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Figure 4 | Stem Cell Research & Therapy

Figure 4

From: The pregnancy hormones human chorionic gonadotropin and progesterone induce human embryonic stem cell proliferation and differentiation into neuroectodermal rosettes

Figure 4

P 4 promotes blastulation and neurulation. (a) hESCs were grown in lithium-free TESR1 medium in the presence of E2 (10 nM) or P4 (2 μM) or E2 (10 nM) + P4 (2 μM) for 6 days, and cell numbers were measured by using the trypan blue assay. Results are expressed as mean ± SEM, n = 4; *P < 0.05; **P < 0.005 compared with 6 day control. (b) Equal amounts of protein from cell lysates of hESCs treated for 9 days as described earlier were analyzed with immunoblot by using a monoclonal antibody against nestin (clone 10C2). (c) EB formation: hESCs were allowed to form colonies by culturing for 4 days on an MEF feeder layer, as described earlier, and then were enzymatically lifted, and the colonies were placed into EB medium (containing serum) in the absence (i; control) or presence (ii) of RU-486 (20 μM) and rocked gently for an additional 10 days to allow EB formation. Structures were then assessed morphologically. Rosette formation: hESCs were allowed to form colonies by culturing for 4 days on an MEF feeder layer; colonies were then enzymatically lifted and placed into EB medium (containing serum) and rocked gently for an additional 4 days. Colonies were then placed in neural induction medium with P4 (2 μM) (iii), without P4 (v) or with P4 (2 μM) + RU-486 (20 μM) (iv) for an additional 11 days. At 19 days, the structures were analyzed morphologically. (d) The structures in (c) were collected, and equal amounts of protein from cell lysates were analyzed for nestin with immunoblot analysis. (e) hESCs were grown in mTeSR1 medium and treated with 500 mIU/ml hCG for 8 days. Cells were collected, protein content determined by using the BCA assay, equal amounts of protein were run on SDS-PAGE, and the immunoblot was probed for human nestin. Molecular-weight markers are shown on the left of immunoblots.

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