Functional characterization of the immunomodulatory properties of human urine-derived stem cells.

BACKGROUND: Urine-derived stem cells (USCs) have been widely researched as a novel cell source for stem cell therapy, but their immunomodulatory characteristics remain to be investigated. This study aimed to characterize the immunomodulatory properties of human USCs. METHODS: Human USCs were isolated from fresh voiding urine samples from healthy male donors and expanded. Their cell surface markers were characterized by flow cytometry analysis and the telomerase activities for several USCs clones were determined. The immunosuppressive potential of USCs was evaluated by the performing the mixed lymphocyte reaction (MLR) [co-culture with peripheral blood mononuclear cells (PBMNCs)] and natural killer cells (NK) cytotoxicity assay. USCs cytokines release profile was determined by using human cytokine proteome array. RESULTS: USCs exhibited high cell surface expression of embryonic/mesenchymal stem cells (MSCs) markers CD29, CD44, CD54, CD73, CD90, CD146, and CD166, while lacked expression of hematopoietic stem cell markers CD11, CD14, CD19, CD31, CD34, CD45, B cell marker CD79, and co-stimulatory factors CD80 and CD86, thus, exhibiting the phenotype of MSCs. MLR indicated that USCs significantly inhibited the proliferation of PBMNCs, as compared to that of the human smooth muscle cells (SMCs). In cell cytotoxicity assays, NK cells displayed less cytotoxicity against USCs than against bone marrow mesenchymal stem cells (BMSCs) and SMCs. Furthermore, upon PBMNCs stimulation, USCs secreted higher levels of immunomodulatory cytokines, including IL-6, IL-8, MCP-1, RANTES, GROα, and GM-CSF, compared to those of BMSCs, especially when directly contact mix-culture with PBMNCs. CONCLUSIONS: USCs secreted immunoregulatory cytokines and possessed immunomodulatory properties, comparable to those of BMSCs.

Regenerative medicine: prospects for the treatment of inflammatory bowel disease.

This article reviews the current understanding of the processes driving the development and progression of inflammatory bowel disease (IBD), discusses how the dynamic crosstalk between resident microorganisms, host cells and the immune system is required in order to maintain immune homeostasis, and considers innovative strategies that allow the modification or modulation of the intestinal microorganismal community as a potential approach for treating IBD. This article next rationalizes the use of cell-based regenerative medicine as treatment for IBD, discusses the obstacles hindering its success, summarizes some of the results of recent clinical trials employing these therapies, and discusses ongoing work to enhance mesenchymal stem/stromal cells, making them better suited to the task of repairing the damage within the IBD gut.

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 fetal sheep. Analysis at 75 d post-transplantation showed 2 of the 6 clones engrafting the intestine at 4- to 5-fold higher levels (5.03±0.089 and 5.04±0.15%, respectively) than the other clones (P<0.01), correlating with the percentage of donor-derived Musashi-1(+) (12.01-14.17 vs. 1.2-3.8%; P<0.01) or leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5)(+) cells within the intestinal stem 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 crypt-like structures in vitro still expressing EphB2. Additional transplants based on BMSC EphB2 expression demonstrated that, at 7 d post-transplant, the EphB2(high) BMSCs engrafted in the ISC region at levels of 2.1 ± 0.2%, while control EphB2(low) 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.

Mesenchymal stem cells contribute to endogenous FVIII:c production.

Besides the liver, it has been difficult to identify which organ(s) and/or cellular component(s) contribute significantly to the production of human FVIII:c (FVIII). Thus far, only endothelial cells have been shown to constitute a robust extrahepatic source of FVIII, possibly explaining both the diverse presence of FVIII mRNA in the body, and the observed increase in FVIII levels during liver failure. Here, we investigate whether human mesenchymal stem cells (MSC), ubiquitously present in different organs, could also contribute to FVIII production. MSC isolated from human lung, liver, brain, and bone marrow expressed FVIII message as determined by quantitative-RT-PCR. Using an antibody specific for FVIII, confocal microscopy, and umbilical cord-derived endothelial cells (HUVEC) as a negative control, we demonstrated that, in MSC, FVIII protein was not stored in granules; rather, it localized to the perinuclear region. Furthermore, functional FVIII was detected in MSC supernatants and cell lysates by aPTT and chromogenic assays. These results demonstrate that MSC can contribute at low levels to the functional FVIII pool, and advance the understanding of the physiology of FVIII production and secretion.

