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DOI: 10.1055/s-0035-1544336
Identification of Cardiac Progenitor Cells of the Second Heart Field by Application of ISLET1-Specific Fluorescent Reporter Probes Across Species Borders
Objectives: Cardiac progenitor (CP) cells provide a promising cell source for regenerative therapies due to their restricted cardiac differentiation potential combined with a reduced risk for teratoma formation. CP cells are characterized by the expression of distinct transcription factors. In the absence of specific surface markers their isolation requires fixation resulting in the loss of cell viability. Here we describe a new approach using fluorescence labeled ISLET1-specific reporter particles allowing the isolation of live second heart field CP cells.
Methods: Optimum conditions to efficiently label cardiac progenitor cells during differentiation of pluripotent cells were established by applying graded concentrations Cy3-labeled GAPDH-specific reporter nanoparticles (SmartFlare™ Probes) directly to living cells. For the identification of cardiac progenitor cells of the second heart field an ISLET1-specific SmartFlare™ Probe was applied. The fluorescent signal was recorded microscopically 24 hours after the application of the probes. The fraction of ISLET1 positive progenitor cells was determined by flow cytometry.
Results: We first determined the labeling efficiency by applying graded concentrations of GAPDH-specific SmartFlare™ Probes to 293 cells growing as monolayers. A concentration of 1.000 pm labeled the majority of 293 cells. The same concentration induced a Cy3-specific signal in 85% of three-dimensionally growing murine ES cell colonies. Furthermore, almost every cell during differentiation of murine and human embryoid bodies (EBs) could be labeled under these conditions. Analysis of ISLET1 gene expression revealed strong upregulation by day 6 in differentiated murine and human pluripotent cells. Thus, cardiac progenitors of the second heart field were visualized by the application of an ISLET1-specific SmartFlare™ Probe on day 5. Cy3-positive cells were clearly visible under a fluorescence microscope 24 hours later. Subsequent flow cytometry revealed a fraction of ∼10% and 8% Cy3-positive cells in differentiated human and murine EBs, respectively.
Conclusion: Our results show that fluorescent ISLET1-specific reporter probes are able to identify cardiac progenitor cells of the second heart field across species borders directly in live cells. This approach may provide a powerful tool to obtain a promising cellular source for potential subsequent applications in regenerative therapies.