Conditionally immortalized stem cell lines from human spinal cord retain regional identity and generate functional V2a interneurons and motorneurons
© Cocks et al.; licensee BioMed Central Ltd. 2013
Received: 17 December 2012
Accepted: 3 June 2013
Published: 7 June 2013
The use of immortalized neural stem cells either as models of neural development in vitro or as cellular therapies in central nervous system (CNS) disorders has been controversial. This controversy has centered on the capacity of immortalized cells to retain characteristic features of the progenitor cells resident in the tissue of origin from which they were derived, and the potential for tumorogenicity as a result of immortalization. Here, we report the generation of conditionally immortalized neural stem cell lines from human fetal spinal cord tissue, which addresses these issues.
Clonal neural stem cell lines were derived from 10-week-old human fetal spinal cord and conditionally immortalized with an inducible form of cMyc. The derived lines were karyotyped, transcriptionally profiled by microarray, and assessed against a panel of spinal cord progenitor markers with immunocytochemistry. In addition, the lines were differentiated and assessed for the presence of neuronal fate markers and functional calcium channels. Finally, a clonal line expressing eGFP was grafted into lesioned rat spinal cord and assessed for survival, differentiation characteristics, and tumorogenicity.
We demonstrate that these clonal lines (a) retain a clear transcriptional signature of ventral spinal cord progenitors and a normal karyotype after extensive propagation in vitro, (b) differentiate into relevant ventral neuronal subtypes with functional T-, L-, N-, and P/Q-type Ca2+ channels and spontaneous calcium oscillations, and (c) stably engraft into lesioned rat spinal cord without tumorogenicity.
We propose that these cells represent a useful tool both for the in vitro study of differentiation into ventral spinal cord neuronal subtypes, and for examining the potential of conditionally immortalized neural stem cells to facilitate functional recovery after spinal cord injury or disease.
KeywordsNeural stem cells Spinal cord V2a interneurons Motoneurons Voltage-operated Ca2+ channels Spontaneous Ca2+ oscillations
Stem cells have received substantial interest both for their potential as in vitro tools to study development and as potential therapeutic agents in a range of degenerative diseases of the nervous system . One area of particularly strong research activity has been spinal cord injury (SCI), for which treatment options are very limited . Stem cells derived from a range of different tissue sources and developmental stages have been studied for their capacity to elicit functional recovery in animal models of SCI [3, 4]. One such approach has been to generate immortalized neural stem cell lines from postmortem human fetal spinal cord tissue for transplantation [5–8]. An important question in the use of tissue-specific immortalized neural stem cell lines as cellular therapies is the extent to which these cells are able to retain the phenotypic characteristics of the tissue of origin after immortalization, prolonged in vitro propagation, and engraftment into lesioned tissue.
In the current study, we generated three clonal neural stem cell lines from human fetal spinal cord, designated SPC-01, SPC-04, and SPC-06, conditionally immortalized with 4-hydroxy tamoxifen (4-OHT)-inducible cMyc (cMycERTAM) . This technology involves transducing primary dissociated cells with a retrovirus containing the gene cMyc fused to a mutated form of the estrogen receptor. This fusion protein is specifically activated by the presence of the synthetic ligand 4-OHT, triggering dimerization and translocation to the nucleus. The nuclear cMycER protein regulates gene expression, and in particular, directly upregulates telomerase , thus allowing the cell to proliferate indefinitely without undergoing replicative senescence. Removal of 4-OHT from the media results in inactivation of cMycER and terminal cellular differentiation .
To assess whether these conditionally immortalized neural stem cells retain the identity of their tissue of origin after prolonged in vitro propagation, we performed a genome-wide transcriptome analysis. This dataset was then analyzed in terms of the expression of homeodomain transcription factors known to play an instructive role in the identity of progenitor subtypes in the developing spinal cord , and the findings validated by immunostaining. The ventral spinal cord has four major interneuron progenitor subdomains (p0, p1, p2, and p3), and one motoneuron progenitor subdomain (pMN) specified by the cross-repressive activities of class I and II homeodomain transcription factors . The ventral p2 domain of the spinal cord, comprising Nkx6.1+/Irx3+ cells, gives rise to two main lineages of interneurons designated V2a and V2b, specified by differential Notch signaling [13, 14]. A third lineage designated V2c, derived from the V2b lineage and dependent on Sox1 expression, has also recently been identified . The genome-wide transcriptome analysis of the conditionally immortalized neural stem cell lines reported here, and subsequently confirmed by immunostaining, revealed a homeodomain transcription-factor profile indicative of the ventral spinal cord p2 and pMN domains.
