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Keywords:

  • TrpC1;
  • TrpC4;
  • Voltage-gated calcium channels;
  • Embryonic stem cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

Spontaneous calcium (Ca2+) transients in the developing nervous system can affect proliferation, migration, neuronal subtype specification, and neurite outgrowth. Here, we show that telencephalic human neuroepithelia (hNE) and postmitotic neurons (PMNs) generated from embryonic stem cells display robust Ca2+ transients. Unlike previous reports in animal models, transients occurred by a Gd3+/La3+-sensitive, but thapsigargin- and Cd2+-insensitive, mechanism, strongly suggestive of a role for transient receptor potential (Trp) channels. Furthermore, Ca2+ transients in PMNs exhibited an additional sensitivity to the canonical Trp (TrpC) antagonist SKF96365 and shRNA-mediated knockdown of the TrpC1 subunit. Functionally, inhibition of Ca2+ transients in dividing hNE cells led to a significant reduction in proliferation, whereas either pharmacological inhibition or shRNA-mediated knockdown of the TrpC1 and TrpC4 subunits significantly reduced neurite extension in PMNs. Primary neurons cultured from fetal human cortex displayed nearly identical Ca2+ transients and pharmacological sensitivities to Trp channel antagonists. Together these data suggest that Trp channels present a novel mechanism for controlling Ca2+ transients in human neurons and may offer a target for regulating proliferation and neurite outgrowth when engineering cells for therapeutic transplantation. STEM CELLS 2009;27:2906–2916


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

In animal models spontaneous calcium (Ca2+) oscillations play a significant role in nervous system development, involved in neural induction, proliferation, migration, axon guidance, and growth cone morphology [1–5]. In Xenopus neurons, the frequency of Ca2+ “spikes” mediated by T-type Ca2+ channels controls neurotransmitter specificity, whereas slow Ca2+ “waves” appear critical for neurite outgrowth [1]. Alternatively, in rodent cortical progenitors, Ca2+ transients are mediated by release from intracellular stores [6], and simultaneous Ca2+ transients between ventricular zone progenitors coordinate cell cycle entry [5]. Precisely how Ca2+ controls these disparate processes remains unknown, but the source of Ca2+ appears to be critical as different Ca2+ entry mechanisms can activate specific downstream signaling cascades [7].

In addition to entry through voltage-gated calcium channels (VGCCs) and release from intracellular stores, members of the transient receptor potential (Trp) channel superfamily present an alternative mechanism for Ca2+ entry and regulate multiple processes in the developing and mature nervous system. Trp channels are a family of 28 nonselective cation channels, and all except TrpM4 and TrpM5 display varying degrees of calcium permeability [8]. Interestingly, members of the canonical Trp (TrpC) family are involved in store operated Ca2+ entry [9], which is thought to be an essential component in the establishment of intracellular Ca2+ fluctuations [10, 11]. In the mature nervous system Trp channels play important roles in the processing of sensory information [12] and fear-related learning and memory [13], and defects in particular channels underlie models of neurodegeneration such as cerebellar ataxia [14]. During early development members of the Trp channel families modulate neural progenitor proliferation [15] whereas at later stages, specific members of the TrpC family have been shown to both positively and negatively regulate neurite extension [16, 17], likely because of the activation of Ca2+-dependent processes [18].

Whether and/or how Ca2+ transients affect human neuronal differentiation is not known. Human embryonic stem cells (hESCs) can be efficiently directed to proliferating neuroepithelial (hNE) cells and then to postmitotic neurons (PMNs) [19], presenting an experimentally accessible tool to explore the regulatory mechanisms that underlie neuronal differentiation. Furthermore, the development of strategies to regulate hNE proliferation and PMN differentiation, including the regulation of intracellular Ca2+ concentrations, may allow us to better control the regenerative potential of stem cells. Therefore, we sought to investigate the role and mechanism(s) of spontaneous increases in intracellular Ca2+ in hNE and PMNs.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

Cell Culture

hESCs (WA09 line, P16-30) were maintained and differentiated as described [19, 20]. After 21 days of differentiation hNE clusters were treated with 0.5% trypsin-EGTA (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) for 5 minutes and trypsin inhibitor (1 mg/ml; Invitrogen) for 2 minutes and then triturated to single cells. Cells were centrifuged (1000 rpm for 5 minutes) and resuspended in neural differentiation media described elsewhere [19]. Cells were then plated onto glass coverslips at a density of 150,000 cells per gridded coverslip (Bellco, Vineland, NJ, http://www.bellcoglass.com/) or 75,000 cells per 10-mm coverslip.

For primary cultures, 15-week-old human fetal brains were obtained from Clive Svendsen, UW-Madison, in accordance with guidelines set by the National Institute of Health and the University of Wisconsin Madison Institutional Review Board for collection and use of such tissue. Cells were enzymatically dissociated with 0.5% trypsin-EGTA, washed three times with Dulbecco's modified Eagle's medium-F12 + 20% fetal bovine serum, and resuspended in plating media described in [19]. After 5 days, cultures were fed with serum-free media that contained 4 μM cytosine 1-β-D-arabinofuranoside (AraC) to inhibit glial growth [21]. Unless noted, all chemicals were obtained from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com).

