从人多能干细胞的黑素细胞的无饲养推导
Summary
这个工作描述了一种在体外分化协议经由神经嵴，以产生自人多能干细胞色素，成熟的黑素细胞，并使用一个无饲养25日实验方案黑素细胞的中间阶段。
Abstract
Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.
Introduction
人多能干细胞（hPSCs）提供一个平台，以模仿正常分化以可伸缩的方式用于疾病建模，药物筛选和细胞替代疗法1-6。特别感兴趣的，hPSCs开拓各种途径，为研究难以分离或在患者样本稀少罕见/短暂性的细胞类型。此外，诱导多能干细胞（iPS细胞）使研究人员研究发育和疾病建模在患者中特定的方式解开独特机制1,2,7-11。用于从hPSCs黑色素细胞的分化的先前公布的协议需要长达6周分化的和涉及培养细胞从L- Wnt3a的单元12的条件培养基。该协议最初由云母等人提出，并说明这里在三周内产生色素细胞和消除歧义和条件培养基相关的不一致。
黑素细胞ARE从神经嵴，独特的脊椎动物细胞的流动人口的。神经嵴是原肠胚形成过程中定义，并在神经板的边缘代表细胞群体，神经和非神经外胚层之间接壤。在神经胚形成，神经组织由神经板的发展，形成神经褶，其中汇聚在导致神经管13,14背中线。
神经嵴细胞的神经管的屋顶板出现，脊索对面，并迁移远离以产生分化细胞的不同群体之前经历上皮至间质转变。的嵴细胞的命运是部分地由沿胚的体轴顶板的解剖位置界定。神经嵴细胞衍生物包括谱系两者中胚层的特征（平滑肌细胞，成骨细胞，脂肪细胞，软骨细胞）和外胚层细胞（黑素细胞，雪旺氏Ç厄尔，神 经元）14。神经嵴干细胞上调转录因子SOX10，可以通过荧光激活细胞用针对P75和HNK1排序来分离。
注定要成为黑素细胞穿过一个黑素细胞阶段和上调KIT和MITF（小眼球相关的转录因子）6,21 MITF是黑素细胞发展的主要调节并是一种转录因子，负责控制多黑素细胞发展的神经嵴细胞22- 24。人黑素细胞迁移到它们驻留无论是在毛发凸起或由角质形成细胞在表皮包围（形成色素单位）作为前体的成熟的，着色的黑素细胞表皮的基底层。黑素细胞的分化和成熟成颜料黑素发生伴随毛球的定植和表达的黑色素生产途径（TYRP1，TYR，OCA2的和PMEL）25,26。
从患者中分离的人类黑色素细胞和黑素细胞是昂贵的，困难的，在数量上的限制。该协议使研究人员能够区分hPSCs（诱发或胚胎）为黑素细胞或黑素细胞前体在一个定义良好的，快速的，可重复的，可扩展的，廉价的无细胞分选方法。协议以前用于从患者的色素沉着紊乱分化的iPSC时标识特定疾病的缺陷。
Protocol
Representative Results
Discussion
用于从hPSCs黑素细胞的成功分化以下建议，应考虑。首先，它是必不可少的在任何时候都无菌培养的条件下工作。此外，为了开始与多能性，完全未分化hPSCs是很重要的;如果起始群体包含分化细胞的产量将不可避免地下降，因为污染物不能对黑素细胞被引导，甚至可能进一步扰乱了正常分化细胞。
为了确保细胞保持多能照顾坚持干细胞维持既定规则;定期饲料和通道和新郎文?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作是由来自乔安娜M.尼古拉基金会研究员黑色素瘤的奖学金和健康的露丝下L. Kirschstein国家研究服务奖F31的国家机构提供支持。这项工作是通过从NYSTEM和三机构干细胞的倡议（斯塔尔基金会）资助的进一步支持。
Materials
| Accutase | Innovative Cell Technologies | AT104 | |
| apo human transferrin | Sigma | T1147 | |
| Ascorbic Acid (L-AA) | Sigma | A4034 | 100 mM |
| B27 (B27 Supplement) | Invitrogen | 17504044 | |
| β-Mercaptoethanol | Gibco-Life Technologies | 21985-023 | 10 mg/ml |
| BMP4 | R&D Systems | 314-bp | |
| CHIR99021 | Tocris-R&D Systems | 4423 | 6 mM |
| Cholera toxin | Sigma | C8052 | 50 mg/ml |
| cAMP (cyclicAMP) | Sigma | D0627 | 100 mM |
| Dexamethasone | Sigma | D2915-100MG | 50 μM |
| DMEM – Dulbecco's Modified Eagle Medium | Gibco-Life Technologies | 11985-092 | |
| DMEM/F12 – Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 | Gibco-Life Technologies | 1133–032 | |
| DMEM/F12 powder | Invitrogen | 12500-096 | |
| EDN3 (Endothelin-3, human) | American Peptide Company | 88-5-10B | 100 μM |
| Fibronectin | BD Biosciences | 356008 | 200 μg/ml |
