Human embryonic stem cells (hESCs) permit unique bioprocessing challenges. Cell-cell and cell-matrix signaling must be tightly regulated to maintain cells in the undifferentiated, self-renewing state during scaleup of hESC culture. If individual colonies become too small, cells replicate slowly or fail to grow. If colonies become too large, spontaneous differentiation can occur.
Phase contrast image of hESC colony overlayed with anti-Oct4 immunofluorescence (green). The hESC colony is cocultured with mouse embryonic fibroblast feeder cells.
Currently, most hESCs are cultured with undefined media that has been conditioned by various types of fibroblasts. These media are unsuitable for use in culturing hESCs for therapeutic applications because of the unknown animal components and the batch-to-batch heterogeneity. Identifying chemical signals that repress differentiation and promote self-renewal is critical for advancing hESC research and development of hESC-based therapies.
Mechanical forces during culture may also affect hESC self-renewal and differentiation decisions in culture. We have shown that cyclic biaxial strain represses differentiation, allowing culture of hESCs to higher densities and for longer times without spontaneous differentiation than is possible in the absence of strain. We working to identify the strain receptors and mechanotransduction pathways stimulated by strain in hESCs. We hope that this information can be coupled with chemical factors to design methods to scale up hESC culture systems.
hESCs cultured for 12 days in the presence of 10% cyclic biaxial strain express Oct4, signifying they are undifferentiated while most hESCs grown under the same conditions in the absence of strain do not express Oct4, indicating that they have differentiated
Inefficient cryopreservation methods also hamper use of hESCs in research and clinical settings. These cells have extremely low viability following freezing and thawing, and many of the cells that do survive undergo spontaneous differentiation. We have identified that maintaining adhesion signals during the cryopreservation process inhibits apoptosis, improving viability by approximately two orders of magnitude and virtually eliminating differentiation. In this method, hESC colonies are frozen and thawed embedded in Matrigel. Current cryopreservation efforts include design of novel chemical cryoprotectants and cryopreservation protocols guided by biological studies to identify the types of damage experienced by hESCs during freezing and thawing.
Schematic for cryopreservation of hESCs embedded in Matrigel.
Copyright 2005 The Board of Regents of the University of Wisconsin System
Date last modified:
12-Sep-2005
Date created: 8-Sep-2005
Content by: palecek@engr.wisc.edu
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