Slow Turning Lateral Vessel Bioreactor Improves Embryoid Body Formation and Cardiogenic Differentiation of Mouse Embryonic Stem Cells

Abstract

Embryonic stem cells (ESCs) have the ability to form aggregates, which are called embryoid bodies (EBs). EBs mimic early embryonic development and are commonly produced for cardiomyogenesis. Here, we describe a method of EB formation in hydrodynamic conditions using a slow-turning lateral vessel (STLV) bioreactor and the subsequent differentiation of EBs into cardiomyocytes. EBs formed in the STLV were compared with conventional techniques, such as hanging drop (HD) or static suspension cell culture (SSC), for homogeneity of EB size, shape, proliferation, apoptosis, and in vitro cardiac differentiation. After 3 days of culture, a four-fold improvement in the yield of EB formation/mL, a six-fold enhancement in total yield of EB/mL, and a nearly 10-fold reduction of cells that failed to incorporate into EBs were achieved in STLV versus SSC. During cardiac differentiation, a 1.5- to 4.2-fold increase in the area of cardiac troponin T (cTnT) per single EB in STLV versus SSC and HD was achieved. These results demonstrate that the STLV method improves the quality and quantity of ES cells to form EBs and enhances the efficiency of cardiac differentiation. We have demonstrated that the mechanical method of cell differentiation creates different microenvironments for the cells and thus influences their lineage commitments, even when genetic origin and the culture medium are the same. Ascorbic acid (ASC) improved further cardiac commitment in differentiation assays. Hence, this culture system is suitable for the production of large numbers of cells for clinical cell replacement therapies and industrial drug testing applications.

Exogenous LIN28 Is Required for the Maintenance of Self-Renewal and Pluripotency in Presumptive Porcine-Induced Pluripotent Stem Cells

“…Spontaneous cardiogenic differentiation of the piPSCs was induced as described in a previously published report ( Rungarunlert et al, 2013 ). In brief, floating embryoid bodies (EBs) were formed in the poly(2-hydroxyethyl methacrylate)-coated wells of 96-well plates using 5,000 piPSCs per well and maintained in differentiation medium for 21 days.…”

Rabbit induced pluripotent stem cells retain capability of <i>in vitro</i> cardiac differentiation

“…The protocol for cardiac differentiation via hanging drop technique was performed as previously described [42] with minor modification. Briefly, iPSC lines R1, R2 and R3 (passage 22–25) were dissociated and allowed to aggregate into three-dimension in EB medium which was composed of DMEM/F12 medium supplemented 1 mM L-glutamine, 1% (v/v) NEAA, 0.1 mM β-mercaptoethanol, BMP4 (10 ng/ml) and 15% (v/v) FBS (HyClone TM , Utah, USA).…”

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].…”