Abstract
Purinergic signaling mediated by P2 receptors (P2Rs) plays important roles in embryonic and stem cell development. However, how it mediates Ca2+ signals in human embryonic stem cells (hESCs) and derived cardiovascular progenitor cells (CVPCs) remains unclear. Here, we aimed to determine the role of P2Rs in mediating Ca2+ mobilizations of these cells. hESCs were induced to differentiate into CVPCs by our recently established methods. Gene expression of P2Rs and inositol 1,4,5-trisphosphate receptors (IP3Rs) was analyzed by quantitative/RT-PCR. IP3R3 knockdown (KD) or IP3R2 knockout (KO) hESCs were established by shRNA- or TALEN-mediated gene manipulations, respectively. Confocal imaging revealed that Ca2+ responses in CVPCs to ATP and UTP were more sensitive and stronger than those in hESCs. Consistently, the gene expression levels of most P2YRs except P2Y1 were increased in CVPCs. Suramin or PPADS blocked ATP-induced Ca2+ transients in hESCs but only partially inhibited those in CVPCs. Moreover, the P2Y1 receptor-specific antagonist MRS2279 abolished most ATP-induced Ca2+ signals in hESCs but not in CVPCs. P2Y1 receptor-specific agonist MRS2365 induced Ca2+ transients only in hESCs but not in CVPCs. Furthermore, IP3R2KO but not IP3R3KD decreased the proportion of hESCs responding to MRS2365. In contrast, both IP3R2 and IP3R3 contributed to UTP-induced Ca2+ responses while ATP-induced Ca2+ responses were more dependent on IP3R2 in the CVPCs. In conclusion, a predominant role of P2Y1 receptors in hESCs and a transition of P2Y-IP3R coupling in derived CVPCs are responsible for the differential Ca2+ mobilization between these cells.
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Introduction
Human embryonic stem cells (hESCs) are pluripotent cells with the properties of self-renewal and differentiation potential into various cell types, including cardiovascular progenitor cells (CVPCs [1–4]). This in vitro differentiation system provides unique tools for the study of early cardiovascular development [5], drug screening [6, 7], cytotoxicity testing [8, 9], and cardiac regenerative therapy [10, 11]. However, the physiological and pharmacological properties of hESCs and CVPCs, such as the Ca2+ signaling and the response to various modulators, are largely unknown. Such knowledge will promote the pharmacological regulation of lineage-specific commitment, the quality control of hESC derivatives from functional perspectives, and subsequently benefit the preclinical and clinical study of these cells.
Nucleotides, such as ATP, have been shown to play a fundamental role in the cardiovascular system under physiological and pathological conditions through the activation of specific purinergic receptors [12–14]. Extracellular nucleotide-activated membrane P2 receptors (P2Rs) are composed of seven mammalian P2X receptors (P2XRs) acting as Ca2+ channels and eight mammalian P2Y receptors (P2YRs) coupling to G proteins [15]. Both P2XRs and P2YRs are widely expressed in early developing embryos, suggesting that they may play roles in various cell types [16]. However, little is known about the difference of P2R subtype-mediated Ca2+ signals between the hESCs and CVPCs, a gap for the application of these cells in drug development and cytotoxicity testing.
P2R activation-mediated Ca2+ signals have been shown to regulate the physiological responses in the cardiovascular system, such as endothelial cells [17, 18], smooth muscle cells [19, 20], and cardiomyocytes [21, 22]. They are also involved in the proliferation and cardiac differentiation of mouse (m) ESCs [23, 24] and the growth of human adult cardiac progenitors [25]. In addition, G protein-mediated Ca2+ signals play a role in the maintenance of undifferentiated state of hESCs [26]. ATP has been verified to induce Ca2+ transients in hESCs and derived cardiomyocytes [27]. However, the contribution of P2Rs to extracellular nucleotide-mediated Ca2+ signals in the hESCs and the derived CVPCs is largely unknown.
The activation of Gq/11-protein coupled P2YR subtypes elicits intracellular Ca2+ release via inositol 1,4,5-trisphosphate receptors (IP3Rs) on the endoplasmic reticulum [28]. IP3Rs consist of three subtypes, termed IP3R1, IP3R2, and IP3R3, and have a tissue-specific distribution relating to their physiological and pharmacological responses in various tissues [29, 30], including cardiovascular systems [31–33]. However, the roles and the functional coupling of P2Rs and IP3R subtypes in hESCs and derived CVPCs remain unclear.
Thus, the aims of this study are to determine (i) the expression pattern of P2Rs and IP3Rs in hESCs and derived CVPCs; (ii) the Ca2+ responses of these cells to multiple extracellular nucleotides (ATP, UTP, ADP, and UDP) and P2R-targeted reagents; and (iii) the contribution of IP3R subtypes to P2R-mediated Ca2+ signals.
