Small molecules supplemented in neuronal induction medium enhance neurogenic differentiation of stem cells from apical papilla

doi:10.3877/cma.j.issn.1674-1366.2019.02.002

Chinese Journal of Stomatological Research(Electronic Edition) ›› 2019, Vol. 13 ›› Issue (02): 71-76. doi: 10.3877/cma.j.issn.1674-1366.2019.02.002

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• Basic Science Research • Previous Articles Next Articles

Small molecules supplemented in neuronal induction medium enhance neurogenic differentiation of stem cells from apical papilla

Qixin Chen¹, Changyong Yuan², Zongxiang Liu³, Shaoyue Zhu⁴, Ganggang Hu¹, Penglai Wang²^,^†()

^1. School of Stomatology, Xuzhou Medical University, Xuzhou 221004, China
^2. School of Stomatology, Xuzhou Medical University, Xuzhou 221004, China; Depaetment of Implant Dentistry, The Affiliated Stomatology Hospital of Xuzhou Medical University, Xuzhou 221002, China
^3. Science and Education Section, The Affiliated Stomatology Hospital of Xuzhou Medical University, Xuzhou 221002, China
^4. Depaetment of Orthodontics, The Affiliated Stomatology Hospital of Xuzhou Medical University, Xuzhou 221002, China

Received:2018-12-21 Online:2019-04-01 Published:2019-04-01
Contact: Penglai Wang
About author:
Corresponding author: Wang Penglai, Email: wpl0771@163.com
Supported by:
Young Scientists Fund of the National Natural Science Foundation of China(81700954)

Abstract

Abstract:

Objective

To observe the effect of small molecules supplemented in neuronal induction medium on the neurogenic differentiation of stem cells from apical papilla (SCAPs) .

Methods

SCAPs were cultured in neuronal induction medium with or without small molecules. After 8 days of culture, the cellular morphology was observed with invert microscope. qRT-PCR and western blot were used to analyze the expression of neurofilament (NFM) , microtubule-associated protein 2 (MAP2) and neuron specific enolase (NSE) . Immunofluorescence was used to detect tubulin beta 3 (TUBB3) . The data was analyzed using SPSS 16.0 software package.

Results

It was observed that SCAPs treated with small molecules exhibited marked difference in cellular morphology compared with the untreated control, which displayed more neurite outgrowth. The mRNA relative expression of NFM, MAP2 and NSE with small molecules (6.608 ± 2.836, 29.40 ± 6.645 and 6.428 ± 1.025) was higher than that of the control (1.074 ± 0.202, 1.092 ± 0.115 and 1.140 ± 0.281) , which was statistically significant (t_NFM= 3.387, P_NFM = 0.0276; t_MAP2 = 7.490, P_MAP2 = 0.0017; t_NSE = 8.615, P_NSE = 0.0010) . In Western blot assay, the expression of NFM, MAP2 and NSE with small molecules was higher than that of the control, too. The immunofluorescence expression of TUBB3 in the experimental group was also higher than that of the control.

Conclusion

Neuronal induction medium with small molecules can enhance neurogenic differentiation of SCAPs.

Key words: Stem cells, apical papilla, Neurogenesis, Small molecules, Neuronal induction medium

Qixin Chen, Changyong Yuan, Zongxiang Liu, Shaoyue Zhu, Ganggang Hu, Penglai Wang. Small molecules supplemented in neuronal induction medium enhance neurogenic differentiation of stem cells from apical papilla[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2019, 13(02): 71-76.

