Human IL12p80 Promotes Murine Oligodendrocyte Differentiation to Repair Nerve Injury.

Nerve injury of the central nervous system and the peripheral nervous system still poses a major challenge in modern clinics. Understanding the roles of neurotrophic factors and their molecular mechanisms on neuro-regeneration will not only benefit patients with neural damage but could potentially treat neurodegenerative disorders, such as amyotrophic lateral sclerosis. In this study, we showed that human IL12 p40-p40 homodimer (hIL12p80) within PLA and PLGA conduits improved sciatic nerve regeneration in mice. As such, the group of conduits with NSCs and hIL12p80 (CNI) showed the best recovery among the groups in the sciatic functional index (SFI), compound muscle action potential (CMAP), and Rotarod performance analyses. In addition, the CNI group had a faster recovery and outperformed the other groups in SFI and Rotarod performance tests beginning in the fourth week post-surgery. Immunohistochemistry showed that the CNI group increased the diameter of the newly regenerated nerve by two-fold (p < 0.01). In vitro studies showed that hIL12p80 stimulated differentiation of mouse NSCs to oligodendrocyte lineages through phosphorylation of Stat3 at Y705 and S727. Furthermore, implantation using PLGA conduits (C2.0 and C2.1) showed better recovery in the Rotarod test and CMAP than using PLA conduits in FVB mice. In B6 mice, the group with C2.1 + NSCs + hIL12p80 (C2.1NI) not only promoted sciatic functional recovery but also reduced the rate of experimental autotomy. These results suggested that hIL12p80, combined with NSCs, enhanced the functional recovery and accelerated the regeneration of damaged nerves in the sciatic nerve injury mice. Our findings could further shed light on IL12's application not only in damaged nerves but also in rectifying the oligodendrocytes' defects in neurodegenerative diseases, such as amyotrophic lateral sclerosis and multiple sclerosis.

Activation of Aurora A kinase through the FGF1/FGFR signaling axis sustains the stem cell characteristics of glioblastoma cells.

UNLABELLED: Fibroblast growth factor 1 (FGF1) binds and activates FGF receptors, thereby regulating cell proliferation and neurogenesis. Human FGF1 gene 1B promoter (-540 to +31)-driven SV40 T antigen has been shown to result in tumorigenesis in the brains of transgenic mice. FGF1B promoter (-540 to +31)-driven green fluorescent protein (F1BGFP) has also been used in isolating neural stem cells (NSCs) with self-renewal and multipotency from developing and adult mouse brains. In this study, we provide six lines of evidence to demonstrate that FGF1/FGFR signaling is implicated in the expression of Aurora A (AurA) and the activation of its kinase domain (Thr288 phosphorylation) in the maintenance of glioblastoma (GBM) cells and NSCs. First, treatment of FGF1 increases AurA expression in human GBM cell lines. Second, using fluorescence-activated cell sorting, we observed that F1BGFP reporter facilitates the isolation of F1BGFP(+) GBM cells with higher expression levels of FGFR and AurA. Third, both FGFR inhibitor (SU5402) and AurA inhibitor (VX680) could down-regulate F1BGFP-dependent AurA activity. Fourth, inhibition of AurA activity by two different AurA inhibitors (VX680 and valproic acid) not only reduced neurosphere formation but also induced neuronal differentiation of F1BGFP(+) GBM cells. Fifth, flow cytometric analyses demonstrated that F1BGFP(+) GBM cells possessed different NSC cell surface markers. Finally, inhibition of AurA by VX680 reduced the neurosphere formation of different types of NSCs. Our results show that activation of AurA kinase through FGF1/FGFR signaling axis sustains the stem cell characteristics of GBM cells. IMPLICATIONS: This study identified a novel mechanism for the malignancy of GBM, which could be a potential therapeutic target for GBM.

The mood stabilizer valproate activates human FGF1 gene promoter through inhibiting HDAC and GSK-3 activities.

