Don-Ching Lee

Neural Stem Cells, Neural Progenitors, and Neurotrophic Factors

Neural stem cells (NSCs) have been proposed as a promising cellular source for the treatment of diseases in nervous systems. NSCs can self-renew and generate major cell types of the mammalian central nervous system throughout adulthood. NSCs exist not only in the embryo, but also in the adult brain neurogenic region: the subventricular zone (SVZ) of the lateral ventricle. Embryonic stem (ES) cells acquire NSC identity with a default mechanism. Under the regulations of leukemia inhibitory factor (LIF) and fibroblast growth factors, the NSCs then become neural progenitors. Neurotrophic and differentiation factors that regulate gene expression for controlling neural cell fate and function determine the differentiation of neural progenitors in the developing mammalian brain. For clinical application of NSCs in neurodegenerative disorders and damaged neurons, there are several critical problems that remain to be resolved: 1) how to obtain enough NSCs from reliable sources for autologous transplantation; 2) how to regulate neural plasticity of different adult stem cells; 3) how to control differentiation of NSCs in the adult nervous system. In order to understand the mechanisms that control NSC differentiation and behavior, we review the ontogeny of NSCs and other stem cell plasticity of neuronal differentiation. The role of NSCs and their regulation by neurotrophic factors in CNS development are also reviewed.

Induction and regulation of differentiation in neural stem cells on ultra-nanocrystalline diamond films

The interaction of ultra-nanocrystalline diamond (UNCD) with neural stem cells (NSCs) has been studied in order to evaluate its potential as a biomaterial. Hydrogen-terminated UNCD (H-UNCD) films were compared with standard grade polystyrene in terms of their impact on the differentiation of NSCs. When NSCs were cultured on these substrates in medium supplemented with low concentration of serum and without any differentiating factors, H-UNCD films spontaneously induced neuronal differentiation on NSCs. By direct suppression of mitogen-activated protein kinase/extracellular signaling-regulated kinase1/2 (MAPK/Erk1/2) signaling pathway in NSCs using U0126, known to inhibit the activation of Erk1/2, we demonstrated that the enhancement of Erk1/2 pathway is one of the effects of H-UNCD-induced NSCs differentiation. Moreover, functional-blocking antibody directed against integrin beta1 subunit inhibited neuronal differentiation on H-UNCD films. This result demonstrated the involvement of integrin beta1 in H-UNCD-mediated neuronal differentiation. Mechanistic studies revealed the cell adhesion to H-UNCD films associated with focal adhesion kinase (Fak) and initiated MAPK/Erk1/2 signaling. Our study demonstrated that H-UNCD films-mediated NSCs differentiation involves fibronectin-integrin beta1 and Fak-MAPK/Erk signaling pathways in the absence of differentiation factors. These observations raise the potential for the use of UNCD as a biomaterial for central nervous system transplantation and tissue engineering.

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.

Hypoxia‐inducible factor‐1α regulates matrix metalloproteinase‐1 activity in human bone marrow‐derived mesenchymal stem cells

We examined the mRNA levels of hypoxia‐inducible factor‐1α (HIF‐1α) in bone marrow mesenchymal stem cells (bmMSCs) of eight osteoarthritis patients. BmMSC‐1, expressing higher HIF‐1α mRNA and protein than bmMSC‐5, elicited higher matrix metalloproteinase‐1 (MMP1) activity and stronger invasive capacity. In vitro invasion assays and quantitative PCR analyses showed that targeted inhibition of HIF‐1α in bmMSC‐1 decreased its invasion and expressions of MMP1 and MMP3, whereas overexpression of HIF‐1α in bmMSC‐5 increased its invasion and expressions of MMP1 and MMP3. Therefore, HIF‐1α can regulate MMP1 and MMP3 expressions in human bmMSCs, which might suggest a pathophysiological role of bmMSC expressing high HIF‐1α in bone diseases.

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.

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.

Single-cell enzyme-free dissociation of neurospheres using a microfluidic chip.

Obtaining single dissociated cells from neurospheres is difficult using nonenzymatic methods. In this paper we report the development of a microfluidic-chip-based approach that utilizes flow and microstructures to dissociate neurospheres. We show that this microfluidic-chip-based neurosphere-dissociation method can generate high yields of single cells from dissociated neurospheres of mouse KT98 and DC115 cell models (passage number, 3-8; diameter range, 40-250 μm): 90% and 95%, respectively. The microfluidic-chip-dissociated cells had high viabilities (80-85%) and the ability to regrow into neurospheres, demonstrating the applicability of this device to neurosphere assay applications. In addition, the dissociated cells retained their normal differentiation potentials, as shown by their capabilities to differentiate into three neural lineages (neurons, astroglia, and oligodendrocytes) when cultured in differentiation culture conditions. Since this microfluidic-chip-based method does not require the use of enzymatic reagents, the risk of contamination from exogenous substances could be reduced, making it an attractive tool for a wide range of applications where neurosphere dissociation is needed.

Neural stem cells promote nerve regeneration through IL12-induced Schwann cell differentiation

&NA; Regeneration of injured peripheral nerves is a slow, complicated process that could be improved by implantation of neural stem cells (NSCs) or nerve conduit. Implantation of NSCs along with conduits promotes the regeneration of damaged nerve, likely because (i) conduit supports and guides axonal growth from one nerve stump to the other, while preventing fibrous tissue ingrowth and retaining neurotrophic factors; and (ii) implanted NSCs differentiate into Schwann cells and maintain a growth factor enriched microenvironment, which promotes nerve regeneration. In this study, we identified IL12p80 (homodimer of IL12p40) in the cell extracts of implanted nerve conduit combined with NSCs by using protein antibody array and Western blotting. Levels of IL12p80 in these conduits are 1.6‐fold higher than those in conduits without NSCs. In the sciatic nerve injury mouse model, implantation of NSCs combined with nerve conduit and IL12p80 improves motor recovery and increases the diameter up to 4.5‐fold, at the medial site of the regenerated nerve. In vitro study further revealed that IL12p80 stimulates the Schwann cell differentiation of mouse NSCs through the phosphorylation of signal transducer and activator of transcription 3 (Stat3). These results suggest that IL12p80 can trigger Schwann cell differentiation of mouse NSCs through Stat3 phosphorylation and enhance the functional recovery and the diameter of regenerated nerves in a mouse sciatic nerve injury model. HighlightsIL12p80 was detected in sciatic nerve regeneration mediated by neural stem cells.Administration of IL12p80 facilitates the repair of sciatic nerve injury.IL12p80 promotes Schwann cell differentiation through Stat3 phosphorylation.

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 Parkinsons disease and Alzheimers disease.

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.