Methods Isolation, Characterization, and Labeling of Rat MSCs

Matthew T. Harting, M.D.1, Fernando Jimenez, M.S.1, Hasan Xue, M.D.1, Uwe M. Fischer, M.D. 1, James Baumgartner, M.D.1, Pramod K. Dash, Ph.D.2,3, and Charles S. Cox Jr., M.D.1 1Departments of Pediatric Surgery, University of Texas Medical School at Houston, Texas 2Neurobiology and Anatomy, University of Texas Medical School at Houston, Texas 3The Vivian L. Smith Center for Neurologic Research, University of Texas Medical School at Houston, Texas

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

Address correspondence to: Charles S. Cox Jr., M.D., Department of Pediatric Surgery, University of Texas Medical School at Houston, 6431 Fannin Street, MSB 5.254, Houston, Texas 77030. [email protected].. Disclosure This work was supported by National Institutes of Health Grant Nos. T32 GM008792-06 (M.T.H.), MO1 RR 02558 (C.S.C.), and R21 HD 04 2659-01A1 (C.S.C.), as well as funds from the Children’s Memorial Hermann Hospital Foundation (C.S.C.) and Texas Higher Education Coordinating Board (C.S.C.). Disclaimer The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

NIH Public Access Author Manuscript J Neurosurg. Author manuscript; available in PMC 2010 June 22.

Published in final edited form as: J Neurosurg. 2009 June ; 110(6): 1189–1197. doi:10.3171/2008.9.JNS08158.

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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.

Keywords adult stem cell; cellular therapy; in vivo tracking; mesenchymal stem cell; traumatic brain injury

CELL therapy has garnered significant interest as a treatment strategy for a wide range of diseases over the last decade. Heart disease, peripheral vascular disease, bone disease, cancer, hepatic disease, and neurological disease have all been the focus of promising cell therapy breakthroughs.7,15,23,24,34,40,48 Traumatic brain injury is one area in which positive preclinical evidence has led to the initiation of early clinical trials (www.clinicaltrials.gov).

Although cell therapy has been hailed as one of the next frontiers in modern medicine, there has been no universal agreement on many of the findings of early cell therapy work. Questions and concerns regarding many aspects of cell therapy, including transdifferentiation,6,8,33,39, 41,44 cell labeling,5 route of administration,37 preferred cell type, and immune privilege,1,26, 35 have been raised. Furthermore, publication bias may have skewed the expectations surrounding this treatment strategy.

Intravenous infusion has been used as the means of cell delivery in a large number of preclinical studies16,19,27,32,43 as well as some early clinical trials.14,17,21 Intravenous delivery has been asserted to be advantageous because of its broad distribution, ability to handle a large-volume infusion, and ease of access.32 Nonetheless, questions have been raised regarding its ability to transport a critical number of cells to the area of injury.28,37,42 Previous work has shown that cell infusion via the intravenous route allows a significant number of cells to reach the traumatically injured brain and to modulate significant functional recovery.25,27,30,32

We report on intravenously infused rat MSCs as therapy for TBI. We hypothesized that intravenously administered rat MSCs could reach the traumatically injured rat brain in critical numbers and improve the recovery of motor and cognitive function. We used well- characterized rat MSCs, non–BrdU-based cell labeling, and a CCI injury model. Behavioral outcomes were evaluated with well-validated measures of motor (rotarod, NSS, balance beam, and foot fault) and cognitive (Morris water maze, learning paradigm) function.