Voltage-Sensitive and Ligand-Gated Channels in Differentiating Neural Stem–Like Cells Derived from the Nonhematopoietic Fraction of Human Um
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《干细胞学杂志》
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@buffalo.edu
ABSTRACT
Neuronal and glial populations in the developing brain are generated from multipotent neural stem cells (NSCs) located predominantly in the subventricular zone and hippocampus . 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 . 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 . 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 . Mouse embryonic NSCs transplanted into animal models of Parkinson’s differentiate into dopamine (DA)–containing neurons with their characteristic electrophysiological properties . Moreover, intravenous or intrathecal injections of adult neural precursor cells into animal models of multiple sclerosis promote multifocal remyelination and functional recovery .
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 and even to differentiate into astrocytes and tyrosine hydroxylase (TH)–expressing neurons that synthesize DA when grafted into the Parkinsonian mouse . Moreover, in patients receiving therapeutic bone marrow transplants, some transplanted cells migrate into the brain and display fusion-independent neuronal phenotype . This suggests that non embryonic stem cells from sources other than the nervous system could be used for neuron-replacement therapy .
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 . Indeed, when transplanted into the brain, the mononuclear fraction of HUCB was shown to survive in brain tissue and reduce motor and neurological deficits . 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 .
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 , 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. 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 . 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 ?-tubulin III and MAP-2, 30% developed astrocyte phenotypes expressing GFAP and S100?, whereas 11% of cells developed oligodendroglial phenotypes expressing galactosylceramide (GalC) . 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
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 . 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. 1A, arrowhead; Fig. 1B). These cells expressed nestin and GFAP but did not express ?-tubulin III or other markers of advanced neuronal (MAP2) or astroglial (S100?) differentiation (data not shown). The second population consisted of flattened cells that attached to the plastic wells (Fig. 1A, arrow; Fig. 1C). These cells expressed early markers of neuronal differentiation, including NF-200, NF-70, and ?-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–1F). These HUCB-NSCDs were immunopositive for ?-tubulin III (80%) (Fig. 1D), neurofilament NF-200 (Fig. 1E), and NF-70 (Fig. 1F), a late, structural, neural marker. Relatively few cells (<19%) with astrocyte-like morphology and GFAP immunoreactivity were observed.
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) ?-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 , (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 1. 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.
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 1, 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) 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, 2C). HUCB-NSCD immunopositive for GluR2 also coexpressed ?-tubulin III (Figs. 2B, 2C), an early neuronal maker . ?-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, 2F). These cells were also immunopositive for ?-tubulin III, seen as thin strands in the soma and in the processes extending from the cell body (Figs. 2E, 2F). 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 2G. The high percentage of HUCB-NSCDs immunopositive for GABA and GABA-AR is consistent with an autocrine/paracrine mechanism.
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 ?-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 ?-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, -aminobutyric acid.
Figure 3 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. 3A). Nicotinic AChR immunolabeling were observed on the soma, often as large puncta, and on neurites (Fig. 3B). 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. 3D). None of the above immunolabeling was observed when the specific primary antibodies were omitted (not shown).
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. 1B) and from HUCB-NSCDs attached to poly-lysine/laminin–coated plates and treated with dBcAMP/CPT for 1–28 days (Fig. 1C). 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 , was present in HUCB-NSCs and HUCB-NSCDs (Fig. 4A). Consistent with the expression of the Kir gene (Table 1), 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. 4A); no current was observed at voltages more positive than –70 mV when the external K+ concentration was 7 mM (Fig. 4B, 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 ,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 ,n = 59), Ba2+ (5 mM, Fig. 4D ,n = 5), or Cd2+ (0.1 mM, Fig. 4E ,n = 10), known antagonists of Kir ; Kir quickly recovered after Cs+, Ba2+, or Cd2+ (0.1 mM, Fig. 4E) was washed out (n = 46). The amplitude of Kir was largely unaffected by the antagonists 4-AP (Fig. 4F) or TEA (15 mM) (data not shown) . 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. 4B). This difference was statistically significant (t-test, p < .01).
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. 5A). To isolate IK+ from Kir, Cs+ was added to the bath solution (Fig. 5B), revealing a slowly activating IK+ at voltages more positive than –10 mV (Figs. 5B, 5C; n = 24). IK+ could be blocked by external TEA (15 mM) (Fig. 5D ,n = 4) or 4-AP (1 mM) (Fig. 5E ,n = 4), consistent with the criteria of a slowly activating, outward-rectifying potassium current . IK+ was partially blocked by Cd2+ (100 μM, Fig. 5F ,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, 5F). This interpretation is consistent with expression of a gene for a calcium-activated potassium channel (Table 1).
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 (); Kir was blocked by Cs+ but not IK+ (). (D): IK+ was activated around –30 mV () and was reversibly blocked by tetraethylammonium (TEA) (15 mM, ). (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 6A. 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.
