MicroRNA and gene expression patterns in the differentiation of human embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>The unique features of human embryonic stem (hES) cells make them the best candidate resource for both cell replacement therapy and development research. However, the molecular mechanisms responsible for the simultaneous maintenance of their self-renewal properties and undifferentiated state remain unclear. Non-coding microRNAs (miRNA) which regulate mRNA cleavage and inhibit encoded protein translation exhibit temporal or tissue-specific expression patterns and they play an important role in development timing.</p> <p>Results</p> <p>In this study, we analyzed miRNA and gene expression profiles among samples from 3 hES cell lines (H9, I6 and BG01v), differentiated embryoid bodies (EB) derived from H9 cells at different time points, and 5 adult cell types including Human Microvascular Endothelial Cells (HMVEC), Human Umbilical Vein Endothelial Cells (HUVEC), Umbilical Artery Smooth Muscle Cells (UASMC), Normal Human Astrocytes (NHA), and Lung Fibroblasts (LFB). This analysis rendered 104 miRNAs and 776 genes differentially expressed among the three cell types. Selected differentially expressed miRNAs and genes were further validated and confirmed by quantitative real-time-PCR (qRT-PCR). Especially, members of the miR-302 cluster on chromosome 4 and miR-520 cluster on chromosome 19 were highly expressed in undifferentiated hES cells. MiRNAs in these two clusters displayed similar expression levels. The members of these two clusters share a consensus 7-mer seed sequence and their targeted genes had overlapping functions. Among the targeted genes, genes with chromatin structure modification function are enriched suggesting a role in the maintenance of chromatin structure. We also found that the expression level of members of the two clusters, miR-520b and miR-302c, were negatively correlated with their targeted genes based on gene expression analysis</p> <p>Conclusion</p> <p>We identified the expression patterns of miRNAs and gene transcripts in the undifferentiation of human embryonic stem cells; among the miRNAs that are highly expressed in undifferentiated embryonic stem cells, the miR-520 cluster may be closely involved in hES cell function and its relevance to chromatin structure warrants further study.</p

Co-administration of a DNA vaccine encoding the prostate specific membrane antigen and CpG oligodeoxynucleotides suppresses tumor growth

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a well characterized prostate-specific tumor associated antigen. Its expression is elevated in prostate carcinoma, particularly in metastatic and recurrent lesions. These observations suggest that PSMA can be used as immune target to induce tumor cell-specific recognition by the host and, consequently tumor rejection. We utilized a DNA-based vaccine to specifically enhance PSMA expression. An immune modulator, such as CpG oligodeoxynucleotides which promote Th1-type immune responses was combined to increase the efficacy of tumor recognition and elimination. METHODS: A eukaryotic expression plasmid pCDNA3.1-PSMA encoding full-length PSMA was constructed. C57BL/6 mice were immunized with endotoxin-free pCDNA3.1-PSMA alone or in combination with CpG oligodeoxynucleotides by intramuscular injection. After 4 immunizations, PSMA specific antibodies and cytotoxic T lymphocyte reactivity were measured. Immunized C57BL/6 mice were also challenged subcutaneously with B16 cells transfected with PSMA to evaluate suppression of tumor growth. RESULTS: Vaccine-specific cytotoxic T lymphocytes reactive with B16 cells expressing PSMA could be induced with this treatment schedule. Immune protection was observed in vaccinated mice as indicated by increased tumor growth in the control group (100%) compared with the groups vaccinated with DNA alone (66.7%) or DNA plus CpG oligodeoxynucleotides (50%) respectively. Average tumor volume was smaller in vaccinated groups and tumor-free survival time was prolonged by the vaccination. CONCLUSION: The current findings suggest that specific anti-tumor immune response can be induced by DNA vaccines expressing PSMA. In addition, the suppression of in vivo growth of tumor cells expressing PSMA was augmented by CpG oligodeoxynucleotides. This strategy may provide a new venue for the treatment of carcinoma of prostate after failure of standard therapy

