Experimental Study on Trace Marking and Oncogenicity of Neural Stem Cells Derived from Bone Marrow

It has been well accredited that the neural stem cells (NSCs) derived from bone marrow stroma cells (BMSCs) can be used as the therapeutic application. However, their efficacy and safety in therapeutic application are uncertain. In this experiment, the trace marking and oncogenicity of NSCs derived from BMSCs (BMSCs-D-NSCs) were studied. The BMSCs were harvested by gradient centrifugation and cultured in "NSCs medium" in vitro. The verified CD133/Nestin-positive BMSCs-D-NSCs were then transplanted into nude mice to detect the oncogenicity, into the right lateral cerebral ventricle or right caudae putamen and substantia nigra to examine, whether the symptoms were improved in Parkinson's Disease (PD) models after transplantation, by both SPECT image assay of dopamine transporter (DAT) in corpus striatum and its average standard uptake value (SUVave) in corpus striatum and thalamus. Tissue samples and surviving model animals were studied at 1, 3, and 6 months post-transplantation. Before transplantation, the cells were labeled with BrdU or rAAV-GFP for the pathological sections, and with Feridex for the in vivo trace by MRI assay. The concanavalin A (ConA) agglutination test, stop-dependence test with soft agar, karyotype analysis of chromosome G zone in BMSCs-D-NSCs, and the nude mouse neoplasia test were also performed. The BrdU, rAAV-GFP or Feridex can be used as trace markers of BMSCs-D-NSCs during transplantation. The transplanted BMSCs-D-NSCs displayed neither toxicity nor neoplasia up to 6 months in vivo, but could play an important role in improving the symptoms of the animals with degenerative diseases like PD.     

Comparative capability of menstrual blood versus bone marrow derived stem cells in neural differentiation

In order to characterize the potency of menstrual blood stem cells (MenSCs) for future cell therapy of neurological disorders instead of bone marrow stem cells (BMSCs) as a well-known and conventional source of adult stem cells, we examined the in vitro differentiation potential of these stem cells into neural-like cells. The differentiation potential of MenSCs to neural cells in comparison with BMSCs was assessed under two step neural differentiation including conversion to neurosphere-like cells and final differentiation. The expression levels of Nestin, Microtubule-associated protein 2, gamma-aminobutyric acid type B receptor subunit 1 and 2, and Tubulin, beta 3 class III mRNA and/or protein were up-regulated during development of MenSCs into neurosphere-like cells (NSCs) and neural-like cells. The up-regulation level of these markers in differentiated neural-like cells from MenSCs was comparable with differentiated cells from BMSCs. Moreover, both differentiated MenSCs and BMSCs expressed high levels of potassium, calcium and sodium channel genes developing functional channels with electrophysiological recording. For the first time, we demonstrated that MenSCs are a unique cell population with differentiation ability into neural-like cells comparable to BMSCs. In addition, we have introduced an approach to generate NSCs from MenSCs and BMSCs and their further differentiation into neural-like cells in vitro. Our results hold a promise to future stem cell therapy of neurological disorders using NSCs derived from menstrual blood, an accessible source in every woman.     

TEA-Sensitive Currents Contribute to Membrane Potential of Organ Surface Primo-node Cells in Rats

The primo-vascular (Bonghan) tissue has been identified in most tissues in the body, but its structure and functions are not yet well understood. We characterized electrophysiological properties of the cells of the primo-nodes (PN) on the surface of abdominal organs using a slice patch clamp technique. The most abundant were small round cells (~10 μm) without processes. These PN cells exhibited low resting membrane potential (−36 mV) and did not fire action potentials. On the basis of the current–voltage (I–V) relationships and kinetics of outward currents, the PN cells can be grouped into four types. Among these, type I cells were the majority (69%); they showed strong outward rectification in I–V relations. The outward current was activated rapidly and sustained without decay. Tetraethylammonium (TEA) dose-dependently blocked both outward and inward current (IC₅₀, 4.3 mM at ±60 mV). In current clamp conditions, TEA dose-dependently depolarized the membrane potential (18.5 mV at 30 mM) with increase in input resistance. The tail current following a depolarizing voltage step was reversed at −27 mV, and transient outward current like A-type K⁺ current was not expressed at holding potential of −80 mV. Taken together, the results demonstrate for the first time that the small round PN cells are heterogenous, and that, in type I cells, TEA-sensitive current with limited selectivity to K⁺ contributed to resting membrane potential of these cells.     

