Figure 3. Human ES cells serially passaged in NM23-H1-MM express pluripotency markers, differentiate down all three germlines and display coordinated differentiation.

H9 hES cells on Matrigel were cultured for at least six passages in either NM23-H1-MM (minimal stem cell media) or bFGF plus MEF conditioned media. a) NM23-H1-MM cultured cells stained positive for the presence of the typical pluripotency markers.. NM23-H1 cultured cells were allowed to differentiate by the embryoid body method (Methods S1) then stained with nuclear marker DAPI and antibodies against markers of the three germlines: b, e) endoderm - alpha feto protein, c, f) ectoderm - nestin, and d, g) mesoderm - smooth muscle actin. Cells cultured in either NM23-H1-MM or bFGF-CM both differentiated down all three germlines. Cells that had been cultured in NM23-H1-MM displayed apparently coordinated differentiation with most cells in a cluster differentiating down the same germline (b–d), whereas cells cultured in bFGF did not (e–g). h) The percentage of cells in each cluster that expressed the same germline marker was quantified (n = 5 to 7). Cells that had been cultured in NM23-H1-MM have a higher percentage of cells in the same cluster differentiating down the same germline than cells that had been cultured in bFGF. i) iPS cells on Matrigel, cultured for six passages in NM23-H1-MM stained positive for the presence of the typical pluripotency markers. All images 4X