Updated publication reference for PubMed record(s): 30845956. The importance of regenerative potential of cardiac stem/progenitor cells (CSCs) after acute myocardial infarction (AMI) has been emphasized in the last decade. Several studies have demonstrated that this cell population has a relevant role in myocardial repair, supported by the establishment of a paracrine cross-talk between CSCs and the injured tissue. However, the majority of current in vitro strategies to study these cells fails to include important modulators of CSC activity, such as the paracrine effect of cardiomyocytes (CMs) and the physicochemical changes occurring in tissue during Ischemia/Reperfusion (I/R) injury. In this work, we established the first human in vitro heterotypic model of myocardial I/R injury with human CSCs (hCSCs) and human induced pluripotent cell-derived cardiomyocytes (hiPSC-CMs) using transwell inserts. hCSCs response to I/R was characterized using advanced whole proteomic mass spectrometry-based tools that allowed us to propose new pathways in hCSCs regenerative process, including: cell cycle regulation inhibition, proliferation through EGF signaling, gluthathione-mediated reactive oxygen species detoxification, and signaling through several paracrine growth factors. The model established in this work contributes with new insights regarding hCSC biology in response to AMI and provides an important tool to study and modulate molecular mechanisms involved in the regenerative potential of CSCs.