br Materials and methods br Results There are a number

Materials and methods
Results There are a number of complex methods for the generation of neural subtypes from hESCs and iPSCs. In most of them, intact pluripotent colonies are first lifted, induced to form EBs, and then grown in the presence of lineage specific morphogens and growth factors to produce a variety of terminally differentiated neurons (dopamine, cholinergic, etc.), astrocytes, and oligodendrocytes (Murry and Keller, 2008). Although successful, this technique was not efficient for all iPSCs. For example, although all iPSCs generated EBs, over 50% of the EBs derived from both healthy and diseased iPSCs displayed an unhealthy appearance, such as tattered edges, fragmented and irregular shapes, membrane blebbing, and dark coloring, and did not efficiently survive neural specification (Fig. 1A). We therefore developed EZ spheres as an alternative to EB formation to consistently transition iPSCs from undifferentiated colonies to differentiated neural subtypes. Furthermore, in order to avoid potential bias in long-term differentiation potential, as shown in previously published protocols (Elkabetz et al., 2008; Koch et al., 2009; Nemati et al., 2011), we aimed to generate an expandable pre-rosette population of neural stem buy UNC0638 that could retain greater plasticity upon differentiation. EZ spheres were generated by gently lifting the undifferentiated colonies from MEFs or Matrigel using enzymatic dissociation (e.g. collagenase, dispase), techniques consistent with EB generation protocols. Clumps of cells were then placed in ultra-low attachment flasks in neural progenitor cell medium consisting of 100ng/ml EGF, 100ng/ml FGF-2 (human or zebrafish), and 5μg/ml heparin; the high level of EGF has previously been shown to increase the proliferation and neuronal potential of human fetal neural progenitor cells (Nelson et al., 2008). In this medium, the cells clustered into tightly compacted spheres with regular edges and a golden color within 3–5days (Fig. 1B). EZ spheres continued to expand (many cultured for over a year), were passaged weekly by mechanical chopping, and could be cryopreserved and subsequently thawed with high efficiency. Cells within EZ spheres doubled in number every 7days with approximately 20% of cells undergoing active proliferation at any one time based on Ki67 labeling (Figs. 1C–E). They also showed chromosomal stability for at least 30 passages (latest passage assessed) (Fig. S1). We have produced and expanded EZ spheres from 19 independent hESC and iPSC lines, including healthy and disease-specific lines generated through multiple reprogramming methods, all of which demonstrates the robustness of the method (Table S1). In order to better characterize the composition and stability of EZ spheres, we undertook a systematic and longitudinal assessment using EZ spheres derived from H9 hESCs (Thomson et al., 1998) and two independent unaffected iPSC lines (Coriell fibroblast line GM003814 (4.2 iPSC), Coriell fibroblast line GM002183 (21.8 iPSC)). These iPSC lines have each been previously characterized for full reprogramming and down-regulation of exogenous transgene expression (Ebert et al., 2009; HD iPSC Consortium, 2012). First, EZ spheres were analyzed for various pluripotency and neural progenitor markers at 0, 2, 4, 8, 15, 20, and 28 passages post-sphere formation by immunocytochemistry. Specifically, we found that nearly 75% of cells derived from all three stem cell lines were positive for OCT4 (endogenous POU5F1) immediately following sphere formation (passage 0), but this dramatically decreased by passage 2 and was virtually absent in subsequent passages (Fig. 2A). Further, immunocytochemical analysis of dissociated EZ spheres showed that there was a significant increase in nestin expression after passage 2, such that >60% of cells were nestin positive after passage 4 and >90% of cells by passage 7 (Fig. 2B). Next, immunocytochemistry of intact EZ spheres showed expression of neural progenitor and radial glial markers such as nestin, PAX6, vimentin, BLBP, SOX1, and SOX2 throughout the spheres, but limited expression of the motor neuron and/or oligodendrocyte progenitor marker OLIG2, the mature neuronal marker MAP2, or pluripotency markers Nanog, SSEA3, and Tra-1-81 (Fig. 2C).