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Conceived and designed the experiments: TG HMCJ RCS HU. Performed the experiments: TG SLBO AC RB PM MF CL HMCJ. Analyzed the data: TG SLBO AC CL. Contributed reagents/materials/analysis tools: HMCJ RCS HU. Wrote the paper: TG RCS HU.
Novel developmental functions have been attributed to the P2X7 receptor (P2X7R) including proliferation stimulation and neural differentiation. Mouse embryonic stem cells (ESC), induced with retinoic acid to neural differentiation, closely assemble processes occurring during neuroectodermal development of the early embryo.
P2X7R expression together with the pluripotency marker Oct-4 was highest in undifferentiated ESC. In undifferentiated cells, the P2X7R agonist Bz-ATP accelerated cell cycle entry, which was blocked by the specific P2X7R inhibitor KN-62. ESC induced to neural differentiation with retinoic acid, reduced Oct-4 and P2X7R expression. P2X7R receptor-promoted intracellular calcium fluxes were obtained at lower Bz-ATP ligand concentrations in undifferentiated and in neural-differentiated cells compared to other studies. The presence of KN-62 led to increased number of cells expressing SSEA-1, Dcx and β3-tubulin, as well as the number of SSEA-1 and β3-tubulin-double-positive cells confirming that onset of neuroectodermal differentiation and neuronal fate determination depends on suppression of P2X7R activity. Moreover, an increase in the number of Ki-67 positive cells in conditions of P2X7R inhibition indicates rescue of progenitors into the cell cycle, augmenting the number of neuroblasts and consequently neurogenesis.
In embryonic cells, P2X7R expression and activity is upregulated, maintaining proliferation, while upon induction to neural differentiation P2X7 receptor expression and activity needs to be suppressed.
Purinergic receptors are classified as P1 adenosine and P2 ATP receptors based on their selectivity for adenosine and nucleotide agonists. While P1 and P2Y subtypes are G-protein-coupled metabotropic receptors, P2X receptors are resembled as homo- or hetero-trimeric ligand-gated ion channels from seven possible subunits. The ion channels formed by P2X1-P2X7 subunits are permeable to Na+, K+ and Ca2+ ions, while at high agonist concentrations P2X7 receptor (P2X7R) subtypes assemble cation ion channels that are capable of pore forming, allowing the unselective flow of compounds with molecular masses of 700Da besides the uncontrolled entry of ions, including Ca2+, into the cell which may induce intrinsic cell death programs
Being expressed in almost every cell and attributed to multiple cellular functions, purinergic receptors have been detected in early embryonic development
Maiken Nedergaard's group showed that neuronal differentiation is accompanied by a marked down-regulation of purinergic signaling and the neural progenitor cells themselves were the source of local ATP secretion
Complex developmental mechanisms are often studied in simplified environment by using stem cell models. Embryonic stem cells (ESC) are isolated from blastocysts inner cell mass maintaining
Extracellular ATP induces proliferation and regulates proliferation in pluripotent stem cell models expressing various purinergic receptor subtypes
P2X7 (−,−) knock-out mice, developed by the method of Dr James Mobley (PGRD, Pfizer Inc, Groton, CT, USA) were housed in controlled temperature of 22±2°C and 60-70% humidity and unlimited access to food and water
The feeder cell independent E14Tg2A embryonic stem (ES) cell line was kindly provided by Dr. Deborah Schechtman, Instituto de Química, University of São Paulo. Cells were first isolated by Hooper et al, 1987
Cells were plated at a 30×104 density in p35 mm previous gelatinized plates. Cells were removed from duplicate plates by use of trypsin and resuspended in 1 ml of Hanks' balanced salt solution containing 0.5% formalin to fix them after 8, 12, 24, 36, 48 and 60 h. Cell numbers were determined with a Neubauer chamber (0.100 mm depth, 0.0025 mm2 area) on an inverted microscope Axiovert 200 (Zeiss).
For preparation of cell lysates, E14Tg2A undifferentiated and differentiated cells were trypsinized, centrifuged for 10 min at 400 g, washed with PBS and centrifuged again. The pellet was then dissolved in lysis buffer (20 mM Tris-HCl, 1 mM EDTA, 0.5% NP40, 20% Glycerol ph = 7,5) plus a protease inhibitor cocktail (Thermo Life Sciences) and phosphatase inhibitors (2 mM orthovanadate and 5 mM sodium fluoride, Thermo Life Sciences), incubated for 15 min on ice, and then centrifuged for 25 min at 2000×g and 4°C. The same procedure was performed with total brain lysates of P2X7R (−/−) animals.
Protein quantification was measured by the Coomassie Blue method
Undifferentiated and differentiated
Changes in [Ca2+]i were determined by microfluorimetry using the FlexStation III (Molecular Devices Corp., Sunny Valley, CA), following the instructions of the manufacturer
Samples were read at 1.52 s intervals for 120 s with a total of 79 read-outs per well. Following 20 s of monitoring basal fluorescence intensity for [Ca2+]i levels of nonstimulated cells, agonists (ATP and Bz-ATP) were applied onto the cells, and induced-[Ca2+]i transients were monitored for up to 200 s. Responses to agonist addition were determined as peak fluorescence minus the basal fluorescence intensity using the SoftMax2Pro software (Molecular Devices Corp.). Data were expressed as mean values ± standard errors (S.E.).
