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Stem Cells Vol. 23 No. 7 August 2005, pp. 931 -945
doi:10.1634/stemcells.2004-0316; www.StemCells.com
© 2005 AlphaMed Press

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Voltage-Sensitive and Ligand-Gated Channels in Differentiating Neural Stem–Like Cells Derived from the Nonhematopoietic Fraction of Human Umbilical Cord Blood

Wei Suna, Leonora Buzanskab,d, Krystyna Domanska-Janikd, Richard J. Salvia, Michal K. Stachowiakc

a Hearing Research Lab,
b Molecular and Structural Neurobiology and Gene Therapy Lab, and
c Department of Pathology and Anatomical Sciences, SUNY University at Buffalo, Buffalo, New York, USA;
d NeuroRepair Department, Medical Research Center, Warsaw, Poland

Key Words. Neural stem cells • Human umbilical cord blood • Neurotransmitter receptors • Patch clamp • Inward rectifying potassium current • Outward rectifying potassium current

Correspondence: Michal K. Stachowiak, Ph.D., Department of Pathology and Anatomical Sciences, 206A Farber Hall, SUNY University at Buffalo, Buffalo, New York 14214, USA. Telephone: 716-829-3540; Fax: 716-829-2911; e-mail: mks4{at}buffalo.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 References
 
Fetal cells with the characteristics of neural stem cells (NSCs) can be derived from the nonhematopoietic fraction of human umbilical cord blood (HUCB), expanded as a nonimmortalized cell line (HUCB-NSC), and further differentiated into neuron-like cells (HUCB-NSCD); however, the functional and neuronal properties of these cells are poorly understood. To address this issue, we used whole-cell patch-clamp recordings, gene microarrays, and immunocytochemistry to identify voltage-gated channels and ligand-gated receptors on HUCB-NSCs and HUCB-NSCDs. Gene microarray analysis identified genes for voltage-dependent potassium and sodium channels and the neurotransmitter receptors acetylcholine (ACh), {gamma}-aminobutyric acid (GABA), glutamate, glycine, 5-hydroxytryptamine (5-HT), and dopamine (DA). Several of these genes (GABA-A, glycine and glutamate receptors, voltage-gated potassium channels, and voltage-gated sodium type XII alpha channels) were not expressed in the HUCB mono-nuclear fraction (HUCB-MC), which served as a starting cell population for HUCB-NSC. HUCB-NSCD acquired neuronal phenotypes and displayed an inward rectifying potassium current (Kir) and an outward rectifying potassium current (IK+). Kir was present on most HUCB-NSCs and HUCB-NSCDs, whereas IK+ was present only on HUCB-NSCDs. Many HUCB-NSCDs were immunopositive for glutamate, glycine, nicotinic ACh, DA, 5-HT, and GABA receptors. Kainic acid (KA), a non–N-methyl-D-asparate (NMDA) glutamate-receptor agonist, induced an inward current in some HUCB-NSCDs. KA, glycine, DA, ACh, GABA, and 5-HT partially blocked Kir through their respective receptors. These results suggest that HUCB-NSCs differentiate toward neuron-like cells, with functional voltage- and ligand-gated channels identified in other neuronal systems.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Summary
 References
 
Neuronal and glial populations in the developing brain are generated from multipotent neural stem cells (NSCs) located predominantly in the subventricular zone and hippocampus [1]. Recent studies have demonstrated that a small number of NSCs provide a source of new neurons in the olfactory bulb, hippocampus, cortex, and basal ganglia [2, 3]. NSCs have been cultured in vitro in the form of neurospheres and used to investigate the molecular mechanisms of lineage determination and mechanisms of neuronal and glial differentiation. Transplantation of NSCs into brain or spinal cord could potentially be used to replace damaged neurons and glial cells and thus treat a wide range of neurodegenerative disorders and central nervous system (CNS) injuries [4–6]. Experiments in rodents and primates show that cultured NSCs or their neuronal-committed progeny from fetal rat or human brain can survive, mature, and develop axonal connections after transplantation into a damaged brain, thereby providing both structural and functional replacements for damaged neurons [7, 8]. Mouse embryonic NSCs transplanted into animal models of Parkinson’s differentiate into dopamine (DA)–containing neurons with their characteristic electrophysiological properties [4]. Moreover, intravenous or intrathecal injections of adult neural precursor cells into animal models of multiple sclerosis promote multifocal remyelination and functional recovery [9, 10].

Although the use of embryonic, fetal, or adult brain-derived NSCs holds great therapeutic potential, their limited supply and evoked immunogenicity in case of allografts limit their usefulness for human therapy. An alternative approach has been to use animal and human bone marrow stromal cells, which have been shown to integrate into rat brain tissue [11, 12] and even to differentiate into astrocytes and tyrosine hydroxylase (TH)–expressing neurons that synthesize DA when grafted into the Parkinsonian mouse [13]. Moreover, in patients receiving therapeutic bone marrow transplants, some transplanted cells migrate into the brain and display fusion-independent neuronal phenotype [14–16]. This suggests that non embryonic stem cells from sources other than the nervous system could be used for neuron-replacement therapy [17].

Human umbilical cord blood (HUCB) mononuclear fraction contains stem cells belonging to hematopoietic and nonhema-topoietic lineages, which may represent a source of multipotent NSCs with relatively low antigenicity [12, 18–22]. Indeed, when transplanted into the brain, the mononuclear fraction of HUCB was shown to survive in brain tissue [23, 24] and reduce motor and neurological deficits [25]. However, due to limited implantation of these cells into brain tissue, the mechanisms underlying the therapeutic effect of these transplanted cord blood cells are still under discussion [26].

Cord blood contains a relatively well-defined population of CD34+ stem cells committed to a hematopoietic lineage. Although it is possible that such cells transdifferentiate into other types of stem cells [27], HUCBs may also contain nonhematopoietic stem cells from the fetus with wider potential, which can give rise to NSCs. Using the CD34–/CD45– mononuclear fraction of HUCB as starting material, Buzanska et al. [22] obtained human, multipotent, neural stem–like cells. From these cells, a clonogenic nonimmortalized cell line (HUCB-NSC) with relatively high self-renewal potency and expressing several NSC markers including nestin and glial fibrillary acidic protein (GFAP) was further developed [28]. HUCB-NSCs can differentiate in vitro and give rise to all three brain cell types. In the presence of the neuromorphogen/retinoic acid, 40% of cells attained a neuronal phenotype expressing ß-tubulin III and MAP-2, 30% developed astrocyte phenotypes expressing GFAP and S100ß, whereas 11% of cells developed oligodendroglial phenotypes expressing galactosylceramide (GalC) [22, 28]. These results suggest that HUCB might be expanded in vitro to produce a population of human fetal NSCs that could be useful clinically. For therapeutic application, HUCB cells must not only develop morphological characteristics of neurons but also voltage- and ligand-gated ion channels that would allow them to function within a neural network and respond to neurotransmitters released from neighboring neurons. To begin to address these issues, we used the whole-cell patch-clamp technique to characterize the electrophysiological properties and ligand-gated receptors on HUCB–differentiated NSCs (NSCDs) and HUCB-NSCs, and gene microarray analysis and immunocytochemistry were used to confirm and further characterize HUCB-NSCD, HUCB-NSC, and HUCB mononuclear cells (HUCB-MCs).


