Development of Functional Neurons from Postnatal Stem Cells In Vitro - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:48 |只看该作者 |倒序浏览 |打印

Department of Biomedical Sciences, Iowa State University, Ames, Iowa, USA: F) |5 M$ \- O$ T3 }( z( |6 G

Key Words. Adult stem cells ? Astrocytes ? Long-term cultures ? Neural stem cell ? Calcium transients

Correspondence: Srdija Jeftinija, D.V.M., Ph.D., Department of Biomedical Sciences, Iowa State University, 1098 Vet Med Bldg, Ames, Iowa 50011, USA. Telephone: 515-294-8494; Fax: 515-294-2315; e-mail: sjeftini@iastate.edu- x0 \& }' i* [" P; u- S3 j
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ABSTRACT

In the past decade, there has been an explosion of research related to stem cells. This has led to the identification and isolation of adult stem cells from many organs, including the brain, spinal cord, bone marrow, liver, intestine, retina, skeletal muscle, pancreas, cornea, and skin . Adult and embryonic stem cells differ from each other in certain aspects. Embryonic stem cells are derived from the inner cell mass of a blastocyst and are pluripotent, whereas adult stem cells are derived from differentiated organs and have varying degrees of plasticity . Adult stem cells, like embryonic stem cells, are capable of self-renewal and can give rise to fully differentiated mature cell types .( c. n& o3 V) i6 j4 L% |
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Neural stem cells (NSCs) can generate cells of glial and neuronal lineages and have been identified in the hippocampus, sub-ventricular zone, olfactory bulb, and spinal cord of adult mammals . They are the object of increasing attention for their potential use in therapies of central nervous system (CNS) disorders . Recent research demonstrates that fetal bovine serum (FBS) promotes differentiation of optic nerve oligodendrocyte precursor cells (OPCs) to a type 2 astrocyte, and when followed by exposure to basic fibroblast growth factor (bFGF), these cells revert to a state in which they cannot only self-renew but are capable of differentiating into oligodendrocytes, astrocytes, and neurons . The possibility of neural progenitor contamination from other neurogenic regions is remote because the optic nerve was harvested rostral to the optic chiasm, eliminating concerns of any neuronal contamination.. W) R# x- W; l2 ?1 T6 H

OPCs are widely distributed in the CNS and constitute a major cycling population in the brain and spinal cord . In some regions of the adult rat CNS, 70% of the dividing cells have been found to be OPCs . In humans, OPCs comprise 3% of the cells in the subcortical white matter . The widespread distribution of these cells and their potential to revert into a cell type that can give rise to all three major cell types of the CNS make them very appealing therapeutic candidates .& x+ t* m" g& i/ L- Y" z2 b* v
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Before stem cells can be used successfully as replacement therapies for neurodegenerative disorders, we must understand the functional properties of these cells. For a stem cell–derived neuron to be considered functional, it must be stably differentiated, polarized showing a single axon and multiple dendrites, capable of generating an action potential, and not only be able to release neurotransmitters but also possess receptors for them . In this paper, we examine whether cells that are differentiated from OPCs and display morphological characteristics of neurons also have functional properties similar to neurons from other brain regions in culture. Specifically, we demonstrate that NSC-derived neurons release glutamate in a calcium-dependent manner and express a set of glutamate receptors.; L9 x! A8 r+ h0 N. H1 r& U

MATERIALS AND METHODS' ^6 A, s) D. ?# n
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Morphological Characterization of NSCs5 l: L7 p' I3 x( V