Methods
hESC culture and CVPC induction
hESC culture and CVPC induction were carried out as described previously [4, 34]. Briefly, hESC lines H7 and H9 (WiCell Research Institute, Madison, WI, USA) were routinely maintained in mTeSR1 media (Stem Cell Technologies, Vancouver, Canada) on Matrigel- (hESC qualified; Corning, New York, NY, USA) coated dishes. For CVPC induction, hESCs with density of 80 % confluence were digested into single cells by Accutase (Stem Cell Technologies, Vancouver, Canada) and seeded onto Matrigel-coated dishes at a density of 3.5 × 104 cells cm−2 in CVPC induction medium (CIM) for 3 days. CIM contained DMEM/F12, 1 × B27 supplement without vitamin A, 1 % L-glutamine, 1 % penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA), and supplemented with 400 μM 1-thioglycerol (Sigma, Saint Louis, MO, USA), 50 μg mL−1 ascorbic acid (Sigma, Santa Clara, CA, USA), 25 ng mL−1 bone morphogenetic protein 4 (R&D Systems, Minneapolis, MN, USA), and 3 μM Glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021 (Axon Medchem, Groningen, Netherlands).
For Ca2+ imaging, hESCs were digested into single cells by Accutase and seeded onto Matrigel-coated 20 mm glass-bottom dishes (Nest Scientific, Rahway, NJ, USA) at a density of 3.5 × 104 cells cm−2 in mTeSR1 or CIM. The medium was changed every day. Of 36–48 h after seeding, hESC clones were used for Ca2+ imaging to allow homogenous loading of the Ca2+ indicator. CVPCs on day 3 of differentiation were used for Ca2+ imaging. To reduce apoptosis, 5 mM Rock inhibitor Y27632 (Stem Cell Technologies, Vancouver, Canada) was added for the first 24 h after cell seeding. The embryoid body (EB) differentiation assay was performed according to the previous report [35].
Immunostaining
Immunostaining assays were performed as previously described [34]. Briefly, cells were fixed with 4 % paraformaldehyde and permeabilized in 0.3 % Triton X-100 for intracellular antigens (Sigma, Saint Louis, MO, USA), blocked in 10 % normal goat serum (Vector Laboratories, Burlingame, CA, USA) and then incubated with primary antibodies against MESP1 (1:100; Aviva Systems Biology, San Diego, CA, USA), MEF2C (1:100; Cell Signaling Technology, Danvers, MA, USA), ISL1 (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), Oct4 (1:200; Abcam, Cambridge, UK), or SSEA4 (1:100; Millipore, Temecula, CA, USA) at 4 °C overnight and detected by DyLight 488- or 549-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Nuclei were stained with DAPI (Sigma, Saint Louis, MO, USA). A Zeiss Observer microscope was used for slide observation and image capture.
Flow cytometry analysis
Cells were harvested and dissociated by Accutase (Stem Cell Technologies, Vancouver, Canada). Samples were blocked by 3 % fetal bovine serum and then stained for the presence of appropriate hESCs or CVPCs markers using antibodies including PE-conjugated SSEA1 (1:20; eBioscience, San Diego, CA, USA), PE-conjugated SSEA4 (1:100; BD Biosciences, San Jose, CA, USA), and un-conjugated SOX2 (1:100; R&D Systems, Minneapolis, MN, USA) with PE-conjugated secondary antibody (1:200; eBioscience, San Diego, CA, USA) or isotype-matched negative controls.
Ca2+ imaging
hESCs or CVPCs were loaded with 2.5 μM Fluo4-AM (Life Technologies, South San Francisco, CA, USA) dissolved in the extracellular bath solution for 30 min at 37 °C [27]. Then the Ca2+ indicator was washed out for three times by the bath solution and the cells were used 30 min later at room temperature for the de-esterification of the dye. The fluorescence of hESCs or CVPCs was measured using a confocal laser scanning microscope (LSM 710, Carl Zeiss, Oberkochen, Germany) with a ×20 objective. The fluorescence intensity was excited with wavelength 488 nm and the emission was collected with wavelength >493 nm. Images were acquired every 2 s.
Analysis of Ca2+ responses
The analysis of Ca2+ responses was based on the customer-modified Interactive Data Language (IDL, ITT corporation, White Plains, NY, USA) Program, Flash Sniper reported previously [36]. Briefly, the images recorded were opened by Flash Sniper and a mask was set up to exclude the noise signals from non-cell regions. The region of interest (ROI) was manually selected for each cell. The normalized amplitude of a Ca2+ transient was expressed as dF/F 0 = (F 1 − F 0)/F 0, where F 0 and F 1 are the values of the fluorescence at rest and the peak time point, respectively. For each ROI, the start-time and end-time of Ca2+ transients were set manually and dF/F 0 was calculated by Flash Sniper. Finally, the traces representing the fluorescence changes were automatically generated by the software. The cell with dF/F 0 > 0.2 was defined as a responding cell. For the calculation of amplitude values, the counted cell number of randomly selected responding cells in each field is commonly >50, while all the responding cells were chosen when the total responding cell number is <50 in the experiment with some reagents. All the cells in each field of imaging were counted for calculating the responding percentage. The responding cell percentage was calculated as responding cell number/total cell number examined. At least three independent experiments were done for each concentration of the reagents.
Reverse transcription (RT)-PCR and quantitative real-time PCR (Q-PCR)
Total RNA was prepared using an RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions and treated with RNase-Free DNAse (Promega, Madison, WI, USA) for 15 min to eliminate the potential contamination of genomic DNA. cDNAs were generated by reverse-transcribed total RNA (1 μg) using ReverTra Ace reverse transcriptase (Toyobo, Osaka, Japan). The RT-PCR was carried out using 2× Taq Plus Master Mix (Vazyme, Piscataway, NJ, USA). GAPDH was used as endogenous control, and samples without reverse transcription were used as negative controls. Q-PCR was performed and analyzed by the ViiA™ 7 Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) with SYBR Green Q-PCR Master Mix (Toyobo, Osaka, Japan). Q-PCR data were expressed as fold changes normalized to internal control GAPDH. The RT-PCR and Q-PCR primers were selected from the Primer Bank and were listed in Supporting Information Table S1.
