Fundamental differentiation and growth characterization of murine embryonic stem cells in varied culture conditions

Although embryonic stem (ES) cells and their pluripotent capability have been elucidated for decades, little study has been done on obtaining the pluripotency profile of ES cells in the incipient stages of differentiation. In this research, an ES cell line with transfected green fluorescent protein (GFP) co-expressed by an Oct-4 promoter was analyzed by fluorescence-activated cell sorter (FACS) to obtain such profile. As Oct-4 is an ES cell differentiation marker whose expression varies with pluripotency, GFP expression could simply be measured in these cells to determine how pluripotent they are as a population. The differentiation characterization of ES cells was also conducted with different culture conditions of reduced serum and glucose concentrations both in the presence and absence of leukemia inhibitory factor (LIF) which prevents spontaneous differentiation, as well as at varied LIF concentrations and seeding densities. In addition, fundamental growth kinetic and metabolic profiles were obtained to get a more complete picture of how ES cells behave under these varied culturing conditions. The doubling time (t[sub]d) of R1 Oct4-GFP cell line was found to be 13 hours in LIF⁺ culture and 8 hours in culture with LIF addition after 7 days of LIF withdrawal, implying that cell proliferation rate is higher for cells receiving a sudden upregulation of genes controlling cell division through LIF addition. Although the upregulation of the genes is rapid, the downregulation of these genes through LIF withdrawal was found to take 6-7 days, while 3-4 days were required to downregulate the pou5f gene (which controls Oct4 expression). Higher concentration of LIF resulted in higher ES cell proliferation rate, but GFP⁺ expression was unaffected by

Department dc:contributor.department

Massachusetts Institute of Technology. Biological Engineering Division.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2004

Author dc:creator

• Hashimura, Yasunori, 1980-

Advisor dc:contributor.advisor

• Jean-François Hamel.

• Biological Engineering Division.

Statement dc:rights

• MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

en_US

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/28529

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/28529