札幌医科大学医学部

   分子医学研究部門
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2004年7月23日

 

 

札幌医科大学 分子医学研究部門 濱田洋文


Recent advances in laboratory:

Bone marrow stem cells (MSC) for clinical applications.

 

Hirofumi Hamada,  Department of  Molecular Medicine, Sapporo Medical University, Sapporo 060-8556, Japan.  MAIL: hhamada@sapmed.ac.jp

 

Examination of the clinical therapeutic efficacy of using bone marrow stromal cells, including mesenchymal stem cells (MSC), has recently been the focus of much investigation.  Here I demonstrate their potential clinical applications, e.g., 1) to support in vitro proliferation of bone marrow stem cells for bone marrow transplantation, 2) to protect the brain tissue from ischemic damage, and 3) to use them as drug delivery vehicles for the treatment of invasive malignant tumors.  

 

Artificial Bone Marrow using MSC-derived Stromal Cells: Indian Hedgehog Gene Transfer Augments Hematopoietic Support of Human Stromal cells Including NOD/SCID-Repopulating Cells.  We have previously shown that telomerized human bone marrow (BM) derived stromal cells (hTERT-stromal cells) exhibited prolonged lifespan and expanded hematopoietic clonogenic cells (Kawano-Y et al. Blood, 2003).  However, it had modest effect on the expansion of primitive and repopulating hematopoietic cells, even though it maintains stem cell activity ex vivo for a long term.  To improve the ability of short-term expansion of human stromal cells, retrovirus mediated-gene transfer of Indian hedgehog (Ihh), which activates primitive hematopoiesis in developing yolk sac, were conducted.  The degree of expansion of CD34+ cells, total number of colony-forming units in culture (CFU-C), CFU-mix and highly proliferative potential colony forming cell (HPP-CFC) upon 2-weeks coculture with the Ihh-transduced stromal cells (Ihh-stromal cells) were significantly higher than those upon 2-weeks co-culture with mock-transduced control stromal cells (CD34, 26±2 vs. 95±6-fold of the initial cell number; CFUs, 26±2 vs. 59±3-fold; CFU-mix, 63±37 vs. 349±116-fold; HPP-CFC, 200±175 vs. 1784±1555-fold).  The expansion of progenitor cells was partially neutralized by hedgehog blocking antibody.  The degree of engraftment of SCID repopulating cells at 8 and 13 weeks after transplantation that had been cocultured with Ihh-stromal cells, was 15 times higher than that of cocultured with control stromal cells (0.74±0.53 vs. 10.6±8.20 at 8 week, 0.06±0.03 vs. 3.23±0.62 at 13 week, p<0.05).  These results suggest that gene transfer of Ihh could enhance the hematopoietic stem cell support of human stromal cells and may have potential for medical application to regulate hematopoiesis and to develop a new modality of hematopoietic stem cells in therapeutic use. 

 

MSC for the Protection of Brain Damage: BDNF gene-modified mesenchymal stem cells promote functional recovery and reduce infarct size in the rat middle cerebral artery occlusion model.  MSC were reported to ameliorate functional deficits after stroke in rats, with some of this improvement possibly resulting from the action of cytokines secreted by these cells. To enhance such cytokine effects, we transfected telomerized human MSC with the BDNF gene using a fiber-mutant F/RGD adenovirus vector and investigated whether these cells contributed to improved functional recovery in a rat transient middle cerebral artery occlusion (MCAO) model. BDNF production by MSC-BDNF cells was 23-fold greater than that seen in uninfected MSC. Rats that received MSC-BDNF showed significantly more functional recovery than did control rats following MCAO. Specifically, MRI analysis revealed that the rats in the MSC-BDNF group exhibited more significant recovery from ischemia after 7 and 14 days. The number of TUNEL-positive cells in the ischemic boundary zone was significantly smaller in animals treated with MSC-BDNF compared to animals in the control group. These data suggest that MSC transfected with the BDNF gene may be useful in the treatment of cerebral ischemia and may represent a new strategy for the treatment of stroke.

 

MSC as a Tool for Drug Delivery: Gene-modified mesenchymal stem cells (MSCs) as a therapeutic tool for malignant brain neoplasms.   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 cells 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 alone caused significant inhibition of 9L tumor growth and increased the survival of 9L glioma-bearing rats.  Due to the lack of the receptor (CAR) on MSCs, the efficiency of gene transfer into MSCs by the adenoviral vector (Adv) with the wild-type Ad5 fiber (Adv-F/wt) was very poor.  In clear contrast, nearly 100% genetic transduction of MSCs was achieved using the integrin-targeting fiber-mutant Adv-F/RGD, which possesses a CDCRGDCFC peptide motif at the HI-loop of the fiber knob.  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, invasive brain tumors.

 

REFERENCES


  1. Kawano Y., Kobune M., Yamaguchi M., Nakamura K., Ito Y., Sasaki K., Takahashi S., Nakamura T., Chiba H., Sato T., Matsunaga T., Azuma H., Ikebuchi K., Ikeda H., Kato J., Niitsu Y. and Hamada H.  Ex vivo expansion of human umbilical cord hematopoietic progenitor cells using a coculture system with human telomerase catalytic subnit (hTERT)-transfected human stromal cells.  Blood,101(2): 532-540, 2003
  2. Tsuda H., Wada T., Ito Y., Uchida H., Dehari H., Nakamura K., Sasaki K., Kobune M., Yamashita T. and Hamada H.  Efficient BMP2 gene transfer and bone formation of mesenchymal stem cells by a fiber-mutant adenoviral vector.  Mol. Ther., 7(3): 354-365, 2003.

  3. Kobune M., Kawano Y., Ito Y., Chiba H., Nakamura K., Tsuda H., Sasaki K., Dehari H., Uchida H., Honmou O., Takahashi S., Bizen A., Takimoto R., Matsunaga T., Kato J., Kato K., Houkin K., Niitsu Y. and Hamada H.  Telomerized human multipotent mesenchymal cells can differentiate into hematopoietic and cobblestone area-supporting cells  Exp Hematol.31(8): 715-722, 2003.

  4. Kurozumi, K., Nakamura, K., Tamiya, T., Kawano, Y., Kobune, M., Hirai, S., Uchida, H., Sasaki, K., Ito, Y., Kato, K., Honmou, O., Houkin, H., Date, I., and Hamada, H.   BDNF gene-modified mesenchymal stem cells promote functional recovery and reduce infarct size in the rat middle cerebral artery occlusion model.  Mol. Ther.  9(2):189-97, 2004.

  5. Nakamura K, Ito Y, Kawano Y, Kurozumi K, Kobune M, Tsuda H, Bizen A, Honmou O, Niitsu Y, and Hamada, H.  Anti-tumor effect of genetically engineered mesenchymal stem cells in a rat glioma model.  Gene Ther.  In press, 2004.

  6. Kobune M, Ito Y, Kawano Y, Sasaki K, Uchida H, Nakamura K, Dehari H, Chiba H, Takimoto R, Matsunaga T, Terui T, Kato J, Niitsu Y, Hamada H.  Indian hedgehog gene transfer augments hematopoietic support of human stromal cells including NOD/SCID-{beta}2m-/- repopulating cells.  Blood in press 2004.





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