Immunochemical Staining

Immunolabeling of hESC-derived neural progenitor cultures was performed according to previously established methods [20, 22]. Primary antibodies used were as follows: polyclonal βIII-tubulin (1:5000; Covance, Princeton, NJ, http://www.covance.com), monoclonal βIII-tubulin (1:1000), monoclonal 3CB2 [1:1000; Developmental Studies Hybridoma Bank (DSHB) at The University of Iowa, Iowa City, IA, http://dshb.biology.uiowa.edu)], monoclonal Pax6 (0.5 μg/ml; DSHB), polyclonal Sox2 (1:1000; R&D Systems Inc., Minneapolis, http://www.rndsystems.com), polyclonal FoxG1 (BF1, 1:1000; gift from Dr. Yoshiki Sasai, RIKEN, Kobe, Japan, http://www.riken.jp), polyclonal glial fibrillary acidic protein (GFAP, 1:5000; DAKO, Glostrup, Denmark, http://www.dako.com), monoclonal N-cadherin (1:5000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), polyclonal nestin (1:300; Millipore, Billerica, MA, http://www. millipore.com), monoclonal vimentin (1:40; DSHB), and polyclonal bromodeoxyuridine (BrdU, 1:500; Accurate Chemical & Scientific Corporation, Westbury, NY, http://accuratechemical.com). Secondary antibodies used were as follows: Alexa-Fluor donkey anti-rabbit 488, Alexa-Fluor donkey anti-mouse 594, Alexa-Fluor donkey anti-goat 594 (Invitrogen), and donkey anti-rat Cy3 (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com), all at a concentration of 1:1000. Topro-3 (1:300) was used as a nuclear stain.

Immunoblotting

Western blots were performed essentially as described [23]. Membranes were probed with the indicated primary TrpC antibodies (Santa Cruz Biotechnology) and horseradish peroxidase-conjugated secondary antibodies (Pierce Biotechnology, Rockford, IL, http://www.piercenet.com). Luminescent membranes were visualized on an Alpha Innotech FluorChem HD2 bioimaging system and quantified using spot densitometry analysis with AlphaEaseFC software (Alpha Innotech Corporation, San Leandro, CA, http://www.alphainnotech.com).

Proliferation/TUNEL Assays and Immunocytochemical Analysis

BrdU-treated cells were processed according to [24] using an anti-BrdU antibody (1:5000; Accurate Chemical & Scientific Corp.). TUNEL staining was performed according to the manufacturer's guidelines (Roche Diagnostics, Indianapolis, IN http://www.roche-applied-science.com). Proliferation of green fluorescent protein (GFP)-expressing cells was assessed using a “Click-iT EdU” (EdU, 5′-ethynyl-2′-deoxyuridine) assay (Invitrogen) according to the manufacturer's instructions.

Acquisition and quantification of fluorescent intensities were determined using a Nikon confocal workstation (D-Eclipse C1) and EZ-C1 software (v3.5). Multiple coverslips (2-3) were prepared following the same experimental conditions and experiments were replicated three times to verify results. For quantification of hNE markers and BrdU and TUNEL, cells were considered positive if more than 20% of the nuclear area (defined as Topro-3+) contained pixels with above-threshold values. Neurite length measurements were performed using IPlab software v4.0 (BD Biosciences, Rockville, MD http://www.bdbiosciences. com), on compressed z-stacks (7-10 images, 0.35 μm per image) taken under ×60 magnification. Length was determined by manually tracing primary neurites from the edge of the soma to the leading edge of the process. Only cells with intact nuclei were used for analysis.

Calcium Imaging

Fluo-4 and Fura-2 imaging were performed as essentially as described in [2, 25], respectively. A detailed description of procedures can be found in the supporting information data.

Quantitative Polymerase Chain Reaction

RNA isolation and detection at days 12 and 21 was performed as described [26]. To calculate the fold change (FC), we used the following equations:

  • equation image
  • equation image

Polymerase chain reaction (PCR) primers are listed in supporting information Table 1.

RNA Inhibition

Short hairpin RNAs (shRNA) were provided by Dr. Mitchell Villereal (University of Chicago, Chicago, IL, http://www. uchicago.edu) and cloned into the LVTHM lentiviral vector provided by Dr. Didier Trono from Tronolab (Lausanne, Switzerland, http://tronolab.epfl.ch). TrpC1 overexpression vector was provided by Dr. Mike Zhu (The Ohio State University, Columbus, OH, http://www.osu.edu) and TrpC4 overexpression vector was provided by Dr. James Putney (NIEHS, NIH, Research Triangle Park, NC, http://www.niehs.nih.gov). Lentiviral production was performed as described [27].

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

Dissociated Human Neuroepithelia Spontaneously Reform Neural Tube-like Rosettes and Differentiate to Neurons

hESCs differentiate to forebrain hNE within 10 days of culture without the addition of growth factors or morphogens [26]. By 3 weeks, spheres contain hNE and βIII-tubulin+ neurons [19], but analysis of these two populations is difficult because of the three-dimensional structure. Therefore, we first asked whether hNE cells grown as a monolayer would retain their rosette-like morphology, a structure that is critical for maintaining cells in a progenitor state [28]. We found that 21-day-old dissociated hNE cells reformed neural tube-like rosettes within hours of plating (Fig. 1A). Rosettes exhibited polarity similar to those observed at earlier time points [26], with strong N-cadherin localization near the inner lumen (Fig. 1B). At this time point βIII-tubulin+ cells were found surrounding rosettes (Fig. 1A, arrows), never coexpressed the cell cycle marker Ki67 (supporting information Fig. 1A, 1B; n = 3), and are referred to as PMNs.

