学位論文内容の要旨（ Summary of dissertation）

博士の専攻分野の名称
博士（医 学）（Degree conferred: Doctor of Philosophy）

氏 名 譚 成博
（Name of recipient: Chengbo Tan）

Studies on regeneration therapy for central nervous system using bone marrow stromal cells（骨髄間質細胞移植による中枢神経再生医療の研究）【Background and Objectives】
Human bone marrow stromal cells (hBMSCs) have been regarded as a potential cell source for ischemic
stroke therapy, owing to their potential to differentiate into multiple cell lineages, their neuroprotective
effects, and their ability to promote functional neural recovery of patients. Although these results areencouraging, several problems still remain unsolved to impede its clinical application. To solve theproblems, we designed new clinical trials called the RAINBOW study, which is a phase 1 study about
autologous BMSC transplantation for acute ischemic stroke. In the present study, we aimed to evaluate thefeasibility and the efficacy of hBMSC culture with allogeneic PL (Chapter I), the safety of SPIO-labeled
BMSCs and the curative effect of cell therapy after stereotactically transplantation (Chapter II) as thepreparatory work for RAINBOW study. We aimed to translate these results to the optimal design of theclinical trials.
【Materials and Methods】
Chapter I: Feasibility and efficiency of human bone marrow stromal cell culture with allogeneic platelet
lysate-supplementation for cell therapy against stroke
Platelet-concentrates (PC) was harvested from nine healthy volunteers and made into single
donor-derived PL (sPL). Three types of PL mixtures (mPL) were made from three different sPLs.
Platelet-derived growth factor (PDGF)-BB, transforming growth factor (TGF)-β1, brain-derivedneurotrophic factor (BDNF), and platelet cell surface antigens (CD41 and CD61) were detected byenzyme-linked immunosorbent assay (ELISA). Cell proliferative potential of 10% PL was compared withfetal calf serum (FCS). Cell surface markers of hBMSC cultured with PL (hBMSC-PL) were analyzed. The
hBMSC-PLs were incubated with superparamagnetic iron oxide (SPIO) agents and injected into a pig brain.
MRI and histological analysis were performed.
Chapter II: Short-, middle- and long-term safety of superparamagnetic iron oxide-labeled allogeneic
bone marrow stromal cell transplantation in rat lacunar infarction model
BMSCs were isolated from transgenic rats expressing green fluorescent protein (GFP), and they werelabeled with SPIO. A Na/K ATPase pump inhibitor ouabain or vehicle was stereotactically injected into theright striatum of wild type rats to induce lacunar lesion (n=22). 7 days after the insult, BMSCs or SPIO
solution were stereotactically transplanted into the left striatum. A 7.0-Tesla MRI was performed to seriallymonitor the behavior of BMSCs in the host brain. The animals were sacrificed 7 days (n=7), 6 weeks (n=6)
or 10 months (n=9) after transplantation, respectively.

【Results】
Chapter I:
MRI and histological analysis were performed. The result showed that twelve types of PLs (sPL: n = 9,mPL: n = 3) were prepared. ELISA analysis showed that PL contained adequate growth factors and a verysmall number of platelet surface antigens. Two weeks after culture, the proliferation capacity ofhBMSC-PLs was equivalent to or higher than that of FCS. No contradiction in cell surface markers werefound in hBMSC-PL. When injected into the pig brain, MRI detected the distribution of SPIO-labeled
hBMSCs similar to histological analysis.
Chapter II:
MRI demonstrated that BMSCs migrated to the damage area through the corpus callosum. Histologicalanalysis showed that activated microglia were found around the bolus of donor cells 7 days after allogeneic
cell transplantation, though immunosuppressive drug was used. The SPIO-labeled BMSCs resided andstarted to proliferate around the route of cell transplantation. Within 6 weeks, a large number ofSPIO-labeled BMSCs had reached the lacunar infarction area from transplantation region through thecorpus callosum. Some SPIO nanoparticles were phagocytized by microglia. In 10 months later, the SPIO
positive cells were less than the numbers in 7-day and 6-week groups. There was no tumorigenicity andsevere injury in all animals.
【Discussion】
In the present study, we found that the content of these growth factors in each PL was relevant to thenumber of platelets in original PC. Furthermore, the ability of cell expansion correlated with the contents ofthe growth factors in each PL. When the contents of platelets in original PC reached more than 15 units, thecell proliferating potential of PL was equivalent or much higher compared with the FCS. This indicated that
the PL-supplement contains adequate essential growth factors and nutrition as well as FCS for theexpansion of hBMSC. Moreover, because there was a smaller difference among mPL compared with sPL
regarding the contents of growth factors, pooled PL should be useful in mass production. The present
results were used for a quality control of PL produced under good manufacturing practice (GMP).
In addition, cell labeling with SPIO was employed to track donor cells in the host brain by means of MRI.
Cultured hBMSCs can uptake SPIO nanoparticles into their cytoplasm when the particles are added to theculture medium. Because they have clearly detectable signal extinctions, the labelled cells were easily
tracked anatomically with QSM and T2 MR images. We believe that MRI cell tracking with SPIO basedlabeling agents is a good resource to monitor cell distribution after hBMSC transplantation. In order toevaluate the safety of SPIO-labeled BMSCs histologically, we used immunohistochemistry with Turnbull
blue staining technique. In 10-month group, SPIO-solution or SPIO-labeled BMSCs caused no severe
inflammation in host brain, regardless of lacunar infarction or not. We hope this technology can be used forcell therapy in clinical applications.
【Conclusion】
hBMSCs cultured with allogeneic PL and labeled with SPIO may be a valuable, feasible, safe, andeffectible for cell therapy against cerebral ischemic stroke. We will translate these results to the protocol ofRAINBOW study, and we hope that it will be helpful for the patients who suffer from stroke.