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.Here, we employ the human cardiac sodium channel (hNa_V1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.The properties of fast activating ion channels, such as hNa_V1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest¹. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.     

神经干细胞/前体细胞分化的功能学鉴定研究进展

神经干细胞／前体细胞分化是神经生物学研究的热点．以往对神经干细胞／前体细胞分化水平的鉴定主要依赖于形态学指标,而随着膜片钳技术的应用,神经干细胞／前体细胞分化过程中膜电特性的改变、离子通道活动等功能学指标越来越受到重视．综述了利用膜片钳技术研究神经干细胞／前体细胞功能分化的最新进展,并对存在的问题做出思考和展望．     

Glutathione Induces Neuronal Differentiation in Rat Bone Marrow Stromal Cells

It has been reported that rat bone marrow stromal cells (BMSCs) are differentiated into neuronal cells by administration of 2-mercaptoethanol [Woodbury et al (2000) J Neurosci Res 61:364–370]. In this study, we examined the effects of various sulfhydryl (SH) compounds on the differentiation of BMSCs obtained from rat femurs. Neuronal differentiation was detected morphologically and immunocytochemically. It was found that the cells treated with reduced glutathione (GSH) apparently differentiated into neurons, showing extensive processes, and expressing neuron-specific enolase and microtubule-associated protein 2. Glutathione monoethyl ester (GEE), which increased the cellular GSH content, showed no effect on the expression of neuronal markers. It is concluded that the neural differentiation of BMSCs occurs by the administration of GSH. It was suggested that extracellular and not intracellular GSH have effects on the induction of the neuronal differentiation of BMSCs.     

Relationship of the Electrophysiological Property of Tobacco Cultured Cells to Their Regeneration Ability

The electrophysiological properties of the high-density fractionof tobacco cultured cells (cell clusters) which formed leafprimordia in high yield in Linsmaier-Skoog's medium containingkinetin were compared with those of low-density cell fractionsin which leaf primordia hardly differentiated. Cells of theformer fraction had a higher membrane potential and an electrogenicmechanism which responded to sugars and amino acids. (Received February 12, 1983; Accepted March 23, 1984)     

Peptide Pheromone Plantaricin A Produced by Lactobacillus plantarum Permeabilizes Liver and Kidney Cells

Certain antimicrobial peptides from multicellular animals kill a variety of tumor cells at concentrations not affecting normal eukaryotic cells. Recently, it was reported that also plantaricin A (PlnA), which is a peptide pheromone with strain-specific antibacterial activity produced by Lactobacillus plantarum, permeabilizes cancerous rat pituitary cells (GH₄ cells), whereas normal rat anterior pituitary cells are resistant to the peptide. To examine whether the preferential permeabilization of cancerous cells is a general feature of PlnA, we studied its effect on primary cultures of cells from rat liver (hepatocytes, endothelial, and Kupffer cells) and rat kidney cortex, as well as two epithelial cell lines of primate kidney origin (Vero cells from green monkey and human Caki-2 cells). The Vero cell line is derived from normal cells, whereas the Caki-2 cell line is derived from a cancerous tumor. The membrane effects were studied by patch clamp recordings and microfluorometric (fura-2) monitoring of the cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) and fluorophore. In all the tested cell types except Kupffer cells, exposure to 10–100 μM PlnA induced a nearly instant permeabilization of the membrane, indicated by the following criteria: increased membrane conductance, membrane depolarization, increased [Ca²⁺]_i, and diffusional loss of fluorophore from the cytosol. At a concentration of 5 μM, PlnA had no effect on any of the cell types. The Kupffer cells were permeabilized by 500 μM PlnA. We conclude that the permeabilizing effect of PlnA is not restricted to cancerous cells.     