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. 6B). 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. 7A) induced an action potential–like response 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. 7B). 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 . 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. 6A) 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).
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. 8A). The KA-induced current was totally blocked by CNQX, a non-NMDA receptor antagonist (Fig. 8B). The KA-induced current recovered when CNQX was washed out (Fig. 8C).
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. 8D). 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. 8E); 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. 9A) 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.
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, -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. 9B). 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. 10A). ACh suppressed the amplitude of Kir (Fig. 10A) 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. 10B) and nicotine (Fig. 10C) suppressed Kir; Kir recovered after washout of these agonists.
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. 11A). 5-HT greatly reduced the amplitude of Kir (Fig. 11A) 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. 11B).
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. 11C); DA gradually reduced the amplitude by 59% ± 16% (n = 5) of Kir in 5 of 13 cells.
DISCUSSION
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 or organotypic brain slices . During early development, neurotransmitters as a class of secreted molecules could also influence proliferation, migration, and differentiation . 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 .
ACKNOWLEDGMENTS
Eriksson PS, Perfilieva E, Bjork-Eriksson T et al. Neurogenesis in the adult human hippocampus. Nat Med 1998;4:1313–1317.
Magavi SS, Leavitt BR, Macklis JD. Induction of neurogenesis in the neo-cortex of adult mice. Nature 2000;405:951–955.
Hallbergson AF, Gnatenco C, Peterson DA. Neurogenesis and brain injury: managing a renewable resource for repair. J Clin Invest 2003;112:1128–1133.
Kim JH, Auerbach JM, Rodriguez-Gomez JA et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 2002;418:50–56.
Pluchino S, Furlan R, Martino G. Cell-based remyelinating therapies in multiple sclerosis: evidence from experimental studies. Curr Opin Neurol 2004;17:247–255.
Richardson RM, Fillmore HL, Holloway KL et al. Progress in cerebral transplantation of expanded neuronal stem cells. J Neurosurg 2004;100:659–671.
Toda H, Takahashi J, Mizoguchi A et al. Neurons generated from adult rat hippocampal stem cells form functional glutamatergic and GABAergic synapses in vitro. Exp Neurol 2000;165:66–76.
Englund U, Bjorklund A, Wictorin K et al. Grafted neural stem cells develop into functional pyramidal neurons and integrate into host cortical circuitry. Proc Natl Acad Sci U S A 2002;99:17089–17094.
Muraro PA, Cassiani Ingoni R, Martin R. Hematopoietic stem cell transplantation for multiple sclerosis: current status and future challenges. Curr Opin Neurol 2003;16:299–305.
Windrem MS, Nunes MC, Rashbaum WK et al. Fetal and adult human oligodendrocyte progenitor cell isolates myelinate the congenitally dys-myelinated brain. Nat Med 2004;10:93–97.
Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci U S A 1999;96:10711–10716.
Sanchez-Ramos J, Song S, Cardozo-Pelaez F et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol 2000;164:247–256.
Li Y, Chen J, Wang L et al. Intracerebral transplantation of bone marrow stromal cells in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson’s disease. Neurosci Lett 2001;316:67–70.
Mezey E, Chandross KJ, Harta G et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000;290:1779–1782.
Weimann JM, Charlton CA, Brazelton TR et al. Contribution of transplanted bone marrow cells to Purkinje neurons in human adult brains. Proc Natl Acad Sci U S A 2003;100:2088–2093.
Cogle CR, Yachnis AT, Laywell ED et al. Bone marrow trans differentiation in brain after transplantation: a retrospective study. Lancet 2004;363:1432–1437.
Corti S, Locatelli F, Strazzer S et al. Neuronal generation from somatic stem cells: current knowledge and perspectives on the treatment of acquired and degenerative central nervous system disorders. Curr Gene Ther 2003;3:247–272.
Kim DK, Fujiki Y, Fukushima T et al. Comparison of hematopoietic activities of human bone marrow and umbilical cord blood CD34 positive and negative cells. STEM CELLS 1999;17:286–294.
Sanchez-Ramos JR, Song S, Kamath SG et al. Expression of neural markers in human umbilical cord blood. Exp Neurol 2001;171:109–115.
Storms RW, Goodell MA, Fisher A et al. Hoechst dye efflux reveals a novel CD7(+)CD34(–) lymphoid progenitor in human umbilical cord blood. Blood 2000;96:2125–2133.
Zigova T, Song S, Willing AE et al. Human umbilical cord blood cells express neural antigens after transplantation into the developing rat brain. Cell Transplant 2002;11:265–274.
Buzanska L, Machaj EK, Zablocka B et al. Human cord blood-derived cells attain neuronal and glial features in vitro. J Cell Sci 2002;115:2131–2138.
Sanchez-Ramos JR. Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res 2002;69:880–893.
Hung SC, Cheng H, Pan CY et al. In vitro differentiation of size-sieved stem cells into electrically active neural cells. STEM CELLS 2002;20:522–529.
