal administration of stem cells and adeno-associated virus in newborn rats^10,11,12. However, a step-by-step presentation of the technique and in vivo tracking of the transplanted stem cells is not available. The newborn rat is appropriate for studying the effects of intratracheal administration on preterm infants with respiratory distress because the saccular stage of the rat lung at birth is equivalent to that of the human lung at 26−28 week of gestation¹³. An effective method for administration into the rat trachea is crucial for successful pulmonary distribution. The technique presented here allows for the study of intratracheal administration of cells and/or drugs for treatment of neonatal pulmonary diseases using rats as a model for humans.

Protocol

This procedure was approved by the Animal Care and Use Committee at Taipei Medical University.

NOTE: Human MSCs stably transfected with green fluorescent protein (GFP) and firefly luciferase genes (Fluc) were obtained from a commercial company (Table of Materials).

1. Characterization of human MSCs with firefly luciferase and green fluorescent protein

1. Maintain human MSCs transfected with GFP and Fluc in complete media (minimum essential medium eagle-alpha modification [αMEM], supplemented with 10−15% fetal bovine serum [FBS], 2 mM L-glutamine, 1 ng/mL basic FGF, and PSF) at 37 °C with saturated humidity and 5% CO₂. Passage cells at ~70−90% confluence.
2. Observe MSCs under a fluorescence phase contrast microscope (Figure 1A) and analyze the expression levels of Fluc and GFP¹⁴.
3. Characterize the MSCs by analyzing the expression of CD markers including CD44, CD73, CD90, CD105 using flow cytometry (Figure 1B). Induce trilineage differentiation of stem cells to adipocytes, chondrocytes, and osteocytes, and confirm trilineage differentiation (Figure 1C) by von Kossa, oil red O, and Alcian blue staining following a commercial protocol^15,16.

2. Anesthetization of rat pups

1. Allow time-dated pregnant Sprague–Dawley rats to deliver vaginally at term.
2. Remove the rat pups from the cage on postnatal day 5.
3. Anesthetize the rat pups using gas anesthesia (i.e., 2% isoflurane) in an anesthesia chamber.
4. Confirm adequate anesthesia by checking breathing and reflexes.
   NOTE: Breathing should become shallow and reflexes should diminish. Rat pups remain unconscious for at least ~10 min with this isoflurane concentration.

3. Intratracheal instillation

1. Once anesthetized, restrain the rat pups on an intubation stand angled at ~60° and hold pups in place with laboratory labeling tape on all four limbs.
2. Apply tape below the nose to fix the head and pinpoint the neck for puncture tracheotomy.
3. Disinfect the incision area (i.e., neck) with a 75% alcohol prep pad.
4. Make a 0.3 cm vertical midline neck incision above the trachea with microscissors to avoid damaging the carotid arteries.
5. Dissect the fat and muscle layers away to locate the trachea with curved tip tapered tweezers without a hook.
6. Hold the trachea with the curved tip tapered tweezers.
7. Hold a 100 μL syringe upright and slowly inject 30 μL of normal saline (i.e., control) or 30 μL of Fluc-GFP labeled MSCs (1 x 10⁵ cells) into the trachea through a 30 G needle syringe during inspiratory phase.
8. Close the incision with one 6-0 silk stitch, tie the knot as small as possible, and cut the ends as short as possible.
9. Place the rats under infrared light or on a heating pad to keep warm and allow them to recover from anesthesia.
10. Confirm the rats are warm, pink, and capable of spontaneous movement before returning the rats to the cage.

4. Monitoring the pulmonary distribution of MSCs

1. To track the transplanted human MSCs, intraperitoneally inject the rats with luciferin potassium salt in phosphate buffered saline (PBS) at a dose of 125 mg/kg of body weight 15 min after MSC injection.
2. Anesthetize the rats using 2% isoflurane through nose cones.
3. Acquire sequential images at 5–15 s intervals 10 min after luciferin administration with medium binning, 1 f/stop, and a 26 cm field of view using a small-animal imaging system (Table of Materials).
4. Quantify the luminescence activity from the lungs based on the automatic regions of interest using imaging software (Table of Materials)¹⁷.

Results

The pulmonary distribution of intratracheal instillation of stem cells in the term neonatal rats was determined by firefly luciferase (Fluc)-labeled stem cells. MSCs were labeled with Fluc and tagged with green fluorescent protein through lentiviral transduction. Figure 1A demonstrates a high level of GFP expression in human MSCs, and 93.7% of the population showed GFP positive expression detected by flow cytometry. MSCs were characterized by analyzing the expression of CD markers (i.e., CD ...

Discussion

Newborn infants with respiratory distress commonly require intratracheal surfactant and/or corticosteroid treatment¹⁹. Human phase I clinical trials have demonstrated the safety of intratracheal MSCs in preterm infants⁸. These studies suggest that intratracheal administration of drugs is an important option for newborn infants with respiratory distress. Animal model studies are most helpful if the model features are directly pertinent to humans. Term...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-0 silk	Ethicon	1916G
Alcohol Prep Pad	CSD	3032
BD Stemflow hMSC Analysis Kit	BD Biosciences	562245	CD markers
CMV-Luciferase-EF1α-copGFP BLIV 2.0 Lentivector for In Vivo Imaging	SBI	BLIV511PA-1
CryoStor10	BioLife Solutions	640222
Human MSCs	Meridigen Biotech Co., Ltd. Taipei, Taiwan
Infrared light	JING SHANG	JS300T
Isoflurane	Halocarbon	26675-46-7
IVIS-200 small animal imaging system	Caliper LifeSciences, Hopkinton, MA
Luciferin potassium salt	Promega, Madison, WI
Micro-scissors, straight	Vannas	H4240
Normal saline	TAIWAN BIOTECH CO., LTD.	113531	Isotonic Sodium Chloride Solution
Small Hub RN Needle, 30 gauge	Hamilton Company, Reno, NV	7799-06
Syringe (100 µl)	Hamilton Company, Reno, NV	81065
Xenogen Living Image 2.5 software	Caliper LifeSciences, Hopkinton, MA	N/A