For immunofluorescence detection of specific marker proteins for respective differentiation stages, E14Tg2A cells were grown and induced to differentiate on rounded coverslips (1 cm diameter). Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, washed three times with PBS and then incubated for 30 min in a blocking solution containing 0.05% Triton-X 100 and 2% FBS. Cells were incubated overnight at 4°C with primary antibodies raised against mouse monoclonal anti stage-specific embryonic antigen-1 (SSEA-1) (1∶200, Chemicon, Bioscience Research Reagents, Temecula, CA), rabbit polyclonal anti-βIII tubulin (1∶200, Millipore, Billerica, MA), Oct-4 rabbit polyclonal (1∶1000 Millipore), rabbit monoclonal P2X7R (1∶2000 AbCam, Cambridge, MA) and nestin (1∶1000 Millipore) antibodies. The slides were washed three times with PBS, followed by one hour incubation with Alexa Fluor 488 or 555 goat anti-mouse (1∶800, Sigma). In control experiments, the primary antibody was omitted, and immunostaining was never observed. Counterstaining of cell nuclei was achieved with 0.1% of 4′,6-diamidino-2-phenylindole (DAPI). After washing with PBS, the slides were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and examined on an Axiovert 200 epifluorescence microscope (Zeiss, Aalen, Germany), equipped with a Nikon DMX1200F camera and Metamorph image analysis program or on a confocal microscope (Zeiss LSM 780-NLO Multiphoton) and analyzed with the LSMib software (Zeiss).
Total RNA was extracted from undifferentiated and ESC subjected to 8 days of neural differentiation using the TRIzol Reagent (Invitrogen) following manufacturer's instruction. All samples were further treated with amplification grade DNase I (Sigma-Aldrich). Reverse transcription for cDNA synthesis was carried out on a thermal cycler using the RevertAid Reverse Transcriptase (Thermo Scientific Fermentas) first strand synthesis system according to the manufacturer's protocol (Invitrogen) in the presence of specific primers listed in (
Gene | Forward (5′-3′) | Reverse (5′-3′) |
Total RNA was extracted from undifferentiated and 8 days differentiated ESC using TRIzol Reagent (Invitrogen) following manufacturer's instruction. All samples were further treated with amplification grade DNase I (Sigma-Aldrich). Reverse transcription for cDNA synthesis was carried out on a thermal cycler using the RevertAid Reverse Transcriptase (Thermo Scientific Fermentas) to first strand synthesis system according to the manufacturer's protocol (Invitrogen). The transcription rates of selected mRNAs were measured by PCR using a thermal cycler. PCR was performed in 20 µl of buffer reaction containing of 2 µg cDNA, Maxima Hot Start Taq DNA Polymerase (Thermo Scientific Fermentas), and 5 pmol of each sequence-specific primers (
Cell proliferation was measured following incubation with 20 µM 5-bromo-2-deoxyuridine (BrdU; Sigma-Aldrich) for 1 hour. The cells were fixed with ice-cold 75% ethanol for 10 minutes, washed with PBS, incubated for 30 min in 2.0 M HCl and neutralized with 0.1 M sodium tetraborate. The cells were blocked with a 2% fetal bovine serum, 0.1% Triton-X solution in PBS for 30 minutes. After washing with PBS, they were incubated for 1 h with rat anti-BrdU antibodies (Abcam; 1∶200 dilution). Alexa Fluor 488 secondary antibodies (Life Technologies) were used at 1∶1000 dilution. After another washing step, propidium iodide solution (Life Technologies) at 50 µg/ml was used as a DNA stain
The cells were fixed with a 4% paraformaldehyde solution for 30 minutes, washed in PBS and blocked with a 2% fetal bovine serum, 0,1% triton-x solution in PBS for 30 minutes. The cells were then incubated in mouse anti-SSEA1 antibodies (Millipore, 1∶500 dilution) and either rabbit anti-Ki67 (Millipore, 1∶500 dilution) or rabbit anti-β3 tubulin antibodies (Sigma, 1∶700 dilution) for 1 hour. The cells were incubated for 45 min. with the Alexa Fluor 488 anti-rabbit and Alexa Fluor 647 anti-mouse secondary antibodies (Life Technologies, 1∶1000 dilution). Cells which had undergone through the whole process, but were not marked with the primary antibodies, were used as negative control. The percentage of marked cells was measured in the Attune flow cytometer (Life Technologies, CA). Alexa Fluor 488 was excited by a 488 nm blue laser and its emission was captured through a 530/30 filter. Alexa Fluor 647 was excited by a 638 nm red laser and its emission was captured through a 660/20 filter.
