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      Replicable Expansion and Differentiation of Neural Precursors from Adult Canine Skin

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          Summary

          Repopulation of brain circuits by neural precursors is a potential therapeutic strategy for neurodegenerative disorders; however, choice of cell is critical. Previously, we introduced a two-step culture system that generates a high yield of neural precursors from small samples of adult canine skin. Here, we probe their gene and protein expression profiles in comparison with dermal fibroblasts and brain-derived neural stem cells and characterize their neuronal potential. To date, we have produced >50 skin-derived neural precursor (SKN) lines. SKNs can be cultured in a highly replicable fashion and uniformly express a panel of identifying markers. Upon differentiation, they self-upregulate neural specification genes, generating neurons with basic electrophysiological functionality. This unique population of neural precursors, derived from mature skin, overcomes many of the practical issues that have limited clinical translation of alternative cell types. Easily accessible, neuronally committed, and patient specific, SKNs may have potential for the treatment of brain disorders.

          Highlights

          • SKNs can be routinely expanded in culture from adult skin

          • SKNs are a distinct population, expressing a homogeneous panel of neurogenic markers

          • SKNs are capable of generating high yields of neurons in vitro

          Abstract

          In this article, the authors characterize a unique population of neurogenic cells derived from small samples of adult canine skin. These cells are shown to be highly homogeneous and capable of generating high yields of neurons that display basic synaptic and electrophysiological functionality. Accordingly, they propose that these cells may have applications in autologous neuroreplacement therapies.

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          Most cited references46

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          Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues.

          Human adult dental pulp stem cells (DPSCs) reside within the perivascular niche of dental pulp and are thought to originate from migrating cranial neural crest (CNC) cells. During embryonic development, CNC cells differentiate into a wide variety of cell types, including neurons of the peripheral nervous system. Previously, we have demonstrated that DPSCs derived from adult human third molar teeth differentiate into cell types reminiscent of CNC embryonic ontology. We hypothesized that DPSCs exposed to the appropriate environmental cues would differentiate into functionally active neurons. The data demonstrated that ex vivo-expanded human adult DPSCs responded to neuronal inductive conditions both in vitro and in vivo. Human adult DPSCs, but not human foreskin fibroblasts (HFFs), acquired a neuronal morphology, and expressed neuronal-specific markers at both the gene and protein levels. Culture-expanded DPSCs also exhibited the capacity to produce a sodium current consistent with functional neuronal cells when exposed to neuronal inductive media. Furthermore, the response of human DPSCs and HFFs to endogenous neuronal environmental cues was determined in vivo using an avian xenotransplantation assay. DPSCs expressed neuronal markers and acquired a neuronal morphology following transplantation into the mesencephalon of embryonic day-2 chicken embryo, whereas HFFs maintained a thin spindle fibroblastic morphology. We propose that adult human DPSCs provide a readily accessible source of exogenous stem/precursor cells that have the potential for use in cell-therapeutic paradigms to treat neurological disease.
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            PROTOCADHERINS MEDIATE DENDRITIC SELF-AVOIDANCE IN THE MAMMALIAN NERVOUS SYSTEM

            Dendritic arbors of many neurons are patterned by a process called self-avoidance, in which branches arising from a single neuron repel each other 1-7 . By minimizing gaps and overlaps within the arbor, self-avoidance facilitates complete coverage of a neuron’s territory by its neurites 1-3 . Remarkably, some neurons that display self-avoidance interact freely with other neurons of the same subtype, implying that they discriminate self from non-self. Here, we demonstrate roles for the clustered protocadherins (Pcdhs) in dendritic self-avoidance and self/non-self discrimination. The Pcdh locus encodes ~60 related cadherin-like transmembrane proteins, at least some of which exhibit isoform-specific homophilic adhesion in heterologous cells and are expressed stochastically and combinatorially in single neurons 7-11 . Deletion of all 22 Pcdhs in the mouse gamma subcluster (Pcdhgs) disrupts self-avoidance of dendrites in retinal starburst amacrine cells (SACs) and cerebellar Purkinje cells. Further genetic analysis of SACs showed that Pcdhgs act cell-autonomously during development, and that replacement of the 22 Pcdhgs with a single isoform restores self-avoidance. Moreover, expression of the same single isoform in all SACs decreases interactions among dendrites of neighboring SACs (heteroneuronal interactions). These results suggest that homophilic Pcdhg interactions between sibling neurites (isoneuronal interactions) generate a repulsive signal that leads to self-avoidance. In this model, heteroneuronal interactions are normally permitted because dendrites seldom encounter a matched set of Pcdhgs unless they emanate from the same soma. In many respects, our results mirror those reported for Dscam1 in Drosophila: this complex gene encodes thousands of recognition molecules that exhibit stochastic expression and isoform-specific interactions, and mediate both self-avoidance and self/non-self discrimination 4-7,12-15 . Thus, although insect Dscams and vertebrate Pcdhs share no sequence homology, they appear to underlie similar strategies for endowing neurons with distinct molecular identities and patterning their arbors.
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              PI3K/Akt and CREB regulate adult neural hippocampal progenitor proliferation and differentiation.

              The phosphoinositide 3-OH kinase (PI3K)/Akt pathway has been implicated in regulating several important cellular processes, including apoptosis, survival, proliferation, and metabolism. Using both pharmacological and genetic means, we demonstrate here that PI3K/Akt plays a crucial role in the proliferation of adult hippocampal neural progenitor cells. PI3K/Akt transduces intracellular signals from multiple mitogens, including basic fibroblast growth factor (FGF-2), Sonic hedgehog (Shh), and insulin-like growth factor 1 (IGF-1). In addition, retroviral vector-mediated over-expression of wild type Akt increased cell proliferation, while a dominant negative Akt inhibited proliferation. Furthermore, wild type Akt over-expression reduced glial (GFAP) and neuronal (beta-tubulin III) marker expression during differentiation, indicating that it inhibits cell differentiation. We also show that activation of the cAMP response element binding protein (CREB), which occurs in cells stimulated by FGF-2, is limited when Akt signaling is inhibited, demonstrating a link between Akt and CREB. Over-expression of wild type CREB increases progenitor proliferation, whereas dominant negative CREB only slightly decreases proliferation. These results indicate that PI3K/Akt signaling integrates extracellular signaling information to promote cellular proliferation and inhibit differentiation in adult neural progenitors.
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                Author and article information

                Contributors
                Journal
                Stem Cell Reports
                Stem Cell Reports
                Stem Cell Reports
                Elsevier
                2213-6711
                08 August 2017
                08 August 2017
                08 August 2017
                : 9
                : 2
                : 557-570
                Affiliations
                [1 ]Regenerative Neuroscience Group, Brain and Mind Centre, University of Sydney, Sydney, NSW 2050, Australia
                [2 ]Stem Cell Laboratory, University of New South Wales, Sydney, NSW 2031, Australia
                [3 ]Centre for Healthy Brain Ageing, University of New South Wales, Sydney, NSW 2031, Australia
                [4 ]School of Psychiatry, University of New South Wales, Sydney, NSW 2052, Australia
                [5 ]School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia
                [6 ]School of Biotechnology and Biomolecular Science, University of New South Wales, Sydney, NSW 2052, Australia
                Author notes
                Article
                S2213-6711(17)30318-1
                10.1016/j.stemcr.2017.07.008
                5550271
                28793248
                80112116-fdaf-470f-a16b-c425fe39ef21
                © 2017 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 15 December 2016
                : 7 July 2017
                : 10 July 2017
                Categories
                Article

                skin,adult stem cells,skn,neural precursors,neurogenic,differentiation,neurons

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