Abstract 137: Ghrhr is a Cell-surface Marker of Human Pluripotent Stem Cell-derived Cardiomyogenic Precursors

Abstract

Introduction: A major roadblock for generating human pluripotent stem cell (hPSCs) derivatives highly enriched in cardiomyogenic precursors (CPCs), has been the lack of CPC-specific cell surface markers. Hypothesis: Based on observations that adult CPCs are responsive to growth hormone-releasing hormone (GHRH) signaling, we hypothesized that the GHRH receptor (GHRHR) is a specific cell-surface marker for hPSC-derived CPCs. Methods: We performed temporal analysis of GHRHR expression in an in-vitro model of human cardiogenesis using induced hPSCs (hiPSCs) and SOX10::GFP embryonic hPSCs (hESCs) ; and mouse ( in-vivo ) cardiogenesis in wild-type (WT), MEF2c-AHF-Cre, Wnt1-Cre2 and cKit-CreERT2/+ reporter mice. Results: Gene expression and confocal immunofluorescence analyses during chemically-defined, stage-specific, cardiac lineage differentiation indicated that GHRHR is not expressed in undifferentiated hiPSCs or during specification into primitive streak-like Brachyury + or Mesp1 + precardiac cells; but is induced in cardiogenic mesoderm-like cells, at the stage of commitment into NKX2.5 + and/or ISL1 + CPCs ( p =0.001) and persists in Troponin-T + cardiomyocytes. Similarly, experiments modeling cardiac neural crest (CNC) with SOX10::GFP hESCs indicated that GHRHR is not expressed by GFP + CNCs but is induced following differentiation into NKX2.5 + and/or ISL1 + derivatives. Importantly, stimulation with 1μm recombinant GHRH during days 5-7 of hiPSCs differentiation increased NKX2.5 expression 2.5-fold, an effect that was abolished by exposure to 1μM Somatostatin, a GHRH antagonist ( p =0.0009). Last, in vivo analyses in WT , MEF2c-AHF-Cre, Wnt1-Cre2 and cKit-CreERT2/+ reporter embryonic and postnatal hearts corroborated that GHRHR specifically marks NKX2.5 + mesoderm- and CNC-lineage descendants in vivo, whereas GHRHR is not expressed by Wnt1-Cre2 and cKit-CreERT2/+ CNCs descendants that are Nkx2.5 – . Conclusions: Together these findings indicate that GHRHR is universally expressed by NKX2.5 + /ISL1 + CPCs and cardiomyocytes of both mesoderm and CNC origin. Therefore, GHRHR appears to be a valuable cell-surface marker for the selection and enrichment of CPCs from hPSCs for biomedical and regenerative medicine applications.