Lu D, Sanberg PR, Mahmood A et al. Intravenous administration of human umbilical cord blood reduces neurological deficit in the rat after traumatic brain injury. Cell Transplant 2002;11:275–281.
Willing AE, Vendrame M, Mallery J et al. Mobilized peripheral blood cells administered intravenously produce functional recovery in stroke. Cell Transplant 2003;12:449–454.
Yoo ES, Lee KE, Seo JW et al. Adherent cells generated during long-term culture of human umbilical cord blood CD34+ cells have characteristics of endothelial cells and beneficial effect on cord blood ex vivo expansion. STEM CELLS 2003;21:228–235.
Buzanska L, Habich A, Jurga M et al. Human cord blood-derived neural stem cell line - possible implementation in studying neurotoxicity. Toxicology In Vitro 2005 (in press).
Seigel GM, Sun W, Salvi R et al. Human corneal stem cells display functional neuronal properties. Mol Vis 2003;9:159–163.
Sun W, Ding D, Wang P et al. Substance P inhibits potassium and calcium currents in inner ear spiral ganglion neurons. Brain Res 2004;1012:82–92.
Sun W, Salvi RJ. Dopamine modulates soduim currents in cochlear spiral ganglion neurons. Neuroreport 2001;12:803–807.
Shirihai O, Attali B, Dagan D et al. Expression of two inward rectifier potassium channels is essential for differentiation of primitive human hematopoietic progenitor cells. J Cell Physiol 1998;177:197–205.
Hille B. Ionic Channels of Excitable Membrane. Sunderlang, MA: Sinauer Associates Inc., 1992.
Shirihai O, Merchav S, Attali B et al. K+ channel antisense oligodeoxy-nucleotides inhibit cytokine-induced expansion of human hemopoietic progenitors. Pflugers Arch 1996;431:632–638.
HuQ, Shi YL. Characterization of an inward-rectifying potassium current in NG108-15 neuroblastoma x glioma cells. Pflugers Arch 1997;433:617–625.
Kuryshev YA, Haak L, Childs GV et al. Corticotropin releasing hormone inhibits an inwardly rectifying potassium current in rat corticotropes. J Physiol 1997;502:265–279.
Tourneur Y. Action potential-like responses due to the inward rectifying potassium channel. J Membr Biol 1986;90:115–122.
Elliott AA, Elliott JR. Characterization of TTX-sensitive and TTX-resistant sodium currents in small cells from adult rat dorsal root ganglia. J Physiol 1993;463:39–56.
Day ML, Pickering SJ, Johnson MH et al. Cell-cycle control of a large-conductance K+ channel in mouse early embryos. Nature 1993;365:560–562.
Patil N, Cox DR, Bhat D et al. A potassium channel mutation in weaver mice implicates membrane excitability in granule cell differentiation. Nat Genet 1995;11:126–129.
Neusch C, Weishaupt JH, Bahr M. Kir channels in the CNS: emerging new roles and implications for neurological diseases. Cell Tissue Res 2003;311:131–138.
Braun GS, Horster MF, Huber SM. Kir1.1 expression in embryonic kidney epithelia. Biochem Biophys Res Commun 2003;312:1191–1195.
Schlichter LC, Sakellaropoulos G, Ballyk B et al. Properties of K+ and Cl– channels and their involvement in proliferation of rat microglial cells. Glia 1996;17:225–236.
Rudy B. Diversity and ubiquity of K channels. Neuroscience 1988;25:729–749.
Feldman DH, Thinschmidt JS, Peel AL et al. Differentiation of ionic currents in CNS progenitor cells: dependence upon substrate attachment and epidermal growth factor. Exp Neurol 1996;140:206–217.
Cho T, Bae JH, Choi HB et al. Human neural stem cells: electrophysiological properties of voltage-gated ion channels. Neuroreport 2002;13:1447–1452.
Wu P, Tarasenko YI, Gu Y et al. Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat. Nat Neurosci 2002;5:1271–1278.
Song H, Stevens CF, Gage FH. Astroglia induce neurogenesis from adult neural stem cells. Nature 2002;417:39–44.
Jiang Y, Henderson D, Blackstad M et al. Neuroectodermal differentiation from mouse multipotent adult progenitor cells. Proc Natl Acad Sci U S A 2003;100(suppl 1):11854–11860.
Hermann A, Gastl R, Liebau S et al. Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells. J Cell Sci 2004;117:4411–4422.
Bertollini L, Biella G, Wanke E et al. Fluoride reversibly blocks HVA calcium current in mammalian thalamic neurones. Neuroreport 1994;5:553–556.
Breakwell NA, Behnisch T, Publicover SJ et al. Attenuation of high-voltage-activated Ca2+ current run-down in rat hippocampal CA1 pyramidal cells by NaF. Exp Brain Res 1995;106:505–508.