Knockout (KO) and knockdown (KD) of IP3Rs
IP3R2KO cell lines were established by electroporation using the hESC Nucleofector® Kit (Lonza, Basel, Switzerland) with the transcription activator-like effector nucleases (TALEN) plasmids (Viewsolid Biotech, Beijing, China) targeting 5′-CCA GCT TCC TCT ACA T-3′ (the left target) and 5′-ACG CGG AGG GCT CGG TC-3′ (the right target) of the first exon of ITPR2 (the gene of IP3R2, Online Resource Fig. S1a). Single clones were selected by puromycin and two clones of IP3R2KO (IP3R2KO-6 and IP3R2KO-12) from H7 hESCs were confirmed by sequencing and Western blot. To establish IP3R3KD hESCs, short hairpin (sh) RNA-mediated mRNA interference were used and the two 19-bp sequences (5′-GAA GTT CCG TGA CTG CCT C-3′ and 5′-GAG GCA GTC ACG GAA CTT C-3′ [37]) were cloned into the pLKO1 vector containing puromycin-resistant gene (Online Resource Fig. S1b). The vector control was pLKO1 vector inserted with non-targeted sh-RNA sequence (Sigma, Saint Louis, MO, USA). The H9 and H7 hESCs were transfected by the lentivirus constructed with the above vectors and selected by puromycin (Life Technologies, Carlsbad, CA, USA) at 1 mg mL−1. Single clones were picked out and amplified. Two clones of IP3R3KD from either H9 (siIP3R3-1 and siIP3R3-2) or H7 (siIP3R3-a and siIP3R3-b) hESCs were used in this study.
Western blot analysis
Total membrane fractions of hESCs and CVPCs were prepared as previously reported [24] and were separated on 8 % SDS-PAGE gels and transferred to Nitrocellulose Blotting Membranes. The antibodies against IP3R1 and IP3R2 were made as previously reported [38]. The antibodies against IP3R1 (1:1000), IP3R2 (1:1000), IP3R3 (1:1000; BD Biosciences, San Jose, CA, USA), and β-actin (1:8000; Sigma, Saint Louis, MO, USA) were used. Then the membranes were incubated with IRDye 680LT Donkey anti-Rabbit IgG or IRDye 800LT Donkey anti-mouse IgG (1:8000; Li-COR Biosciences, Lincoln, NE, USA) as secondary antibodies and visualized on an Odyssey Infrared Imager (Li-COR Biosciences, Lincoln, NE, USA).
Reagents and solutions
(N)-Methanocarba-2-MeSADP (MRS2365), 2-methylthio-ATP (2-MeSATP), and 2-chloro-N 6-methyl-(N)-methanocarba-2′-deoxyadenosione-3′,5′-bisphosphate (MRS2279) were purchased from Tocris Biosciences Ltd (Bristol, UK). All the other reagents, including ATP, UTP, ADP, UDP, 2′(3′)-O-(4-Benzoylbenzoyl)-ATP (BzATP), pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), suramin, and 2-aminoethoxydiphenyl borate (2-APB) were purchased from Sigma (Saint Louis, MO, USA). Reagents were all directly dissolved in distilled water except PPADS and 2-APB which were dissolved in DMSO. The extracellular bath solution contains (in mM) NaCl, 135; KCl, 5.4; CaCl2 1.8; MgCl2 1.0; glucose, 10; and HEPES, 10 (pH adjusted to 7.4). For Ca2+-free solution, extracellular CaCl2 was removed and 1 mM EGTA was added.
Statistical analysis
The data were presented as means ± SEM. Two-tail t test was used to analyze the data obtained from Q-PCR assays; one-way ANOVA with Dunnett’s multiple comparison tests were used to analyze the responding cell percentage of hESCs or CVPCs pre-treated with Ca2+-free solution, suramin, PPADS, or 2-APB vs. ATP alone, and to analyze the responding cell percentage to MRS2365 in hESCs vs. CVPCs. Two-way ANOVA with Bonferroni post-test was used to analyze the responses to ATP or 2MeSATP in hESCs with or without MRS2279. Two-way ANOVA with Dunnett’s multiple comparison tests were used to analyze the data of responding cell percentage to ADP in hESCs vs. that in CVPCs and to MRS2365 in IP3R2KO hESCs vs. wild type (WT) cells; the Hill slopes and EC50 values of the concentration-response curves were calculated using nonlinear regression (variable slope), and the slopes of concentration-amplitude curves were calculated using linear regression. All the statistical analyses were performed by GraphPad Prism5 (San Diego, CA, USA). P < 0.05 was considered statistically significant.
Results
Differential Ca2+ responses to the P2R stimulation in hESCs and derived CVPCs
H9 and H7 hESCs had typical morphology and pluripotent characteristics, such as alkaline phosphatase activity (ALP, Fig. 1a) and the expression of pluripotent markers SSEA4 and SOX2 (Fig. 1b), while the derived CVPCs expressed ISL1/MESP1/MEF2C/SSEA1 (Fig. 1c) as previously reported [4, 34].