Furthermore, we demonstrated that on removal of growth factors and 4-OHT, these cells differentiate into V2 interneurons and motoneurons, consistent with the expression of p2 and pMN domain markers in the progenitor cells.
To study the functional properties of neurons derived from these conditionally immortalized neural stem cells, we assessed the Ca2+ responses induced by high K+ and specific Ca2+ channel blockers. Intracellular Ca2+ changes control many neuronal functions including neurotransmitter release , membrane excitability , gene transcription , and growth . It was shown previously that, during the period of synaptogenesis, acutely dissociated embryonic motoneurons express a great variety of voltage-operated Ca2+ channels (VOCCs), able to induce a Ca2+-induced Ca2+ release (CICR) through a new type of intracellular Ca2+ pathway functionally linked to P-type Cav2.1 Ca2+ channel subunits [20–22]. We found that neurons derived from the clonal lines described here express functional T-, L-, N-, and P/Q-type Ca2+ channels. Furthermore, we demonstrated that a subset of these neurons exhibit spontaneous calcium oscillations typically observed in dissociated embryonic rat motoneurons cultures .
Finally, in a series of grafting experiments into lesioned rat spinal cord, we demonstrated that these cells are able to stably engraft, differentiate into choline acetyltransferase positive (ChAT+) motoneurons, and show robust survival after 4 months without tumorogenicity.
Materials and methods
Generation of clonal lines
Ten-week-old fetal tissue was obtained from Advanced Bioscience Resources (Alameda, CA, USA) after normal terminations and in accordance with nationally (UK and USA) approved ethical and legal guidelines [24, 25]. Primary cells were prepared by finely chopping the cervical region of the fetal spinal cord with a scalpel and dissociation at 37°C with 0.25% trypsin (BioWhittaker) in DMEM:F12 (Gibco), followed by 0.25 mg/ml soybean trypsin inhibitor (Gibco). Clonal conditionally immortalized cell lines were generated by using MMLV-type retrovirus encoding the gene cMYC-ERTAM, as previously described [9, 11]. In brief, primary spinal cord cells transduced with cMYC-ERTAM were plated at clonal density, and individual colonies were passaged by using glass cloning cylinders (Sigma-Aldrich). Spinal cord clones SPC-01, SPC-04, and SPC-06 were initially selected based on uniform growth over 20 population doublings before further characterization. A version of spinal cord clonal line 1 (SPC-01) expressing eGFP was also generated by using a lentiviral vector containing a ubiquitous chromatin opening element to prevent silencing on engraftment, as previously described .
Cell growth and differentiation
Cell lines were routinely cultured, as previously described in Pollock et al. . In brief, cells were grown on laminin-coated (Sigma-Aldrich), tissue-culture flasks in DMEM/F12 supplemented with bFGF (10 ng/ml), EGF (20 ng/ml) (PeproTech, UK); human serum albumin (0.03%) (Baxter Healthcare); L-glutamine (2 mM) (Gibco); human transferrin (100 μg/ml), putrescine dihydrochloride (16.2 μg/ml), human insulin (5 μg/ml), progesterone (60 ng/ml), sodium selenite (selenium) (40 ng/ml), and 4-OHT (100 nM) (Sigma-Aldrich). Cell differentiation was triggered by the removal of growth factors and 4-OHT from the media with or without the addition of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (10 μM) or all-trans retinoic acid (ATRA), as indicated in the text. Long-term growth and population doublings were monitored by recording the total number of cells at each passage.
Cells at 70% to 80% confluence were treated with 100 ng/ml colcemid (Life Technologies), for 3 hours, and subjected to hypotonic lysis in 0.075 M potassium chloride for 10 minutes at 37°C. Samples were then fixed in methanol/glacial acetic acid (ratio 3:1) and stained with Giemsa on glass slides for analysis.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, washed with PBS, and permeabilized with 0.1% Triton-X100/tris-buffered saline (TBS) for 30 minutes. Nonspecific binding was then blocked with 10% normal donkey serum in TBS for 1 hour at room temperature. Cells were then probed with primary antibodies to Nestin (1:500; Chemicon), Sox2, and ChAT (1:1,000; Millipore), βIII tubulin and Irx3 (1:1,000; Sigma-Aldrich), Tau and S100Β (1:2,000; DAKO), Olig2, and MASH1 (1:200; Millipore), Pax6, Nkx6.1, Lhx3, En1, and Isl1 (all Developmental Studies Hybridoma Bank (University of Iowa), 1:200), and Chx10 and GATA3 (1:250; Abcam) at 4°C overnight. Secondary antibodies used were donkey anti-mouse Alexa 488, donkey anti-rabbit Alexa 594, donkey anti-sheep Alexa 488, donkey anti-goat Alexa 488, and donkey anti-rabbit Alexa 680 (all 1:300, Molecular Probes), as appropriate, in 1% NDS/TBS for 1 hour at room temperature. Cells were then washed with TBS and counterstained with 1 μM Hoechst 33342 (Sigma-Aldrich).
