Sujeong Jang

Functional neural differentiation of human adipose tissue-derived stem cells using bFGF and forskolin.

BackgroundAdult mesenchymal stem cells (MSCs) derived from adipose tissue have the capacity to differentiate into mesenchymal as well as endodermal and ectodermal cell lineage in vitro. We characterized the multipotent ability of human adipose tissue-derived stem cells (hADSCs) as MSCs and investigated the neural differentiation potential of these cells.ResultsHuman ADSCs from earlobe fat maintained self-renewing capacity and differentiated into adipocytes, osteoblasts, or chondrocytes under specific culture conditions. Following neural induction with bFGF and forskolin, hADSCs were differentiated into various types of neural cells including neurons and glia in vitro. In neural differentiated-hADSCs (NI-hADSCs), the immunoreactivities for neural stem cell marker (nestin), neuronal markers (Tuj1, MAP2, NFL, NFM, NFH, NSE, and NeuN), astrocyte marker (GFAP), and oligodendrocyte marker (CNPase) were significantly increased than in the primary hADSCs. RT-PCR analysis demonstrated that the mRNA levels encoding for ABCG2, nestin, Tuj1, MAP2, NFL, NFM, NSE, GAP43, SNAP25, GFAP, and CNPase were also highly increased in NI-hADSCs. Moreover, NI-hADSCs acquired neuron-like functions characterized by the display of voltage-dependent tetrodotoxin (TTX)-sensitive sodium currents, outward potassium currents, and prominent negative resting membrane potentials under whole-cell patch clamp recordings. Further examination by RT-PCR showed that NI-hADSCs expressed high level of ionic channel genes for sodium (SCN5A), potassium (MaxiK, Kv4.2, and EAG2), and calcium channels (CACNA1C and CACNA1G), which were expressed constitutively in the primary hADSCs. In addition, we demonstrated that Kv4.3 and Eag1, potassium channel genes, and NE-Na, a TTX-sensitive sodium channel gene, were highly induced following neural differentiation.ConclusionsThese combined results indicate that hADSCs have the same self-renewing capacity and multipotency as stem cells, and can be differentiated into functional neurons using bFGF and forskolin.

Effect of neural-induced mesenchymal stem cells and platelet-rich plasma on facial nerve regeneration in an acute nerve injury model.

The purpose of this study was to investigate the effects of platelet‐rich plasma (PRP) and neural‐induced human mesenchymal stem cells (nMSCs) on axonal regeneration from a facial nerve axotomy injury in a guinea pig model.

Neuroprotective effects of (-)-epigallocatechin-3-gallate against quinolinic acid-induced excitotoxicity via PI3K pathway and NO inhibition.

Excessive stimulation of the NMDA receptor induces neuronal cell death and is implicated in the development of several neurodegenerative diseases. While EGCG suppresses apoptosis induced by NMDA receptor-mediated excitotoxicity, the mechanisms underlying this process have yet to be completely determined. This study was designed to investigate whether (-)-epigallocatechin-3-gallate (EGCG) plays a neuroprotective role by inhibiting nitric oxide (NO) production and activating cellular signaling mechanisms including MAP kinase, PI3K, and GSK-3beta and acting on the antiapoptotic and the proapoptotic genes in N18D3 neural cells. The cells were pretreated with EGCG for 2 h and then exposed to quinolinic acid (QUIN), a NMDA receptor agonist, 30 mM for 24 h. MTT assay and DAPI staining were used to identify cell viability and apoptosis, respectively, and demonstrated that EGCG significantly increased cell viability and protected the cells from apoptotic death. In addition, EGCG had a capacity to reduce QUIN-induced excitotoxic cell death not only by blocking increase of intracellular calcium levels but also by inhibiting NO production. Gene expression analysis revealed that EGCG prevented the QUIN-induced expression of the proapoptotic gene, caspase-9, and increased that of the antiapoptotic genes, Bcl-XL, Bcl-2, and Bcl-w. Further examination about potential cell signaling candidate involved in this neuroprotective effect showed that immunoreacitivity of PI3K was significantly increased in the cells treated with EGCG. These results suggest that the neuroprotective mechanism of EGCG against QUIN-induced excitotoxic cell death includes regulation of PI3K and modulation of cell survival and death genes through decreasing of intracellular calcium levels and controlling of NO production.

