An overview and synopsis of techniques for directing stem cell differentiation in vitro

The majority of studies on stem cell differentiation have so far been based in vivo, on live animal models. The usefulness of such models is limited, since it is much more technically challenging to conduct molecular studies and genetic manipulation on live animal models compared to in vitro cell culture. Hence, it is imperative that efficient protocols for directing stem cell differentiation into well-defined lineages in vitro are developed. The development of such protocols would also be useful for clinical therapy, since it is likely that the transplantation of differentiated stem cells would result in higher engraftment efficiency and enhanced clinical efficacy, compared to the transplantation of undifferentiated stem cells. The in vitro differentiation of stem cells, prior to transplantation in vivo, would also avoid spontaneous differentiation into undesired lineages at the transplantation site, as well as reduce the risk of teratoma formation, in the case of embryonic stem cells. Hence, this review critically examines the various strategies that could be employed to direct and control stem cell differentiation in vitro.     

IgA immune complex blunts the contraction of cultured mesangial cells through the inhibition of protein kinase C and intracellular calcium

The effects of IgA immune complex (IgA-IC) on the contractile function of cultured mesangial cells were measured by the changes in planar surface area in response to treatment with agonists. Incubation of mesangial cells with IgA-IC for 24 hours significantly decreased the contractile responses to angiotensin II (10(-6) M) and phorbol 12-myristate 13-acetate (PMA, 10(-6) M). Pretreatment of mesangial cells with the protein kinase C (PKC) inhibitor, chelerythrine (10(-6) M), eliminated the difference in contractile responses to angiotensin II or PMA between the control and IgA-IC groups indicating IgA-IC may inhibit the activity of PKC. The contractile responses to ionomycin were not significantly different between IgA-IC treated and control mesangial cells, suggesting that the contractile machinery is not impaired by IgA-IC. Intracellular calcium, [Ca2+]i measured by changes in fura-2 level in response to ATP or bradykinin, was significantly inhibited in IgA-IC treated mesangial cells, compared to control cells. In contrast, treatment with thapsigargin did not result in significant differences in [Ca2+]i between IgA-IC and control mesangial cells, suggesting that a negligible role of endoplasmic reticulum in the effects of IgA-IC. Using PKC specific antibodies, IgA-IC significantly increased the particulate fraction of PKC-iota of mesangial cells to 141+/-13% of control, without significantly changing the protein content of PKC-alpha, -delta and -lambda in the cytosolic and particulate fractions. In summary, IgA-IC inhibits the contractile responses of cultured mesangial cells to agonists by inhibiting the activation of PKC and [Ca2+]i.     

Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples

Analysis of genomic DNA derived from cells and fresh or fixed tissues often requires whole genome amplification prior to microarray screening. Technical hurdles to this process are the introduction of amplification bias and/or the inhibitory effects of formalin fixation on DNA amplification. Here we demonstrate a balanced-PCR procedure that allows unbiased amplification of genomic DNA from fresh or modestly degraded paraffin-embedded DNA samples. Following digestion and ligation of a target and a control genome with distinct linkers, the two are mixed and amplified in a single PCR, thereby avoiding biases associated with PCR saturation and impurities. We demonstrate genome-wide retention of allelic differences following balanced-PCR amplification of DNA from breast cancer and normal human cells and genomic profiling by array-CGH (cDNA arrays, 100 kb resolution) and by real-time PCR (single gene resolution). Comparison of balanced-PCR with multiple displacement amplification (MDA) demonstrates equivalent performance between the two when intact genomic DNA is used. When DNA from paraffin-embedded samples is used, balanced PCR overcomes problems associated with modest DNA degradation and produces unbiased amplification whereas MDA does not. Balanced-PCR allows amplification and recovery of modestly degraded genomic DNA for subsequent retrospective analysis of human tumors with known outcomes.     

