Pluripotent marker expression and differentiation of human second trimester Mesenchymal Stem Cells.

Mesenchymal stem cells are an easily obtainable stem cell source from bone marrow. Presently, they are the most widely used cell type for cellular replacement strategies in humans as a result of extensive research that has demonstrated that these cells are capable of self-renewal, able to undergo multi-lineage differentiation, engraft, and ameliorate symptoms in numerous animal models. In this study, we comprehensively characterize human second trimester mesenchymal stem cells (STMSCs). We demonstrate that STMSCs are easily expandable to clinical relevance and express pluripotent markers such as Oct-4, Nanog, Sox-2, and SSEA-4 at the cellular and molecular level. Moreover, we directionally differentiate STMSCs into osteogenic, chondrogenic, adipogenic, neurogenic, and cardiogenic cell lineages. These studies demonstrate the plasticity of STMSCs and the potential for their use in cellular replacement therapy.

HIV-1 Nef alters the expression of betaII and epsilon isoforms of protein kinase C and the activation of the long terminal repeat promoter in human astrocytoma cells.

In the human immunodeficiency virus type 1 (HIV-1)-infected brain, the virus does not replicate in astrocytes, but a synthesis of viral regulatory proteins occurs in these cells, leading to accumulation of Nef. As an approach to understand the effects of Nef on astrocyte functional activity, we analyzed whether intracellular Nef interferes with the expression and activation of the enzyme protein kinase C (PKC), which is an important regulator of astroglial functions and HIV-1 replication. Astrocytoma clones (U251 MG) not expressing Nef (Neo), or expressing wild-type Nef (Bru) or nonmyristoylated Nef (TH) were used to monitor the expression and activation of 10 PKC isoforms. The same clones were used to evaluate the effect of Nef on the viral long terminal repeat (LTR) promoter after activation of PKC with the phorbol ester 12-myristate 13-acetate (PMA). PKC intracellular distribution and activation were evaluated by Western blot analysis of cytosolic and membrane fractions of control and Nef-expressing clones. PMA-induced LTR activation was analyzed in clones transfected with a plasmid encoding for the CAT reporter gene controlled by the LTR promoter, by using an enzyme-linked immunosorbent assay to measure CAT expression. Nef selectively downregulated the expression and activation of betaII and epsilon PKC isoforms in astrocytoma cells. Such downregulation correlated with an inhibition of LTR activation after PMA stimulation. The myristoylation of Nef and its membrane localization were essential for these effects. These results suggest that Nef may alter astrocytic functions by interfering with PKC expression and activation and contribute to the restriction of HIV-1 replication in astrocytes.

Expression and translocation of protein kinase C isoforms in rat microglial and astroglial cultures.

Cellular distribution and activation by phorbol myristate acetate (PMA) of classical (alpha, betaI, betaII,gamma), novel (delta, epsilon, theta, eta), and atypical (zeta, iota) protein kinase C (PKC) isoforms were studied in cultured rat neonatal microglial and astroglial cells by Western blot analysis. Among the classical isoforms, only betaII was expressed in microglia and astrocytes in the same abundance. The expression of betaI in microglia was less abundant, while PKCalpha was not detectable in this cell type. PKCgamma was absent in both cell populations. A different pattern of expression was also found for novel and atypical isoenzymes: Both cell types expressed delta, theta, eta, zeta, and iota isoforms, but PKCepsilon was absent in microglia and the expression of PKCzeta and PKCiota in these cells was low compared to astrocytes. The pattern of PKC distribution in cytosolic and particulate fractions as well as activation by short (10 min) and prolonged (4 hr) PMA treatment in both cell types were similar. On the whole, in comparison with astrocytes, PKC in microglial cells was less expressed, both in terms of number of isoforms and level of expression. The microglial profile of PKC isoforms differed from that of rat peritoneal macrophages, which did express PKCalpha. Preliminary evidence suggests that the ability of PMA to enhance cyclic AMP responses in astrocytes, but not in microglia, is related to the different pattern of expression of PKCalpha and PKCepsilon in the two cell types.

