OVER EXPRESSION OF Mash-1 GENE IN PROMOTING DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO NEURAL CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XULeqin ^1,2 , DINGDaofang ³ , LIXiaofeng ² , SHIQi ² , WANGYongjun ² ,  ZHOUChongjian ²

1. Department of Science & Education, Xiamen Hospital of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine, Xiamen Fujian, 361009, P. R. China;
2. Institute of Spine, Shanghai University of Traditional Chinese Medicine;
3. Institute of Traumatology & Orthopedics, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine;

Corresponding author：

ZHOUChongjian, Email: zhouchongjian@hotmail.com

Keywords：

Embryonic stem cells; Mash-1; Neural differentiation; Mouse

DOI：

10.7507/1002-1892.20150331

Video：

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Objective To investigate the effects of over expression of Mash-1 gene on the differentiation of embryonic stem cells (ESC) into neural cells in vitro. Methods The ESC of rats (CE3 cells) were transfected with MSCVMash- 1 (MSCV-Mash-1-CE3 group) or MSCV (MSCV-CE3 group). The expression of Mash-1 gene was detected by RT-PCR. After transfection, hanging-drop culture was used to form embryonic bodies, and then embryonic bodies were cultured with neural induction medium. The cell morphology was observed under inverted phase contrast microscopy at 7 and 21 days; the positive rates of neural stem cells marker protein (nestin) and neuron marker protein (β-tubulin Ⅲ) were measured by immunofluorescence staining after cell attachment; and the gene expressions of α-fetal protein (AFP), Brachyury, fibroblast growth factor 5 (FGF-5), Oct3/4, nestin, and β-tubulin Ⅲ were detected by real-time fluorescence quantitative PCR at 0, 1, 7, 14, and 21 days after culture. The CE3 cells were used as control (CE3 group). Results Compared with MSCV-CE3 and CE3 groups, the expression of Mash-1 gene in MSCV-Mash-1-CE3 group was significantly increased. At 7 and 21 days after neural induction cultured, cells in MSCV-Mash-1-CE3 group had axons growth and showed neural stem cell-like and neuron cell-like morphology (unipolar, bipolar, and multipolar neurons), but few cells had axons growth in MSCV-CE3 and CE3 groups. The positive rates of nestin at 7 days and β-tubulin Ⅲ at 21 days in MSCV-Mash-1-CE3 group were significantly higher than those in MSCV-CE3 and CE3 groups (P<0.05). Real-time fluorescence quatitative PCR results showed that the gene expression of Brachyury was significantly decreased after 1 day (P<0.05), and the gene expressions of FGF-5 and nestin were significantly increased after 1 day (P<0.05) in MSCV-Mash- 1-CE3 group when compared with CE3 and MSCV-CE3 groups; the gene expression of β-tubulin Ⅲ was significantly increased after 7 days (P<0.05). There was no significant difference in above indexes between CE3 and MSCV-CE3 groups (P>0.05). The expressions of AFP and Oct3/4 showed no significant difference among groups at each time point (P>0.05). Conclusion Over expression of Mash-1 gene can promote differentiation of ESC into neural cells in vitro.

Citation： XULeqin, DINGDaofang, LIXiaofeng, SHIQi, WANGYongjun, ZHOUChongjian. OVER EXPRESSION OF Mash-1 GENE IN PROMOTING DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO NEURAL CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(12): 1553-1559. doi: 10.7507/1002-1892.20150331 Copy

