Lhx8 promote differentiation of hippocampal neural stem/progenitor cells into cholinergic neurons in vitro

Lhx8, also named L3, is a recently identified member of the LIM homeobox gene family. Previously, we found acetylcholinesterase (AChE)-positive cells in fimbria?Cfornix (FF) transected rat hippocampal subgranular zone (SGZ). In the present study, we detected choline acetyltransferase (ChAT)-positive cholinergic cells in hippocampal SGZ after FF transaction, and these ChAT-positive cells were double labeled by Lhx8. Then we overexpressed Lhx8 during neural differentiation of hippocampal neural stem/progenitor cells on adherent conditions using lentivirus Lenti6.3-Lhx8. The result indicated that overexpression of Lhx8 did not affect the proportion of MAP2-positive neurons, but increased the proportion of ChAT-positive cells in vitro. These results suggested that FF-transected hippocampal niche promoted the ChAT/Lhx8-positive cholinergic neurons generation in rodent hippocampus, and Lhx8 was not associated with the MAP2-positive neurons differentiation on adherent conditions, but played a role in the specification of cholinergic neurons derived from hippocampal neural stem/progenitor cells in vitro.     

Structural characterization of Escherichia coli sialic acid synthase

Wnt-1, the vertebrate counterpart of the Drosophila wingless gene, plays an important role in the early morphogenesis of neural tissues. In this report, we have shown that overexpression of Wnt-1 can direct embryonic carcinoma P19 cells to differentiate into neuron-like cells in the absence of retinoic acid. Immunocytochemistry showed that these cells expressed neuronal markers, such as the neurofilament (NF) and microtubule-associated protein 2 (MAP2), but failed to express the glial cell marker, glial fibrillary acidic protein (GFAP). RT-PCR revealed that two basic helix-loop-helix (bHLH) genes, Mash-1 and Ngn-1, were up-regulated during the differentiation stage of Wnt-1-overexpressing P19 cells. These results suggest that the Wnt-1 gene promotes neuronal differentiation and inhibits gliogenesis during the neural differentiation of P19 cells, and that neural bHLH genes might be involved in this process.     

肝胚瘤细胞株HepG_2中HBx蛋白对SOCS3表达的影响及其机制

目的探讨乙型肝炎病毒X蛋白(Hepatitis B virus X protein,HBx)对肝胚瘤细胞株HepG2表达信号抑制因子3(SOCS3)的影响及其机制。方法将表达HBx蛋白的重组质粒FL1-145HBx转染HepG2细胞,以不同浓度的SRC抑制剂PP2处理转染细胞,运用Western blot和RT-PCR技术分析HBx、SOCS3 mRNA和蛋白的表达情况,并以免疫细胞化学分析转染细胞中p-SRC的表达水平。结果重组质粒FL1-145HBx转染HepG2细胞后,转染细胞HBx能够表达HBx蛋白,并且细胞中SOCS3 mRNA和蛋白表达水平随着HBx蛋白的表达而显著增加(P0.05),同时p-SRC蛋白的表达增强;而SRC抑制剂PP2处理转染细胞后,随着p-SRC蛋白表达的减少SOCS3蛋白表达逐渐下降。结论在肝胚瘤细胞株HepG2中,HBx蛋白很可能通过促进SRC蛋白的磷酸化而诱导SOCS3蛋白的表达。     

Adult peripheral blood mononuclear cells transdifferentiate in vitro and integrate into the retina in vivo

