Human Embryonic Stem Cells (hESCs) and Teratoma: Evidence of Stemness and Research Tool for Tissue Engineering

Human embryonic stem cells (hESCs) and recently introduced induced pluripotent stem cells (iPSCs) are stem cells with an indefinite self-renewal and the potential to generate any mature cell type. Because of these properties, pluripotent stem cells hold great promise for regenerative medicine. One of the critical issues, however, is the risk of teratoma formation after transplantation of derivatives of these pluriopotent stem cells. Experimentally, transplantation of hESC and iPS cells into immunodeificient animal led to the formation of tumors composed of all three germ layers, i.e. teratoma. This property is considered the gold standard for demonstrating pluripotency of a given cell type. At the same time, the same type of assay hold promise as a standard for assessing safety of the therapeutic cells intended for clinical applications. This review article describes the key aspects of pluripotent stem cellderived teratoma formation and utilization of teratoma as a research tool for human embryonic development or disease pathogenesis. The promises and limitations of current ES cell-based therapy are also discussed.ope

Fast and Efficient Isolation of Mouse Bone Marrow-Derived Mesenchymal Stem Cells by Using a Biocompatible Polymer

Mesenchymal stem cells (MSCs) differentiate into bone, fat, cartilage, tendon, and other organ progenitor cells. The rarity of MSCs in bone marrow necessitates fast and efficient isolation and/or in vitro expansion prior to clinical and biomedical applications. Previously, we reported that UV-exposed diphenylamino-s-triazine bridged p-phenylene vinylene (DTOPV-UV) with a hydrophilic and negative surface-containing carboxyl group is highly biocompatible and provides a substrate for efficient human bone marrow-derived MSC attachment. In this study, we applied this polymeric film to early adhesion and enrichment of MSCs from mouse bone marrow. With its high protein- binding capacity, DTOPV-UV film was more efficient in early capture of adherent bone marrow cells than conventional tissue culture polystyrene (TCPS). Cell binding to DTOPV-UV reached full capacity within 1 hr, whereas cell attachment to TCPS gradually increased over time. The isolated and culture-expanded MSCs from mouse bone marrow displayed typical morphology, phenotype, and differentiation into osteoblasts, adipocytes, and chondrocytes. Here, we demonstrate a novel method for isolating MSCs from mouse bone marrow using a biocompatible polymer. This method will aid the development of rapid and efficient isolation and in vitro expansion protocols for rare adherent cells.ope

Aging-Associated Decline in Innate Immunity and Therapeutic Strategies to Counteract It

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Isolation of Mesenchymal Stem Cells from the Mononuclear Cells Remaining in the Bone Marrow Processing Kit

Background: Mesenchymal stem cells (MSCs) that are capable of extensive self renewal and differentiation have attracted great attention as a promising tool for regenerative medicine and tissue engineering. Although MSCs can be isolated from various tissues, bone marrow currently represents one of the most reliable sources for providing a sufficient yield of cells in a good quality. Herein, used bone marrow processing kits were evaluated as a valuable source of MSCs. Methods: Bone marrow mononuclear cells (MNCs) were recovered from used bone marrow processing kits after routine bone marrow processing by using the COBE 2991 Cell Processor (CaridianBCT Inc.). The MSCs were isolated from the recovered MNCs using a standard plastic adherence method. Immunophenotyping and differentiation assays were performed to clarify the characteristics of the isolated MSCs. Results: An average of 1×108 bone marrow MNCs was collected, and the MSCs were successfully isolated from the recovered bone marrow MNCs in all case. The isolated MSCs were positive for essential MSC surface molecules (CD29, CD44, CD73, CD90, CD105) and they were negative for most hematopoietic and endothelial cell markers (CD34, CD45, CD31, CD14). The isolated MSCs were capable of differentiation along the osteogenic, adipogenic and chondrogenic pathways. Conclusion: MSCs isolated from used bone marrow processing kits are an alternative and ethical source of bone marrow derived MSCs, and they can be used for research purposesope

Selective Depletion of SSEA-3- and TRA-1-60-Positive Undifferentiated Human Embryonic Stem Cells by Magnetic Activated Cell Sorter (MACS)