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2.	Ying L, Mills JA, French DL, et al. OCT4 coordinates with WNT signaling to pre-pattern chromatin at the SOX17 locus during human ES cell differentiation into definitive endoderm. Stem Cell Reports, 2015, 5(4):490-498.
3.	Morey L, Santanach A, Blanco E, et al. Polycomb regulates mesoderm cell fate-specification in embryonic stem cells through activation and repression mechanisms. Cell Stem Cell, 2015, 17(3):300-315.
4.	Ochiai H, Suga H, Yamada T, et al. BMP4 and FGF strongly induce differentiation of mouse ES cells into oral ectoderm. Stem Cell Res, 2015, 15(2):290-298.
5.	Lee-Kubli CA, Lu P. Induced pluripotent stem cell-derived neural stem cell therapies for spinal cord injury. Neural Regen Res, 2015, 10(1):10-16.
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7.	Chuang JH, Tung LC, Lin Y. Neural differentiation from embryonic stem cells in vitro:An overview of the signaling pathways. World J Stem Cells, 2015, 7(2):437-447.
8.	Liu S, Li C, Xing Y, et al. Effect of transplantation of human embryonic stem cell-derived neural progenitor cells on adult neurogenesis in aged hippocampus. Am J Stem Cells, 2014, 3(1):21-26.
9.	Ambasudhan R, Dolatabadi N, Nutter A, et al. Potential for cell therapy in Parkinson's disease using genetically programmed human embryonic stem cell-derived neural progenitor cells. J Comp Neurol, 2014, 522(12):2845-2856.
10.	Dottori M, Tay C, Hughes SM. Neural development in human embryonic stem cells-applications of lentiviral vectors. J Cell Biochem, 2011, 112(8):1955-1962.
11.	Peljto M, Wichterle H. Programming embryonic stem cells to neuronal subtypes. Curr Opin Neurobiol, 2011, 21(1):43-51.
12.	Guan K, Chang H, Rolletschek A, et al. Embryonic stem cell-derived neurogenesis. Retinoic acid induction and lineage selection of neuronal cells. Cell Tissue Res, 2001, 305(2):171-176.
13.	Battiste J, Helms AW, Kim EJ, et al. Ascl1 defines sequentially generated lineage-restricted neuronal and oligodendrocyte precursor cells in the spinal cord. Development, 2007, 134(2):285-293.
14.	Wang B, Waclaw RR, Allen ZJ 2nd, et al. Ascl1 is a required downstream effector of Gsx gene function in the embryonic mouse telencephalon. Neural Dev, 2009, 4:5.
15.	Ding D, Xu L, Xu H, et al. Mash1 efficiently reprograms rat astrocytes into neurons. Neural Regen Res, 2014, 9(1):25-32.
16.	Schwarz SC, Schwarz J. Translation of stem cell therapy for neurological diseases. Transl Res, 2010, 156(3):155-160.
17.	Willerth SM. Neural tissue engineering using embryonic and induced pluripotent stem cells. Stem Cell Res Ther, 2011, 2(2):17.
18.	Titushkin IA, Shin J, Cho M. A new perspective for stem-cell mechanobiology:biomechanical control of stem-cell behavior and fate. Crit Rev Biomed Eng, 2010, 38(5):393-433.
19.	Bertrand N, Castro DS, Guillemot F. Proneural genes and the specification of neural cell types. Nat Rev Neurosci, 2002, 3(7):517-530.
20.	Ross SE, Greenberg ME, Stiles CD. Basic helix-loop-helix factors in cortical development. Neuron, 2003, 39(1):13-25.
21.	Guillemot F, Lo LC, Johnson JE, et al. Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons. Cell, 1993, 75(3):463-476.
22.	Skerjanc IS, Wilton S. Myocyte enhancer factor 2C upregulates MASH-1 expression and induces neurogenesis in P19 cells. FEBS Letters, 2000, 472(1):53-56.
23.	Wang K, Long Q, Jia C, et al. Over-expression of Mash1 improves the GABAergic differentiation of bone marrow mesenchymal stem cells in vitro. Brain Res Bull, 2013, 99:84-94.
24.	Tomita T, Akimoto J, Haraoka J, et al. Clinicopathological significance of expression of nestin, a neural stem/progenitor cell marker, in human glioma tissue. Brain Tumor Pathol, 2014, 31(3):162-171.
25.	Locher H, de Rooij KE, de Groot JC, et al. Class Ⅲ β-tubulin, a novel biomarker in the human melanocyte lineage. Differentiation, 2013, 85(4-5):173-181.
26.	Foudah D, Monfrini M, Donzelli E, et al. Expression of neural markers by undifferentiated mesenchymal-like stem cells from different sources. J Immunol Res, 2014, 2014:987678.