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.

Cyclophilin A is required for efficient human cytomegalovirus DNA replication and reactivation.

Human cytomegalovirus (HCMV) is a large DNA virus belonging to the subfamily Betaherpesvirinae. Haematopoietic cells of the myeloid lineage have been shown to harbour latent HCMV. However, following terminal differentiation of these cells, virus is reactivated, and in an immunocompromised host acute infection can occur. It is currently unknown which viral and cellular factors are involved in regulating the switch between lytic and latent infections. Cyclophilin A (CyPA) is a cellular protein that acts as a major factor in virus replication and/or virion maturation for a number of different viruses, including human immunodeficiency virus, hepatitis C virus, murine cytomegalovirus, influenza A virus and vaccinia virus. This study investigated the role of CyPA during HCMV infection. CyPA expression was silenced in human foreskin fibroblast (HF) and THP-1 cells using small interfering RNA (siRNA) technology, or the cells were treated with cyclosporin A (CsA) to inhibit CyPA activity. Silencing CyPA in HF cells with siRNA resulted in an overall reduction in virus production characterized by delayed expression of immediate-early (IE) proteins, decreased viral DNA loads and reduced titres. Furthermore, silencing of CyPA in THP-1 cells pre- and post-differentiation prevented IE protein expression and virus reactivation from a non-productive state. Interestingly, it was observed that treatment of THP-1 cells with CsA prevented the cells from establishing a fully latent infection. In summary, these results demonstrate that CyPA expression is an important factor in HCMV IE protein expression and virus production in lytically infected HF cells, and is a major component in virus reactivation from infected THP-1 cells.

HCMV protein LUNA is required for viral reactivation from latently infected primary CD14⁺ cells.

Human cytomegalovirus (HCMV) is a member of the Herpesviridae family that infects individuals throughout the world. Following an initial lytic stage, HCMV can persist in the individual for life in a non-active (or latent) form. During latency, the virus resides within cells of the myeloid lineage. The mechanisms controlling HCMV latency are not completely understood. A latency associated transcript, UL81-82ast, encoding the protein LUNA (Latency Unique Natural Antigen) was identified from latently infected donors in vivo. To address the role of the UL81-82ast protein product LUNA, in the context of the viral genome, we developed a recombinant HCMV bacterial artificial chromosome (BAC) that does not express LUNA. This construct, LUNA knockout FIX virus (FIX-ΔLUNA), was used to evaluate LUNA's role in HCMV latency. The FIX-ΔLUNA virus was able to lytically infect Human Fibroblast (HF) cells, showing that LUNA is not required to establish a lytic infection. Interestingly, we observed significantly higher viral copy numbers in HF cells infected with FIX-ΔLUNA when compared to FIX-WT virus. Furthermore, FIX-WT and FIX-ΔLUNA genomic DNA and transcription of UL81-82ast persisted over time in primary monocytes. In contrast, the levels of UL138 transcript expression in FIX-ΔLUNA infected HF and CD14⁺ cells was 100 and 1000 fold lower (respectively) when compared to the levels observed for FIX-WT infection. Moreover, FIX-ΔLUNA virus failed to reactivate from infected CD14⁺ cells following differentiation. This lack of viral reactivation was accompanied by a lack of lytic gene expression, increase in viral copy numbers, and lack of the production of infectious units following differentiation of the cells. Our study suggests that the LUNA protein is involved in regulating HCMV reactivation, and that in the absence of LUNA, HCMV may not be able to enter a proper latent state and therefore cannot be rescued from the established persistent infection in CD14⁺ cells.