The capacity of indefinite self-renewal and pluripotency make human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) an attractive source for potential regenerative medicine. However, teratoma formation is one of the major obstacles for implementing hESC-based therapeutics in a clinical setting. While this tumorigenic capacity is known to be lost upon lineage differentiation in vitro, there is a potential risk that any residual undifferentiated hESCs or progenitor cells may form tumors upon transplantation. To ensure the safety of hESCs in clinical application, we thus explored the magnetic activated cell sorter (MACS) as a tool for separating undifferentiated hESCs from a mixed population of hESCs and human blood mononuclear cells. Labeled with single or combinations of two fluorsecein-labeled monoclonal antibodies (SSEA-3 and TRA-1-60) and subsequently stained with anti-FITC and/or anti-PE magnetic microbeads, cells were subjected to MACS separation. While a reduction of hESCs by depletion with a single marker was observed, there was still a significant fraction of residual hESCs in the flow-through fraction. However, two consecutive MACS separations upon simultaneous staining of two different hESC markers, SSEA-3 and TRA-1-60, completely depleted hESCs, as validated by flow cytometer, real-time PCR and immunofluoresence microscopic analyses. The maximum efficacy of hESC removal using this protocol was higher than 99.9%. No teratoma formation was observed in hESC-depleted cell injection to NOD/SCID mice. These results indicate that the current MACS protocol with two antibodies can efficiently eliminate undifferentiated cells from differentiated cells and greatly alleviate concerns about tumor formation by hESC-derived cellular therapeuticsope

The Genetically Modified Polysialylated Form of Neural Cell Adhesion Molecule-Positive Cells for Potential Treatment of X-Linked Adrenoleukodystrophy

PURPOSE: Cell transplantation of myelin-producing exogenous cells is being extensively explored as a means of remyelinating axons in X-linked adrenoleukodystrophy. We determined whether 3,3',5-Triiodo-L-thyronine (T3) overexpresses the ABCD2 gene in the polysialylated (PSA) form of neural cell adhesion molecule (NCAM)-positive cells and promotes cell proliferation and favors oligodendrocyte lineage differentiation. MATERIALS AND METHODS: PSA-NCAM+ cells from newborn Sprague-Dawley rats were grown for five days on uncoated dishes in defined medium with or without supplementation of basic fibroblast growth factor (bFGF) and/or T3. Then, PSA-NCAM+ spheres were prepared in single cells and transferred to polyornithine/fibronectin-coated glass coverslips for five days to determine the fate of the cells according to the supplementation of these molecules. T3 responsiveness of ABCD2 was analyzed using real-time quantitative polymerase chain reaction, the growth and fate of cells were determined using 5-bromo-2-deoxyuridine incorporation and immunocytochemistry, respectively. RESULTS: Results demonstrated that T3 induces overexpression of the ABCD2 gene in PSA-NCAM+ cells, and can enhance PSA-NCAM+ cell growth in the presence of bFGF, favoring an oligodendrocyte fate. CONCLUSION: These results may provide new insights into investigation of PSA-NCAM+ cells for therapeutic application to X-linked adrenoleukodystrophy.ope

Enhanced Neovascularization by Simultaneous Transplantation of Peripheral Blood CD34+ Hematopoietic Stem Cells and CD14+ Monocytes

Formation of new blood vessels is required for normal embryonic development and healing of damaged tissues, but also is essential for tumor growth. Although CD34+VEGF-R2(KDR)+ endothelial progenitor cells and CD14+ monocytes in human peripheral blood are known to actively participate in angiogenesis and vasculogenesis, the clear role of monocytes in the process of neovascularization is matter of debate. Here, we investigated whether a combination of two types of cells shows synergism in tumor-induced neovascularization. Fluorescently labeled purified CD34+ HSCs, CD14+ monocytes or combination of CD34+ HSCs and CD14+ monocytes were intratumorally injected into nude mice bearing human tumor of pancreatic adenocarcinoma. CD14+ monocytes or combination of CD34+ HSCs and CD14+ monocytes. Injection of a mixture of the 2 subsets resulted in improved neovascularization in vivo to any single-cell-type transplantation. These data demonstrate that human CD14+ monocytes as well as CD34+ HSCs can differentiate along the endothelial lineage in a specific permissive environment and thus this combination represent an autologous transplantable cell source for therapeutic neovasculogenesisope

High Dose Chemotherapy and Autologous Peripheral Blood Stem Cell Transplantation in Pediatric Patients with High-risk Neuroblastoma