G-protein-coupled receptor signaling components localize in both sarcolemmal and intracellular caveolin-3-associated microdomains in adult cardiac myocytes

This study tests the hypothesis that G-protein-coupled receptor (GPCR) signaling components involved in the regulation of adenylyl cyclase (AC) localize with caveolin (Cav), a protein marker for caveolae, in both cell-surface and intracellular membrane regions. Using sucrose density fractionation of adult cardiac myocytes, we detected Cav-3 in both buoyant membrane fractions (BF) and heavy/non-buoyant fractions (HF); beta2-adrenergic receptors (AR) in BF; and AC5/6, beta1-AR, M4-muscarinic acetylcholine receptors (mAChR), mu-opioid receptors, and Galpha(s) in both BF and HF. In contrast, M2-mAChR, Galpha(i3), and Galpha(i2) were found only in HF. Immunofluorescence microscopy showed co-localization of Cav-3 with AC5/6, Galpha(s), beta2-AR, and mu-opioid receptors in both sarcolemmal and intracellular membranes, whereas M2-mAChR were detected only intracellularly. Immunofluorescence of adult heart revealed a distribution of Cav-3 identical to that in isolated adult cardiac myocytes. Upon immunoelectron microscopy, Cav-3 co-localized with AC5/6 and Galpha(s) in sarcolemmal and intracellular vesicles, the latter closely allied with T-tubules. Cav-3 immunoprecipitates possessed components that were necessary and sufficient for GPCR agonist-promoted stimulation and inhibition of cAMP formation. The distribution of GPCR, G-proteins, and AC with Cav-3 in both sarcolemmal and intracellular T-tubule-associated regions indicates the existence of multiple Cav-3-localized cellular microdomains for signaling by hormones and drugs in the heart.     

Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a widely used model organism for studies of oxygenic photosynthesis in eukaryotes. Here we describe the development of a resource for functional genomics of photosynthesis using insertional mutagenesis of the Chlamydomonas nuclear genome. Chlamydomonas cells were transformed with either of two plasmids conferring zeocin resistance, and insertional mutants were selected in the dark on acetate-containing medium to recover light-sensitive and nonphotosynthetic mutants. The population of insertional mutants was subjected to a battery of primary and secondary phenotypic screens to identify photosynthesis-related mutants that were pigment deficient, light sensitive, nonphotosynthetic, or hypersensitive to reactive oxygen species. Approximately 9% of the insertional mutants exhibited 1 or more of these phenotypes. Molecular analysis showed that each mutant line contains an average of 1.4 insertions, and genetic analysis indicated that approximately 50% of the mutations are tagged by the transforming DNA. Flanking DNA was isolated from the mutants, and sequence data for the insertion sites in 50 mutants are presented and discussed.     

Melatonin,immune function and aging

Aging is associated with a decline in immune function (immunosenescence), a situation known to correlate with increased incidence of cancer, infectious and degenerative diseases. Innate, cellular and humoral immunity all exhibit increased deterioration with age. A decrease in functional competence of individual natural killer (NK) cells is found with advancing age. Macrophages and granulocytes show functional decline in aging as evidenced by their diminished phagocytic activity and impairment of superoxide generation. There is also marked shift in cytokine profile as age advances, e.g., CD3+ and CD4+ cells decline in number whereas CD8+ cells increase in elderly individuals. A decline in organ specific antibodies occurs causing reduced humoral responsiveness. Circulating melatonin decreases with age and in recent years much interest has been focused on its immunomodulatory effect. Melatonin stimulates the production of progenitor cells for granulocytes-macrophages. It also stimulates the production of NK cells and CD4+ cells and inhibits CD8+ cells. The production and release of various cytokines from NK cells and T-helper lymphocytes also are enhanced by melatonin. Melatonin presumably regulates immune function by acting on the immune-opioid network, by affecting G protein-cAMP signal pathway and by regulating intracellular glutathione levels. Melatonin has the potential therapeutic value to enhance immune function in aged individuals and in patients in an immunocompromised state.