Tracking Neural Stem Cells in Patients with Brain Trauma

Tracking Neural Stem Cells in Patients with Brain Trauma

To the Editor: Regeneration of damaged brain tissue with neural stem cells is a promising strategy for reversing neurologic deficits.1 Superparamagnetic iron oxide nanoparticles have been used to label and track dendritic cells in the experimental treatment of melanoma2 and in experiments in animals.3 We report the feasibility of labeling neural stem cells from humans (two patients for whom written informed consent was provided by next of kin) with superparamagnetic iron oxide nanoparticles and tracking them with the use of magnetic resonance imaging (MRI).

A 34-year-old man had brain trauma in the left temporal lobe in February 2004. During an emergency operation, exposed neural tissue from his brain was collected and cultured in a medium previously shown to select for neural stem cells (for more details, see the Supplementary Appendix, available with the full text of this letter at www.nejm.org ). The day before implantation of the cultured neural stem cells, we incubated them in Feridex I.V.4 (a contrast agent containing superparamagnetic iron oxide nanoparticles that is approved by the Food and Drug Administration5) and Effectene (a lipofection reagent) in medium for 60 minutes to allow the Feridex I.V. to infuse into the cells and thereby label them (Figure 1). These autologous cultured neural stem cells were then implanted stereotactically around the region of brain damage. We performed MRI with a 3.0-T system (Signa, General Electric) and gradient reflection echo with a recovery time of 200 msec, an echo time of 20 msec, and a flip angle of 20 degrees at 24 hours and then every 7 days after transplantation for 10 weeks.

Figure 1

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Figure 1. Photomicrograph (Panel A) and Transmission Electron Photomicrograph (Panel B) of Neural Stem Cells Labeled with Iron Oxide Nanoparticles.

Panel A shows the nanoparticles (blue) in the cells after Prussian blue staining and counterstaining with neutral red. Panel B shows clusters of iron nanoparticles in nuclear and cell membranes (arrows) close to the Golgi apparatus; these clusters confirm the presence of iron inside the cell.

We observed marked signal dampening on T2-weighted MRI images. Pronounced hypointense signals were not found at the injection sites before implantation (Figure 2A). The injection sites were visible as circular areas of dark tissue on the first day after implantation (Figure 2B). The hypointense signal at each injection site faded thereafter (Figure 2C through 2F). One week after implantation, the change in signal was consistent with cell accumulation and proliferation around the lesion (Figure 2D). The signal at the periphery of the lesion intensified during the second and third weeks (Figure 2E and 2F), suggesting that the neural stem cells had migrated from the primary sites of injection to the border of the damaged tissue. We did not observe a hypointense signal after 7 weeks, which we attribute to a dilution of signal due to cell proliferation.

Figure 2

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Figure 2. MRI Scans from the Patient Who Received Neural Stem Cells Labeled with Iron Oxide Nanoparticles (Panels A through F) and the Patient Who Received Unlabeled Cells (Panels G through L).

The scan obtained before the implantation of the labeled neural stem cells (Panel A) did not show a pronounced hypointense signal around the lesion (asterisk) in the left temporal lobe, whereas circular areas of hypointense signal were visible at the injection sites 1 day after implantation (Panel B). Magnified images are shown in Panels C through F. Four hypointense signals (black arrows) were observed at injection sites around the lesion on day 1 (Panel C), day 7 (Panel D), day 14 (Panel E), and day 21 (Panel F). On day 7 (Panel D), we observed dark signals (white arrow) posterior to the lesion, a finding that was consistent with the presence of the labeled cells. By day 14 (Panel E), the hypointense signals at the injection sites had faded, and another dark signal (white arrowhead) had appeared and spread along the border of the damaged brain tissue.

By day 21 (Panel F), the dark signal had thickened and extended further along the lesion (white arrow). The scans in Panels G and H, from the patient who underwent implantation of unlabeled cells, were obtained on days 0 and 1, respectively, and the magnified views in Panels I, J, K, and L were obtained on days 1, 7, 14, and 21, respectively. A slightly hypointense signal is present around the injection sites in Panels I, J, K, and L. In these panels, the black arrows indicate the hypointense signal, and the asterisks indicate the lesion.

