Arinobu Tojo

Comparison of mesenchymal stem cells derived from arterial, venous, and Wharton's jelly explants of human umbilical cord.

We isolated mesenchymal stem cells (MSC) from arteries (UCA), veins (UCV), and Wharton’s jelly (UCWJ) of human umbilical cords (UC) and determined their relative capacities for sustained proliferation and multilineage differentiation. Individual UC components were dissected, diced into 1–2 mm³ fragments, and aligned in explant cultures from which migrating cells were isolated using trypsinization. Preparations from 13 UCs produced 13 UCWJ, 11 UCV, and 10 UCA cultures of fibroblast-like, spindle-shaped cells negative for CD31, CD34, CD45, CD271, and HLA-class II, but positive for CD13, CD29, CD44, CD73, CD90, CD105, and HLA-class I. UCV cells exhibited a significantly higher frequency of colony-forming units fibroblasts than did UCWJ and UCA cells. Individual MSCs could be selectively differentiated into osteoblasts, chondrocytes, and adipocytes. When compared for osteogenic potential, UCWJ cells were the least effective precursors, whereas UCA-derived cells developed alkaline phosphatase activity with or without an osteogenic stimulus. UC components, especially blood vessels, could provide a promising source of MSCs with important clinical applications.

A highly conserved tyrosine residue at codon 845 within the kinase domain is not required for the transforming activity of human epidermal growth factor receptor

Epidermal growth factor receptor (EGF-R) is a widely expressed ligand-dependent tyrosine kinase. The tyrosine residue at 845 in EGF-R corresponds to Y416 of v/c-src kinase, which is highly conserved and functionally important in many tyrosine kinases. To clarify the functional role of Y845, we constructed a mutant human EGF-R in which this tyrosine was replaced with phenylalanine and transfected it to NIH3T3 cells. EGF-R F845 induced EGF-dependent cellular transformation and revealed tyrosine-autophosphorylation of a 170 kDa protein, and initiated DNA synthesis similar to the wild-type EGF-R. We conclude here that Y845 is dispensable in the above mentioned functions of EGF-R tyrosine kinase.

Effective transduction and stable transgene expression in human blood cells by a third-generation lentiviral vector

Difficulty in gene transduction of human blood cells, including hematopoietic stem cells, has hampered the development of gene therapy applications for hematological disorders, encouraging the development and use of new gene delivery systems. In this study, we used a third-generation self-inactivating (SIN) lentiviral vector system based on human immunodeficiency virus type 1 (HIV-1) to improve transduction efficiency and prevent vector-related toxicity. The transduction efficiency of the HIV-1-based vector was compared directly with the Moloney murine leukemia virus (MLV) SIN vector in human leukemia cell lines. Initial transduction efficiencies were almost 100% for the HIV and less than 50% for the MLV vectors. Similar results were observed in 11 types of primary cells obtained from leukemia or myeloma patients. Transgene expression persisted for 8 weeks in cells transduced with the HIV vector, but declined with the MLV vector. In addition, resting peripheral blood lymphocytes and CD34+ hematopoietic cells were transduced successfully with the HIV vector, but not with the MLV vector. Finally, we confirmed vector gene integration in almost all colony-forming cells transduced with the HIV vector, but not with the MLV vector. In conclusion, this lentiviral vector is an excellent gene transduction system for human blood cells because of its high gene transduction and host chromosome integration efficiency.

Cytomegalovirus infection following unrelated cord blood transplantation for adult patients : a single institute experience in Japan

Summary. Cytomegalovirus (CMV) infection in 28 adult patients after cord blood transplantation (CBT) from unrelated donors was compared with that after bone marrow transplantation from HLA (human leucocyte antigen)‐matched related (R‐BMT) and unrelated (U‐BMT) donors. Positive CMV antigenaemia was seen in 19 (79%) of 24 CMV‐seropositive patients at a median of 42 d (range 29–85 d) after CBT, but in zero of four CMV‐seronegative patients. This did not differ significantly from values observed after R‐BMT and U‐BMT (66%, P = 0·22, and 60%, P = 0·15 respectively). Based on the antigenaemia results, 16 patients (67%) received pre‐emptive ganciclovir therapy from a median of 47 d (range 36–67 d) after CBT. This proportion was higher than that observed after R‐BMT (28%, P = 0·0048), but did not differ from that after U‐BMT (50%, P = 0·21). In addition, the probability of requiring more than two courses of ganciclovir therapy after CBT (21%) was higher than after R‐BMT and U‐BMT (0%, P = 0·015 and 0·039 respectively). One patient (5%) developed CMV disease after U‐BMT, whereas no patients developed CMV disease after CBT or R‐BMT. The CMV serostatus, use of a steroid and HLA disparity affected the probability of requiring ganciclovir therapy after CBT (P = 0·024, 0·032 and 0·017 respectively). These results suggest that recovery of CMV‐specific immunity after CBT is delayed when compared with BMT.

Feasibility and efficacy of bone tissue engineering using human bone marrow stromal cells cultivated in serum-free conditions.

Current standard techniques for bone tissue engineering utilize ex vivo expanded osteogenic cells. However, ex vivo expansion requires serum, which may hinder clinical applications. Here, we report the feasibility and efficacy of bone tissue engineering with human bone marrow stromal cells (BMSCs) expanded in serum-free conditions. Bone marrow was aspirated from 4 healthy donors and adherent cells were cultured in either serum-free medium (STEMPRO((R)) MSC SFM) or conventional serum-containing medium (alpha-MEM supplemented with 10% serum). Efficacy of expansion was greater in serum-free medium. Phenotypically, serum-free expanded BMSCs were smaller in cell-size and showed expression of CD105(++) and CD146(dim). After osteogenic induction, serum-free expanded BMSCs showed lower alkaline phosphatase activity. However, they showed higher responsiveness to induction. In vivo bone-forming ability was also confirmed. In conclusion, bone tissue engineering with serum-free expanded BMSCs is feasible and as efficient as that obtained with BMSCs expanded in conventional serum-containing medium.

