臺灣博碩士論文加值系統

神經脊細胞是一種多能性的細胞，可分化成周邊神經、硬骨細胞、軟骨細胞、肌細胞、黑色素細胞，且頭部的神經脊細胞可分化為間質幹細胞。從萬能幹細胞分化成神經脊細胞的分化方法大約需要9至14天，且培養的方法也多是使用血清來培養，使得這些神經脊細胞的誘導過程與細胞特性於不同實驗間變異較大。本研究嘗試以人類胚胎幹細胞與誘導萬能幹細胞透過懸浮分化與貼附分化配合BiSP來分化神經脊細胞，並比較4種不同培養液與SEFH的組合對於神經脊細胞的培養效果。我們發現貼附分化搭配上述的訊號調控可以在誘導兩天後高效率的分化出神經脊細胞，且該細胞可以在N培養液+SEFH的環境中培養至少23代，並維持神經脊細胞的多能性與蛋白標記的表現。本研究展示了一個非常快速且簡便的分化方法，並且找出適合神經脊細胞長時間培養的確定培養基，希望能夠降低神經脊細胞在未來應用上的門檻，促進再生醫學的發展。

Neural crest cells (NCCs) are a multipotent cell population with the ability to differentiate into a wide variety of cell types, like peripheral neurons, osteocytes, chondrocytes, myocytes and melanocytes. Cranial NCCs can also become the lineage of mesenchymal stem cells. Methods of NCC differentiation from pluripotent stem cells have been investigated, showing that the induction of NCCs usually takes 9 to 14 days, and bovine serum is necessary to maintain NCCs proliferation. To facilitate the NCC induction from pluripotent stem cells, the effects of BiSP were evaluated. We further compared the culture effects of four different kinds of medium with or without SEFH. We found that both hESC and iPSC can efficiently differentiate into NCCs after two days of induction in a 2D culture situation. In addition, a defined medium consisting of N medium with SEFH can maintain NCCs growth up to 23 passages. We have thus demonstrated a robust and efficient NCCs differentiation method and a chemically defined culture medium for long term expansion of NCCs, which may pave the road for future clinical applications of pluripotent stem cells-derived NCCs.

目次

中文摘要………………………………………………..………i
英文摘要………………………………………………….……ii
目次……………………………………………………………iii
圖表目次………………………………………………………iv

前言………………………………………………………….…1
實驗材料與方法…………………………………………….…8
實驗結果…………………………………………………………...13
討論……………………………………………………..…….22
參考文獻…………………………………………………..….26
圖表………………………………………………………...…30

