Simple and efficient method for generation of induced pluripotent stem cells using piggyBac transposition of doxycycline-inducible factors and an EOS reporter system

PiggyBac (PB) transposition of reprogramming factors (Oct3/4 (O), Sox2 (S), Klf4 (K) and c-Myc) is a safe, nonviral method for generating induced pluripotent stem cells (iPSCs). However, compared with retroviral methods, the reprogramming efficiency of the PB-mediated methods is relatively low. In this study, we describe a simple and efficient system for generating high-quality iPSCs by a single transfection of multiple plasmids that does not require the use of a virus, special instruments or skilled techniques. To improve reprogramming efficiency, we modified the components of the polycistronic 2A vectors used in this study and also investigated the combination of another reprogramming-related factor (L-Myc). By simultaneous transposition of multiple PB vectors containing an EOS (early transposon promoter and Oct3/4 and Sox2 enhancers) reporter and modified polycistronic doxycycline (Dox)-inducible factors, we reprogrammed mouse somatic cells with an efficiency higher than is usually obtained with retroviral methods and we established some iPSC lines that contributed highly to chimeras. By using the Dox-inducible system, we also showed that the appropriate elimination of exogenous-factor expression at appropriate time accelerated the induction of Oct3/4 when a combination of OKS and c-Myc vectors were used.     

Myeloid neoplasm-related gene abnormalities differentially affect dendritic cell differentiation from murine hematopoietic stem/progenitor cells

Dendritic cells (DCs) play important roles in tumor immunology. Leukemic cells in patients with myeloid neoplasms can differentiate into DCs in vivo (referred to as in vivo leukemic DCs), which are postulated to affect anti-leukemia immune responses. We established a reproducible culture system of in vitro FLT3 ligand-mediated DC (FL-DC) differentiation from murine lineage(-) Sca-1(+) c-Kit(high) cells (LSKs), which made it possible to analyse the effects of target genes on steady-state DC differentiation from hematopoietic stem/progenitor cells. Using this system, we analysed the effects of various myeloid neoplasm-related gene abnormalities, termed class I and class II mutations, on FL-DC differentiation from LSKs. All class II mutations uniformly impaired FL-DC differentiation maintaining a plasmacytoid DC (pDC)/conventional DC (cDC) ratio comparable to the control cells. In contrast, class I mutations differentially affected FL-DC differentiation from LSKs. FLT3-ITD and a constitutively active form of Ras (CA-N-Ras) yielded more FL-DCs than the control, whereas the other class I mutations tested yielded less FL-DCs. Both FLT3-ITD and FLT3-tyrosine kinase domain (TKD) mutation showed a comparable pDC/cDC ratio as the control. CA-N-Ras, c-Kit-TKD, TEL/PDGFRβ, and FIP1L1/PDGFRα showed a severe decrease in the pDC/cDC ratio. CA-STAT5 and CA-MEK1 severely inhibited pDC differentiation. FLT3-ITD, CA-N-Ras, and TEL/PDGFRβ aberrantly induced programmed death ligand-1 (PD-L1)-expressing DCs. In conclusion, we have established a simple, efficient, and reproducible in vitro FL-DC differentiation system from LSKs. This system could uncover novel findings on how myeloid neoplasm-related gene abnormalities differentially affect FL-DC differentiation from murine hematopoietic stem/progenitor cells in a gene-specific manner.     

Identification of CD56dim subpopulation marked with high expression of GZMB/PRF1/PI-9 in CD56+ interferon-α-induced dendritic cells

As the sentinels of innate and adaptive immune system, dendritic cells (DCs) have been considered to hold a great promise for medical application. Among the diverse types of DCs, monocyte-derived DCs (mo-DCs) generated in vitro have been most commonly employed. We have been improving the culture protocol and devised a protocol to produce mature interferon-α-induced DCs (IFN-DCs), hereinafter called (mat)IFN-DCs. While exploring the relationship between the expression of CD56 and the cytotoxic activity of (mat)IFN-DCs, we unexpectedly found that sorting of (mat)IFN-DCs with CD56 antibody-coated microbeads (MB) resulted in fractionating cells with tumoricidal activity into the flow-through (FT) but not MB-bound fraction. We uncovered that the FT fraction contains cells expressing low but substantial level of CD56. Moreover, those cells express granzyme B (GrB), perforin (PFN), and serpin B9 at high levels. By employing a specific inhibitor of PFN, we confirmed that direct tumoricidal activity relies on the GrB/PFN pathway. We designated subpopulation in FT fraction as CD56^dim and that in CD56 positively sorted fraction as CD56^bright, respectively. This is the first time, to our knowledge, to identify subpopulations of CD56-positive IFN-DCs with distinct tumoricidal activity which is ascribed to high expression of the components of GrB/PFN pathway.     