Nguyen L, Rigo JM, Rocher V et al. Neurotransmitters as early signals for central nervous system development. Cell Tissue Res 2001;305:187–202.
Schroder W, Seifert G, Huttmann K et al. AMPA receptor-mediated modulation of inward rectifier K+ channels in astrocytes of mouse hippocampus. Mol Cell Neurosci 2002;19:447–458.
Maric D, Liu QY, Grant GM et al. Functional ionotropic glutamate receptors emerge during terminal cell division and early neuronal differentiation of rat neuroepithelial cells. J Neurosci Res 2000;61:652–662.
Metzger F, Wiese S, Sendtner M. Effect of glutamate on dendritic growth in embryonic rat motorneurons. J Neurosci 1998;18:1735–1742.
Gallo V, Zhou JM, McBain CJ et al. Oligodendrocyte progenitor cell proliferation and lineage progression are regulated by glutamate receptor-mediated K+ channel block. J Neurosci 1996;16:2659–2670.
Gerber U. Metabotropic glutamate receptors in vertebrate retina. Doc Ophthalmol 2003;106:83–87.
Dyka FM, May CA, Enz R. Metabotropic glutamate receptors are differentially regulated under elevated intraocular pressure. J Neurochem 2004;90:190–202.
Knopfel T, Grandes P. Metabo tropic glutamate receptors in the cerebellum with a focus on their function in Purkinje cells. Cerebellum 2002;1:19–26.
Nguyen L, Malgrange B, Belachew S et al. Functional glycine receptors are expressed by postnatal nestin-positive neural stem/progenitor cells. Eur J Neurosci 2002;15:1299–1305.
Belachew S, Rogister B, Rigo JM et al. Cultured oligodendrocyte progenitors derived from cerebral cortex express a glycine receptor which is pharmacologically distinct from the neuronal isoform. Eur J Neurosci 1998;10:3556–3564.
Young TL, Cepko CL. A role for ligand-gated ion channels in rod photoreceptor development. Neuron 2004;41:867–879.
Renteria RC, Johnson J, Copenhagen DR. Need rods? Get glycine receptors and taurine. Neuron 2004;41:839–841.
Owens DF, Kriegstein AR. Is there more to GABA than synaptic inhibition? Nat Rev Neurosci 2002;3:715–727.
LoTurco JJ, Owens DF, Heath MJ et al. GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis. Neuron 1995;15:1287–1298.
Behar TN, Li YX, Tran HT et al. GABA stimulates chemotaxis and chemokinesis of embryonic cortical neurons via calcium-dependent mechanisms. J Neurosci 1996;16:1808–1818.
Nguyen L, Malgrange B, Breuskin I et al. Autocrine/paracrine activation of the GABA(A) receptor inhibits the proliferation of neurogenic polysi-alylated neural cell adhesion molecule-positive (PSA-NCAM+) precursor cells from postnatal striatum. J Neurosci 2003;23:3278–3294.
Dooley AE, Pappas IS, Parnavelas JG. Serotonin promotes the survival of cortical glutamatergic neurons in vitro. Exp Neurol 1997;148:205–214.
Malberg JE, Duman RS. Cell proliferation in adult hippocampus is decreased by inescapable stress: reversal by fluoxetine treatment. Neuro-psychopharmacology 2003;28:1562–1571.
Banasr M, Hery M, Printemps R et al. Serotonin-induced increases in adult cell proliferation and neurogenesis are mediated through different and common 5-HT receptor subtypes in the dentate gyrus and the subven-tricular zone. Neuropsychopharmacology 2004;29:450–460.
Merzak A, Koochekpour S, Fillion MP et al. Expression of serotonin receptors in human fetal astrocytes and glioma cell lines: a possible role in glioma cell proliferation and migration. Brain Res Mol Brain Res 1996;41:1–7.
Schneider AS, Atluri P, Shen Q et al. Functional nicotinic acetylcholine receptor expression on stem and progenitor cells of the early embryonic nervous system. Ann N Y Acad Sci 2002;971:135–138.
Rogers SW, Gregori NZ, Carlson N et al. Neuronal nicotinic acetylcholine receptor expression by O2A/oligodendrocyte progenitor cells. Glia 2001;33:306–313.
Ohtani N, Goto T, Waeber C et al. Dopamine modulates cell cycle in the lateral ganglionic eminence. J Neurosci 2003;23:2840–2850.
Schmidt U, Pilgrim C, Beyer C. Differentiative effects of dopamine on striatal neurons involve stimulation of the cAMP/PKA pathway. Mol Cell Neurosci 1998;11:9–18.
Schmidt U, Beyer C, Oestreicher AB et al. Activation of dopaminergic D1 receptors promotes morphogenesis of developing striatal neurons. Neuroscience 1996;74:453–460.