Background: High dose chemotherapy followed by autologous peripheral blood stem cell transplantation (PBSCT) has become standard therapy for high-risk neuroblastoma patients. We performed a retrospective analysis to assess the characteristics of peripheral blood stem cell harvest (PBSCH) and PBSCT and its clinical outcome.Methods: We reviewed 17 cases of patients with high-risk neuroblastoma that underwent PBSCH and/or high dose chemotherapy followed by PBSCT.Results: Sixteen patients had stage IV neuroblastoma and one patient had a stage III neuroblastoma with MYCN amplification. The median age of the 17 patients was 43 months (range 22∼114 months) and the median body weight was 15 kg (range 10∼24 kg). After induction chemotherapy using a modified N7 protocol, 34 PBSCHs (1.5 leukapheresis per PBSCH) were performed. A statistically significant correlation was found between the pre-leukapheresis CD34+ cell count and the total number CD34+ cells of the harvested products (P＜0.0001). Tyrosine hydroxylase mRNA was not detected by RT-PCR in all of the leukapheresis products. High dose chemotherapy followed by PBSCT was performed in 24 cases. The mean infused CD34+ cell dose was 4.01×106/kg and WBC and platelet engraftment was performed on day 12.0 and 21.5, respectively. Eleven patients died, and six patients are surviving 11 to 68 months after PBSCT (median survival time, 35 months).Conclusion: In this single institution study, treatment with high dose chemotherapy and PBSCT was performed successfully for children with high-risk neuroblastoma.ope

Analysis of Characteristics of Mononuclear Cells Remaining in the Leukoreduction System Chamber of Trima Accel(R) and Their Differentiation Into Dendritic Cells

BACKGROUND: We investigated the characteristics of the mononuclear cells remaining in the leukoreduction system (LRS) chambers of Trima Accel(R) in comparison with those of standard buffy coat cells, and evaluated their potential for differentiation into dendritic cells. METHODS: Twenty-six LRS chambers of Trima Accel(R) were collected after platelet pheresis from healthy adults. Flow cytometric analysis for T, B, NK, and CD14+ cells was performed and the number of CD34+ cells was counted. Differentiation and maturation into dendritic cells were induced using CD14+ cells seperated via Magnetic cell sorting (MACS(R)) Seperation (Miltenyi Biotec Inc., USA). RESULTS: Total white blood cell (WBC) count in LRS chambers was 10.8x108 (range 7.7-18.0x108). The median values (range) of proportions of each cells were CD4+ T cell 29.6% (18.7-37.6), CD8+ T cell 27.7% (19.2-40.0), B cell 5.5% (2.2-12.1), NK cell 15.7% (13.7-19.9), and CD14+ cells 12.4% (8.6-32.3) respectively. Although total WBC count was significantly higher in the buffy coat (whole blood of 400 mL) than the LRS chambers, the numbers of lymphocytes and monocytes were not statistically different. The numbers of B cells and CD4+ cells were significantly higher in the buffy coat than the LRS chambers (P<0.05). The median value (range) of CD34+ cells obtained from the LRS chambers was 0.9x10(6) (0.2-2.6x10(6)). After 7 days of cytokine-supplemented culture, the CD14+ cells were successfully differentiated into dendritic cells. CONCLUSIONS: The mononuclear cells in LRS chambers of Trima Accel(R) are an excellent alternative source of viable and functional human blood cells, which can be used for research purposesope

ABCD2 Is a Direct Target of β-Catenin and TCF-4: Implications for X-Linked Adrenoleukodystrophy Therapy

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene that encodes the peroxisomal ATP-binding cassette (ABC) transporter subfamily D member 1 protein (ABCD1), which is referred to as the adrenoleukodystrophy protein (ALDP). Induction of the ABCD2 gene, the closest homolog of ABCD1, has been mentioned as a possible therapeutic option for the defective ABCD1 protein in X-ALD. However, little is known about the transcriptional regulation of ABCD2 gene expression. Here, through in silico analysis, we found two putative TCF-4 binding elements between nucleotide positions −360 and −260 of the promoter region of the ABCD2 gene. The transcriptional activity of the ABCD2 promoter was strongly increased by ectopic expression of β-catenin and TCF-4. In addition, mutation of either or both TCF-4 binding elements by site-directed mutagenesis decreased promoter activity. This was further validated by the finding that β-catenin and the promoter of the ABCD2 gene were pulled down with a β-catenin antibody in a chromatin immunoprecipitation assay. Moreover, real-time PCR analysis revealed that β-catenin and TCF-4 increased mRNA levels of ABCD2 in both a hepatocellular carcinoma cell line and primary fibroblasts from an X-ALD patient. Interestingly, we found that the levels of very long chain fatty acids were decreased by ectopic expression of ABCD2-GFP as well as β-catenin and TCF-4. Taken together, our results demonstrate for the first time the direct regulation of ABCD2 by β-catenin and TCF-4.ope