Induction of NK1.1(+) alpha beta TCR(+) T cells by bypassing TCR signals in ZAP-70 deficient mice

The mechanism of development of a unique subset of T cells, thymic NK1.1(+) alpha beta T cells, has been poorly understood. We found that the development of thymic NK1.1(+) alpha beta T cells was defective in mice deficient in ZAP-70. Instead, an accumulation of NK1.1(+) TCR beta(-) NK-like population was detected in the thymus and spleen of the ZAP-70 deficient (ZAP -/-) mouse. In the present report, we examined whether biochemical treatments that replace TCR-mediated positive selection signals could restore the generation of thymic NK1.1(+) alpha beta T cells in ZAP -/- mice using the thymus organ culture. We found that a higher concentration of phorbol ester (PMA) than that required for CD4(+) T cell generation and ionomycin induced the generation of NK1.1(+) alpha beta T cells. Phenotypic analysis of the induced NK1.1(+) alpha beta T cell population suggested that these cells expressed CD8 but not CD4 molecules, which is a different characteristic from ordinary thymic NK1.1(+) alpha beta T cells. These results suggest that differential signaling is required for the generation of mainstream T cells and thymic NK1.1(+) alpha beta T cells.     

Immunoregulatory defects of V alpha 24V+ beta 11+ NKT cells in development of Wegener's granulomatosis and relapsing polychondritis

The frequency of either CD4(-)8(-) (double negative; DN) or CD4(+) V alpha 24(+)V beta 11(+) NKT cells, the expression of CD1d and the binding of CD1d-tetramer loaded with alpha-galactosylceramide (alpha-GalCer) to NKT cells were analysed in peripheral blood mononuclear cells (PBMCs) of patients with Wegener's granulomatosis (WG), relapsing polychondritis (RP) and healthy subjects (HS). DN and CD4(+) V alpha 24(+)V beta 11(+) NKT cells as well as CD1d-alpha-GalCer tetramer-positive NKT cells, were significantly decreased in number in both WG and RP patients compared to those from HS. When cytokine profiles were analysed in these PBMCs upon stimulation with phorbol ester and calcium ionophore, CD4(+) T cells from patients with WG and RP exhibited a Th1 bias, whereas CD4(+) NKT cells from WG patients in remission showed a Th2 bias. These findings suggest that NKT cells (especially CD4(+) NKT cells) play a regulatory role in Th1 autoimmunity in patients with WG and RP. The reduction in NKT cell counts appears to be associated with the low responsiveness to alpha-GalCer. The dysfunction of NKT cells to recognize ligands such as alpha-GalCer may also contribute to the defects observed in NKT cells from WG and RP patients.     

Laughter and humor as complementary and alternative medicines for dementia patients

Background  

The number of dementia patients has increased worldwide, with an estimated 13.7 million dementia patients in the Asia Pacific region alone. This number is expected to increase to 64.6 million by the year 2050.     

Effects of histamine on functional maturation of dendritic cells

There is increasing evidence that histamine affects dendritic cell (DC) activation, maturation, and preference for Th1/Th2 differentiation. In this paper we report that histamine affects interleukin (IL)-12 and IL-6 production in an immature DC (iDC) line derived from murine spleen. Histamine treatment of iDC significantly increased the IL-12 p40 mRNA and protein levels compared to histamine untreated iDC. In the presence of tumor necrosis factor (TNF)-alpha histamine also increased IL-12 p40 and IL-6 production. However, histamine significantly decreased IL-12 p40 production by lipopolysaccharide (LPS)-stimulated DC in a concentration dependent manner. When expressions of histamine H1 (H1R) and H2 (H2R) receptors in DC were analyzed by RT-PCR, both receptors were down-regulated after LPS or TNF-alpha stimulation compared to unstimulated iDC. Histamine treatment significantly increased the expression of H2R mRNA in iDC and H1R mRNA in LPS-activated DC. However, histamine treatment decreased the expression of both histamine receptors in TNF-alpha-stimulated DC. Similar results were obtained by flow cytometry with FITC-conjugated histamine. These results demonstrate that histamine can regulate the expression of its own receptors and activate iDC, which may influence subsequent functional states of mature DC in a maturation signal-dependent manner. Consequently, histamine may contribute to an immune response outcome.