Name: intratracheal instillation of stem cells in term neonatal rats
Uploaded: 2020-05-04T14:30:00
Description: Watch this Scientific Journal Video about Intratracheal Instillation of Stem Cells in Term Neonatal Rats at JoVE.com

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

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
<ol>
	<li>Maintain human MSCs transfected with GFP and Fluc in complete media (minimum essential ...

<ol>
	<li>Ramanathan, R., Bhatia, J. J., Sekar, K., Ernst, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+in+preterm+infants+with+respiratory+distress+syndrome+treated+with+poractant+alfa,+calfactant+or+beractant:+a+retrospective+study.">Mortality in preterm infants with respiratory distress syndrome treated with poractant alfa, calfactant or beractant: a retrospective study.</a> Journal of Perinatology. 33, 119-125 (2013).</li><li>Gien, J., Kinsella, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+treatment+of+bronchopulmonary+dysplasia.">Pathogenesis and treatment of bronchopulmonary dysplasia.</a> Current Opinion in Pediatrics. 23, 305-313 (2011).</li><li>Pasha, A. B., Chen, X. Q., Zhou, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bronchopulmonary+dysplasia:+Pathogenesis+and+treatment.">Bronchopulmonary dysplasia: Pathogenesis and treatment.</a> Experimental and Therapeutic. 16, 4315-4321 (2018).</li><li>Committee on Fetus and Newborn. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+corticosteroids+to+treat+or+prevent+chronic+lung+disease+in+preterm+infants.">Postnatal corticosteroids to treat or prevent chronic lung disease in preterm infants.</a> Pediatrics. 109, 330-338 (2002).</li><li>Watterberg, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Academy+of+Pediatrics;+Committee+on+Fetus+and+Newborn.+Policy+statement-postnatal+corticosteroids+to+prevent+or+treat+bronchopulmonary+dysplasia.">American Academy of Pediatrics; Committee on Fetus and Newborn. Policy statement-postnatal corticosteroids to prevent or treat bronchopulmonary dysplasia.</a> Pediatrics. 126, 800-808 (2010).</li><li>Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marrow+stromal+cells+as+stem+cells+for+nonhematopoietic+tissues.">Marrow stromal cells as stem cells for nonhematopoietic tissues.</a> Science. 276, 71-74 (1997).</li><li>Nemeth, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+attenuate+sepsis+via+prostaglandin+E(2)-+dependent+reprogramming+of+host+macrophages+to+increase+their+interleukin-10+production.">Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)- dependent reprogramming of host macrophages to increase their interleukin-10 production.</a> Nature Medicine. 15 (2), 42-49 (2009).</li><li>Chou, H. C., Li, Y. T., Chen, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells+attenuate+experimental+bronchopulmonary+dysplasia+induced+by+perinatal+inflammation+and+hyperoxia.">Human mesenchymal stem cells attenuate experimental bronchopulmonary dysplasia induced by perinatal inflammation and hyperoxia.</a> American Journal of Translational Research. 8, 342-353 (2016).</li><li>Chen, C. M., Chou, H. C., Lin, W., Tseng, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant+effects+on+the+viability+and+function+of+human+mesenchymal+stem+cells:+in+vitro+and+in+vivo+assessment.">Surfactant effects on the viability and function of human mesenchymal stem cells: in vitro and in vivo assessment.</a> Stem Cell Research &amp; Therapy. 8, 180 (2017).</li><li>Kim, Y. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratracheal+transplantation+of+mesenchymal+stem+cells+simultaneously+attenuates+both+lung+and+brain+injuries+in+hyperoxic+newborn+rats.">Intratracheal transplantation of mesenchymal stem cells simultaneously attenuates both lung and brain injuries in hyperoxic newborn rats.</a> Pediatric Research. 80, 415-424 (2016).</li><li>Fleurence, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+efficacy+of+intratracheal+adeno-associated+virus+administration+to+newborn+rats.">Comparative efficacy of intratracheal adeno-associated virus administration to newborn rats.</a> Human Gene Therapy. 16, 1298-1306 (2005).</li><li>Waszak, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+intratracheal+adenoviral+vector+administration+on+lung+development+in+newborn+rats.">Effect of intratracheal adenoviral vector administration on lung development in newborn rats.</a> Human Gene Therapy. 13, 1873-1885 (2002).</li><li>O'Reilly, M., Th&#233;baud, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+bronchopulmonary+dysplasia.+The+term+rat+models.">Animal models of bronchopulmonary dysplasia. The term rat models.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 307, 948 (2014).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP+labeling+and+hepatic+differentiation+potential+of+human+placenta-derived+mesenchymal+stem+cells.">GFP labeling and hepatic differentiation potential of human placenta-derived mesenchymal stem cells.</a> Cellular Physiology and Biochemistry. 35, 2299-2308 (2015).</li><li>Dominici, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8, 315-317 (2006).</li><li>Zhang, Z. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superior+osteogenic+capacity+for+bone+tissue+engineering+of+fetal+compared+with+perinatal+and+adult+mesenchymal+stem+cells.">Superior osteogenic capacity for bone tissue engineering of fetal compared with perinatal and adult mesenchymal stem cells.</a> Stem Cells. 27, 126-137 (2009).</li><li>Huang, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+surfactant+and+budesonide+on+pulmonary+distribution+of+fluorescent+dye+in+mice.">Effect of surfactant and budesonide on pulmonary distribution of fluorescent dye in mice.</a> Pediatric and Neonatology. 56, 19-24 (2015).</li><li>Keyaerts, M., Caveliers, V., Lahoutte, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging:+looking+beyond+the+light.">Bioluminescence imaging: looking beyond the light.</a> Trends in Molecular Medicine. 18, 164-172 (2012).</li><li>Yeh, T. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-tracheal+administration+of+budesonide/surfactant+to+prevent+bronchopulmonary+dysplasia.">Intra-tracheal administration of budesonide/surfactant to prevent bronchopulmonary dysplasia.</a> American Journal of Respiratory and Critical Care Medicine. 193, 86-95 (2016).</li><li>Nguyen, J. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratracheal+inoculation+of+Fischer+344+rats+with+Francisella+tularensis.">Intratracheal inoculation of Fischer 344 rats with Francisella tularensis.</a> Journal of Visualized Experiments. (127), e56123 (2017).</li><li>de Almeida, P. E., van Rappard, J. R., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+for+tracking+cell+fate+and+function.">In vivo bioluminescence for tracking cell fate and function.</a> American Journal of Physiology-Heart and Circulatory Physiology. 301, 663-671 (2011).</li><li>Kim, J. E., Kalimuthu, S., Ahn, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cell+tracking+with+bioluminescence+imaging.">In vivo cell tracking with bioluminescence imaging.</a> Nuclear Medicine and Molecular Imaging. 49, 3-10 (2011).</li></ol>

