Parul Trivedi

Human embryonic stem cells (hESCs) could potentially provide a renewable source of different types of cells for cell therapy applications. Recently, mesenchymal stem cells (MSCs) have been derived from hESCs either through co-culturing with murine OP9 bone marrow stromal cell line or directly from hESCs without co-culturing with OP9 cells. Although the latter methodology is clinically advantageous over co-culturing with an animal cell layer those mesenchymal cells were reported to be positive for SSEA4. SSEA4 is a marker of undifferentiated hESCs and thus the presence of this marker on hESC-derived cells could potentially be problematic for clinical applications. We have recently achieved a novel and reproducible methodology for deriving a pure population of SSEA4-/CD73+ MSCs from federally approved hESC lines H1 and H9. To initiate the differentiation of hESCs to MSCs, we cultured undifferentiated hESCs on matrigel plates in murine embryonic fibroblast conditioned media with media ...

Background Components of the microenvironment such as bone marrow stromal cells (BMSCs) are well known to support multiple myeloma (MM) disease progression and resistance to chemotherapy including the proteasome inhibitor bortezomib. However, functional distinctions between BMSCs in MM patients and those in disease-free marrow are not completely understood. We and other investigators have recently reported that NF-κB activity in primary MM cells is largely resistant to the proteasome inhibitor bortezomib, and that further enhancement of NF-κB by BMSCs is similarly resistant to bortezomib and may mediate resistance to this therapy. The mediating factor(s) of this bortezomib-resistant NF-κB activity is induced by BMSCs is not currently understood. Results Here we report that BMSCs specifically derived from MM patients are capable of further activating bortezomib-resistant NF-κB activity in MM cells. This induced activity is mediated by soluble proteinaceous factors secreted by MM BMSC...

Intrarectal inoculation of rhesus monkeys with low doses of SIVmac led to a prolonged clinical and virological latency that was not observed for high intrarectal doses or for intravenous inoculation. Animals infected intrarectally with low virus doses remained negative for serum antibody responses to SIV for at least one year even though they readily transferred SIV to naive recipients via transfusion of whole blood.

Human embryonic stem cells (hESCs) have been shown to generate CD34(+) primitive hematopoietic cells after several days of coculturing with the OP9 murine stromal cell line. CD73(+) multipotent mesenchymal cells have also been isolated from hESC/OP9 cocultures after several weeks. We hypothesized that generation of CD34(+) hematopoietic cells and CD73(+) mesenchymal stem cells (MSCs) may follow similar kinetics, so we investigated the generation of CD73(+) cells in the first 2 weeks of hESC/OP9 cocultures, at a time when CD34(+) cells are generated. We cocultured hESCs with OP9 cells and examined the time course of appearance of human CD34(+) and CD73(+) cells using flow cytometry. We tested the hematopoietic progenitor potentials of CD34(+) cells generated using hematopoietic colony-forming assays, and the multipotent mesenchymal properties of CD73(+) cells generated using in vitro differentiation assays. We observed that in the first 2 weeks of the hESC/OP9 coculture system CD34(+...

Mesenchymal stroma cells (MSCs) have a high potential for novel cell therapy approaches in clinical transplantation. Commonly used bone marrow-derived MSCs (BM-MSCs), however, have a restricted proliferative capacity and cultures are difficult to standardize. Recently developed human embryonic stem cell-derived mesenchymal stroma cells (hES-MSCs) might represent an alternative and unlimited source of hMSCs. We therefore compared human ES-cell-derived MSCs (hES-MP002.5 cells) to normal human bone marrow-derived MSCs (BM-MSCs). hES-MP002.5 cells had lower yet reasonable CFU-F capacity compared with BM-MSC (8±3 versus 29±13 CFU-F per 100 cells). Both cell types showed similar immunophenotypic properties, i.e. cells were positive for CD105, CD73, CD166, HLA-ABC, CD44, CD146, CD90, and negative for CD45, CD34, CD14, CD31, CD117, CD19, CD 271, SSEA-4 and HLA-DR. hES-MP002.5 cells, like BM-MSCs, could be differentiated into adipocytes, osteoblasts and chondrocytes in vitro. Neither hES-MP002.5 cells nor BM-MSCs homed to the bone marrow of immune-deficient NSG mice following intravenous transplantation, whereas intra-femoral transplantation into NSG mice resulted in engraftment for both cell types. In vitro long-term culture-initiating cell assays and in vivo co-transplantation experiments with cord blood CD34+ hematopoietic cells demonstrated furthermore that hES-MP002.5 cells, like BM-MSCs, possess potent stroma support function. In contrast to BM-MSCs, however, hES-MP002.5 cells showed no or only little activity in mixed lymphocyte cultures and phytohemagglutinin (PHA) lymphocyte stimulation assays. In summary, ES-cell derived MSCs might be an attractive unlimited source for stroma transplantation approaches without suppressing immune function.

Intrarectal simian immunodeficiency virus (SIV) infection in rhesus macaques is a model for sexual transmission of primate retroviruses. Phylogenetic studies on envelope gene sequences that were present in blood following intrarectal SIV inoculation provided evidence for selective amplification of a subset of viruses present in the inoculum and defined one amino acid sequence uniquely associated with intrarectal infection. Both persistent and transient viremia states were observed after intrarectal infection. Immune responses in persistently infected animals accounted for slower rates of disease progression despite the presence of highly pathogenic viruses that were documented by transfusion studies. Transient viremia elicited protective immunity against subsequent intrarectal virus challenge but did not protect against intravenous virus challenge. Transient viremia usually but not always led to self-limiting infection. In one animal, we documented a relapse to active viremia long a...

The unintegrated viral DNA found in human immunodeficiency virus type 1 infection includes linear and circular forms. We targeted the circular form containing two copies of the viral long terminal repeat (2-LTR circle) and developed specific assays to detect this molecule in peripheral blood mononuclear cells from HIV-infected patients. In vitro HIV-1 infection of peripheral blood mononuclear cells showed rapid accumulation and rapid decay of 2-LTR circular viral DNA. Examination of 2-LTR circular viral DNA levels provides a view of spreading infection based on a viral DNA form that is structurally distinct and has a known, short half-life in infected cells. In patients not receiving antiviral therapy, the levels of 2-LTR circular viral DNA and total viral DNA were significantly correlated to CD4 cell counts. Similar correlations were not observed in patients receiving zidovudine (AZT), didanosine (ddA), or zalcitabine (ddC).