Removal Of Myeloid Cells From Autologous Leukocytes Used For Chimeric Antigen Receptor (Car) T Cell Manufacturing Improves Final Product Consistency And Yields

Early phase clinic trails of T cells genetically engineered to express Chimeric Antigen Receptors (CAR) have been promising. CD19-CAR T cells have been used successfully in a number of clinical trials to treat non-Hodgkin’s lymphoma and acute lymphocytic leukemia (ALL) and clinical trials of GD2-CAR T cells for the treatment of osteosarcoma and neuroblastoma are underway. Most CAR T cell manufacturing protocols make use of autologous peripheral blood mononuclear cell (PBMC) concentrates collected by apheresis, however, the lymphocyte-rich PBMC concentrates are also enriched for monocytes and contain small but variable quantities of red blood cells, platelets and neutrophils and prior to beginning CAR T cell manufacturing the PBMC concentrates are generally enriched for lymphocytes or CD3+ cells. We initially manufactured CD19- and GD2-CAR T cells using autologous PBMC concentrates enriched for T cells by selection with the anti-CD3/CD28 beads. These same anti-CD3/CD28 beads were used to stimulated T cell expansion. While the method was, in general, effective, we found that the quantities of GD2-CAR T cells produced were less than the quantities of CD19 CAR T cells produced. In addition, T cells from some patients failed to expand at all. Further investigation found that the presence of large quantities of monocytes or granulocytes in some PBMC concentrates which was associated with poor in vitro CAR T cell expansion. Myeloid derived suppressor cells (MDSCs) that inhibit T cell proliferation are present in sarcoma and ALL patients. These MDSCs may have a monocyte or neutrophil phenotype. We hypothesized that MDSCs in the PBSC concentrates bound non-specifically to the anti-CD3/CD28 beads and more rigorous enrichment of the starting material for lymphocytes would improve CAR T cell yields and reduce the incidence of manufacturing failures. We modified the T cell enrichment method to include a monocyte-depleting plastic adherence step. This change improved T cell expansion, but it was not completely effective at removing contaminating monocytes and granulocytes and did not completely eliminate manufacturing failures. To provide better depletion of monocytes and granulocytes we subjected PBMC concentrates to counter flow elutriation instrument. We manufactured 8 CD19- and 5 GD2-CAR T cell products from elutriated lymphocytes. All 13 CAR T cell manufacturing procedures yielded sufficient quantities of T cells to meet the dose criteria. The 13 CAR T cell products contained 2,166±1,113 x106 CD3+ cells and 1,064±877 x106 transduced CD3+ T cells. The CAR T cell products manufactured from elutriated lymphocytes yielded significantly more CD3+ cells and transduced CD3+ cells than that of those manufactured from anti-CD3/CD28 bead selected and plastic adherence selected cells. These results show CAR T cell manufacturing yields are greater and more consistent when manufacturing is initiated with lymphocytes that have few contaminating myeloid cells

Pancreatic islet cell therapy for type I diabetes: understanding the effects of glucose stimulation on islets in order to produce better islets for transplantation

While insulin replacement remains the cornerstone treatment for type I diabetes mellitus (T1DM), the transplantation of pancreatic islets of Langerhans has the potential to become an important alternative. And yet, islet transplant therapy is limited by several factors, including far too few donor pancreases. Attempts to expand mature islets or to produce islets from stem cells are far from clinical application. The production and expansion of the insulin-producing cells within the islet (so called β cells), or even creating cells that secrete insulin under appropriate physiological control, has proven difficult. The difficulty is explained, in part, because insulin synthesis and release is complex, unique, and not entirely characterized. Understanding β-cell function at the molecular level will likely facilitate the development of techniques to manufacture β-cells from stem cells. We will review islet transplantation, as well as the mechanisms underlying insulin transcription, translation and glucose stimulated insulin release

Differentiation of two types of mobilized peripheral blood stem cells by microRNA and cDNA expression analysis

