Effects of Nanog overexpression on the proliferation and multipotent differentiation potentiality of human dental pulp cells

doi:10.3877/cma.j.issn.1674-1366.2015.05.002

Abstract

Abstract:

Objective

To investigate the effect of overexpression Nanog on the cell proliferationand multipotent differentiation potentiality of human dental pulp cells (hDPCs) .

Methods

Primary human pulp cells were transfected with plasmid pSIN-EF2-Nanog-IRES-GFP-puro by lentiviral transfection and stable nanog-expressing cell line was screened under fluorescence microscope. The effect of Nanog on the proliferation of hDPCs was examined with BrdU assay. The expressions levels of pluripotency factor Oct4 and Sox2 were detected using real-time PCR and western blotting. Nanog-hDPCs were cultured in odontogenic and adipogenic induction media respectively. The odontogenic and adipogenic differentiation potentiality were evaluated by Alizarin red S staining and Oil red O staining, and the odontogenic and adipogenic markers were detected using real-time PCR.

Results

The recombinant lentiviral vector containing Nanog was successfully constructed and showed high efficiency during infection in hDPCs. The BrdU assay showed that the growth rate of Nanog-hDPCs was higher compared with the controls (F = 90.421, P = 0.000) . The expressions of Oct4 and Sox2 were increased in Nanog-hDPCs in comparison with the controls (F_{Oct4 mRNA}= 71.649, P_{Oct4 mRNA} = 0.000; F_{Sox2 mRNA}= 106.278, P_{Sox2 mRNA} = 0.000; F_Oct4 = 41.632, P_Oct4 = 0.002; F_Sox2 = 38.962, P_Sox2 = 0.002) . Furthermore, the mRNA levels of DSPP, DMP1, and OCN were increased, and the number of mineralized nodules was significantly higher in Nanog-hDPCs (F_DSPP = 15.261, P_DSPP < 0.05; F_DMP1 = 16.235, P_DMP1 < 0.05; F_OCN = 17.019, P_OCN < 0.05) . Meanwhile, significantly higher mRNA expression levels of LPL and PPARγ2 and more lipid droplets were observed in Nanog-hDPCs compared to the controls (F_LPL = 15.542, P_LPL < 0.05; F_PPARγ2 = 10.437, P_PPARγ2 < 0.05) .

Conclusion

Nanog overexpression enhanced cell proliferation, upregulated the expression of pluripotent markers, and promoted the multipotent differentiation potential (odontogenic and adipogenic differentiation) of hDPCs.

Key words: Dental pulp cells, Nanog, Cell proliferation, Multipotent differentiation, Pluripotency-associated transcription factors

Yanyang Ning, Jinling Li, Qiong Xu. Effects of Nanog overexpression on the proliferation and multipotent differentiation potentiality of human dental pulp cells[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2015, 09(05): 357-363.

/ / Recommend

Add to citation manager EndNote|Ris|BibTeX

URL: https://zhkqyxyjzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-1366.2015.05.002

