Masayoshi Kobune

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.

Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.

Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone

There are currently no approved antifibrotic therapies for liver cirrhosis. We used vitamin A–coupled liposomes to deliver small interfering RNA (siRNA) against gp46, the rat homolog of human heat shock protein 47, to hepatic stellate cells. Our approach exploits the key roles of these cells in both fibrogenesis as well as uptake and storage of vitamin A. Five treatments with the siRNA-bearing vitamin A–coupled liposomes almost completely resolved liver fibrosis and prolonged survival in rats with otherwise lethal dimethylnitrosamine-induced liver cirrhosis in a dose- and duration-dependent manner. Rescue was not related to off-target effects or associated with recruitment of innate immunity. Receptor-specific siRNA delivery was similarly effective in suppressing collagen secretion and treating fibrosis induced by CCl4 or bile duct ligation. The efficacy of the approach using both acute and chronic models of liver fibrosis suggests its therapeutic potential for reversing human liver cirrhosis.

Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy

We developed human mesenchymal stem cell (MSC) lines that could differentiate into various tissue cells including bone, neural cells, bone marrow (BM) stromal cells supporting the growth of hematopoietic stem cell (HSC), and so‐called ‘tumor stromal cells’ mixing with tumor cells. We investigated the applicability of MSC as therapeutic cell transplanting reagents (cytoreagents). Telomerized human BM derived stromal cells exhibited a prolonged lifespan and supported the growth of hematopoietic clonogenic cells. The gene transfer of Indian hedgehog (Ihh) remarkably enhanced the HSC expansion supported by the human BM stromal cells. Gene‐modified MSC are useful as therapeutic tools for brain tissue damage (e.g. brain infarction) and malignant brain neoplasms. MSC transplantation protected the brain tissue from acute ischemic damage in the midcerebral artery occlusion (MCAO) animal model. Brain‐derived neurotrophic factor (BDNF)‐gene transduction further enhanced the protective efficacy against the ischemic damage. MSC possessed excellent migratory ability and exerted inhibitory effects on the proliferation of glioma cells. Gene‐modification of MSC with therapeutic cytokines clearly augmented the antitumor effect and prolonged the survival of tumor‐bearing animals. Gene therapy employing MSC as a tissue‐protecting and targeting cytoreagent would be a promising approach. (Cancer Sci 2005; 96: 149–156)

Long-term phlebotomy with low-iron diet therapy lowers risk of development of hepatocellular carcinoma from chronic hepatitis C.

BackgroundWe have previously demonstrated that in patients with chronic hepatitis C (CHC), iron depletion improves serum alanine aminotransferase (ALT) levels as well as hepatic oxidative DNA damage. However, it has not been determined whether continuation of iron depletion therapy for CHC favorably influences its progression to hepatocellular carcinoma (HCC).MethodsWe conducted a cohort study on biopsy-proven CHC patients with moderate or severe liver fibrosis who failed to respond to previous interferon (IFN) therapy or had conditions for which IFN is contradicted. Patients were divided into two groups: subjects in group A (n = 35) underwent weekly phlebotomy (200 g) until they reached a state of mild iron deficiency, followed by monthly maintenance phlebotomy for 44–144 months (median, 107 months), and they were advised to consume a low-iron diet (5–7 mg iron/day); group B (n = 40) comprised CHC patients who declined to receive iron depletion therapy.ResultsIn group A, during the maintenance phase, serum ALT levels decreased to less than 60 IU/l in all patients and normalized (<40 IU/l) in 24 patients (69%), whereas in group B no spontaneous decrease in serum ALT occurred. Hepatocarcinogenesis rates in groups A and B were 5.7% and 17.5% at the end of the fifth year, and 8.6% and 39% in the tenth year, respectively. Multivariate analysis revealed that iron depletion therapy significantly lowered the risk of HCC (odds ratio, 0.57) compared with that of untreated patients (P = 0.0337).ConclusionsLong-term iron depletion for CHC patients is a promising modality for lowering the risk of progression to HCC.

