Generation and Characterization of Functional Cardiomyocytes from Rhesus Monkey Embryonic Stem Cells

Abstract

Embryonic stem cells (ESCs) from mice and humans (hESCs) have been shown to be able to efficiently differentiate toward cardiomyocytes (CMs). Because murineESCs and hESCs do not allow for establishment of preclinical allogeneic transplantation models, the aim of our study was to generate functional CMs from rhesus monkey ESCs (rESCs). Although formation of ectodermal and neuronal/glial cells appears to be the default pathway of the rESC line R366.4, we were able to change this commitment and to direct generation of endodermal/mesodermal cells and further differentiation toward CMs. Differentiation of rESCs resulted in an average of 18% of spontaneously contracting embryoid bodies (EBs) from rESCs. Semiquantitative reverse transcription-polymerase chain reaction analyses demonstrated expression of marker genes typical for endoderm, mesoderm, cardiac mesoderm, and CMs, including brachyury, goosecoid, Tbx-5, Tbx-20, Mesp1, Nkx2.5, GATA-4, FOG-2, Mlc2a, MLC2v, ANF, and ␣-MHC in rESC-derived CMs. Immunohistological and ultrastructural studies showed expression of CM-typical proteins, including sarcomeric actinin, troponin T, titin, connexin 43, and cross-striated muscle fibrils. Electrophysiological studies by means of multielectrode arrays revealed evidence of functionality, electrical coupling, and ␤-adrenergic signaling of the generated CMs. This is the first study demonstrating generation of functional CMs derived from rESCs. In contrast to hESCs, rESCs allow for establishment of preclinical allogeneic transplantation models. Moreover, rESC-derived CMs represent a cell source for the development of high-throughput assays for cardiac safety pharmacology.

Generation of Functional Murine Cardiac Myocytes From Induced Pluripotent Stem Cells

“…Interestingly, RT-PCR analyses revealed that both the undifferentiated iPS cells and the ES cells expressed significant amounts of mRNA for the mesodermal markers Brachyury and Mesp1 and for cardiac markers including the MLC (Figure 3). Similar results have been reported for both human and rhesus monkey ES cells 26,27 and are consistent with the reports that undifferentiated ES cell cultures often contain some colonies with a "differentiated" morphology. 27 Further RT-PCR analyses detected expression of the marker genes typical for mesoderm, endoderm, cardiac mesoderm, and cardiomyocytes in the differentiating iPS cells (Figure 3).…”

A Novel Miniaturized Multimodal Bioreactor for Continuous In Situ Assessment of Bioartificial Cardiac Tissue During Stimulation and Maturation

“…With this in mind, the ideal cell source for cardiac tissue engineering would be patient-specific stem cell-derived cardiomyocytes. Cardiac 1 differentiation has been demonstrated successfully for both embryonic stem cells 5,6 and induced pluripotent stem cells [7][8][9][10] from various species; however, these cells are currently not suitable for clinical application in patients due to the methods used in their generation. Despite recent promising results regarding scalable expansion 11,12 and differentiation 13 of embryonic and pluripotent stem cells, the available numbers of stem cell-derived cardiac cells have not been sufficient for the generation of BCT of appropriate sizes for therapeutic application so far.…”

Cardiomyogenesis of embryonic stem cells upon purinergic receptor activation by ADP and ATP

“…Considering the prominent role of Ca 2+ as a cellular second messenger in directing the fate of stem cells towards cardiac [19][20][21]. We postulated that one of the critical targets f o r i n d u c t i o n o f [ C a 2 + ] i s i g n a l i n g i n v o l v e d i n cardiomyogenesis could be the activation of purinergic receptors.…”

“…Intracellular [Ca +2 ] i exerts multiple functions in the process of cardiac cell differentiation and early heart development depending upon the amplitude, space, and time of [Ca 2+ ] i signaling. Decoding the molecular mechanism of the multifaceted roles of [Ca 2+ ] i signaling will therefore not only contribute to decipher molecular pathways of cardiogenesis but also help to elaborate protocols for highly efficient cardiomyocyte differentiation from ES cells [18][19][20][21]. During embryonic heart formation, a couple of cardiogenic factors turn on Ca 2+ -dependent signaling pathways in cardiac progenitor cells.…”