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Summary
 References
 
Cell Culture
The nonimmortalized HUCB-NSC line, immuno-negative for CD34 and CD45 hematopoietic cell markers [22], was derived from the MC fraction as described previously [22, 28]. The cell line was established through sequential in vitro passaging and further selection in a culture of nonadherent proliferating cells (unpublished data). The HUCB-NSC line was expanded as a culture of only nonadherent and nondifferentiated cells in serum-free medium that consisted of Dulbecco’s modified Eagle’s medium (DMEM)/F12, antibiotic-antimycotic solution (AAS) (diluted x100; Sigma, St. Louis, http://www.sigmaaldrich.com), supplemented with B27 (1:50; Invitrogen, Grand Island, NY, http://www.invitrogen.com), and mitogens (epidermal growth factor [EGF] [10 ng/ml], basic fibroblast growth factor [bFGF] [10 ng/ml], and leukemia inhibitory factor [10 ng/ml]; Sigma). Alternatively HUCB-NSCs were expanded in a culture containing adherent as well as floating, rounded cells using DMEM/F12, supplemented with 2% fetal bovine serum (FBS), insulin-transferrin-selenium (ITS) (1:100; Invitrogen), and AAS (diluted x100; Sigma) (unpublished data). In the present study, HUCB-NSCs were cultured in DMEM/F12, 2% FBS, supplemented with ITS and AAS. Nonadherent cells, which were undifferentiated, are here after referred to as HUCB-NSCs. To induce differentiation, nonadherent HUCB-NSCs were collected and plated on glass coated with poly-L-lysine plus laminin (0.5–1 µg/cm2) in 12-well plates at a density of 104 cells per cm2. Attached cells were treated with dBcAMP/CPT (300 µM; Sigma) and placed in an incubator (5% CO2, 37°C) for 1 day to 4 weeks.

Gene Microarray Analysis
Total RNA was isolated using Trizol Reagent (Invitrogen) from approximately 4 million cells for three experimental groups: (a) control HUCB-MCs, (b) nonattached HUCB-NSCs, and (c) attached HUCB-NSCDs differentiated for 4 weeks on poly-L-lysine and laminin-coated coverslips and treated with dBcAMP/CPT (300µM;Sigma). cRNA samples were hybridized to Affymetrix HG-U133 Set A and B Gene Chips with probes for approximately 33,000 human genes (Gene Core Laboratory, Roswell Park Cancer Institute, Buffalo, NY, http://www.roswellpark.org). Streptavidin-phycoerytrin–stained gene chips were scanned with a Gene Chip System confocal scanner (Agilent, Affymetrix Inc., Santa Clara, CA, http://www.affymetrix.com) at 3-µm resolution. Fluorescence intensity for each gene was normalized to the average fluorescence intensity of the entire chip. Relative abundance of each gene was assessed according to its p value, calculated according to the statistical algorithms provided by Affymetrix Microarray Suite (Affymetrix Inc.). Present (P), marginal (M), and absent (A) calls for gene expression were defined as p < .05, .05 ≤ p ≤ .06, and p > .06, respectively. Mean values from two separate experimental runs were calculated. GeneChip absolute analysis and comparisons among the three experimental conditions were performed using Data Mining Tool (Affymetrix Inc.). Data were transferred to Excel files for later analysis. Genes of interest in this study were genes associated with voltage-gated and ligand-gated ion channels. Identification of relevant genes in the data set was found by searching the Affymetrix data set for key words such as receptors, neural, and neuronal. Results of all remaining genes will be published elsewhere.

Immunocytochemistry
HUCB-NSCDs, attached to poly- L-lysine/laminin–covered glass and treated with dBcAMP/CPT (300 µM), were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate (pH 7.2) for 20 minutes at room temperature. Cultures were washed three times with 0.1 M phosphate-buffered saline (PBS), blocked in 5% normal goat serum for 1–2 hours. Samples were incubated with primary antibody (Ab) overnight at 4°C. For cytoskeletal staining, cells were additionally permeabilized in PBS plus 0.1%–0.25% Triton X-100 for 20 minutes. Primary antibodies against cytoskeletal proteins were monoclonal anti–ß-tubulin III (1:300; Sigma), monoclonal neurofilament NF-200 (1:400; Chemicon, Temecula, CA, http://www.chemicon.com), and monoclonal anti–light chain of neurofilament NF-70 (1:100; Chemicon). The antibodies used for neurotransmitter receptors were anti-glutamate receptor 2 Ab (polyclonal, 1:100; Chemicon), anti–5-HT1C (5-HT1CR, 1:500, polyclonal Ab; Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), anti–{gamma}-aminobutyric acid (GABA)–A receptor (GABA-AR, beta chain 1:100, monoclonal; Research Diagnostic Inc., Flanders, NJ, http://www.researchd.com), glycine receptor Ab (GlyR, 1:100, polyclonal; Chemicon), acetylcholine (ACh) nicotinic receptor beta subunit Ab (1:100, monoclonal; Transduction Laboratories, http://www.bdbiosciences.com), and anti–DA D2 receptor polyclonal Ab (D2; 1:100; Chemicon). An Ab for the neurotransmitter GABA was also evaluated (anti-GABA, 1:200, monoclonal; Chemicon). In some experiments, cell nuclei were stained with ToPro-1 (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) at a final concentration of 2 µM. After rinsing in PBS, cells were incubated with secondary Ab (goat anti-mouse immunoglobulin G [IgG] Alexa-555, goat anti-rabbit IgG Alexa 488, 1:300 [Molecular Probes]; goat anti-mouse IgG FITC, 1:300 [Sigma]; or goat anti-rabbit Cy3, 1:1000 [Jackson ImmunoResearch, West Grove, PA, http://www.jacksonimmuno.com]) for 1 hour at room temperature. Double immunostaining of neurotransmitter receptors and cytoskeletal proteins was performed sequentially. Cells were incubated first with primary anti-receptor Ab overnight at 4°C, followed by 30-minute incubation with secondary Ab at 37°C. Cells were subsequently permeabilized for 20 minutes in 0.25% Triton X-100 and incubated with primary Ab against cytoskeletal proteins for 2 hours at room temperature and with secondary Ab at 37°C for 30 minutes. As a negative control, the primary Ab was omitted, with other steps remaining the same. Images of cells mounted on Fluoromont G (SouthernBio-tech, Birmingham, AL, http://www.southernbiotech.com) were taken using a fluorescence microscope (Axioskop; Carl Zeiss, Jena, Germany, http://www.zeiss.com) or confocal Bio-Rad MRC microscope (Bio-Rad Laboratories, Hercules, CA, http://www.bio-rad.com), processed with Adobe PhotoShop (Adobe Systems, San Jose, CA, http://www.adobe.com), and printed on a dye sublimation printer (Kodak 8670; Eastman Kodak Company, Rochester, NY, http://www.kodak.com).

