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EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES |
aLaboratory of Cellular and Molecular Biophysics and
bNASA/NIH Center for Three-Dimensional Tissue Culture, National Institute of Child Health and Human Development and
cCell Biology Section, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland, USA
Key Words. Embryonic stem cell biology • Rhesus • Real-time reverse transcription-polymerase chain reaction • Lineage analysis
Correspondence: Hynda Kleinman, Ph.D., NIH/NIDCR, 30 Convent Drive, Building 30, Room 407, Bethesda, Maryland 20892, USA. Telephone: 301-728-2380; Fax: 301-402-0897; e-mail: hkleinman{at}dir.nidcr.nih.gov or Leonid Margolis, Ph.D., NIH/NICHD, 10 Center Drive, Building 10, Room 9D58, Bethesda, Maryland 20892, USA. Telephone: 301-768-5805; Fax: 301-480-0857; e-mail: margolil{at}mail.nih.gov
Received July 11, 2006;
accepted for publication October 31, 2006.
Cell interactions with the extracellular matrix (ECM) play a critical role in their physiology. Here, we sought to determine the role of exogenous and endogenous ECM in the differentiation of nonhuman primate ESCs. We evaluated cell differentiation from expression of lineage gene mRNA and proteins using real-time polymerase chain reaction and immunohistochemistry. We found that ESCs that attached to and spread upon highly adhesive collagen do not differentiate efficiently, whereas on the less adhesive Matrigel, ESCs form aggregates and differentiate along mesoderm and especially endoderm lineages. To further decrease ESC attachment to the substrate, we cultured them either on nonadhesive agarose or in suspension. In both cases, ESCs formed aggregates and efficiently differentiated along endoderm and mesoderm lineages, most strikingly into cardiomyocytes. Aggregates formed by thus-differentiated ESCs started to beat with a frequency of 50–100 beats per minute and continued to beat for approximately a month. In spite of the presence of exogenous ECM, ESCs were dependent on endogenous ECM for their survival and differentiation, as the inhibition of endogenous collagen induced a gradual loss of ESCs and neither a simple matrix, such as type I collagen, nor the complex matrix Matrigel was able to rescue these cells. In conclusion, adhesiveness to various ECM and nonbiological substrates determines the differentiation of ESCs in such a way that efficient cell-cell aggregation, together with less efficient cell attachment and spreading, results in more efficient cell differentiation.
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