The aims of our study were to verify whether it was possible to generate in vitro, from different adult human tissues, a population of cells that behaved, in culture, as multipotent stem cells and if these latter shared common properties. To this purpose, we grew and cloned finite cell lines obtained from adult human liver, heart, and bone marrow and named them human multipotent adult stem cells (hMASCs). Cloned hMASCs, obtained from the 3 different tissues, expressed the pluripotent state-specific transcription factors Oct-4, NANOG, and REX1, displayed telomerase activity, and exhibited a wide range of differentiation potential, as shown both at a morphologic and functional level. hMASCs maintained a human diploid DNA content, and shared a common gene expression signature, compared with several somatic cell lines and irrespectively of the tissue of isolation. In particular, the pathways regulating stem cell self-renewal/maintenance, such as Wnt, Hedgehog, and Notch, were transcriptionally active. Our findings demonstrate that we have optimized an in vitro protocol to generate and expand cells from multiple organs that could be induced to acquire morphologic and func- IntroductionThe presently accumulated evidence indicates that adult bone marrow (BM) contains at least 2 populations of stem cells: hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), responsible for the generation of the BM microenvironment. 1 Intriguingly, several reports have demonstrated the ability of MSCs to differentiate toward derivatives of germ layers other than mesoderm. [2][3][4][5][6] Although it is still unclear whether widely multipotent cells do exist in vivo and if they play a significant role in tissue repair and turnover, the ability to generate in vitro cells that, under defined culture conditions, display a very high developmental plasticity is nonetheless of important clinical relevance.Until now, the most convincing evidence, although debated, 7 of the possibility to grow in culture a population of widely multipotent cells in humans has been obtained only for BM, 8 while a similar feature has been just postulated for other adult human tissues. 9 We therefore planned to verify if human multipotent adult stem cells (hMASCs) could be produced from other adult human organs on top of BM, and we used this latter as a control/reference tissue.By systematically using a highly reproducible method, we were able to grow in culture cell lines from adult human liver, heart, and BM. These cell lines, once cloned at single-cell level, maintained the in vitro properties of parental lines, including the capability to differentiate into morphologically mature and functionally competent cells, even of tissues embryologically not related to the one of origin.Finally, we performed a comparative in vitro analysis on hMASCs originated from the 3 different sources with respect to immunophenotype, growth kinetics, specific transcriptional settings, telomerase activity, and global gene expression profile. Altogether the obtained result...

Adult mesenchymal stem cells derived from adipose tissue (A-MSC) have the capacity to differentiate in vitro into mesenchymal as well as endodermal and ectodermal cell lineages. We investigated the neuronal differentiation potential of human A-MSC with a protocol which included sphere formation and sequential culture in brain-derived neurotrophic factor (BDNF) and retinoic acid (RA). After 30 days, about 57% A-MSC showed morphological, immunocytochemical and electrophysiological evidence of initial neuronal differentiation. In fact, A-MSC displayed elongated shape with protrusion of two or three cellular processes, selectively expressed nestin and neuronal molecules (including GABA receptor and tyroxine hydroxilase) in the absence of glial phenotypic markers. Differentiated cells showed negative membrane potential (-60 mV), delayed rectifier potassium currents and TTX-sensitive sodium currents. Such changes were stable for at least 7 days after removal of differentiation medium. In view of these results and the easy availability of adipose tissue, A-MSC may be a ready source of adult MSC with neuronal differentiation potential, an useful tool to treat neurodegenerative diseases.

The olfactory bulb of mammals contains a large population of dopaminergic interneurones within the glomerular layer. Dopamine has been shown both in vivo and in vitro to modulate several aspects of olfactory information processing, but the functional properties of dopaminergic neurones have never been described due to the inability to recognize these cells in living preparations. To overcome this difficulty, we used a transgenic mouse strain harbouring an eGFP (enhanced green fluorescent protein) reporter construct under the promoter of tyrosine hydroxylase, the rate-limiting enzyme for cathecolamine synthesis. As a result, we were able to identify dopaminergic neurones (TH-GFP cells) in living preparations and, for the first time, we could study the functional properties of such neurones in the olfactory bulb, in both slices and dissociated cells. The most prominent feature of these cells was the autorhythmicity. In these cells we identified five main voltage-dependent conductances: the two having largest amplitude were a fast transient Na + current and a delayed rectifier K + current. In addition, we observed three smaller inward currents, sustained by Na + ions (persistent type) and by Ca 2+ ions (LVA and HVA). Using pharmacological tools and ion substitution methods we showed that the pacemaking process is supported by the interplay of the persistent Na + current and of a T-type Ca 2+ current. We carried out a complete kinetical analysis of the five conductances present in these cells, and developed a Hodgkin-Huxley model of TH-GFP cells, capable of reproducing accurately the properties of living cells, including autorhytmicity, and allowing a precise understanding of the process.

A significant fraction of the interneurons added in adulthood to the glomerular layer (GL) of the olfactory bulb (OB) are dopaminergic (DA). In the OB, DA neurons are restricted to the GL, but using transgenic mice expressing eGFP under the tyrosine hydroxylase (TH) promoter, we also detected the presence of TH-GFP+ cells in the mitral and external plexiform layers. We hypothesized that these could be adult-generated neurons committed to become DA but not yet entirely differentiated. Accordingly, TH-GFP+ cells outside the GL exhibit functional properties (appearance of pacemaker currents, synaptic connection with the olfactory nerve, intracellular chloride concentration, and other) marking a gradient of maturity toward the dopaminergic phenotype along the mitral-glomerular axis. Finally, we propose that the establishment of a synaptic contact with the olfactory nerve is the key event allowing these cells to complete their differentiation toward the DA phenotype and to reach their final destination.

Vertebrates can detect light intensity changes in vastly different photic environments, in part, because postreceptoral neurons undergo "network adaptation." Previous data implicated dopaminergic, cAMP-dependent inhibition of retinal ganglion cells in this process yet left unclear how this occurs and whether this occurs in darkness versus light. To test for light- and dopamine-dependent changes in ganglion cell cAMP levels in situ, we immunostained dark- and light-adapted retinas with anti-cAMP antisera in the presence and absence of various dopamine receptor ligands. To test for direct effects of dopamine receptor ligands and membrane-permeable protein kinase ligands on ganglion cell excitability, we recorded spikes from isolated ganglion cells in perforated-patch whole-cell mode before and during application of these agents by microperfusion. Our immunostainings show that light, endogenous dopamine, and exogenous dopamine elevate ganglion cell cAMP levels in situ by activating D1-type dopamine receptors. Our spike recordings show that D1-type agonists and 8-bromo cAMP reduce spike frequency and curtail sustained spike firing and that these effects entail protein kinase A activation. These effects resemble those of background light on ganglion cell responses to light flashes. Network adaptation could thus be produced, to some extent, by dopaminergic modulation of ganglion cell spike generation, a mechanism distinct from modulation of transmitter release onto ganglion cells or of transmitter-gated currents in ganglion cells. Combining these observations with results obtained in studies of photoreceptor, bipolar, and horizontal cells indicates that all three layers of neurons in the retina are equipped with mechanisms for adaptation to ambient light intensity.