Comparison of osteogenesis of human embryonic stem cells within 2D and 3D culture systems

Abstract

The objective of this study was to compare the osteogenic potential of human embryonic stem cells (hESCs) within two- and three-dimensional (2D and 3D) culture systems. hESCs of the H1 line (Wicell Inc., Madison, Wisc., USA) were induced to form embryoid bodies (EBs) through 5 days of suspension culture within non-adherent culture dishes. Following enzymatic dissociation, the EB-derived single cells were seeded on either novel 3D porous PLGA scaffolds or 2D culture dishes with the same total cell number. Osteogenic differentiation was induced through culture media supplemented with dexamethasone, L-ascorbic acid and beta-glycerophosphate. After 3 weeks of in vitro culture, quantitative and qualitative assays of osteogenic differentiation were conducted. Osteocalcin secretion and alkaline phosphatase (AP) activities were detected at significantly higher levels within 3D culture compared with the 2D system. Subsequently, the cell-scaffold constructs were implanted in iliac crest defects of immunosuppressed rabbits. After 4 weeks, the constructs were subsequently explanted and characterized by histology and X-ray analysis. Formation of new bone was detected within and around the implanted scaffolds. The results demonstrate that the osteogenic differentiation of human embryonic stem cells is enhanced in a 3D culture system compared to a 2D culture environment. Upon implantation in situ, the differentiating human embryonic stem cells can contribute positively to the repair and regeneration of bone defects.

Engineering Embryonic Stem-Cell Aggregation Allows an Enhanced Osteogenic Differentiation In Vitro

“…Osteogenic differentiation has been shown in samples following EB dissociation before osteogenic culture. 63,64 Results showed that settled EB cultures exhibited greater osteogenic differentiation than dissociated EB cultures, demonstrating that continued 3D cell-cell and cellmatrix interaction benefited osteogenic differentiation, which has been previously reported 63,65 (Fig. 4E, G).…”

Finding the winning combination

“…These observations are supported by several studies that have shown cellular function significantly differs in 2D microenvironments relative to their 3D counterparts. [24][25][26][27] Therefore, developing 3D microarray platforms is important for studying stem cell differentiation in a high-throughput manner. 10,23,28 In one such study Dordick and colleagues 28 developed a 3D cellular microarray platform that could be used to track and quantify stem cell behavior.…”

Differentiation of Hepatocytes from Mice Embryonic Stem Cells in Three-Dimensional Culture System Imitating in vivo Environment

“…One is that 3D culture systems using scaffolds can provide cell-to-cell and/or cell-to-matrix interactions. These interactions promote differentiation of ES cells and construction of tissue-like structures (Carlberg et al, 2009, Elisseeff et al, 2006, Gao et al, 2010, Tian et al, 2008, Yim et al, 2006. Another benefit of the 3D culture system is that it can be manipulated to retrieve the cells or tissues derived from EBs without injuring or destroying them.…”