Hajime Tsuda

Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model

The prognosis of patients with malignant glioma is extremely poor, despite the extensive surgical treatment that they receive and recent improvements in adjuvant radio- and chemotherapy. In the present study, we propose the use of gene-modified mesenchymal stem cells (MSCs) as a new tool for gene therapy of malignant brain neoplasms. Primary MSCs isolated from Fischer 344 rats possessed excellent migratory ability and exerted inhibitory effects on the proliferation of 9L glioma cell in vitro. We also confirmed the migratory capacity of MSCs in vivo and showed that when they were inoculated into the contralateral hemisphere, they migrated towards 9L glioma cells through the corpus callosum. MSCs implanted directly into the tumor localized mainly at the border between the 9L tumor cells and normal brain parenchyma, and also infiltrated into the tumor bed. Intratumoral injection of MSCs caused significant inhibition of 9L tumor growth and increased the survival of 9L glioma-bearing rats. Gene-modification of MSCs by infection with an adenoviral vector encoding human interleukin-2 (IL-2) clearly augmented the antitumor effect and further prolonged the survival of tumor-bearing rats. Thus, gene therapy employing MSCs as a targeting vehicle would be promising as a new therapeutic approach for refractory brain tumor.

Efficient BMP2 gene transfer and bone formation of mesenchymal stem cells by a fiber-mutant adenoviral vector

Strategies using mesenchymal stem cell (MSC)-mediated gene therapy have been developed to improve bone healing. However, transduction efficiency into MSCs by each vector is not always high. To overcome this problem, we used a modified adenoviral vector (Adv-F/RGD) with an RGD-containing peptide in the HI loop of the fiber knob domain of adenovirus type 5 (Ad5). Transduction efficiency into bone marrow-derived MSCs with Adv-F/RGD increased 12-fold compared with a vector containing the wild-type fiber (Adv-F/wt) by beta-galactosidase chemiluminescent assay. As a next step, we constructed AxCAhBMP2-F/RGD and AxCAhBMP2-F/wt carrying human bone morphogenetic protein 2 (BMP2). At the same multiplicity of infection, MSCs infected with AxCAhBMP2-F/RGD produced higher amounts of BMP2 than cells infected with AxCAhBMP2-F/wt, and also differentiated towards the osteogenic lineage more efficiently in vitro. Furthermore, using ex vivo gene transduction, we evaluated the potential for ectopic bone formation by the transduced MSCs in vivo. Transduction with AxCAhBMP2-F/RGD exhibited greatly enhanced new bone formation. These data suggest that Adv-F/RGD is useful for introducing foreign genes into MSCs and that it will be a powerful gene therapy tool for bone regeneration and other tissue engineering.

Telomerized human multipotent mesenchymal cells can differentiate into hematopoietic and cobblestone area-supporting cells.

OBJECTIVE To compare the hematopoietic support provided by telomerized human mesenchymal stem cells (MSCs) and telomerized MSC-derived stromal cells. METHODS We transfected the human telomerase catalytic subunit (hTERT) gene into primary MSCs to establish hTERT-transduced MSCs (hTERT-MSCs). Stromal induction of hTERT-MSCs was performed by replacing the culture medium with Dexter-type culture medium. Hematopoietic support was examined by coculture with cord blood CD34(+) cells. RESULTS The hTERT-MSCs were morphologically identical with the primary MSCs and expressed surface antigens including CD105, CD73, and CD166. hTERT-MSCs showed a similar doubling time as primary MSCs and continued to proliferate to over 80 population doublings (PD), although the primary MSCs underwent crisis in vitro at 16 PD. The osteogenic, chondrogenic, adipogenic, neurogenic, and stromal differentiation potential of hTERT-MSCs were maintained up to at least 40 PD. The degree of expansion of CD34(+) cells and total number of colony-forming units in culture (CFU-C) upon 12-day coculture with the hTERT-MSC-derived stromal cells were nearly the same as those upon 12-day coculture with hTERT-MSCs (CD34, 33.0-fold+/-2.8-fold vs 36.1-fold+/-1.7-fold of the initial cell number; CFUs, 344.4-fold+/-62.5-fold vs 239.3-fold+/-87.0-fold; CFU-mix, 368.4-fold+/-113.7-fold vs 341.3-fold+/-234.3-fold). However, on day 18 of coculture, the number of cobblestone areas (CA) observed beneath the stromal cells was 15 times higher than that beneath hTERT-MSCs (CA, 146.9+/-54.6 vs 9.4+/-8.1, p<0.01). CONCLUSION Stromal induction of hTERT-MSCs exclusively enhanced the support of CA formation provided by hTERT-MSCs. Our human hTERT-MSCs will be useful for elucidating the mechanism of the formation of CAs.

Enhanced osteoinduction by mesenchymal stem cells transfected with a fiber-mutant adenoviral BMP2 gene.

Bone regeneration therapy using mesenchymal stem cells (MSCs) is beginning to come into clinical use. To overcome the difficulty of healing large bone defects, we previously reported the efficacy of using rat mesenchymal stem cells (rMSCs) carrying a modified adenoviral vector (Adv‐F/RGD) with an RGD‐containing peptide in the HI loop of the fiber knob domain of adenovirus type 5 (Ad5).

Clinical Presentation of Cervical Myelopathy at C1-2 Level

Study Design Single-center retrospective study. Purpose To clarify the clinical features of cervical myelopathy at the C1–2 level. Overview of Literature Methods for distinguishing the affected level based on myelomere symptoms or dysfunction of the conducting pathway were established. However, no symptoms have been identified as being specific to the C1–2 level segment. Methods We evaluated 24 patients with cervical myelopathy due to spinal cord compression at the C1–2 level. Preoperative neurological assessment were investigated and compared with the rate and site of compression of the spinal cord using computed tomography-myelography. Results Impaired temperature and pain sensation were confirmed in 18 of the 24 patients with that localized to the upper arms (n=3), forearm (n=9), both (n=2), and whole body (n=4). Muscle weakness was observed in 18 patients, muscle weakness extended from the biceps brachii to the abductor digiti minimi in 10 patients, and in the whole body in 8 patients. Deep tendon reflexes were normal in 10 patients, whereas hyperactive deep tendon reflexes were noted in 14 patients. The rate of spinal cord compression was significantly higher in patients with perceptual dysfunction and muscle weakness compared with those with no dysfunction. However, no significant difference in the rate and site of compression was identified in those with dysfunction. Conclusions Perceptual dysfunction and muscle weakness localized to the upper limbs was observed in 58% and 42% of patients, respectively. Neurological abnormalities, such as perceptual dysfunction and muscle weakness, were visualized in patients with marked compression.