Purification of Immature Neuronal Cells from Neural Stem Cell Progeny

Abstract

Large-scale proliferation and multi-lineage differentiation capabilities make neural stem cells (NSCs) a promising renewable source of cells for therapeutic applications. However, the practical application for neuronal cell replacement is limited by heterogeneity of NSC progeny, relatively low yield of neurons, predominance of astrocytes, poor survival of donor cells following transplantation and the potential for uncontrolled proliferation of precursor cells. To address these impediments, we have developed a method for the generation of highly enriched immature neurons from murine NSC progeny. Adaptation of the standard differentiation procedure in concert with flow cytometry selection, using scattered light and positive fluorescent light selection based on cell surface antibody binding, provided a near pure (97%) immature neuron population. Using the purified neurons, we screened a panel of growth factors and found that bone morphogenetic protein-4 (BMP-4) demonstrated a strong survival effect on the cells in vitro, and enhanced their functional maturity. This effect was maintained following transplantation into the adult mouse striatum where we observed a 2-fold increase in the survival of the implanted cells and a 3-fold increase in NeuN expression. Additionally, based on the neural-colony forming cell assay (N-CFCA), we noted a 64 fold reduction of the bona fide NSC frequency in neuronal cell population and that implanted donor cells showed no signs of excessive or uncontrolled proliferation. The ability to provide defined neural cell populations from renewable sources such as NSC may find application for cell replacement therapies in the central nervous system.

Direct Conversion of Human Fibroblasts into Neuronal Restricted Progenitors

“…The small amount of residual pluripotent stem cells among the induced cells can be a safety concern because of the risk of their tumorigenic tendency (29,30). NRPs could be sorted from NSC progeny as well (31), but the resource is limited and the procedures are cumbersome. We provide a simple method to obtain high-pu-FIGURE 3.…”

Equine Epidermis: A Source of Epithelial-Like Stem/Progenitor Cells with In Vitro and In Vivo Regenerative Capacities

“…Subsequently, these cells were designated as stem cells because of their bipotent properties [21,22]. To date, different stem cell types have been isolated, such as mammary stem/progenitor cells [23], corneal stem/progenitor cells [24], and neural stem cells [25]. The use of non-coated flasks was later employed to isolate spherical cell clusters derived from human hair follicles [16].…”

In Vitro Models for Neurogenesis

“…1A). As this was reminiscent of previously described SVZ in vivo neurogenesis, we further characterized this observation and developed an assay that not only would recapitulate the sequence of events from a neural stem cell to neuron but could also be used as a method to obtain large quantities of purified neurons for both in vitro study and as a donor source for cell-replacement therapies (Azari et al 2011b(Azari et al , 2012(Azari et al , 2014.…”

“…1C). Further differentiation of these immature A2B5 þ / nestin þ /bIII tubulin þ cells in situ using RA or using BMP4 after isolation from their glial bed led to complete maturation and neurite outgrowth and arborization (Scheffler et al 2005;Azari et al 2011b). On maturation, these neurons express GAD-65/67 and GABA demonstrating GABAergic interneuron phenotype.…”