Name: epigenetic conversion as a safe and simple method to obtain insulin-secreting cells from adult skin fibroblasts
Uploaded: 2016-03-18T15:00:00.000+00:00
Duration: 8 min 45 s
Description: Watch this Scientific Journal Video about Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts at JoVE.com

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。

注意:下面描述的所有的程序必须在无菌条件下层流罩下进行。确保所有培养过程在其整个处理上恒温控制的阶段进行，并维持细胞在37℃。 1.皮肤成纤维细胞分离<ol><li>准备培养皿涂料解决方案<ol><li>将0.1g猪明胶的在100ml水中（最终浓度0.1％）。用消毒液高压灭菌。 </li><li>加入1.5毫升无菌0.1％猪的明胶为35 mm的培养皿。等待2小时，大衣，保持它们在室温下。 注意:人的皮肤活检通过在局部麻醉下切除从前臂的前方面的无血管区域收集并存储在在+ 4℃下补充有2％抗生素抗真菌溶液，使用前的Dulbecco磷酸盐缓冲盐水（PBS）中。 </li></ol></li><li>洗涤切片用新的PBS补充有2％antibi耳真菌溶液。 </li><li>在100毫米培养皿地方活检切成用无菌手术刀约2mm 3的片段。 </li><li>除去涂布溶液过量之前立即镀片段。 </li><li>放置5-6皮碎片进入预涂35毫米培养皿。 </li><li>制备成纤维细胞的培养基:77％的Dulbecco改良的Eagle培养基（DMEM）高葡萄糖，20％胎牛血清（FBS），1％L-谷氨酰胺溶液和2％抗生素抗真菌溶液。 </li><li>在37℃下在5％CO 2在每个片段（通常为100微升每片段）和培养它们添加成纤维介质的液滴。 </li><li> 24小时后，加入500微升的成纤维细胞培养基中过度的片段以保持湿润，在37℃在5％CO 2。 </li><li>改变用吸管至少每48小时的培养基。 </li><li>孵化后第6天，小心地取出组织碎片和丢弃它们。 注:6天之后，成纤维细胞先从生长出组织碎片，并开始以形成细胞单层。 </li><li>刷新介质，加2 ml成纤维细胞的培养基，继续细胞单层培养在37℃下在5％CO 2培养箱中。 </li></ol> 2，成纤维细胞培养<ol><li>在37℃下培养的成纤维细胞在5％CO 2，直到80％汇合</li><li>用于传代，从组织培养皿抽吸成纤维细胞的培养基。洗涤细胞三次用4ml的PBS补充有1％抗生素 - 抗真菌溶液。 </li><li>加的胰蛋白酶-EDTA溶液（0.5克/升的猪胰蛋白酶和0.2g / L EDTA）的薄层（培养基体积的10％），在37℃下孵育直至单层细胞开始从组织的底部分离培养皿和细胞分离。 </li><li>稀释细胞悬浮液以9份的成纤维细胞的培养基中和胰蛋白酶作用。离心是没有必要的。 </li><li>板细胞在新的培养皿（用出明胶），在37℃，5％CO 2培养箱培养。保持1之间的通路比:2和1:4，这取决于生长速度。 </li><li>当细胞达到约80％汇合，他们通过（通常每周两次）。 </li></ol> 3.成纤维细胞为电镀表观遗传转化<ol><li>添加0.26毫升/ 0.1％猪的明胶厘米2（在1.1的介绍准备），以细胞培养皿。等待2小时，外套。 </li><li>除去之前电镀的成纤维细胞的涂布溶液过量10-30分钟。 </li><li>从培养皿中取出成纤维细胞培养液中。