Intravenous mesenchymal stem cell therapy for traumatic brain injury

Abstract

Object: Cell therapy has shown preclinical promise in the treatment of many diseases, and its application is being translated to the clinical arena. Intravenous mesenchymal stem cell (MSC) therapy has been shown to improve functional recovery after traumatic brain injury (TBI). Herein, the authors report on their attempts to reproduce such observations, including detailed characterizations of the MSC population, non-bromodeoxyuridine-based cell labeling, macroscopic and microscopic cell tracking, quantification of cells traversing the pulmonary microvasculature, and well-validated measurement of motor and cognitive function recovery.

Methods: Rat MSCs were isolated, expanded in vitro, immunophenotyped, and labeled. Four million MSCs were intravenously infused into Sprague-Dawley rats 24 hours after receiving a moderate, unilateral controlled cortical impact TBI. Infrared macroscopic cell tracking was used to identify cell distribution. Immunohistochemical analysis of brain and lung tissues 48 hours and 2 weeks postinfusion revealed transplanted cells in these locations, and these cells were quantified. Intraarterial blood sampling and flow cytometry were used to quantify the number of transplanted cells reaching the arterial circulation. Motor and cognitive behavioral testing was performed to evaluate functional recovery.

Results: At 48 hours post-MSC infusion, the majority of cells were localized to the lungs. Between 1.5 and 3.7% of the infused cells were estimated to traverse the lungs and reach the arterial circulation, 0.295% reached the carotid artery, and a very small percentage reached the cerebral parenchyma (0.0005%) and remained there. Almost no cells were identified in the brain tissue at 2 weeks postinfusion. No motor or cognitive functional improvements in recovery were identified.

Conclusions: The intravenous infusion of MSCs appeared neither to result in significant acute or prolonged cerebral engraftment of cells nor to modify the recovery of motor or cognitive function. Less than 4% of the infused cells were likely to traverse the pulmonary microvasculature and reach the arterial circulation, a phenomenon termed the "pulmonary first-pass effect," which may limit the efficacy of this therapeutic approach. The data in this study contradict the findings of previous reports and highlight the potential shortcomings of acute, single-dose, intravenous MSC therapy for TBI.

Fig. 1

Upper: Infrared images (image resolution: 84 μm; focus offset: 1 mm) demonstrating intravenously infused MSCs in the lungs, kidney, spleen, and liver of rats 48 hours after the infusion of 4 × 10⁶ MSCs (experimental rats 1–4) or PBS vehicle (Control). Lower: Bar graph showing the lung integrated intensities in the experimental animals, which were significantly different from those in controls (p = 0.003, t-test).

Fig. 2

A and B: Photomicrographs showing numerous MSCs (Qtracker labeled) in the pulmonary tissue 48 hours after intravenous infusion. Cell nuclei are revealed by DAPI staining (blue) and MSCs by Qtracker 655 (red). Arrowheads indicate representative MSCs.

Fig. 3

Upper: Infrared images (image resolution: 84 μm; focus offset: 1 mm) revealing the lungs, kidney, spleen, and liver of rats 2 weeks after the intravenous infusion of 4 × 10⁶ MSCs (experimental rats 1–3) or vehicle (Control). Although some cells remain in the pulmonary tissue, there are significantly fewer than those seen 48 hours postinfusion. Lower: Bar graph showing lung integrated intensities of the experimental animals, which were not significantly different from those from controls (p = 0.10, t-test).

Fig. 4

Flow cytometric scatterplot demonstrating rat whole blood cells (A) and Qtracker labeled rat MSCs (B). The red line shows the positive threshold (10²) used to distinguish infused MSCs from other cells (> 95% of MSCs). The clear difference in intensity of rat blood (A) versus MSCs (B) allowed recognition of infused cells by flow cytometry. To generate a standard curve, MSC counts were obtained after adding known quantities of MSCs to control samples. Example scatterplots showing the effects of 100,000 MSCs (C) and 10,000 MSCs (D) added to control samples of rat blood. The number of cells with a fluorescent intensity > 10², after collecting 20,000 total events, was plotted against the known MSC number to generate the standard curve. APC-A = allophycocyanine–A channel; SSC-A = side scatter–A channel.

Fig. 5

Bar graph showing Qtracker-labeled MSCs in intraarterial blood samples run through a flow cytometer. Four million rat MSCs (Qtracker labeled) were infused via the internal jugular vein (5 rats), and blood was continuously sampled from the common carotid artery over the subsequent 10 minutes. IV = intravenous.

Fig. 6

Graphs showing the results of rotarod (A), NSS (B), balance beam (C), and foot fault (D) testing. There were no significant differences between the CCI groups. Rotarod maximum speed is the highest rpm during which the rat could stay on the top of the rotating rod. Time on beam was measured from the time the animal was placed on the beam until the animal fell off and hit the safe landing area. Foot faults were counted when the left rear paw dropped below the chicken wire (out of 25 steps). Sham, 6 rats; CCI + vehicle, 5 rats; CCI + 2 × 10⁶ MSCs, 4 rats; CCI + 4 × 10⁶ MSCs, 6 rats. s = seconds.