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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.
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.
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 k....
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
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 .......
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.......
This study was partly supported by a grant from Meridigen Biotech Co., Ltd. Taipei, Taiwan (A-109-008).
....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 |
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