The maintenance of hESCs and directed differentiation to CVPCs. a Morphology and alkaline phosphatase (ALP) activity of H9 and H7 hESCs on day 2 and day 6 after passage. Scale bar, 100 μm. b Flow cytometry analysis of the SSEA4+ and SOX2+ cell percentage in H9 and H7 hESCs. c Immunostaining of CVPC markers ISL1, MESP1, or MEF2C (red) with a nucleus marker DAPI (blue) and flow cytometry analysis of the SSEA1+ H9 and H7 CVPCs. Scale bar, 50 μm
To determine the function of P2Rs in hESCs and derived CVPCs, we measured intracellular Ca2+ responses to various nucleotides in these cells. hESCs and CVPCs showed different responding patterns of intracellular Ca2+ activities to extracellular nucleotides in a concentration-dependent manner. UTP, a P2Y2,4 receptor agonist [39], up to 300 μM only induced Ca2+ signals in 34.6 ± 6.3 % (H9) and 18.4 % ± 3.4 % (H7) of hESCs but in almost all CVPCs (Fig. 2a, b), while the non-specific P2R agonist ATP (100 μM) triggered intracellular Ca2+ responses in almost all hESCs and CVPCs (Fig. 2a, c).
Characteristics of Ca2+ signals induced by UTP and ATP in hESCs and derived CVPCs. a Representative confocal images showing the Ca2+ responses to UTP and ATP (both 100 μM) in hESCs (left panel) and derived CVPCs (right panel). Scale bar, 50 μm. b, c The concentration-response curves of UTP (b) and ATP (c) in hESCs and CVPCs. d, e The concentration-amplitude curves of Ca2+ transients to UTP (d) or ATP (e) in hESCs and CVPCs. n = 5–9 independent experiments for each concentration; 50–150 hESCs and 200–400 CVPCs in each experiment. *** P < 0.001 (H9) and ### P < 0.001 (H7) CVPCs vs. corresponding hESCs
The concentration-response curves for UTP (Fig. 2b) and ATP (Fig. 2c) significantly shifted to left in the CVPCs compared with those in the hESCs, indicating that the CVPCs are more sensitive to UTP and ATP than hESCs. The EC50 values of UTP were more than 40-fold higher in hESCs than those in CVPCs (Fig. 2b). The EC50 values of ATP were more than ninefold higher in hESCs than those in CVPCs (Fig. 2c). Moreover, the amplitude of Ca2+ transients induced by UTP or ATP was concentration-dependently increased in both hESCs and CVPCs, but the amplitude was significantly higher in the CVPCs than in the hESCs (Fig. 2d, e). Further, the slopes of concentration-amplitude curves to UTP in CVPCs were nearly twofold higher than those in hESCs (Fig. 2d), while those to ATP were slightly higher in CVPCs than in hESCs (Fig. 2e).
ADP, a P2Y1,12,13 receptor agonist [39], at 100 or 500 μM induced the Ca2+ responses in most hESCs and CVPCs (Fig. 3a). However, hESCs showed higher Ca2+ responding percentage than that of CVPCs at lower concentration of ADP (10 μM, Fig. 3b). There is no Ca2+ response to UDP (up to 500 μM), a P2Y6 receptor agonist [39], in hESCs (Fig. 3c, d), while CVPCs were concentration-dependently activated by UDP (Fig. 3d).
Characteristics of Ca2+ signals induced by ADP and UDP in hESCs and derived CVPCs. a Representative confocal images of the Ca2+ responses to ADP (100 μM for hESCs, 500 μM for CVPCs). b The cell percentages with Ca2+ responses to ADP stimulation (10–500 μM) in hESCs and CVPCs. c Representative confocal images of the Ca2+ responses to UDP (500 μM) in hESCs and CVPCs. d The cell percentages with Ca2+ responses to UDP stimulation (10–500 μM) in hESCs and CVPCs. n = 5 independent experiments; 50–150 hESCs and 200–400 CVPCs in each experiment; * P < 0.05, *** P < 0.001 vs. hESCs at the corresponding concentrations. Scale bar, 50 μm
These results demonstrate that the hESCs, at least H7 and H9 cell lines, and their derived CVPCs respond different Ca2+ activities to extracellular nucleotides. Especially, CVPCs have significantly higher sensitivity and amplitude of Ca2+ transients responding to UTP and ATP comparing with the hESCs.
Gene expression patterns of P2Rs and IP3Rs in hESCs and CVPCs
To determine whether the different nucleotide-induced Ca2+ activities between hESCs and CVPCs are related to the transition of P2Rs, we analyzed their gene expression patterns. RNA samples from day 18 EBs differentiated from hESCs were used as positive controls. The expression of P2RX2, P2RX5, P2RX7, P2RY1, P2RY2, P2RY6, and P2RY12 was detected in both H9 and H7 hESCs (Fig. 4a). The expression patterns of P2Rs were different in the CVPCs. P2RX1, P2RX2, and P2RX6 were hardly detected, while P2RX3 and P2RX4 were increased but P2RX5 and P2RX7 were decreased in the CVPCs compared with the hESCs (Fig. 4a). Q-PCR analysis further confirmed the significantly lower expression levels of P2RX2, P2RX5, and P2RX7 and higher expression levels of P2RX3 and P2RX4 in the CVPCs than in the hESCs (Fig. 4b). The expression of most P2RY genes were 7-fold to 60-fold higher in the CVPCs than those in the hESCs, including those hardly detected in the hESCs, such as the genes encoding P2Y4,11,14 receptors (Fig. 4a, b). In contrast, only the expression of P2RY1 was reduced by 75 % in the CVPCs compared with that in the hESCs (Fig. 4b). These data indicate the transition of P2R gene expression patterns between the hESCs and CVPCs.