Opposite regulation of adenylyl cyclase by protein kinase C in astrocyte and microglia cultures.

We studied the regulation of cyclic AMP responses by protein kinase C (PKC) in purified astrocyte and microglia cultures obtained from the neonatal rat brain. In astrocytes, a 10-min treatment with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and 4beta-phorbol 12,13-didecanoate (4beta-PDD) (but not with 4alpha-PDD) or with diacylglycerol, which activate PKC, dose-dependently enhanced cyclic AMP accumulation induced by the beta-adrenergic agonist isoproterenol and the adenylyl cyclase activator forskolin. Such enhancement was prevented by the PKC inhibitors staurosporine and calphostin-C and by down-regulation of PKC and was not related to activation of membrane receptors or Gs proteins or to inhibition of Gi proteins or phosphodiesterases. Instead, the activity of adenylyl cyclase doubled in PMA-treated astrocytes. In microglia, a 10-min treatment with PMA or PKC inhibitors did not affect cyclic AMP accumulation, whereas longer treatments with PMA or 4beta-PDD (but not 4alpha-PDD) inhibited the cyclic AMP response in a time- and dose-dependent manner. Such inhibition was mimicked by staurosporine and calphostin-C. Also, in the case of microglia, the modulation of cyclic AMP responses appeared to occur at the level of adenylyl cyclase, and not elsewhere in the cyclic AMP cascade. The inhibition of microglial adenylyl cyclase was apparently not due to aspecific cytotoxicity. A differential regulation of adenylyl cyclase by PKC in astrocytes and microglia may help to explain qualitative and quantitative differences in the response of these cells to various physiological and pathological stimuli.

Reorientation of prostanoid production accompanies "activation" of adult microglial cells in culture.

Using morphological, immunocytochemical, and functional parameters we have previously shown that highly purified adult rat microglial cells undergo a process of "activation" when cultured in a serum-containing medium in the absence of added proinflammatory substances or other factors (Slepko and Levi: Glia 16:241-246, 1996). Here we studied the lipopolysaccharide (LPS)-evoked production of two prostanoids, thromboxane A2 (measured as thromboxane B2) (TXB2) and prostaglandin E2 (PGE2), as a function of microglial "activation." LPS induced a greater time- and dose-dependent release of TXB2, compared to PGE2, in the less "activated" cells. Further "activation" led to amplified synthesis of PGE2 and not of TXB2, so that the TXB2/PGE2 ratio changed from 2.2 to 0.25 between the 2nd and 4th day in culture. Western blot experiments showed that the LPS-evoked expression of the inducible form of cyclooxygenase (COX) was markedly higher in cells exhibiting a more "activated" phenotype. The expression of the constitutive isoform of COX was low in all conditions, was slightly greater in more "activated" cells, and was not affected by LPS. Neither progression in microglial "activation" nor LPS treatment enhanced thromboxane synthase activity. We hypothesize that reorientation of prostanoid synthesis toward a major production of PGE2 in the more "activated" cells can be largely attributed to an increased inducibility of cellular COX expression, combined with the inability of thromboxane synthase to cope with the increased availability of the COX product prostaglandin H2 (PGH2), the common precursor of TXA2 and PGE2. In view of the different, and at times opposite, functional activity of TXB2 and PGE2, the described change in prostanoid production pattern may contribute to the role of "activated" microglia in inflammation and host defense.

Progressive activation of adult microglial cells in vitro.