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Figure 1. Dissociated hNE and PMNs reform rosettes and display spontaneous Ca2+ transients. (A): Twenty-four hours after plating, hNE cells reformed rosette structures (DIC) whereas βIII-tubulin+ PMNs (arrows) were observed adjacent to rosettes. (B): N-Cadherin (red) expression was observed at the inner lumen of nestin+ cells (green) within rosettes. (C): Many cells within rosette structures expressed the radial glial marker 3CB2, whereas PMNs did not. (D): The majority of hNE cells, but not PMNs, expressed the neuroepithelial markers Sox2 and Pax6. FoxG1 was expressed in more than 90% of hNE and PMNs. (Ei–iv): Sequential images of cells loaded with Fluo-4; a subset displayed spontaneous increases in [Ca2+]i. (F): Overlay of cells imaged using Fluo-4 and stained for βIII-tubulin for post hoc analysis (hNE cells = arrows; PMN = arrowhead). (G): Representative traces from images taken every second of cells shown in (E). Large [Ca2+]i fluctuations exhibited complex kinetics and variable amplitudes. (H): Representative traces of small-amplitude transients. (I): Mean exponential decay curve derived from small-amplitude transients (17 cells from 3 cultures). Scale bars represent 50 μm. Error bars represent ± SEM. Abbreviations: hNE, human neuroepithelia; PMNs, postmitotic neurons.

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Interestingly, robust expression of the radial glial markers 3CB2 and vimentin was observed in hNE cells, most often associated with rosette structures (Fig. 1C and data not shown). Expression of the NE markers Sox2 and Pax6 was evident in the majority of cells within rosettes and in cell aggregates without distinct morphology, but rarely in PMNs (Fig. 1D). Consistent with previous work, almost all cells at this time point expressed the forebrain marker FoxG1 (Fig. 1D, [19]). Importantly, hNE progenitors are thought to remain exclusively neurogenic until 6-7 weeks, when some gliogenic precursors begin to give rise to astrocytes [19]. Thus, 21-day-old monolayer cultures of hNE cells give rise to PMNs, express forebrain markers, 3CB2 and vimentin, reminiscent of developing radial glial cells that occupy the ventricular zone of mammalian cortices early in development [29–31].

Spontaneous Calcium Transients Exhibit Prolonged Durations in hNE and PMNs

To test for the presence of spontaneous Ca2+ transients, we performed Fluo-4 Ca2+ imaging once per second for 10 minutes. Figure 1Ei–iv shows a representative field of cells, some of which underwent Ca2+ transients during the imaging period. All transients observed appeared asynchronous and isolated to single cells, unlike some reports in mice that displayed coupled transients between adjacent cells [5]. Following imaging, cultures were stained for βIII-tubulin to distinguish between hNE and PMNs (Fig. 1F). Figure 1G shows traces from cells in (Fig. 1E, 1F), illustrating the variety of amplitudes and durations of Ca2+ transients, and also their highly complex kinetics (Fig. 1G, peaks 1-3). However, transients with smaller amplitudes exhibited more regular kinetics (Fig. 1H). These traces were averaged and fit with a single exponential decay that resulted in a τ value of 22.2 ± 3.6 seconds and a mean duration of approximately 60 seconds (Fig. 1I). Interestingly, this is almost 6 times longer than the mean duration of transients observed in mouse cortex [6], suggestive of a unique mechanism for their production. Using these data, we calculated that sampling at 10-second intervals would detect most Ca2+ transients.

Spontaneous Calcium Transients in Human NE and PMNs Are Dependent on Extracellular Calcium

To determine the mechanism(s) underlying these transients, we manipulated the extracellular milieu or applied standard pharmacological inhibitors. Removal of extracellular Ca2+ (n = 4), or addition of the calcium chelators EGTA-AM (50 μM, n = 3) or BAPTA-AM (50 μM, n = 3), completely eliminated transients in both hNE and PMNs, whereas depletion of intracellular stores with thapsigargin had no effect (Fig. 2B, 2C, and data not shown; hNE: n = 4, PMNs: n = 4, p > .05). However, robust increases in [Ca2+]i were observed in both hNE and PMNs within minutes of thapsigargin addition, indicating that functional Ca2+ stores were present. Similar to findings in Xenopus neurons [1], high concentrations of Ni2+ (5 mM), a nonspecific inhibitor of VGCCs, almost completely abolished transients (Fig. 2A–2C; hNE: n = 3, PMNs: n = 3, p < .001). Furthermore, whereas the combined application of inhibitors of L-, P/Q-, N-, or T-type Ca2+ channels did not significantly reduce the incidence of transients in hNE (n = 3, p > .05), they did significantly reduce transient incidence in PMNs (Fig. 2A–2C; n = 3, p < .05). This effect appeared to be due to the inhibition of L-type Ca2+ channels as only the application of nifedipine significantly reduced transient incidence in PMNs (Fig. 2C and data not shown; n = 5, p < .05). In addition, the sodium channel antagonist tetrodotoxin caused a small but significant reduction in Ca2+ transients in PMNs (Fig. 2C; n = 6, p < .05). Thus, Ca2+ transients in hESC-derived neuronal cultures require Ca2+ entry from the extracellular environment and multiple Ca2+ entry mechanisms exist in PMNs whereas hNE cells may utilize a single mechanism.

