Ing-Ming Chiu

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 adaptor protein SH2B3 (Lnk) negatively regulates neurite outgrowth of PC12 cells and cortical neurons.

SH2B adaptor protein family members (SH2B1-3) regulate various physiological responses through affecting signaling, gene expression, and cell adhesion. SH2B1 and SH2B2 were reported to enhance nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells, a well-established neuronal model system. In contrast, SH2B3 was reported to inhibit cell proliferation during the development of immune system. No study so far addresses the role of SH2B3 in the nervous system. In this study, we provide evidence suggesting that SH2B3 is expressed in the cortex of embryonic rat brain. Overexpression of SH2B3 not only inhibits NGF-induced differentiation of PC12 cells but also reduces neurite outgrowth of primary cortical neurons. SH2B3 does so by repressing NGF-induced activation of PLCγ, MEK-ERK1/2 and PI3K-AKT pathways and the expression of Egr-1. SH2B3 is capable of binding to phosphorylated NGF receptor, TrkA, as well as SH2B1β. Our data further demonstrate that overexpression of SH2B3 reduces the interaction between SH2B1β and TrkA. Consistent with this finding, overexpressing the SH2 domain of SH2B3 is sufficient to inhibit NGF-induced neurite outgrowth. Together, our data demonstrate that SH2B3, unlike the other two family members, inhibits neuronal differentiation of PC12 cells and primary cortical neurons. Its inhibitory mechanism is likely through the competition of TrkA binding with the positive-acting SH2B1 and SH2B2.

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.

SH2B1β interacts with STAT3 and enhances fibroblast growth factor 1-induced gene expression during neuronal differentiation

ABSTRACT Neurite outgrowth is an essential process during neuronal differentiation as well as neuroregeneration. Thus, understanding the molecular and cellular control of neurite outgrowth will benefit patients with neurological diseases. We have previously shown that overexpression of the signaling adaptor protein SH2B1β promotes fibroblast growth factor 1 (FGF1)-induced neurite outgrowth (W. F. Lin, C. J. Chen, Y. J. Chang, S. L. Chen, I. M. Chiu, and L. Chen, Cell. Signal. 21:1060–1072, 2009). SH2B1β also undergoes nucleocytoplasmic shuttling and regulates a subset of neurotrophin-induced genes. Although these findings suggest that SH2B1β regulates gene expression, the nuclear role of SH2B1β was not known. In this study, we show that SH2B1β interacts with the transcription factor, signal transducer, and activator of transcription 3 (STAT3) in neuronal PC12 cells, cortical neurons, and COS7 fibroblasts. By affecting the subcellular distribution of STAT3, SH2B1β increased serine phosphorylation and the concomitant transcriptional activity of STAT3. As a result, overexpressing SH2B1β enhanced FGF1-induced expression of STAT3 target genes Egr1 and Cdh2. Chromatin immunoprecipitation assays further reveal that, in response to FGF1, overexpression of SH2B1β promotes the in vivo occupancy of STAT3-Sp1 heterodimers at the promoter of Egr1 and Cdh2. These findings establish a central role of SH2B1β in orchestrating signaling events to transcriptional activation through interacting and regulating STAT3-containing complexes during neuronal differentiation.

Signaling Adaptor Protein SH2B1 Enhances Neurite Outgrowth and Accelerates the Maturation of Human Induced Neurons

Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of adult mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) and induced neurons (iNs) under defined conditions. However, human cells appear to be less plastic and have a higher epigenetic hurdle for reprogramming to both iPSCs and iNs. Here, we show that SH2B adaptor protein 1β (SH2B1) can enhance neurite outgrowth of iNs reprogrammed from human fibroblasts as early as day 14, when combined with miR124 and transcription factors BRN2 and MYT1L (IBM) under defined conditions. These SH2B1‐enhanced iNs (S‐IBM) showed canonical neuronal morphology, and expressed multiple neuronal markers, such as TuJ1, NeuN, and synapsin, and functional proteins for neurotransmitter release, such as GABA, vGluT2, and tyrosine hydroxylase. Importantly, SH2B1 accelerated mature process of functional neurons and exhibited action potentials as early as day 14; without SH2B1, the IBM iNs do not exhibit action potentials until day 21. Our data demonstrate that SH2B1 can enhance neurite outgrowth and accelerate the maturation of human iNs under defined conditions. This approach will facilitate the application of iNs in regenerative medicine and in vitro disease modeling.

The EP300, KDM5A, KDM6A and KDM6B Chromatin Regulators Cooperate with KLF4 in the Transcriptional Activation of POU5F1

POU5F1 is essential for maintaining pluripotency in embryonic stem cells (ESCs). It has been reported that the constitutive activation of POU5F1 is sustained by the core transcriptional regulatory circuitry in ESCs; however, the means by which POU5F1 is epigenetically regulated remains enigmatic. In this study a fluorescence-based reporter system was used to monitor the interplay of 5 reprogramming-associated TFs and 17 chromatin regulators in the transcription of POU5F1. We show the existence of a stoichiometric effect for SOX2, POU5F1, NANOG, MYC and KLF4, in regulating POU5F1 transcription. Chromatin regulators EP300, KDM5A, KDM6A and KDM6B cooperate with KLF4 in promoting the transcription of POU5F1. Moreover, inhibiting HDAC activities induced the expression of Pou5f1 in mouse neural stem cells (NSCs) in a spatial- and temporal- dependent manner. Quantitative chromatin immunoprecipitation-PCR (ChIP-qPCR) shows that treatment with valproic acid (VPA) increases the recruitment of Kdm5a and Kdm6a to proximal promoter (PP) and proximal enhancer (PE) of Pou5f1 whereas enrichment of Ep300 and Kdm6b was seen in PP but not PE of Pou5f1 promoter. These findings reveal the interplay between the chromatin regulators and histone modifications in the expression of POU5F1.

Nanocrystalline Diamond Films: Applications and Advances in Nanomedicine

Ying-Chieh Chen1,2, Don-Ching Lee1 and Ing-Ming Chiu1,3,4* 1Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, 2Center for Biomedical Engineering, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, 3Department of Life Sciences, National Chung Hsing University, Taichung 4Department of Internal Medicine, The Ohio State University, Columbus 1,3Taiwan 2,4USA

Enzyme-Free Dissociation of Neurospheres by a Microfluidic Chip-Based Method

Neurosphere assay is a common and robust method for identification of neural stem/progenitor cells, but obtaining large numbers of live single cells from dissociated neurospheres is difficult using nonenzymatic methods. Here, we present an enzyme-free method for high-efficiency neurosphere dissociation into single cells using microfluidic device technology. This method allows single cell dissociation of DC115 and KT98 cells with high cell viabilities (80-85u2009%), single-cell yield (91-95u2009%), and recovery (75-93u2009%).