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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 1, 2005 as doi:10.1096/fj.05-4082fje. |
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,¶
Department of
* Physiology,
Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan;
Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan;
Subteam for Manipulation of Cell Fate, BioResource Center, RIKEN, Tsukuba Institute, Ibaraki, Japan;
¶ Department of Pediatrics, Stanford University School of Medicine, The Bio-X program at the James H. Clark Center, Stanford, California, USA;
|| Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology, Saitama, Japan
1 Correspondence: Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, 160-8582, Japan. E-mail: hidokano{at}sc.itc.keio.ac.jp
SPECIFIC AIMS
In the research of cell transplantation therapy, understanding the survival time of grafted cells and determining the extent of migration away from transplantation sites are essential for optimizing treatment regimens. This study was designed to noninvasively assess primary neural stem/progenitor cells (NSPCs) that were transplanted into injured spinal cord and to investigate the effect of timing of NSPCs transplantation.
PRINCIPAL FINDINGS
1. Sufficient expression of bioluminescence and GFP in the lentivirally transduced NSPCs
While the feasibility and usefulness of in vivo bioluminescence imaging (BLI) have been demonstrated in several studies, long-term tracking of primary NSPCs has been difficult because, unlike tumor cells and immortalized cell lines, a large percentage of NSPCs was lost shortly after transplantation. To follow primary cells for long periods, high levels of sustained reporter gene expression are necessary. We used third-generation lentiviral vectors to transfer the reporter genes (luciferase and GFP) to primary cultured NSPCs. These lentivirally transduced NSPCs expressed intense luminescence as well as fluorescence and a population of 100 of these NSPCs was sufficient to produce a detectable luminescent signal in vitro. There were no differences between untreated and lentivirally transduced NSPCs in cell viability and in the pattern of phenotypes after differentiation. These results demonstrated the ability of lentiviral vectors to confer high levels of specific gene expression while preserving the character of NSPCs.
2. In vivo BLI of transplanted NSPCs and viability studies in spinal cord injury
We next examined whether NSPCs that were transplanted into the spinal cord were detectable. Different numbers (ranging from 25,000 to 500,000) of labeled NSPCs were stereotactically transplanted into the normal spinal cord of adult C57BL/6J mice, and cell distribution and signal intensity were assessed in living animals using BLI. An intense focal spot of bioluminescence was observed at the transplantation site without leaking to the surrounding tissue and quantitative analysis revealed that the luminescent intensity was clearly in direct proportion to transplanted cell numbers in vivo as well as in vitro. The luminescence from these NSPCs was sufficient in intensity to allow for observation of the signals in C57BL/6J mice, whose dark fur and skin had been believed inappropriate for optical imaging due to absorbance of the signals.
The sequential evaluation of grafted cell viability is possible with the BLI system since the luciferin-lusiferase reaction depends on ATP and only living cells release photons. To examine the viability of transplanted NSPCs in the injured spinal cord, contusion injury was induced at the 10th thoracic vertebrae level of C57BL/6J mice and transplantation of NSPCs (5x105) was performed immediately after injury. Images were obtained daily for 1 wk, then weekly over a 6 wk period. We observed drastic reductions (nearly 80%) in signal intensity within the first 4 days after transplantation, then relatively stable bioluminescent signal for the next 6 wk (Fig. 1
). Similar results were observed when transplantation was performed at 9 days after injury, and quantitative comparison between acute and delayed transplantation revealed no apparent differences in photon emission.
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3. Timing of NSPC transplantation was a key determinant of the fate and function of integrated cells for in the injured spinal cord
Although no significant differences in cell viability were observed between acute and delayed transplantation, there were apparent differences in the location and morphology of the integrated cells. Histological examination and BLI revealed that the majority of acutely transplanted NSPCs were distributed within the scar area of the lesion epicenter, contributing to the formation of the astrocytic glial scar at 6 wk after injury. On the other hand, NSPCs transplanted in the delayed phase were found around the scar area and a portion of these cells migrated caudally with extended neurites (Fig. 2
). The number of integrated neurons and oligodendrocytes was ten times greater in the delayed transplantation group than that of the acute transplantation. We also evaluated the recovery of locomotor function for 6 wk. While improved recovery was found in both acute and delayed transplantation groups compared with the medium-injected control group, the mice in the delayed transplantation group showed a tendency of long-lasting recovery, and the only statistically significant difference was between the control and delayed transplantation group with the post hoc test.
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CONCLUSIONS AND SIGNIFICANCE
The fate of transplanted NSPCs in the host central nervous system is of great scientific interest. Currently the research of cell transplantation therapy is limited to histological investigation when evaluating cell viability and migration. This approach only provides data from a single time point and requires large numbers of animal sacrifices. In order to evaluate the progress of transplanted cells, an animal must be killed at each time point. The data obtained from multiple animals may be misleading, being subject to a range of variable factors. The BLI system makes continuous monitoring of integrated cells in a specific animal possible, significantly contributing to the study of cell transplantation. In the present study, the high transduction efficacy of reporter genes through the use of lentiviral vectors made long-term monitoring of NSPCs transplanted into the injured spinal cord possible. Lentiviruses, a type of retrovirus are ideal for labeling graft cells because they can integrate transgenes into the host cell genome and provide sustained expression of the delivered transgenes. Moreover, lentiviruses allow high transduction efficiency because they can be concentrated to high titers owing to their modified envelop glycoprotein, and unlike retroviral vectors, also have the ability to transduce any type of cell including quiescent nondividing cells. We also manipulated NSPCs to express green fluorescence by placing GFP gene downstream of luciferase gene with the IRES in order to investigate morphology and phenotype of the integrated cells in histological sections.
In this study, we demonstrated several utilities of the BLI system in the field of the cell transplantation research (Fig. 3
). First, it can be used to confirm the accurate and successful transplantation by evaluating the location and intensity of luminescence of transplanted cells immediately after transplantation. Second, grafted cell viability can be quantified in living animals while simultaneously checking for tumorigenic transformations. Finally, the extent of cell migration and translocation can be visualized clearly. These advantages will greatly contribute to the research of cell therapy mechanism.
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Using both BLI and histological examination, we investigated the relation between the timing of transplantation and cell viability, migration, morphology, and function, by observing differences of NSPCs when transplanted at different times relative to injury. There were striking differences in the location, migration, and phenotypes of integrated cells between the acute and delayed transplants. NSPCs transplanted in the delayed phase differentiated into more neurons and oligodendrocytes and had better recovery than that of the acute phase, suggesting that the microenvironment of injured spinal cord has a great influence on the fate of grafted NSPCs. Although the precise mechanism of improvement by NSPCs transplantation remains to be elucidated, delayed transplantation could have more significant clinical efficacy.
The methods in the present study can be widely applied to various research fields of regeneration medicine, including cell transplantation therapy and the study of primary cultured cells.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4082fje;
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