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Figure 2. Spontaneous calcium transients are dependent on extracellular calcium but not release from intracellular stores. (A): Representative traces of transient elevations of [Ca2+]i in untreated hNE cells (top), upon application of 5 mM Ni2+ (middle), or those treated with a cocktail of voltage-gated calcium channel inhibitors (bottom). (B,C): Pooled data illustrating the change in the percentage of cells that underwent Ca2+ transients under various conditions. (C): PMNs were uniquely sensitive to nifedipine and tetrodotoxin. Error bars represent ± SEM. *, p < .05; **, p < .01; Student's t tests. Abbreviations: hNE, human neuroepithelia; PMNs, postmitotic neurons; TTX, tetrodotoxin.

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Trp Channel Antagonists Differentially Inhibit Spontaneous Calcium Transients in hNE and PMNs

Because internal store release and VGCC activation did not account for the majority of spontaneous Ca2+ transients in hNE and PMNs (Fig. 2B, 2C), we probed for additional mechanisms. Members of the Trp channel family are nonselective cation channels that display varying degrees of calcium permeability [8, 12]. The majority of these channels are inhibited by trivalent metal ions gadolinium (Gd3+) and lanthanum (La3+). We found that application of 10 μM Gd3+ or La3+ significantly attenuated transient incidence in hNE (Fig. 3A; control: n = 7, Gd3+: n = 5, p < .01 and data not shown). Ca2+ transients remaining after Gd3+ treatment also had significantly smaller amplitudes (control: 189 ± 4% F/Fo, n = 7, Gd3+: 139 ± 6% F/Fo, n = 5, p < .01). A concentration-response curve revealed the EC50 of Gd3+ to be 3.8 μM (Fig. 3B). Whereas application of more than 10 μM Gd3+ (Fig. 3B) almost completely abolished spontaneous Ca2+ transients, it tended to slowly precipitate in our culture media, making long-term incubations unfeasible (see below). Therefore, 10 μM Gd3+ was used for the remainder of our experiments except where indicated.

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Figure 3. Spontaneous calcium transients in hNE cells and PMNs are differentially sensitive to antagonists of transient receptor potential (Trp) channels. (A): Pooled data demonstrate that the percentage of hNE cells that displayed Ca2+ transients was significantly decreased upon application of the Trp channel antagonist Gd3+ (10 μM). (B): Concentration-response curve for the effect of Gd3+ on the percentage of cells that underwent Ca2+ transients during a 15-minute period. (C): Pooled data demonstrate that application of Gd3+ and SKF96365 significantly reduced the percentage of PMNs with Ca2+ transients. (D): Representative images displaying maximum Fluo-4 fluorescence intensity over a 15-minute imaging period with and without Trp channel antagonists. (E,F): Representative traces of Ca2+ transients in hNE cells (E) and PMNs (F) that received control treatment, Gd3+, or SKF96365. (G): Representative confocal images of primary human cortical cultures of 15 gestational week-old tissue show differentiated astrocytes (GFAP; green) and PMNs (βIII-tubulin; red). (H): Representative traces of Ca2+ transients in human primary neurons with and without SKF96365 (5 μM). (F): Pooled data demonstrate that Gd3+, SKF96365, and nifedipine (5 μM) decreased the percentage of cells with Ca2+ transients in human primary neurons. Scale bars represent 50 μm. Error bars represent ± SEM. *, p < .05; **, p < .01; ***, p < .001; Student's t tests. Abbreviations: GFAP, glial fibrillary acidic protein; hNE, human neuroepithelia; PMNs, postmitotic neurons; RuR, Ruthenium Red.

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To determine which specific Trp channel(s) mediate the observed Ca2+ transients, we applied two antagonists believed to inhibit specific subfamilies. Ruthenium Red is used as an inhibitor of members of the TrpV family [32, 33], whereas SKF96365 is used to inhibit TrpC family members [34, 35]. Application of 5 μM Ruthenium Red (n = 3) or 5 μM SKF96365 (n = 5) had no significant effect on the percentage of hNE cells that displayed Ca2+ transients (Fig. 3A, 3D arrowheads, 3E; p > .05). Interestingly, although Ca2+ transients in PMNs displayed the same sensitivity to Gd3+ as hNE cells (compare Fig. 5A, 5C; n = 5, p < .01), they were also significantly inhibited by SKF96365 (Fig. 3C, 3D arrow, 3F; n = 5, p < .01), suggestive of a role for TrpC channels in PMNs (see below). Ruthenium Red, however, was without effect in PMNs (Fig. 3C; n = 3, p > .05).

To further characterize the Trp channel(s) and/or signaling pathways responsible for the induction of Ca2+ transients in hNE cells, we applied a host of pharmacological agents (Table 1). However, no treatment resulted in visible inhibition or augmentation of Ca2+ transients, ruling out the involvement of connexin hemichannels, purinergic receptors, arachidonic acid channels, ryanodine receptors, stretch-activated channels, nicotinic acetylcholine receptors, γ-Aminobutyric acid (GABA)/glutamate receptors, and other VGCCs, all of which can cause direct or indirect Ca2+ influx. Thus, for hNE cells we were unable to identify specific channels responsible for triggering Ca2+ transients.