https://zhkqyxyjzz.cma-cmc.com.cn/EN/Y2015/V09/I05/357

Figures/Tables 5

References 22

[1]	Gronthos S，Brahim J，Li W，et al. Stem cell properties of human dental pulp stem cells[J]. J Dent Res，2002，81(8):531-535.
[2]	Papaccio G，Graziano A，d′Aquino R，et al. Long-term cryopreservation of dental pulp stem cells（SBP-DPSCs）and their differentiated osteoblasts：a cell source for tissue repair[J]. J Cell Physiol，2006，208(2):319-325.
[3]	Ballini A，De Frenza G，Cantore S，et al. In vitro stem cell cultures from human dental pulp and periodontal ligament：new prospects in dentistry[J]. Int J Immunopathol Pharmacol，2007，20(1):9-16.
[4]	Toriumi T，Takayama N，Murakami M，et al. Characterization of mesenchymal progenitor cells in the crown and root pulp of primary teeth[J]. Biomed Res，2015，36(1):31-45.
[5]	Mitsui K，Tokuzawa Y，Itoh H，et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[J]. Cell，2003，113(5):631-642.
[6]	Chambers I，Colby D，Robertson M，et al. Functional expression cloning of Nanog，a pluripotency sustaining factor in embryonic stem cells[J]. Cell，2003，113(5):643-655.
[7]	Kerkis I，Kerkis A，Dozortsev D，et al. Isolation and characterization of a population of immature dental pulp stem cells expressing OCT-4 and other embryonic stem cell markers[J]. Cells Tissues Organs，2006，184(3-4):105-116.
[8]	Ponnaiyan D，Bhat KM，Bhat GS. Comparison of immuno-phenotypes of stem cells from human dental pulp and periodontal ligament[J]. Int J Immunopathol Pharmacol，2012，25(1):127-134.
[9]	宁艳洋，徐琼，王彤，等. 多能性转录因子Nanog在体外培养牙髓细胞中的表达[J]. 牙体牙髓牙周病学杂志，2013，23(6):362-365.
[10]	Yu J，Vodyanik MA，Smuga-Otto K，et al. Induced pluripotent stem cell lines derived from human somatic cells[J]. Science，2007，318(5858):1917-1920.
[11]	Yan X，Qin H，Qu C，et al. iPS cells reprogrammed from human mesenchymal- like stem/progenitor cells of dental tissue origin[J]. Stem Cells Dev，2010，19(4):469-480.
[12]	Theunissen TW，van Oosten AL，Castelo-Branco G，et al. Nanog overcomes reprogramming barriers and induces pluripotency in minimal conditions[J]. Curr Biol，2011，21(1):65-71.
[13]	Wu J，Tzanakakis ES. Contribution of stochastic partitioning at human embryonic stem cell division to NANOG heterogeneity[J]. PLoS One，2012，7(11):e50715.
[14]	Huang CE，Hu FW，Yu CH，et al. Concurrent expression of Oct4 and Nanog maintains mesenchymal stem-like property of human dental pulp cells[J]. Int J Mol Sci，2014，15(10):18623-18639.
[15]	Boyer LA，Lee TI，Cole MF，et al. Core transcriptional regulatory circuitry in human embryonic stem cells[J]. Cell，2005，122(6):947-956.
[16]	Pan G，Thomson JA. Nanog and transcriptional networks in embryonic stem cell pluripotency[J]. Cell Res，2007，17(1):42-49.
[17]	Liu L，Wei X，Ling J，et al. Expression pattern of Oct-4，Sox2，and c-Myc in the primary culture of human dental pulp derived cells[J]. J Endod，2011，37(4):466-472.
[18]	韦曦，张芳，刘路，等. Oct4基因转染对人牙髓细胞多能相关转录因子表达的影响[J/CD]. 中华口腔医学研究杂志：电子版，2011，5(4):347-355.
[19]	Liu L，Wu L，Wei X，et al. Induced overexpression of Oct4A in human dental pulp cells enhances pluripotency and multilineage differentiation capability[J]. Stem Cells Dev，2015，24(8):962-972.
[20]	Wang Z，Oron E，Nelson B，et al. Distinct lineage specification roles for NANOG，OCT4，and SOX2 in human embryonic stem cells[J]. Cell Stem Cell，2012，10(4):440-454.
[21]	Bertero A，Madrigal P，Galli A，et al. Activin/nodal signaling and NANOG orchestrate human embryonic stem cell fate decisions by controlling the H3K4me3 chromatin mark[J]. Genes Dev，2015，29(7):702-717.
[22]	Katano M，Ema M，Nakachi Y，et al. Forced expression of Nanog or Esrrb preserves the ESC status in the absence of nucleostemin expression[J]. Stem Cells，2015，33(4):1089-1101.

基因	引物序列
Nanog	F:5′-GATTTGTGGGCCTGAAGAAA-3′
?	R:5′-ATGGAGGAGGGAAGAGGAGA-3′
Oct4	F:5′-GTGGAGGAAGCTGACAACAA-3′
?	R:5′-GGTTCTCGATACTGGTTCGC-3′
Sox2	F:5′-AACCCCAAGATGCACAACTC-3′
?	R:5′-GCTTAGCCTCGTCGATGAAC-3′
DSPP	F:5′-GCCACTTTCAGTCTTCAA AGAGA-3′
?	R:5′-GCCCAAATGCAAAAATATGTA-3′
DMP1	F:5′-TGGGCATAGATTTCCTCTTTG-3′
?	R:5′-TGAGCAGGATGCTGATCTTC-3′
OCN	F:5′-CTCACACTCCTCGCCCTATT-3′
?	R:5′-TTGGACACAAAGGCTGCAC-3′
LPL	F:5′-CCCCGAGATGGAGAGCAAAG-3′
?	R:5′-ATCTCTTCTTTGGTCGGCGG-3′
PPARγ2	F:5′-GCAAACCCCTATTCCATGCTG-3′
?	R:5′-CACGGAGCTGATCCCAAAGT-3′
GAPDH	F:5′-AAGGTGAAGGTCGGAGTCAA-3′
?	R:5′-AATGAAGGGGTCATTGATGG-3′

基因	引物序列
Nanog	F:5′-GATTTGTGGGCCTGAAGAAA-3′
?	R:5′-ATGGAGGAGGGAAGAGGAGA-3′
Oct4	F:5′-GTGGAGGAAGCTGACAACAA-3′
?	R:5′-GGTTCTCGATACTGGTTCGC-3′
Sox2	F:5′-AACCCCAAGATGCACAACTC-3′
?	R:5′-GCTTAGCCTCGTCGATGAAC-3′
DSPP	F:5′-GCCACTTTCAGTCTTCAA AGAGA-3′
?	R:5′-GCCCAAATGCAAAAATATGTA-3′
DMP1	F:5′-TGGGCATAGATTTCCTCTTTG-3′
?	R:5′-TGAGCAGGATGCTGATCTTC-3′
OCN	F:5′-CTCACACTCCTCGCCCTATT-3′
?	R:5′-TTGGACACAAAGGCTGCAC-3′
LPL	F:5′-CCCCGAGATGGAGAGCAAAG-3′
?	R:5′-ATCTCTTCTTTGGTCGGCGG-3′
PPARγ2	F:5′-GCAAACCCCTATTCCATGCTG-3′
?	R:5′-CACGGAGCTGATCCCAAAGT-3′
GAPDH	F:5′-AAGGTGAAGGTCGGAGTCAA-3′
?	R:5′-AATGAAGGGGTCATTGATGG-3′

Please choose a citation manager

Content to export