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Figures/Tables 6

References 24

[1]	Parisi L, Manfredi E. Applicability of tooth derived stem cells in neural regeneration[J]. Neural Regen Res，2016，11(11):1704-1707. DOI：10.4103/1673-5374.194705.
[2]	Panagopoulos GN, Megaloikonomos PD, Mavrogenis AF. The Present and Future for Peripheral Nerve Regeneration[J]. Orthopedics，2017，40(1):e141-e156. DOI：10.3928/01477447-20161019-01.
[3]	De Berdt P, Vanacker J, Ucakar B，et al. Dental Apical Papilla as Therapy for Spinal Cord Injury[J]. J Dent Res，2015，94(11):1575-1581. DOI：10.1177/0022034515604612.
[4]	Kolar MK, Itte VN, Kingham PJ，et al. The neurotrophic effects of different human dental mesenchymal stem cells[J]. Sci Rep，2017，7(1):12605. DOI：10.1038/s41598-017-12969-1.
[5]	Hu W, Qiu B, Guan W，et al. Direct Conversion of Normal and Alzheimer′s Disease Human Fibroblasts into Neuronal Cells by Small Molecules[J]. Cell Stem Cell，2015，17(2):204-212. DOI：10.1016/j.stem.2015.07.006.
[6]	Yuan C, Wang P, Zhu S，et al. EphrinB2 Stabilizes Vascularlike Structures Generated by Endothelial Cells and Stem Cells from Apical Papilla[J]. J Endod，2016，42(9):1362-1370. DOI：10.1016/j.joen.2016.05.012.
[7]	Pall O, Varga B, Collart-Dutilleul PY，et al. Comments on："An Overview of Protocols for the Neural Induction of Dental and Oral Stem Cells In Vitro" Article in Tissue Engineering Part B，Heng et al.，February 2016. DOI：10.1089/ten.teb.2015.0488[J]. Tissue Engineering Part B：Reviews，2017. DOI：10.1089/ten.TEB.2016.0512.
[8]	Ladewig J, Mertens J, Kesavan J，et al. Small molecules enable highly efficient neuronal conversion of human fibroblasts[J]. Nature Methods，2012，9(6):575-578. DOI：10.1038/NMETH.1972.
[9]	Liu ML, Zang T, Zou Y，et al. Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons[J]. Nat Commun，2013，4(4):2183. DOI：10.1038/ncomms3183.
[10]	Gafni O, Weinberger L, Mansour AA，et al. Derivation of novel human ground state naive pluripotent stem cells[J]. Nature，2013，504(7479):282-286. DOI：10.1038/nature12745.
[11]	Li X, Meng G, Krawetz R，et al. The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation[J]. Stem Cells Dev，2008，17(6):1079-1085. DOI：10.1089/scd.2007.0247.
[12]	Staff PO. Correction：Neurotrophic requirements of human motor neurons defined using amplified and purified stem cell-derived cultures[J]. Plos One，2015，10(3):e0119195. DOI：10.1371/journal.pone.0119195.
[13]	Li X, Zuo X, Jing J，et al. Small-Molecule-Driven Direct Reprogramming of Mouse Fibroblasts into Functional Neurons[J]. Cell Stem Cell，2015，17(2):195-203. DOI：10.1016/j.stem.2015.06.003.
[14]	Pfisterer U, Ek F, Lang S，et al. Small molecules increase direct neural conversion of human fibroblasts[J]. Scientific Reports，2016(6):38290. DOI：10.1038/srep38290.
[15]	Isgrò MA, Bottoni P, Scatena R. Neuron-Specific Enolase as a Biomarker：Biochemical and Clinical Aspects[M]//Scatena. R. Advances in Cancer Biomarkers：From biochemistry to clinic for a critical revision. Dordrecht：Springer Netherlands，2015：125-143.
[16]	Mirshafiey A, Mohsenzadegan M. TGF-beta as a promising option in the treatment of multiple sclerosis[J]. Neuropharmacology，2009，56(6-7):929-936. DOI：10.1016/j.neuropharm.2009.02.007.
[17]	Jahromi M, Razavi S, Amirpour N，et al. Paroxetine Can Enhance Neurogenesis during Neurogenic Differentiation of Human Adipose-derived Stem Cells[J]. Avicenna J Med Biotechnol，2016，8(4):152-158.
[18]	卓本慧，江和碧，瞿平，等.骨髓间充质干细胞向神经细胞定向分化的体外研究[J].中国修复重建外科杂志，2005，19(5):373-376.
[19]	杨乃龙，杨芬，徐丽丽.共培养法神经细胞诱导骨髓间充质干细胞向神经元的分化[J].中国组织工程研究与临床康复，2008，12(29):5611-5614.
[20]	Kwon SKC, Kovesdi E, Gyorgy AB，et al. Stress and Traumatic Brain Injury：A Behavioral，Proteomics，and Histological Study[J]. Front Neurol，2011，2(12):12. DOI：10.3389/fneur.2011.00012.
[21]	Huangfu D, Maehr R, Guo W，et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds[J]. Nature Biotechnology，2008，26(7):795-797. DOI：10.1038/nbt1418.
[22]	Huangfu D, Osafune K, Maehr R，et al. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2[J]. Nat Biotechnol，2008，26(11):1269-1275. DOI：10.1038/nbt.1502.
[23]	Mikkelsen TS, Hanna J, Zhang X，et al. Dissecting direct reprogramming through integrative genomic analysis[J]. Nature，2008，454(7200):49-55. DOI：10.1038/nature07196.
[24]	Shi Y, Do JT, Desponts C，et al. A Combined Chemical and Genetic Approach for the Generation of Induced Pluripotent Stem Cells[J]. Cell Stem Cell，2008，2(6):525-528. DOI：10.1016/j.stem.2008.05.011.