Table 1. Pharmacological treatments with no observable effects on the incidence of calcium transients in human embryonic stem cell-derived neuronal cultures
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To determine whether the SKF96365-sensitive Ca2+ transients in PMNs were an artifact of the differentiation of neurons from hESCs, we cultured human embryonic cortical neurons from a 15-week-old fetus. Figure 3G shows fetal cultures after 1 week, which were comprised mainly of PMNs (red) and astrocytes (green). Fluo-4 Ca2+ imaging revealed the presence of spontaneous transients that displayed a similar sensitivity to Trp antagonists when compared to hESC-derived neurons (Fig. 3H, 3I; control: 225 cells from 9 coverslips, Gd3+: 153 cells from 6 coverslips, p < .001; SKF96365: 313 cells from 8 coverslips, p < .001). In addition, nifedipine significantly reduced the incidence of Ca2+ transients in human primary cultures, whereas thapsigargin was without effect (Fig. 3I; nifedipine: 175 cells from 5 coverslips, p < .05, thapsigargin: 179 cells from 4 coverslips, p > .05). SKF96365 appeared to have no effect on spontaneous Ca2+ fluctuations in glial cells, which were similar to those observed in primary rodent cells [36]. Together, these data show that spontaneous Ca2+ transients in hESC-derived neurons are nearly identical to those taken directly from human embryonic tissue and that neither the presence of glia nor treatment with AraC modified their pharmacological sensitivity.

Inhibition of Trp Channels Significantly Attenuates Proliferation in hNE Cells

In rodent and chick neural progenitor cells, Ca2+ has been shown to act at different stages of the cell cycle to promote cellular proliferation [5, 37]. In human cells, application of 10 μM Gd3+ significantly reduced the number of cells that incorporated BrdU during a 12-hour treatment (Fig. 4A, 4B; 1,606 cells from 3 coverslips, p < .001). Similar data were obtained using the cell-cycle marker Ki67 (supporting information Fig. 1), suggesting that Gd3+ was not simply interfering with BrdU incorporation.

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Figure 4. Trp channel inhibition significantly reduces proliferation of hNE cells. (A): Representative confocal images showing BrdU incorporation after 12-hour control, Gd3+ (10 μM), or Ni2+ (500 μM) treatments. (B): Pooled data demonstrate that 10 μM Gd3+ and 500 μM Ni2+ significantly decreased BrdU incorporation. (C): Representative confocal images of TUNEL-stained cells that were untreated or treated with 10 μM Gd3+ or 500 μM Ni2+. (D): 5 μM SKF96365 and 500 μM Ni2+ significantly increased cell death. Scale bars represent 50 μm. Error bars represent ± SEM. **, p < .01; ***, p < 0.001; Student's t tests. Abbreviations: BrdU, bromodeoxyuridine; hNE, human neuroepithelia.

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The Gd3+ effect appeared to be specific, as Trp antagonists that did not affect Ca2+ transients (1 μM Gd3+, SKF96365, and Ruthenium Red (Fig. 3A, 3B)) also did not affect the number of hNEs that incorporated BrdU (Fig. 4B; 1 μM Gd3+: 1,084 cells from 3 coverslips, SKF96365: 1,185 cells from 3 coverslips, Ruthenium Red: 1,338 cells from 3 coverslips, p > .05). TUNEL staining revealed no significant difference between cells treated with 10 μM Gd3+ and untreated controls (Fig. 4C, 4D; control: 1,150 cells from 3 coverslips, Gd3+: 1,599 cells from 3 coverslips, p > .05), suggesting that this was not due to increased cell death. Ni2+ was used at two different concentrations to control for nonspecific effects of long-term incubations of extracellular metals. Ten micromolar Ni2+ had no effect on BrdU incorporation (1,485 cells from 3 coverslips, p > .05) or TUNEL staining (1,261 cells from 2 coverslips, p > .05). However, 500 μM Ni2+ caused a small but significant decrease in BrdU incorporation (Fig. 4B; 1,220 cells from 2 coverslips, p < .01), although this concentration also led to a fourfold increase in cell death (Fig. 4D, 4E; 1,139 cells from 2 coverslips, p < .001). This suggests that high concentrations of Ni2+, like those that are used to inhibit Ca2+ transients ([1], Fig. 3), likely have nonspecific toxic effects that result in changes in BrdU incorporation.

Blockade of TrpCs Reduces Neurite Outgrowth from Human Neurons

In PMNs, TrpCs have been shown to serve many functions during development including the control of growth cone guidance and neurite extension [16, 38]. We found that incubation of hESC-derived cultures for 12 hours with SKF96365 significantly reduced mean neurite length compared to controls (Fig. 5A–5C; control: n = 5, SKF96365: n = 5, p < .001). In contrast, although nifedipine significantly reduced Ca2+ transient incidence (Fig. 2C), it did not affect neurite length (Fig. 5C; n = 5, p > .05). Furthermore, this was likely not due to a nonspecific effect of SKF96365 on cell health (Fig. 4D; 1,140 cells from 3 coverslips, p < .01), as 500 μM Ni2+, which also caused a significant increase in cell death (Fig. 4D), did not affect neurite length (supporting information Fig. 2). These results were verified in human primary cultures, where 12-hour treatments with SKF96365 reduced mean neurite length by approximately 50% compared with untreated or nifedipine-treated neurons (Fig. 5D–5F; control: 123 cells from 3 coverslips, SKF96365: 182 cells from 3 coverslips, p < .001; nifedipine: 95 cells from 3 coverslips, p > .05).