Whole-Cell Patch-Clamp Recording
Our procedures for making whole-cell, patch-clamp recordings have been described in detail previously [29–31]. Before recording from HUCB-NSC or HUCB-NSC-NSCD, the culture medium was replaced with Hanks’ balanced salt solution (HBSS), which contained (in mM) NaCl 137, Na2HPO4 0.2, KCl 5.4, KH2PO4 0.4, MgSO2 0.8, CaCl2 1.3, glucose 5.6, and HEPES 10. The recording pipette was filled with a solution containing (in mM) KCl 120, KF 20, NaCl 2, MgCl2 2, EGTA 10, and HEPES 10; pH was buffered to 7.3 with NaOH, and osmolarity was adjusted with sucrose to 290 mOsm. The pipette resistance in the bath solution was typically 2–6 Mµ. The whole-cell recording configuration was established on the soma of HUCB-NSC or HUCB-NSCD (1–3 Gµ), and recordings were made at 22°C. In voltage-clamp configuration, series resistance and series compensation were applied as reported in our previous publications. Current and voltage signals were amplified using a patch-clamp amplifier (Multiclamp 700A), digitized (DigiData 1200; Axon Instruments, Union City, CA, http://www.axon.com), and analyzed by pCLAMP (version 8.01; Axon Instruments). Kainic acid (KA), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), DA, glycine, strychnine, 4-aminopridine (4-AP), tetraethylammonium (TEA), CdCl2, BaCl2, and CsCl were all purchased from Sigma. The solutions were made in HBSS and applied alone or mixed together through a puffer electrode connected to a DAD-12 super fusion system (ALA Instruments, Westbury, NY, http://www.alascience.com). Electrophysiological results were evaluated using Student’s t-test (Sigma Stat, SPSS, version 2) as indicated in the Results section.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Summary
 References
 
Expression of Neural Markers
In our previous study, HUCB-NSCs were shown to express several structural or cytoskeletal proteins characteristic of cells of neural lineage while maintained in DMEM, 10% FBS (Invitrogen), and 10 ng/ml EGF [22]. The same characteristics were observed in the present study when HUCB-NSCs were maintained in 2% FBS medium, without growth factors, but supplemented with ITS (1:100; Sigma). HUCB-NSC cultures proliferated at a high rate and consisted of two cell populations. The first population was represented by round, floating cell aggregates consisting of cells of approximately 8–10 µm in diameter with relatively large nuclei and scant cytoplasm (Fig. 1AGo, arrowhead; Fig. 1BGo). These cells expressed nestin and GFAP but did not express ß-tubulin III or other markers of advanced neuronal (MAP2) or astroglial (S100ß) differentiation (data not shown). The second population consisted of flattened cells that attached to the plastic wells (Fig. 1AGo, arrow; Fig. 1CGo). These cells expressed early markers of neuronal differentiation, including NF-200, NF-70, and ß-tubulin III (not shown). When HUCB-NSCs were plated on polylysine/laminin-coated glass plates and maintained in medium containing dBcAMP/CPT for 1–24 days, they readily attached to the substratum and developed long, neurite-like processes after 5 or more days in culture (Figs. 1D–1FGo). These HUCB-NSCDs were immunopositive for ß-tubulin III (80%) (Fig. 1DGo), neurofilament NF-200 (Fig. 1EGo), and NF-70 (Fig. 1FGo), a late, structural, neural marker. Relatively few cells (<19%) with astrocyte-like morphology and GFAP immunoreactivity were observed.



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  		Figure 1. (A): Cultures of HUCB-NSCs maintained in 2% FBS medium contained flattened cells that attached to plastic dish (arrow) and round, floating aggregate-forming cells with relatively large nuclei and scant cytoplasm (arrowhead). Phase-contrast photomicrograph showing whole-cell recording electrode on a (B) unattached HUCB-NSC and (C) attached HUCB-NSCD cultured with 2% FBS and dBcAMP/CPT for 12 days. Note processes (arrows) extending from soma of HUCB-NSCD. HUCB-NSCDs immunopositive for (D) ß-tubulin III mouse primary Ab, anti-mouse FITC-conjugated secondary Ab (differentiated 4 days in the presence of dBcAMP/CPT; arrows show neurites extending from soma of some cells), (E) neurofilament 200-kD mouse primary Ab, goat anti-mouse Alexa-555–conjugated secondary antibody (differentiated 12 days in the presence of dBcAMP/CPT, arrows show neurites extending from soma), and (F) neurofilament 70-kD mouse primary Ab, goat anti-mouse FITC-conjugated secondary antibody (differentiated 12 days, arrows show neurites extending from soma). Scale bar for A, D, E, F: 50 µm; for B and C: 20 µm. Abbreviations: Ab, antibody; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HUCB, human umbilical cord blood; NSC, neural stem cell; NSCD, differentiated neural stem cell.

 
Genes Associated with Ion Channels and Neurotransmitters
Microarray RNA analysis was applied to (a) the starting population of HUCB-MCs from which our neural progenitors had been selected and expanded [22], (b) floating HUCB-NSCs, and (c) HUCB-NSCDs attached to poly- L -lysine/laminin–coated cover-slips and differentiated for 4 weeks in dBcAMP/CPT. Expression of approximately 33,000 genes was analyzed using Affymetrix HG-U133 Set A and B Gene Chips. Receptor and ion channel genes important for neuronal function, the focus of the present study, are listed in Table 1Go. Their relative expression in HUCB-MCs, HUCB-NSCs, and HUCB-NSCDs was evaluated using statistical algorithms provided by Affymetrix Microarray Suite as described above. The results with the remaining genes will be reported elsewhere.


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  		Table 1. Expression of neuronal voltage-gated or ligand-gated receptor genes in dBcAMP/CPT differentiated with HUCB-MC, HUCB-NSC, and HUCB-NSCD
 
As shown in Table 1Go, 14 neurotransmitter receptors genes and 7 ion channel genes considered important for neural development and function were expressed (present or marginal calls for expression; see Materials and Methods) in HUCB-NSCDs. The neurotransmitter receptor genes expressed in HUCB-NSCDs consisted of three cholinergic receptor subtypes, two dopaminergic receptor subtypes, three GABAergic receptors subtypes, two glutamate receptor subtypes, one glycinergic subtype, and three serotonergic subtypes. None of the three GABAergic receptor subtypes was expressed in MCs, whereas two of three GABAergic receptor subtypes were expressed in HUCB-NSCs. Only one of two glutamate receptor subtypes, metabotropic 6, was expressed in HUCB-MCs; however, both the metabotropic and kainate 4 subtypes were expressed in HUCB-NSCs and HUCB-NSCDs. Glycine receptor beta was not expressed in HUCB-MCs but was detected in HUCB-NSCs and HUCB-NSCDs. Thus, the transition from HUCB-MC to HUCB-NSC was associated with increased expression of GABA, glycine, and glutamate receptor subtypes. No change in expression of cholin-ergic, dopaminergic, and serotonergic receptor subtypes was seen from MC to HUCB-NSC or from HUCB-NSC to HUCB-NSCD.

