Optimization of induced differentiation of human neural stem cells into dopaminergic neurons by factorial design

doi:10.3877/cma.j.issn.2095-1221.2017.04.002

Abstract

Abstract:

Objective

To induce the differentiation of human neural stem cells (hNSCs) into dopaminergic neurons.

Methods

From a total of 5 kinds of inducing factors including aFGF, dbcAMP, pramipexole (PRX) , TPA (phorbol-12-myristae-13-acetate) , forskolin, single factor experiments were used to screen significant factors which play an important role in inducing hNSCs into dopaminergic neurons, and significance tests were tested by student's t. Factorial design was used to evaluate the inducing effect of the selected significant factors and to find out how these factors affect the induced dopaminergic neuron positive ratio. Statistical significance was tested by analysis of variance. Finally, the optimal concentration of each significant factor was determined by using the steepest-ascent method.

Results

According to single factor experiments, aFGF, dbcAMP and TPA were considered to be important factors in inducing differentiation of dopaminergic neurons form hNSCs. Inducing effect of aFGF, dbcAMP and TPA was modeled by factorial design. These results showed that aFGF and dbcAMP were positive factors and TPA was negative factor at the initial concentrations. The ratio of concentration changes in aFGF, dbcAMP and TPA should be 0.29 : 0.90 : -0.39. The stepwise of aFGF, dbcAMP and TPA used in the steepest-ascent method was selected as 11 ng/ml, 35 μmol/L and -15 nmol/L, respectively. Then, the optimal concentration of aFGF, dbcAMP and TPA was achieved at 166 ng/ml, 310 μmol/L and 10 nmol/L. The optimal dopaminergic neuron positive ratio was (6.56±0.07) ﹪, which increased by 23.8﹪ comparing to control group.

Conclusion

Factorial design can effectively optimize the differentiation of hNSCs into dopaminergic neurons.

Key words: Neural stem cells, Dopamine, Neurons

Zhentao Song, Heng Chen, Shuai Wang, Zhenhui Tian, Xiaodun Liu, Tingyu Qu. Optimization of induced differentiation of human neural stem cells into dopaminergic neurons by factorial design[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2017, 07(04): 195-201.

/ / Recommend

Add to citation manager EndNote|Ris|BibTeX

URL: https://zhxbygxbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-1221.2017.04.002

