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Cellular and Molecular Neurobiology

, Volume 30, Issue 3, pp 415–426 | Cite as

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

  • Jun-ichi SatohEmail author
  • Shinya Obayashi
  • Hiroko Tabunoki
  • Taeko Wakana
  • Seung U. Kim
Original Research

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.

Keywords

HB1.F3 KeyMolnet LGR5 Microarray Neural stem cells Neurogenin 1 

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

Notes

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.

Supplementary material

10571_2009_9466_MOESM1_ESM.ppt (280 kb)
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
10571_2009_9466_MOESM2_ESM.xls (61 kb)
Supplementary material 2 (XLS 61 kb)
10571_2009_9466_MOESM3_ESM.xls (73 kb)
Supplementary material 3 (XLS 73 kb)

References

  1. Ahn SM, Byun K, Kim D, Lee K, Yoo JS, Kim SU, Jho EH, Simpson RJ, Lee B (2008) Olig2-induced neural stem cell differentiation involves downregulation of Wnt signaling and induction of Dickkopf-1 expression. PLoS One 3:e3917CrossRefPubMedGoogle Scholar
  2. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ, Clevers H (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449:1003–1007CrossRefPubMedGoogle Scholar
  3. Barkho BZ, Munoz AE, Li X, Li L, Cunningham LA, Zhao X (2008) Endogenous matrix metalloproteinase (MMP)-3 and MMP-9 promote the differentiation and migration of adult neural progenitor cells in response to chemokines. Stem Cells 26:3139–3149CrossRefPubMedGoogle Scholar
  4. Garcia MI, Ghiani M, Lefort A, Libert F, Strollo S, Vassart G (2009) LGR5 deficiency deregulates Wnt signaling and leads to precocious Paneth cell differentiation in the fetal intestine. Dev Biol 331:58–67CrossRefPubMedGoogle Scholar
  5. Hendrix ND, Wu R, Kuick R, Schwartz DR, Fearon ER, Cho KR (2006) Fibroblast growth factor 9 has oncogenic activity and is a downstream target of Wnt signaling in ovarian endometrioid adenocarcinomas. Cancer Res 66:1354–1362CrossRefPubMedGoogle Scholar
  6. Hirabayashi Y, Itoh Y, Tabata H, Nakajima K, Akiyama T, Masuyama N, Gotoh Y (2004) The Wnt/β-catenin pathway directs neuronal differentiation of cortical neural precursor cells. Development 131:2791–2801CrossRefPubMedGoogle Scholar
  7. Hsu SY, Liang SG, Hsueh AJ (1998) Characterization of two LGR genes homologous to gonadotropin and thyrotropin receptors with extracellular leucine-rich repeats and a G protein-coupled, seven-transmembrane region. Mol Endocrinol 12:1830–1845CrossRefPubMedGoogle Scholar
  8. Huang da W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57CrossRefPubMedGoogle Scholar
  9. Jaks V, Barker N, Kasper M, van Es JH, Snippert HJ, Clevers H, Toftgård R (2008) Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet 40:1291–1299CrossRefPubMedGoogle Scholar
  10. Kim SU (2004) Human neural stem cells genetically modified for brain repair in neurological disorders. Neuropathology 24:159–174CrossRefPubMedGoogle Scholar
  11. Kim SU, de Vellis J (2009) Stem cell-based cell therapy in neurological diseases: a review. J Neurosci Res 87:2183–2200CrossRefPubMedGoogle Scholar
  12. Kim S, Ghil SH, Kim SS, Myeong HH, Lee YD, Suh-Kim H (2002) Overexpression of neurogenin1 induces neurite outgrowth in F11 neuroblastoma cells. Exp Mol Med 34:469–475PubMedGoogle Scholar
  13. Kim S, Yoon YS, Kim JW, Jung M, Kim SU, Lee YD, Suh-Kim H (2004) Neurogenin1 is sufficient to induce neuronal differentiation of embryonal carcinoma P19 cells in the absence of retinoic acid. Cell Mol Neurobiol 24:343–356CrossRefPubMedGoogle Scholar
  14. Kim SU, Park IH, Kim TH, Kim KS, Choi HB, Hong SH, Bang JH, Lee MA, Joo IS, Lee CS, Kim YS (2006) Brain transplantation of human neural stem cells transduced with tyrosine hydroxylase and GTP cyclohydrolase 1 provides functional improvement in animal models of Parkinson disease. Neuropathology 26:129–140CrossRefPubMedGoogle Scholar
  15. Lum M, Turbic A, Mitrovic B, Turnley AM (2009) Fibroblast growth factor-9 inhibits astrocyte differentiation of adult mouse neural progenitor cells. J Neurosci Res 87:2201–2210CrossRefPubMedGoogle Scholar
