Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems

Abstract

Embryonic stem (ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that in¯uence cell fate decisions and to develop scalable methods of cell production. We compared four standard ES cell differentiation culture systems by measuring aspects of embryoid body (EB) formation ef®ciency and cell proliferation, and by tracking development of a speci®c differentiated tissue typeÐbloodÐusing functional (colony-forming cell) and phenotypic (Flk-1 and CD34 expression) assays. We report that individual murine 1 1 1 1 1 1 1 1 1 1 ES cells form EBs with an ef®ciency of 2 2 2 2 2 2 2 2 2 2 42 9%, but this value is rarely obtained because of EB aggregationÐa process whereby two or more individual ES cells or EBs fuse to form a single, larger cell aggregate. Regardless of whether EBs were generated from a single ES cell in methylcellulose or liquid suspension culture, or aggregates of ES cells in hanging drop culture, they grew to a similar maximum cell number of 28,000 9,000 cells per EB. Among the three methods for EB generation in suspension culture there were no differences in the kinetics or frequency of hematopoietic development. Thus, initiating EBs with a single ES cell and preventing EB aggregation should allow for maximum yield of differentiated cells in the EB system. EB differentiation cultures were also compared to attached differentiation culture using the same outputs. Attached colonies were not similarly limited in cell number; however, hematopoietic development in attached culture was impaired. The percentage of early Flk-1 and CD34 expressing cells was dramatically lower than in EBs cultured in suspension, whereas hematopoietic colony formation was almost completely inhibited. These results provide a foundation for development of ef®cient, scalable bioprocesses for ES cell differentiation, and inform novel methods for the production of hematopoietic tissues.

Hydrophobic surfaces for enhanced differentiation of embryonic stem cell-derived embryoid bodies

“…Although EBs can be generated through several methodologies, the suspension culture technique allows for easy access to the cultured EBs and can be scaled for expansion (9). In this method, EBs are formed when ES cells are removed from feeder contact and dispersed on low-attachment tissue culture plates, supplemented by culture medium absent of key factors necessary for the maintenance of undifferentiated ES cell growth.…”

Enhanced Chondrogenic Differentiation of Embryonic Stem Cells by Coculture with Hepatic Cells

“…Once cultured in suspension without LIF, ES cells aggregate and form clusters, mimicking the structure of natural blastocysts. Conventionally, EBs are plated onto tissue culture dishes, and the dissociated EBs are differentiated into various specialized cells in a monolayer [14,29,30]. Bilayer co-culture systems, alternatively, provide three-dimensional environments to the cells, mimicking the native tissue environment.…”

Cardiac Repair by Embryonic Stem-Derived Cells

“…Maltsev and colleagues developed the hanging drop technique, which permitted reproducible generation of uniformly sized EBs (Maltsev et al 1993). Dang and colleagues subsequently showed that cell-cell interactions in EBs was largely mediated by E-cadherin, and furthermore that E-cadherin expression decreased as EBs began to differentiate (Dang et al 2002(Dang et al , 2004). Zandstra and colleagues took advantage of these observations: EBs generated with the hanging drop technique and subsequently transferred into stirred suspension cultures yielded highdensity cultures comprised of well-differentiated cells (Zandstra et al 2003).…”