Of the seven ion channel receptor subtypes seen in HUCB-NSCDs, three were members of the potassium family, three were members of the sodium family, and one was a member of the transient receptor potential cation channel. Inward-rectifying potassium channels (Kir) and calcium-activated potassium (IK[Ca]) were consistently expressed in HUCB-MC, HUCB-NSC, and HUCB-NSCD, but the KQT potassium channel subtype was not expressed in MCs. Voltage-gated sodium channels type X alpha and type XII alpha and non–voltage-gated sodium channel type 1 beta subtypes were present in HUCB-NSCs and HUCB-NSCDs; however, the voltage-gated type XII and non–voltage-gated 1 beta subtypes were not expressed in HUCB-MCs. The transient receptor potential cation channel was not expressed in HUCB-MCs but was present in HUCB-NSCs and HUCB-NSCDs.

Immunolabeling of Neurotransmitter Receptors
HUCB-NSCDs were maintained under differentiating conditions for 14 days and then immunolabeled for neurotransmitter receptors commonly expressed in the CNS, namely glutamate, GABA, glycine, serotonin, DA, and acetylcholine receptors. Many (88% ± 6.5%) HUCB-NSCD cells showed strong immunolabeling for kainate GluR2 receptor subunit; small puncta were observed over the soma and over the long, thin processes extending from the cell body (Figs. 2A, 2CGo). HUCB-NSCD immunopositive for GluR2 also coexpressed ß-tubulin III (Figs. 2B, 2CGo), an early neuronal maker [22]. ß-Tubulin III immunolabeling was strongly expressed in the processes extending from the soma, whereas minimal labeling was seen in the nuclear region. Almost all (93% ± 7%) HUCB-NSCDs were immunopositive for GABA-AR (Figs. 2D, 2FGo). These cells were also immunopositive for ß-tubulin III, seen as thin strands in the soma and in the processes extending from the cell body (Figs. 2E, 2FGo). Because immature neurons in the CNS often express GABA, we examined whether HUCB-NSCDs express this neurotransmitter and thus could be regulated by GABA in an autocrine or paracrine fashion. Strong GABA immunolabeling was observed in 90% ± 2.5% of NSCD, as shown in Figure 2GGo. The high percentage of HUCB-NSCDs immunopositive for GABA and GABA-AR is consistent with an autocrine/paracrine mechanism.



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  		Figure 2. (A): Photomicrograph showing immunolabeling of glutamate receptor using polyclonal antiglutamate receptor 2 antibody, goat anti-rabbit Cy3 secondary Ab. (B): Same cell as in (A) immunolabeled for ß-tubulin III using monoclonal Ab and goat anti-mouse FITC secondary Ab. (C): Merger of (A) and (B). (D): Immunolabeling of GABA-A receptor using polyclonal antibody and goat anti-rabbit Cy3 secondary Ab. (E): Same cell as in (D) immunolabeled for ß-tubulin III using monoclonal Ab and goat anti-mouse FITC secondary Ab. (F): Merger of (D) and (E). (G): Immunolabeling of GABA using a monoclonal anti-GABA and goat anti-mouse Alexa-555 secondary Ab. Scale bar: 50 µM. Abbreviations: Ab, antibody, FITC, fluorescein isothiocyanate; GABA, {gamma}-aminobutyric acid.

 
Figure 3Go shows HUCB-NSCD cells double-labeled with To-Pro 1, which stains the nuclei, and antibodies against GlyR (A), nicotinic AChR (B), 5-HT1CR (C), and DA (D) receptors. Immunolabeling for all neurotransmitter receptors examined was absent from negative controls in which the primary Ab was omitted (data not shown). GlyR immunolabeling was present in approximately 20% ± 2.5% of HUCB-NSCD. Labeling appeared as fine or granular puncta on the soma as well as on the processes extending from the cell body (Fig. 3AGo). Nicotinic AChR immunolabeling were observed on the soma, often as large puncta, and on neurites (Fig. 3BGo). Nicotinic AChR immunolabeling was seen on approximately 39.3% ± 2.5% of HUCB-NSCDs. 5HT1CR immunolabeling was present as granular puncta on the soma and fine labeling on neurites; immunolabeling was observed on 90.3% ± 3.5% of HUCB-NSCD. DA receptor D2 immunolabeling was present on 85% ± 2.5% of HUCB-NSCD cells; patches of granular labeling were often observed on the soma and neurites (Fig. 3DGo). None of the above immunolabeling was observed when the specific primary antibodies were omitted (not shown).



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  		Figure 3. Immunolabeling of (A) glycine, (B) acetylcholine-nicotinic, (C) 5-HT, and (D) D2 dopamine receptors in human umbilical cord blood differentiated neural stem cells. Goat anti-mouse Alexa 555 secondary antibody used to detect acetylcholine and 5-HT receptors; goat anti-rabbit Cy3 used to detect glycine and D2 receptors. Nuclei stained with ToPro-1. Scale bar: 50 µM. Abbreviation: 5-HT, 5-hydroxytryptamine.

 
Electrophysiology of Nondifferentiated and Differentiated HUCB-NSCs
Whole-cell patch-clamp recordings were made from floating HUCB-NSCs (Fig. 1BGo) and from HUCB-NSCDs attached to poly-lysine/laminin–coated plates and treated with dBcAMP/CPT for 1–28 days (Fig. 1CGo). The mean membrane potential of HUCB-NSCs was –48 ± 23 mV (n = 14), whereas the mean membrane potential of HUCB-NSCDs treated with dBcAMP/CPT (Sigma, 300 µM) acid for 5–28 days was –51 ± 20 mV (n = 57). Although the membrane potential of HUCB-NSCD was slightly greater than HUCB-NSC, this difference was not statistically significant (t-test).