Expression patterns of genes encoding P2Rs and IP3Rs in hESCs and CVPCs. a RT-PCR analysis of gene expression of all mammalian subtypes of P2Rs and IP3Rs in hESCs and CVPCs. EB, embryoid body. b, c Q-PCR analysis of the gene expression of P2Rs and IP3Rs in hESCs and CVPCs. n = 4–5 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. the corresponding hESCs
Because some P2YRs elicit Ca2+ release from intracellular Ca2+ store via activating IP3Rs [39], we then compared the expression of three genes encoding for IP3R subtypes [29, 40]. It is noteworthy that the expression levels of all three ITPRs encoding IP3Rs were significantly lower in CVPCs than in hESCs (Fig. 4a, c), which was contradictory to the observed stronger Ca2+ response in the CVPCs. These results suggest that the gene expression patterns of P2Rs and IP3Rs may account for the different intracellular Ca2+ responses to the above nucleotides in the two cell types examined.
Differential P2YR subtypes linking to IP3Rs account for distinct Ca2+ responses in hESCs and CVPCs
To further clarify the contribution of P2Rs to the different kinetics of nucleotide-stimulated Ca2+ mobilization in hESCs and CVPCs, we pre-incubated the cells with extracellular Ca2+-free solution to block the Ca2+ entry through membrane P2XRs [41] as shown in Fig. 5a. The extracellular Ca2+-free condition did not significantly alter the percentage of cells responding to ATP and only slightly decreased the amplitude of Ca2+ transients in H9 hESCs and derived CVPCs (Fig. 5b–d), indicating that little effects of P2XRs are involved. Then suramin, a nonselective antagonist of P2Rs except P2Y4 receptor, and PPADS, a nonselective antagonist of most P2XRs and P2Y1,4,6,13 receptors [42], were applied to detect the involvement of P2YRs (Fig. 5a). Pretreatment of 100 μM suramin or 50 μM PPADS reversibly blocked almost all the Ca2+ events induced by ATP in hESCs (Fig. 5b–d). In contrast, they only partially inhibited the proportion of Ca2+ responding cells (Fig. 5c) and the amplitude of Ca2+ transients (Fig. 5d) in the CVPCs, even up to 200 μM suramin or 100 μM PPADS (data not shown). Similar results were observed in H7 hESCs and CVPCs (Online Resource Fig. S2). These results suggest that distinct P2YR subtypes regulate Ca2+ signals in hESCs and derived CVPCs, while the contribution of P2XRs is trivial.
Analysis of P2YRs and IP3Rs involved in ATP-mediated Ca2+ events in H9 hESCs and CVPCs. a An outline of the experimental protocol. b Representative images showing the effects of extracellular Ca2+-free solution, suramin, PPADS, and 2-APB on the Ca2+ responses induced by ATP in hESCs (left) and CVPCs (right). Scale bar, 50 μm. c The percentage of Ca2+ responding cells to ATP with or without various reagents. d Box whisker plots showing the median and 10–90 percentiles of the amplitudes (dF/F 0) of Ca2+ transients from responding cells to ATP with various pretreatments. Dots represent the outlier data of individual cells out of 10–90 percentiles. n = 4–5 independent experiments; 50–150 hESCs and 200–400 CVPCs in each experiment. ** P < 0.01, *** P < 0.001 vs. ATP alone
Since IP3Rs were the critical downstream mediators of the Gq/11-coupled P2Y1,2,4,6,11 receptors [39], we then examined whether IP3Rs were involved in the ATP-induced Ca2+ activities. The IP3R inhibitor 2-APB at 100 μM [43] did not affect the baseline of Ca2+ signals in H9 hESCs and CVPCs (Fig. 5b), while it completely and reversibly inhibited the percentage of ATP-activated Ca2+ events in the hESCs and largely attenuated the proportion of Ca2+ responding CVPCs by about 90 % (Fig. 5c) accompanied with a significant decrease of the amplitudes of Ca2+ transients (Fig. 5d). Similar results were observed in H7 hESCs and derived CVPCs (Online Resource Fig. S2). These results suggest an important role of IP3Rs in the generation of P2YR-mediated Ca2+ signals following the ATP stimulation in both hESCs and CVPCs.
P2Y1 receptors are predominant in hESCs but not in CVPCs
Based on the above observations, we hypothesized that P2Y1 receptors would be critically responsible for the intracellular Ca2+ response in hESCs but not in CVPCs. To test it, we used a P2Y1 receptor selective agonist (MRS2365), a P2X and P2Y1 receptor nonselective agonist (2-MeSATP [44, 45]), and a P2Y1 receptor selective antagonist (MRS2279 [46]). Firstly, the ATP-induced Ca2+ transients in hESCs were blocked by pre-incubation of 20 μM MRS2279 (Fig. 6a, b) but were not affected in CVPCs (Fig. 6c, d). MRS2279 pre-incubation decreased the percentage of responding cells to ATP by 91 % in H9 and 82 % in H7 hESCs compared with ATP alone but it was ineffective in CVPCs (Fig. 6e). Secondly, 2-MeSATP (50 μM) induced Ca2+ transients in more than 90 % hESCs, and these responses were almost completely blocked by MRS2279 (20 μM) (Fig. 6a, b, f). These data further confirmed that the function of P2XRs is trivial in hESCs. In contrast, only few CVPCs responded to 2-MeSATP (100 μM) stimulation (Fig. 6g). Thirdly, MRS2365 (50 μM), a P2Y1 receptor selective agonist, induced Ca2+ transients in almost all hESCs (Fig. 6a, b, h), while no responses to MRS2365 were detected in the CVPCs up to 100 μM (Fig. 6c, d, h). Thus, P2Y1 receptors appear to be a dominant P2R subtype in hESCs but not in CVPCs.