Valproic acid (VPA) is the primary mood-stabilizing drug to exert neuroprotective effects and to treat bipolar disorder in clinic. Fibroblast growth factor 1 (FGF1) has been shown to regulate cell proliferation, cell division, and neurogenesis. Human FGF1 gene 1B promoter (-540 to +31)-driven green fluorescence (F1BGFP) has been shown to recapitulate endogenous FGF1 gene expression and facilitates the isolation of neural stem/progenitor cells (NSPCs) from developing and adult mouse brains. In this study, we provide several lines of evidence to demonstrate the underlying mechanisms of VPA in activating FGF-1B promoter activity: (i) VPA significantly increased the FGF-1B mRNA expression and the percentage of F1BGFP(+) cells; (ii) the increase of F1BGFP expression by VPA involves changes of regulatory factor X (RFX) 1-3 transcriptional complexes and the increase of histone H3 acetylation on the 18-bp cis-element of FGF-1B promoter; (iii) treatments of other histone deacetylases (HDAC) inhibitors, sodium butyrate and trichostatin A, significantly increased the expression levels of FGF-1B, RFX2, and RFX3 transcripts; (iv) treatments of glycogen synthase kinase 3 (GSK-3) inhibitor, lithium, or GSK-3 siRNAs also significantly activated FGF-1B promoter; (v) VPA specifically enhanced neuronal differentiation in F1BGFP(+) embryonic stem cells and NSPCs rather than GFP(-) cells. This study suggested, for the first time, that VPA activates human FGF1 gene promoter through inhibiting HDAC and GSK-3 activities.

Ciliogenic RFX transcription factors regulate FGF1 gene promoter.

Fibroblast growth factor 1 (FGF1) has been shown to regulate cell proliferation, cell division, and neurogenesis. Human FGF1 gene 1B promoter (-540 to +31)-driven green fluorescence (F1BGFP) was shown to recapitulate endogenous FGF1 gene expression. It can also be used to isolate neural stem/progenitor cells (NSPCs) and glioblastoma stem cells (GBM-SCs) from developing mouse brains and human glioblastoma tissues, respectively. However, the regulatory mechanisms of FGF-1B promoter and F1BGFP(+) cells are not clear. In this study, we present several lines of evidence to show the roles of ciliogenic RFX transcription factors in the regulation of FGF-1B gene promoter and F1BGFP(+) cells: (i) RFX1, RFX2, and RFX3 transcription factors could directly bind the 18-bp cis-element (-484 to -467), and contribute to the regulation of FGF1 promoter and neurosphere formation. (ii) We demonstrated RFX2/RFX3 complex could only be detected in the nuclear extract of FGF-1B positive cells, but not in FGF-1B negative cells. (iii) Protein kinase C inhibitors, staurosporine and rottlerin, could decrease the percentage of F1BGFP(+) cells and their neurosphere formation efficiency through reducing the RFX2/3 complex. (iv) RNA interference knockdown of RFX2 could significantly reduce the percentage of F1BGFP(+) cells and their neurosphere formation efficiency whereas overexpression of RFX2 resulted in the opposite effects. Taken together, this study suggests ciliogenic RFX transcription factors regulate FGF-1B promoter activity and the maintenance of F1BGFP(+) NSPCs and GBM-SCs.

Establishing F1A-CreERT2 Mice to Trace Fgf1 Expression in Adult Mouse Cardiomyocytes.