Developmental regulation of a novel outwardly rectifying mechanosensitive anion channel in Caenorhabditis elegans

The nematode Caenorhabditis elegans offers unique experimental advantages for defining the molecular basis of anion channel function and regulation. However, the relative inaccessibility of somatic cells in adult animals greatly limits direct electrophysiological studies of channel activity. We developed methods to routinely isolate and patch clamp C. elegans embryo cells and oocytes and to culture and patch clamp neurons and muscle cells. Dissociated embryonic cells express a robust outwardly rectifying anion current that is activated by membrane stretch and depolarization. This current, termed I(Cl,mec), is inhibited by anion and mechanosensitive channel inhibitors. I(Cl,mec) has broad anion selectivity and the channel has a unitary conductance of 5-7 picosiemens. I(Cl,mec) is not detectable in whole-cell or isolated patch recordings from oocytes, cultured muscle cells, and cultured neurons but is expressed in single cell and later embryos. Channel density is high, and the current is observed in >80% of membrane patches. Macroscopic currents of 40-120 pA at +100 mV are typically observed in inside-out membrane patches formed using low resistance patch pipettes. Isolated membrane patches of early embryonic cells therefore contain 60-200 I(Cl,mec) channels. The apparent activation of I(Cl,mec) shortly after fertilization and its down-regulation in terminally differentiated cells suggests that the channel may play important roles in embryogenesis and/or cytokinesis.     

低氧促进神经干细胞向多巴胺能神经元分化

神经干细胞（neural stem cells,NSCs）作为具有多向分化潜能的神经前体细胞,被广泛应用于细胞移植等研究,而低氧不但调节干细胞的体外增殖,在干细胞分化中也具有重要的作用。本文着重探讨了低氧对NSCs分化的调节作用。采用Wistar孕大鼠（E13．5d）,分离胚胎中脑NSCs,加入无血清DMEM／F12培养液（含20ng／mL EGF、20ng／mL bFGF、1％ N2和B27）,3～5d后传代,细胞培养至第三代进行诱导分化,分别在低氧（3％O2）和常氧（20％O2）条件下诱导分化3d,然后在常氧条件下分化成熟5～7d（DMEM／F12含1％FBS、N2和B27）后进行检测。Nestin、NeuN以及TH免疫组织化学鉴定NSCs;流式细胞术分析测定NSCs向TH阳性神经元方向的分化;高效液相色谱测定细胞培养上清液中多巴胺（dopamine,DA）含量。结果显示,分离培养的NSCs均为nestin阳性细胞;低氧可明显促进NSCs向神经元方向的分化;TH阳性神经元比例在常氧和低氧组分别为（10．25±1．03）％和（19．88±1．44）％。NSCs诱导分化7d后,低氧组细胞培养上清液中DA浓度明显增加,约为常氧组的2倍（P〈0．05,n=8）。上述结果表明,3％低氧可促进NSCs向神经元方向,特别是向DA能神经元方向分化。这为NSCs应用于临床治疗帕金森病提供了基础。     

Single Mechanosensitive and Ca2+-Sensitive Channel Currents Recorded from Mouse and Human Embryonic Stem Cells

Cell-attached and inside-out patch clamp recording was used to compare the functional expression of membrane ion channels in mouse and human embryonic stem cells (ESCs). Both ESCs express mechanosensitive Ca²⁺ permeant cation channels (MscCa) and large conductance (200 pS) Ca²⁺-sensitive K⁺ (BK_Ca2+) channels but with markedly different patch densities. MscCa is expressed at higher density in mESCs compared with hESCs (70 % vs. 3 % of patches), whereas the BK_Ca2+ channel is more highly expressed in hESCs compared with mESCs (~50 % vs. 1 % of patches). ESCs of both species express a smaller conductance (25 pS) nonselective cation channel that is activated upon inside-out patch formation but is neither mechanosensitive nor strictly Ca²⁺-dependent. The finding that mouse and human ESCs express different channels that sense membrane tension and intracellular [Ca²⁺] may contribute to their different patterns of growth and differentiation in response to mechanical and chemical cues.     

