Infection of donor lymphocytes with human T lymphotrophic virus type 1 (HTLV-I) following allogeneic bone marrow transplantation for HTLV-I positive adult T-cell leukaemia

Human T lymphotrophic virus type 1 (HTLV-I) associated leukaemia has a poor prognosis even with chemotherapy. We describe a patient with adult T-cell leukaemia treated with allogeneic bone marrow transplantation from an HTLV-I negative identical sibling donor. During follow-up after bone marrow transplantation, HTLV-I could be repeatedly isolated inspite of anti-viral prophylaxis. The patient died of an acute encephalitis and HTLV-I could be detected in autopsy material from the brain. By a PCR-based technique using short tandem repeats (STRs) it was shown that the patient's haemopoiesis was of donor origin. This shows the infection of donor cells in vivo by an aetiological agent which has been implicated in the leukaemogenic process for adult T-cell leukaemia.

Three-dimensional cultures of normal human osteoblasts: proliferation and differentiation potential in vitro and upon ectopic implantation in nude mice.

This paper is novel andreports on the in vitro establishment of 3-D cultures of human osteoblasts. These were evaluated for protein markers of bone cells. Sequentially alkaline phosphatase, calcium incorporation for matrix mineralisation and then finally osteocalcin expression were detected in cultures. The extracellular matrix was composed of type 1 collagen and as it mineralised, needle shaped crystals were often associated with matrix vesicles initiating mineralisation. In vivo implantation in nude mice showed progression of mineralisation from the inner region outward with peripheral cells in a non-mineralised matrix. Host vessels invaded the implanted cell area. The research has relevance to musculoskeletal tissue engineering.

Chondrogenic Differentiation Alters the Immunosuppressive Property of Bone Marrow-Derived Mesenchymal Stem Cells, and the Effect is Partially due to the Upregulated Expression of B7 Molecules

To investigate the immunosuppressive properties of mesenchymal stem cells (MSCs), in the present study we examined the immunogenicity of undifferentiated and tri-lineage (chondrocytes, osteoblasts and adipocytes) differentiated rat bone marrow-derived MSCs under xenogeneic conditions. After chondrogenic-differentiation, rat bone marrow-derived MSCs stimulated human peripheral blood monocyte-derived DCs (hDCs), leading to 8- and 4-fold higher lymphocyte proliferation and cytotoxicity than that of undifferentiated MSCs. The Chondrogenic-differentiated MSCs were chemotactic to hDCs in Dunn chamber chemotaxis system and were rosetted by hDCs inrosette assays. Flow cytometry analysis revealed that chondrogenic-differentiated MSCs had promoted hDCs maturation causing higher CD83 expression in hDCs, whereas undifferentiated MSCs, osteogenic-and adipogenic-differentiated MSCs showed inhibitory effect on hDCs maturation. The co-stimulatory molecules B7 were up-regulated only in the chondrogenic-differentiated MSCs. After blocking B7 molecules with specific monoclonal antibodies in the chondrogenic-differentiated MSCs, CD83 expression of co-cultured hDCs was greatly reduced. In conclusion, chondrogenic differentiation may increase the immunogenicity of MSCs, leading to stimulation of DCs. The up-regulated expression of B7 molecules on the chondrogenic differentiated MSCs may be partially responsible for this event.

Allogenic Peripheral Blood Derived Mesenchymal Stem Cells (MSCs) Enhance Bone Regeneration in Rabbit Ulna Critical-Sized Bone Defect Model

Mesenchymal stem cells (MSCs) were demonstrated to exist within peripheral blood (PB) of several mammalian species including human, guinea pig, mice, rat, and rabbit. Whether or not the PB derived MSCs (PBMSCs) could enhance the regeneration of large bone defects have not been reported. In this study, rabbit MSCs were obtained from mononuclear cells (MNCs) cultures of both the PB and bone marrow (BM) origin. The number of PBMSCs was relatively lower, with the colony forming efficiency (CFE) ranging from 1.2-13 per million MNCs. Under specific inductive conditions, PBMSCs differentiated into osteoblasts, chondrocytes, and adipocytes, showing multi- differentiation ability similar to BMMSCs. Bilateral 20 mm critical-sized bone defects were created in the ulnae of twelve 6-month old New Zealand white rabbits. The defects were treated with allogenic PBMSCs/Skelite (porous calcium phosphate resorbable substitute), BMMSCs/Skelite, PBMNCs/Skelite, Skelite alone and left empty for 12 weeks. Bone regeneration was evaluated by serial radiography, peripheral quantitative computed tomography (pQCT), and histological examinations. The x-ray scores and the pQCT total bone mineral density in the PBMSCs/Skelite and BMMSCs/Skelite treated groups were significantly greater than those of the PBMNCs/Skelite and Skelite alone groups (p

Human marrow mesenchymal stem Cells (MSCs) in the peplated non-adherent cell population serve as a complementary source of osteogenic cells

To obtain enough quantity of osteogenic cells is a challenge for successful cell therapy in bone defect treatment, and cell numbers were usually achieved by culturing bone marrow cells in a relatively long duration. This study reported a simple and cost effective method to enhance the number of MSCs by collecting and replating the non-adherent cell population of marrow MSCs culture. Bone marrow MSCs were isolated from 11 patients, cultured at a density of 1×105/cm2 to 1×106/cm2 in flasks. For the first three times of media change, the floating cells were centrifuged and replated in separate flasks. The total number of cells in both the primary and replating flasks were counted at day 21. Cell proliferation rate, potentials for osteogenic, chondrognenic, and adipogenic differentiation were examined in both cell types in vitro. In-vivo osteogenic potentials of the cells were also tested in mice implantation model. The results showed that MSCs derived from non-adherent cell population of marrow cell cultures have similar cell proliferation and differentiation potentials as the originally attached MSCs in vitro. When implanted with HA-TCP materials subcutaneously in SCID mice, newly formed bony tissues were found in both cell type groups with osteocalcin expression. We have obtained 36.6% (20.70%-44.97%) more MSCs in the same culture period when the non-adherent cell populations were collected. The findings confirmed that the non-adherent cell population in the bone marrow culture is a complementary source of MSCs, collecting these cells is a simple and cost-effective way to increase MSCs numbers and reduce the time required for culturing MSCs for clinical applications.