洗涤细胞三次，用PBS补充有1％抗生素 - 抗真菌溶液。 </li><li>加的胰蛋白酶-EDTA溶液（0.5克/升的猪胰蛋白酶和0.2g / L EDTA）的薄层（培养基体积的10％），在37℃下孵育直至单层细胞开始从组织的底部分离培养皿和细胞分离。 </li><li>稀释细胞悬液，用9份fibrobl的AST培养基中和胰蛋白酶作用。 </li><li>算在室温下在显微镜下使用计数室的细胞。计算成纤维细胞培养基的所需体积重悬细胞，以得到7.8×10 4的成纤维细胞/ cm 2的细胞浓度。这将取决于所使用的特定类型的腔。 细胞/μL=每小格×90（倍频系数）×稀释电池的平均数量</li><li>在150克，在室温下5分钟离心细胞悬浮液。除去上清，重悬细胞与成纤维细胞培养基的先前计算的体积。 </li><li>板单元上0.1％明胶预涂覆菜肴和培养它们在37℃，5％CO 2培养箱24小时。 </li></ol> 4.增加细胞可塑性使用德甲基化剂5-氮杂CR <ol><li> 0天<ol><li>通过在10毫升的DMEM高糖中介溶解2.44毫克5-氮杂CR的制备5-氮杂CR原液嗯。通过过滤消毒。制备5-氮杂CR库存即将使用前。 </li><li>稀释1微升5氮杂CR原液在1 ml成纤维细胞培养基中（终浓度1μM）的。 </li><li>为了增加细胞的可塑性，细胞电镀（子目3.8）24小时后，从接种的成纤维细胞除去培养基，并在37℃，5％CO 2培养箱中添加1μM的5-氮杂CR原液并培养18小时。 </li></ol></li><li> 1天<ol><li>如表1中所述制备的新鲜人多能干（HP）平台。 </li><li>用1μM-5-氮杂 - CR温育后，除去培养基并洗涤细胞三次，用PBS以确保5-氮杂CR被漂洗掉。 </li><li>孵育-5-氮杂- CR处理的成纤维细胞用3小时（恢复期）中于37℃下在5％CO 2的HP介质。 </li><li>恢复期后，除去培养基，用PBS洗涤三次。 </li><li>为了监测5-氮杂CR处理的效率，为您在这个步骤f或形态变化的存在（在结果部分详细说明）。细胞失去成纤维细胞的典型的细长的形态，并获得一个圆形或椭圆形，成为尺寸小，与细胞核增大。 </li><li>胰腺癌的分化继续。 </li></ol></li></ol> 5.胰腺分化协议<ol><li> 1-6天<ol><li>如表2中所述制备胰腺癌基础培养基。 </li><li>准备激活素原液:溶于5微克166.6微升无菌水激活素A的重组人蛋白。 </li><li>培养-5-氮杂- CR在5％CO 2培养箱中处理的成纤维细胞0.26毫升/厘米胰腺癌基础培养基的2，补充有1微升/ ml激活素A原液（见5.1.2）6天，在37℃。改变中的日常。 </li></ol></li><li> 7-8天<ol><li>通过加入16.6毫升二甲基亚砜（DMSO）中的至50mg视黄ACI的制备视黄酸原液ð。 </li><li>在胰腺癌基础培养基0.26毫升/厘米2培养细胞补充有1微升/毫升，在37℃激活素A原液（见5.1.2）和1μl/ ml的视黄酸储备溶液（见5.2.1）2天Ç在5％CO 2。改变中的日常。 </li></ol></li><li> 9-36天<ol><li> 0.26毫升/厘米2胰腺癌基础培养基的培养细胞的补充有1％（体积/体积）胰岛素转铁硒（ITS），2％（V / V）B27和0.1％（体积/体积）重组人FGF基本（bFGF）的储备溶液（ 见表1）在37℃在5％CO 2培养箱中。每天改变介质的第15天。 </li><li>从向前16天，刷新介质隔日，在显微镜下，由于形成聚集体细胞可以从培养皿的底部脱离。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

