Coupling switch of P2Y-IP3 receptors mediates differential Ca(2+) signaling in human embryonic stem cells and derived cardiovascular progenitor cells

Abstract

Purinergic signaling mediated by P2 receptors (P2Rs) plays important roles in embryonic and stem cell development. However, how it mediates Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) transients in hESCs but only partially inhibited those in CVPCs. Moreover, the P2Y1 receptor-specific antagonist MRS2279 abolished most ATP-induced Ca(2+) signals in hESCs but not in CVPCs. P2Y1 receptor-specific agonist MRS2365 induced Ca(2+) 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 Ca(2+) responses while ATP-induced Ca(2+) 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 Ca(2+) mobilization between these cells.

Keywords: Ca2+ signaling; IP3 receptors; Lineage progenitors; P2Y receptors; Pluripotent stem cells.

Fig. 1

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

Fig. 2

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

Fig. 3

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

Fig. 4

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

Fig. 5

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

Fig. 6

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

Fig. 7

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

Fig. 8

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 P2Y_2,4 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