Efficient In Vitro Labeling Rabbit Bone Marrow-Derived Mesenchymal Stem Cells with SPIO and Differentiating into Neural-Like Cells

Mesenchymal stem cells (MSCs) can differentiate into neural cells to treat nervous system diseases. Magnetic resonance is an ideal means for cell tracking through labeling cells with superparamagnetic iron oxide (SPIO). However, no studies have described the neural differentiation ability of SPIO-labeled MSCs, which is the foundation for cell therapy and cell tracking in vivo. Our results showed that bone marrow-derived mesenchymal stem cells (BM-MSCs) labeled in vitro with SPIO can be induced into neural-like cells without affecting the viability and labeling efficiency. The cellular uptake of SPIO was maintained after labeled BM-MSCs differentiated into neural-like cells, which were the basis for transplanted cells that can be dynamically and non-invasively tracked in vivo by MRI. Moreover, the SPIO-labeled induced neural-like cells showed neural cell morphology and expressed related markers such as NSE, MAP-2. Furthermore, whole-cell patch clamp recording demonstrated that these neural-like cells exhibited electrophysiological properties of neurons. More importantly, there was no significant difference in the cellular viability and [Ca²⁺]i between the induced labeled and unlabeled neural-like cells. In this study, we show for the first time that SPIO-labeled MSCs retained their differentiation capacity and could differentiate into neural-like cells with high cell viability and a good cellular state in vitro.     

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously^1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.     

Cells from the adult corneal stroma can be reprogrammed to a neuron-like cell using exogenous growth factors

Cells thought to be stem cells isolated from the cornea of the eye have been shown to exhibit neurogenic potential. We set out to uncover the identity and location of these cells within the cornea and to elucidate their neuronal protein and gene expression profile during the process of switching to a neuron-like cell. Here we report that every cell of the adult human and rat corneal stroma is capable of differentiating into a neuron-like cell when treated with neurogenic differentiation specifying growth factors. Furthermore, the expression of genes regulating neurogenesis and mature neuronal structure and function was increased. The switch from a corneal stromal cell to a neuron-like cell was also shown to occur in vivo in intact corneas of living rats. Our results clearly indicate that lineage specifying growth factors can affect changes in the protein and gene expression profiles of adult cells, suggesting that possibly many adult cell populations can be made to switch into another type of mature cell by simply modifying the growth factor environment.     

Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins

Giant protoplasts of Saccharomyces cerevisiae of 10-35 µm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and “patchability” of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 ± 0.07 µF/cm²) and conductance (23-44 µS/cm²) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels.     

Enhancement of Sodium Current in NG108-15 Cells during Neural Differentiation is Mainly due to an Increase in NaV1.7 Expression

It is well known that morphological and functional changes during neural differentiation sometimes accompany the expression of various voltage-gated ion channels. In this work, we investigated whether the enhancement of sodium current in differentiated neuroblastoma × glioma NG108-15 cells treated with dibutyryl cAMP is related to the expression of voltage-gated sodium channels. The results were as follows. (1) Sodium current density on peak voltage in differentiated cells was significantly enhanced compared with that in undifferentiated cells, as detected by the whole-cell patch clamp method. The steady-state inactivation curve in differentiated cells was similar to that for undifferentiated cells, but a hyperpolarized shift in the activation curve for differentiated cells was observed. The sodium currents of differentiated and undifferentiated cells were completely inhibited by 10⁻⁷ M tetrodotoxin (TTX). (2) The only Na_V mRNA with an increased expression level during neuronal differentiation was that for Na_V1.7, as observed by real-time PCR analysis. (3) The increase in the level of Na_V1.7 α subunit expression during neuronal differentiation was also observed by immunocytochemistry; in particular, the localization of Na_V1.7 α subunits on the soma, varicosities and growth cone was significant. These results suggest that the enhancement of TTX-sensitive sodium current density in differentiated NG108-15 cells is mainly due to the increase in the expression of the TTX-sensitive voltage-gated Na⁺ channel, Na_V1.7.     