  16. Ma Q, Fode C, Guillemot F, Anderson DJ (1999) Neurogenin1 and neurogenin2 control two distinct waves of neurogenesis in developing dorsal root ganglia. Genes Dev 13:1717–1728CrossRefPubMedGoogle Scholar
  17. May R, Sureban SM, Hoang N, Riehl TE, Lightfoot SA, Ramanujam R, Wyche JH, Anant S, Houchen CW (2009) DCAMKL-1 and LGR5 mark quiescent and cycling intestinal stem cells respectively. Stem Cells. doi: 10.1002/stem.193
  18. Morita H, Mazerbourg S, Bouley DM, Luo CW, Kawamura K, Kuwabara Y, Baribault H, Tian H, Hsueh AJ (2004) Neonatal lethality of LGR5 null mice is associated with ankyloglossia and gastrointestinal distension. Mol Cell Biol 24:9736–9743CrossRefPubMedGoogle Scholar
  19. Morrison SJ (2001) Neuronal differentiation: proneural genes inhibit gliogenesis. Curr Biol 11:R349–R351CrossRefPubMedGoogle Scholar
  20. Niida A, Hiroko T, Kasai M, Furukawa Y, Nakamura Y, Suzuki Y, Sugano S, Akiyama T (2004) DKK1, a negative regulator of Wnt signaling, is a target of the β-catenin/TCF pathway. Oncogene 23:8520–8526CrossRefPubMedGoogle Scholar
  21. Obayashi S, Tabunoki H, Kim SU, Satoh J (2009) Gene expression profiling of human neural progenitor cells following the serum-induced astrocyte differentiation. Cell Mol Neurobiol 29:423–438CrossRefPubMedGoogle Scholar
  22. Ootani A, Li X, Sangiorgi E, Ho QT, Ueno H, Toda S, Sugihara H, Fujimoto K, Weissman IL, Capecchi MR, Kuo CJ (2009) Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat Med 15:701–706CrossRefPubMedGoogle Scholar
  23. Ota M, Ito K (2003) Induction of neurogenin-1 expression by sonic hedgehog: its role in development of trigeminal sensory neurons. Dev Dyn 227:544–551CrossRefPubMedGoogle Scholar
  24. Prakash N, Wurst W (2007) A Wnt signal regulates stem cell fate and differentiation in vivo. Neurodegener Dis 4:333–338CrossRefPubMedGoogle Scholar
  25. Sato H, Ishida S, Toda K, Matsuda R, Hayashi Y, Shigetaka M, Fukuda M, Wakamatsu Y, Itai A (2005) New approaches to mechanism analysis for drug discovery using DNA microarray data combined with KeyMolnet. Curr Drug Discov Technol 2:89–98CrossRefPubMedGoogle Scholar
  26. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265CrossRefPubMedGoogle Scholar
  27. Sommer L, Ma Q, Anderson DJ (1996) Neurogenins, a novel family of atonal-related bHLH transcription factors, are putative mammalian neuronal determination genes that reveal progenitor cell heterogeneity in the developing CNS and PNS. Mol Cell Neurosci 8:221–241CrossRefPubMedGoogle Scholar
  28. Sun Y, Nadal-Vicens M, Misono S, Lin MZ, Zubiaga A, Hua X, Fan G, Greenberg ME (2001) Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms. Cell 104:365–376CrossRefPubMedGoogle Scholar
  29. Tanese K, Fukuma M, Yamada T, Mori T, Yoshikawa T, Watanabe W, Ishiko A, Amagai M, Nishikawa T, Sakamoto M (2008) G-protein-coupled receptor GPR49 is up-regulated in basal cell carcinoma and promotes cell proliferation and tumor formation. Am J Pathol 173:835–843CrossRefPubMedGoogle Scholar
  30. Wu B, Crampton SP, Hughes CC (2007) Wnt signaling induces matrix metalloproteinase expression and regulates T cell transmigration. Immunity 26:227–239CrossRefPubMedGoogle Scholar
  31. Yamamoto Y, Sakamoto M, Fujii G, Tsuiji H, Kenetaka K, Asaka M, Hirohashi S (2003) Overexpression of orphan G-protein-coupled receptor, Gpr49, in human hepatocellular carcinomas with β-catenin mutations. Hepatology 37:528–533CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Jun-ichi Satoh
    • 1
    Email author
  • Shinya Obayashi
    • 1
  • Hiroko Tabunoki
    • 1
  • Taeko Wakana
    • 1
  • Seung U. Kim
    • 2
    • 3
  1. 1.Department of Bioinformatics and Molecular NeuropathologyMeiji Pharmaceutical UniversityTokyoJapan
  2. 2.Division of Neurology, Department of MedicineUniversity of British Columbia Hospital, University of British ColumbiaVancouverCanada
  3. 3.Medical Research InstituteChung-Ang University College of MedicineSeoulKorea

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