Benninger F, Beck H, Wernig M et al. Functional integration of embryonic stem cell-derived neurons in hippocampal slice cultures. J Neurosci 2003;23:7075–7083.(Wei Suna, Leonora Buzansk)
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@buffalo.edu
ABSTRACT
Neuronal and glial populations in the developing brain are generated from multipotent neural stem cells (NSCs) located predominantly in the subventricular zone and hippocampus . 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 . 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 . 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 . Mouse embryonic NSCs transplanted into animal models of Parkinson’s differentiate into dopamine (DA)–containing neurons with their characteristic electrophysiological properties . Moreover, intravenous or intrathecal injections of adult neural precursor cells into animal models of multiple sclerosis promote multifocal remyelination and functional recovery .
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 and even to differentiate into astrocytes and tyrosine hydroxylase (TH)–expressing neurons that synthesize DA when grafted into the Parkinsonian mouse . Moreover, in patients receiving therapeutic bone marrow transplants, some transplanted cells migrate into the brain and display fusion-independent neuronal phenotype . This suggests that non embryonic stem cells from sources other than the nervous system could be used for neuron-replacement therapy .
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 . Indeed, when transplanted into the brain, the mononuclear fraction of HUCB was shown to survive in brain tissue and reduce motor and neurological deficits . 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 .
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 , 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. 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 . 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 ?-tubulin III and MAP-2, 30% developed astrocyte phenotypes expressing GFAP and S100?, whereas 11% of cells developed oligodendroglial phenotypes expressing galactosylceramide (GalC) . 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
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 . 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. 1A, arrowhead; Fig. 1B). These cells expressed nestin and GFAP but did not express ?-tubulin III or other markers of advanced neuronal (MAP2) or astroglial (S100?) differentiation (data not shown). The second population consisted of flattened cells that attached to the plastic wells (Fig. 1A, arrow; Fig. 1C). These cells expressed early markers of neuronal differentiation, including NF-200, NF-70, and ?-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–1F). These HUCB-NSCDs were immunopositive for ?-tubulin III (80%) (Fig. 1D), neurofilament NF-200 (Fig. 1E), and NF-70 (Fig. 1F), a late, structural, neural marker. Relatively few cells (<19%) with astrocyte-like morphology and GFAP immunoreactivity were observed.
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) ?-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 , (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 1. 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.
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 1, 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) 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, 2C). HUCB-NSCD immunopositive for GluR2 also coexpressed ?-tubulin III (Figs. 2B, 2C), an early neuronal maker . ?-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, 2F). These cells were also immunopositive for ?-tubulin III, seen as thin strands in the soma and in the processes extending from the cell body (Figs. 2E, 2F). 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 2G. The high percentage of HUCB-NSCDs immunopositive for GABA and GABA-AR is consistent with an autocrine/paracrine mechanism.
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 ?-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 ?-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, -aminobutyric acid.
Figure 3 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. 3A). Nicotinic AChR immunolabeling were observed on the soma, often as large puncta, and on neurites (Fig. 3B). 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. 3D). None of the above immunolabeling was observed when the specific primary antibodies were omitted (not shown).
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. 1B) and from HUCB-NSCDs attached to poly-lysine/laminin–coated plates and treated with dBcAMP/CPT for 1–28 days (Fig. 1C). 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 , was present in HUCB-NSCs and HUCB-NSCDs (Fig. 4A). Consistent with the expression of the Kir gene (Table 1), 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. 4A); no current was observed at voltages more positive than –70 mV when the external K+ concentration was 7 mM (Fig. 4B, 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 ,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 ,n = 59), Ba2+ (5 mM, Fig. 4D ,n = 5), or Cd2+ (0.1 mM, Fig. 4E ,n = 10), known antagonists of Kir ; Kir quickly recovered after Cs+, Ba2+, or Cd2+ (0.1 mM, Fig. 4E) was washed out (n = 46). The amplitude of Kir was largely unaffected by the antagonists 4-AP (Fig. 4F) or TEA (15 mM) (data not shown) . 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. 4B). This difference was statistically significant (t-test, p < .01).
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. 5A). To isolate IK+ from Kir, Cs+ was added to the bath solution (Fig. 5B), revealing a slowly activating IK+ at voltages more positive than –10 mV (Figs. 5B, 5C; n = 24). IK+ could be blocked by external TEA (15 mM) (Fig. 5D ,n = 4) or 4-AP (1 mM) (Fig. 5E ,n = 4), consistent with the criteria of a slowly activating, outward-rectifying potassium current . IK+ was partially blocked by Cd2+ (100 μM, Fig. 5F ,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, 5F). This interpretation is consistent with expression of a gene for a calcium-activated potassium channel (Table 1).
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 (); Kir was blocked by Cs+ but not IK+ (). (D): IK+ was activated around –30 mV () and was reversibly blocked by tetraethylammonium (TEA) (15 mM, ). (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 6A. 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.
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. 6B). 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. 7A) induced an action potential–like response 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. 7B). 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 . 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. 6A) 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).