Fibroblast growth factor 1 (FGF1) regulates many biological and physiological processes. In mice, Fgf1 gene contains at least three upstream promoters and are alternatively spliced to the first protein coding exon, giving rise to different Fgf1 mRNA variants (1A, 1B and 1G). Among them, the Fgf1A transcript is predominantly expressed in the heart. FGF1 can induce cardiomyocyte regeneration and cardiogenesis in vitro and in vivo. Here, we generated a novel mouse line using the Fgf1A promoter (F1A) driving the expression of the inducible Cre recombinase (CreERT2). We firstly demonstrated that the highest mRNA expression of CreERT2 were detected in the heart specifically of F1A-CreERT2 mice, similar to that of Fgf1A mRNA. The F1A-CreERT2 mice were crossed with ROSA26 mice, and the F1 mice were analyzed. The LacZ-positive signals were detected exclusively in the heart after tamoxifen administration. The CreERT2-mediated recombination in the tissues is monitored through LacZ-positive signals, indicating the in situ localization of F1A-positive cells. Consistently, these F1A-positive cells with RFP-positive signals or LacZ-positive blue signals were co-localized with cardiomyocytes expressing cardiac troponin T, suggesting cardiomyocyte-specific activation of Fgf1A promoter. Our data suggested that the F1A-CreERT2 mouse line could be used for time-dependent and lineage tracing of Fgf1A-expressing cells in vivo.

Isolation of neural stem/progenitor cells by using EGF/FGF1 and FGF1B promoter-driven green fluorescence from embryonic and adult mouse brains.

Fibroblast growth factor 1 (FGF1) and FGF2 have been shown to maintain the proliferation, self-renewal and multipotent capacities of neural stem/progenitor cells (NSPCs) in vitro. FGF1 is unique for binding to all known FGF receptors. In this study, we investigated if exogenous EGF and FGF1 could be used in the isolation of NSPCs from embryonic mouse brains. We demonstrated that EGF/FGF1-responsive cells exhibited lower proliferation rate and neurosphere formation efficiency than EGF/FGF2-responsive NSPCs. However, EGF/FGF1-responsive mouse brain cells exhibited better neural differentiation capacities than EGF/FGF2-responsive NSPCs at E11.5. Using F1BGFP reporter, we further demonstrated that F1BGFP+ cells showed similar multipotent capacities to CD133+ NSPCs, and could be induced more efficiently toward neuronal differentiation. Our results suggested that EGF/FGF1-responsive cells from E11.5 mouse brains could self-renew and have better multipotency than EGF/FGF2-responsive NSPCs. Further, CD133+ and F1BGFP+ NSPCs may also represent different subsets of NSPCs during neural development and adult neurogenesis.

Human FGF1 promoter is active in ependymal cells and dopaminergic neurons in the brains of F1B-GFP transgenic mice.

FGF1 is involved in multiple biological functions and exhibits the importance in neuroprotective effects. Our previous studies indicated that, in human brain and retina, the FGF1B promoter controlled the expression of FGF1. However, the exact function and regulation of FGF1 in brain is still unclear. Here, we generated F1B-GFP transgenic mice that expressed the GFP reporter gene under the control of human FGF1B promoter (-540 to +31). Using the fresh brain sections of F1B-GFP transgenic mice, we found that the F1B-GFP cells expressed strong fluorescent signals in the ventricular system throughout the brain. The results of immunohistochemistry further showed that two distinct populations of F1B-GFP(+) cells existed in the brains of F1B-GFP transgenic mice. We demonstrated that one population of F1B-GFP(+) cells was ependymal cells, which distributed along the entire ventricles, and the second population of F1B-GFP(+) cells was neuronal cells that projected their long processes into multiple directions in specific areas of the brain. The double labeling of F1B-GFP(+) cells and tyrosine hydroxylase indicated that a subpopulation of F1B-GFP(+) -neuronal cells was dopaminergic neurons. Importantly, these F1B-GFP(+) /TH(+) cells were distributed in the main dopaminergic neuronal groups including hypothalamus, ventral tegmental area, and raphe nuclei. These results suggested that human FGF1B promoter was active in ependymal cells, neurons, and a portion of dopaminergic neurons. Thus, the F1B-GFP transgenic mice provide an animal model not only for studying FGF1 gene expression in vivo but also for understanding the role of FGF1 contribution in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease.

The effect of ultra-nanocrystalline diamond films on the proliferation and differentiation of neural stem cells.