Adult peripheral blood-derived cells are able to differentiate into a variety of cell types, including nerve cells, liver-like cells and epithelial cells. However, their differentiation into retina-like cells is controversial. In the present study, transdifferentiation potential of human adult peripheral blood mononuclear cells into retina-like cells and integration into the retina of mice were investigated. Freshly isolated adult peripheral blood mononuclear cells were divided into two groups: cells in group I were cultured in neural stem cell medium, and cells in group II were exposed to conditioned medium from rat retinal tissue culture. After 5 days, several distinct cell morphologies were observed, including standard mononuclear, neurons with one or two axons and elongated glial-like cells. Immunohistochemical analysis of neural stem cell, neuron and retina cell markers demonstrated that cells in both groups were nestin-, MAP2 (microtubule-associated protein)- and GFAP (glial fibrillary acidic protein)-positive. Flow cytometry results suggested a significant increase in nestin-, MAP2- and CD16-positive cells in group I and nestin-, GFAP-, MAP2-, vimentin- and rhodopsin-positive cells in group II. To determine survival, migration and integration in vivo, cell suspensions (containing group I or group II cells) were injected into the vitreous or the peritoneum. Tissue specimens were obtained and immunostained 4 weeks after transplantation. We found that cells delivered by intravitreal injection integrated into the retina. Labelled cells were not detected in the retina of mice receiving differentiated cells by intraperitoneal injection, but cells (groups I and II) were detected in the liver and spleen. Our findings revealed that human adult peripheral blood mononuclear cells could be induced to transdifferentiate into neural precursor cells and retinal progenitor cells in vitro, and the differentiated peripheral blood mononuclear cells can migrate and integrate into the retina in vivo.     

Effects of low-intensity pulsed ultrasound on cell viability, proliferation and neural differentiation of induced pluripotent stem cells-derived neural crest stem cells

Low-intensity pulsed ultrasound (LIPUS) acting on induced pluripotent stem cells–derived neural crest stem cells (iPSCs–NCSCs) is considered a promising therapy to improve the efficacy of injured peripheral nerve regeneration. Effects of LIPUS on cell viability, proliferation and neural differentiation of iPSCs–NCSCs were examined respectively in this study. LIPUS at 500 mW cm^?2 enhanced the viability and proliferation of iPSCs–NCSCs after 2 days and, after 4 days, up-regulated gene and protein expressions of NF-M, Tuj1, S100β and GFAP in iPSCs–NCSCs whereas after 7 days expression of only NF-M, S100β and GFAP were up-regulated. LIPUS treatment at an appropriate intensity can, therefore, be an efficient and cost-effective method to enhance cell viability, proliferation and neural differentiation of iPSCs–NCSCs in vitro for peripheral nerve tissue engineering.     

Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow

Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH) stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10), was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1), neurons (neurofilament protein, synapsin and MAP2), astrocytes (glial fibrillary acidic protein, GFAP) and oligodendrocytes (myelin basic protein, MBP) as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells), neurofilament protein and beta-tubulin III (neurons) GFAP (astrocytes), and galactocerebroside (oligodendrocytes). Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.     

MicroRNA-7 promotes neural differentiation of trabecular meshwork mesenchymal stem cell on nanofibrous scaffold

The purpose of this study was to investigate miR-7 overexpression effects on neural differentiation of mesenchymal stem cells (MSCs) on both two-dimensional (2D) and three-dimensional (3D) culture systems. We upregulated miR-7 through lentiviral vector in trabecular meshwork MSCs (TMMSCs) and polymers of poly l -lactic acid/polycaprolactone fibrous scaffold were fabricated by electrospinning and characterized using scanning electron microscopy (SEM) and Fourier transform infrared (FTIR). Neural markers expression was evaluated through quantitative-polymerase chain reaction (q-PCR) and immunostaining. The results showed that the high percentage of cell transduction (84.9%) and miR-7 expression (folds: 677.93 and 556.4) was detected in TMMSCs-miR-7(+). SEM and FTIR established the fabrication of the hybrid scaffold. q-PCR analysis showed that on days 14 and 21 of transduction, the expression level of Nestin and glial fibrillary acidic protein (GFAP) genes were significantly higher in the scaffold (3D) compared with tissue culture polystyrene (2D) culture. The expression of microtubule-associated protein-2 (MAP-2) and GFAP genes in TMMSCs-miR-7(+) cells were significantly higher than those miR-7(−) cells after 21 days of cell culture. Also, MAP-2 and Nestin proteins were detected in TMMSCs-miR-7(+) cells. Our results demonstrate that miR-7 is involved in neural differentiation of TMMSCs and scaffold can improve differentiate into glial and neural progenitor cells. These findings provided some information for future use of microRNAs and scaffold in tissue engineering and cell therapy for neurological diseases.     