All cell counting was carried out in biologic triplicate, in which the experiment was replicated with cells plated several passages apart. Each biologic replicate consisted of three wells (technical replicates) for each condition. Each technical replicate consisted of three randomly placed nonoverlapping images taken per well with the 40× objective. Images were imported into ImageJ, and nuclei and target-positive cells were counted manually. The three-well images were averaged to generate one technical replicate. The three technical replicates (wells) were averaged to generate one biologic replicate (plate), which was then used for statistical analysis. All cell counting was carried out from images taken from the 1×70 inverted microscope (Olympus) and processed with the Axio Vision Digital Image Processing Software (Carl Zeiss Inc.). Exposure times were kept consistent for each target. Differences in the proportion of marker-positive cells between cell lines were tested for statistical significance by using a one-way ANOVA for each marker (Prism).
RNA was extracted from cultured cells by using Trizol (Life Technologies), and DNase treated with Turbo DNase (Life Technologies). RNA was assessed for quality with an Agilent Bioanalyzer ensuring an RNA integrity number greater than 9. Genome-wide gene-expression profiling was performed with the Illumina HumanWG-6 v3.0 expression beadchip array, at the Welcome Trust Centre for Human Genetics (Oxford, UK). Sample probe profiles were exported from GenomeStudio into lumi (bioconductor). Variance-stabilizing transformation was applied to datasets, quantile normalized, and log2 transformed. All microarray data from this study are available through the Gene Expression Omnibus , with accession number GSE37282.
Drugs and solutions
Unless otherwise stated, all standard chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sandimmune (Novartis Pharma AG, Basel, Switzerland); Immuran (GlaxoSmith-Kline); Solu-Medrol (Pfizer, Puurs, Belgium); Fura-2 AM 1 mM solution in anhydrous DMSO and Pluronic F-127 (30% stock in distilled water) (Molecular Probes, USA); ω-Aga Toxin IVA (ω-Aga IVA), and ω-conotoxin GVIA (ω-GVIA) (Alomone Labs Ltd. Jerusalem, Israel). Concentrated stock solutions of nicardipine were prepared in DMSO, whereas the remaining stock solutions of Ca2+-channel toxins were dissolved in dH2O. All concentrated stock solutions were stored at −20°C. Test solutions were prepared daily by using aliquots from frozen stocks to obtain the working concentrations. All buffers and solutions used for the Ca2+-measurement studies were made by using ion-free dH2O from Merck-Germany.
Solutions for [Ca2+]i measurements
Normal Locke (NL) buffer was used for [Ca2+]i measurements on single cells in culture, containing (mM): NaCl, 140; KCl, 5; MgCl2, 1.2; CaCl2, 2.2; glucose, 10; HEPES-Tris, 10; BSA, 0.02%; pH 7.25. The osmolarity of the solutions used ranged between 298 and 300 mosmol/l-1. High-K+ buffer contained (mM): NaCl, 90; KCl, 50; MgCl2, 1.2; CaCl2, 2.2; glucose, 10; HEPES, 10; at pH 7.25. For other K+ concentrations, KCl was added at the desired concentration and was adjusted with NaCl appropriately to bring the osmolarity to the required range. The Ca2+-channel blockers ω-GVIA and ω-Aga IVA were prepared as concentrated stocks in distilled water, stored at −70°C, and diluted to working concentrations before use. The control and test solutions were applied by using a multiple capillary perfusion system (200 μm inner-diameter capillary tubing; flow rate, 250 μl/min), and the cells were subjected to a constant fast-flow control buffer. Each capillary was fed by a reservoir 30 cm above the bath and connected to a temperature-control device (Harvard-France). In this approach, switching the flow from one capillary to the next resulted in complete solution changes within 2 to 5 seconds. After each application of the tested drug, the cells were washed with control buffer. This method allowed fast and reliable exchange of the solution surrounding the cells.
