BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses.

Background:Osteosarcoma cancer stem cells (CSCs) are defined as a subpopulation of osteosarcoma cells, which have the ability of self-renewal, proliferation and differentiation. This study aimed to identify CSCs from human osteosarcomain vitro.Methods:Osteosarcoma CSCs were isolated and cultured with sphere-forming assay technique on an ultra-low well attachment surface plate. After sarcosphere colonies were formed, we conducted reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the expression of genes of embryonic stem cells such asNANOG, Oct3/4, STAT3 and gene of MSC CD133. Immunofluorescence analysis (IFA) of alkaline phosphatase (ALP), osteocalcin, and CD 133 was also performed to see the expression of osteosarcoma CSC surface protein with immuno-enzymatic staining principle. We also performed alizarin red staining to evaluate calcification in osteosarcoma CSCs.Results:The culture sphere-of the osteosarcoma cells showed three dimension round shaped colonies (sarcospheres) in slightly hypoxicand serum free condition which was not attached to the substrate with tight density. RT-PCR demonstrated that sarcospheres expressed genes which encodeNANOG, Oct3/4 STAT 3, but not for CD 133. IFA showed positive protein expression of ALP, osteocalcin and CD 133 which was moderate, strong, and weak positive respectively. Sarcospheres also had a positive reaction toward alizarin red staining.Conclusion:Osteosarcoma CSCs could be isolated from human osteosarcoma by sphere-forming assay technique and characterized by the expression of genes of embryonic stem cells,such asNANOG, Oct3/4, STAT3 and IFA of ALP, osteocalcin, and CD 133.

Currently available prophylactic vaccines have no therapeutic efficacy for preexisting human papillomavirus (HPVs) infections, do not target all oncogenic HPVs and are insufficient to eliminate the burden of HPV induced cancer. We aim to develop an alternative HPV vaccine which is broadly effective and capable of clearing preexisting infection. In an initial attempt to develop a broadly reactive therapeutic vaccine, we designed a putative papillomavirus (PV) ancestor antigen (circulating sequence derived antigenic sequences E1E2—CDSE1E2) based on the conserved E1 and E2 protein sequences from existing oncogenic HPV strains. This antigen was found to be as related to circulating oncogenic Macaca fascicularis papillomaviruses (MfPVs) as to oncogenic HPVs. The CDSE1E2 antigen was fused to a T-cell adjuvant and encoded in chimpanzee 3 and 63 adenoviral vectors. We first showed that the combination of these 2 vaccines induced long-lasting potent CDSE1E2 specific T cell responses in outbred mice. This prime-boost regimen was then tested in female macaques naturally infected with MfPVs. All immunized animals (16/16) responded to the vaccine antigen but with reduced cross-reactivity against existing PVs. Preexisting MfPV infections did not prime vaccine inducible immune responses. Importantly, immunized oncogenic MfPV type 3 (MfPV3) infected animals that responded toward MfPV3 were able to diminish cervical MfPV3 DNA content. Although insufficient breadth was achieved, our results suggest that a relevant level of E1E2 specific T cell immunity is achievable and might be sufficient for the elimination of PV infection. Importantly, naturally infected macaques, offer a relevant model for testing vaccines aimed at eliminating mucosal PV infections.

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Background and Aim: Cynomolgus monkeys (Macaca fascicularis) develop spontaneous infection of Papillomavirus (PV); thus, potentially beneficial for modeling human PV (HPV) infection study. Contrary to human origin, infection in cynomolgus monkeys does not always show evident clinical symptoms of cervical cancer. The absence of cervical cancer clinical symptoms leads us to investigate the molecular mechanism of the HPV infection in cynomolgus monkeys. This study aimed to investigate the messenger ribonucleic acid (mRNA) expression levels of KI67 and P53 genes, majorly known as biomarker oncogenesis of PV infection. Materials and Methods: The polymerase chain reaction (PCR) technique was used with MY11/MY09 primer to screen PV in cynomolgus monkey, further grouped as positive-PV and negative-PV infection groups. Real-time quantitative PCR was also applied to quantify the mRNA expression levels of KI67 and P53 genes in animals. Results: Increased expression of mRNA level of KI67 genes was significantly higher in Positive- PV group than negative-PV group. In contrast, the P53 mRNA expression level increased markedly higher in the negative-PV group than in the positive-PV group. Conclusion: Our study describes the potential of cynomolgus monkeys as a spontaneous oncogenesis model of PV infection-type. However, we used a limited number of cancer genetic markers. So, further study of other genetic markers is required to prove that cervical cancer could be developed naturally in cynomolgus monkeys.

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Objectives: Mesenchymal stem cells (MSC) are primarily isolated from bone marrow. Peripheral blood is also reported as an alternative source of MSC. This study compared MSC which were isolated and cultured from bone marrow and those from peripheral blood of rats. Methods: MSC from bone marrow and peripheral blood were harvested from 5 male Sprague Dawley rats. After isolation, the cells were grown on tissue culture plates with concentration of 10 7 cells per well. Observations were conducted to evaluate the attachment of nucleated cells with fibroblast-like morphology. Characterization of MSC was done using reverse transcriptasepolymerase chain reaction (RT-PCR) and immunocytochemistry assay. Results: The mean number of nucleated cells isolated from the bone marrow on day 0 was higher than those isolated from the peripheral blood. Bone marrow MSC with typical fibroblast-like morphology proliferated rapidly and reached 80% confluency on day 14. Subcultures were able to be conducted on day 15 (first passage) and day 28 (second passage). On days 15 and 28, no nucleated cells remained in peripheral blood cultures. Conclusions: Bone marrow derived MSC are more enriched and grow more efficiently than those derived from peripheral blood.

PurposeA major question remaining in approaches to tissue engineering and organ replacement is the role of native mobilized native cells in the regeneration process of damaged tissues and organs. The goal of this study was to compare the cell mobilizing effects of the chemokine CXCL12 and cell therapy on the urinary sphincter of nonhuman primates (NHP) with chronic intrinsic urinary sphincter dysfunction.MethodsEither autologous lenti-M-cherry labeled skeletal muscle precursor cells (skMPCs) or CXCL12 were injected directly into the sphincter complex of female NHPs with or without surgery-induced chronic urinary sphincter dysfunction (n=4/treatment condition). All monkeys had partial bone marrow transplantation with autologous lenti-green fluorescent protein (GFP) bone marrow cells prior to treatment. Labeled cells were identified, characterized and quantified using computer-assisted immunohistochemistry 6 months posttreatment.ResultsGFP-labeled bone marrow cells (BMCs) were identified in the bone marrow and both BMCs and skMPCs were found in the urinary sphincter at 6-month postinjection. BMCs and skMPCs were present in the striated muscle, smooth muscle, and lamina propria/urothelium of the sphincter tissue. Sphincter injury increased the sphincter content of BMCs when analyzed 6-month postinjection. CXCL12 treatment, but not skMPCs, increased the number of BMCs in all layers of the sphincter complex (P<0.05). CXCL12 only modestly (P=0.15) increased the number of skMPCs in the sphincter complex.ConclusionsThis dual labeling methodology now provides us with the tools to measure the relative number of locally injected cells versus bone marrow transplanted cells. The results of this study suggest that CXCL12 promotes mobilization of cells to the sphincter, which may contribute more to sphincter regeneration than injected cells.