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...

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&gt;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。 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。

<table><tbody><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma</td><td>D5652</td><td>PBS; for cell wash and solution preparation</td></tr><tr><td>Antibiotic Antimycotic Solution</td><td>Sigma</td><td>A5955</td><td>Component of Fibroblast, HP and Pancreatic media</td></tr><tr><td>100 mm Petri dish</td><td>Sarstedt</td><td>83.3902</td><td>For Fibroblast isolation</td></tr><tr><td>Porcine Gelatin</td><td>Sigma</td><td>G1890</td><td>For dish coating</td></tr><tr><td>Water</td><td>Sigma</td><td>W3500</td><td>For solution preparation</td></tr><tr><td>35 mm Petri dishes</td><td>Sarstedt</td><td>83.39</td><td>For Fibroblast isolation</td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Life Technologies</td><td>41966052</td><td>For Fibroblast culture medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>10500064</td><td>FBS; Component of Fibroblast and HP media</td></tr><tr><td>L-Glutamine solution</td><td>Sigma</td><td>G7513</td><td>Component of Fibroblast, HP and Pancreatic media</td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma</td><td>T3924</td><td>For Fibroblast dissociation</td></tr><tr><td>KOVA GLASSTIC SLIDE 10 WITH GRIDS</td><td>Hycor Biomedical</td><td>87144</td><td>Cell counting</td></tr><tr><td>5-Azacytidine</td><td>Sigma</td><td>A2385</td><td>5-aza-CR, for increrase cell plasticity in fibroblasts</td></tr><tr><td>Ham&#039;s F-10 Nutrient Mix</td><td>Life Technologies</td><td>31550031</td><td>For HP medium</td></tr><tr><td>DMEM, low glucose, pyruvate</td><td>Life Technologies</td><td>31885023</td><td>For HP medium</td></tr><tr><td>KnockOut Serum Replacement</td><td>Life Technologies</td><td>10828028</td><td>Component of HP medium</td></tr><tr><td>MEM Non-Essential Amino Acids Solution</td><td>Life Technologies</td><td>11140035</td><td>Component of HP and Pancreatic Basal media</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M7522</td><td>Component of HP and Pancreatic Basal media</td></tr><tr><td>Guanosine</td><td>Sigma</td><td>G6264</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Adenosine</td><td>Sigma</td><td>A4036</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Cytidine</td><td>Sigma</td><td>C4654</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Uridine</td><td>Sigma</td><td>U3003</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Thymidine</td><td>Sigma</td><td>T1895</td><td>Nucleoside mix stock component of HP medium</td></tr><tr><td>Millex-GS 0,22 &amp;micro;m</td><td>Millipore</td><td>SLGS033SB</td><td>For sterilizing of solution</td></tr><tr><td>FGF-Basic (AA 1-155) Recombinant Human Protein</td><td>Life Technologies</td><td>PHG0261</td><td>bFGF; Component of HP and Pancreatic Basal medium</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A3311</td><td>BSA; Component of Pancreatic Basal medium</td></tr><tr><td>DMEM/F-12</td><td>Life Technologies</td><td>11320074</td><td>For Pancreatic Basal medium</td></tr><tr><td>B-27 Supplement Minus Vitamin A</td><td>Life Technologies</td><td>12587010</td><td>Component of Pancreatic medium</td></tr><tr><td>N-2 Supplement</td><td>Life Technologies</td><td>17502048</td><td>Component of Pancreatic Basal medium</td></tr><tr><td>Activin A Recombinant Human Protein</td><td>Life Technologies</td><td>PHG9014</td><td>For Pancreatic medium</td></tr><tr><td>Retinoic Acid</td><td>Sigma</td><td>R2625</td><td>For Pancreatic medium</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D2650</td><td>DMSO; for Retinoic Acid stock preparation</td></tr><tr><td>Insulin-Transferrin-Selenium</td><td>Life Technologies</td><td>41400045</td><td>ITS; for Pancreatic Final medium</td></tr><tr><td>Anti-Vimentin antibody&amp;nbsp;</td><td>Abcam</td><td>ab8069</td><td>For immunocytochemical analisys. Working dilution 1:100</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride</td><td>Sigma</td><td>32670</td><td>DAPI. For immunocytochemical analisys. Working dilution&amp;nbsp; 1 &amp;micro;g/ml</td></tr><tr><td>5-Methylcytidine</td><td>Eurogentec</td><td>MMS-900P-B</td><td>For immunocytochemical analisys. Working dilution 1:500</td></tr><tr><td>Anti-C Peptide antibody&amp;nbsp;</td><td>Abcam</td><td>ab14181</td><td>For immunocytochemical analisys. Working dilution 1:100</td></tr><tr><td>Anti-PDX1 antibody&amp;nbsp;</td><td>Abcam</td><td>ab47267</td><td>For immunocytochemical analisys. Working dilution 1:500</td></tr><tr><td>Mercodia Insulin ELISA</td><td>Mercodia</td><td>10-1113-10</td><td>For insulin release detection</td></tr></tbody></table>

epigenetic conversion as a safe and simple method to obtain insulin-secreting cells from adult skin fibroblasts

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.

从皮肤活检原代培养的建立 皮肤切片切成小碎片，并放置在明胶预涂覆的菜肴。 6天后，成纤维细胞开始生长出组织碎片和形成的细胞单层（ 图1A）。细胞显示典型的细长的形状，正如预期的那样，显示的均匀免疫阳性的成纤维细胞特异性标记波形蛋白（Vim的， 图1B）。 形态学变化和甲基化皮肤?...

Watch this Scientific Journal Video about Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts at JoVE.com

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

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.

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

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

epigenetic-conversion-as-safe-simple-method-to-obtain-insulin

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

6.7K 次观看.  Università degli Studi di Milano. 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.

视频: Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

科研

发育生物学

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

医学

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

将小鼠成纤维细胞直接重编程为黑素细胞

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

神经科学

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

人类多能干细胞分化为产生胰岛素的胰岛簇

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

生物工程

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

使用从纤维细胞直接转换为神经元祖先细胞和分化为星形细胞的神经系统疾病体外建模

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

结合表观遗传修饰和生物力学线索的两步策略，以产生哺乳动物多能细胞

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

生物学

使用皮肤活检生成患者来源的足细胞

Generation of Patient-Derived Podocytes from Skin Biopsies

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes.&#160;Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients&#39; mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.

作者聚焦：从皮肤活检中生成患者来源的足细胞  

This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes.

从皮肤活检中生成患者来源的足细胞

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the ...