Neurogenic neuroepithelial and radial glial cells generated from six human embryonic stem cell lines in serum‐free suspension and adherent cultures

Abstract

The great potential of human embryonic stem (hES) cells offers the opportunity both for studying basic developmental processes in vitro as well as for drug screening, modeling diseases, or future cell therapy. Defining protocols for the generation of human neural progenies represents a most important prerequisite. Here, we have used six hES cell lines to evaluate defined conditions for neural differentiation in suspension and adherent culture systems. Our protocol does not require fetal serum, feeder cells, or retinoic acid at any step, to induce neural fate decisions in hES cells. We monitored neurogenesis in differentiating cultures using morphological (including on-line follow up), immunocytochemical, and RT-PCR assays. For each hES cell line, in suspension or adherent culture, the same longitudinal progression of neural differentiation occurs. We showed the dynamic transitions from hES cells to neuroepithelial (NE) cells, to radial glial (RG) cells, and to neurons. Thus, 7 days after neural induction the majority of cells were NE, expressing nestin, Sox1, and Pax6. During neural proliferation and differentiation, NE cells transformed in RG cells, which acquired vimentin, BLBP, GLAST, and GFAP, proliferated and formed radial scaffolds. gamma-Aminobutyric acid (GABA)-positive and glutamate positive neurons, few oligodendrocyte progenitors and astrocytes were formed in our conditions and timing. Our system successfully generates human RG cells and could be an effective source for neuronal replacement, since RG cells predominantly generate neurons and provide them with support and guidance.

The cellular form of the prion protein guides the differentiation of human embryonic stem cells into neuron-, oligodendrocyte-, and astrocyte-committed lineages

“…Three experimental protocols that were previously shown to induce differentiation of hESCs into predominantly neuronal cells, oligodendrocytes and astrocytes were employed. [41][42][43] In all three protocols, PrP C expression was suppressed starting from day zero and the suppression was continued until the experimental end-point, day 40.…”

“…43 Briefly, after culturing in feeder free conditions, hESC colonies were incubated in neural induction medium (NIM, DMEM:Neurobasal (1:1) supplemented with 1x N2 and 1x B27 supplements and 2mM Glutamax) for one day. Then, hESCs were cultured on non-adherent culture dishes in NIM until they form uniformed aggregates for 2-4 d. Floating aggregates were transferred to neural proliferation medium (NPM, DMEM:Neurobasal (1:1) supplemented with 0.5x N2 and 0.5x B27 supplements, 2mM Glutamax and 20 ng/ml FGF2) on day 5.…”

Neural differentiation from human embryonic stem cells in a defined adherent culture condition

“…In contrast, it was suggested that neuroepithelial formation may be a default process during neural differentiation of ESCs in vitro (Liour et al, 2006). Nat et al, (2007) have also shown that the addition of RA is not mandatory for the induction of neuroepithelial phenotype. Further, RA in concert with bFGF signaling leads to the differentiation of spinal motor neurons (Mizuseki et al, 2003, Wichterle et al, 2002.…”

Human stem cells for modeling neurological disorders: Accelerating the drug discovery pipeline

“…Hence they can be expanded to large numbers [Phillips et al, 2008] and be differentiated into most cells of the human body, including the CNS. These properties make them an ideal resource for highthroughput cell-based assayology, including a potentially unlimited supply of neural derivatives [Nat et al, 2007].…”