Expression of neural markers through differentiation. Undifferentiated human embryonic stem cells (hESCs) were negative for Pax6 or Sox1 (A, B), and N-Cadherin was present in a diffuse pattern (C). All hESCs were positive for Sox2 (D). After 10 days, Pax6, but not Sox1, was expressed in columnar cells in the center of colonies, whereas peripheral flat cells did not express the neural markers (E, F). N-Cadherin was expressed in the membrane of columnar cells (G) and Sox2 was expressed, but most notably in the columnar neuroepithelia at 10 days (H). At 17 days, Sox1 was coexpressed by neuroectoderm cells when they form neural tube-like rosettes along with Pax6 (I, J). N-Cadherin was specifically concentrated in cells lining the lumens of forming rosettes (K, L), and Sox2 expression persisted (L). (M): At 8 days of differentiation, the majority of cells were Otx2 +, and a subset also expressed Pax6. Flow cytometry of cells immunostained for Pax6, showed that in cultures at 8 days, 54% of the cells express Pax6 (N); after 11 days, 95% were definitively positive for Pax6 (O). Scale bars = 100 µm.

Regional identity of early and late neuroepithelial progenitors and neurons. Cells at 10 days expressed the anterior markers Lhx2 (A), BF1 (B), and Otx2 (C), but not the posterior marker Hoxb4 (D); arrows mark positive cells and arrowheads identify cells at the periphery of colonies that do not express anterior markers. Neural differentiation in the presence of 20 ng/ml FGF2, resulted in the uniform formation of Otx2 +/Hoxb4 − anterior-patterned neuroepithelia at 21 days (E, F), and continued differentiation of these cells resulted in neurons with an anterior identity (G). Switching to 0.1 µM RA during days 10–21 of culture resulted in downregulation of Otx2 (H) and widespread expression of Hoxb4 (I). Continued culture with RA further eliminated anterior cells and maintained posterioralized Hoxb4 + cells after 4 weeks. Abbreviation: RA, retinoic acid. Scale bars = 100 µm.

Cellular and molecular transformation along human embryonic stem cell (hESC) differentiation to neuroepithelia. (A): hESCs (inset photo shows typical morphology) were enzymatically separated from the supportive feeder and grown as free-floating aggregates for 6 days (B). After 10–11 days of differentiation, cells in the center of colonies elongated and organized into columnar epithelia (C). At 14–16 days, cells organized around an inner lumen to take on a neural tube-like morphology (D). Scale bars = 100 µm. (E): Reverse transcriptase polymerase chain reaction (PCR) analysis showed that inner cell mass markers (Fgf4, Zfp42, TDGF1, Oct4, and Nanog) are downregulated, whereas neuroectodermal markers (Pax6, Zic1, Sox1, Sox3, NCAD, and Churchill) are turned on or upregulated after 6 days, with Sox1 expression mainly turned on at day 15. The epiblast stage marker FGF5 was induced at 6 days, whereas AFP and Brachyury were barely detectable. Sox2 was detected throughout differentiation. (F): Quantitative PCR analyses showed fold changes of Pax6 (empty column) and Sox1 (filled column) relative to ESCs (day 0). Mean ± SEM for Pax6: day 0 = 1.00 ± 0.18, day 6 = 0.51 ± 0.13, day 10 = 48.28 ± 4.66, day 15 = 40.22 ± 8.49; Sox1 day 0 = 1.00 ± 0.25, day 6 = 1.30 ± 0.34, day 10 = 15.96 ± 2.85, day 15 = 38.59 ± 8.35. (G): Cells expressed Pax6 after 10 days when the aggregates were suspended in ESCM, but the expression of Pax6 was diminished, and these cells failed to express Sox1 after 17 days even after attachment. Sox1 expression was still induced after 17 days if cells continued to float, but only if they were transferred to neural medium. (H): Rhesus ESCs differentiated to neuroepithelia express Pax6 before Sox1 as in human cells, but earlier in differentiation. Abbreviations: ESCM, embryonic stem cell growth medium; NM, neural medium.

Microarray analysis. (A): Left to right lanes are day-0, -6, -10, and -17 samples (each lane is a composite of at least two arrayed samples). Red indicates highest levels of expression, green lowest. Cluster analysis showed that the largest overall shift in gene expression was at the transition from aggregates to 10-day epithelia. A full list of genes can be found in supplemental online Table 2. (B): Polymerase chain reaction confirmation with replicate samples of genes identified through array analysis with expression patterns mirroring important morphological changes at 6, 10, and 17 days of differentiation. Immunocytochemical analyses indicate that FABP7 was not expressed in human embryonic stem cells (C) but was turned on in neuroepithelia at 17 days (D). Scale bars = 100 µm.

Model of neuroectoderm development from human embryonic stem cells (hESCs). (A): A temporal comparison of critical events during neural development in the embryo and hESCs differentiating to NE. The dashed line tracks morphological landmarks of neural development in a human embryo. The solid line tracks the same time in hESCs differentiating to NE, taking into consideration the embryonic stage at which hESCs are isolated (5.5 days). Colored bars summarize profiles of gene expression during NE specification in vitro. This comparison indicates the recapitulation of neuroectodermal development using in vitro hESC differentiation and places the beginning of neuroectodermal specification near the primitive streak stage. (B): Hypothesized model of neuroectodermal development in the human. We propose that neuroectodermal development goes through a stage of transient PAN that can be further differentiated to definitive NE (progenitors) with specific anterior or posterior identities. To shift PAN cells to a posterior fate, caudalizing factors such as RA must be provided. Abbreviations: ESC, embryonic stem cell; ICM, inner cell mass; PAN, primitive anterior neuroepithelia; NE, neuroepithelia; RA, retinoic acid.