Tracking of Neural Stem Cells in Rats with Intracerebral Hemorrhage by the Use of 3T MRI

Objective To access the feasibility of clinically available 3T MRI to detect the migration of labeled neural stem cells (NSCs) in intracerebral hemorrhage (ICH) in a rat model. Materials and Methods The ethics committee of our institution approved this study. ICH was induced by the injection of collagenase type IV into the right striatum of ten Sprague-Dawley rats. Human NSCs conjugated with Feridex (super-paramagnetic iron oxide: SPIO) were transplanted into the left striatum one week after ICH induction. MRI was performed on a 3T scanner during the first, second, third, fourth, and sixth weeks post-transplantation. MRI was obtained using coronal T2- and T2*-weighted sequences. Two rats were sacrificed every week after in vivo MRI in order to analyze the histological findings. Results ICH in the right striatum was detected by MRI one and two weeks after transplantation without migration of the NSCs. There was no migration of the NSCs as seen on the histological findings one week after transplantation. The histological findings two weeks after transplantation showed a small number of NSCs along the corpus callosum. On MRI three weeks after transplantation, there was a hypointense line along the corpus callosum and decreased signal intensity in the right periventricular region. Histological findings three weeks after transplantation confirmed the presence of the hypointense line representing SPIO-labeled NSCs. MRI four and six weeks after transplantation showed a hypointense spot in the right periventricular region. The histological findings four and six weeks after transplantation showed the presence of prominent NSCs in the right periventricular region. Conclusion 3T MRI can detect the migration of NSCs in rats with ICH along the corpus callosum. Therefore, 3T MRI could be feasible for detecting the migration of NSCs in the clinical setting of stem cell therapy.

Transplantation of Neural Differentiated Human Mesenchymal Stem Cells into the Cochlea of an Auditory-neuropathy Guinea Pig Model

The aim of this study was to determine the effects of transplanted neural differentiated human mesenchymal stem cells (hMSCs) in a guinea pig model of auditory neuropathy. In this study, hMSCs were pretreated with a neural-induction protocol and transplanted into the scala tympani of the guinea pig cochlea 7 days after ouabain injury. A control model was made by injection of Hanks balanced salt solution alone into the scala tympani of the guinea pig cochlea 7 days after ouabain injury. We established the auditory neuropathy guinea pig model using 1 mM ouabain application to the round window niche. After application of ouabain to the round window niche, degeneration of most spiral ganglion neurons (SGNs) without the loss of hair cells within the organ of Corti and increasing the auditory brain responses (ABR) threshold were found. After transplantation of neural differentiated hMSCs, the number of SGNs was increased, and some of the SGNs expressed immunoreactivity with human nuclear antibody under confocal laser scanning microscopy. ABR results showed mild hearing recovery after transplantation. Based on an auditory neuropathy animal model, these findings suggest that it may be possible to replace degenerated SGNs by grafting stem cells into the scala tympani.

Spontaneous Electrical Activity of Cultured Interstitial Cells of Cajal from Mouse Urinary Bladder

Interstitial cells of Cajal (ICCs) from the urinary bladder regulate detrusor smooth muscle activities. We cultured ICCs from the urinary bladder of mice and performed patch clamp and intracellular Ca2+ ([Ca2+]i) imaging to investigate whether cultured ICCs can be a valuable tool for cellular functional studies. The cultured ICCs displayed two types of spontaneous electrical activities which are similar to those recorded in intact bladder tissues. Spontaneous electrical activities of cultured ICCs were nifedipine-sensitive. Carbachol and ATP, both excitatory neurotransmitters in the urinary bladder, depolarized the membrane and increased the frequency of spike potentials. Carbachol increased [Ca2+]i oscillations and basal Ca2+ levels, which were blocked by atropine. These results suggest that cultured ICCs from the urinary bladder retain rhythmic phenotypes similar to the spontaneous electrical activities recorded from the intact urinary bladder. Therefore, we suggest that cultured ICCs from the urinary bladder may be useful for cellular and molecular studies of ICCs.

