Embryonic stem cell sphere: a controlled method for production of mouse embryonic stem cell aggregates for differentiation.

摘要

OBJECTIVES: Embryonic stem cells (ESCs) are of significant interest as a renewable source of nonproliferating cells. Differentiation of ESCs is initiated by the formation of embryoid bodies (EBs). Standard methods of EB formation are limited in their production capacity, in any variations in EB size and formation of EBs through frequent passages. Here we have reported the utility of a microencapsulation technique for overcoming these limitations by mass production of mouse ESCs in alginate beads called ESC spheres. METHODS: The mouse ESCs were encapsulated in 1.2% alginate solution and cocultured on a feeder layer. The cells were evaluated by flow cytometry, in vitro differentiation, immunofluorescence, and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Analysis of encapsulated ESC spheres by flow cytometry showed similar percentages of Oct-4 and stage-specific embryonic antigen-1 (SSEA-1) expression in comparison with routine culture of ESCs. Moreover, the ESC spheres maintained a pluripotency potential which was comparable with ESCs cultured on feeder cells directly, as demonstrated by immunofluorescence and RT-PCR. CONCLUSIONS: The results demonstrated that alginate encapsulation as a simple bioreactor, provides a scalable system for mass undifferentiated ESC sphere production with similar sizes and without the need for frequent passages for differentiation and clinical and pharmaceutical applications.