An inward rectifier potassium current, Kir [32], was present in HUCB-NSCs and HUCB-NSCDs (Fig. 4AGo). Consistent with the expression of the Kir gene (Table 1Go), we recorded the Kir current in almost all HUCB-NSCs and HUC-NSCDs (n = 116). Under whole-cell recording conditions, Kir was activated during hyper-polarizing voltage steps (–50 mV holding potential, –140 to +30 mV, 10-mV step). Kir increased in amplitude with voltage steps more negative than –70 mV (Fig. 4AGo); no current was observed at voltages more positive than –70 mV when the external K+ concentration was 7 mM (Fig. 4BGo, open circle). To confirm that Kir was selectively permeable to K+, we varied the external K+ and measured the reversal potential. When the external K+ concentration was changed from 7 to 30 mM, the reversal potential of Kir shifted from –70 to –40 mV (Fig. 4B Go,n = 4). These data are consistent with the predicted reversal potential for potassium calculated from the Nernst equation. Kir was completely eliminated by external Cs+ (5 mM, Fig. 4C Go,n = 59), Ba2+ (5 mM, Fig. 4D Go,n = 5), or Cd2+ (0.1 mM, Fig. 4E Go,n = 10), known antagonists of Kir [33–35]; Kir quickly recovered after Cs+, Ba2+, or Cd2+ (0.1 mM, Fig. 4EGo) was washed out (n = 46). The amplitude of Kir was largely unaffected by the antagonists 4-AP (Fig. 4FGo) or TEA (15 mM) (data not shown) [36]. The average amplitude of Kir induced by stepping the voltage from –50 to –140 mV was –0.33 ± 0.18 nA (n = 14) in floating HUCB-NSCs and –0.7 ± 0.5 nA (n = 80) in attached HUCB-NSCDs that had been differentiated for 5 days or more (Fig. 4BGo). This difference was statistically significant (t-test, p < .01).



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  		Figure 4. Inward rectifier potassium current (Kir) induced from human umbilical cord blood differentiated neural stem cells. (A): Typical Kir current traces induced by negative voltage steps (–50 to –140 mV, 10-mV step). Command voltage shown below. (B): I/V curve of Kir recorded from cell with 7 mM or 30 mM K+ in the bath solution. Note reversal potential of Kir switches from approximately –70 mV with 7 mM K+ to –30 mV with 30 mM K+. (C): Kir was reversibly blocked by external Cs+ (5 mM). (D): Kir was reversibly blocked by external Ba2+ (5 mM). (E): Kir was reversibly blocked by external Cd2+ (0.1 mM). (F): Addition of 4-AP did not change the I/V curve for Kir.

 
Outward Rectifying IK+ Increases with Differentiation
IK+ was not detected in floating HUCB-NSCs. However, when HUCB-NSCDs were differentiated for 5–28 days, IK+ was observed along with Kir. IK+ was present in approximately 40% (40 of 105) of HUCB-NSCD when the voltage was stepped from –50 to +80 mV (–50-mV holding potential, 10-mV step, 50 ms) (Fig. 5AGo). To isolate IK+ from Kir, Cs+ was added to the bath solution (Fig. 5BGo), revealing a slowly activating IK+ at voltages more positive than –10 mV (Figs. 5B, 5CGo; n = 24). IK+ could be blocked by external TEA (15 mM) (Fig. 5D Go,n = 4) or 4-AP (1 mM) (Fig. 5E Go,n = 4), consistent with the criteria of a slowly activating, outward-rectifying potassium current [33]. IK+ was partially blocked by Cd2+ (100 µM, Fig. 5F Go,n = 10), suggesting that a calcium-activated potassium current makes a small contribution to the current measured at voltages more positive than +30 mV (Figs. 5D, 5FGo). This interpretation is consistent with expression of a gene for a calcium-activated potassium channel (Table 1Go).



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  		Figure 5. Outward potassium current (IK+) and Kir current present in human umbilical cord blood differentiated neural stem cells. (A): Kir and IK+ were induced in voltage clamp (holding potential –50 mV, voltage step from –140 to +50 mV, 10-mV step). (B): Cs+ (5 mM) can block Kir but not IK+. (C): I/V curve of IK+ and Kir currents ({bullet}); Kir was blocked by Cs+ but not IK+ ({circ}). (D): IK+ was activated around –30 mV ({bullet}) and was reversibly blocked by tetraethylammonium (TEA) (15 mM, {circ}). (E): Ik+ was reversibly blocked by external 4-AP (1 mM). (F): Ik+ was partly blocked by external Cd2+ (0.1 mM).

 
The percentages of HUCB-NSCs and HUCB-NSCDs expressing Kir and IK+ under undifferentiated conditions and differentiated conditions (attached and treated with dBcAMP/CPT) for 2–5 days or more are shown in Figure 6AGo. The percentage of cells expressing Kir showed little change over time; however, the percentage of cells expressing IK+ increased from 0% (0 of 14) in the undifferentiated condition to approximately 19% (7 of 36) in differentiation conditions.



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  		Figure 6. Comparison of Kir and IK+ in HUCB-NSC versus HUCB-NSCD. (A): Kir was recorded from nearly all (~95%) HUCB-NSCs and HUCB-NSCDs. IK+ only expressed in HUCB-NSCD. IK+ was absent from HUCB-NSC; only a few HUCB-NSCDs (7 of 36) expressed IK+ after 2–5 days of attachment, whereas approximately 46% (35 of 73) of HUCB-NSCD expressed IK+ after more then 5 days of attachment. (B): The average amplitude of Kir from HUCB-NSC was approximately 0.3 nA versus 0.7 nA for HUCB-NSCD (t-test, p < .01). IK+ amplitude increased from 0.4 ± 0.6 nA (n = 7) to 1.5 ± 1.9 nA (n = 35) in cells differentiated for 2–5 days versus >5 days. Abbreviations: HUCB, human umbilical cord blood; NSC, neural stem cell; NSCD, differentiated neural stem cell.

 
The amplitude of Kir showed only a slight increase from undifferentiated to differentiated conditions; Kir amplitude was 0.33 ± 0.18 nA (n = 14) in undifferentiated cells and increased to 0.61 ± 0.5 nA (n = 28) in cells differentiated 2–5 days and to 0.69 ± 0.5 nA (n = 71) in cells differentiated >5 days (Fig. 6BGo). Cells expressing IK+ were first observed after being maintained under differentiating conditions for 2–5 days. IK+ amplitude was 0.4 ± 0.6 nA (n = 7) after being differentiated for 2–5 days and 1.5 ± 1.9 nA (n = 35) when differentiated for more than 5 days. These results indicate that differentiating conditions enhanced the expression of IK+ but have little effect on Kir expression or amplitude.

Excitability of NSCs
In some HUCB-NSCDs (n = 8), current pulses (0 to +200 pA, 140 ms; Fig. 7AGo) induced an action potential–like response [37] consisting of a rapid depolarization followed by a partial repolarization. To identify the ionic conductance mediating this action potential–like response, TEA was added to the bath solution to block IK+; this eliminated the repolarizing phase, leaving behind a steady depolarization (Fig. 7BGo). The depolarization could not be blocked by 300 nM tetrodotoxin (TTX) (n = 8), a sodium channel blocker (data not shown) that blocks TTX-sensitive INa+ but not TTX-resistant INa+ at the concentration used here [38]. Although NSCDs were differentiated for up to 4 weeks, we failed to detect INa+, a hallmark of mature neurons that generate action potentials. These results suggest that the action potential–like response seen in HUCB-NSCDs (Fig. 6AGo) arises from the interaction of Kir and a slowly activating IK+. We cannot completely exclude the possibility that a TTX-resistant sodium current or voltage-gated calcium current is involved in this action potential–like response; however, this seems unlikely given that the response activates slowly (~7 ms).



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  		Figure 7. (A): Depolarizing response of human umbilical cord blood differentiated neural stem cells in response to a current pulse (0 to +200 pA, 140 ms). (B): Tetraethylammonium (TEA), which blocks IK+, eliminates the onset spike and increases the width of the depolarizing response.

 
Neurotransmitter Receptors
To determine if the neurotransmitter receptors identified by gene arrays and immunocytochemistry were functional, we performed whole-cell patch-clamp recordings while applying receptor agonists and antagonists. To test for N-methyl-D-asparate(NMDA) receptors, we applied NMDA (1 mM) to HUCB-NSCDs at a holding potential of –50 mV. Application of NMDA failed to induce a current in the four cells tested (data not shown). Application of NMDA also failed to alter the amplitude of Kir (data not shown). To test for non-NMDA glutamate receptors, we applied KA to HUCB-NSCDs. At a holding potential of –70 mV, KA induced a non desensitizing, inward current in some HUCB-NSCDs (4 of 45; Fig. 8AGo). The KA-induced current was totally blocked by CNQX, a non-NMDA receptor antagonist (Fig. 8BGo). The KA-induced current recovered when CNQX was washed out (Fig. 8CGo).



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  		Figure 8. (A): KA (0.5 mM) induced an inward current from human umbilical cord blood differentiated neural stem cells. (B): The inward current was totally blocked by CNQX, a non-NMDA receptor antagonist. (C): CNQX-induced suppression was eliminated when KA was applied alone. (D): KA (0.5 mM) partly blocked the Kir induced by voltage step (below). (E): CNQX (0.1 mM) blocked the KA (0.5 mM)-induced suppression of Kir induced by voltage step (below). Abbreviations: CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; KA, kainic acid; NMDA, N-methyl-D-asparate.

 
Application of KA (0.5 mM) onto HUCB-NSCD suppressed the amplitude of Kir by 22% ± 7% (n = 8, 8 of 10) induced by a voltage step (–50 to –140 mV) (Fig. 8DGo). To confirm that this effect was mediated through non-NMDA receptors, we applied KA in the presence of CNQX (0.1 mM). CNQX completely blocked the suppressive effect of KA on Kir amplitude (Fig. 8EGo); Kir quickly recovered when CNQX was washed out (data not shown).

To test for the presence of functional glycine receptors, HUCB-NSCDs were maintained at a holding potential of –50 mV to avoid activating Kir. Under these conditions, glycine (1 mM) failed to induce a current (data not shown). To determine if glycine had an effect on the amplitude of Kir, glycine was perfused onto cells while stepping the voltage from a holding potential of –50 to –140 mV. Glycine suppressed the amplitude of Kir by up to 80% (Fig. 9AGo) in most cells (five of six). To confirm that this effect was mediated by glycine receptors, we perfused glycine in the presence of strychnine (0.1 mM), a glycine receptor antagonist. Strychnine completely blocked the effect of glycine on Kir amplitude (data not shown); Kir quickly recovered when strychnine was washed out.



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  		Figure 9. Both (A) glycine (1 mM) and (B) GABA (1 mM) suppressed the Kir induced by a negative voltage step (–50 to –140 mV). Abbreviation: GABA, {gamma}-aminobutyric acid.

 
To test for the presence of GABA receptors, HUCB-NSCDs were maintained at –50 to avoid activating Kir. Under these conditions, GABA (1 mM) failed to induce a current in any cells (n = 8, data not shown). To determine if GABA had an effect on the amplitude of Kir, GABA was perfused onto cells while stepping the voltage from a holding potential of –50 to –140 mV (Fig. 9BGo). Application of GABA (1 mM) reduced the amplitude of Kir by 58% ± 30% in most cells (five of eight).

To test for the presence of functional ACh receptors, HUCB-NSCDs were maintained at a holding potential of –50 mV to avoid activating Kir. Under these conditions, ACh (1 mM) failed to induce a current (data not shown). To determine if ACh had an effect on the amplitude of Kir, ACh was perfused onto cells while stepping the voltage from a holding potential of –50 to –140 mV (Fig. 10AGo). ACh suppressed the amplitude of Kir (Fig. 10AGo) in 5 of 13 cells; the average reduction was 33% ± 22%. To assess the effect of ACh in more detail, the holding potential was set to –100 mV to produce a sustained activation of Kir, and then ACh or nicotine was perfused onto cells. ACh (Fig. 10BGo) and nicotine (Fig. 10CGo) suppressed Kir; Kir recovered after washout of these agonists.



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  		Figure 10. Human umbilical cord blood differentiated neural stem cells clamped to –140 mV to induce Kir. (A): ACh (1 mM) partially blocks Kir; Kir partly recovers after ACh was washed out. (B): Nicotine (1 mM) greatly reduces Kir; Kir recovers after nicotine washed out. Abbreviation: ACh, acetylcholine.

 
To test for the presence of serotonin receptors, HUCB-NSCD was maintained at a holding potential of –50 mV to avoid activating Kir. Under these conditions, 5-HT failed to induce a current (data not shown). To determine if 5-HT had an effect on the amplitude of Kir, 5-HT was perfused onto cells while repeatedly stepping the voltage from a holding potential of –50 to –140 mV (Fig. 11AGo). 5-HT greatly reduced the amplitude of Kir (Fig. 11AGo) in 5 of 13 cells; the average reduction was 65% ± 22%. To confirm that this effect was mediated through 5-HT receptors, 5-HT was applied in the presence of L-278,276, a 5-HT receptor antagonist (0.5 mM). L-278,276 greatly reduced the suppressive effect of 5-HT on Kir amplitude (Fig. 11BGo).



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  		Figure 11. Human umbilical cord blood differentiated neural stem cells. (A): 5-HT suppresses Kir amplitude induced by negative voltage step (–50 to –140 mV); Kir amplitude recovers after 5-HT washed out. (B): 5-HT–induced suppression of Kir largely blocked by L-278,276, a 5-HT receptor antagonist. (C): Kir activated at holding potential of –100 mV. DA (1 mM) suppresses Kir amplitude; amplitude recovers after DA washed out. Abbreviations: 5-HT, 5-hydroxytryptamine; DA, dopamine.

