RESUMO
While the mammalian heart has low, but functionally significant, levels of telomerase expression, the cellular population responsible remains incompletely characterized. This study aimed to identify the cell types responsible for cardiac telomerase activity in neonatal, adult, and cryoinjured adult hearts using transgenic mice expressing green fluorescent protein (GFP), driven by the promoter for murine telomerase reverse transcriptase (mTert), which is a necessary and rate-limiting component of telomerase. A rare population of mTert-GFP-expressing cells was identified that possessed all detectable cardiac telomerase RNA and telomerase activity. It was heterogeneous and included cells coexpressing markers of cardiomyocytic, endothelial, and mesenchymal lineages, putative cardiac stem cell markers, and, interestingly, cardiomyocytes with a differentiated phenotype. Quantification using both flow cytometry and immunofluorescence identified a significant decline in mTert-GFP cells in adult animals compared to neonates (â¼9- and â¼20-fold, respectively). Cardiac injury resulted in a â¼6.45-fold expansion of this population (P<0.005) compared with sham-operated controls. This study identifies the cells responsible for cardiac telomerase activity, demonstrates a significant diminution with age but a marked response to injury, and, given the relationship between telomerase activity and stem cell populations, suggests that they represent a potential target for further investigation of cardiac regenerative potential.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Miocárdio/metabolismo , Telomerase/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Antígenos Ly/genética , Antígenos Ly/metabolismo , Citometria de Fluxo , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Miocárdio/citologia , Miocárdio/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Embryonal carcinoma (EC) stem cells derived from germ cell tumours are valuable tools for the study of embryogenesis and closely resemble embryonic stem cells. When human TERA2.cl.SP12 EC cells are exposed to retinoic acid and grown as adherent monolayers, approximately 10-15% of cells commit toward becoming neurons whilst the remainder of cells produce non-neuronal cell types. Using established protocols it is possible to isolate and purify neurons from these cultures but such a process takes several weeks and the numbers of neurons produced are relatively low. In this study, we describe the development of novel procedures to enhance neuronal productivity with dramatically increased efficiency, which will be of value for research purposes and drug discovery programmes.