1. Chen Y, Lai D. Pluripotent states of human embryonic stem cells. Cell Reprogram, 2015, 17(1):1-6.
2. Ying L, Mills JA, French DL, et al. OCT4 coordinates with WNT signaling to pre-pattern chromatin at the SOX17 locus during human ES cell differentiation into definitive endoderm. Stem Cell Reports, 2015, 5(4):490-498.
3. Morey L, Santanach A, Blanco E, et al. Polycomb regulates mesoderm cell fate-specification in embryonic stem cells through activation and repression mechanisms. Cell Stem Cell, 2015, 17(3):300-315.
4. Ochiai H, Suga H, Yamada T, et al. BMP4 and FGF strongly induce differentiation of mouse ES cells into oral ectoderm. Stem Cell Res, 2015, 15(2):290-298.
5. Lee-Kubli CA, Lu P. Induced pluripotent stem cell-derived neural stem cell therapies for spinal cord injury. Neural Regen Res, 2015, 10(1):10-16.
6. Otsu M, Nakayama T, Inoue N. Pluripotent stem cell-derived neural stem cells:From basic research to applications. World J Stem Cells, 2014, 6(5):651-657.
7. Chuang JH, Tung LC, Lin Y. Neural differentiation from embryonic stem cells in vitro:An overview of the signaling pathways. World J Stem Cells, 2015, 7(2):437-447.
8. Liu S, Li C, Xing Y, et al. Effect of transplantation of human embryonic stem cell-derived neural progenitor cells on adult neurogenesis in aged hippocampus. Am J Stem Cells, 2014, 3(1):21-26.
9. Ambasudhan R, Dolatabadi N, Nutter A, et al. Potential for cell therapy in Parkinson's disease using genetically programmed human embryonic stem cell-derived neural progenitor cells. J Comp Neurol, 2014, 522(12):2845-2856.
10. Dottori M, Tay C, Hughes SM. Neural development in human embryonic stem cells-applications of lentiviral vectors. J Cell Biochem, 2011, 112(8):1955-1962.
11. Peljto M, Wichterle H. Programming embryonic stem cells to neuronal subtypes. Curr Opin Neurobiol, 2011, 21(1):43-51.
12. Guan K, Chang H, Rolletschek A, et al. Embryonic stem cell-derived neurogenesis. Retinoic acid induction and lineage selection of neuronal cells. Cell Tissue Res, 2001, 305(2):171-176.
13. Battiste J, Helms AW, Kim EJ, et al. Ascl1 defines sequentially generated lineage-restricted neuronal and oligodendrocyte precursor cells in the spinal cord. Development, 2007, 134(2):285-293.
14. Wang B, Waclaw RR, Allen ZJ 2nd, et al. Ascl1 is a required downstream effector of Gsx gene function in the embryonic mouse telencephalon. Neural Dev, 2009, 4:5.
15. Ding D, Xu L, Xu H, et al. Mash1 efficiently reprograms rat astrocytes into neurons. Neural Regen Res, 2014, 9(1):25-32.
16. Schwarz SC, Schwarz J. Translation of stem cell therapy for neurological diseases. Transl Res, 2010, 156(3):155-160.
17. Willerth SM. Neural tissue engineering using embryonic and induced pluripotent stem cells. Stem Cell Res Ther, 2011, 2(2):17.
18. Titushkin IA, Shin J, Cho M. A new perspective for stem-cell mechanobiology:biomechanical control of stem-cell behavior and fate. Crit Rev Biomed Eng, 2010, 38(5):393-433.
19. Bertrand N, Castro DS, Guillemot F. Proneural genes and the specification of neural cell types. Nat Rev Neurosci, 2002, 3(7):517-530.
20. Ross SE, Greenberg ME, Stiles CD. Basic helix-loop-helix factors in cortical development. Neuron, 2003, 39(1):13-25.
21. Guillemot F, Lo LC, Johnson JE, et al. Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons. Cell, 1993, 75(3):463-476.
22. Skerjanc IS, Wilton S. Myocyte enhancer factor 2C upregulates MASH-1 expression and induces neurogenesis in P19 cells. FEBS Letters, 2000, 472(1):53-56.
23. Wang K, Long Q, Jia C, et al. Over-expression of Mash1 improves the GABAergic differentiation of bone marrow mesenchymal stem cells in vitro. Brain Res Bull, 2013, 99:84-94.
24. Tomita T, Akimoto J, Haraoka J, et al. Clinicopathological significance of expression of nestin, a neural stem/progenitor cell marker, in human glioma tissue. Brain Tumor Pathol, 2014, 31(3):162-171.
25. Locher H, de Rooij KE, de Groot JC, et al. Class Ⅲ β-tubulin, a novel biomarker in the human melanocyte lineage. Differentiation, 2013, 85(4-5):173-181.
26. Foudah D, Monfrini M, Donzelli E, et al. Expression of neural markers by undifferentiated mesenchymal-like stem cells from different sources. J Immunol Res, 2014, 2014:987678.

Chinese Journal of Reparative and Reconstructive Surgery

OVER EXPRESSION OF Mash-1 GENE IN PROMOTING DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO NEURAL CELLS

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