It is important to prepare the cell culture devices with functional polymer surfaces. Notch ligand delta-1 is considered as one of the important membrane proteins for self-renewal of hematopoietic stem cells. In this study, a polymer matrix immobilized with Notch ligand delta-1 was prepared by photo-immobilization technique for culture the stem cells. It has been reported that photo-reactive polymers bearing azidophenyl groups could immobilize functional proteins without losing their biological activity. Therefore, a novel photo-reactive phospholipid polymer, 2-methacryloyloxyethyl phosphorylcholine polymer bearing azidophenyl groups, was applied as the matrix to conjugate with Notch ligand delta-1. A leukemia cell line, TMD7, was cultured on the bioconjugated polymer surface. The polymer surface immobilized with Notch ligand delta-1 was recognized by TMD7, and the cells efficiently grow on the phospholipid polar group concentrated surface (PC surface) with protein. It was considered that the PC surface provided a suitable environment around the membrane proteins without denaturation. The photo-reactive phospholipid polymer was expected to constitute on in vitro niche to culture hematopoietic stem cells.
Heme oxygenase(HO)-1 is an enzyme, an inducible form of HO, which catalyzes heme into carbon monoxide(CO), Fe2+, and biliverdin. CO suppresses apoptosis and macrophage activation, whereas biliverdin is converted into bilirubin, an antioxidant. Fe2+ stimulates the production of ferritin, a protective protein. Various noxious stimuli such as hypoxia or heavy metal loading induce expression of HO-1, which possesses cytoprotective effects mediated by the heme degradation products. Indeed, forced or chemically induced expression of HO-1 has beneficial effects on respiratory, inflammatory, renal diseases, and inflammatory disorders in animal models. On the other hand, HO-1 deficiency leads to systemic inflammation in mice and a patient. HO-1 is involved in pathogenesis of various diseases. Some of malignant tumors express abundant HO-1, resulting in suppressed apoptosis of tumor cells, whereas reduced expression of HO-1 is implicated in degenerative diseases such as Alzheimer dementia. Our preliminary results showed increased expression of HO-1 in joint lesions of rheumatoid arthritis and circulating leukocytes from patients with adult-onset Still's disease and hemophagocytic syndrome, though the role of HO-1 remains unknown in these diseases. Excessively expressed HO-1 appears toxic in some conditions. HO-1 induction by auranofin and statins has been shown to contribute to the pharmacological effects at least in part. To optimize HO-1 expression may lead to development of novel therapeutic strategies in various diseases including inflammatory disorders.
Atopic dermatitis, pollinosis, and bronchial asthma allergic immune disorders are characterized by a predominance of T helper 2(Th2) cells, the resulting elevation of allergen-specific IgE, and mast cell-and basophile-associated inflammation. The cytokine environment at the site of the initial antigen stimulation determines the direction of differentiation into Th1 or Th2 cells. The SOCS(suppressor of cytokine signaling) proteins are known to act as a negative regulator for many aspect of cytokine signaling and play a precise role to control the balance between Th1 and Th2 cells. SOCS3 and SOCS5 are predominantly expressed in Th2 and Th1 cells, respectively, and they reciprocally inhibit the Th1 and Th2 differentiation processes. Here, We discuss the role of SOCS proteins in helper T cell differentiation and explore the potential of SOCS proteins as targets for therapeutic strategies in allergic disorders.
Embryonic stem(ES) cells have a potential to differentiate into various progenitor cells. In this review we investigated the capacity of mouse ES cells to differentiate into renal tubular cells. After stably transfecting Wnt4 cDNA to mouse ES cells(Wnt4-ES cells) by electroporation, the ES cells were incubated by the hanging drop culture method without leukemia inhibitory factor(LIF) to induce differentiation to embryoid bodies(EBs). During the culture of the EBs derived from the Wnt4-ES cells, aquaporin-2(AQP2) mRNA and protein were expressed within 15-20 days after the removal of LIF. The expression of AQP2 in Wnt4-EBs was enhanced in the presence of hepatocyte growth factor(HGF) and activin A. We observed AQP2 positive tubular-like formation from Wnt4-EBs in three-dimensional culture. Our result shows that two new findings: firstly, that cultured Wnt4-EBs have an ability to differentiate into renal tubular-like cells; and secondly, that Wnt4, HGF, and activin A may promote the differentiation of ES cells to renal tubular-like cells.
Efficient gene transfer into stem cells which are able to self-renew and differenciate into certain type of cell is essential for not only difining the precise molecular mechanism of self-renewal and differenciation, but the supplying of the cells for regenerative medicine. In this paper, we review our approach to the efficient gene transfer into hematopoietic stem cell, mesenchymal stem cell, and ES cell by modified adenovirus vectors.
Intracellular free Ca2+ movements in neural progenitor cells obtained by transfection of embryonic stem(ES) cells with MASH1 transcription factor gene were studied. The MASH1 transfected cells, majority of which were Islet1 positive, have been shown to improve motor functions of hemiplegic mice when transplanted into the injured brain. In this study RT-PCR was conducted to detect Ca2+ channel mRNAs. We found that mRNAs of L-type, N-type and T-type Ca2+ channel mRNAs were expressed in the MASH1 transfected cells. Next, a Ca2+-sensitive fluorescence probe, fluo-3, and a scanning confocal laser microscope were used to detect any changes of intracellular Ca2+ concentration. Enhanced fluo-3 fluorescence was observed when the cells were stimulated with increased extracellular potassium, a depolarization signal. Depletion of extracellular Ca2+ abrogated the increase of fluorescence intensity upon depolarization. A Ca2+ channel blocker, lead, inhibited the increase of fluorescence intensity upon depolarization. A L-type channel inhibitor, nifedipine, but not a N-type channel inhibitor, omega-conotoxin GVIA, reduced the increase of fluorescence intensity upon depolarization. Thapsigargin, which depletes intracellular Ca2+ stores, did not attenuate the depolarization-induced signals. Potassium channel inhibitors, tetraethylammonium and 4-aminopyridine inhibited the fluorescence signals. These results indicate that depolarization-induced intracellular Ca2+ increase is mainly due to inward flow (influx) of Ca2+ via Ca2+ channels and marginally due to the release (mobilization) of Ca2+ from intracellular stores in the MASH1 transfected neural progenitor cells. The calcium signals may regulate various aspects of neural cell function such as authentic cellular activity including neurotransmitter secretion and cell differentiation. Our data add the possibility that calcium signals may play roles even in the ectopically transplanted MASH1-transfected neural progenitor cells reconstituting neural network to improve motor functions of hemiplegic mice.