To determine whether there were progenitors in the optic nerve with a latent ability to generate neurons, the optic nerve was harvested, dissociated, and fractionated. As described by Kondo and Raff , short-term exposure of acutely dissociated OPCs to FBS followed by culturing in serum-free medium with bFGF predictably resulted in cells expressing neuronal markers. In contrast, less than 5% of cells cultured in platelet-derived growth factor or FBS for 1 month or longer were MAP2-positive . These results demonstrate that OPCs cultured sequentially in FBS and bFGF will generate NSCs as a result of treatment rather than the effect of time in culture. Using this protocol, but in the tissue culture flasks without poly-L-lysine coating, the cells proliferated in bFGF and created neurosphere bodies for many weeks and months. After several weeks in culture, a small but significant minority of cells were MAP2-positive neurons when induced to differentiate. Neurons were often found in small clusters, suggesting a clonal derivation. Figure 1A illustrates MAP2 antibody–stained cells that were exposed to differentiation media, indicating that the cells were neurons and that few if any non-neuronal cells were present. The high percentage of MAP2-positive cells grown under these conditions is consistent with the findings of Kondo and Raff . In addition to expressing neuronal microtubule marker, most of the cells (more than 90%) reacted with secretory protein synaptotegmin antibody (Fig. 1B). Most of the synaptotegmin immunoproduct labeling was confined to the cell bodies of the differentiated cells, which is a characteristic of young neurons. The fact that these neurons were generated from cells isolated from the optic nerve dispels any concerns of contamination from other known neurogenic zones within the CNS.
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Figure 1. Neuronal marker expression in optic nerve–derived NSCs cultured for 6 months. (A): Ninety percent of the cells grown in differentiation media show immunoreactivity for MAP2 antibody. Both individual and cell clusters were stained. (B): In addition to expressing MAP2, more than 90% of the cells reacted with secretory protein synaptotagmin. Note the higher concentration of immunoproduct in the cell body, which is characteristic of young neurons. Arrowheads indicate cells outside the cluster that expressed immunoproduct in bodies only. (C): Fluorescein isothiocyanate–labeled heavy chain tetanus toxin has the same tropism for the NSC-differentiated neurons as it does for neurons. Scale bar = 20 μm. Abbreviations: MAP2, microtubule-associated protein 2; NSC, neural stem cell.
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To further assess the morphological characteristics of neurons derived from NSCs, we exposed these cells to the heavy chain (HC) component of tetanus toxin. The C-terminal portion of the HC is responsible for the neurospecificity of tetanus toxin. Recent studies demonstrated that the HC of the tetanus toxin is a multivalent oligosaccharide-binding protein, and the sugar-binding site in the C-terminal domain ensures specific binding of tetanus toxin to carbohydrate-containing receptors on the neuronal membrane (for review, see ). Differentiated NSCs were exposed to the HC component of tetanus toxin that was labeled with fluorescein isothiocyanate (FITC) (Fig. 1C). The cultures were then examined using confocal microscopy, and more than 90% of the cells in culture were FITC-labeled. This demonstrates that the tetanus toxin has a high affinity for the differentiated NSC neurons and further illustrates that these cells are morphologically similar to neurons.( b* ?+ P$ f, n: }' }* p

Functional Identification of NSCs
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Several independent criteria were used to functionally identify NSCs as cells that possess functional properties similar to those of functional neurons. These included the presence of voltage-gated calcium channels, ligand-gated calcium channels, and ionotropic glutamate AMPA (-amino-3-hydroxy-5-methyl isoxazole propionic acid) receptors. Coverslips containing NSCs were mounted onto a fast-rate exchange perfusion chamber for imaging experiments. Fura-2 calcium imaging was used to examine the stimulatory effects of a nonselective depolarizing stimulus, 50 mM K , and two neurotransmitters, ATP and AMPA.
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To determine the presence of voltage-gated calcium channels in cultured cells, 50 mM K  was used. Elevated potassium concentrations are commonly used to functionally identify neurons . In resting conditions, the cytoplasmic calcium level of NSCs was 91 ± 2 nM (n = 201). Brief perfusion application of 50 mM K  (2 minutes) produced an increase in the level of calcium in 97% of the cells studied (n = 194; Fig. 2). This increase of intracellular calcium reached the peak level of 207 ± 7 nM (n = 189; Fig. 2) approximately 80 seconds (mechanical delay) after the initiation of 50 mM K  application and was sustained for several minutes. Removal of external calcium from the bathing medium abolished the potassium-induced calcium transients in NSC-derived neurons (Fig. 3), indicating that the increase in intracellular calcium was dependent on external calcium sources. It has been well documented that perfusion application of 50 mM K  on astrocytes was without effect on intracellular calcium concentration . These data demonstrate that NSC-derived neurons express voltage-dependent calcium channels similar to those of functional neurons.
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Figure 2. Kinetic changes of i and glutamate release in differentiated neural stem cells (NSCs) in response to perfusion application of 50 mM K  and ATP. (A): Perfusion application of 50 mM K  and 100 μM ATP in the presence of low Ca2  was without effect. (B): The increase in intracellular calcium induced by perfusion application of ATP coincided with the increase in the release of glutamate from differentiated NSCs.' R5 k5 f( @+ ?, Q, z! O

