No abstract

Efficient 3D cell systems for neuronal induction are needed for future use in tissue regeneration. In this study, we have characterized the ability of neural stem/progenitor cells (NS/PC) to survive, proliferate, and differentiate in a collagen type I-hyaluronan scaffold. Embryonic, postnatal, and adult NS/PC were seeded in the present 3D scaffold and cultured in medium containing epidermal growth factor and fibroblast growth factor-2, a condition that stimulates NS/PC proliferation. Progenitor cells from the embryonic brain had the highest proliferation rate, and adult cells the lowest, indicating a difference in mitogenic responsiveness. NS/PC from postnatal stages down-regulated nestin expression more rapidly than both embryonic and adult NS/PC, indicating a faster differentiation process. After 6 days of differentiation in the 3D scaffold, NS/PC from the postnatal brain had generated up to 70% neurons, compared with 14% in 2D. NS/PC from other ages gave rise to approximately the same proportion of neurons in 3D as in 2D (9-26% depending on the source for NS/PC). In the postnatal NS/PC cultures, the majority of betaIII-tubulin-positive cells expressed glutamate, gamma-aminobutyric acid, and synapsin I after 11 days of differentiation, indicating differentiation to mature neurons. Here we report that postnatal NS/PC survive, proliferate, and efficiently form synapsin I-positive neurons in a biocompatible hydrogel.

Abuse of androgenic anabolic steroids can affect brain function leading to behavioural changes. In this study, the effects of the testosterone analogue, 19-nortestosterone, on rat neural stem cells was examined. The androgen receptor is expressed by cultured embryonic and adult neural stem cells, and is also present in the ventricular epithelium during development and in the adult brain in, among others, dentate gyrus. In neural stem cells stimulated with epidermal growth factor, nandrolone reduced cell proliferation, especially in adult ones. The decrease was abolished by flutamide, a receptor antagonist. Nandrolone also decreased the BrdU labelling of neural stem cells in the dentate gyrus, demonstrating an effect of the hormone on cell proliferation in vivo. The effect of nandrolone was observed with both female and male rats but it was more pronounced in pregnant rats, indicating an involvement of oestrogen in nandrolone action. Nandrolone also decreased the number of newly born neuronal cells in the dentate gyrus of male rats. The results show that nandrolone has important effects on the proliferation and differentiation of neural stem cells expressing the cognate androgen receptor. The data show that the use of nandrolone may severely affect the formation of neural stem cells and could therefore have long-term negative consequences in the brain.

In this study we describe the development of an injectable, in situ chemical hydrogel forming system. The gelation occurs under neutral pH and at room temperature immediately upon mixing of the two aqueous poly (vinyl alcohol) components specifically derivatized through carbamate linkages with aldehyde (PVA-AL) and hydrazide (PVA-HY) functional groups, respectively. Aldehyde and hydrazide pendant groups were incorporated with a low degree of substitution (DS) into the PVA backbone to keep PVA structural homogeneity minimally altered. As a result, the hydrazone crosslinks are formed rapidly between aldehyde and hydrazide pendant groups when the correspondingly modified PVA components are brought in contact as water solutions. To assess in situ hydrazone crosslinks formation for in vitro cytocompatibility, murine neuroblastoma N2a cells were suspended in cell culture medium with the dissolved PVA-HY prior to addition to the PVA-AL aqueous solution. Thus, the cells were chemically encapsulated in a polymer network that was formed by mixing of the corresponding aqueous solutions of PVA functional precursors. Biochemical analysis revealed that cells survived chemical crosslinking and remained viable in the hydrogel for 4 days of culture.

Growth factors play an important role in regulating neural stem cell proliferation and differentiation. This study shows that platelet-derived growth factor (PDGF) induces a partial differentiation of neural stem/progenitor cells (NSPCs) in the absence of other mitogens in vitro. NSPCs thus acquire an immature morphology and display markers for both neurons and glia. In addition, these cells do not readily mature in the absence of further stimuli. When NSPC cultures treated with PDGF were exposed to additional differentiation factors, however, the differentiation proceeded into neurons, astrocytes, and oligodendrocytes. We find that NSPC cultures are endowed with an endogenous PDGF-BB production. The PDGF-BB expression peaks during early differentiation and is present both in cell lysates and in conditioned medium, allowing for autocrine as well as paracrine signaling. When the NSPC-derived PDGF was inhibited, progenitor cell numbers decreased, showing that PDGF is involved in NSPC expansion. Addition of a PDGF receptor (PDGFR) inhibitor resulted in a more rapid differentiation. Neurons and oligodendrocytes appeared earlier and had more elaborate processes than in control cultures where endogenous PDGFR signaling was not blocked. Our observations point to PDGF as an inducer of partial differentiation of NSPC that also sustains progenitor cell division. Such an intermediate stage in stem cell differentiation is of relevance for the understanding of brain tumor development because autocrine PDGF stimulation is believed to drive malignant conversion of central nervous system progenitor cells. (Cancer Res 2006; 66(16): 8042-8)