PMC

Intratracheal bone marrow–derived mesenchymal stem cell (BM-MSC) administration on Postnatal Day 4 improves survival and exercise capacity. (A) Kaplan-Meier survival curve showing significant improvement in survival of MSC-treated animals as compared with untreated or pulmonary artery smooth muscle cell (PASMC)-treated animals. (B) Exercise capacity assessed according to a predetermined protocol shows that MSC-treated animals run for longer distances as compared with untreated or PASMC-treated animals.

(A) Exposure of rat pups to hyperoxia during the critical period of alveolar development results in arrested alveolar growth. Representative hematoxylin and eosin–stained lung sections show larger and fewer alveoli in hyperoxia-exposed lungs as compared with room air–housed control animals. (B) Representative figure of a colony-forming unit-fibroblast (CFU-F). (C) CFU-F frequency. Shown is the number of CFU-F (mean ± SEM) per 10⁶ cells from bone marrow (BM), blood, and lung in normoxia or hypoxia. *P < 0.001.

Bone marrow–derived mesenchymal stem cells (BMSCs) administered intratracheally engraft and adopt a type II alveolar epithelial cell (AEC2) phenotype. BMSCs were labeled with the green fluorescent marker 5()-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (inset) and injected intratracheally into 4-day-old rat pups. Immunofluorescence of frozen lung sections examined by confocal microscopy reveals nuclear staining (4′,6-diamidino-2-phenylindole [DAPI], blue) and colocalization of BMSCs (green) and the distal AEC2 marker surfactant protein (SP)-C (red).

Intratracheal bone marrow–derived mesenchymal stem cell (MSC) administration on Postnatal Day 4 improves lung structure. O₂-exposed lungs display the characteristic features of alveolar simplification that are unchanged with pulmonary artery smooth muscle cells (PASMCs). Conversely, lungs treated with intratracheal bone marrow–derived mesenchymal stem cells (BMSCs) have smaller and more numerous alveoli. Quantification of alveolar structures, using the mean linear intercept (Lm), confirms improved alveolarization in MSC-treated animals as compared with the other O₂-exposed groups.

Intratracheal bone marrow–derived mesenchymal stem cells restore the hyperoxia-induced decrease in lung capillary density. (A) Microangiograph computed tomography (CT) scan and Mercox casts examined by scanning electron microscopy of the lung capillary bed show that intratracheal mesenchymal stem cells (MSCs) promote lung angiogenesis and restore a denser capillary network in the lungs of O₂-exposed rats. (B) Representative hematoxylin and eosin–stained barium angiograms and mean capillary count show decreased capillary density in O₂-exposed lungs. Intratracheal MSCs, but not pulmonary artery smooth muscle cells (PASMCs), restore arterial density to control values.

Intratracheal bone marrow–derived mesenchymal stem cell (BMSC) administration prevents pulmonary hypertension associated with O₂-induced lung injury. (A) Pulmonary arterial acceleration time (PAAT). PAAT was significantly decreased in chronic hyperoxia–induced lung injury and showed a characteristic notch indicating pulmonary hypertension (arrows). Intratracheal BMSCs, but not pulmonary artery smooth muscle cells (PASMCs), restored the PAAT almost to control levels. (B) Right ventricular hypertrophy (RVH). Hyperoxic-exposed rats had significant RVH as indicated by the increase in RV/LV+S (right ventricle/left ventricle plus septum) ratio compared with normoxic controls. Intratracheal BMSCs, but not PASMCs, reduced RVH.

Protective effect of bone marrow–derived mesenchymal stem cell (BMSC) conditioned medium (CdM) in vitro. (A) BMSC CdM protects type II alveolar epithelial cells (AEC2) from O₂-induced DNA damage. AEC2 undergo DNA damage (brown) when exposed to hyperoxia. BMSC CdM prevented O₂-induced AEC2 DNA damage as compared with control medium. (B) BMSC CdM protects AEC2 from O₂-induced apoptosis. AEC2 exposed to hyperoxia expressed cleaved caspase-3. BMSC CdM prevented O₂-induced AEC2 apoptosis as compared with control medium. (C) BMSC CdM accelerates AEC2 wound closure. Confluent monolayers of AEC2 were damaged with a pipette tip, washed to remove damaged cells, and incubated in Dulbecco's modified Eagle's medium (DMEM) or BMSC CdM. CdM accelerated AEC2 wound closure as compared with DMEM. (D) BMSC CdM promotes endothelial network formation. Quantitative assessment of cordlike structure formation shows a significant decrease in the number of intersects and the total length of cordlike structures in hyperoxia. BMSC CdM preserved the number of intersects and total cord-structure length. RLMVEC = rat lung microvascular endothelial cells.

(A) Characterization of mesenchymal stem cell (MSC) immunophenotype. Shown are fluorescence intensity histograms with specific antibodies for membrane antigens (red line) and irrelevant isotypic-matched antibody as negative control (gray area). Experiments were performed in triplicate. MSC differentiation potential was assessed by (B) histochemistry and (C) mRNA expression of lineage-specific genes for adipogenic (Ad), osteogenic (Os), and chondrogenic (Ch) differentiation. Passage 2 bone marrow (BM)–derived adherent cells, after culture in differentiation medium (Ad, Os, or Ch), were stained for oil red O (Ad staining), alizarin red (Os staining), and safranin O (Ch staining). Ctl = control proliferation medium; Neg = polymerase chain reaction without cDNA. All experiments were performed in triplicate. DMEM = Dulbecco's modified Eagle's medium; H = hypoxic; N = normoxic. (D) Migration assay in Boyden chamber. In vitro, MSCs placed in the upper chamber migrate preferentially to lungs from O₂-exposed rats than to culture medium only or lungs from rats housed in room air. PLSD = Fisher's probable least significant difference; RFU = relative fluorescence units. (E) In vitro, bone marrow–derived mesenchymal stem cells (BMSCs) differentiate into type II alveolar epithelial cells (AEC2). The lung microenvironment induces BMSCs to adopt an AEC2 phenotype. Co-culture experiments of BMSCs with O₂-injured lung, but not with culture medium alone, show molecular (surfactant protein [SP]-C mRNA and protein expression) and ultrastructural (lamellar bodies in electron microscopy) features of AEC2. SAGM = small airway growth medium.