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.

Ex Vivo Large‐Scale Generation of Human Platelets from Cord Blood CD34+ Cells

In the present investigation, we generated platelets (PLTs) from cord blood (CB) CD34+ cells using a three‐phase culture system. We first cultured 500 CB CD34+ cells on telomerase gene‐transduced human stromal cells (hTERT stroma) in serum‐free medium supplemented with stem cell factor (SCF), Flt‐3/Flk‐2 ligand (FL), and thrombopoietin (TPO) for 14 days. We then transferred the cells to hTERT stroma and cultured for another 14 days with fresh medium containing interleukin‐11 (IL‐11) in addition to the original cytokine cocktail. Subsequently, we cultured the cells in a liquid culture medium containing SCF, FL, TPO, and IL‐11 for another 5 days to recover PLT fractions from the supernatant, which were then gel‐filtered to purify the PLTs. The calculated yield of PLTs from 1.0 unit of CB (5 × 106 CD34+ cells) was 1.26 × 1011−1.68 × 1011 PLTs. These numbers of PLTs are equivalent to 2.5–3.4 units of random donor‐derived PLTs or 2/5–6/10 of single‐apheresis PLTs. The CB‐derived PLTs exhibited features quite similar to those from peripheral blood in morphology, as revealed by electron micrographs, and in function, as revealed by fibrinogen/ADP aggregation, with the appearance of P‐selectin and activated glycoprotein IIb‐IIIa antigens. Thus, this culture system may be applicable for large‐scale generation of PLTs for future clinical use.

Drug resistance is dramatically restored by hedgehog inhibitors in CD34+ leukemic cells.

Aberrant reactivation of hedgehog (Hh) signaling has been described in a wide variety of human cancers and in cancer stem cells. However, the contribution of Hh signaling to leukemic cell regulation has remained unclear. In this study, we assessed the possibility that Hh pathway activation contributes to the survival and drug resistance of cluster of differentiation (CD)34+ leukemia cells. Hh signaling in leukemic cell lines and primary leukemic cells was screened by reverse transcription – polymerase chain reaction (RT‐PCR) and a Hh signaling reporter assay. We found that Hh signaling is active in several human acute myeloid leukemia (AML) cells, especially primary CD34+ leukemic cells and cytokine‐responsive CD34+ cell lines such as Kasumi‐1, Kasumi‐3 and TF‐1. These CD34+ cells express the downstream effectors glioma‐associated oncogene homolog (GLI)1 or GLI2, indicative of active Hh signaling. Moreover, inhibition of Hh signaling with the naturally derived Smoothened antagonist cyclopamine, endogenous Hh inhibitor hedgehog‐interacting protein or anti‐hedgehog neutralizing antibody induced apoptosis after 48 h of exposure, although these CD34+ cell lines exhibited resistance to cytarabine (Ara‐C). In contrast, cyclopamine failed to affect growth or survival in U937 and HL‐60 cell lines that lack expression of Hh receptor components, confirming that the effect of Hh inhibition is specific. Furthermore, combination with 10 µM cyclopamine significantly reduced drug resistance of CD34+ cell lines and primary CD34+ leukemic cells to Ara‐C. These results suggest that aberrant Hh pathway activation is a feature of some CD34+ myeloid leukemic cells and Hh inhibitors may have a therapeutic role in the treatment of AML. (Cancer Sci 2009; 100: 948–955)

Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages

We generated red blood cells (RBC) from cord blood (CB) CD34+ cells using a four-phase culture system. We first cultured CB CD34+ cells on telomerase gene-transduced human stromal cells in serum-free medium containing stem cell factor (SCF), Flt-3/Flk-2 ligand, and thrombopoietin to expand CD34+ cells (980-fold) and the total cells (10,400-fold) (first phase). Expanded cells from the first phase were liquid-cultured with SCF, interleukin-3 (IL-3), and erythropoietin (EPO) to expand (113-fold) and differentiate them into erythroblasts (second phase). To obtain macrophages for the next phase, we expanded CD34+ cells from a different donor using the same co-culture system. Expanded cells from the first phase were liquid-cultured with granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor (M-CSF), IL-3, and SCF to generate monocytes/macrophages (75-fold), which were incubated with type AB serum and M-CSF to fully differentiate them into macrophages. Erythroblasts were then co-cultured with macrophages in the presence of EPO to expand (threefold) and fully differentiate them (61% orthochromatic erythroblasts plus 39% RBC) (third phase). RBC were purified from erythroblasts and debris through a deleukocyting filter to generate 6.0 × 1012 RBC from 1.0 unit of CB (3.0 transfusable units). Qualitatively, these RBC showed a hemoglobin content, oxygenation of hemoglobin, and in vivo clearance similar to those of adult peripheral RBC. Finally, an almost complete enucleation of orthochromatic erythroblasts (99.4%) was achieved by the cultivation method recently described by Miharada et al. in the absence of macrophages and cytokines (fourth phase). RBC were purified from remnant erythroblasts and debris by passage through a deleukocyting filter to generate 1.76 × 1013 RBC from 1.0 unit of CB (8.8 transfusable units), the highest yield ever reported. Thus, this method may be useful for generating an alternative RBC supply for transfusions, investigating infectious agents that target erythroid cells, and as a general in vitro hematopoietic model system.

Hepatitis C virus core protein promotes proliferation of human hepatoma cells through enhancement of transforming growth factor α expression via activation of nuclear factor-κB

Background: Hepatitis C virus (HCV) infection is a major cause of human hepatocellular carcinoma (HCC). The precise mechanism of hepatocarcinogenesis in humans by HCV is currently unclear. It was recently shown, however, that transgenic mice with the HCV core gene often develop HCC, suggesting tumorigenic activity of the HCV core protein. Further, the HCV core protein expressed in HepG2 cells transfected with the core gene was shown to stimulate proliferation of transfectants through activation of nuclear factor-κB (NF-κB). The downstream target molecule(s) of NF-κB activated by the HCV core protein to evoke cell proliferation is not yet identified. Transforming growth factor (TGF) α, which is often overexpressed in various tumour tissues such as HCC, has been shown to stimulate hepatocyte proliferation through activation of the mitogen-activated protein kinase or extracellular signal-related protein kinase (MAPK/ERK) cascade. Aims: To explore the possibility that TGFα might be a target molecule for NF-κB activated by the HCV core, and that TGFα participates in the growth promotion of the core transfectants in an autocrine manner, activating the MAPK/ERK pathway. Methods: A HCV core expression vector was transfected into human hepatoma Huh-7, HepG2 and Hep3B cells. NF-κB activity was examined by an electrophoretic mobility shift assay. TGFα transcription was assessed by a luciferase reporter assay. TGFα protein was determined by immunoblot and ELISA. MAPK/ERK activity was examined by an in vitro kinase assay. Cell proliferation was assessed by a water-soluble tetrazolium salt-1 assay. Results: In the HCV core transfectants, NF-κB bound to the κB site in the TGFα proximal promoter region, resulting in an increase in TGFα transcription. Immunoblot as well as ELISA showed increased TGFα expression in the HCV core transfectants. SN50, a specific inhibitory peptide for NF-κB, cancelled HCV core-induced TGFα expression. HCV core protein increased cell proliferation as well as ERK activity of the HCV core transfectants as compared with the mock transfectants. The growth-promoting activity and activation of ERK by the HCV core protein were negated by treatment with anti-TGFα antibodies. Conclusions: These results suggest that the HCV core protein promotes proliferation of human hepatoma cells by activation of the MAPK/ERK pathway through up regulation of TGFα transcription via activation of NF-κB. Our finding provides a new insight into the mechanism of hepatocarcinogenesis by HCV infection.