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Figure 5. TrpC channels contribute to neurite outgrowth in human neurons. (A,B): Confocal images of human embryonic stem cell-derived postmitotic neurons (PMNs) from control and SKF96365-treated cultures. (C): Pooled data demonstrate that SKF96365 treatments significantly reduced mean neurite length. (D,E): Confocal images of human primary cultures from control and SKF96365-treated cultures. (F): Pooled data show that neurite extension in human primary neurons was also significantly inhibited by SKF96365. (G): Pooled data from quantitative polymerase chain reaction experiments illustrate the relative amounts of mRNA for individual TrpC subunits in human neuroepithelia (hNE) cultures (day 12) and mixed hNE + PMN cultures (day 21). (H): Immunoblots of various TrpC proteins performed on samples from postnatal day 1 mouse brain (Brain), hNE cells (day 12), and PMNs + hNE (day 21); 40 μg of protein per lane. Scale bars represent 50 μm. Error bars represent ± SEM. **, p < .01; Student's t tests. Abbreviation: TrpC, canonical transient receptor potential.

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To determine which TrpCs may be involved, we examined transcript expression levels in both hNE cells (day 12) and after PMNs began to differentiate (day 21, [19, 26]). Quantitative reverse transcription-polymerase chain reaction (qPCR) revealed mRNA expression of TrpC1, TrpC3, and TrpC4 in both populations, and low levels of TrpC6 transcripts were detected at day 21 (Fig. 5G). In contrast, Western blots revealed no expression of TrpC1 and moderate levels of TrpC4 protein at day 12. However, both subunits were strongly expressed at day 21 (Fig. 5H), indicating an upregulation of both proteins in PMNs. Although mRNA was detected for other TrpCs (e.g., TrpC3 and TrpC6) at various time points during differentiation (Fig. 5G), little or no protein expression was detected (Fig. 5H). Together these data demonstrate that TrpC-dependent regulation of neurite outgrowth in human neurons appears to be reproduced in hESC-derived neurons and that TrpC1 and TrpC4 are the primary subunits expressed in early human forebrain neurons.

TrpC1 and TrpC4 Regulate Neurite Extension in hESC-Derived Neurons

To test the roles of specific TrpC subunits, targeted reduction of either TrpC1 or TrpC4 was performed using shRNA [39] incorporated into a lentiviral vector. In TrpC1 or TrpC4-transfected HEK293 cells, qPCR demonstrated an 11.3 ± 1.2 (n = 3)-fold decrease of TrpC1 mRNA and a 9.0 ± 0.6 (n = 3)-fold decrease of TrpC4 mRNA in cells that received the respective shRNA. A scrambled shRNA was without effect. Western blots also demonstrated a significant attenuation of targeted TrpC isoforms without concomitant loss of nonspecific subunits (Fig. 6A; TrpC1 shRNA: 20 ± 2.9%, n = 4, p < .001; TrpC4 shRNA: 17.4 ± 4.7%, n = 4, p < .001). Compared to nontransfected cells, a scrambled shRNA control construct was without effect (TrpC1: 97.9 ± 1.6%, n = 3, p > .05; TrpC4: 99.4 ± 0.9%, n = 3, p > .05).

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Figure 6. TrpC1 and TrpC4 contribute to neurite outgrowth in hESC-derived neurons. (A): Western blots performed on TrpC-transfected HEK cells that also received shRNA constructs directed toward either TrpC1 or TrpC4 demonstrate specific knockdown of each construct whereas a scrambled shRNA construct had no effect (p > .05): 10 μg of protein per lane (TrpC rows); 40 μg of protein per lane (actin). (B): Representative confocal images of control shRNA green fluorescent protein expression in hNE cells (arrowheads) and PMNs (arrows) 10 days after viral infection. (C): Confocal images of hNE cells expressing control, TrpC1, or TrpC4 shRNA (green) that incorporated EdU during a 12-hour exposure. Pooled data revealed no significant differences between groups (p > .05). (D): Confocal images of PMNs (βIII-tubulin+) that expressed control, TrpC1, or TrpC4 shRNA (green). Pooled data demonstrate a significant decrease in mean neurite length for cells that expressed either TrpC1 or TrpC4 shRNA, whereas the control shRNA was without effect. Scale bars represent 50 μm. Error bars represent ± SEM. *, p < .05; Student's t tests. Abbreviation: EdU, 5-ethyl-2′-deoxyuridine; TrpC, canonical transient receptor potential.

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The addition of shRNA-containing lentiviruses to hESC-derived forebrain cells revealed robust infection of hNE cells within rosette structures (Fig. 6B, arrowheads), with reduced efficiency in βIII-tubulin+ PMNs (Fig. 6B, arrows). Proliferation analyses revealed no significant difference in EdU incorporation (see Materials and Methods) between control-infected cells and those that did not incorporate virus (Fig. 6C; p > .05). Cells that expressed either TrpC1 or TrpC4 shRNA also showed no significant difference in proliferation rates compared to controls (Fig. 6C; p > .05). These findings were not surprising as TrpC1 protein did not appear to be expressed in hNE cells, and TrpC4 has not been previously implicated in regulating cell proliferation. Importantly, these data suggest that neither the virus itself nor shRNA hairpins caused alterations in cell viability.