[Ca2+]i measurements on individual SPC-01-derived neurons
Intracellular calcium ([Ca2+]i) measurements on single cells were performed after 10 days of differentiation by using fast fluorescence spectrofluorimetry. SPC-01 cells differentiated on 22-mm glass-bottom dishes (WillCo Wells BV) were incubated with 2.5 μM Fura-2 AM plus 0.02% Pluronic F-127 at 24°C for 50 minutes. The preparations were then washed with dye-free solution and kept at 37°C until used. Fluorescence measurements of [Ca2+]i were performed with the Zeiss Microscope Photometer System (Fast Fluorescence Photometer (FFP), Zeiss, Germany), based on an inverted microscope (Axiovert; Zeiss) equipped for epifluorescence (objective, Plan-Neofluar 100 ×/1.30 oil immersion). The cells were alternately illuminated (200 Hz) at 340 ± 10 and 380 ± 10 nm. To minimize the background noise of the Fura-2 signal, successive values were averaged to a final time resolution of 308 milliseconds. For fast switching between different excitation wavelengths, a rotating filter wheel was mounted in the excitation light path. A measuring amplifier was synchronized to the filter wheel to measure the fluorescence intensities resulting from different wavelengths. The FFP software controlled the acquisition of intensity data and provided functions for adjusting the signal values, the display and storage of the measured data, and calculations of ion concentrations. A CCD camera was used to visualize the cells. With fluorescence values corrected for background and dark current, [Ca2+]i calculations were carried out from the ratio between 340- and 380-nm recordings. Fura-2 calibration was performed with the actual instrument by following the same procedure described previously , which yielded Rmin = 0.16; Rmax = 3.173; β = 2.968; and Kd = 224 at 37°C.
Origin 8.5.1 was used for plotting and statistical procedures (OriginLab). The results are expressed as mean ± SEM. The number of the sample size (n) given is the number of cells tested according to the same protocol (control, test drug, recovery) for each group. The figures (traces) show on-line single-cell measurements of the [Ca2+]i levels before and after the application of test substances, whereas bar diagrams and numeric data are given as mean ± SEM and present the peak amplitude of the [Ca2+]i increase as concentration (in nM) calculated fluorescence values of 340/380 nm excitation wavelengths. The results were analyzed by using one-way ANOVA. Differences were considered statistically significant if P ≤ 0.05.
Spinal cord compression lesion and cell transplantation
All animal experiments were approved by the Animal Committee of the Czech Republic and the Animal Care and Use of Animals Committee of the Institute of Experimental Medicine AS CR. Adult male Wistar rats weighing 280 to 300 g were anesthetized with isofluorane vapor inhalation (3% to 5%), and a balloon-induced spinal cord compression lesion was performed at the Th8 to Th9 level of the spinal cord, according to protocols previously described . The animals were assisted with manual urination twice a day until the reflex returned, and gentamicin was administered by intramuscular injection twice a day for 3 days. Cell transplantation was performed 7 days after SCI, according to a previously published procedure . For transplantation, SPC-01 cells were detached by TrypZean (Lonza). Harvested cells were grafted by using a stereotaxic injection instrument (Stoelting Co., Wood Dale, IL, USA) and a nanoinjector pump (KD Scientific Inc, Hillstone, MA, USA). In total, of 5 × 105 cells suspended in 5-μl growth media were injected into the epicenter of the lesion, at a depth of 2 mm. All grafted rats (n = 22) were immunosuppressed with Sandimmun (Novartis Pharama AG, Basel, Switzerland; 10 mg/kg intraperitoneally), Immuran (GlaxoSmith-Kline, 4 mg/kg intraperitoneally), and Solu-Medrol (Pfizer, Puurs, Belgium; 2 mg/kg intramuscularly) 24 hours before transplantation and then daily until the end of the experiment.
Histology and immunohistochemistry
The animals were killed and perfused either 8 weeks (n = 16) or 4 months (n = 6) after cell transplantation for histologic examination. The rats were deeply anesthetized, and 200 ml of PBS was perfused intracardially into the left ventricle, followed by 300 ml of ice-cold 4% (vol/vol) PFA in 0.1 M PBS. The spinal cords were dissected, immersed in 4% (vol/vol) PFA at 4°C for 24 hours, and then placed in 30% (wt/vol) sucrose for 3 days. After freezing, spinal cords were cryosectioned longitudinally in 14-mm-thick slices. To identify SPC-01 cells transplanted into the rat spinal cord, antibodies directed against human mitochondria (MTCO2; mouse monoclonal, 1:125, Abcam), human nuclei (HuNu; mouse monoclonal 1:40, Millipore), choline acetyltransferase ChAT (rabbit polyclonal, 1:100, Abcam), nestin (rabbit polyclonal, 1:200 Millipore), Islet2, Nkx 6.1 (both mouse monoclonal, 1:20, Developmental Studies Hybridoma Bank), and GFAP (mouse monoclonal 1:200, Sigma-Aldrich) were used. The Ki67 index and the number of Nkx 6.1-positive cells were calculated as the ratio of Ki67/HuNu-positive cells or Nkx 6.1/HuNu-positive cells to the total number of HuNu-positive cells.