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. 8A). The KA-induced current was totally blocked by CNQX, a non-NMDA receptor antagonist (Fig. 8B). The KA-induced current recovered when CNQX was washed out (Fig. 8C).
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. 8D). 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. 8E); 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. 9A) 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.
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, -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. 9B). 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. 10A). ACh suppressed the amplitude of Kir (Fig. 10A) 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. 10B) and nicotine (Fig. 10C) suppressed Kir; Kir recovered after washout of these agonists.
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. 11A). 5-HT greatly reduced the amplitude of Kir (Fig. 11A) 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. 11B).
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. 11C); DA gradually reduced the amplitude by 59% ± 16% (n = 5) of Kir in 5 of 13 cells.
DISCUSSION
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 or organotypic brain slices . During early development, neurotransmitters as a class of secreted molecules could also influence proliferation, migration, and differentiation . 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 .
ACKNOWLEDGMENTS
Eriksson PS, Perfilieva E, Bjork-Eriksson T et al. Neurogenesis in the adult human hippocampus. Nat Med 1998;4:1313–1317.
Magavi SS, Leavitt BR, Macklis JD. Induction of neurogenesis in the neo-cortex of adult mice. Nature 2000;405:951–955.
Hallbergson AF, Gnatenco C, Peterson DA. Neurogenesis and brain injury: managing a renewable resource for repair. J Clin Invest 2003;112:1128–1133.
Kim JH, Auerbach JM, Rodriguez-Gomez JA et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature 2002;418:50–56.
Pluchino S, Furlan R, Martino G. Cell-based remyelinating therapies in multiple sclerosis: evidence from experimental studies. Curr Opin Neurol 2004;17:247–255.
Richardson RM, Fillmore HL, Holloway KL et al. Progress in cerebral transplantation of expanded neuronal stem cells. J Neurosurg 2004;100:659–671.
Toda H, Takahashi J, Mizoguchi A et al. Neurons generated from adult rat hippocampal stem cells form functional glutamatergic and GABAergic synapses in vitro. Exp Neurol 2000;165:66–76.
Englund U, Bjorklund A, Wictorin K et al. Grafted neural stem cells develop into functional pyramidal neurons and integrate into host cortical circuitry. Proc Natl Acad Sci U S A 2002;99:17089–17094.
Muraro PA, Cassiani Ingoni R, Martin R. Hematopoietic stem cell transplantation for multiple sclerosis: current status and future challenges. Curr Opin Neurol 2003;16:299–305.
Windrem MS, Nunes MC, Rashbaum WK et al. Fetal and adult human oligodendrocyte progenitor cell isolates myelinate the congenitally dys-myelinated brain. Nat Med 2004;10:93–97.
Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci U S A 1999;96:10711–10716.
Sanchez-Ramos J, Song S, Cardozo-Pelaez F et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol 2000;164:247–256.
Li Y, Chen J, Wang L et al. Intracerebral transplantation of bone marrow stromal cells in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson’s disease. Neurosci Lett 2001;316:67–70.
Mezey E, Chandross KJ, Harta G et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000;290:1779–1782.
Weimann JM, Charlton CA, Brazelton TR et al. Contribution of transplanted bone marrow cells to Purkinje neurons in human adult brains. Proc Natl Acad Sci U S A 2003;100:2088–2093.
Cogle CR, Yachnis AT, Laywell ED et al. Bone marrow trans differentiation in brain after transplantation: a retrospective study. Lancet 2004;363:1432–1437.
Corti S, Locatelli F, Strazzer S et al. Neuronal generation from somatic stem cells: current knowledge and perspectives on the treatment of acquired and degenerative central nervous system disorders. Curr Gene Ther 2003;3:247–272.
Kim DK, Fujiki Y, Fukushima T et al. Comparison of hematopoietic activities of human bone marrow and umbilical cord blood CD34 positive and negative cells. STEM CELLS 1999;17:286–294.
Sanchez-Ramos JR, Song S, Kamath SG et al. Expression of neural markers in human umbilical cord blood. Exp Neurol 2001;171:109–115.
Storms RW, Goodell MA, Fisher A et al. Hoechst dye efflux reveals a novel CD7(+)CD34(–) lymphoid progenitor in human umbilical cord blood. Blood 2000;96:2125–2133.
Zigova T, Song S, Willing AE et al. Human umbilical cord blood cells express neural antigens after transplantation into the developing rat brain. Cell Transplant 2002;11:265–274.
Buzanska L, Machaj EK, Zablocka B et al. Human cord blood-derived cells attain neuronal and glial features in vitro. J Cell Sci 2002;115:2131–2138.
Sanchez-Ramos JR. Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res 2002;69:880–893.
Hung SC, Cheng H, Pan CY et al. In vitro differentiation of size-sieved stem cells into electrically active neural cells. STEM CELLS 2002;20:522–529.
