Abstract 132: CD13 and ROR2 Mediated Isoloation of hESC Derived Cardiac Mesoderm
The human heart has a limited regenerative capacity and there is an unmet demand for improved therapies for myocardial disease. Human embryonic stem cell (hESC) derived cardiomyocytes have potential to facilitate the development of both cell-based and pharmaceutical treatments. To date many studies have focused on the efficient generation of cardiomyocytes from hESCs. However, its clinical use has been limited by technical challenges, including the inability to isolate a pure population of cardiovascular progenitors capable of robust engraftment and regeneration. To this end, we have attempted to identify surface markers that facilitate isolation of cardiac mesoderm and cardiovascular progenitors from differentiating hESCs.
Lineage-specific reporter hESC lines (MIXL1-GFP and NKX2.5-GFP) were utilized to test prospective isolation of early cardiac progenitors using two novel cell surface markers, CD13 and ROR2. A distinct population of cells co-expressing CD13 and ROR2 was isolated after 3 days of hESC differentiation which exhibited a transcriptional profile similar to primitive streak/mesoderm cells. Gene expression analysis and immunostaining confirmed the enrichment of mesodermal cells and near complete removal of endodermal, ectodermal and undifferentiated cells. To quantify the extent of cardiovascular progenitors generated from CD13/ROR2 expressing cells, an NKX2.5-GFP reporter hESC line was used and the double positive cells were sorted after 3 days and maintained in culture for 14 days. The majority of these cells showed enrichment for cardiovascular cells based on gene and protein expression, phenotype, and transplantation studies. By enabling retrieval of early mesoderm precursors from heterogeneous differentiated hESC populations, we anticipate CD13 and ROR2 should have utility in enriching for subsequent cardiac potential_the necessary first developmental step towards creating purified cardiomyocytes from hESC.
- Cardiomyocyte lineage specification
- Cardiac mesoderm isolation
- Cardiac progenitor cell characterization
- © 2013 by American Heart Association, Inc.