Generation and Applications of Human Pluripotent Stem Cells Induced into Neural Lineages and Neural Tissues

Human pluripotent stem cells (hPSCs) represent a new and exciting field in modern medicine, now the focus of many researchers and media outlets. The hype is well-earned because of the potential of stem cells to contribute to disease modeling, drug screening, and even therapeutic approaches. In this review, we focus first on neural differentiation of these cells. In a second part we compare the various cell types available and their advantages for in vitro modeling. Then we provide a “state-of-the-art” report about two major biomedical applications: (1) the drug and toxicity screening and (2) the neural tissue replacement. Finally, we made an overview about current biomedical research using differentiated hPSCs

Rapid generation of stable transgenic embryonic stem cell lines using modular lentivectors

Generation of stable transgenic embryonic stem (ES) cell lines by classic transfection is still a difficult task, requiring time-consuming clonal selection, and hampered by clonal artifacts and gene silencing. Here we describe a novel system that allows construction of lentivectors and generation of stable ES cell lines with > 99% transgene expression within a very short time frame. Rapid insertion of promoters and genes of interest is obtained through a modular recombinational cloning system. Vectors contain central polypurine tract from HIV-1 element and woodchuck hepatitis virus post-transcriptional regulatory element as well as antibiotic resistance to achieve optimal and homogenous transgene expression. We show that the system 1) is functional in mouse and human ES cells, 2) allows the generation of ES cells expressing genes of interest under the control of ubiquitous or tissue-specific promoters, and 3) allows ES cells expressing two constructs through selection with different antibiotics to be obtained. The technology described herein should become a useful tool in stem cell research

Nicotinic acetylcholine receptors in neonatal motoneurons are regulated by axotomy: an electrophysiological and immunohistochemical study in human bcl-2 transgenic mice

Motoneuron axotomy was exploited as a model system for studying functional and morphological changes caused in motoneuron cell bodies by peripheral axon injury. Rodent facial motoneurons express functional nicotinic acetylcholine receptors. We have determined the effect of neonatal unilateral facial nerve transection on these receptors by using electrophysiological and immunohistochemical techniques. To avoid rapid apoptotic cell death of axotomized motoneurons, the study was done in mice overexpressing the human bcl-2 transgene. Intact motoneurons responded to acetylcholine by generating a rapidly rising inward current, which was insensitive to methyllycaconitine, a selective antagonist of alpha7-containing nicotinic receptors, but was suppressed by dihydro-beta-erythroidine, a broad-spectrum antagonist. This indicates that mouse facial motoneurons possess nicotinic receptors which are probably devoid of the alpha7 subunit. In striking contrast, axotomized motoneurons displayed little or no sensitivity to acetylcholine. Axotomy did not affect the sensitivity of facial motoneurons to the selective glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxaxolepropionic acid. Immunohistochemical studies revealed that the alpha4 nicotinic receptor subunit was present in intact motoneurons but was undetectable in axotomized motoneurons. By contrast, the beta2 subunit was comparable in intact and axotomized motoneurons. alpha3 immunoreactivity was undetectable, both in intact and in axotomized motoneurons.Thus, mouse facial nicotinic receptors are possibly of the alpha4beta2 type and axotomy interferes negatively with the expression of the alpha4 subunit. By down-regulating nicotinic receptors, peripheral nerve injury may facilitate motoneuron degeneration. Alternatively, nicotinic receptor downregulation and motoneuron degeneration may be independent consequences of peripheral axotomy

Genetic engineering of embryonic stem cells

We describe a novel generation oflentiviral vectors that are particularly well suited for work with embryonic stem cells. The possibility of selecting cell lines with antibiotics and the rapid insertion of any combination of promoters and genes of interest makes them a powerful tool in the generation of transgenic ES cell lines. This vector can also greatly facilitate studies aimed at the improvement of neuronal engineering from ES cells, by making it possible to monitor the emergence and differentiation of neurons

Genetic engineering of embryonic stem cells

We describe a novel generation oflentiviral vectors that are particularly well suited for work with embryonic stem cells. The possibility of selecting cell lines with antibiotics and the rapid insertion of any combination of promoters and genes of interest makes them a powerful tool in the generation of transgenic ES cell lines. This vector can also greatly facilitate studies aimed at the improvement of neuronal engineering from ES cells, by making it possible to monitor the emergence and differentiation of neurons

The DBP gene is expressed according to a circadian rhythm in the suprachiasmatic nucleus and influences circadian behavior.

DBP, a PAR leucine zipper transcription factor, accumulates according to a robust circadian rhythm in liver and several other tissues of mouse and rat. Here we report that DBP mRNA levels also oscillate strongly in the suprachiasmatic nucleus (SCN) of the hypothalamus, believed to harbor the central mammalian pacemaker. However, peak and minimum levels of DBP mRNA are reached about 4 h earlier in the SCN than in liver, suggesting that circadian DBP expression is controlled by different mechanisms in SCN and in peripheral tissues. Mice homozygous for a DBP-null allele display less locomotor activity and free-run with a shorter period than otherwise isogenic wild-type animals. The altered locomotor activity in DBP mutant mice and the highly rhythmic expression of the DBP gene in SCN neurons suggest that DBP is involved in controlling circadian behavior. However, since DBP-/- mice are still rhythmic and since DBP protein is not required for the circadian expression of its own gene, dbp is more likely to be a component of the circadian output pathway than a master gene of the clock