Cymerus™ iPSC-MSCs significantly prolong survival in a pre-clinical, humanized mouse model of Graft-vs-host disease.

The immune-mediated tissue destruction of graft-vs-host disease (GvHD) remains a major barrier to greater use of hematopoietic stem cell transplantation (HSCT). Mesenchymal stem cells (MSCs) have intrinsic immunosuppressive qualities and are being actively investigated as a therapeutic strategy for treating GvHD. We characterized Cymerus™ MSCs, which are derived from adult, induced pluripotent stem cells (iPSCs), and show they display surface markers and tri-lineage differentiation consistent with MSCs isolated from bone marrow (BM). Administering iPSC-MSCs altered phosphorylation and cellular localization of the T cell-specific kinase, Protein Kinase C theta (PKCθ), attenuated disease severity, and prolonged survival in a humanized mouse model of GvHD. Finally, we evaluated a constellation of pro-inflammatory molecules on circulating PBMCs that correlated closely with disease progression and which may serve as biomarkers to monitor therapeutic response. Altogether, our data suggest Cymerus iPSC-MSCs offer the potential for an off-the-shelf, cell-based therapy to treat GvHD.

Differentiation of Pathogenic Th17 Cells Is Negatively Regulated by Let-7 MicroRNAs in a Mouse Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a disabling demyelinating autoimmune disorder of the central nervous system (CNS) which is driven by IL-23- and IL-1ß-induced autoreactive Th17 cells that traffic to the CNS and secrete proinflammatory cytokines. Th17 pathogenicity in MS has been correlated with the dysregulation of microRNA (miRNA) expression, and specific miRNAs have been shown to promote the pathogenic Th17 phenotype. In the present study, we demonstrate, using the animal model of MS, experimental autoimmune encephalomyelitis (EAE), that let-7 miRNAs confer protection against EAE by negatively regulating the proliferation, differentiation and chemokine-mediated migration of pathogenic Th17 cells to the CNS. Specifically, we found that let-7 miRNAs may directly target the cytokine receptors Il1r1 and Il23r, as well as the chemokine receptors Ccr2 and Ccr5. Therefore, our results identify a novel regulatory role for let-7 miRNAs in pathogenic Th17 differentiation during EAE development, suggesting a promising therapeutic application for disease treatment.

Notch and T Cell Function - A Complex Tale.

Notch drives critical decisions in a multitude of developmental decisions in many invertebrate and vertebrate organisms including flies, worms, fish, mice and humans. Therefore, it is not surprising that Notch family members also play a key role in cell fate choices in the vertebrate immune system. This review highlights the critical function of Notch in the development of mature T lymphocytes from hematopoietic precursors and describes the role of Notch in mature T cell activation, proliferation and differentiation.

Acquisition of an oncogenic fusion protein serves as an initial driving mutation by inducing aneuploidy and overriding proliferative defects.

While many solid tumors are defined by the presence of a particular oncogene, the role that this oncogene plays in driving transformation through the acquisition of aneuploidy and overcoming growth arrest are often not known. Further, although aneuploidy is present in many solid tumors, it is not clear whether it is the cause or effect of malignant transformation. The childhood sarcoma, Alveolar Rhabdomyosarcoma (ARMS), is primarily defined by the t(2;13)(q35;q14) translocation, creating the PAX3-FOXO1 fusion protein. It is unclear what role PAX3-FOXO1 plays in the initial stages of tumor development through the acquisition and persistence of aneuploidy. In this study we demonstrate that PAX3-FOXO1 serves as a driver mutation to initiate a cascade of mRNA and miRNA changes that ultimately reprogram proliferating myoblasts to induce the formation of ARMS. We present evidence that cells containing PAX3-FOXO1 have changes in the expression of mRNA and miRNA essential for maintaining proper chromosome number and structure thereby promoting aneuploidy. Further, we demonstrate that the presence of PAX3-FOXO1 alters the expression of growth factor related mRNA and miRNA, thereby overriding aneuploid-dependent growth arrest. Finally, we present evidence that phosphorylation of PAX3-FOXO1 contributes to these changes. This is one of the first studies describing how an oncogene and post-translational modifications drive the development of a tumor through the acquisition and persistence of aneuploidy. This mechanism has implications for other solid tumors where large-scale genomics studies may elucidate how global alterations contribute to tumor phenotypes allowing the development of much needed multi-faceted tumor-specific therapeutic regimens.

Intracellular Delivery of Anti-pPKCθ (Thr538) via Protein Transduction Domain Mimics for Immunomodulation.

Targeting cellular proteins with antibodies, to better understand cellular signaling pathways in the context of disease modulation, is a fast-growing area of investigation. Humanized antibodies are increasingly gaining attention for their therapeutic potential, but the collection of cellular targets is limited to those secreted from cells or expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as putative targets for antibody binding. Protein kinase Cθ (PKCθ) is essential to T cell activation, proliferation, and differentiation, and its phosphorylation at specific residues is required for its activity. Here we report on the design, synthesis, and characterization of a protein transduction domain mimic capable of efficiently delivering an antibody against phosphorylated PKCθ (Thr538) into human peripheral mononuclear blood cells and altering expression of downstream indicators of T cell activation and differentiation. We used a humanized, lymphocyte transfer model of graft-versus-host disease, to evaluate the durability of protein transduction domain mimic:Anti-pPKCθ modulation, when delivered into human peripheral mononuclear blood cells ex vivo. We demonstrate that protein transduction domain mimic:Antibody complexes can be readily introduced with high efficacy into hard-to-transfect human peripheral mononuclear blood cells, eliciting a biological response sufficient to alter disease progression. Thus, protein transduction domain mimic:Antibody delivery may represent an efficient ex vivo approach to manipulating cellular responses by targeting intracellular proteins.

miR-24 regulates menin in the endocrine pancreas.

Menin, the product of the MEN1 gene, functions as a tumor suppressor and was first identified in 1997 due to its causative role in the endocrine tumor disorder multiple endocrine neoplasia, type 1 (MEN1). More recently, menin has been identified as a key player in pancreatic islet biology with the observation of an inverse relationship between menin levels and pancreatic islet proliferation. However, the factors regulating menin and the MEN1 gene in the pancreas are poorly understood. Here, we describe the regulation of menin by miR-24 and demonstrate that miR-24 directly decreases menin levels and impacts downstream cell cycle inhibitors in MIN6 insulinoma cells and in ßlox5 immortalized ß-cells. This regulation of menin impacts cell viability and proliferation in ßlox5 cells. Furthermore, our data show a feedback regulation between miR-24 and menin that is present in the pancreas, suggesting that miR-24 regulates menin levels in the pancreatic islet.