组别	基础培养基	其他添加物	生长因子	小分子混合物
神经诱导液	Advance DMEM/F12：	0.5%（v/v）N2+1%（v/v）	20 ng/mL bFGF	无
（对照组）	Neurobasal A（1∶1）	B27+100 mmol/L cAMP	?	?
神经诱导液添加	Advance DMEM/F12：	0.5%（v/v）N2+1%（v/v）	20 ng/mL bFGF	0.5 mmol/L VPA，+3 μmol/L CHIR99021+1 μmol/L
小分子混合物	Neurobasal A（1∶1）	B27+100 mmol/L cAMP	?	Repsox，+10 μmol/L Forskolin+10 μmol/L SP600125,+5 μmol/L GO6983,+5 μmol/L Y-27632
（实验组）	?	?	?

组别	基础培养基	其他添加物	生长因子	小分子混合物
神经诱导液	Advance DMEM/F12：	0.5%（v/v）N2+1%（v/v）	20 ng/mL bFGF	无
（对照组）	Neurobasal A（1∶1）	B27+100 mmol/L cAMP	?	?
神经诱导液添加	Advance DMEM/F12：	0.5%（v/v）N2+1%（v/v）	20 ng/mL bFGF	0.5 mmol/L VPA，+3 μmol/L CHIR99021+1 μmol/L
小分子混合物	Neurobasal A（1∶1）	B27+100 mmol/L cAMP	?	Repsox，+10 μmol/L Forskolin+10 μmol/L SP600125,+5 μmol/L GO6983,+5 μmol/L Y-27632
（实验组）	?	?	?

基因	引物序列
NSE	F：5′-GTCCCACGTGTCTTCCACTT-3′
?	R：5′-TGGGATCTACAGCCACATGA-3′
MAP2	F：5′-TTGGTGCCGAGTGAGAAGAA-3′
?	R：5′-GGTCTGGCAGTGGTTGGTTAA-3′
NFM	F：5′-GTCAAGATGGCTCTGGATATAGAAATC-3′
?	R：5′-TACAGTGGCCCAGTGATGCTT-3′

基因	引物序列
NSE	F：5′-GTCCCACGTGTCTTCCACTT-3′
?	R：5′-TGGGATCTACAGCCACATGA-3′
MAP2	F：5′-TTGGTGCCGAGTGAGAAGAA-3′
?	R：5′-GGTCTGGCAGTGGTTGGTTAA-3′
NFM	F：5′-GTCAAGATGGCTCTGGATATAGAAATC-3′
?	R：5′-TACAGTGGCCCAGTGATGCTT-3′

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