High density cultures of embryoid bodies enhanced cardiac differentiation of murine embryonic stem cells

Abstract

Murine embryonic stem cell (mESC)-derived cardiomyocytes represent a promising source of cells for use in the development of models for studying early cardiac development as well as cell-based therapies in postnatal pathologies. Here, we report a highly efficient cardiac differentiation system in which high density embyoid body (EB) cultures leads to a marked increase of cardiomyocytes production from multiple mESC lines without the addition of any cardiogenic growth factors. Our results show that high density EB cultures significantly increase the yield of functional cardiomyocytes, which express typical cardiac markers, exhibit normal rhythmic Ca2+ transients, and respond to both β-adrenergic and electric stimulations. During the differentiation period, the inhibition of bone morphogenetic protein (BMP) signaling significantly attenuates the increase of cardiac differentiation as well as the increased expression of cardiac-specific genes, NK2 transcription factor related 5 (Nkx2.5) and myosin light chain 2v (Mlc2v) by high density EB cultures. Therefore, we believe that we offer a novel and efficient means of cardiomyocyte production for practical use of mESCs in cardiac regenerative medicine.

An inactivating mutation in the histone deacetylase SIRT6 causes human perinatal lethality

“…1A), we tested the capacity of D63H mutant mESCs to differentiate into functional cardiomyocytes. To address this question, we used an EB-directed differentiation protocol in mESCs to aid in mesoderm layer specification (Lee et al 2011). From day 0 to day 4, mESCs were cultured under hanging drop conditions as EBs.…”

“…Subsequently, from day 4 to day 25, cells were placed in EGM-2 (endothelial cell type) medium containing growth factors essential for normal cardiac development ( Fig. 4A, top panel; Lee et al 2011). On day 25, wild-type SIRT6 differentiated cardiomyocytes morphologically resembled cardiomyocyte foci and exhibited regular spontaneous contraction patterns (Supplemental Fig.…”

Making cardiomyocytes: How mechanical stimulation can influence differentiation of pluripotent stem cells

“…This method is very effective in monolayer culture, as cardiomyocyte yields of up to 90–95% have been observed following this regimen with human ESCs . Cardiomyocyte differentiation from embryoid body (EB) culture is significantly lower, averaging only 1–27% yields as indicated by gene markers such as cardiomyocyte‐specific gene myosin light chain 6 (Myl6) and contractile activity . These relatively low yields confirm the necessity to improve EB differentiation protocols for more high‐throughput systems.…”

Differentiation of Human Atrial Myocytes From Endothelial Progenitor Cell-Derived Induced Pluripotent Stem Cells

“…Embryoid bodies are three-dimensional aggregates of pluripotent stem cells that undergo differentiation and cell specification into all the three-germ layers including CM in a manner similar to the early embryo [216][217][218][219]. In this technique, small clumps of pluripotent stem cell are dispersed in a suspension culture containing serum media to form spherical aggregates.…”

“…Over the years that followed, a series of optimization studies examined the capacity of first generation iPSC lines to differentiate into CM lineages [214,215]. These approaches include Embryoid Body formation (EB) [216][217][218][219] co-culture with stromal layer [220] and direct cytokine-guided differentiation [221].…”