Background:
Although studies have demonstrated the feasibility of
in vivo
cardiac transplantation of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) using large animals, it requires large quantities of purified PSC-CMs. Moreover, genetic modification and contamination of non-CMs are inappropriate for clinical application. Using antibodies on the surface of the transplanted cells is one of the useful methods, but can be immunogenic and cause local inflammation or graft failure. We have shown the synthetic mRNAs encoding a fluorescent protein tagged with sequences targeted by microRNAs (miRNAs) expressed in specific cell types can efficiently detect and purify the particular cell populations. Using a miRNA switch and magnetic-activated cell sorting (MACS), we evaluated the efficiency of purification of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in a large scale.
Methods:
We used CD4 as a selection marker for MACS and miR-208a as a specific mRNA of CMs. We synthesized CD4 mRNA and transfected it into differentiated cells from iPSCs to confirm CD4 expressing on the surface of the transfected cells. We also synthesized a miRNA switch encoding CD4 tagged with sequences targeted by miR-208a (CD4-208a switch) and transfected it into differentiated cells to demonstrate transfected non-CMs expressing CD4 and transfected CMs non-expressing CD4. Finally, we transfected the CD4-208a switch and puromycin resistance mRNA simultaneously, and purified iPSC-CMs by eliminating CD4+ cells using MACS and untransfected cells using puromycin. Purified cells were transplanted into NOG mouse hearts with myocardial infarction by direct injections into the myocardium.
Results:
After transfecting CD4 mRNA into differentiated cells from iPSCs, 78±5% expressed CD4 on the surface. We also confirmed that the CD4-208a switch separated CMs and non-CMs. Using MACS and puromycin selection, we purified iPS-CMs 69±5% to 97±2% assessed by troponin T. Purified cells were also engrafted as CMs in mouse hearts.
Conclusions:
We demonstrated that CD4-208a switch purifies iPSC-CMs efficiently in a large scale. Synthetic microRNA switches can apply for many studies of stem cell-based cell replacement therapy for clinical application.
Large-scale immuno-magnetic cell sorting of T cells based on a self-designed high-throughput system for potential clinical application