Transient hematopoietic stem cell rescue using umbilical cord blood for a lethally irradiated nuclear accident victim.

We performed stem cell rescue and allogeneic skin transplantation on a lethally neutron-irradiated nuclear accident victim. HLA-DRB1 mismatched unrelated umbilical cord blood cells (2.08 × 107/kg recipient body weight) were transplanted to an 8–10 Gy equivalent neutron-irradiated patient because of a lack of a suitable bone marrow or peripheral blood donor. Pre-transplant conditioning consisted of anti-thymocyte γ-globulin alone, and GVHD prophylaxis was a combination of cyclosporine (CYA) and methylprednisolone (mPSL). Granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), and thrombopoietin (TPO) were concurrently administered after transplantation. The absolute neutrophil count reached 0.5 × 109/l on day 15, the reticulocyte count rose above 1% on day 23, and the platelet count was over 50 × 109/l on day 27, respectively. Cytogenetic studies of blood and marrow showed donor/recipient mixed chimerism. Rapid autologous hematopoietic recovery was recognized after withdrawal of CYA and mPSL. Repeated pathological examinations of the skin revealed no evidence of acute GVHD. Eighty-two days after the irradiation, skin transplantation was performed to treat radiation burns. Almost 90% of the transplanted skin engrafted. Immunological examination after autologous hematopoietic recovery revealed an almost normal T cell count. However, immune functions were severely impaired. The patient died from infectious complication 210 days after the accident.Bone Marrow Transplantation (2002) 29, 197–204. doi:10.1038/sj.bmt.1703356

A clinical comparison of unrelated cord blood transplantation and unrelated bone marrow transplantation for adult patients with acute leukaemia in complete remission

Summary.  We performed a clinical comparison of unrelated cord blood transplantation (UCBT) and unrelated bone marrow transplantation in adult acute leukaemia patients in complete remission (CR) who received the same conditioning regimen, graft‐versus‐host disease (GvHD) prophylaxis and supportive treatment. The incidence of acute GvHD was almost the same between the two groups, but the haematopoietic recovery was delayed and the incidence of chronic GvHD was higher in the UCBT group. The probability of 2 year disease‐free survival was similar between the two groups. These results suggest that adult acute leukaemia patients in CR without a suitable donor should be considered as candidates for UCBT.

Effects of type β transforming growth factors on haematopoietic progenitor cells

The effects of type β transforming growth factors (TGF‐βs) on normal human and murine haematopoietic progenitor cells were examined using bone marrow colony assays. In erythroid colony assays, TGF‐β1 inhibited human CFU‐E derived colony formation, BFU‐E derived burst formation, and murine BFU‐E derived burst formation in a dose dependent manner between 0.1 and 5.0 ng/ml. However, murine CFU‐E derived colony formation was unaffected even at a concentration of 5.0 ng/ml TGF‐β1. In myeloid colony assays, different sensitivity of progenitor cells to the inhibitory effects of TGF‐βs was observed between both species. TGF‐β1 inhibited murine granulocyte‐macrophage colony (GM‐colony) formation and granulocyte colony (G‐colony) formation in a dose dependent manner between 0.1 and 5.0 ng/ml, but had no remarkable effects on human GM‐colony and G‐colony formation. TGF‐β2 also had similar inhibitory effects on haematopoietic progenitor cells, while its inhibitory effect was less potent than that of TGF‐β1. Thus our data suggest that TGF‐β may be involved in negative regulation of haematopoiesis and that its inhibitory action may be restricted in lineage and/or species specific manner.

Bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for bone tissue engineering: basic science to clinical translation.

Bone tissue engineering is a promising field of regenerative medicine in which cultured cells, scaffolds, and osteogenic inductive signals are used to regenerate bone. This technology has already been used in several clinical studies and its efficacy has been reported. In this review, we focus on bone marrow stromal cells, which are the most commonly used cell source for bone tissue engineering. The nature of the cells, suitable culture conditions for bone tissue engineering, and their potential therapeutic applications are reviewed with possible caveats. Furthermore, recent advances in bone marrow stromal cell biology are discussed with reference to clinical translation.

ErbB receptor tyrosine kinase/NF-κB signaling controls mammosphere formation in human breast cancer.

Breast cancer is one of the most common cancers in humans. However, our understanding of the cellular and molecular mechanisms underlying tumorigenesis in breast tissues is limited. Here, we identified a molecular mechanism that controls the ability of breast cancer cells to form multicellular spheroids (mammospheres). We found that heregulin (HRG), a ligand for ErbB3, induced mammosphere formation of a breast cancer stem cell (BCSC)–enriched population as well as in breast cancer cell lines. HRG-induced mammosphere formation was reduced by treatment with inhibitors for phosphatidyl inositol 3-kinase (PI3K) or NF-κB and by expression of IκBα-Super Repressor (IκBαSR), a dominant-negative inhibitor for NF-κB. Moreover, the overexpression of IκBαSR in breast cancer cells inhibited tumorigenesis in NOD/SCID mice. Furthermore, we found that the expression of IL8, a regulator of self-renewal in BCSC-enriched populations, was induced by HRG through the activation of the PI3K/NF-κB pathway. These findings illustrate that HRG/ErbB3 signaling appears to maintain mammosphere formation through a PI3K/NF-κB pathway in human breast cancer.