Results and discussion
cMycER conditionally immortalized spinal cord neural stem cells retain a normal karyotype and regional identity after prolonged culture
Log 2 -transformed data from Illumina beadchip expression analysis of SPC-01, SPC-04, and SPC-06 cell lines
Log2expression values (detection P value)
Dorsal spinal cord
Ventral spinal cord
SPC-01 generates V2a interneurons and motoneurons
Differential Notch signaling has been demonstrated to specify the binary-fate decision of p2 progenitors between V2a excitatory and V2b inhibitory interneurons [13, 14]. We therefore speculated that inhibition of Notch should drive a V2a excitatory fate in these clonal lines. Inhibition of Notch for 48 hours by the γ-secretase inhibitor DAPT upregulated Mash1 in undifferentiated SPC-01 (Figure 4a) and, on differentiation, resulted in a significant increase in the proportion of Chx10+ neurons (Figure 4b and 4c). Notch inhibition therefore drives a V2a interneuronal fate in these cells. Significant progress has been made in identifying the components and stoichiometric interactions of the transcription factor complexes involved in specifying the fate of p2 progenitors into these different interneuron subtypes [33, 34]. The ability of SPC-01 to rapidly and reproducibly differentiate into V2a Chx10+ interneurons represents a useful tool to study the acquisition of V2 interneuronal fates in vitro.
Given that SPC-01 also expresses low levels of OLIG2, a marker of the pMN domain, we asked if these cells were also competent to give rise to motoneurons. It was shown previously that retinoid signaling is required for the specification of motoneuron fate in the ventral spinal cord . We therefore sought to drive the fate of SPC-01 cells along a motoneuron lineage by the addition of 100 nM ATRA for the first 2 days of differentiation. We found that SPC-01 did indeed give rise to ISL1+ putative motoneurons (Additional file 3: Figure S3a). However, these Isl1+ neurons represent only a small subpopulation (<5%) of the total neurons generated (Additional file 3: Figure S3b), suggesting that the default differentiation of these cells is toward a V2 interneuronal fate.
[Ca2+]i responses in SPC-01-derived neurons
In another set of experiments, we tested the [Ca2+]i responses induced by high K+ in the presence of the specific P/Q-type Ca2+ channel blocker Ω-Aga-IVA. The P/Q-type Ca2+ channel is typically expressed in rat embryonic and adult motoneurons . Ω-Aga-IVA (300 nM) was found significantly to block the [Ca2+]i responses induced by high K+ by 76% ± 24% (n = 9; P = 0.001; Figure 5d), suggesting the importance of functional P/Q-type Ca2+ channels in SPC-01 neurons. These results suggest the expression of T, L-, N- and P/Q-type Ca2+ channels in motoneuron-like cells in differentiated SPC-01.
SPC-01 stably engrafts in the lesioned rat spinal cord without tumorogenicity
We generated immortalized neural stem cell lines from human fetal spinal cord; these retain the phenotypic characteristics of the tissue of origin even after prolonged in vitro propagation and engraftment into lesioned rodent spinal cord. These cell lines therefore represent a useful tool for studying V2 interneuron differentiation in vitro and for further examining the potential of human neural stem cells as cellular therapies for spinal cord injury.
All-trans retinoic acid
N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester
Enhanced green fluorescent protein
Normal Locke buffer
Spinal cord neural stem cell clone 01
Voltage-operated Ca2+ channels.
Grants: AV0Z50390703, GA AV: IAA500390902, The Charles Wolfson Charitable trust, EU 6th Framework Programme. G. Dayanithi and O. Forostyak were supported by the grant P304/11/2373 from the Grant Agency of the Czech Republic.
The antibodies Isl1, Lhx3, and En1 were developed by TM Jessell and S Brenner-Morton. Pax6 was developed by A Kawakami, and Nkx6.1, developed by OD Madsen, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242, USA.
We thank John Sindon and Erik Miljan for facilitating the generation of the SPC cell lines at ReNeuron plc, Dr. Michael Antoniou for providing the UCOE lentiviral construct, and Dr. James Dutt, IEM, ASCR, for helpful discussions and critical reading of the manuscript.
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