Pulmonary passage is a major obstacle for intravenous stem cell delivery: the pulmonary first-pass effect

Abstract

Intravenous (IV) stem cell delivery for regenerative tissue therapy has been increasingly used in both experimental and clinical trials. However, recent data suggest that the majority of administered stem cells are initially trapped in the lungs. We sought to investigate variables that may affect this pulmonary first-pass effect. In anesthetized Sprague-Dawley rats, silicone tubing catheters were placed in the left internal jugular vein and common carotid artery. We investigated four different cell types: mesenchymal stromal cells (MSC), multipotent adult progenitor cells (MAPCs), bone marrow-derived mononuclear cells (BMMC), and neural stem cells (NSC). Cells were co-labeled with Qtracker 655 (for flow cytometry) and Qtracker 800 (for infrared imaging) and infused intravenously with continual arterial sample collection. Samples were analyzed via flow cytometry to detect labeled cells reaching the arterial circulation. Following sampling and exsanguination, heart, lungs, spleen, kidney, and liver were harvested and placed on an infrared imaging system to identify the presence of labeled cells. The majority of MSCs were trapped inside the lungs following intravenous infusion. NSC and MAPC pulmonary passage was 2-fold and BMMC passage was 30-fold increased as compared to MSCs. Inhibition of MSC CD49d significantly increased MSC pulmonary passage. Infusion via two boluses increased pulmonary MSC passage as compared to single bolus administration. Infrared imaging revealed stem cells evenly distributed over all lung fields. Larger stem and progenitor cells are initially trapped inside the lungs following intravenous administration with a therapeutically questionable number of cells reaching the arterial system acutely.

FIG. 1.

A Coulter Counter Z1 Series particle counter (Beckman Coulter Inc., Hialeah, FL) was used to determine the size range of our cell populations. Prior to taking measurements, we optimized the dynamic range of size measurement by adjusting gain and current settings using methods specified in the Beckman Counter Z1 Series user manual. The distribution of cell sizes was determined by recording the total particle count within a range of increasing specific volume (μm³) limits. Cell population was distinguished from debris by observing the generated minima on both sides of the curve. Abbreviations: MSC, mesenchymal stem cell; MAPC, multipotent adult progenitor cell; NSC, neural stem cell; BMMC, mononuclear cell fraction.

FIG. 2.

Labeled MSCs were identified in the arterial blood samples using flow cytometry. The flow cytometric dual parameter dot plots show rat whole blood cells (A) and Qtracker^®-labeled MSCs (B). The y-axis is side scatter (SSC) and the x-axis is the channel (APC) in which the Qtracker^® can be detected. A histogram overlay of the whole blood (gray) and MSC (white) population is shown (C). A representative dual parameter dot plot identifying cells that transverse the lungs in vivo (D). The vertical line shows the positive threshold (10²) used to distinguish infused MSCs from other cells (>90% of MSCs). The clear difference in intensity of rat blood (A) versus MSCs (B) allows recognition of infused cells by flow cytometry.

FIG. 3.

Pulmonary stem cell passage. Total number (A) and percentage (B) of stem cells reaching the carotid artery are shown for groups A–J. *P < 0.05 versus A.

FIG. 4.

Intra-arterial (IA) MSC collection after intravenous (IV) infusion (n = 5 rats/group). Stem cells (Qtracker^®-labeled) were infused via the internal jugular vein and blood was continuously sampled from the common carotid artery over the subsequent 10 min (35–40 samples). IA blood samples were run through a flow cytometer to detect the Qtracker^®-labeled stem cells. Number of MSCs per sample is shown on the y-axis and sample numbers are shown on the x-axis.

FIG. 5.

Infrared imaging of organs immediately after intravenous stem cell infusion. Lungs, heart, kidney, spleen, and liver of rats immediately after intravenous stem cell infusion (B–K) or vehicle (A, control) imaged ex vivo on the LI-COR Odyssey^® infrared imaging system.

FIG. 6.

Intravenous injection of two MSC boluses (2 million each) labeled with two different colors. Infrared imaging of lungs, heart, kidney, spleen, and liver immediately after intravenous stem cell infusion (A), total number of stem cells reaching the carotid artery for bolus 1 and bolus 2, respectively (B), and number of MSCs per sample is shown (C; see Fig. 4 for details). *P < 0.05 versus bolus 1.