Comparisons between experimental data were made by one- or two-way analysis of variance following the Bonferroni post-test using GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA). Criteria for statistical significance were set at p<0.05 (*), p<0.01 (**), or p<0.001 (***).
ESC induced for 8–16 days with
P2X7 receptor and Oct-4 expression levels in undifferentiated (und) and cells induced to differentiation (days 0–8) were determined by (
In undifferentiated ESC, P2X7R expression pattern showed one band with a molecular weight of 75 KDa and another one with 100 KDa. During the progress of differentiation the expression of both isoforms decayed, but remained expressed (
In order to determine the expression pattern for the different P2X7 splice variants along the neuronal differentiation of E14Tg2a cells, RT-PCR with specific sets of primers (
(
Undifferentiated ESC express P2X7R altogether with SSEA-1, which is a membrane carbohydrate typically found in stem cells
P2X7R expression and co-localization with differentiation-stage specific proteins was determined by confocal microscopy and immunofluorescence assays as described in Materials and
In agreement with functional expression of purinergic receptors, we analyzed the induction of intracellular calcium transients ([Ca2+]i) in undifferentiated and neural-differentiated cells by the purinergic agonists ATP and Bz-ATP, which is a selective P2X7R agonist
ATP- and Bz-ATP-evoked P2X7R responses were determined by single-cell calcium imaging and microfluorimetry assays as described in Materials and
Neural-differentiated cells revealed EC50 values for ATP and Bz-ATP that are not statistically different from those observed in undifferentiated cells (
Many studies have shown that activation of P2X7R needs higher concentration of agonists (100–1000 µM of ATP)
Expression of P2X7R in neural-committed cells has been documented previously. For instance, embryonic rats (E15.5) express P2X7R mRNA together with the neural stem and progenitor cell marker nestin
ES cells are derived without the intervention of any immortalizing agent, do not undergo senescence, they proliferate without apparent limit and are not subject to contact inhibition or anchorage dependence. In fact, no means of inducing cell-cycle arrest and quiescence in ES cells are known
As already cited above, Di Virgilio and co-workers found that P2X7R promoted proliferation in tumor and microglial cells when stimulated by agonist
In order to understand modulation of ESC proliferation by P2X7R, we analyzed cell cycle distributions and cell cycle entry rates of undifferentiated cells cultured for 96 h in the absence or presence of Bz-ATP (0.1 and 1 µM) or the selective P2X7R inhibitor KN-62 (10 µM)
Cell cycle analysis based on flow cytometric analysis of BrdU incorporation and propidium iodide DNA-staining were performed as described in Materials and
Cells were plated at 3X105 cell/ml density and P2X7R was inhibited daily with 10 µM KN-62 or 1 µM A438079 and then the number of cells were counted. Data represent mean values ±S.E. of three independent experiments performed in triplicate (*p<0,05 compared to control).
A possible underlying mechanism could be that the P2X7R contributes to cell cycle-dependent [Ca2+]i transients, which are required for G1/S progression of mouse embryonic stem cells
The importance of P2X7R on ESC survival at the undifferentiated state has been reported previously
For this purpose, cells were induced to neural differentiation in the absence or presence of Bz-ATP (10 µM) or KN-62 (10 µM) along differentiation followed by gene expression analysis of the following differentiation stage markers: SSEA-1, expressed by stem cells undergoing neuroectoermal differentiation
Total RNA was isolated from cell cultures, and real-time PCR reactions were performed as described in Materials and
We observed by flow cytometry and by immunofluorescence assays that the population of differentiating cells is heterogeneous with around 70% of cells expressing only β3-tubulin and revealing neuronal morphology and neurite extensions (
(
P2X7R inhibition promoted neuronal differentiation, as the presence of KN-62 led to an increase of the number of β3-tubulin-positive cells within the SSEA-1 positive cells and decreased the β3-tubulin negative cells (
SSEA-1 is a carbohydrate adhesion molecule
Co-expression analysis of SSEA-1 and Ki-67, a marker of proliferating cells, by flow cytometry revealed that a few cells were SSEA-1+/Ki67−, while the larger percentage of the population represents proliferating neuroblasts in agreement with their characteristics as dividing cells
(
We have provided experimental evidence for novel functions of the P2X7R in ESC biology: P2X7R expression and activity is upregulated in embryonic cells, maintaining ESC proliferation, while upon induction to neural differentiation P2X7 receptor expression and activity needs to be suppressed. Pharmacological inhibition of P2X7R activity results in a higher percentage of ESC undergoing neuroectodermal differentiation, in the rescue of quiescent progenitors into cell cycle and in the promotion of differentiation into neurons. Our data suggest that extracellular nucleotides may provide a novel and powerful tool for modulating ESC functions.
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We thank Denise Yamamoto and Zilda Mendonça Izzo for technical assistance, Dr. Isis C. Nascimento for helping with cell culture, and Prof. Deborah Schechtman, Department of Biochemistry, Institute of Chemistry, University of São Paulo, for giving us the E14Tg2A cell line.