Figure 3. Kinetic changes of i in a mixed neural stem cell astrocyte culture in response to perfusion application of 50 mM K  and 100 μM ATP. The group of cells in the upper set of graphs responded to both K  and ATP under normal conditions but failed to respond in the presence of a low extracellular calcium solution. This type of response is characteristic of neurons. The cell kinetic tracing in the lower set of graphs failed to respond to 50 mM K  under both conditions but responded to ATP, which is characteristic of an astrocyte. The separation of the graphs is done artificially using software to make the effect more obvious.
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Activation of purinoceptors leads to an increase in intracellular calcium in a variety of excitable and unexcitable cells . Two mechanisms are involved in ATP-evoked intracellular calcium increase. First, ATP can activate ATP-gated cation channels . This first mechanism is consistent with the P2X subtype of receptor . Second, extracellular ATP can stimulate the breakdown of inositol phospholipids, and the resulting increase in inositol 1,4,5,-triphosphate (IP3) is responsible for the elevation of cytosolic Ca2  . This mechanism is coupled to the P2Y type of receptor. Astrocytes have a relatively small population of P2X receptors but a large population of P2Y receptors that mediate increases in intracellular calcium . Neurons also display both the P2X and P2Y receptor, but the P2Y receptor mediates slow changes in membrane potential . This evidence prompted us to use ATP in combination with low extracellular calcium to functionally discriminate astrocyte-like from neuron-like cells.

Figure 2 illustrates a typical response of differentiated NSC cultures to perfusion application of 50 mM K  followed by perfusion application of 100 μM ATP for 1 minute. Application of ATP induced an increase in calcium level in 83% of cells (180 of 216 cells in eight experiments), and this effect was completely abolished in 87% of the cells bathed in low calcium (121 of 139 cells, six independent cultures). This finding strongly suggests that the differentiated cells have a P2X type of purinergic receptor that is consistent with neurons and that few non-neuronal cells are present.+ R2 V. V$ t/ Z/ M$ X6 d
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To further assess the functional characteristics of differentiated cells, we investigated whether these cells were capable of releasing glutamate in response to ATP stimulation. The release of glutamate from NSC cultures was assayed using HPLC on the superfusate. The basal release of glutamate into the superfusate was 19 ± 2 nM (p
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To confirm that optic nerve–derived stem cells were mainly of neuronal phenotype, we plated NSCs onto established cortical astrocyte cultures and stimulated them with potassium and ATP. As can be seen in Figure 3, there were two kinds of cells. One group of cells responded to potassium and ATP in normal calcium but failed to respond to either in low calcium (Fig. 3; Ca2  transients of cells in upper set of tracings). The second group of cells did not respond to potassium but responded to ATP in both normal (2 mM) calcium and low (26 nM) calcium HEPES buffer (Fig. 3; lower set of tracings). For functional identification, the cells in upper tracings were identified as neuron-like cells and lower tracing cells were identified as astrocytes.  A, G- r! t0 y9 k
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Fast excitatory transmission between the neurons of the CNS occurs when glutamate directly activates AMPA and kainate receptors. AMPA receptors lacking the GluR2 subunit are permeable to Ca2  . To further functionally characterize NSCs, we applied 10 μM AMPA for 1 minute. Figure 4 illustrates the typical response of differentiated NSCs to applications of potassium and AMPA. Stimulatory effects of AMPA were abolished in low calcium, indicating that AMPA receptors in NSCs are permeable to extracellular Ca2 . Perfusion applications of potassium and 10 μM AMPA were without effect on enriched cortical astrocyte cultures (Fig. 4B).

Figure 4. Kinetic changes of i in a neural stem cell culture and enriched astrocyte culture in response to perfusion application of 50 mM K  and 10 μM AMPA (-amino-3-hydroxy-5-methyl isoxazole propionic acid). (A): The AMPA response is abolished in low-calcium HEPES, indicating that the increase in i is dependent on extracellular calcium. (B): Both 50 mM K and AMPA failed to induce calcium increase in enriched astrocyte culture, whereas cells responded to application of ATP in low calcium.
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