| gelatin (PBS without Mg/Ca) | in house | 0.1% in PBS | |
| Glucose | Sigma | G7021 | |
| Human insulin | Sigma | I2643 | |
| ITS+ Universal Culture Supplement Premix | BD Biosciences | 354352 | |
| KSR (Knockout Serum Replacement) | Gibco-Life Technologies | 10828-028 | |
| Knockout DMEM | Gibco-Life Technologies | 10829-018 | |
| L-Glutamine | Gibco-Life Technologies | 25030-081 | |
| LDN193189 | Stemgent | 04-0074 | 100 mM |
| Low glucose DMEM | Invitrogen | 11885-084 | |
| Matrigel matrix | BD Biosciences | 354234 | Dissolve 1:20 in DMEM/F12 |
| MCDB201 Medium | Sigma | M6770 | |
| MEM minimum essential amino acids solution | Gibco-Life Technologies | 11140-080 | |
| Mouse embryonic fibroblasts (7 million cells/vial) | GlobalStem | GSC-8105M | |
| Mouse Laminin-I | R&D Systems | 3400-010-01 | 1 mg/ml |
| Neurobasal medium | Invitrogen | 21103049 | |
| Penicilin/Streptomycin | Gibco-Life Technologies | 15140-122 | 10,000 U/ml |
| Poly-L Omithin hydrobromide | Sigma | P3655 | 15 mg/ml |
| Progesterone | Sigma | P8783 | 0.032g in 100ml 100% ethanol |
| Putrescine dihydrochloride | Sigma | P5780 | |
| FGF2 (Recombinant human FGF basic) | R&D Systems | 233-FB-001MG/CF | 10 mg/ml |
| SB431542 | Tocris-R&D Systems | 1814 | 10 mM |
| Selenite | Sigma | S5261 | |
| Sodium Bicarbonate | Sigma | S5761 | |
| SCF (Stem Cell Factor, recombinant Human) | Peprotech Inc. | 300-07 | 50 μg/ml |
| TYRP1 (G-17) Antibody | Santa Cruz | 10443 | 1:200 |
| TYRP2 Antibody | Abcam | 74073 | 1:200 |
| Trypsin-EDTA (0.05%) | Gibco-Life Technologies | 25300-054 | |
| Y-27632 dihydrochloride | Tocris-R&D Systems | 1254 | 10 mM |
Referenzen
- Ebert, A. D., et al. Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature. 457, 277-280 (2009).
- Lee, G., et al. Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature. 461, 402-406 (2009).
- Lee, G., et al. Large-scale screening using familial dysautonomia induced pluripotent stem cells identifies compounds that rescue IKBKAP expression. Nat biotechnol. 30, 1244-1248 (2012).
- Lee, G., Studer, L. Induced pluripotent stem cell technology for the study of human disease. Nat methods. 7, 25-27 (2010).
- Zimmer, B., et al. Evaluation of developmental toxicants and signaling pathways in a functional test based on the migration of human neural crest cells. Environ health persp. 120, 1116-1122 (2012).