Effect of Calcitonin Gene-Related Peptide on the Neurogenesis of Rat Adipose-Derived Stem Cells In Vitro

Calcitonin gene-related peptide (CGRP) promotes neuron recruitment and neurogenic activity. However, no evidence suggests that CGRP affects the ability of stem cells to differentiate toward neurogenesis. In this study, we genetically modified rat adipose-derived stem cells (ADSCs) with the CGRP gene (CGRP-ADSCs) and subsequently cultured in complete neural-induced medium. The formation of neurospheres, cellular morphology, and proliferative capacity of ADSCs were observed. In addition, the expression of the anti-apoptotic protein Bcl-2 and special markers of neural cells, such as Nestin, MAP2, RIP and GFAP, were evaluated using Western blot and immunocytochemistry analysis. The CGRP-ADSCs displayed a greater proliferation than un-transduced (ADSCs) and Vector-transduced (Vector-ADSCs) ADSCs (p<0.05), and lower rates of apoptosis, associated with the incremental expression of Bcl-2, were also observed for CGRP-ADSCs. Moreover, upon neural induction, CGRP-ADSCs formed markedly more and larger neurospheres and showed round cell bodies with more branching extensions contacted with neighboring cells widely. Furthermore, the expression levels of Nestin, MAP2, and RIP in CGRP-ADSCs were markedly increased, resulting in higher levels than the other groups (p<0.05); however, GFAP was distinctly undetectable until day 7, when slight GFAP expression was detected among all groups. Wnt signals, primarily Wnt 3a, Wnt 5a and β-catenin, regulate the neural differentiation of ADSCs, and CGRP gene expression apparently depends on canonical Wnt signals to promote the neurogenesis of ADSCs. Consequently, ADSCs genetically modified with CGRP exhibit stronger potential for differentiation and neurogenesis in vitro, potentially reflecting the usefulness of ADSCs as seed cells in therapeutic strategies for spinal cord injury.     

Neural stimulation on human bone marrow‐derived mesenchymal stem cells by extremely low frequency electromagnetic fields

Adult stem cells are considered multipotent. Especially, human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) have the potential to differentiate into nerve type cells. Electromagnetic fields (EMFs) are widely distributed in the environment, and recently there have been many reports on the biological effects of EMFs. hBM‐MSCs are weak and sensitive pluripotent stem cells, therefore extremely low frequency‐electromagnetic fields (ELF‐EMFs) could be affect the changes of biological functions within the cells. In our experiments, ELF‐EMFs inhibited the growth of hBM‐MSCs in 12 days exposure. Their gene level was changed and expression of the neural stem cell marker like nestin was decreased but the neural cell markers like MAP2, NEUROD1, NF‐L, and Tau were induced. In immunofluorescence study, we confirmed the expression of each protein of neural cells. And also both oligodendrocyte and astrocyte related proteins like O4 and GFAP were expressed by ELF‐EMFs. We suggest that EMFs can induce neural differentiation in BM‐MSCs without any chemicals or differentiation factors. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

LIF and BMP signaling generate separate and discrete types of GFAP-expressing cells

Bone morphogenetic protein (BMP) and leukemia inhibitory factor (LIF) signaling both promote the differentiation of neural stem/progenitor cells into glial fibrillary acidic protein (GFAP) immunoreactive cells. This study compares the cellular and molecular characteristics, and the potentiality, of GFAP(+) cells generated by these different signaling pathways. Treatment of cultured embryonic subventricular zone (SVZ) progenitor cells with LIF generates GFAP(+) cells that have a bipolar/tripolar morphology, remain in cell cycle, contain progenitor cell markers and demonstrate self-renewal with enhanced neurogenesis - characteristics that are typical of adult SVZ and subgranular zone (SGZ) stem cells/astrocytes. By contrast, BMP-induced GFAP(+) cells are stellate, exit the cell cycle, and lack progenitor traits and self-renewal--characteristics that are typical of astrocytes in the non-neurogenic adult cortex. In vivo, transgenic overexpression of BMP4 increases the number of GFAP(+) astrocytes but depletes the GFAP(+) progenitor cell pool, whereas transgenic inhibition of BMP signaling increases the size of the GFAP(+) progenitor cell pool but reduces the overall numbers of astrocytes. We conclude that LIF and BMP signaling generate different astrocytic cell types, and propose that these cells are, respectively, adult progenitor cells and mature astrocytes.