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Controlling aggregate formation and stability: A. Pre-differentiation improves aggregate formation and stability.

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posted on 2008-02-13, 02:13 authored by Mark D. Ungrin, Chirag Joshi, Andra Nica, Céline Bauwens, Peter W. Zandstra

hESC cultured on mouse embryonic fibroblast (MEF) feeders were pre-differentiated with 20% serum for 72 hours prior to aggregate formation, resulting an overall reduction in the population level of Oct4 expression [left panel, red line: standard maintenance culture; blue line: pre-differentiated; black: control (unstained)]. Aggregates formed from 2,000 input cells were substantially larger with treatment (blue bar) than without (red bar). Y axis represents aggregate cross-sectional area in microns2, error bars represent one standard deviation. B. The ROCK inhibitor Y-27632 promotes aggregate stability. hESC cells cultured on Matrigel in MEF-conditioned medium with and without pre-differentiation [left panel, red line: standard maintenance culture; blue line: pre-differentiated; black: control (unstained)] were used to form SISO-aggregates in the presence or absence of 10 µM Y-27632. Under these culture conditions, in the absence of both, no aggregates were formed (N.D. - size not determined). With 48 hours pre-differentiation in 20% serum, consistent aggregates were formed (first blue bar). When Y-27632 was added to the suspension of cells without (red bar) or with (second blue bar) pre-differentiation immediately prior to dispensing into the well plate, sizeable aggregates resulted.

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