- Mica, 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 rep. 3, 1140-1152 (2013).
- Mariani, J., et al. FOXG1-Dependent Dysregulation of GABA/Glutamate Neuron Differentiation in Autism Spectrum Disorders. Cell. 162, 375-390 (2015).
- Doulatov, S., et al. Modeling Diamond Blackfan Anemia in Vivo Using Human Induced Pluripotent Stem Cells. Blood. 124, (2014).
- Cherry, A. B. C., Daley, G. Q. Reprogrammed Cells for Disease Modeling and Regenerative Medicine. Annu Rev Med. 64, 277-290 (2013).
- Inoue, H., Nagata, N., Kurokawa, H., Yamanaka, S. iPS cells: a game changer for future medicine. Embo J. 33, 409-417 (2014).
- Kim, C., et al. Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs. Nature. 494, 105-110 (2013).
- Fang, D., et al. Defining the conditions for the generation of melanocytes from human embryonic stem cells. Stem cells. 24, 1668-1677 (2006).
- Huang, X., Saint-Jeannet, J. P. Induction of the neural crest and the opportunities of life on the edge. Dev biol. 275, 1-11 (2004).
- White, R. M., Zon, L. I. Melanocytes in development, regeneration, and cancer. Cell stem cell. 3, 242-252 (2008).
- Morrison, S. J., White, P. M., Zock, C., Anderson, D. J. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell. 96, 737-749 (1999).
- Elworthy, S., Pinto, J. P., Pettifer, A., Cancela, M. L., Kelsh, R. N. Phox2b function in the enteric nervous system is conserved in zebrafish and is sox10-dependent. Mech develop. 122, 659-669 (2005).
- Harris, M. L., Baxter, L. L., Loftus, S. K., Pavan, W. J. Sox proteins in melanocyte development and melanoma. Pigm cell melanoma r. 23, 496-513 (2010).
- Herbarth, B., et al. Mutation of the Sry-related Sox10 gene in Dominant megacolon, a mouse model for human Hirschsprung disease. PNAS. 95, 5161-5165 (1998).
- Southard-Smith, E. M., Kos, L., Pavan, W. J. Sox10 mutation disrupts neural crest development in Dom Hirschsprung mouse model. Nat genetics. 18, 60-64 (1998).
- Dupin, E., Glavieux, C., Vaigot, P., Le Douarin, N. M. Endothelin 3 induces the reversion of melanocytes to glia through a neural crest-derived glial-melanocytic progenitor. PNAS. 97, 7882-7887 (2000).
- Lister, J. A., Robertson, C. P., Lepage, T., Johnson, S. L., Raible, D. W. nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. Development. 126, 3757-3767 (1999).
- Hou, L., Panthier, J. J., Arnheiter, H. Signaling and transcriptional regulation in the neural crest-derived melanocyte lineage: interactions between KIT and MITF. Development. 127, 5379-5389 (2000).
- Levy, C., Khaled, M., Fisher, D. E. MITF: master regulator of melanocyte development and melanoma oncogene. Trends mol med. 12, 406-414 (2006).
- Phung, B., Sun, J., Schepsky, A., Steingrimsson, E., Ronnstrand, L. C-KIT signaling depends on microphthalmia-associated transcription factor for effects on cell proliferation. PloS one. 6, e24064 (2011).
- Grichnik, J. M., et al. KIT expression reveals a population of precursor melanocytes in human skin. J invest dermatol. 106, 967-971 (1996).
- Li, L., et al. Human dermal stem cells differentiate into functional epidermal melanocytes. J cell sci. 123, 853-860 (2010).
- Nishimura, E. K., et al. Dominant role of the niche in melanocyte stem-cell fate determination. Nature. 416, 854-860 (2002).
- Zur Nieden, N. I. . Embryonic stem cell therapy for osteo-degenerative diseases : methods and protocols. , (2011).
- Lee, J. H., et al. high-content screening for novel pigmentation regulators using a keratinocyte/melanocyte co-culture system. Exp dermatol. 23, 125-129 (2014).
