Efficient and Fast Differentiation of Human Neural Stem Cells from Human Embryonic Stem Cells for Cell Therapy

Figure 2

Neural stem cell-like cells were differentiated from human embryonic stem cells. (a) Human embryonic stem cells were cultured and digested into single cell, then the feeder cells (MEF cells) were removed, and single-layer cells were cultured in MEF conditional media until they were confluent. (A) hES cells grew on MEF feeder cells and will be digested after 7 days culture to start differentiation. (B) hES cells had grown on MEF feeder cells for 7 days and will be digested immediately. (C) hES cells were digested to gelatin-coated plates to remove MEF cells. (D) The nonadherent hES cells expanded in MEF-C medium about 4 days until confluent. (b) Noggin and SB431542 induced human ES cells into hNSCs. (A) The confluent single-layer hES cells were cultured in differentiation medium including KSR medium with TGF-beta inhibitor and Noggin for 1 day. (B) Differentiation for 5 days in differentiation medium. (C) Differentiation for 10 days and changing to KSR medium only with Noggin from the 6th day. (D) After 12 days, cells grew in 25% N2 media with 75% KSR medium for 2 days. (c) Cells crawled out from the assembled ES cells. After cell passage, the clone was visible. (A) Cells climbed from hESCs at 15 days. (B–D) State of cells around passage. (d) Differentiated cells from human ES grew homogeneously and fast. Scale bar = 50 μm. (A, B) Different generations from passage 0 to passage 1.

(a)

(b)

(c)

(d)