后生转换为安全和简单的方法来获得成人皮肤成纤维细胞胰岛素分泌细胞
Summary
Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.
Abstract
Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a “highly permissive” state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated “epigenetic cell conversion”. It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.
Introduction
再生医学的一个基本目的是一种可用于修复或替换受损新，功能细胞的产生，退化的组织。重塑容易获得的成人细胞进入新的，通过从一种细胞类型转换到另一个，是一个特别有吸引力的方法，特别是当所需要的细胞群不丰富或难以进入。然而，成体细胞是相当稳定的。他们通过自己的选择逐渐限制获得他们分化 状态，一旦他们达到成熟的终端专业化，他们稳定地保持它1。
在过去几年中的一些协议已被开发，即使重新编程到的体细胞（IPS）的多能性通过的一组转录因子2,3的强制表达来实现。另外，电池的转换可以通过直接的血统转获得，引进了4单</suP>或转录的组合因子5-7。这种策略不涉及通过去分化状态的转变，但需要高表达的特异性转录因子8。
最近，我们开发了一种基于成人细胞的短暂暴露于胞苷类似物的5-氮杂胞苷（5-氮杂CR）充分表征的DNA甲基转移酶抑制剂的去甲基化性质的转换协议。去甲基化步骤之后紧接着一个特定分化方案9-11，它允许以获得所需的终端的表型。这种方法能够成熟，分化的细胞转换成不同谱系的细胞，并具有相当大的优势，以避免同时使用病毒载体和任何外源性转录因子的转染。此次收购一个稳定的多能状态的，以及相关的易感性增加细胞的不稳定也是可以避免的。
<p class="“jove_content”">具体协议，允许成人皮肤成纤维细胞转化为全功能胰岛素分泌细胞是这里提出。然而，值得注意的是，该技术已被应用到不同类型的细胞，并产生积极的结果，解决向各种分化途径的细胞时。此外，后生转化已经成功在人类和猪物种9-13以及在狗中使用（手稿提交），表明该方法的广泛有效性和鲁棒性。Protocol
Representative Results
Discussion
本手稿描述了一种方法，其允许人皮肤成纤维细胞转化成产生胰岛素的细胞，通过瞬时和短暂暴露于5-氮杂CR，随后组织特异性诱导方案。这种方法允许中胚层内胚层到相关细胞的开关，无需转录因子或小分子RNA的强制表达，也不是收购一个稳定的多能状态中，使细胞更不稳定，容易出错14。
在第一步骤中，细胞可塑性增加由于在终末分化细胞诱导瞬态，可逆允许状态…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是由Carraresi基金会和欧洲基金会糖尿病研究学会（EFSD）的资助。 GP是由米兰大学的博士后奖学金支持。作者是成本行动FA1201 Epiconcept成员：表观遗传学和围孕期环境和COST行动BM1308共享大型动物模型的进步（SALAAM）。 TALB是COST行动CM1406后生化学生物学（EPICHEM）的成员。
Materials
| Dulbecco's Phosphate Buffered Saline | Sigma | D5652 | PBS; for cell wash and solution preparation |
| Antibiotic Antimycotic Solution | Sigma | A5955 | Component of Fibroblast, HP and Pancreatic media |
| 100 mm petri dish | Sarstedt | 83.3902 | For Fibroblast isolation |
| Porcine Gelatin | Sigma | G1890 | For dish coating |
| Water | Sigma | W3500 | For solution preparation |
| 35 mm petri dishes | Sarstedt | 83.39 | For Fibroblast isolation |
| DMEM, high glucose, pyruvate | Life Technologies | 41966052 | For Fibroblast culture medium |
| Fetal Bovine Serum | Life Technologies | 10500064 | FBS; Component of Fibroblast and HP media |
| L-Glutamine solution | Sigma | G7513 | Component of Fibroblast, HP and Pancreatic media |
| Trypsin-EDTA solution | Sigma | T3924 | For Fibroblast dissociation |
| KOVA GLASSTIC SLIDE 10 WITH GRIDS | Hycor Biomedical | 87144 | Cell counting |
| 5-Azacytidine | Sigma | A2385 | 5-aza-CR, for increrase cell plasticity in fibroblasts |
| Ham's F-10 Nutrient Mix | Life Technologies | 31550031 | For HP medium |
| DMEM, low glucose, pyruvate | Life Technologies | 31885023 | For HP medium |
| KnockOut Serum Replacement | Life Technologies | 10828028 | Component of HP medium |
| MEM Non-Essential Amino Acids Solution | Life Technologies | 11140035 | Component of HP and Pancreatic Basal media |
| 2-Mercaptoethanol | Sigma | M7522 | Component of HP and Pancreatic Basal media |
| Guanosine | Sigma | G6264 | Nucleoside mix stock component of HP medium |
| Adenosine | Sigma | A4036 | Nucleoside mix stock component of HP medium |
| Cytidine | Sigma | C4654 | Nucleoside mix stock component of HP medium |
| Uridine | Sigma | U3003 | Nucleoside mix stock component of HP medium |
| Thymidine | Sigma | T1895 | Nucleoside mix stock component of HP medium |
| Millex-GS 0,22 µm | Millipore | SLGS033SB | For sterilizing of solution |
| FGF-Basic (AA 1-155) Recombinant Human Protein | Life Technologies | PHG0261 | bFGF; Component of HP and Pancreatic Basal medium |
| Bovine Serum Albumin | Sigma | A3311 | BSA; Component of Pancreatic Basal medium |
| DMEM/F-12 | Life Technologies | 11320074 | For Pancreatic Basal medium |
| B-27 Supplement Minus Vitamin A | Life Technologies | 12587010 | Component of Pancreatic medium |
| N-2 Supplement | Life Technologies | 17502048 | Component of Pancreatic Basal medium |
| Activin A Recombinant Human Protein | Life Technologies | PHG9014 | For Pancreatic medium |
| Retinoic Acid | Sigma | R2625 | For Pancreatic medium |
| Dimethyl sulfoxide | Sigma | D2650 | DMSO; for Retinoic Acid stock preparation |
| Insulin-Transferrin-Selenium | Life Technologies | 41400045 | ITS; for Pancreatic Final medium |
| Anti-Vimentin antibody | Abcam | ab8069 | For immunocytochemical analisys. Working dilution 1:100 |
| 4′,6-Diamidino-2-phenylindole dihydrochloride | Sigma | 32670 | DAPI. For immunocytochemical analisys. Working dilution 1µg/ml |
| 5-Methylcytidine | Eurogentec | MMS-900P-B | For immunocytochemical analisys. Working dilution 1:500 |
| Anti-C Peptide antibody | Abcam | ab14181 | For immunocytochemical analisys. Working dilution 1:100 |
| Anti-PDX1 antibody | Abcam | ab47267 | For immunocytochemical analisys. Working dilution 1:500 |
| Mercodia Insulin ELISA | Mercodia | 10-1113-10 | For insulin release detection |
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