The system for directional and stable differentiation of
amniotic fluid stem cells into neuron-like cells

doi:10.3969/j.issn.2095-4344.2059

Abstract

Abstract:

BACKGROUND: Neuronal regeneration using stem cell differentiation has gained a lot of attentions from researchers. Although embryonic stem cells and induced pluripotent stem cells have good potential for neuronal differentiation, a high risk of tumor development in vivo limits the further study.

OBJECTIVE: To establish a stable system for sorting, culture and neuronal differentiation of amniotic fluid stem cells, and to explore the feasibility as seed cells for neuronal regeneration.

METHODS: Amniotic fluid sample (10 mL) was obtained at 19-22 weeks of pregnancy under B-ultrasound guidance, and amniotic fluid stem cells were isolated by c-Kit magnetic beads. The markers Oct-4 and Sox2 of amniotic fluid stem cells were identified by immunofluorescence. The expression levels of c-Kit, Oct-4, Sox2 and Nestin in amniotic fluid stem cells after multiple passages were detected by RT-PCR. Then, the cells were cultured by hanging drop for 4 days to observe the embryoid bodies-like structures. Amniotic fluid stem cells were induced to differentiate into neurons using two-stage method. The expression levels of Neuro D and Tuj1 were observed by immunofluorescence.

RESULTS AND CONCLUSION: (1) About 1% of amniotic fluid stem cells were positive for c-Kit. (2) (75.0±4.6)% of amniotic fluid stem cells expressed Oct-4 and (86.0±2.8)% of the cells expressed Sox2. (3) The expression levels of c-Kit, Oct-4, Sox2 and Nestin detected by RT-PCR did not change with passage times. (4) Embryoid bodies-like structures formed after hanging drop culture. (5) Immunofluorescence results showed that amniotic fluid stem cells expressed neuronal marker Tuj1, but without the typical morphological features. RT-PCR detected the expression of Tuj1 in different amniotic fluid stem cell specimens as well as in the same sample after several passages. (6) Amniotic fluid stem cells could have the characteristics of neuron-like cells after induction with basic fibroblast growth factor, brain-derived neurotrophic factor, and neurotrophin factor 3 in two stages, and could express neural stem cell marker Neuro D and neuronal marker Tuj1.

Key words: amniotic fluid stem cells, embryonic body, hanging drop, neuronal differentiation, neurotrophic factor

CLC Number:

Zong Ling, Chen Guangui, Zhang Lanzhen, Zhai Jinming, Long Yanbo, Liu Xiaolan. The system for directional and stable differentiation of amniotic fluid stem cells into neuron-like cells[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(13): 2055-2060.