The control patient was a 42-year-old man who had brain trauma in the right temporal lobe. We cultured neural stem cells from this patient without labeling them with superparamagnetic iron oxide nanoparticles and then implanted them. We did not observe a pronounced change in signal around his lesion after implantation, but we did observe a slightly hypointense signal around the injection sites (Figure 2G through 2L), which showed no significant change during the observation period.

To explore the possibility that the magnetic signal was generated by macrophages that engulfed the labeled neural stem cells, we implanted neural stem cells that had been double-labeled (with green fluorescent protein and superparamagnetic iron oxide nanoparticles) in a rat model of traumatic brain injury. Three weeks after implantation, the regions containing the cells labeled with superparamagnetic iron oxide nanoparticles were identical to those containing cells labeled with green fluorescent protein. This finding suggests that the hypointense signals were not generated by engulfment of the implanted cells by macrophages (for more details, see the Supplementary Appendix). Our pilot clinical study shows that stem-cell engraftment and migration after implantation can be detected noninvasively with the use of MRI.

Jianhong Zhu, M.D., Ph.D.
Liangfu Zhou, M.D.
Fudan University Huashan Hospital
Shanghai 200040, China
jzhu@fudan.edu.cn

FengGe XingWu, M.D.
Shanghai National Key Laboratory for Medical Neurobiology
Shanghai 200032, China

References

1. Lindvall O, Kokaia Z, Martinez-Serrano A. Stem cell therapy for human neurodegenerative disorders -- how to make it work. Nat Med 2004;10:Suppl:S42-S50. [CrossRef][Medline]
2. de Vries IJ, Lesterhuis WJ, Barentsz JO, et al. Magnetic resonance tracking of dendritic cells in melanoma patients for monitoring of cellular therapy. Nat Biotechnol 2005;23:1407-1413. [CrossRef][ISI][Medline]
3. Arbab AS, Yocum GT, Kalish H, et al. Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI. Blood 2004;104:1217-1223. [Free Full Text]
http://content.nejm.org/cgi/ijlink?linkType=ABST&journalCode=bloodjournal&resid=104/4/1217
4. Arbab AS, Bashaw LA, Miller BR, et al. Characterization of biophysical and metabolic properties of cells labeled with superparamagnetic iron oxide nanoparticles and transfection agent for cellular MR imaging. Radiology 2003;229:838-846. [Free Full Text]
http://content.nejm.org/cgi/ijlink?linkType=ABST&journalCode=radiology&resid=229/3/838
5. Harisinghani MG, Barentsz J, Hahn PF, et al. Noninvasive detection of clinically occult lymph-node metastases in prostate cancer. N Engl J Med 2003;348:2491-2499. [Erratum, N Engl J Med 2003;349:1010.] [Free Full Text]
http://content.nejm.org/cgi/ijlink?linkType=ABST&journalCode=nejm&resid=348/25/2491

Thanks for the post. I can't believe the number of people I know who are angry at the Republicans for opposing "stem cell" research when they mean "embryonic stem cell" research.

Neural stem cells show great promise. As do Umbilical Stem cells.

7 days ago I listened to a presentation given by a friend who who was diagnosed with an extremely agressive form of leukemia called AML. 1 year later she is not only alive but has no vestige of the disease left in her body.

She was given what is called a dual donor umbilical stem cell transplant. Her blood type changed, and her skin is beginning to darken to a shade closer to that of the donor (Spanish).

My mind is still spinning trying to grasp what the doctors accomplished. I am astounded.

Wow! We don't hear these kinds of things on LMSM.

And we need to get the word out by writing letters to editors, and/or calling talk shows, that there are two kinds of stem cells - adult and embryonic.

Her doctor is in Boston, but she lives here in Maine. She went to the local hospital for a routine transfusion as part of her treatment, and they wouldn't perform the procedure at first because her tested type didn't match their records.

They had to call the doctor in Boston to confirm what had happened before they would proceed. The hospital staff was incredulous.

She is literally more than 40 years older than her own blood factory. And it appears that the stem cells haven't only been busy on replacing her bone marrow.

I will say it again, it boggles my mind. I am still having a difficult time absorbing the implications of what she has experienced. In 7 years, is the genetic code for most of her self-replacing tissues going to test out as being from another individual? If so, what does that portend?

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