Neural Differentiation of Human Adipose Tissue-Derived Stem Cells Involves Activation of the Wnt5a/JNK Signalling

Stem cells are a powerful resource for cell-based transplantation therapies, but understanding of stem cell differentiation at the molecular level is not clear yet. We hypothesized that the Wnt pathway controls stem cell maintenance and neural differentiation. We have characterized the transcriptional expression of Wnt during the neural differentiation of hADSCs. After neural induction, the expressions of Wnt2, Wnt4, and Wnt11 were decreased, but the expression of Wnt5a was increased compared with primary hADSCs in RT-PCR analysis. In addition, the expression levels of most Fzds and LRP5/6 ligand were decreased, but not Fzd3 and Fzd5. Furthermore, Dvl1 and RYK expression levels were downregulated in NI-hADSCs. There were no changes in the expression of ß-catenin and GSK3ß. Interestingly, Wnt5a expression was highly increased in NI-hADSCs by real time RT-PCR analysis and western blot. Wnt5a level was upregulated after neural differentiation and Wnt3, Dvl2, and Naked1 levels were downregulated. Finally, we found that the JNK expression was increased after neural induction and ERK level was decreased. Thus, this study shows for the first time how a single Wnt5a ligand can activate the neural differentiation pathway through the activation of Wnt5a/JNK pathway by binding Fzd3 and Fzd5 and directing Axin/GSK-3ß in hADSCs.

The Expression of Toll-Like Receptors (TLRs) in Cultured Human Skin Fibroblast is Modulated by Histamine

Fibroblasts are responsible for the synthesis and degradation of various connective tissue components and soluble mediators of extracellular matrix metabolism. Few studies have been conducted concerning the expression of toll-like receptors (TLRs) in fibroblasts until now. This study aimed first to determine the quantitative expression of TLRs 1 to 10 in human skin fibroblasts and secondarily to explore any influence of expression by histamine, which is a well-known factor engaged in dermal inflammation. It was found that all 10 TLRs were expressed in fibroblasts. Interestingly, the expression of TLRs 4, 5, and 10 was increased after 2 and 6 hours of histamine treatment during culture. However, the expression of TLRs 2, 3, 6, 7, 8, and 9 was decreased after 6 hours of histamine treatment. Among the TLRs with a decreasing expression pattern, TLRs 7 and 8 showed a persistent tendency to decrease. All of these changes in TLR expression with histamine treatment were antagonized by treatment with diphenhydramine, a well-known antihistamine. Thus, these results suggest a role of histamine in the early phase of the dermal inflammatory reaction mediated by TLRs.

Effects of Sphingosine-1-Phosphate on Neural Differentiation and Neurite Outgrowth in Neuroblastoma Cells

Sphingosine-1-phosphate (S1P) is emerging as a new class of second messenger involved in cellular proliferation, differentiation, and apoptosis and is implicated in diverse physiological functions. Despite many studies on the biological functions of S1P, however, little is known about its role in neuronal differentiation. By use of reverse transcription-polymerase chain reaction and immunostaining, this study aimed to explore whether S1P can differentiate neuroblastoma cells into neural cells. After incubation with 1 uM or 10 uM S1P, the number of neurite-bearing cells increased. Furthermore, the neuroblastoma cells revealed immunoreactivity for neural-specific markers such as GAP43, NFH, and SYP by immunostaining. The expression of NFH, MAP2, SYP, NeuroD1, and SYT mRNA, which is specific for neurons, was increased as shown by RT-PCR studies. The results of this study suggest that that S1P can induce neuronal differentiation and may be a good candidate for the treatment of neurodegenerative diseases.

Effects of (-)-epigallocatechin-3-gallate on the activity of substantia nigra dopaminergic neurons.

Despite many studies on the biological and pharmacological properties of (-)-epigallocatechin-3-gallate (EGCG), an active component of green tea, information on neuronal modulation by EGCG is limited. This study was designed to investigate the effects of EGCG on the electrical activity of rat substantia nigra dopaminergic neurons using whole-cell patch clamp recordings. The spike frequency was increased to 6.33+/-0.23 (p<0.05) and 7.15+/-0.29 (p<0.05) by 5 and 10 microM EGCG, respectively, from the control level of 5.49+/-0.19 spikes/second, respectively (n=18). The resting membrane potential of the cells was decreased to -45.66+/-0.45 and -43.99+/-0.87 (p<0.05), by 5 and 10 microM EGCG, respectively, from -47.82+/-0.57 mV. The amplitude of afterhyperpolarization was decreased to 12.73+/-0.45 (p<0.05) and 11.60+/-0.57 (p<0.05) by 5 and 10 microM EGCG, respectively, from 13.80+/-0.31 mV. The neuronal activity of dopaminergic neurons is closely linked to dopamine release. When neurons switch from a single-spike firing to bursts of action potentials, the release of dopamine increases. The above experimental results suggest that EGCG increases the neuronal activity via inhibition of calcium-dependent potassium currents underlying the afterhyperpolarization, and it could act as a facilitating factor that elicits NMDA-dependent bursts of action potentials like apamin or bicuculline methiodide.