Cell marking in Arabidopsis thaliana and its application to patch–clamp studies

Ion transport processes at the plasma membrane of plant cells are frequently studied by applying membrane-patch voltage-clamp (patch–clamp) electrophysiological techniques to isolated protoplasts. As plants are composed of many tissues and cell types, and each tissue and cell type may be specialized to a particular function and possess a unique complement of transport proteins, it is important to certify the anatomical origin of the protoplasts used for patch–clamp studies. This paper describes a general molecular genetic approach to marking specific cell types for subsequent patch–clamp studies and presents a specific example: a comparison of the K⁺ currents in protoplasts from cortical and stelar cells of Arabidopsis roots. Transgenic Arabidopsis were generated in which the expression of green fluorescent protein (GFP) from Aequoria victoria was driven by the CaMV 35S promoter (line mGFP3). In roots of the transgenic mGFP3 line, visible fluorescence was restricted to the stele. Protoplasts were generated from roots of the mGFP3 line and K⁺ currents in non-fluorescent (cortical/epidermal) and fluorescent (stelar) protoplasts were assayed using patch–clamp techniques. It was found that both the frequency of observing inward rectifying K⁺ channel (IRC) activity and the relative occurrence of IRC compared to outward rectifying K⁺ channels were significantly lower in protoplasts from cortical/epidermal cells compared to cells of the stele. The presence of GFP did not affect the occurrence or biophysical properties of K⁺ channels. It is concluded that the generation of transgenic Arabidopsis expressing GFP in a cell-specific fashion is a convenient and reliable way to mark protoplasts derived from contrasting cell types for subsequent patch–clamp studies.     

Electrophysiological Characterisation of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Induced by Olfactory Ensheathing Cell-Conditioned Medium

Umbilical cord blood-derived marrow stromal cells (UCB-MSCs) with high proliferation capacity and immunomodulatory properties are considered to be a good candidate for cell-based therapies. But until now, little work has been focused on the differentiation of UCB-MSCs. In this work, UCB-MSCs were demonstrated to be negative for CD34 and CD45 expression but positive for CD90 and CD105 expression. The gate values of UCB-MSCs for CD90 and CD105 were 99.3 and 98.6 %, respectively. Two weeks after treatment, the percentage of neuron-like cells differentiated from UCB-MSCs was increased to 84 ± 12 % in the experimental group [treated with olfactory ensheathing cells (OECs)-conditioned medium] and they were neuron-specific enolase positive; few neuron-like cells were found in the control group (without OECs-conditioned medium). Using whole-cell recording, sodium and potassium currents were recorded in UCB-MSCs after differentiation by OECs. Thus, human UCB-MSCs could be differentiated to neural cells by secreted secretion from OECs and exhibited electrophysiological properties similar to mature neurons after 2 weeks post-induction. These results imply that OECs can be used as a new strategy for stem cell differentiation and provide an alternative neurogenesis pathway for generating sufficient numbers of neural cells for cell therapy.     

Basolateral K+ Conductance Establishes Driving Force for Cation Absorption by Outer Sulcus Epithelial Cells

Outer sulcus epithelial cells were recently found to actively reabsorb cations from the cochlear luminal fluid, endolymph, via nonselective cation channels in the apical membrane. Here we determined the transport properties of the basolateral membrane with the whole-cell patch clamp technique; the apical membrane contributed insignificantly to the recordings. Outer sulcus epithelial cells exhibited both outward and inward currents and had a resting membrane potential of −90.4 ± 0.7 mV (n= 78), close to the Nernst potential for K⁺ (−95 mV). The reversal potential depolarized by 54 mV for a tenfold increase in extracellular K⁺ concentration with a K⁺/Na⁺ permeability ratio of 36. The most frequently observed K⁺ current was voltage independent over a broad range of membrane potentials. The current was reduced by extracellular barium (10⁻⁵ to 10⁻³ m), amiloride (0.5 mm), quinine (1 mm), lidocaine (5 mm) and ouabain (1 mm). On the other hand, TEA (20 mm), charybdotoxin (100 nm), apamin (100 nm), glibenclamide (10 μm), 4-aminopyridine (1 mm) and gadolinium (1 mm) had no significant effect. These data suggest that the large K⁺ conductance, in concert with the Na⁺,K⁺-ATPase, of the basolateral membrane of outer sulcus cells provides the driving force for cation entry across the apical membrane, thereby energizing vectorial cation absorption by this epithelium and contributing to the homeostasis of endolymph.