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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 k....

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 .......

Representative 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.......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
6-0 silkEthicon1916G
Alcohol Prep PadCSD3032
BD Stemflow hMSC Analysis KitBD Biosciences562245CD markers
CMV-Luciferase-EF1α-copGFP BLIV 2.0 Lentivector for In Vivo ImagingSBIBLIV511PA-1
CryoStor10BioLife Solutions640222
Human MSCsMeridigen Biotech Co., Ltd. Taipei, Taiwan
Infrared lightJING SHANGJS300T
IsofluraneHalocarbon26675-46-7
IVIS-200 small animal imaging systemCaliper LifeSciences, Hopkinton, MA
Luciferin potassium saltPromega, Madison, WI
Micro-scissors, straightVannasH4240
Normal salineTAIWAN BIOTECH CO., LTD.113531Isotonic Sodium Chloride Solution
Small Hub RN Needle, 30 gaugeHamilton Company, Reno, NV7799-06
Syringe (100 µl)Hamilton Company, Reno, NV81065
Xenogen Living Image 2.5 softwareCaliper LifeSciences, Hopkinton, MAN/A

References

  1. Ramanathan, R., Bhatia, J. J., Sekar, K., Ernst, F. R. Mortality in preterm infants with respiratory distress syndrome treated with poractant alfa, calfactant or beractant: a retrospective study. Journal of Perinatology. 33, 119-125 (2013).
  2. Gien, J., Kinsella, J. P.

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Intratracheal InstillationStem CellsNeonatal RatsPulmonary DeliveryMesenchymal Stromal CellsLuciferaseGFPCell TransplantationImagingCell Characterization

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