Figures/Tables 9

References

[1] BAS E, VAN DE WATER TR, LUMBRERAS V, et al. Adult human nasal mesenchymal-like stem cells restore cochlear spiral ganglion neurons after experimental lesion. Stem Cells Dev. 2014;23(5):502-514.
[2] CHEN W, JONGKAMONWIWAT N, ABBAS L, et al. Restoration of auditory evoked responses by human ES-cell-derived otic progenitors. Nature. 2012;490(7419):278-282.
[3] PANDIT SR, SULLIVAN JM, EGGER V, et al. Functional effects of adult human olfactory stem cells on early-onset sensorineural hearing loss. Stem Cells. 2011;29(4):670-677.
[4] ALHATTAB D, JAMALI F, ALI D, et al. An insight into the whole transcriptome profile of four tissue-specific human mesenchymal stem cells. Regen Med. 2019;14(9):841-865.
[5] JIANG B, YAN L, WANG X, et al. Concise Review: Mesenchymal Stem Cells Derived from Human Pluripotent Cells, an Unlimited and Quality-Controllable Source for Therapeutic Applications. Stem Cells. 2019;37(5):572-581.
[6] XU J. Therapeutic Applications of Mesenchymal Stem Cells for Systemic Lupus Erythematosus. Adv Exp Med Biol. 2018;1089:73-85.
[7] GUTIÉRREZ-FERNÁNDEZ M, OTERO-ORTEGA L, RAMOS-CEJUDO J, et al. Adipose tissue-derived mesenchymal stem cells as a strategy to improve recovery after stroke. Expert Opin Biol Ther. 2015;15(6): 873-881.
[8] BAIN G, KITCHENS D, YAO M, et al. Embryonic stem cells express neuronal properties in vitro. Dev Biol. 1995;168(2):342-357.
[9] OKABE S, FORSBERG-NILSSON K, SPIRO AC, et al. Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech Dev. 1996;59(1):89-102.
[10] DE COPPI P, BARTSCH G JR, SIDDIQUI MM, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol. 2007;25(1): 100-106.
[11] MCLAUGHLIN D, TSIRIMONAKI E, VALLIANATOS G, et al. Stable expression of a neuronal dopaminergic progenitor phenotype in cell lines derived from human amniotic fluid cells. J Neurosci Res. 2006;83(7): 1190-1200.
[12] FAUZA D. Amniotic fluid and placental stem cells. Best Pract Res Clin Obstet Gynaecol. 2004;18(6):877-891.
[13] IN 'T ANKER PS, SCHERJON SA, KLEIJBURG-VAN DER KEUR C, et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood. 2003;102(4):1548-1549.
[14] PRUSA AR, HENGSTSCHLAGER M. Amniotic fluid cells and human stem cell research: a new connection. Med Sci Monit. 2002;8(11): RA253-257.
[15] ZONG L, CHEN K, ZHOU W, et al. Inner ear stem cells derived feeder layer promote directional differentiation of amniotic fluid stem cells into functional neurons. Hear Res. 2014;316:57-64.
[16] TSAI MS, LEE JL, CHANG YJ, et al. Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum Reprod. 2004;19(6):1450-1456.
[17] DONALDSON AE, CAI J, YANG M, et al. Human amniotic fluid stem cells do not differentiate into dopamine neurons in vitro or after transplantation in vivo. Stem Cells Dev. 2009;18(7):1003-1012.
[18] ZHANG M, MA Q, HU H, et al. Stem cell factor/c-kit signaling enhances invasion of pancreatic cancer cells via HIF-1α under normoxic condition. Cancer Lett. 2011;303(2):108-117.
[19] VALLI A, ROSNER M, FUCHS C, et al. Embryoid body formation of human amniotic fluid stem cells depends on mTOR. Oncogene. 2010;29(7):966-977.
[20] 张胜利, 范应中. Transwell 3-D联合共培养促使人羊水克隆样细胞来源干细胞向肝样细胞分化的研究[J].中华实验外科杂志, 2015,32(6):1323-1328.
[21] 张胜利, 范应中. 人羊水克隆样细胞来源干细胞用于侧脑室损伤动物模型细胞治疗的研究[J].中华实验外科杂志, 2017,34(8):1336-1338.
[22] SANTOS D, GONZALEZ-PEREZ F, NAVARRO X, et al. Dose-Dependent Differential Effect of Neurotrophic Factors on In Vitro and In Vivo Regeneration of Motor and Sensory Neurons. Neural Plast. 2016;2016:4969523.
[23] PEVNY LH, NICOLIS SK. Sox2 roles in neural stem cells. Int J Biochem Cell Biol. 2010;42(3):421-424.
[24] FRAICHARD A, CHASSANDE O, BILBAUT G, et al. In vitro differentiation of embryonic stem cells into glial cells and functional neurons. J Cell Sci. 1995;108 ( Pt 10):3181-3188.
[25] KIM TS, NAKAGAWA T, KITA T, et al. Neural connections between embryonic stem cell-derived neurons and vestibular hair cells in vitro. Brain Res. 2005;1057(1-2):127-133.
[26] MOSCHIDOU D, MUKHERJEE S, BLUNDELL MP, et al. Valproic acid confers functional pluripotency to human amniotic fluid stem cells in a transgene-free approach. Mol Ther. 2012;20(10):1953-1967.
[27] CIPRIANI S, BONINI D, MARCHINA E, et al. Mesenchymal cells from human amniotic fluid survive and migrate after transplantation into adult rat brain. Cell Biol Int. 2007;31(8):845-850.
[28] KAKISHITA K, ELWAN MA, NAKAO N, et al. Human amniotic epithelial cells produce dopamine and survive after implantation into the striatum of a rat model of Parkinson's disease: a potential source of donor for transplantation therapy. Exp Neurol. 2000;165(1):27-34.
[29] PAN HC, CHIN CS, YANG DY, et al. Human amniotic fluid mesenchymal stem cells in combination with hyperbaric oxygen augment peripheral nerve regeneration. Neurochem Res. 2009;34(7):1304-1316.