Generation of human cortical neurons from a new immortal fetal neural stem cell line

Abstract

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia. and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology. (c) 2006 Elsevier Inc. All rights reserved

Stem and progenitor cell-mediated tumor selective gene therapy

“…With respect to the first mechanism, several genes have been evaluated for their potential utility in immortalizing stem cells for use as delivery vehicles: the proto-oncogene v-myc, 3,70,72,[89][90][91] the catalytic component of the telomerase complex (human telomerase reverse transcriptase (hTERT)), human papillomavirus type 16 E6/E7 genes, Bmi-1 [92][93][94][95][96][97][98][99] and an N-terminal fragment of SV40 large T-antigen. 2,100 Characteristics of the resultant cell lines, each of which had extended life spans in culture, have been reviewed.…”

DNA-damage response, survival and differentiation in vitro of a human neural stem cell line in relation to ATM expression

“…Such cell lines, recently established from fetal brains using v-myc immortalization, 17,19,30,31 retain self-renewal potential and capacity to differentiate into the three major neural lineages, astrocytes, oligodendrocytes and neurons. Interestingly, transplantation of a conditionally immortal human neural stem cell line into the brain has allowed functional recovery in a rat model of stroke disability.…”

Human midbrain precursors activate the expected developmental genetic program and differentiate long-term to functional A9 dopamine neurons in vitro. Enhancement by Bcl-XL

“…Patterning preservation has been demonstrated in cortical, striatal and midbrain neurospheres [57,58,63], but there is only indirect evidence for FB or VM v-myc hNSC lines [34][35][36][40][41][42]64], and the cell lines have never been studied head-to-head. In the present work we first clarified that long-term cultured FB and VM hNSCs differentially express early neuroectodermal markers (PAX6, EN1), and generate neurons showing differential expression of A9-DAn genes (such as GIRK2) (Fig.…”

“…1A). In the mouse embryo, Pax6 is expressed in the neuroectoderm of the FB and hindbrain [64]. In human stem cells PAX6 acts as a determinant of neuroectoderm fate [65][66][67].…”