<p>Abstract</p> <p>Background</p> <p>Mobilized-peripheral blood hematopoietic stem cells (HSCs) have been used for transplantation, immunotherapy, and cardiovascular regenerative medicine. Agents used for HSC mobilization include G-CSF and the CXCR4 inhibitor AMD3100 (plerixafor). The HSCs cells mobilized by each agent may contain different subtypes and have different functions. To characterize mobilized HSCs used for clinical applications, microRNA (miRNA) profiling and gene expression profiling were used to compare AMD3100-mobilized CD133+ cells from 4 subjects, AMD3100 plus G-CSF-mobilized CD133+ cells from 4 subjects and G-CSF-mobilized CD34+ cells from 5 subjects. The HSCs were compared to peripheral blood leukocytes (PBLs) from 7 subjects.</p> <p>Results</p> <p>Hierarchical clustering of miRNAs separated HSCs from PBLs. miRNAs up-regulated in all HSCs included hematopoiesis-associated miRNA; miR-126, miR-10a, miR-221 and miR-17-92 cluster. miRNAs up-regulated in PBLs included miR-142-3p, -218, -21, and -379. Hierarchical clustering analysis of miRNA expression separated the AMD3100-mobilized CD133+ cells from G-CSF-mobilized CD34+ cells. Gene expression analysis of the HSCs naturally segregated samples according to mobilization and isolation protocol and cell differentiation status.</p> <p>Conclusion</p> <p>HSCs and PBLs have unique miRNA and gene expression profiles. miRNA and gene expression microarrays maybe useful for assessing differences in HSCs.</p

Molecular signatures induced by interleukin-2 on peripheral blood mononuclear cells and T cell subsets

Experimentally, interleukin-2 (IL-2) exerts complex immunological functions promoting the proliferation, survival and activation of T cells on one hand and inducing immune regulatory mechanisms on the other. This complexity results from a cross talk among immune cells which sways the effects of IL-2 according to the experimental or clinical condition tested. Recombinant IL-2 (rIL-2) stimulation of peripheral blood mononuclear cells (PBMC) from 47 donors of different genetic background induced generalized T cell activation and anti-apoptotic effects. Most effects were dependent upon interactions among immune cells. Specialized functions of CD4 and CD8 T cells were less dependent upon and often dampened by the presence of other PBMC populations. In particular, cytotoxic T cell effector function was variably affected with a component strictly dependent upon the direct stimulation of CD8 T cells in the absence of other PBMC. This observation may provide a roadmap for the interpretation of the discrepant biological activities of rIL-2 observed in distinct pathological conditions or treatment modalities

Intra-subject variability in human bone marrow stromal cell (BMSC) replicative senescence: Molecular changes associated with BMSC senescence

AbstractThe outcomes of clinical trials using bone marrow stromal cell (BMSC) are variable; the degree of the expansion of BMSCs during clinical manufacturing may contribute to this variability since cell expansion is limited by senescence. Human BMSCs from aspirates of healthy subjects were subcultured serially until cell growth stopped. Phenotype and functional measurements of BMSCs from two subjects including senescence-associated beta-galactosidase staining and colony formation efficiency changed from an early to a senescence pattern at passage 6 or 7. Transcriptome analysis of 10 early and 15 late passage BMSC samples from 5 subjects revealed 2122 differentially expressed genes, which were associated with immune response, development, and cell proliferation pathways. Analysis of 57 serial BMSC samples from 7 donors revealed that the change from an early to senescent profile was variable among subjects and occurred prior to changes in phenotypes. BMSC age expressed as a percentage of maximum population doublings (PDs) was a good indicator for an early or senescence transcription signature but this measure of BMSC life span can only be calculated after expanding BMSCs to senescence. In order to find a more useful surrogate measure of BMSC age, we used a computational biology approach to identify a set of genes whose expression at each passage would predict elapsed age of BMSCs. A total of 155 genes were highly correlated with BMSC age. A least angle regression algorithm identified a set of 24 BMSC age-predictive genes. In conclusion, the onset of senescence-associated molecular changes was variable and preceded changes in other indicators of BMSC quality and senescence. The 24 BMSC age predictive genes will be useful in assessing the quality of clinical BMSC products