Experimental study
Stem Cell Transplantation: The Lung Barrier

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Abstract

Background

Mesenchymal stem cells (MSCs) show differentiation capacity along mesenchymal lineages and have the potential to aid tissue regeneration. MSC transplantation strategies are therefore currently being assessed following injury to various organs. However, potential MSC migration to these organs after intravenous (IV) MSC injection continues to be impeded by cell trapping within the lung.

Methods

Mouse MSCs were isolated, purified, transfected with firefly luciferase, and labeled with CSFE. Their size was assessed in vitro. To estimate the diameter of mouse pulmonary capillaries, fluorescence-labeled microspheres of different sizes were injected with or without sodium nitroprusside (SN) pretreatment. The lungs were harvested after 30 seconds and mean numbers of trapped microspheres per high-power field (HPF) were calculated. After IV injection of MSC suspensions (with or without SN), their dynamic distribution was monitored by in vivo luciferine imaging as well as by histopathology.

Results

The diameter of suspended MSCs in vitro was 15 to 19 μm. Whereas nearly no 4-μm microspheres could be detected in lung sections, the numbers of trapped 10- and 15-μm microspheres could be significantly decreased by prior SN injection from 19.3 ± 11.1 to 6.0 ± 1.6 cells/HPF (P = .004) and from 34.9 ± 11.9 to 25.6 ± 8.1 cells/HPF (P = .028), respectively. Within seconds after MSC IV injection, the vast majority of cells was found in the lungs. However, cell trapping in the pulmonary microvasculature was significantly reduced by pre-treatment with SN.

Conclusions

We demonstrate that cell trapping in lungs can be reduced with IV SN pretreatment, increasing MSC passage through the lung capillaries, and potentially facilitating cell access to injured organs.

Section snippets

Cell Isolation and Labeling

MSCs were isolated from the long bones of Balb/c mice, purified, and characterized as CD34, CD45 CD90+, CD44+, CD14, c-kit, sca-1+, and CD105+. Cells were made to express firefly luciferase under cytomegalovirus (CMV) promoter control by transfecting Phoenix cells with 10 μg of pRluc DNA9 as previously described10 and infecting wild-type MSC with the resultant viral supernatants. Stable transfectants were enriched by puromycin selection at 0.5 μg/mL, which was the concentration determined

Cell Injection

After administration of 0.5 × 106 MSCs, we noticed episodes of tachypnea, apnea, and hemodynamic alterations characteristic of pulmonary embolism in the group without SN pretreatment. These symptoms could be markedly reduced with prior vasodilator treatment.

In Vivo Imaging, Microsphere Trapping, and Histopathology

After MSC IV infusion without pretreatment, bioluminiscensce signals associated with MSCs were detected primarily in the lungs (Fig 1A). Ex vivo assessments of isolated organs confirmed high lung signals in contrast to only trace signals

Discussion

Using microspheres, we demonstrated that the mean size of suspended MSCs is larger than the size of pulmonary capillaries. Thus, because of their large size, major amounts of IV-injected MSCs are trapped within the pulmonary capillaries, causing pulmonary and hemodynamic alterations, and preventing the intended access to other organs. Because MSCs are naturally located within the bone marrow and are only occasionally liberated into the circulation, their size is usually not a barrier to their

Acknowledgment

S.S. and T.D. equally contributed to this work.

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Supported by a research grant from the Deutsche Forschungsgemeinschaft (DFG) (SCHR992/2-1) to S.S.

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