A New Human Somatic Stem Cell from Placental Cord Blood with Intrinsic Pluripotent Differentiation Potential

Here a new, intrinsically pluripotent, CD45-negative population from human cord blood, termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 1015 cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, and hematopoietic and neural cells including astrocytes and neurons that express neurofilament, sodium channel protein, and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model, the preimmune fetal sheep, resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals

Modulation of Human Mesenchymal Stem Cell Immunogenicity through Forced Expression of Human Cytomegalovirus US Proteins

BACKGROUND: Mesenchymal stem cells (MSC) are promising candidates for cell therapy, as they migrate to areas of injury, differentiate into a broad range of specialized cells, and have immunomodulatory properties. However, MSC are not invisible to the recipient's immune system, and upon in vivo administration, allogeneic MSC are able to trigger immune responses, resulting in rejection of the transplanted cells, precluding their full therapeutic potential. Human cytomegalovirus (HCMV) has developed several strategies to evade cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell recognition. Our goal is to exploit HCMV immunological evasion strategies to reduce MSC immunogenicity. METHODOLOGY/PRINCIPAL FINDINGS: We genetically engineered human MSC to express HCMV proteins known to downregulate HLA-I expression, and investigated whether modified MSC were protected from CTL and NK attack. Flow cytometric analysis showed that amongst the US proteins tested, US6 and US11 efficiently reduced MSC HLA-I expression, and mixed lymphocyte reaction demonstrated a corresponding decrease in human and sheep mononuclear cell proliferation. NK killing assays showed that the decrease in HLA-I expression did not result in increased NK cytotoxicity, and that at certain NK∶MSC ratios, US11 conferred protection from NK cytotoxic effects. Transplantation of MSC-US6 or MSC-US11 into pre-immune fetal sheep resulted in increased liver engraftment when compared to control MSC, as demonstrated by qPCR and immunofluorescence analyses. CONCLUSIONS AND SIGNIFICANCE: These data demonstrate that engineering MSC to express US6 and US11 can be used as a means of decreasing recognition of MSC by the immune system, allowing higher levels of engraftment in an allogeneic transplantation setting. Since one of the major factors responsible for the failure of allogeneic-donor MSC to engraft is the mismatch of HLA-I molecules between the donor and the recipient, MSC-US6 and MSC-US11 could constitute an off-the-shelf product to overcome donor-recipient HLA-I mismatch

Effect of Audiovisual Training on Monaural Spatial Hearing in Horizontal Plane

The article aims to test the hypothesis that audiovisual integration can improve spatial hearing in monaural conditions when interaural difference cues are not available. We trained one group of subjects with an audiovisual task, where a flash was presented in parallel with the sound and another group in an auditory task, where only sound from different spatial locations was presented. To check whether the observed audiovisual effect was similar to feedback, the third group was trained using the visual feedback paradigm. Training sessions were administered once per day, for 5 days. The performance level in each group was compared for auditory only stimulation on the first and the last day of practice. Improvement after audiovisual training was several times higher than after auditory practice. The group trained with visual feedback demonstrated a different effect of training with the improvement smaller than the group with audiovisual training. We conclude that cross-modal facilitation is highly important to improve spatial hearing in monaural conditions and may be applied to the rehabilitation of patients with unilateral deafness and after unilateral cochlear implantation

Mesenchymal stem cells engineered to inhibit complement-mediated damage.

Mesenchymal stem cells (MSC) preferentially migrate to damaged tissues and, due to their immunomodulatory and trophic properties, contribute to tissue repair. Although MSC express molecules, such as membrane cofactor protein (CD46), complement decay-accelerating factor (CD55), and protectin (CD59), which confer protection from complement-mediated lysis, MSC are recruited and activated by anaphylatoxins after transplantation, potentially causing MSC death and limiting therapeutic benefit. We have previously demonstrated that transduction of MSC with a retrovirus encoding HCMV-US proteins resulted in higher levels of MSC engraftment due to decreased HLA-I expression. Here, we investigate whether engineering MSC to express US2 (MSC-US2), US3 (MSC-US3), US6 (MSC-US6), or US11 (MSC-US11) HCMV proteins can alter complement recognition, thereby better protecting MSC from complement attack and lysis. HCMV-US proteins increased MSC CD59 expression at different levels as determined by flow cytometric evaluation of the median fluorescence intensity ratio (MFI). A significant increase in CD59 expression was seen in MSC-US2, MSC-US3, and MSC-US6, but not in MSC-US11. Only MSC-US2 displayed increased expression of CD46, while US2 and US3 proteins were both able to augment the percentage of MSC expressing this molecule. Regardless of the HCMV protein expressed, none changed CD55 MFI; however, expression of US6, US11, and US2 each increased the percentage of MSC that were positive for this molecule. Because US2 protein was the most efficient in up-regulating all three complement regulatory proteins, we used a functional complement-mediated cytotoxicity assay to investigate whether MSC-US2 were protected from complement-mediated lysis. We demonstrated that over-expression of the US2 protein reduced complement lysis by 59.10±12.89% when compared to untransduced MSC. This is the first report, to our knowledge, describing a role of HCMV-US proteins in complement evasion, and our data shows that over-expression of US2 protein on MSC could serve as a strategy to protect these cells from complement lysis

EphB2 isolates a human marrow stromal cell subpopulation with enhanced ability to contribute to the resident intestinal cellular pool.