參考文獻
1Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. science 282, 1145-1147 (1998).
2Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. cell 126, 663-676 (2006).
3Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. cell 131, 861-872 (2007).
4Schwartz, S. D. et al. Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt's macular dystrophy: follow-up of two open-label phase 1/2 studies. The Lancet 385, 509-516 (2015).
5Theveneau, E. & Mayor, R. Neural crest delamination and migration: from epithelium-to-mesenchyme transition to collective cell migration. Developmental biology 366, 34-54 (2012).
6Kulesa, P. M. & Gammill, L. S. Neural crest migration: patterns, phases and signals. Developmental biology 344, 566-568 (2010).
7Neirinckx, V., Marquet, A., Coste, C., Rogister, B. & Wislet-Gendebien, S. Adult bone marrow neural crest stem cells and mesenchymal stem cells are not able to replace lost neurons in acute MPTP-lesioned mice. PLoS One 8, e64723 (2013).
8Neirinckx, V., Coste, C., Rogister, B. & Wislet-Gendebien, S. Concise review: adult mesenchymal stem cells, adult neural crest stem cells, and therapy of neurological pathologies: a state of play. Stem cells translational medicine 2, 284-296 (2013).
9Huang, X. & Saint-Jeannet, J.-P. Induction of the neural crest and the opportunities of life on the edge. Developmental biology 275, 1-11 (2004).
10Meulemans, D. & Bronner-Fraser, M. Gene-regulatory interactions in neural crest evolution and development. Developmental cell 7, 291-299 (2004).
11Vallin, J. et al. Cloning and characterization of three Xenopus slug promoters reveal direct regulation by Lef/beta-catenin signaling. J Biol Chem 276, 30350-30358, doi:10.1074/jbc.M103167200 (2001).
12Merrill, B. J. Wnt pathway regulation of embryonic stem cell self-renewal. Cold Spring Harbor perspectives in biology 4, a007971 (2012).
13Massagué, J. TGFβ signalling in context. Nature reviews Molecular cell biology 13, 616-630 (2012).
14Nakao, A. et al. Identification of Smad7, a TGFβ-inducible antagonist of TGF-β signalling. Nature 389, 631-635 (1997).
15Xu, R.-H. et al. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nature methods 2, 185-190 (2005).
16Minoux, M. & Rijli, F. M. Molecular mechanisms of cranial neural crest cell migration and patterning in craniofacial development. Development 137, 2605-2621, doi:10.1242/dev.040048 (2010).
17Liu, Q. et al. Human neural crest stem cells derived from human ESCs and induced pluripotent stem cells: induction, maintenance, and differentiation into functional schwann cells. Stem cells translational medicine 1, 266-278 (2012).
18Chimge, N. O. & Bayarsaihan, D. Generation of neural crest progenitors from human embryonic stem cells. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution 314, 95-103 (2010).
19Milet, C. & Monsoro-Burq, A. H. Embryonic stem cell strategies to explore neural crest development in human embryos. Developmental biology 366, 96-99 (2012).
20Lee, G. et al. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nature biotechnology 25 (2007).
21Mica, Y., Lee, G., Chambers, S. M., Tomishima, M. J. & Studer, L. Modeling neural crest induction, melanocyte specification, and disease-related pigmentation defects in hESCs and patient-specific iPSCs. Cell reports 3, 1140-1152 (2013).
22Menendez, L., Yatskievych, T. A., Antin, P. B. & Dalton, S. Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells. Proceedings of the National Academy of Sciences 108, 19240-19245 (2011).
23Fukuta, M. et al. Derivation of mesenchymal stromal cells from pluripotent stem cells through a neural crest lineage using small molecule compounds with defined media. PLoS One 9, e112291 (2014).
24Greber, B. et al. FGF signalling inhibits neural induction in human embryonic stem cells. The EMBO journal 30, 4874-4884 (2011).
25Lee, G., Chambers, S. M., Tomishima, M. J. & Studer, L. Derivation of neural crest cells from human pluripotent stem cells. Nature protocols 5, 688-701 (2010).
26Colleoni, S. et al. Long-term culture and differentiation of CNS precursors derived from anterior human neural rosettes following exposure to ventralizing factors. Experimental cell research 316, 1148-1158 (2010).
27Mitchell, P. J., Timmons, P. M., Hébert, J. M., Rigby, P. & Tjian, R. Transcription factor AP-2 is expressed in neural crest cell lineages during mouse embryogenesis. Genes & Development 5, 105-119 (1991).
28Williams, T. & Tjian, R. Analysis of the DNA-binding and activation properties of the human transcription factor AP-2. Genes & development 5, 670-682 (1991).
29Le Douarin, N. M., Ziller, C. & Couly, G. F. Patterning of neural crest derivatives in the avian embryo: in vivo and in vitro studies. Developmental biology 159, 24-49 (1993).
30Schorle, H., Meier, P., Buchert, M., Jaenisch, R. & Mitchell, P. J. Transcription factor AP-2 essential for cranial closure and craniofacial development. Nature 381, 235 (1996).
31Kim, J., Lo, L., Dormand, E. & Anderson, D. J. SOX10 maintains multipotency and inhibits neuronal differentiation of neural crest stem cells. Neuron 38, 17-31 (2003).
32Joseph, N. M. et al. Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells. Development 131, 5599-5612 (2004).
33Sauka-Spengler, T. & Bronner-Fraser, M. A gene regulatory network orchestrates neural crest formation. Nature reviews Molecular cell biology 9, 557-568 (2008).
34Bajpai, R. et al. CHD7 cooperates with PBAF to control multipotent neural crest formation. Nature 463, 958-962 (2010).
35Lee, G. et al. Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. nature 461, 402-406 (2009).