Abstract 14077: Human Placental CDX2 Cells Are Multipotent and Differentiate Robustly to Cardiomyocytes

Regenerative biology hinges on exploration of evolutionarily preserved healing mechanisms to maintain and restore homeostasis. CDX2 is one such conserved factor that regulates trophoblast stem cell differentiation in the placenta. We previously demonstrated that murine placental Cdx2 lineage cells are multipotent and restored cardiac function even in male mice after myocardial infarction. We have now isolated CDX2 cells from over 35 patients’ placentas and studied their properties. Using huCDX2 promoter-driven mCherry lentivirus, CDX2 cells were isolated and multi-omics analyses were carried out versus H9ES cells. In vitro cardiac and vascular differentiation was examined. CDX2 cells expressed HLAG (50.5%±5) and cytokeratin7 (95%±5) supporting a trophoblast progenitor identity. Bulk RNA-seq showed 1065 upregulated genes. Differentially expressed genes like NKX2.5, GATA4 , and TBX5 attribute a cardiac committed progenitor feature to CDX2 cells. CDX2 cells downregulated pluripotency markers negating concerns of any undesirable proliferation. Whole-cell proteome of CDX2 cells revealed 252 unique proteins of which 73 were upregulated involving development and immune modulation including FGF, EFGR, and the Map kinase/ERK pathway crucial for cardiac development, in addition to unique cell surface proteins, which can be utilized in place of lentivirus for live-cell selection. When plated on neonatal murine cardiomyocyte feeders, CDX2 cells robustly differentiated into beating cardiomyocytes expressing cTnT and alpha-actinin. Using an alternate feeder-free method, we observed 80-90% cTnT expression in differentiated CDX2 cardiomyocytes. Additionally, CDX2 cells gave rise to endothelial lineage cells expressing vWF. CDX2 cells further formed tube-like structures on matrigel-based endothelial assays and demonstrated uptake of acetylated LDL suggesting functional endothelial generation. We further demonstrated that CDX2 cells can be passaged and significantly expanded ex vivo (50,000 to 1million cells), while retaining cardiovascular potential. These results bring us closer to translation as placenta-derived CDX2 cells may form the basis for a novel clinical approach to cardiovascular regeneration.