https://zhxbygxbzz.cma-cmc.com.cn/EN/Y2017/V07/I04/195

Figures/Tables 6

References 21

[1]	Suzuki S, Kawamata J, Iwahara N, et al. Intravenous mesenchymal stem cell administration exhibits therapeutic effects against 6-hydroxydopamine-induced dopaminergic neurodegeneration and glial activation in rats[J]. Neurosci Lett, 2015, 584:276-281.
[2]	Peng SP, Copray S. Comparison of Human Primary with Human iPS Cell-Derived Dopaminergic Neuron Grafts in the Rat Model for Parkinson's Disease[J]. Stem Cell Rev, 2016,12(1):105-120.
[3]	Tian LP, Zhang S, Zhang YJ, et al. Lmx1b can promote the differentiation of embryonic stem cells to dopaminergic neurons associated with Parkinson's disease[J]. Biotechnol Lett, 2012, 34(7):1167-1174.
[4]	Christophersen NS, Meijer X, Jørgensen JR, et al. Induction of dopaminergic neurons from growth factor expanded neural stem/progenitor cell cultures derived from human first trimester forebrain[J]. Brain Research Bulletin, 2006, 70(4-6):457-466.
[5]	Mariani M, Karki R, Spennato M, et al. Class III β-tubulin in normal and cancer tissues[J]. Gene, 2015, 563(2):109-114.
[6]	White RB, Thomas MG. Moving beyond tyrosine hydroxylase to define dopaminergic neurons for use in cell replacement therapies for Parkinson's disease[J]. CNS & neurological disorders drug targets, 2012, 11(4):340.
[7]	Moreno EL, Hachi S, Hemmer K, et al. Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture[J]. Lab on a Chip, 2015, 15(11):2419-2428.
[8]	Chen M, Lin H, Chiu H, et al. Human FGF1 promoter is active in ependymal cells and dopaminergic neurons in the brains of F1B-GFP transgenic mice[J]. Dev Neurobiol, 2015, 75(3):232-248.
[9]	Yang H, Wang J, Wang F, et al. Dopaminergic neuronal differentiation from the forebrain-derived human neural stem cells induced in cultures by using a combination of BMP-7 and pramipexole with growth factors[J]. Front Neural Circuits, 2016, 10:29.
[10]	Bono F, Savoia P, Guglielmi A, et al. Role of dopamine D2/D3 receptors in development, plasticity, and neuroprotection in human iPSC-Derived midbrain dopaminergic neurons[J]. Mol Neurobiol, 2017.
[11]	Box GE, Hunter JS, Hunter WG. Statistics for experimenters:design, innovation, and discovery[M]. Vol. 2. New York: Wiley-Interscience, 2005.
[12]	Iacovitti L, Stull ND, Jin H. Differentiation of human dopamine neurons from an embryonic carcinomal stem cell line[J]. Brain Research, 2001, 912(1):99-104.
[13]	Scintu F, Reali C, Pillai R, et al. Differentiation of human bone marrow stem cells into cells with a neural phenotype:diverse effects of two specific treatments[J]. BMC Neuroscience, 2006, 7:14.
[14]	Cabell L, Audesirk G. Effects of selective inhibition of protein kinase C, cyclic AMP-dependent protein kinase, and Ca(2+)-calmodulin-dependent protein kinase on neurite development in cultured rat hippocampal neurons[J]. Int J Dev Neurosci, 1993, 11(3):357-368.
[15]	Sakaguchi DS, Janick LM, Reh TA. Basic fibroblast growth factor (FGF-2) induced transdifferentiation of retinal pigment epithelium: generation of retinal neurons and glia[J]. Dev Dyn, 1997, 209(4):387-398.
[16]	Du X, Stull N, Iacovitti L. Novel expression of the tyrosine hydroxylase gene requires both acidic fibroblast growth factor and an activator[J]. J Neurosci, 1994, 14(12):7688-7694.
[17]	Pan T, Xie W, Jankovic J, et al. Biological effects of pramipexole on dopaminergic neuron-associated genes: relevance to neuroprotection[J]. Neuroscience Letters, 2005, 377(2):106-109.
[18]	黄立，宋振涛，朱明军. 联合试验设计法优化辣蓼饲料添加剂生产工艺[J]. 江苏农业科学, 2016, 44(11):272-276.
[19]	Song ZT, Zhu MJ. Feed additive production by fermentation of herb Polygonum hydropiper L. and cassava pulp with simultaneous flavonoid dissolution[J]. Biotechnol Appl Biochem, 2017, 64(2):290-300.
[20]	Liu C, Wu M, Hwang S. Optimization of serum free medium for cord blood mesenchymal stem cells[J]. Biochemical Engineering Journal, 2007, 33(1):1-9.
[21]	Chang KH, Zandstra PW. Quantitative screening of embryonic stem cell differentiation:Endoderm formation as a model[J]. Biotechnol Bioeng, 2004 , 88(3):287-298.

因素	编码	水平
因素	编码	-1	+1
aFGF（ng/ml）	A	50	150
dbcAMP（μmol/L）	B	50	150
TPA（nmol/L）	C	50	150

因素	编码	水平
因素	编码	-1	+1
aFGF（ng/ml）	A	50	150
dbcAMP（μmol/L）	B	50	150
TPA（nmol/L）	C	50	150

标准序	运行序	aFGF（ng/ml）	dbcAMP（μmol/L）	TPA（nmol/L）	TH阳性率（﹪）
1	4	50	50	50	1.38
2	5	50	50	50	1.04
3	11	150	50	50	1.64
4	9	150	50	50	1.52
5	2	50	150	50	1.43
6	16	50	150	50	2.86
7	7	150	150	50	4.10
8	12	150	150	50	6.00
9	10	50	50	150	1.41
10	14	50	50	150	1.33
11	15	150	50	150	0.58
12	8	150	50	150	0.79
13	3	50	150	150	3.15
14	1	50	150	150	1.95
15	13	150	150	150	2.44
16	6	150	150	150	2.11

标准序	运行序	aFGF（ng/ml）	dbcAMP（μmol/L）	TPA（nmol/L）	TH阳性率（﹪）
1	4	50	50	50	1.38
2	5	50	50	50	1.04
3	11	150	50	50	1.64
4	9	150	50	50	1.52
5	2	50	150	50	1.43
6	16	50	150	50	2.86
7	7	150	150	50	4.10
8	12	150	150	50	6.00
9	10	50	50	150	1.41
10	14	50	50	150	1.33
11	15	150	50	150	0.58
12	8	150	50	150	0.79
13	3	50	150	150	3.15
14	1	50	150	150	1.95
15	13	150	150	150	2.44
16	6	150	150	150	2.11

?	aFGF（ng/ml）	dbcAMP（μmol/L）	TPA（nmol/L）	TH阳性率（﹪）
第1步	100	100	100	1.48±0.10
第2步	111	135	85	1.19±0.96
第3步	122	170	70	1.32±1.86
第4步	133	205	55	2.85±1.64
第5步	144	240	40	3.46±2.48
第6步	155	275	25	5.62±0.57
第7步	166	310	10	6.56±0.07

Please choose a citation manager

Content to export