P2Y1 receptors primarily contributed to P2R-induced Ca2+ activity in hESCs not in CVPCs. a–d Representative traces of the Ca2+ transients from individual cells of hESCs (a, b) or CVPCs (c, d) treated with ATP (100 μM), 2-MeSATP (50 μM), or MRS2365 (50 μM) alone or combined with pre-incubation of MRS2279 (20 μM). e The cell percentages responding to ATP (100 μM) alone or pre-incubation with MRS2279 (20 μM) in hESCs and CVPCs. n = 4–6. *** P < 0.001 vs. ATP alone. f The cell percentages responding to 2-MeSATP (50 μM) alone or pre-incubation with MRS2279 (20 μM) in hESCs. n = 4–5. *** P < 0.001 vs. 2MeSATP alone. g The cell percentages responding to 2-MeSATP (100 μM) in CVPCs. n = 4–5. h The cell percentages responding to MRS2365 (50 μM) in hESCs and CVPCs. n = 5. *** P < 0.001 vs. the corresponding hESCs. n, number of independent experiments; 50–150 hESCs and 200–400 CVPCs in each experiment
P2Y1 receptor-mediated Ca2+ activities through IP3R2 in hESCs
To identify the downstream IP3Rs in the P2Y1 receptor-activated Ca2+ signals in hESCs, we used downregulation and loss of function strategies. Since IP3R1 was hardly detected at protein level in hESCs (Fig. 7a), we then examined the involvement of other two subtypes by establishment of IP3R2KO and stable IP3R3KD hESCs (Online Resource Fig. S1a, b). TALEN-mediated IP3R2KO and shRNA-mediated IP3R3KD (siIP3R3) were confirmed by Western blot analysis (Fig. 7a, b). These cells showed pluripotent markers and ALP activity similar to the vector control and WT hESCs (Online Resource Fig. S1c). The P2Y1 receptor-specific agonist MRS2365 concentration-dependently triggered Ca2+ responses in WT and vector control hESCs (Fig. 7c, d). These responses were not significantly altered in IP3R3KD hESCs (Fig. 7c, d) but were rightward and downward shifted in IP3R2KO cell lines (Fig. 7d). These data reveal that IP3R2 plays an important role in P2Y1 receptor-mediated Ca2+ activities in hESCs.
Contributions of IP3R2 and IP3R3 to P2YR-mediated Ca2+ activity in hESCs and CVPCs. a, b The confirmation of IP3R2KO (a) and IP3R3KD (b) by Western blot analysis. E, hESCs; C, CVPCs. c, d Concentration-response curves of MRS2365 in the H9 (c) and H7 (d) IP3R3KD hESCs or the IP3R2KO hESCs (d). e–h The concentration-response curves of ATP (e, f) and UTP (g, h) in IP3R3KD hESC-derived CVPCs. i, j The concentration-response curves of ATP (i) and UTP (j) in IP3R2KO CVPCs. n = 3–4 independent experiments for each concentration; 50–150 hESCs and 200–400 CVPCs in each experiment. * P < 0.05, *** P < 0.001 (IP3R2KO-6) and # P < 0.05, ### P < 0.001 (IP3R2KO-12) vs. WT hESCs
Differential coupling of IP3R3 and IP3R2 to ATP- and UTP-targeted P2YRs in CVPCs
Due to the negligible function of P2Y1 receptor in CVPCs, the coupling of IP3R2 and IP3R3 to other P2YR-mediated Ca2+ signals in the CVPCs was examined. Our results showed that IP3R3KD or IP3R2KO hESC-derived CVPCs had comparable expression of CVPC marker SSEA1 between vector control and WT CVPCs (Online Resource Fig. S1d, e). IP3R3KD did not alter the concentration-response curves of ATP in CVPCs (Fig. 7e, f), but right-shifted the curves of UTP (Fig. 7g, h), with the EC50 values 7-fold to 9.5-fold (siIP3R3-1/2) and 3.5-fold to 7-fold (siIP3R3-a/b) higher than those of vector control CVPCs (Table 1). However, IP3R2KO right-shifted the concentration-response curves of both ATP (Fig. 7i) and UTP (Fig. 7j). The EC50 values of ATP were 17-fold (IP3R2KO-6) and 12-fold (IP3R2KO-12) higher than those of WT CVPCs, and the values of UTP were 38-fold (IP3R2KO-6) and 17-fold (IP3R2KO-12) higher than those of WT CVPCs (Table 1). In addition, the slopes of the curves were decreased in the IP3R2KO CVPCs (Table 1). Furthermore, the averaged amplitudes of Ca2+ transients induced by ATP (300 nM to 10 μM) and UTP (100 nM to 3 μM) were decreased in IP3R2KO CVPCs compared with those in WT CVPCs (Online Resource Fig. S3). These results suggest that both IP3R2 and IP3R3 contribute to the UTP/P2Y2,4-mediated Ca2+ activities in the CVPCs, while ATP/P2YR-mediated Ca2+ signals are more dependent on the IP3R2 in these cells.
