质粒重编程诱导多潜能干细胞及定向分化神经干细胞

doi:10.13418/j.issn.1001-165x.2016.06.011

Abstract

Abstract:

Objective　To study the induction of non-intergrated induced pluripotent stem cells (iPSCs) with episomal plasmid vectors, and differentiating into neural stem cells in vitro.　Methods　Non-integrating mouse iPSCs were induced from mouse embryonic fibroblasts (MEFs) with plasmid vectors pCEP4-EO2S-ET2K. For the differentiation of neural stem cells (NSCs), iPSCs were cultured in suspension and adherent cultural plate for 2 times with different mediums. The characterization of iPSCs and NSCs were then investigated.　Results　Our results demonstrated that mouse iPSC could be effectively generated and the pluripotency of iPSC was similar to that of mESCs. In addition, no vector integration was found in iPSCs of 10th passages. Quantitative real-time PCR showed that the neural stem cell marker gene expression was significantly improved in iPSC-derived NSCs, which was similar to that of wild-type NSCs. Immunofluorescence demonstrated that iPSC-derived NSCs expressed NESTIN and PAX6 and could differentiated into neurons, oligodendrocytes and astrocytes in vitro.　Conclusions　 Non-integrated iPSCs could be generated with episomal plasmid vectors and differentiated into NSCs which could be the ideal seeding cells for the treatment of spinal cord injury.

Key words: Episomal plasmid vectors, Non-integrating induced pluripotent stem cells, Neural stem cells

LIN Cheng-kai, RONG Li-ming, LIU Bin. Generation of non-integrating induced pluripotent stem cells with episomal plasmids and differentiation into neural stem cells[J]. Chinese Journal Of Clinical Anatomy, 2016, 34(6): 647-654.

References

[1] Fehlings MG, Perrin RG. The role and timing of early decompression for cervical spinal cord injury: update with a review of recent clinical evidence[J]. Injury, 2005, 36(Suppl 2):B13-26.
[2] Tetzlaff W, Okon EB, Karimi-Abdolrezaee S, et al. A systematic review of cellular transplantation therapies for spinal cord injury[J]. J Neurotrauma, 2011, 28(8):1611-1682.
[3] Ogawa Y, Sawamoto K, Miyata T, et al. Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats[J]. J Neurosci Res, 2002, 69(6):925-33.
[4] 鲁俊, 撒亚莲, 严新民. 脐带间充质干细胞移植治疗脊髓损伤的研究进展[J]. 中国临床解剖学杂志, 2014, 32 (2):234-236.
[5] Lu Y, Wang MY. Neural stem cell grafts for complete spinal cord injury[J]. Neurosurgery, 2012, 71(6):N13-15.
[6] Piltti KM, Salazar DL, Uchida N, et al. Safety of human neural stem cell transplantation in chronic spinal cord injury[J]. Stem Cells Transl Med, 2013, 2(12):961-974.
[7] 曹中伟,刘洪文,曾倩, 等. 神经干细胞移植修复大鼠脊髓半切伤的研究[J]. 中国临床解剖学杂志, 2006, 24 (3):315-319.
[8] Suter DM, Krause KH. Neural commitment of embryonic stem cells: molecules, pathways and potential for cell therapy[J]. J Pathol, 2008,215(4):355-368.
[9] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J]. Cell, 2006,126(4):663-676.
[10]Choi HW, Kim JS, Choi S, et al. Neural stem cells differentiated from iPS cells spontaneously regain pluripotency[J]. Stem cells (Dayton, Ohio), 2014, 32(10):2596-2604.
[11]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.
[12]Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells[J]. Nature, 2007, 448(7151):313-317.
[13]Okita K, Yamakawa T, Matsumura Y, et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells[J]. Stem cells (Dayton, Ohio), 2013,31(3):458-466.
[14]Xue Y, Cai X, Wang L, et al. Generating a non-integrating human induced pluripotent stem cell bank from urine-derived cells[J]. PloS one, 2013,8(8):e70573.
[15]Fusaki N, Ban H, Nishiyama A, et al. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome[J]. Proc Jpn Acad Ser B Phys Biol Sci, 2009,85(8):348-362.
[16]Kim D, Kim CH, Moon JI, et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins[J]. Cell Stem Cell, 2009, 4(6):472-476.
[17]Warren L, Manos PD, Ahfeldt T, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA[J]. Cell Stem Cell, 2010,7(5):618-630.
[18]Jia F, Wilson KD, Sun N, et al. A nonviral minicircle vector for deriving human iPS cells[J]. Nat Methods. 2010,7(3):197-199.
[19]Subramanyam D, Lamouille S, Judson RL, et al. Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells[J]. Nat Biotechnol, 2011, 29(5):443-448.

Generation of non-integrating induced pluripotent stem cells with episomal plasmids and differentiation into neural stem cells

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 4

Metrics

Comments

Recommended 0

[1]	LIU Fang, MAO Zhi-rong, DENG Li, CHEN Bo, YUAN Qiong-lan, GAO Xiao-qing. Neuroprotection of GDNF gene-modified NSCs on temporary ischemic stroke in rats [J]. Chinese Journal of Clinical Anatomy, 2020, 38(4): 408-413.
[2]	TUN Pei-Hua, DAN Jian, CHEN Jian, TAN Cheng. The correlation between non-diabetic cerebral infarction and acute hyperglycemia and its mechanism [J]. Chinese Journal Of Clinical Anatomy, 2015, 33(2): 199-203.
[3]	ZHONG De-Jun, LI Sen, KANG Min, XU Shuang, WEI Shu-Yi, WANG Qing, WANG Song. Expression and significance of BMP-2 during rat NSCs induced by ATRA differentiating into neurons in vitro [J]. Chinese Journal Of Clinical Anatomy, 2012, 30(4): 426-430.
[4]	TAN 　Cheng, CHEN 　Jian, GUO 　Yang, CHEN Rui-Qing, LI 　Can, CHEN Tian-Zhou. Effects of glucose concentration on proliferation of hypoxic adult neural stem cells (ANSCs) in vitro [J]. Chinese Journal Of Clinical Anatomy, 2011, 29(6): 672-676.