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Stable Expression of Neurogenin 1 Induces LGR5, a Novel Stem Cell Marker, in an Immortalized Human Neural Stem Cell Line HB1.F3

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Abstract

Neural stem cells (NSC) with self-renewal and multipotent properties serve as an ideal cell source for transplantation to treat spinal cord injury, stroke, and neurodegenerative diseases. To efficiently induce neuronal lineage cells from NSC for neuron replacement therapy, we should clarify the intrinsic genetic programs involved in a time- and place-specific regulation of human NSC differentiation. Recently, we established an immortalized human NSC clone HB1.F3 to provide an unlimited NSC source applicable to genetic manipulation for cell-based therapy. To investigate a role of neurogenin 1 (Ngn1), a proneural basic helix-loop-helix (bHLH) transcription factor, in human NSC differentiation, we established a clone derived from F3 stably overexpressing Ngn1. Genome-wide gene expression profiling identified 250 upregulated genes and 338 downregulated genes in Ngn1-overexpressing F3 cells (F3-Ngn1) versus wild-type F3 cells (F3-WT). Notably, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a novel stem cell marker, showed an 167-fold increase in F3-Ngn1, although transient overexpression of Ngn1 did not induce upregulation of LGR5, suggesting that LGR5 is not a direct transcriptional target of Ngn1. KeyMolnet, a bioinformatics tool for analyzing molecular relations on a comprehensive knowledgebase, suggests that the molecular network of differentially expressed genes involves the complex interaction of networks regulated by multiple transcription factors. Gene ontology (GO) terms of development and morphogenesis are enriched in upregulated genes, while those of extracellular matrix and adhesion are enriched in downregulated genes. These results suggest that stable expression of a single gene Ngn1 in F3 cells induces not simply neurogenic but multifunctional changes that potentially affect the differentiation of human NSC via a reorganization of complex gene regulatory networks.

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Abbreviations

bHLH:

Basic helix-loop-helix

CNS:

Central nervous system

DAVID:

Database for annotation visualization and integrated discovery

DEG:

Differentially expressed genes

FBS:

Fetal bovine serum

GAS2:

Growth arrest-specific 2

GO:

Gene ontology

HAS2:

Hyaluronan synthase 2

LGR5:

Leucine-rich repeat-containing G protein-coupled receptor 5

MMP9:

Matrix metallopeptidase 9

Ngn1:

Neurogenin 1

NPC:

Neural progenitor cells

NSC:

Neural stem cells

ORF:

Open-reading frame

RMA:

Robust multiarray average

RT-PCR:

Reverse transcription-polymerase chain reaction

SHH:

Sonic hedgehog homolog

Wnt:

Wingless-type MMTV integration site family

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Acknowledgments

This work was supported by a research grant to J-IS from the High-Tech Research Center Project, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (S0801043), and from Research on Intractable Diseases, the Ministry of Health, Labour and Welfare of Japan. The microarray data are available from Gene Expression Omnibus (GEO) under the accession number GSE18296.

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Authors and Affiliations

  1. Department of Bioinformatics and Molecular Neuropathology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo, 204-8588, Japan

    Jun-ichi Satoh, Shinya Obayashi, Hiroko Tabunoki & Taeko Wakana

  2. Division of Neurology, Department of Medicine, University of British Columbia Hospital, University of British Columbia, Vancouver, BC, Canada

    Seung U. Kim

  3. Medical Research Institute, Chung-Ang University College of Medicine, Seoul, Korea

    Seung U. Kim

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  1. Jun-ichi Satoh
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  2. Shinya Obayashi
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  3. Hiroko Tabunoki
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  4. Taeko Wakana
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  5. Seung U. Kim
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Corresponding author

Correspondence to Jun-ichi Satoh.

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10571_2009_9466_MOESM1_ESM.ppt

Supplementary material 1 (PPT 280 kb) Supplementary Fig. 1. Upregulation of Ngn1 ORF expression in F3-Ngn1 cells. Genome-wide gene expression profiling of F3-WT and F3-Ngn1 was performed by using two sets of Human Gene 1.0 ST Array for each, followed by two comparisons composed of WT array-1 (F3-WT-1) versus Ngn1 array-1 (F3-Ngn1-1) and WT array-2 (F3-WT-2) versus Ngn1 array-2 (F3-Ngn1-2). The distribution pattern of individual probe-based signal intensities of NEUROG1 (Ngn1) on the array is shown. Among 26 probes spreading across the full-length Ngn1 gene, the position of the ORF is indicated by a purple box

Supplementary material 2 (XLS 61 kb)

Supplementary material 3 (XLS 73 kb)

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Satoh, Ji., Obayashi, S., Tabunoki, H. et al. Stable Expression of Neurogenin 1 Induces LGR5, a Novel Stem Cell Marker, in an Immortalized Human Neural Stem Cell Line HB1.F3. Cell Mol Neurobiol 30, 415–426 (2010). https://doi.org/10.1007/s10571-009-9466-3

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