 
To test for the presence of DA receptors, HUCB-NSCDs were maintained at a holding potential of –50 mV to avoid activating Kir. Under these conditions, DA failed to induce a current (data not shown). To determine if DA had an effect on Kir amplitude, cells were maintained at a holding potential of –100 mV to activate Kir, and DA (1 mM) was perfused onto cells (Fig. 11CGo); DA gradually reduced the amplitude by 59% ± 16% (n = 5) of Kir in 5 of 13 cells.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Summary
 References
 
The main finding of this study is that when HUCB-NSCs are maintained under differentiating conditions, they express neu-rotransmitter receptors, ion channels, and electrophysiological properties similar to those seen in immature neurons or glia. The results provide additional evidence on the neural-like features of HUCB-NSCs and HUCB-NSCDs. Our immunocytochemical results show that polylysine/laminin-attached HUCB-NSCDs grown in 2% FBS and treated with dBcAMP/CPT differentiate almost exclusively (>80%) into neuron-shaped cells expressing ß-tubulin III, a neuron-specific filament protein characteristic of developing neurons (unpublished data). Only a few cells expressed astrocytic (GFAP) or oligodendrocytic (GalC) markers, indicating that under these conditions, HUCB-NSCDs preferentially develop toward a neuronal phenotype. The preferential differentiation of HUCB-NSCs to neuronal-like cells could be related to their fetal origin, because ontogenetically younger stem cells give rise more frequently to neurons than to astrocytes. Microarray analysis (Table 1Go) identified other neuronal markers in proliferating, nonattached HUCB-NSCs and polylysine/laminin-attached HUCB-NSCDs; these included genes encoding a diverse set of voltage-dependent potassium and sodium channels and neurotransmitter receptors (ACh, GABA, glutamate, glycine, 5-HT, and DA). Many of these genes (GABA-A and glycine A receptors, glutamate kainite 4 receptor, potassium voltage-gated KQT-like channel, sodium voltage-gated type XII alpha channel) were not expressed in HUCB-MCs, which served as a starting cell population for HUCB-NSCs. In addition, expression of some neuronal genes (GABA-A alpha 2 and glutamatergic kainate receptor 4) increased in HUCB-NSCDs, indicating further differentiation toward a neuronal phenotype under differentiation conditions.

Voltage-Gated Ion Channels
There is growing evidence that potassium channels play an important role in early embryonic development [39] and later stages of differentiation [40, 41]. We observed two types of potassium channels, Kir and IK+, in HUCB-NSCs. Kir channels were constitutively expressed in both proliferating HUCB-NSCs (93%) and attached (94%–99%) HUCB-NSCDs (Fig. 4Go), results consistent with the gene expression data in Table 1Go. Kir channels play an important role in cell proliferation during early kidney development [42]. Treatment of primitive human hematopoietic progenitor cells (CD34+, CD38–) with stem cell factor (SCF) and interleukin-3 (IL-3) enhances the expression of Kir channels and promotes the expansion of these cells into lineage-restricted precursors [32, 34]. Antisense oligonucleotides against Kir blocked the expression of Kir channels in primitive hematopoietic progenitors stimulated with SCF and IL-3 and prevented the expansion and differentiation of these cells. Similarly, growth-stimulated proliferation of microglia was inhibited by the Kir blocker Ba2+ [43]. These results suggest that Kir channels play an important role in proliferation and differentiation of stem cells. Future studies directed at blocking Kir channels would help to elucidate the role of these potassium channels in the differentiation of HUCB-NSCs.

Outward potassium currents are present in many cell types [44]; however, we did not observe outward-rectifying IK+ in undifferentiated HUCB-NSCs. Interestingly, treatment of attached cells with dBcAMP/CPT resulted in the expression of outward IK+ in approximately half of the cells cultured for >5 days (Fig. 4Go). The outward-rectifying IK+ was partially blocked by 4-AP and TEA, criteria consistent with a delayed, outward-rectifying potassium current [33]. In our dBcAMP/CPT-differentiated HUCB-NSCDs, more than 80% expressed ß-tubulin III; these results suggest that many of the HUCB-NSCDs expressing the delayed, outward IK+ had differentiated toward neural progenitors. Our results are similar to those seen in stem cells derived from the subventricular zone of postnatal rat; potassium currents were observed only in attached, EGF-differentiated cells with neuronal morphology and not in dividing, undifferentiated cells [45].

The IK+ seen in differentiated HUCB-NSCs was partially blocked by Cd2+, suggesting that calcium-activated potassium current may make a small contribution to the outward-rectifying potassium current. The gene expression data (Table 1Go), however, showed evidence for a large-conductance, calcium-activated potassium channel in both undifferentiated and differentiated HUCB-NSCs. It is unclear why calcium-activated potassium currents were not seen in undifferentiated HUCB-NSCs, but their absence suggests that other factors are required for these channels to be functionally expressed in HUCB-NSCs.

Although our gene microarray data provide evidence for voltage-gated sodium channels (types X and XII) in HUCB-NSCDs, we did not find any evidence of voltage-gated sodium currents or action potentials, a hallmark of mature neurons, in HUCB-NSCs or HUCB-NSCDs. Although HUCB-NSCD expressed many neuronal markers, the absence of rapidly activating inward sodium currents after differentiation with dBcAMP/CPT suggests that other factors or conditions are required before these cells can generate action potentials. Human NSCs also lack voltage-gated sodium current; however, sodium currents could be induced in these cells after transfection with Neuro D, aneurogenic transcription factor [46], or after priming for 2 weeks with the cocktail of bFGF, heparin, and laminin [47]. Rat embryonic stem cells fired action potential exclusively after transfection with Nurr1, and when transplanted into the rat striatum, they differentiate toward functional dopaminergic neurons [4]. Adult NSCs isolated from rat hippocampus also developed action potentials when cocul-tured with primary neurons and astrocytes [48]. In bone marrow–derived, multipotent adult progenitor cells, spiking behavior and voltage-gated currents were observed after coculture of neural-committed progenitors with fetal mouse brain astrocytes [49]. On the other hand, human bone marrow–derived NSCs induced to differentiate using neuromorphogens developed voltage-dependent potassium channels but failed to develop voltage-dependent sodium current required for action potentials [50]. Further studies are needed to determine if other factors or conditions can lead to the further differentiation and generation of sodium currents and action potentials in HUCB-NSCs.

In preliminary studies, we looked for evidence of voltage-gated calcium current (data not shown) but failed to detect any. This result is consistent with the absence of voltage-gated calcium channels in our gene microarray data (Table 1Go). Our inability to detect high voltage–activated calcium currents could be due to the recording conditions and, specifically, the presence of fluoride in the recording pipette, which blocks high voltage–activated calcium channels in some cells [51]. However, in other cells, fluoride has been reported to block the rundown of high voltage–activated calcium currents [52].

Ligand-Gated Ion Channels and Neurotransmitters
Differentiated HUCB-NSCs could conceivably be used to repair damaged neural circuits in the brain and spinal cord; however, the integration of HUCB-NSCs into a neural network requires that cells express ligand-gated receptors that can respond to neurotransmitters released from presynaptic inputs. Neurotransmitters acting though their cognate receptors can also regulate cell proliferation, direct neuronal migration, and promote differentiation in the developing nervous system [53].