RUNX3 protein is overexpressed in human basal cell carcinomas

Basal cell carcinomas (BCC), which are the most common form of skin malignancy, are invariably associated with the deregulation of the Sonic Hedgehog (Shh) signalling pathway. As such, BCC represent a unique model for the study of interactions of the Shh pathway with other genes and pathways. We constructed a tissue microarray (TMA) of 75 paired BCC and normal skin and analysed the expression of beta-catenin and RUNX3, nuclear effectors of the wingless-Int (Wnt) and bone morphogenetic protein/transforming growth factor-beta pathways, respectively. In line with previous reports, we observed varying subcellular expression pattern of beta-catenin in BCC, with 31 cases (41%) showing nuclear accumulation. In contrast, all the BCC cases tested by the TMA showed RUNX3 protein uniformly overexpressed in the nuclei of the cancer cells. Analysis by Western blotting and DNA sequencing indicates that the overexpressed protein is normal and full-length, containing no mutation in the coding region, implicating RUNX3 as an oncogene in certain human cancers. Our results indicate that although the deregulation of Wnt signalling could contribute to the pathogenesis of a subset of BCC, RUNX3 appears to be a universal downstream mediator of a constitutively active Shh pathway in BCC.

An in vitro Study to Assess the Potential of a Unique Micro porous Algal Derived Cap Bone Void Filler in Comparison with Clinically-Used Bone Void Fillers

Macroporosity(>100µm) in bone void fillers is a known prerequisite for tissue regeneration, but recent literature has highlighted the added benefit of microporosity(0.5 - 10µm). The aim of this study was to compare the in vitro performances of a novel interconnective microporous hydroxyapatite (HA) derived from red algae to four clinically available macroporous calcium phosphate (CaP) bone void fillers. The use of algae as a starting material for this novel void filler overcomes the issue of sustainability, which overshadows continued use of scleractinian coral in the production of some commercially available materials, namely Pro-OsteonTM and Bio-Coral®. This study investigated the physicochemical properties of each bone voidfiller material using x-ray diffraction, fourier transform infrared spectroscopy, inductive coupled plasma, and nitrogen gas absorption and mercury porosimetry. Biochemical analysis, XTT, picogreen and alkaline phosphatase assays were used to evaluate the biological performances of the five materials. Results showed that algal HA is non-toxic to human foetal osteoblast (hFOB) cells and supports cell proliferation and differentiation. The preliminary in vitro testing of microporous algal-HA suggests that it is comparable to the four clinically approved macroporous bone void fillers tested. The results demonstrate that microporous algal HA has good potential for use in vivo and in new tissue engineered strategies for hard tissue repair.

Isolation of a bone marrow-derived stem cell line with high proliferation potential and its application for preventing acute fatal liver failure

Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. Clonal cell lines were established from the bone marrow of two different rat strains. One of these cell lines, rBM25/S3 cells, grew rapidly (doubling time, approximately 24 hours) without any appreciable changes in cell properties for at least 300 population doubling levels over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin, and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with hepatocyte growth factor and fibroblast growth factor-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, cytochrome P450 (CYP) 1A1, CYP1A2, glucose 6-phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF)1 alpha, and HNF4alpha. Intrasplenic transplantation of the differentiated cells prevented fatal liver failure in 90%-hepatectomized rats. In conclusion, a clonal stem cell line derived from adult rat bone marrow could differentiate into hepatocyte-like cells, and transplantation of the differentiated cells could prevent fatal liver failure in 90%-hepatectomized rats. The present results indicate a promising strategy for treating human fatal liver diseases.

Human mesenchymal stem cells reduce mortality and bacteremia in gram-negative sepsis in mice in part by enhancing the phagocytic activity of blood monocytes

The potential therapeutic value of cell-based therapy with mesenchymal stem cells (MSC) has been reported in mouse models of polymicrobial peritoneal sepsis. However, the mechanisms responsible for the beneficial effects of MSC have not been well defined. Therefore, we tested the therapeutic effect of intravenous bone marrow-derived human MSC in peritoneal sepsis induced by gram-negative bacteria. At 48 h, survival was significantly increased in mice treated with intravenous MSC compared with control mice treated with intravenous fibroblasts (3T3) or intravenous PBS. There were no significant differences in the levels of TNF-a, macrophage inflammatory protein 2, or IL-10 in the plasma. However, there was a marked reduction in the number of bacterial colony-forming units of Pseudomonas aeruginosa in the blood of MSC-treated mice compared with the 3T3 and PBS control groups. In addition, phagocytic activity was increased in blood monocytes isolated from mice treated with MSC compared with the 3T3 and PBS groups. Furthermore, levels of C5a anaphylotoxin were elevated in the blood of mice treated with MSC, a finding that was associated with upregulation of the phagocytosis receptor CD11b on monocytes. The phagocytic activity of neutrophils was not different among the groups. There was also an increase in alternately activated monocytes/macrophages (CD163- and CD206-positive) in the spleen of the MSC-treated mice compared with the two controls. Thus intravenous MSC increased survival from gram-negative peritoneal sepsis, in part by a monocyte-dependent increase in bacterial phagocytosis.