To identify human bone marrow stromal cell(BMSC) subsets with enhanced ability to engraft/contribute to the resident intestinal cellular pool, we transplanted clonally derived BMSCs into fetalsheep. Analysis at 75 d posttransplantation showed 2 of the 6 clones engrafting the intestine at 4­ to 5­fold higher levels (5.03±0.089 and 5.04±0.15%, respectively) than the other clones (P<0.01), correlating with the percentage of donor­derived Musashi­1 (12.01–14.17 vs. 1.2–3.8%; P<0.01) or leucine­rich repeat­containing G­protein coupled receptor 5 (Lgr5) cells within the intestinalstem cell(ISC) region. Phenotypic and transcriptome analysis determined that the clones with enhanced intestinal contribution expressed high levels of Ephrin type B receptor 2 (EphB2). Intestinal explants demonstrated proliferation of the engrafted cells and ability to generate crypt­like structures in vitro still expressing EphB2. Additional transplants based on BMSC EphB2 expression demonstrated that, at 7 d post­transplant, the EphB2 BMSCs engrafted in the ISC region at levels of 2.1 ± 0.2%, while control EphB2 BMSCs engrafted at 0.3 ± 0.1% (P<0.01). Therefore we identified a marker for isolating and culturing an expandable subpopulation of BMSCs with enhanced intestinal homing and contribution to the ISC region.— Colletti, E., El Shabrawy, D., Soland, M., Yamagami, T., Mokhtari, S., Osborne, C., Schlauch, K., Zanjani, E. D., Porada, C. D., Almeida­Porada, G. EphB2 isolates a human marrow stromal cellsubpopulation with enhanced ability to contribute to the resident intestinal cellular pool

Up-regulation of CD59 surface expression on MSC by HCMV US proteins.

<p>(A) MSC, MSC-E, MSC-US2, MSC-US3, MSC-US6, and MSC-US11 were analyzed by flow cytometry for expression of CD59. Each panel depicts representative data of at least three independent experiments. Black filled histograms correspond to different MSC cell populations stained with antibody against CD59, and unfilled histograms are the corresponding isotype control staining. The MFI ratio for each MSC cell line was obtained by performing the following calculation: MFI ratio  =  (Median Fluorescence Intensity for CD59/Median Fluorescence Intensity for isotype control). (B) MFI ratio for each MSC population is shown. The results represent the mean ± SEM from at least three independent experiments. * indicates p<0.05 when comparing MSC-US cells with non-transduced MSC.</p

Up-regulation of CD55 surface expression on MSC by HCMV US proteins.

<p>(A) MSC, MSC-E, MSC-US2, MSC-US3, MSC-US6, and MSC-US11 were analyzed for CD55 surface expression by flow cytometry. Top- Percentage of CD55 positive cells for each MSC population. Bottom- MFI ratio for CD55 expression on different MSC populations. MFI ratio  =  Median Fluorescence Intensity for CD55/Median Fluorescence Intensity for isotype control. The results represent the mean ± SEM from at least three independent experiments. * indicates p<0.05 when comparing MSC-US cells with non-transduced MSC. (B) Each panel depicts representative data of at least three independent experiments. Black filled histograms correspond to each MSC cell population stained with antibody against CD55 and unfilled histograms correspond to isotype staining.</p

Up-regulation of CD46 surface expression on MSC by HCMV US proteins.

<p>(A) MSC, MSC-E, MSC-US2, MSC-US3, MSC-US6, and MSC-US11 were analyzed by flow cytometry for CD46 expression. Top- Percentage of CD46 positive cells for each MSC population. Bottom- MFI ratio for CD46 expression on each MSC population. MFI ratio  =  (Median Fluorescence Intensity for CD46/Median Fluorescence Intensity for isotype control). The results represent the mean ± SEM from at least three independent experiments. * indicates p<0.05 when comparing MSC-US cells with non-transduced MSC. 0(B) Each panel depicts representative data of at least three independent experiments. Black filled histograms correspond to different MSC cell populations stained with antibody against CD46 and unfilled histograms are the staining with the corresponding isotype control.</p

Effect of overexpression of HCMV US2 protein on MSC in a complement-mediated cytotoxicity assay.

<p>(A) The results depict representative data from a flow cytometry-based complement-mediated cytotoxicity assay from at least 3 independent experiments. The assay was performed in the presence of human IgM and rabbit serum complement (experimental) or in the presence of human IgM and absence of the rabbit serum complement (control). MSC stained with calcein were counted as viable, cells stained with Ethidium homodimer-1 were counted as dead, and cells doubly positive were counted as having membrane damage. Doubly negative cells were excluded from the analysis. A live/dead control (top) was used in order to set up the quadrant gates for alive, dead, and membrane-damaged populations and to set parameters for compensation between the 2 fluorescent channels. The experimental assay provided the percentage of dead cells under experimental conditions. The control assay provided the percentage of spontaneously dead cells. The same conditions were applied for non-transduced MSC and MSC-US2. (B) The percentage of cytotoxicity for each cell line was calculated as follows: (percent of dead cells under experimental conditions – percent of spontaneously dead cells)/(100 – spontaneously dead)*100. The percentage of experimental or spontaneously dead cells was determined by calculating the percentage of cells that were positive for Ethidium homodimer-1, and negative for calcein. The results depict the mean ± SEM of three independent experiments. * indicates p<0.05.</p