Abstract 18573: Unmasking Phenotypic Micro-heterogeneities in Adult Cardiac Progenitor Cells: Clonal Analysis, Single Cell QRT-PCR, and Fate Mapping
Characterization of adult cardiac progenitor cells (CPC) is incompletely resolved and their potential heterogeneity needs further investigation. Here, we assess the molecular signature, heterogeneity, and origin of adult heart-derived Lin-/Sca-1+ mouse CPCs by clonal analysis, single cell QRT-PCR and fate-mapping. Clonal growth was predicted by the side population (SP) phenotype. Cloned cardiac Lin-/Sca-1+/SP+ CPCs (cSPC) were propagated for >10 months without senescence or loss of the Sca-1/SP phenotype. QRT-PCR of 43 genes in >30 clones showed a consensus signature defined by stem cell-associated markers and key cardiac transcription factors, but lacking markers of pluripotency, primitive or pre-cardiac mesoderm, hematopoiesis, and cardiomyocytes. Clonal analysis revealed mutually exclusive patterns of three core factors (Gata4, Mef2c, Tbx5), masked in the mixed population. Cloned cSPCs showed cardiac (cTnI, MLC2v, sarc MyHC, sarc α-actin), endothelial (vWF) and smooth muscle (SM-MyHC) differentiation after grafting to the heart. Fresh (non-propagated) cSPCs were profiled with microarrays, compared to 3 clones at passage 40, heart, and ESCs: clones largely resembled fresh cSPCs, excepting an increase in proliferation-associated GO categories. Single cell QRT-PCR (Fluidigm) on >40 single fresh cSPCs confirmed both the transcriptional signature of the clones and heterogeneous expression of core cardiac factors. Thus, cSPCs resemble an incomplete but stable form of heart-forming mesoderm. By Cre/lox fate-mapping, we traced cSPCs’ embryological origin. Cardiac SP cells are derived from pre-cardiac mesoderm (98% labelled in Mesp1-Cre/R26R-tdTomato mice) and are not “lapsed” myocytes, neural crest, or hematopoietic in origin (0.2%, 1.2%, 0.2% with Myh6-, Wnt1-, or Vav-Cre). Half are derived from cells formerly expressing Nkx2.5 or Isl1 (52%, 57%), though these are rarely detected in fresh adult single cells (<2%). Thus, our analyses unmask micro-heterogeneities in adult cSPCs, which may represent different states of differentiation or lineage. Expression of key cardiogenic transcription factors in mutually exclusive patterns may represent a mechanism to maintain the stem cell pool and prevent precocious differentiation.
- © 2012 by American Heart Association, Inc.