Many neurotransmitter receptors were identified on HUCB-NSCs. Microarray analysis indicated that kainate receptors (Table 1Go) but not NMDA receptors were expressed on differentiated and non differentiated HUCB-NSCs but not on HUCB-MCs. Whole-cell recordings revealed functional, kainate-sensitive, non-NMDA glutamate receptors on differentiated HUCB-NSCs, consistent with immunolabeling results (Fig. 2Go). NMDA-induced currents were not detected in whole-cell recordings, consistent with our microarray data. KA induced inward currents in 10% of differentiated HUCB-NSCs and suppressed Kir in most HUCB-NSCs. The latter effect has been attributed to a receptor-mediated influx of sodium, which blocks the intracellular face of the channel [54]. Functional ionotropic glutamate receptors emerge during terminal cell division and early neuronal differentiation of rat neuroepi-thelial cells [55] and NSCs from hippocampus [48]. The influx of calcium through kainate receptors promotes dendrite outgrowth [56]. In addition to their obvious role in rapid neurotransmission, kainate receptors inhibit the proliferation and lineage progression in oligodendrocyte progenitors by increasing intracellular sodium and inhibiting outward potassium currents [57].

Surprisingly, our microarray analysis revealed metabotropic glutamate receptor 6 (mGluR6) in both HUCB-NSCs and HUCB-MCs (Table 1Go). mGluR6 is thought to be exclusively expressed in the retina, where it induces a hyperpolarization in bipolar cells in response to glutamate [58]. In addition, mGluR6 expression is upregulated in the retina under conditions that induce apoptosis [59]. Activation of mGluR6 inhibits adenylate cyclase, leading to a decrease in cAMP, one of the factors added to the cultures to enhance differentiation [60].

Glycine receptor beta mRNA was expressed in undifferentiated and differentiated HUCB-NSCs. In addition, differentiated HUCB-NSCs coexpressed GlyR and ß-tubulin III, an early neural marker, and responded to exogenous glycine by suppressing Kir amplitude. Glycine receptors and their mRNAs have been identified on stem cells derived from rodent striatum [61], neonatal oligodendrocyte progenitors [62], and human corneal limbal stem cells [29]. The functional role of glycine receptors in HUCB-NSCs is unclear; however, in the retina, taurine or the combination of glycine and GABA, acting through glycine receptors expressed on neonatal retinal progenitor cells, promotes exit from the cell cycle and significantly increases the number of rod photoreceptors [63, 64]. These results suggest that glycine receptors could regulate cell proliferation and influence the fate of HUCB-NSCs.

GABA-A receptor mRNAs ({alpha}2, 1, and {gamma}3) were identified in differentiated HUCB-NSCs. Moreover, nearly all HUCB-NSCDs immunopositive for ß-tubulin III, an early neural marker, were also immunopositive for GABA-AR and GABA, suggesting an autocrine/paracrine role for GABA. Exogenous GABA also suppressed the voltage-gated Kir in HUCB-NSCDs. The function of GABA in the developing nervous system is poorly understood; however, there is growing evidence that GABA, acting through GABA-A receptors, serves as a trophic factor and influences cell proliferation, differentiation, migration, and synapse development [65]. Activation of GABA-A receptors depolarizes cells in proliferative zones of the neocortex and suppresses cell division [66], and GABA promotes chemotactic and chemokinetic responses in embryonic cortical cells [67]. Moreover, postnatal cells in the striatum synthesize and release GABA, creating an autocrine/paracrine mechanism that controls their proliferation [68].

All studied cell populations (HUCB-MC, HUCB-NSC, and HUCB-NSCD) expressed mRNAs for three types of serotonin receptors (1C, 2A, and 3A). In addition, nearly all differentiated HUCB-NSCs were immunopositive for the 5-HT1C receptor, and application of serotonin suppressed Kir through its receptor. The function role of serotonin receptors on HUCB cells is not well understood; however, serotonin has been shown to promote the survival of cortical progenitor cells with glutamatergic characteristics [69]. In the adult hippocampus, serotonin acting through 5HT2A receptors regulates cell proliferation [70, 71]. 5-HT1C receptors are expressed on human fetal glioma cell lines, and application of serotonin was shown to modulate proliferation, migration, and tumor invasion [72].

Nicotinic AChR (alpha 5, beta 3, and epsilon) were expressed on HUCB-MC, HUCB-NSC, and HUCB-NSCD (Table 1Go). HUCB-NSCDs were immunopositive for nicotinic AChR beta, and perfusion of ACh and nicotine suppressed Kir. The functional roles of these AChRs are not well understood; however, ACh has been implicated in the proliferation and differentiation of neural progenitor cells. ACh and functional nAChR are present on neural progenitors in embryonic mouse cerebral cortex, suggesting a role in development [73]. Oligodendrocyte progenitor cells also express nAChR, but expression disappears after cells differentiate into oligodendrocytes or astrocytes [74].

Because of the therapeutic potential in Parkinson’s disease, there is considerable interest in stem or progenitor cells that express DA and its receptors. DA D2 and D5 receptor mRNAs were expressed on HUCB-MCs as well as HUCB-NSCs and HUCB-NSCDs. DA receptor D2 immunolabeling was also seen on HUCB-NSCDs, and DA application suppressed Kir amplitude in these cells. In the neostriatum, DA D1 and D2 receptors repress and enhance neurogenesis, respectively [75]. Moreover, DA acting through D1 receptors promotes neural differentiation by stimulating neurite outgrowth and growth cone formation [76, 77]. All together, these results suggest that the DA receptors on HUCB-NSCs may be involved in cell proliferation and differentiation.


   SUMMARY
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
References
 
Our results show that neural stem–like cells derived from the nonhematopoietic fraction of HUCB and expand as a stable, clonogenic line over a long time, retaining their capacity to differentiate into neuron-like cells that express neuron-specific cytoskeletal markers, numerous neurotransmitter receptors, and several functional voltage-gated channels. Kir channels are expressed in most HUCB-NSCs and HUCB-NSCDs; however, IK+ was observed only in some HUCB-NSCDs. Although the bulk of the evidence suggests that HUCB-NSCDs are progressing along a neuronal lineage, the absence of voltage-gated sodium channels clearly indicates that this process is incomplete and that other factors or conditions are required for these cells to fully differentiate into neurons. One approach that might further advance differentiation of HUCB-NSCDs toward more mature neurons would be to coculture these cells with astrocytes [48] or organotypic brain slices [78]. During early development, neurotransmitters as a class of secreted molecules could also influence proliferation, migration, and differentiation [63, 64, 67, 70–72]. An alternative method that could provide important clues regarding the specific roles neurotransmitters play in proliferation and development would be to culture HUCB-NSCs or HUCB-NSCDs with neurotransmitter agonists or antagonists for ACh, GABA, glutamate, glycine, serotonin, and DA. Finally, a third approach would be to transfect HUCB-NSCs with neuro-genic transcription factors or treat them with other morphogens that promote neuronal phenotypes [4, 46].


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 References
 
W.S. and L.B. contributed equally to this study. This research was supported by grants from the NIH (P01 DC03600 to R.J.S., R01 DC06630 to R.J.S., NS46321-01 to M.K.S.), Oishei Foundation (to M.K.S.), NSF (IBN-9728923 to M.K.S.), American Parkinson Disease Association (to M.K.S.), and Polish State Committee for Scientific Research (K053/P05/2003 to K.D.-J.).


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 References
 

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