Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells

Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were sepa- rated and cultured using the ＂pour-off＂ method. Non-adherent bone marrow cell-derived mesen- chymal stem ceils developed colony-forming unit-fibroblasts, and could be expanded by supple- mentation with epidermal growth factor. Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers, neurofilament-200 and NeuN, in vitro. Non-adherent bone marrow cell-derived mesenchymal stem cells from 13-galactosidase transgenic mice were also transplanted into focal ischemic brain （right corpus striatum） of C57BL/6J mice. At 8 weeks, cells positive for LacZ and 13-galactosidase staining were observed in the ischemic tissues, and cells co-labeled with both 13-galactosidase and NeuN were seen by double immunohistochemical staining. These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo.

How old is too old? In vivo engraftment of human peripheral blood stem cells cryopreserved for up to 18 years-implications for clinical transplantation and stability programs

BACKGROUND Peripheral blood stem cells(PBSC)are commonly cryopreserved awaiting clinical use for hematopoietic stem cell transplant.Long term cryopreservation is commonly defined as five years or longer,and limited data exists regarding how long PBSC can be cryopreserved and retain the ability to successfully engraft.Clinical programs,stem cell banks,and regulatory and accrediting agencies interested in product stability would benefit from such data.Thus,we assessed recovery and colony forming ability of PBSC following long-term cryopreservation as well as their ability to engraft in NOD/SCID/IL-2 Rγnull(NSG)mice.AIM To investigate the in vivo engraftment potential of long-term cryopreserved PBSC units.METHODS PBSC units which were collected and frozen using validated clinical protocols were obtained for research use from the Cellular Therapy Laboratory at Indiana University Health.These units were thawed in the Cellular Therapy Laboratory using clinical standards of practice,and the pre-freeze and post-thaw characteristics of the units were compared.Progenitor function was assessed using standard colony-forming assays.CD34-selected cells were transplanted into immunodeficient mice to assess stem cell function.RESULTS Ten PBSC units with mean of 17 years in cryopreservation(range 13.6-18.3 years)demonstrated a mean total cell recovery of 88%±12%(range 68%-110%)and post-thaw viability of 69%±17%(range 34%-86%).BFU-E growth was shown in 9 of 10 units and CFU-GM growth in 7 of 10 units post-thaw.Immunodeficient mice were transplanted with CD34-selected cells from four randomly chosen PBSC units.All mice demonstrated long-term engraftment at 12 wk with mean34%±24%human CD45+cells,and differentiation with presence of human CD19+,CD3+and CD33+cells.Harvested bone marrow from all mice demonstrated growth of erythroid and myeloid colonies.CONCLUSION We demonstrated engraftment of clinically-collected and thawed PBSC following cryopreservation up to 18 years in NSG mice,signifying likely successful clinical transplantation of PBSC following long-term cryopreservation.

Evaluation of the relationship between two different methods for enumeration fecal indicator bacteria: Colony-forming unit and most probable number

Most probable number （MPN） and colony-forming unit （CFU） estimates of fecal indicator bacteria （FIB） concentration are common measures of water quality in aquatic environments. Thus, FIB intensively monitored in Yeongsan Watershed in an attempt to compare two different methods and to develop a statistical model to convert from CFU to MPN estimates or vice versa. As a result, the significant difference was found in the MPN and CFU estimates. The enumerated Escherichia coli concentrations in MPN are greater than those in CFU, except for the measurement in winter. Especially in fall, E. coli concentrations in MPN are one order of magnitude greater than that in CFU. Contrarily, enterococci bacteria in MPN are lower than those in CFU. However, in general, a strongly positive relationship are found between MPN and CFU estimates. Therefore, the statistical models were developed, and showed the reasonable converting FIB concentrations from CFU estimates to MPN estimates. We expect this study will provide preliminary information towards future research on whether different analysis methods may result in different water quality standard violation frequencies for the same water sample.

