Introduction: Current efforts in cardiac regenerative medicine have utilized different stem cell products to institute resuscitation of cardiac function. One example is cardiopoietic stem cells, which has shown signs of efficacy in improving cardiac function for ischemic heart failure. However, the manufacturing cost and inherent variability of cardiac transcription factor activity in this regenerative platform impairs its broad-based use in practice. To address this issue, we developed a novel approach using microencapsulated-modified-messenger RNA (M3-RNA) in conjunction with small molecule inhibitors to create an engineered cardiopoietic stem cell lineage from human adipose-derived mesenchymal stem cells (AMSC).
Hypothesis: Transfection of human AMSC with M3-RNA encoding cardiogenic transcription factors with small molecular inhibitors will induce an engineered cardiopoietic stem cell lineage.
Methods: Human AMSC were transfected with mRNA encoding brachyury (T) and/or Oct4 for up to 48 hours prior to the addition of GSK inhibitor (CHIR99021) and/or Wnt inhibitor (IWP-2) for another 24-48 hours. The cells were probed for cardiac mesoderm marker Mesp1 and cardiopoietic markers Nkx2.5 and Mef2c expression using immunocytochemistry.
Results: Transfection of human AMSC with Oct4 mRNA induced nuclear Oct4 expression 4 hours post-transfection that peaked at 24 hours. Co-transfection with Oct4 and T mRNA induced Mesp1 expression. We then employed a combined or sequential transfection of AMSC with either Oct4 and/or T, along with the addition of CHIR99021 and/or IWP-2 to assess cardiopoietic markers Nkx2.5 and Mef2c expression. While many permutations yielded comparable results, transfection with T alone for 48 hours followed by CHIR99021 for 48 hours was the most effective approach in inducing maximal Nkx2.5 and Mef2c expression.
Conclusions: We here demonstrate a novel approach to engineer cardiopoietic stem cells using Oct4 and T mRNA with small molecule inhibitors in vitro using human AMSC in as few as 5 days. This approach circumvents many limitations of current cocktail-based cardiopoiesis protocols and serves as a highly translatable platform to ease the cost burden in inducing cardiopoiesis for human clinical applications.