Interestingly, significant reductions in mean neurite length were detected in PMNs that received either TrpC1 or TrpC4 shRNA (Fig. 6D; control: 120.9 ± 11.4 μm; TrpC1 shRNA: 93.5 ± 14.7 μm; TrpC4: 80.2 ± 10.2 μm, n = 3, p < .05), but not in cells that received control shRNA (Fig. 6D; 122.5 ± 7.8 μm, n = 3, p > .05). The modest reduction in neurite length in the TrpC1 RNAi condition appeared to be due to large reductions in a subset of cells (Fig. 6D, arrowheads), whereas others appeared to be unaffected (Fig. 6D, arrows).

To determine whether these differences were associated with changes in intracellular Ca2+ dynamics, we performed calcium imaging on GFP+ cells using Fura-2. Spontaneous Ca2+ transients observed using Fura-2 were similar in duration to those measured with Fluo-4, but had diminished amplitudes (supporting information Fig. 3), likely as a result of the differences in Ca2+ affinity inherent to the dyes. No significant differences were found between cells infected with control shRNA and unstransfected cells (supporting information Fig. 3B; untransfected: 20.3 ± 2.0%, 425 cells from 4 coverslips; Control shRNA: 23.3 ± 4.5%, 154 cells from 3 coverslips, p > .05). However, the percentage of cells that displayed transients was significantly reduced in the population that expressed TrpC1 shRNA (supporting information Fig. 3A, 3B; 11.3 ± 3.3%, 279 cells from 4 coverslips, p < .05), but not in cells that expressed TrpC4 shRNA (17.0 ± 4.5%, 172 cells from 3 coverslips, p > .05).

The fact that both TrpC1 and TrpC4 shRNA independently reduced neurite outgrowth, whereas only TrpC1 shRNA reduced global calcium transients, suggested that whole cell Ca2+ signals are likely not the most critical factor for human neurite extension. Previous studies have shown that local Ca2+ signals within growth cones are critical for morphology, steering, and extension/retraction (reviewed in [40]). We found that 1-hour treatment of cells with BAPTA, which is able to chelate Ca2+ proximal to the plasma membrane [41, 42], significantly reduced neurite outgrowth compared to untreated or EGTA-treated controls (control: 101.6 ± 4.7 μm; EGTA: 106.2 ± 5.9 μm; BAPTA: 80.0 ± 4.9 μm, n = 4 ea., p < .05), consistent with previous studies shown above. Furthermore, this occurred despite the fact that both BAPTA and EGTA completely blocked global calcium transients (above). Taken together, these data suggest that TrpC1 plays a role in both local and global Ca2+ signals in human neurons, but that only local Ca2+ signals via TrpC1 and TrpC4 channels can regulate neurite extension.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

Here we show that hNE and PMNs undergo spontaneous Ca2+ transients mediated by Gd3+/La3+-sensitive channels, suggestive of a role for Trp channels. Ca2+ transients displayed relatively long durations, similar to Ca2+ “waves” observed in animal models [1]. Although blockade of Ca2+ transients in hNE cells led to a significant reduction in progenitor cell proliferation, they were inhibited only by broad spectrum antagonists and their precise mechanism remains unknown. In contrast, PMNs from hESCs or primary fetal tissue exhibited L-type Ca2+ channel- and TrpC1-dependent Ca2+ transients. Furthermore, pharmacological inhibition or downregulation of TrpC1 and TrpC4 proteins caused significant reductions in neurite outgrowth. Collectively, our data demonstrate that Trp channels play a critical role in the generation of spontaneous Ca2+ transients in human neurons and their progenitors, and that Trp channels regulate multiple aspects of human neural development.

In the nervous system Trp channels are critically important for somatosensory transduction of a wide array of stimuli [8, 12], fear-related learning and memory [13], visual information processing [43, 44], and the survival of multiple cerebellar neurons [14, 45]. However, whereas the importance of spontaneous whole cell Ca2+ fluctuations in central nervous system development has been demonstrated in animal models, to our knowledge this is the first report of a Trp channel-dependent mechanism. For instance, neural induction in Xenopus and Pleurodeles requires Ca2+ entry through L-type VGCCs, the expression of which correlates with the development of spontaneous Ca2+ oscillations [46]. Calcium fluctuations found in the developing ventricular zone (VZ) of the mouse cortex are dependent upon IP3-mediated Ca2+ release from intracellular stores [6]. Furthermore, Gu et al. described two distinct types of Ca2+ transients in the developing Xenopus spinal cord. Short-duration Ca2+ “spikes” mediated by T-type Ca2+ channels regulate neurotransmitter specification, whereas long-duration Ca2+ waves, which are blocked by millimolar concentrations of Ni2+, appear necessary for normal neurite extension [1]. However, the induction mechanism for Ca2+ waves has remained uncharacterized. It is interesting to speculate that the Trp channel-dependent Ca2+ transients we observed in human cells are the same as Ca2+ waves observed in Xenopus neurons as their pharmacological sensitivity appears similar.

Although evidence in neurons is limited, Trp channel-dependent Ca2+ oscillations have been observed in many nonexcitable cells. In rabbit vascular smooth muscle, phenylephrine- or endothelin-1-induced Ca2+ oscillations occur by an SKF96365-sensitive mechanism [47, 48]. In a stimulated T-lymphocyte line, expression of dominant negative TrpM4 subunits converted oscillatory Ca2+ signals to sustained increases in [Ca2+]i [49]. Similarly, an immortalized B-cell line lacking TrpC1 demonstrated significantly reduced Ca2+ oscillations and capacitative Ca2+ entry in response to antibody stimulation [50]. Thus, it appears that Ca2+ transients in human forebrain cells may occur by similar mechanisms that exist in nonexcitable tissues. However, although knockdown of TrpC1 did significantly reduce transient incidence and remaining transient amplitude in human neurons, TrpC1 expression did not account for all spontaneous Ca2+ influx. Therefore, further investigation is required to identify other channels that mediate Ca2+ transients in hESC-derived neurons.