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...

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 effects4,5.
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 rodents8,9. 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 rats10,11,12. 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&#8722;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.

<table>
	<tbody>
		<tr>
			<td>6-0 silk</td>
			<td>Ethicon</td>
			<td>1916G</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol Prep Pad</td>
			<td>CSD</td>
			<td>3032</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Stemflow hMSC Analysis Kit</td>
			<td>BD Biosciences</td>
			<td>562245</td>
			<td>CD markers</td>
		</tr>
		<tr>
			<td>CMV-Luciferase-EF1&#945;-copGFP BLIV 2.0 Lentivector for In Vivo Imaging</td>
			<td>SBI</td>
			<td>BLIV511PA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor10</td>
			<td>BioLife Solutions</td>
			<td>640222</td>
			<td></td>
		</tr>
		<tr>
			<td>Human MSCs</td>
			<td>Meridigen Biotech Co., Ltd. Taipei, Taiwan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Infrared light</td>
			<td>JING SHANG</td>
			<td>JS300T</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Halocarbon</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS-200 small animal imaging system</td>
			<td>Caliper LifeSciences, Hopkinton, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferin potassium salt</td>
			<td>Promega, Madison, WI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-scissors, straight</td>
			<td>Vannas</td>
			<td>H4240</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>TAIWAN BIOTECH CO., LTD.</td>
			<td>113531</td>
			<td>Isotonic Sodium Chloride Solution</td>
		</tr>
		<tr>
			<td>Small Hub RN Needle, 30 gauge</td>
			<td>Hamilton Company, Reno, NV</td>
			<td>7799-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (100 &#181;l)</td>
			<td>Hamilton Company, Reno, NV</td>
			<td>81065</td>
			<td></td>
		</tr>
		<tr>
			<td>Xenogen Living Image 2.5 software</td>
			<td>Caliper LifeSciences, Hopkinton, MA</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

intratracheal instillation of stem cells in term neonatal rats

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&#8722;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.

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 ...

Watch this Scientific Journal Video about Intratracheal Instillation of Stem Cells in Term Neonatal Rats at JoVE.com

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

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 ...

Research

JoVE Journal

Medicine

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...