Allogeneic compact bone-derived mesenchymal stem cell transplantation increases survival of mice exposed to lethal total body irradiation： a potential immunological mechanism

Background Radiation-induced injury after accidental or therapeutic total body exposure to ionizing radiation has serious pathophysiological consequences,and currently no effective therapy exists.This study was designed to investigate whether transplantation of allogeneic murine compact bone derived-mesenchymal stem cells （CB-MSCs） could improve the survival of mice exposed to lethal dosage total body irradiation （TBI）,and to explore the potential immunoprotective role of MSCs.Methods BALB/c mice were treated with 8 Gy TBI,and then some were administered CB-MSCs isolated from C57BL/6 mice.Survival rates and body weight were analyzed for 14 days post-irradiation.At three days post-irradiation,we evaluated IFN-Y and IL-4 concentrations; CD4＋CD25＋Foxp3＋ regulatory T cell （Treg） percentage; CXCR3,CCR5,and CCR7 expressions on CD3＋T cells; and splenocyte T-bet and GATA-3 mRNA levels.CB-MSC effects on bone marrow hemopoiesis were assessed via colony-forming unit granulocyte/macrophage （CFU-GM） assay.Results After lethal TBI,compared to non-transplanted mice,CB-MSC-transplanted mice exhibited significantly increased survival,body weight,and CFU-GM counts of bone marrow cells （P＜0.05）,as well as higher Treg percentages,reduced IFN-Y,CXCR3 and CCR5 down-regulation,and CCR7 up-regulation.CB-MSC transplantation suppressed Th1 immunity.Irradiated splenocytes directly suppressed CFU-GM formation from bone marrow cells,and CB-MSC co-culture reversed this inhibition.Conclusion Allogeneic CB-MSC transplantation attenuated radiation-induced hematopoietic toxicity,and provided immunoprotection by alleviating lymphocyte-mediated CFU-GM inhibition,expanding Tregs,regulating T cell chemokine receptor expressions,and skewing the Th1/Th2 balance toward anti-inflammatory Th2 polarization.

Adenovirus-mediated and tumor-specific transgene expression of the sodium-iodide symporter from the human telomerase reverse transcriptase promoter enhances killing of lung cancer cell line in vitro

Background The sodium-iodide symporter （NIS） protein can mediate the active radioiodine uptake.The human telomerase reverse transcriptase （hTERT） promoter is known to be selectively reactivated in majority of tumors and hence could be used for tumor targeting.We constructed a recombinant adenovirus containing the human sodium iodide symporter （hNIS） gene directed by the hTERT promoter, characterized the ability of infected cells in uptaking iodide, and explored the therapeutic efficacy of 131I in a lung cancer cell line in vitro.Methods The hTERT promoter was amplified by PCR from DNA isolated from log-phase HepG2 cells, subcloned into lineralized FL＊-hNIS/pcDNA3, and then the hTERT-hNIS sequence was subcloned into the shuttle plasmid pAdTrack.The recombinant adenovirus Ad-hTERT-hNIS was constructed by AdEasy system.A positive control adenovirusAd-CMV-hNIS and a negative control adenovirus Ad-CMV were created similarly.A549 cells were transduced with recombinant adenoviruses.125I uptake studies and sodium perchlorate suppression studies were used to confirm hNIS expression and function.Toxic effects of 131I on tumor cells were studied by in vitro clonogenic assay.Results We first successfully constructed an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter.When infected with recombinant adenovirus constructs expressing hNIS directed by hTERTand CMV-promoters （Ad-hTERT-hNIS and Ad-CMV-hNIS, respectively）, the lung cancer cell line A549 had increased ability to uptake radioiodide up to 23- and 30- fold compared to the control parental cells, respectively.The radioiodide uptake ability of both the Ad-CMV-hNIS and Ad-hTERT-hNIS transduced cell lines were repressed 11-fold by sodium perchlorate （NaCIO4）.The subsequent in vitro clonogenic assay of the infected A549 cell line was further repressed to 23% （Ad-CMV-hNIS） and 30% （Ad-hTERT-hNIS） of the control group after receiving radioiodide for 7 hours （P 〈0.001）.Conclusion Our preliminary study indicates that an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter has the potential to become an effective wide-spectrum yet highly specific anti-cancer strategy.