The interaction of ultra-nanocrystalline diamond (UNCD) with neural stem cells (NSCs) has been studied along with its surface modification in order to improve its function as a biomaterial. Hydrogen- and oxygen-terminated UNCD films were compared with standard grade polystyrene in terms of their impact on the growth, expansion and differentiation of NSCs. When NSCs were cultured on these substrates in low serum and without any differentiating factors, hydrogen-terminated UNCD films spontaneously induced cell proliferation and neuronal differentiation. Oxygen-terminated UNCD films were also shown to further improve neural differentiation, with a preference to differentiate into oligodendrocytes. Hence, controlling the surface properties of UNCD could manipulate the differentiation of NSCs for different biomedical applications. These observations raise the potential for the use of UNCD as a biomaterial for central nervous system transplantation and tissue engineering.

Inducing long-term survival with lasting anti-tumor immunity in treating B cell lymphoma by a combined dendritic cell-based and hydrodynamic plasmid-encoding IL-12 gene therapy.

In a previous study we showed that immunization with dendritic cells (DC) pulsed with idiotype (Id) fused with CD40 ligand (CD40L) could break the tolerance to Id which is expressed on B lymphoma cells and restored the responsiveness of T(h) cells, and, subsequently, induced IgG antibody response. However, this treatment had no therapeutic effect. In the present study, we found that using a hydrodynamic transfection-based technique, a high level of IL-12 production was noticed as early as 7 h after administering plasmid encoding IL-12 (pIL-12) and persisted at a detectable level for at least 9 days. In evaluating the efficacy of DC-based and/or IL-12 gene-based therapy in the treatment of 38C13 B cell lymphoma, it was found that either treatment alone was ineffective. However, a combined treatment induced 100% long-term survival. Furthermore, a long-lasting anti-tumor immunity was induced in these mice which resisted further tumor challenge at 58 days after initial inoculation. The surviving mice showed a strong IFN-gamma-producing T(h) cell response and humoral antibody response, but there were no detectable cytotoxic T lymphocytes. The antibody from the immune sera mediated a complement-dependent lysis of tumor cells that was tumor specific. Furthermore, immunization of mice with DC-based vaccine and pIL-12 treatment elicited higher levels of anti-Id IgG titer and an enhanced IgG2a response which increased the efficacy in mediating 38C13 tumor lysis. On examining the mechanism for this isotype change, we found that IFN-gamma production by CD4(+) T cells is not the only determining factor for achieving a successful therapy. DC-based treatment alone could induce the increase of IFN-gamma production, but lacked any therapeutic effect. The deciding factor appears to be the abrogation of IL-4 production that was achieved by combing with IL-12 gene therapy. Our study provides a basis for exploring the combined use of cytokines or cytokine genes in DC-based treatment for achieving effective cancer immunotherapy.

In vitro hepatic differentiation of human mesenchymal stem cells.

This study examined whether mesenchymal stem cells (MSCs), which are stem cells originated from embryonic mesoderm, are able to differentiate into functional hepatocyte-like cells in vitro. MSCs were isolated from human bone marrow and umbilical cord blood, and the surface phenotype and the mesodermal multilineage differentiation potentials of these cells were characterized and tested. To effectively induce hepatic differentiation, we designed a novel 2-step protocol with the use of hepatocyte growth factor and oncostatin M. After 4 weeks of induction, cuboidal morphology, which is characteristic of hepatocytes, was observed, and cells also expressed marker genes specific of liver cells in a time-dependent manner. Differentiated cells further demonstrated in vitro functions characteristic of liver cells, including albumin production, glycogen storage, urea secretion, uptake of low-density lipoprotein, and phenobarbital-inducible cytochrome P450 activity. In conclusion, human MSCs from different sources are able to differentiate into functional hepatocyte-like cells and, hence, may serve as a cell source for tissue engineering and cell therapy of hepatic tissues. Furthermore, the broad differentiation potential of MSCs indicates that a revision of the definition may be required.