Bulk-isolated microglial cells from the adult rat brain grown in N2 medium supplemented with 10% fetal calf serum survived for at least 2 weeks, and their purity was >99% at day 1 and >93% at day 7. The phenotype of freshly plated cells was comparable to that of "resting," ramified microglia in vivo. With time in culture and with different schedules, depending on the parameter considered, microglia acquired antigenic (e.g., positivity for vimentin, ED1, major histocompatibility complex class I antigens, leukocyte common antigen, and to a lesser extent CD4) and functional (e.g., proliferation, phagocytosis) features characteristic of "activated" microglia as described in situ. Production of nitrite and prostaglandin E2 in response to lipopolysaccharide increased greatly with time in culture. Phagocytosis was also accompanied by increased release of nitrite and prostaglandin E2, the latter being more affected than the first by the age of the cultures. The culture system described may be suitable to study the factors that can modulate "activation" of adult microglia.

Swelling-induced K+ influx in cultured primary astrocytes.

The effect of swelling of cultured primary astrocytes from rat brain in hypotonic medium on K+ influx has been studied. A decrease in osmolality from 310 to 180 mOsm increased the activity of sodium pump (ouabain-inhibited 86Rb+ influx) and Na+,K+,2Cl- cotransport (ouabain-insensitive bumetanide-inhibited 86Rb+ influx) by 70 and 35%, respectively. It is suggested that activation of these transport systems makes it possible to retain a high potassium concentration in the cells under regulatory volume decrease.

[The effect of opioid peptides on a decreased proliferation and on an increased maturation rate of glioma C-6 cells manifested by enhanced marker enzyme activity]. / Vliianie opioidnykh peptidov na snizhenie proliferatsii i uvelichenie skorosti sozrevaniia kletok gliomy C-6, proiavliaemoe povysheniem aktivnosti markirovannykh fermentov.

A study was made of the effect of opioid peptides, (Leu)-enkephalin and its synthetic analog dalargin, on the maturation speed (differentiation) and proliferation activity of glioma C-6 cells. This effect on phenotypes of glioma C-6 cells was determined using some biochemical parameters: changes in the activity of glutamine synthetase (astrocytic marker) and cyclic nucleotide phosphorohydrolase (oligodendrocytic marker) in the culture of glioma C-6 cells in the early and late passages. The biochemical analysis was made at the Laboratory in Denver, USA, and we thank Prof. A. Vernadakis for the possibility to carry out a part of this work that helped us to find the growth activity of opioid peptides on the C-6 cells. Proliferation was examined in cultivation conditions approximately conforming the conditions of cultivation for the primary glial cell cultures. The control proliferation level was high in this case. It is demonstrated that opioid peptides accelerate (or strengthen) the expression of phenotypic signs in C-6 glioma cells in early and late passages changing specific activity of the marker enzymes, i.e. operating as a growth factor. Opioid peptides show glial growth factor characteristics on the glioma C-6 glial cell model as well, for glioma C-6 is known to be a perfect model to analyse the action of different substances on the glia.

[The effect of the synthetic leu-enkephalin analog dalargin on the proliferative activity of glioma C6 cells and on the intensity of their DNA synthesis]. / Issledovanie vliianiia sinteticheskogo analoga lei-énkefalina dalargina na proliferativnuiu aktivnost' kletok gliomy C6 i intensivnost' sinteza v nikh DNK.

Effect of dalargin, an opioid peptide (a synthetic analogue of Leu-enkephalin), on proliferation and intensity of DNA synthesis of C6 glioma cells was studied. Specific conditions of cultivation were selected, with a low control value of proliferation, which permitted to assess growth-stimulating effect of the peptide. Growth curves were plotted to assess peptide activity, which demonstrated that reaction was a many-phase process: a significant increase in cell number under peptide effect was observed only at the beginning of the logarithm phase and at the beginning of the prestationary phase of the growth curve. Cell number increased on average by 25-27% in the presence of dalargin as compared to control. Reaction of glioma DNA synthesis to dalargin also demonstrates complexity of the process: the peptide changes DNA synthesis, but as a rule, this process has a three-phase character and is not directly associated with the duration of cultivation in the presence of dalargin. Effect of naloxone, an opiate receptor blocker, was analysed to assess the receptor mechanism. It was found that reaction for naloxone and for combined effect of naloxone and dalargin was not the same.