Lu D, Sanberg PR, Mahmood A et al. Intravenous administration of human umbilical cord blood reduces neurological deficit in the rat after traumatic brain injury. Cell Transplant 2002;11:275–281.
Willing AE, Vendrame M, Mallery J et al. Mobilized peripheral blood cells administered intravenously produce functional recovery in stroke. Cell Transplant 2003;12:449–454.
Yoo ES, Lee KE, Seo JW et al. Adherent cells generated during long-term culture of human umbilical cord blood CD34+ cells have characteristics of endothelial cells and beneficial effect on cord blood ex vivo expansion. STEM CELLS 2003;21:228–235.
Buzanska L, Habich A, Jurga M et al. Human cord blood-derived neural stem cell line - possible implementation in studying neurotoxicity. Toxicology In Vitro 2005 (in press).
Seigel GM, Sun W, Salvi R et al. Human corneal stem cells display functional neuronal properties. Mol Vis 2003;9:159–163.
Sun W, Ding D, Wang P et al. Substance P inhibits potassium and calcium currents in inner ear spiral ganglion neurons. Brain Res 2004;1012:82–92.
Sun W, Salvi RJ. Dopamine modulates soduim currents in cochlear spiral ganglion neurons. Neuroreport 2001;12:803–807.
Shirihai O, Attali B, Dagan D et al. Expression of two inward rectifier potassium channels is essential for differentiation of primitive human hematopoietic progenitor cells. J Cell Physiol 1998;177:197–205.
Hille B. Ionic Channels of Excitable Membrane. Sunderlang, MA: Sinauer Associates Inc., 1992.
Shirihai O, Merchav S, Attali B et al. K+ channel antisense oligodeoxy-nucleotides inhibit cytokine-induced expansion of human hemopoietic progenitors. Pflugers Arch 1996;431:632–638.
HuQ, Shi YL. Characterization of an inward-rectifying potassium current in NG108-15 neuroblastoma x glioma cells. Pflugers Arch 1997;433:617–625.
Kuryshev YA, Haak L, Childs GV et al. Corticotropin releasing hormone inhibits an inwardly rectifying potassium current in rat corticotropes. J Physiol 1997;502:265–279.
Tourneur Y. Action potential-like responses due to the inward rectifying potassium channel. J Membr Biol 1986;90:115–122.
Elliott AA, Elliott JR. Characterization of TTX-sensitive and TTX-resistant sodium currents in small cells from adult rat dorsal root ganglia. J Physiol 1993;463:39–56.
Day ML, Pickering SJ, Johnson MH et al. Cell-cycle control of a large-conductance K+ channel in mouse early embryos. Nature 1993;365:560–562.
Patil N, Cox DR, Bhat D et al. A potassium channel mutation in weaver mice implicates membrane excitability in granule cell differentiation. Nat Genet 1995;11:126–129.
Neusch C, Weishaupt JH, Bahr M. Kir channels in the CNS: emerging new roles and implications for neurological diseases. Cell Tissue Res 2003;311:131–138.
Braun GS, Horster MF, Huber SM. Kir1.1 expression in embryonic kidney epithelia. Biochem Biophys Res Commun 2003;312:1191–1195.
Schlichter LC, Sakellaropoulos G, Ballyk B et al. Properties of K+ and Cl– channels and their involvement in proliferation of rat microglial cells. Glia 1996;17:225–236.
Rudy B. Diversity and ubiquity of K channels. Neuroscience 1988;25:729–749.
Feldman DH, Thinschmidt JS, Peel AL et al. Differentiation of ionic currents in CNS progenitor cells: dependence upon substrate attachment and epidermal growth factor. Exp Neurol 1996;140:206–217.
Cho T, Bae JH, Choi HB et al. Human neural stem cells: electrophysiological properties of voltage-gated ion channels. Neuroreport 2002;13:1447–1452.
Wu P, Tarasenko YI, Gu Y et al. Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat. Nat Neurosci 2002;5:1271–1278.
Song H, Stevens CF, Gage FH. Astroglia induce neurogenesis from adult neural stem cells. Nature 2002;417:39–44.
Jiang Y, Henderson D, Blackstad M et al. Neuroectodermal differentiation from mouse multipotent adult progenitor cells. Proc Natl Acad Sci U S A 2003;100(suppl 1):11854–11860.
Hermann A, Gastl R, Liebau S et al. Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells. J Cell Sci 2004;117:4411–4422.
Bertollini L, Biella G, Wanke E et al. Fluoride reversibly blocks HVA calcium current in mammalian thalamic neurones. Neuroreport 1994;5:553–556.
Breakwell NA, Behnisch T, Publicover SJ et al. Attenuation of high-voltage-activated Ca2+ current run-down in rat hippocampal CA1 pyramidal cells by NaF. Exp Brain Res 1995;106:505–508.
Nguyen L, Rigo JM, Rocher V et al. Neurotransmitters as early signals for central nervous system development. Cell Tissue Res 2001;305:187–202.
