Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures

doi:10.1111/j.1432-0436.2007.00256.x

Differentiation

Volume 76, Issue 5, June 2008, Pages 454-464

https://doi.org/10.1111/j.1432-0436.2007.00256.x Get rights and content

Abstract

Derivation of human neural progenitors (hNP) from human embryonic stem (hES) cells in culture has been reported with the use of feeder cells or conditioned media. This introduces undefined components into the system, limiting the ability to precisely investigate the requirement for factors that control the process. Also, the use of feeder cells of non-human origin introduces the potential for zoonotic transmission, limiting its clinical usefulness. Here we report a feeder-free system to produce hNP from hES cells and test the effects of various media components involved in the process. Five protocols using defined media components were compared for efficiency of hNP generation. Based on this analysis, we discuss the role of basic fibroblast growth factor (FGF2), N2 supplement, non-essential amino acids (NEAA), and knock-out serum replacement (KSR) on the process of hNP generation. All protocols led to down-regulation of Oct4/POU5F1 expression (from 90.5% to <3%), and up-regulation of neural progenitor markers to varying degrees. Media with N2 but not KSR and NEAA produced cultures with significantly higher (p<0.05) expression of the neural progenitor marker Musashi 1 (MSI1). Approximately 89% of these cells were Nestin (NES)+ after 3 weeks, but they did not proliferate. In contrast, differentiation media supplemented with KSR and NEAA produced fewer NES+ (75%) cells, but these cells were proliferative, and by five passages the culture consisted of >97% NES+ cells. This suggests that KSR and NEAA supplements did not enhance early differentiation but did promote proliferating of hNP cell cultures. This resulted in an efficient, robust, repeatable differentiation system suitable for generating large populations of hNP cells. This will facilitate further study of molecular and biochemical mechanisms in early human neural differentiation and potentially produce uniform neuronal cells for therapeutic uses without concern of zoonotic transmission from feeder layers.

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ORIGINAL ARTICLEHuman neural progenitor cells derived from embryonic stem cells in feeder-free cultures

Abstract

Blood

J Biol Chem

Mol Cell Neurosci

Cell

Dev Biol

Dev Biol

Mech Dev

Dev Biol

Brain Res

Dev Biol

Neuron

Mech Dev

Mech Dev

Neurogenesis and neuroprotection induced by peripheral immunomodulatory treatment of experimental autoimmune encephalomyelitis

J Neurosci

Synergistic activation of the fibroblast growth factor 4 enhancer by Sox2 and Oct-3 depends on protein-protein interactions facilitated by a specific spatial arrangement of factor binding sites

Mol Cell Biol

Multipotent cell lineages in early mouse development depend on SOX2 function

Genes Dev

Growth of a rat neuroblastoma cell line in serum-free supplemented medium

Proc Natl Acad Sci USA

Vertebrate neurogenesis is counteracted by Sox1–3 activity

Nat Neurosci

Proliferation and differentiation of neuronal stem cells regulated by nerve growth factor

Nature

Neural induction in Xenopus requires early FGF signalling in addition to BMP inhibition

Development

Localization and synthesis of alphafoetoprotein in post-implantation mouse embryos

J Embryol Exp Morphol

SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult

Dev Neurosci

Regulation of neuronal differentiation in human CNS stem cell progeny by leukemia inhibitory factor

Dev Neurosci

Ectodermal Wnt function as a neural crest inducer

Science

Differentiation of human embryonic stem cells to neural lineages in adherent culture by blocking bone morphogenetic protein signaling

Stem Cells

Ciliary neurotrophic factor induces type-2 astrocyte differentiation in culture

Nature

FGF and EGF are mitogens for immortalized neural progenitors

J Neurobiol

ORIGINAL ARTICLE
Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures