Human chromosome 11p15.5 harbors an intriguing imprinted gene cluster of 1 Mb. This imprinted domain is implicated in a wide variety of malignancies and Beckwith-Wiedemann syndrome (BWS). Recently, several lines of evidence have suggested that the BWS-associated imprinting cluster consists of separate chromosomal domains. We have previously identified LIT1, a paternally expressed antisense RNA within the KvLQT1 locus through a positional screening approach using human monochromosomal hybrids. KvLQT1 encompasses the translocation breakpoint cluster in BWS and patients exhibit frequent loss of maternal methylation at the LIT1 CpG island, implying a regulatory role for the LIT1 locus in coordinate control of the imprinting cluster. Here we generated modified human chromosomes carrying a targeted deletion of the LIT1 CpG island using recombination-proficient chicken DT40 cells. Consistent with the prediction, this mutation abolished LIT1 expression on the paternal chromosome, accompanied by activation of the normally silent paternal alleles of multiple imprinted loci at the centromeric domain including KvLQT1 and p57(KIP2). The deletion had no effect on imprinting of H19 located at the telomeric end of the cluster. Our findings demonstrate that the LIT1 CpG island can act as a negative regulator in cis for coordinate imprinting at the centromeric domain, thereby suggesting a role for the LIT1 locus in a BWS pathway leading to functional inactivation of p57(KIP2). Thus, the targeting and precise modification of human chromosomal alleles using the DT40 cell shuttle system can be used to define regulatory elements that confer long-range control of gene activity within chromosomal domains.

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A novel approach to the treatment of bone tumors using tissue-engineered implants is reported in this study. The number of mesenchymal stem cells (MSCs) obtained from each patient's bone marrow cells was first increased, and the MSCs were forced to differentiate into osteoblasts followed by bone matrix formation on hydroxyapatite (HA) ceramics. The strong osteogenic ability of the implants, as evidenced by high osteoblastic activity, was confirmed. Consequently, the HA surface was covered with the patient's derived cultured osteoblast/bone matrix. The tissue-engineered HA was used to fill the patient's bone cavity after tumor curettage. Immediate healing potential was found by serial plain radiographs and computed tomograhy images, and no adverse reactions were noted in these patients. The results indicate that tissue-engineered osteogenic ceramics might be an alternative to autologous bone grafts.

There is an increasing interest in developing novel macromolecular vehicles for the intracellular and controlled delivery of bioactive molecules, since they can allow modulation of the cellular functions in a more effective manner ex vivo, and maintain the cellular phenotype in vivo upon re-implantation. The present study was designed to investigate the effect of combining novel dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer (Dex-loaded CMCht/PAMAM) nanoparticles and, both HA and SPCL scaffolds (3D system) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells (RBMSCs) in vitro. A luminescent cell viability assay using RBMSCs was performed for screening cytotoxicity of the developed HA and SPCL scaffolds. Results corroborated previous ones which have demonstrated in vitro, the superior performance of the HA and SPCL scaffolds on supporting cells adhesion and proliferation. Furthermore, this work showed that RBMSCs seeded onto the surface of both HA and SPCL scaffolds differentiate into osteoblasts when cultured in the presence of 0.01 mg ml(-1) Dex-loaded CMCht/PAMAM dendrimer nanoparticles. In addition, results demonstrated that Dex-loaded CMCht/PAMAM dendrimer nanoparticles combined with the HA enhance osteogenesis by increasing ALP activity and mineralization of the extra-cellular matrix. The pre-incubation of stem cells with these kinds of nanoparticles allows the delivery of Dex inside the cells and directly influences their cellular fate, being a promising new tool to be used in cells and tissue engineering strategies.

Abstract:Mesenchymal stem cells (MSCs) are multipotent cells and can be induced in vitro and in vivo to differentiate not only into the variety of mesodermal cells, but into either ectodermal or endodermal cells. This capability indicates the usefulness of MSCs for tissue engineering. Cell surface antigen analyses using various types of CD antibodies demonstrated that adherent fibroblastic cells derived from fresh human bone marrow are mesenchymal types and the cells showed extensive capability for proliferation and/or differentiation. We labeled the adherent cultured marrow cells as MSCs and, significantly, found the MSCs could even proliferate from aged marrow cells. After about sixteen days of culturing, we were able to harvest 100 million MSCs from only 3 ml of fresh human marrow. Moreover, the MSCs could be cryopreserved at -80 ∞ C without noticeable loss of viability and capability of osteoblastic differentiation. These results indicate that MSCs hold promise for utilization in hard tissue regeneration.

Autologous mesenchymal stem cells (MSCs) cultured with beta-tricalcium phosphate (beta-TCP) ceramics and with a free vascularized fibula were transplanted into three patients with steroid-induced osteonecrosis of the femoral head. The average follow-up period was 34 months and the average patient age at the time of surgery was 28 years old. Fifteen milliliters of bone marrow was obtained from the patients 4 weeks before surgery, and was used for in vitro proliferation of MSCs. beta-TCP granules were immersed in the MSC suspension and the cells were further cultured for 2 weeks. Cultured MSCs/beta-TCP composite granules were implanted into the cavity that remained after curettage of necrotic bone; and finally, a free vascularized fibula was grafted. All hips showed preoperative collapse and radiographic progression was observed in two hips postoperatively. Osteonecrosis did not progress any further and early bone regeneration was observed. This tissue-engineered approach has potentials for the treatment of osteonecrosis. However, our results suggested that the present procedure could not be used for cases with severe preoperative collapse.

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