Our results suggest that hNE cells and PMNs utilize disparate channels to control various developmental processes. This is highlighted by the temporal expression pattern and physiological role of TrpC1, which represents a critical discrepancy between our data and previous reports. A study by Fiorio Pla et al. demonstrated that bFGF-mediated increases in Ca2+ and cellular proliferation were significantly reduced in TrpC1 knockout animals [15]. However, here we show that TrpC1 is not expressed in hESC-derived forebrain progenitor cells, but is upregulated upon neuronal differentiation. Furthermore, TrpC1 knockdown did not affect proliferation, suggesting that TrpC1 does not regulate proliferation of our cultures. This discrepancy may reflect a difference in gestational age. Fiorio Pla et al. used neural stem cells derived from the E13 rat cortex, a stage at which progenitors from both the VZ and subventricular zone (SVZ) are present. Our hNE cells may represent an earlier developmental time point, before the formation of the SVZ, when VZ progenitors predominate [51, 52]. Interestingly, we found that blockade of Ca2+ transients in hNE cells with Gd3+/La3+ did significantly reduce proliferation that was independent of TrpC channels, suggestive of a role for other Trp channel family members controlling neural progenitor cell proliferation.

Lastly, our data suggest that TrpC1 and TrpC4 act as molecular targets for controlling neurite elongation in human neurons. A recent report demonstrated that TrpC4 was upregulated under conditions of axonal regeneration and that shRNA-mediated inhibition of TrpC4 in dorsal root ganglion neurons reduced neurite outgrowth [17]. Current studies also point to stretch-activated receptors as key players in neurite outgrowth as their inhibition led to the acceleration of neurite extension [2]. In one study, TrpC1 was shown to be activated directly by membrane stretch [53] and TrpC1 knockdown in Xenopus spinal neurons eliminated growth cone turning in response to netrin-1 [35]. In human cells, only TrpC1 and TrpC4 subunits appeared to be expressed in differentiating neurons. Pharmacological or shRNA-mediated inhibition of either TrpC1 or TrpC4, but not inhibition of L-type Ca2+ channels, significantly inhibited neurite extension. Surprisingly, only inhibition of TrpC1 led to reductions in global Ca2+ transients. This suggests that TrpC1 may act independently of TrpC4 in the cell soma to partially regulate global spontaneous Ca2+ transients, perhaps through interactions with non-TrpC family members [54]. However, both TrpC1 and TrpC4 may be acting at the growth cone to regulate small local Ca2+ transients that control neurite outgrowth, possibly as a single heteromeric channel [55]. This idea is supported by the fact that only BAPTA, a Ca2+ chelator with fast kinetics and high affinity, could reduce both calcium transients and neurite outgrowth, whereas EGTA (having slower kinetics and lower affinity) could reduce global calcium transients but not neurite outgrowth. It should be noted, however, that other Ca2+ regulatory mechanisms such as glutamate receptors [56, 57] and IP3 receptors [58] may work in concert with TrpC channels in growth cones to regulate neurite extension as TrpC knockdown did not fully inhibit outgrowth.

In addition to the basic insights of the Ca2+-dependent regulatory mechanisms that exist during human neural development, our data suggest new methods for engineering cells for therapeutic transplantation. Although significant advancements have been made [59], tumor formation and excess proliferation remain critical concerns for stem cell-based replacement therapies [60]. First, we found that Ca2+ transient inhibition by Trp channel antagonists resulted in a significant reduction of BrdU incorporation in progenitor cells with no observable effect on PMNs. Thus, inhibition of Trp channels may limit unwanted proliferation of transplantable cells. Second, the ability to guide neurites to their appropriate targets or modulate neurite outgrowth rates may help to accelerate synaptic integration and, thus, the desired effects of neural transplants. Here we show that TrpC family members regulate neurite length in human neurons. Therefore, modulating the activity of specific members of the TrpC family may aid in the creation of safe and effective cell-replacement therapies for neurodegenerative disorders.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

We would like to thank Clive Svendsen for generously providing human fetal tissue, Tim Gomez, PhD, and Tim LaVaute, PhD, for their intellectual contributions, Paul Mermelstein, PhD, for a critical reading of this manuscript, and Lydia Kotecki and Erich Zeiss for technical assistance. The project was supported by NINDS, NS 045926, and partly by a core grant to the Waisman Center from the National Institute of Child Health and Human Development (P30 HD03352). Austin Johnson was supported by Rath Distinguished Graduate Student Fellowship. Jason Weick was supported by NIH Stem Cell Training Grant (NIH T32AG027566-01).

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  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

Additional supporting information available online.

FilenameFormatSizeDescription
STEM_212_sm_suppinfo.doc29KSupporting Information
STEM_212_sm_suppinfofigure1.tif819KSupporting Information Figure 1
STEM_212_sm_suppinfofigure2.tif439KSupporting Information Figure 2
STEM_212_sm_suppinfofigure3.tif71KSupporting Information Figure 3
STEM_212_sm_suppinfotable2.doc32KTable 2: Quantitative PCR primers for human TrpC channels and VGCCs

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.