Discussion
The main findings of the present study are that (i) extracellular nucleotides elicit distinct intracellular Ca2+ responses between hESCs and derived CVPCs, with more sensitive and stronger Ca2+ responses to ATP and UTP in CVPCs than those in hESCs; (ii) the expression of most genes encoding P2YRs except P2Y1 receptors are higher in the CVPCs than those in the hESCs; (iii) P2Y1 receptors primarily mediate nucleotide-induced Ca2+ activities in hESCs but not in CVPCs; and (iv) the different functional coupling between the P2YR subtypes and IP3R subtypes contributes to the distinct intracellular Ca2+ signals in hESCs and CVPCs. These findings provide new insights into the physiological and pharmacological properties of hESCs and derived CVPCs by revealing critical roles of P2YRs and contributions of IP3R subtypes in the regulation of Ca2+ signals via purinergic signaling pathways.
Here, we detect the previously unrevealed contribution of P2R subtypes to Ca2+ responses in hESCs, though ATP, the nonselective agonist of P2Rs, has been found to be involved in the Ca2+ responses in mESCs [43] and hESCs [27]. In this study, we reveal that UTP and ADP but not UDP also trigger Ca2+ activities in hESCs. In contrast, all the nucleotides tested induce robust Ca2+ signals in the CVPCs. Moreover, distinct patterns of Ca2+ responses to these nucleotides are observed between the hESCs and CVPCs. The Ca2+ responses to ATP and UTP are more sensitive and stronger in the CVPCs than those in the hESCs, which may explain the recent finding that ATP treatment enhances cardiogenesis during mESC differentiation [23]. The differences could be interpreted by the distinct gene expression levels and selectivity of P2R subtypes in these cells (Fig. 4). Notably, only part of hESCs respond to UTP (Fig. 2b), a P2Y2,4 receptor selective agonist [39]. Considering the correlation of cell surface proteins and pluripotency within hESCs [47], it is worthy to further investigate the physiological relevance of the UTP responding hESC subpopulation on the self-renewal and differentiation.
The Ca2+ signals mediated by the P2XR activation are observed in various cell types [48, 49]. The P2rx3 and P2rx4 genes are expressed in mESCs [24]. In our study, the gene expression of P2RX2, P2RX5, and P2RX7 are detected in hESCs and P2RX3 and P2RX4 are detected in CVPCs (Fig. 4a). However, P2XRs seem to play a limited role in the Ca2+ responses of hESCs and CVPCs. This is supported by the following observations: (i) the responsive cell proportion remains unchanged in hESCs and CVPCs by removing extracellular Ca2+ which blocks the P2XR-mediated Ca2+ entry; (ii) almost no responses are detected in hESCs when triggered by the P2XR and P2Y1 receptor nonselective agonist 2-MeSATP at the presence of P2Y1 receptor selective antagonist MRS2279 (Fig. 6f) and few responses are triggered by 2-MeSATP in CVPCs (Fig. 6g); (iii) no responses are induced by the P2X7 receptor agonist BzATP (data not shown) in hESCs and CVPCs. In contrast, the P2Y receptor agonists, such as ADP, UDP, UTP, and MRS2365, trigger distinct Ca2+ responses between hESCs and CVPCs, suggesting the important role of P2YR during the transition of hESCs to CVPCs (Fig. 3b). Taken together, our data here extend the previous findings by showing that the P2YRs but not P2XRs play crucial roles in nucleotide-mediated Ca2+ signals in hESCs and CVPCs. This is further confirmed by the inhibitory effect of 2-APB, an IP3R antagonist [43], on the ATP-mediated Ca2+ signals in both cell types, suggesting the Ca2+ responses are mediated by P2YR-IP3R coupling (Fig. 5c).
Notably, the contributions of P2YR subtypes are different between hESCs and CVPCs. Among most of the eight P2YR subtypes, P2Y1 receptors predominantly mediate Ca2+ responses in the hESCs but not in the CVPCs. This is supported by the following evidence: firstly, P2Y1 is the only P2YR subtype significantly reduced in the CVPCs compared with the hESCs (Fig. 4a, b); and secondly, the P2Y1 receptor agonist MRS2365 [46] elicits Ca2+ transients in most hESCs. Meanwhile, the majority of the hESCs responding to ATP and 2MeSATP are blocked by MRS2279, a P2Y1 receptor-specific antagonist [46]. Therefore, P2Y1 receptors dominantly contribute to nucleotide-induced Ca2+ signals in the hESCs. Since the activation of P2Y1 receptors in P19 embryonic carcinoma cells, a pluripotent stem cell line [50], has been reported to enhance the cell proliferation [51], it would be interesting to further determine the impact of this P2YR subtype in the regulation of proliferation of hESCs.
