Poor survival of human embryonic stem (hES) cells after cell dissociation is an obstacle to research, hindering manipulations such as subcloning. Here we show that application of a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, to hES cells markedly diminishes dissociation-induced apoptosis, increases cloning efficiency (from approximately 1% to approximately 27%) and facilitates subcloning after gene transfer. Furthermore, dissociated hES cells treated with Y-27632 are protected from apoptosis even in serum-free suspension (SFEB) culture and form floating aggregates. We demonstrate that the protective ability of Y-27632 enables SFEB-cultured hES cells to survive and differentiate into Bf1(+) cortical and basal telencephalic progenitors, as do SFEB-cultured mouse ES cells.
Here, we demonstrate self-organized formation of apico-basally polarized cortical tissues from ESCs using an efficient three-dimensional aggregation culture (SFEBq culture). The generated cortical neurons are functional, transplantable, and capable of forming proper long-range connections in vivo and in vitro. The regional identity of the generated pallial tissues can be selectively controlled (into olfactory bulb, rostral and caudal cortices, hem, and choroid plexus) by secreted patterning factors such as Fgf, Wnt, and BMP. In addition, the in vivo-mimicking birth order of distinct cortical neurons permits the selective generation of particular layer-specific neurons by timed induction of cell-cycle exit. Importantly, cortical tissues generated from mouse and human ESCs form a self-organized structure that includes four distinct zones (ventricular, early and late cortical-plate, and Cajal-Retzius cell zones) along the apico-basal direction. Thus, spatial and temporal aspects of early corticogenesis are recapitulated and can be manipulated in this ESC culture.
We demonstrate directed differentiation of telencephalic precursors from mouse embryonic stem (ES) cells using optimized serum-free suspension culture (SFEB culture). Treatment with Wnt and Nodal antagonists (Dkk1 and LeftyA) during the first 5 d of SFEB culture causes nearly selective neural differentiation in ES cells ( approximately 90%). In the presence of Dkk1, with or without LeftyA, SFEB induces efficient generation ( approximately 35%) of cells expressing telencephalic marker Bf1. Wnt3a treatment during the late culture period increases the pallial telencephalic population (Pax6(+) cells yield up to 75% of Bf1(+) cells), whereas Shh promotes basal telencephalic differentiation (into Nkx2.1(+) and/or Islet1/2(+) cells) at the cost of pallial telencephalic differentiation. Thus, in the absence of caudalizing signals, floating aggregates of ES cells generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.
During cerebellar development, the main portion of the cerebellar plate neuroepithelium gives birth to Purkinje cells and interneurons, whereas the rhombic lip, the germinal zone at its dorsal edge, generates granule cells and cerebellar nuclei neurons. However, it remains elusive how these components cooperate to form the intricate cerebellar structure. Here, we found that a polarized cerebellar structure self-organizes in 3D human embryonic stem cell (ESC) culture. The self-organized neuroepithelium differentiates into electrophysiologically functional Purkinje cells. The addition of fibroblast growth factor 19 (FGF19) promotes spontaneous generation of dorsoventrally polarized neural-tube-like structures at the level of the cerebellum. Furthermore, addition of SDF1 and FGF19 promotes the generation of a continuous cerebellar plate neuroepithelium with rhombic-lip-like structure at one end and a three-layer cytoarchitecture similar to the embryonic cerebellum. Thus, human-ESC-derived cerebellar progenitors exhibit substantial self-organizing potential for generating a polarized structure reminiscent of the early human cerebellum at the first trimester.
Human embryonic stem cells (hESCs), unlike mouse ones (mESCs), are vulnerable to apoptosis upon dissociation. Here, we show that the apoptosis, which is of a nonanoikis type, is caused by ROCK-dependent hyperactivation of actomyosin and efficiently suppressed by the myosin inhibitor Blebbistatin. The actomyosin hyperactivation is triggered by the loss of E-cadherin-dependent intercellular contact and also observed in dissociated mouse epiblast-derived pluripotent cells but not in mESCs. We reveal that Abr, a unique Rho-GEF family factor containing a functional Rac-GAP domain, is an indispensable upstream regulator of the apoptosis and ROCK/myosin hyperactivation. Rho activation coupled with Rac inhibition is induced in hESCs upon dissociation, but not in Abr-depleted hESCs or mESCs. Furthermore, artificial Rho or ROCK activation with Rac inhibition restores the vulnerability of Abr-depleted hESCs to dissociation-induced apoptosis. Thus, the Abr-dependent "Rho-high/Rac-low" state plays a decisive role in initiating the dissociation-induced actomyosin hyperactivation and apoptosis in hESCs.
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