Neuronal Transdifferentiation Potential of Human Mesenchymal Stem Cells from Neonatal and Adult Sources by a Small Molecule Cocktail.

Human mesenchymal stem cells (MSCs) are good candidates for brain cell replacement strategies and have already been used as adjuvant treatments in neurological disorders. MSCs can be obtained from many different sources, and the present study compares the potential of neuronal transdifferentiation in MSCs from adult and neonatal sources (Wharton's jelly (WhJ), dental pulp (DP), periodontal ligament (PDL), gingival tissue (GT), dermis (SK), placenta (PLAC), and umbilical cord blood (UCB)) with a protocol previously tested in bone marrow- (BM-) MSCs consisting of a cocktail of six small molecules: I-BET151, CHIR99021, forskolin, RepSox, Y-27632, and dbcAMP (ICFRYA). Neuronal morphology and the presence of cells positive for neuronal markers (TUJ1 and MAP2) were considered attributes of neuronal induction. The ICFRYA cocktail did not induce neuronal features in WhJ-MSCs, and these features were only partial in the MSCs from dental tissues, SK-MSCs, and PLAC-MSCs. The best response was found in UCB-MSCs, which was comparable to the response of BM-MSCs. The addition of neurotrophic factors to the ICFRYA cocktail significantly increased the number of cells with complex neuron-like morphology and increased the number of cells positive for mature neuronal markers in BM- and UCB-MSCs. The neuronal cells generated from UCB-MSCs and BM-MSCs showed increased reactivity of the neuronal genes TUJ1, MAP2, NF-H, NCAM, ND1, TAU, ENO2, GABA, and NeuN as well as down- and upregulation of MSC and neuronal genes, respectively. The present study showed marked differences between the MSCs from different sources in response to the transdifferentiation protocol used here. These results may contribute to identifying the best source of MSCs for potential cell replacement therapies.

Contribution of Automated Technologies to Ion Channel Drug Discovery.

Automated technologies are now resolving the historical relegation that ion channels have endured as targets for the new drug discovery and development global efforts. The richness and adequacy of functional assay methodologies, remarkably fluorescence-based detection of ions fluxes and patch-clamp electrophysiology recording of ionic currents, are now automated and increasingly employed for the analysis of ion channel activity. While the former is currently the most commonly applied, the latter is finally reaching the throughput capacity to be engaged in the primary screening of chemical libraries conformed by hundreds of thousands of compounds. The use of automated instrumentation for the study of ion channel functionality (and dysfunctionality), particularly in the search for novel pharmacological agents with therapeutic purposes, has now reached out beyond the industrial setting, its original natural enclave, and is making its way into a growing number of academic labs and core facilities. The present chapter reviews the increasing contributions accomplished by a variety of different key automated technologies which have revolutionized the strategies to approach the discovery and development of new drugs targeting ion channels.

A chloride conductance exhibiting bicarbonate conductivity in renal inner medullary collecting duct cells.

The anion conductance in primary cultures of rat inner medullary collecting duct cells was studied using perforated-patch whole-cell clamp technique. Depolarizations above 0 mv induced an outward anionic current with a time-dependent activation (Iovt) exhibiting a similar conductivity to Cl- and HCO3-. Iovt showed half-maximal activation around 32 mV with a slope factor of 23 mV, and showed a voltage-dependent activation time course that was well fitted by a sum of two exponential functions. Iovt was potentiated when external pH or external Ca2+ was increased and was blocked by external DIDS, DPC and furosemide. These characteristics of Iovt resemble that of the ClC-K1 channels mediated currents; however, anion substitution studies showed that Iovt exhibits a Br->Cl->I->NO3- conductivity sequence, different from that observed in the ClC-K1 channels-mediated conductance. We suggest that, in inner medullary collecting duct cells, ClC-K channels of an unidentified type give rise to this Cl- and HCO3- conductance. This is the first study of a channel-mediated HCO3- current in kidney tubular cells.