Schroder W, Seifert G, Huttmann K et al. AMPA receptor-mediated modulation of inward rectifier K+ channels in astrocytes of mouse hippocampus. Mol Cell Neurosci 2002;19:447–458.
Maric D, Liu QY, Grant GM et al. Functional ionotropic glutamate receptors emerge during terminal cell division and early neuronal differentiation of rat neuroepithelial cells. J Neurosci Res 2000;61:652–662.
Metzger F, Wiese S, Sendtner M. Effect of glutamate on dendritic growth in embryonic rat motorneurons. J Neurosci 1998;18:1735–1742.
Gallo V, Zhou JM, McBain CJ et al. Oligodendrocyte progenitor cell proliferation and lineage progression are regulated by glutamate receptor-mediated K+ channel block. J Neurosci 1996;16:2659–2670.
Gerber U. Metabotropic glutamate receptors in vertebrate retina. Doc Ophthalmol 2003;106:83–87.
Dyka FM, May CA, Enz R. Metabotropic glutamate receptors are differentially regulated under elevated intraocular pressure. J Neurochem 2004;90:190–202.
Knopfel T, Grandes P. Metabo tropic glutamate receptors in the cerebellum with a focus on their function in Purkinje cells. Cerebellum 2002;1:19–26.
Nguyen L, Malgrange B, Belachew S et al. Functional glycine receptors are expressed by postnatal nestin-positive neural stem/progenitor cells. Eur J Neurosci 2002;15:1299–1305.
Belachew S, Rogister B, Rigo JM et al. Cultured oligodendrocyte progenitors derived from cerebral cortex express a glycine receptor which is pharmacologically distinct from the neuronal isoform. Eur J Neurosci 1998;10:3556–3564.
Young TL, Cepko CL. A role for ligand-gated ion channels in rod photoreceptor development. Neuron 2004;41:867–879.
Renteria RC, Johnson J, Copenhagen DR. Need rods? Get glycine receptors and taurine. Neuron 2004;41:839–841.
Owens DF, Kriegstein AR. Is there more to GABA than synaptic inhibition? Nat Rev Neurosci 2002;3:715–727.
LoTurco JJ, Owens DF, Heath MJ et al. GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis. Neuron 1995;15:1287–1298.
Behar TN, Li YX, Tran HT et al. GABA stimulates chemotaxis and chemokinesis of embryonic cortical neurons via calcium-dependent mechanisms. J Neurosci 1996;16:1808–1818.
Nguyen L, Malgrange B, Breuskin I et al. Autocrine/paracrine activation of the GABA(A) receptor inhibits the proliferation of neurogenic polysi-alylated neural cell adhesion molecule-positive (PSA-NCAM+) precursor cells from postnatal striatum. J Neurosci 2003;23:3278–3294.
Dooley AE, Pappas IS, Parnavelas JG. Serotonin promotes the survival of cortical glutamatergic neurons in vitro. Exp Neurol 1997;148:205–214.
Malberg JE, Duman RS. Cell proliferation in adult hippocampus is decreased by inescapable stress: reversal by fluoxetine treatment. Neuro-psychopharmacology 2003;28:1562–1571.
Banasr M, Hery M, Printemps R et al. Serotonin-induced increases in adult cell proliferation and neurogenesis are mediated through different and common 5-HT receptor subtypes in the dentate gyrus and the subven-tricular zone. Neuropsychopharmacology 2004;29:450–460.
Merzak A, Koochekpour S, Fillion MP et al. Expression of serotonin receptors in human fetal astrocytes and glioma cell lines: a possible role in glioma cell proliferation and migration. Brain Res Mol Brain Res 1996;41:1–7.
Schneider AS, Atluri P, Shen Q et al. Functional nicotinic acetylcholine receptor expression on stem and progenitor cells of the early embryonic nervous system. Ann N Y Acad Sci 2002;971:135–138.
Rogers SW, Gregori NZ, Carlson N et al. Neuronal nicotinic acetylcholine receptor expression by O2A/oligodendrocyte progenitor cells. Glia 2001;33:306–313.
Ohtani N, Goto T, Waeber C et al. Dopamine modulates cell cycle in the lateral ganglionic eminence. J Neurosci 2003;23:2840–2850.
Schmidt U, Pilgrim C, Beyer C. Differentiative effects of dopamine on striatal neurons involve stimulation of the cAMP/PKA pathway. Mol Cell Neurosci 1998;11:9–18.
Schmidt U, Beyer C, Oestreicher AB et al. Activation of dopaminergic D1 receptors promotes morphogenesis of developing striatal neurons. Neuroscience 1996;74:453–460.
Benninger F, Beck H, Wernig M et al. Functional integration of embryonic stem cell-derived neurons in hippocampal slice cultures. J Neurosci 2003;23:7075–7083.(Wei Suna, Leonora Buzansk)