All the genes encoding P2YRs are detectable in the CVPCs (Fig. 4a), while some of them may not functionally expressed like P2Y1 and P2Y11 (BzATP target [52]). Moreover, based on the different pharmacological profiles of P2YRs [28], the P2Y2,4 receptor agonist UTP and the P2Y6 receptor agonist UDP induce Ca2+ signals in almost 100 % CVPCs (Fig. 2a, b, Fig. 3c, d). Thus, the P2Y2,4,6 receptors are significantly enhanced in the CVPCs compared with the hESCs, though other P2YR subtypes may also involve in the regulation of Ca2+ signals in CVPCs. These results indicate that the hESC-derived CVPCs are useful for the screening and pharmacological study of drugs targeting P2YRs.
Another finding here is the establishment of functional coupling of P2YRs and IP3Rs in the regulation of intracellular Ca2+ release between the hESCs and CVPCs. The blockage of Ca2+ response by the IP3R inhibitor 2-APB in hESCs and CVPCs suggests a crucial role of IP3Rs in the purinergic signaling pathways of these cells. Since IP3Rs are the downstream of P2Y1,2,4,6,11 receptor subtypes [15, 39] and the protein expression of IP3R2 and IP3R3 but not IP3R1 are detected in hESCs in this study (Fig. 7a), it is conceivable to establish the coupling between IP3R2/3 and P2YRs in the hESCs using gene manipulation. There are no changes of the P2Y1 receptor agonist MRS2365-induced concentration-response curves in IP3R3KD hESCs compared with the vector control and WT hESCs, while IP3R2KO significantly decreases the responsive cell proportion to MRS2365 in hESCs, indicating a coupling of IP3R2 to the P2Y1 receptors in the hESCs. Thus, IP3R2 but not IP3R3 is essential for the Ca2+ signals mediated by the activation of P2Y1 receptors in hESCs. Moreover, both IP3R2 and IP3R3 are involved in the regulation of Ca2+ responses in the CVPCs, while they differentially contribute to ATP- and UTP-induced Ca2+ activities (Fig. 7). The functional switch of IP3Rs between hESCs and CVPCs may provide new clues for the dynamics of the intracellular Ca2+ mobilization in the early differentiating hESCs. Notably, although the gene expression of all IP3R subtypes are lower in CVPCs than those in hESCs (Fig. 4a, c), the nucleotide-mediated Ca2+ transients are stronger (Fig. 2d, e). This may additionally due to the enhanced coupling between P2YRs and IP3Rs or post-translational modifications on IP3Rs [53]. The precise mechanisms underlying the heterogeneity of the Ca2+ responses and function of IP3Rs in hESCs and CVPCs remain to be investigated.
Limitation: although our data are collected from two well-documented and wildly used cell lines, we cannot exclude the possibility that other hESCs and their derived CVPCs could display different P2Y and IP3R expression patterns. Further studies are needed to determine if the findings here can be extended to other hESC lines and their derived CVPCs.
Conclusion
Our results reveal that (i) a functional switch of P2YRs occurs between the hESCs and CVPCs; (ii) Ca2+ responses to various nucleotides are more sensitive and stronger in the CVPCs than those in the hESCs; (iii) P2Y1 receptors coupling to IP3R2 are the predominant P2YR subtype in hESCs, while P2Y2,4 receptors coupled to IP3R2/3 are enhanced in the CVPCs (Fig. 8). Our findings suggest that subtype-specific coupling and functional switch of P2YRs and IP3Rs might play pivotal roles in the P2R-mediated Ca2+ signals in hESCs and CVPCs. The findings here would lead to a better understanding of the physiological and pharmacological properties of Ca2+ signal regulatory receptors in these cells.
A working model for the switch of functional coupling between P2YRs and IP3Rs in hESCs and derived CVPCs. Ca2+ mobilization in hESCs and derived CVPCs is regulated by purinergic signals through the specific P2YR-IP3R coupling, which results in the differential sensitivity and strength of Ca2+ responses between those cells. P2Y1 receptors coupling to IP3R2 are predominant in hESCs, while P2Y2,4 receptors coupling to both IP3R2 and IP3R3 are enhanced in the CVPCs with a significantly decreased function of P2Y1 receptors. In addition, P2Y6 receptors are uniquely functional in the CVPCs but not in the hESCs. Little effect of P2XRs exists in the nucleotide-induced Ca2+ activities of hESCs and CVPCs
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Acknowledgments
This work was supported by the grants from the National Natural Science of China Grants (Nos. 31030050 and 81520108004 to HTY, No. 31401167 to MZ); the Strategic Priority Research Program of CAS (No. XDA01020204 to HTY); the National Basic Research Program of China (No. 2014CB965100 to HTY); the National Science and Technology Major Project (No. 2012ZX09501001 to HTY); and the Major Program of Development Fund for Shanghai Zhangjiang National Innovation Demonstration Zone (No. ZJ2014-ZD-002 to HTY). We thank WiCell Research Institute for providing the H7 and H9 hESCs, and Dr. Heping Cheng (Peking University, Beijing, China) for providing the Flash Sniper software and for constructive discussion.
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Jijun Huang and Min Zhang are joint first co-authors.
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Huang, J., Zhang, M., Zhang, P. et al. Coupling switch of P2Y-IP3 receptors mediates differential Ca2+ signaling in human embryonic stem cells and derived cardiovascular progenitor cells. Purinergic Signalling 12, 465–478 (2016). https://doi.org/10.1007/s11302-016-9512-9
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DOI: https://doi.org/10.1007/s11302-016-9512-9