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，可逆允许状态的合成后生改性剂。特别是，5-氮杂CR以便引起在皮肤成纤维细胞的全球甲基减少使用。 5-氮杂CR被称为直接抑制甲基转移酶活性，并防止从头甲基化在新合成的DNA。由于它的作用，这种分子以前已经用来激活沉默的基因，以及修改的真核细胞15,16的分化状态。与此相一致，张贴5-氮杂CR皮肤成纤维细胞表现出全球DNA去甲基化（ 图2A），表明以增加在用于本实验的细胞可塑性-5-氮杂-CR能力。这也是在与5-氮杂CR便于在神经球细胞（NSCs）17的高可塑性相关标记物Oct-4的表达的观察一致。然而，有趣的是要注意，交5-氮杂CR皮肤成纤维细胞恢复到它们的原始表型除去-5-氮杂 - CR之后。事实上，我们以前表明，成纤维细胞恢复到原来的培养基，对多能性相关的因素-9,10-向下调节表达，这表明更高的塑性状态获得的，响应于后生改性剂，是短暂的，可逆的和不涉及的t永久性修改他的细胞。 细胞形态发生明显变化伴随着较高的塑性状态（ 图2A）的诱导。未处理的成纤维细胞的典型的细长细胞体是由圆形或椭圆形的细胞呈现更小的尺寸和增加的核体积，从而出现比分化的细胞的较大的置换。丹羽此相关的核增大被描述为一个多能性相关的功能18轻松的染色质结构。空泡核和颗粒状胞质，以及增加的扁平化的形态存在，是显而易见的。描述可实际用作用于转换协议的第一部分的成功完成的标记的所有形态学变化;根据我们的经验，当变化都存在，5-氮杂CR确实帮助细胞获得一种更加宽容的状态。 有可能利用这个短暂的"高PERMIS的优点sivity时间窗口"来驱动细胞向一个完全不同的表型。本协议表明交5-氮杂CR的成纤维细胞可以被重新寻址到胰腺β细胞样细胞，在响应于特定分化培养基。使用的协议允许细胞从胚层的细胞类型切换到属于所述内胚层谱系的细胞群。 胰岛素，这原本在未处理皮肤成纤维细胞检测不到，是在分化方案（ 图3B）的端部正面。这是伴随着的其它因素，如PDX1，参与整个胰腺的分化同时表达 - 其外分泌，内分泌和导管细胞群，特定的β-细胞谱系的旁边。这是与小鼠胚胎干细胞，指示一个适当的分化成β细胞达到仅当未成熟细胞与水净化分层以前的工作（ESC）相一致- [R内分泌细胞19，这表明由朗格汉斯架构的胰岛提供的局部微环境具有很强的功能性作用。 的确，EpiCC达到成熟分化表型并呈现给至20mM葡萄糖曝光（ 图3）的反应能力。胰岛素积极分泌在培养基中后D-葡萄糖刺激1小时后，在确认EpiCC的善意作为产生胰岛素的那些（ 图3C）。 对于一个成功的过程的不同的要求是细胞在37℃的严格的维护，在所有的步骤，包括无菌的层流和在显微镜下它们的处理。根据我们的经验，它也强烈建议新鲜制备的试剂，其在培养使用之前和中等严格刷新按照协议。该操作必须在显微镜下，因为形成聚集体的细胞中进行可以从培养皿的底部分离，并且在介质变化丢失。 后生转换协议也已成功地应用于猪物种以及对狗（手稿提交），使用用于人描述的相同的细胞数/厘米2和脱甲基化剂的浓度。 最后，一​​个协议，允许的皮肤成纤维细胞转化为另一种细胞类型这里提出。所描述的战略有一个表观遗传学为基础的电池转换的优势:即避免多能状态，有可能使可致癌细胞背后的可能性;消除可能导致细胞中的变化缠绵外源性遗传因素。这些优点使本方法的转化医学的应用非常有前途，并允许患者特定的细胞治疗。

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 border="0" cellpadding="0" cellspacing="0" width="1019">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">D5652</td>
			<td style="width:408px;">PBS; for cell wash and solution preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Antibiotic Antimycotic Solution</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">A5955</td>
			<td style="width:408px;">Component of Fibroblast, HP and Pancreatic media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">100 mm petri dish</td>
			<td style="width:155px;">Sarstedt</td>
			<td style="width:119px;">83.3902</td>
			<td style="width:408px;">For Fibroblast isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Porcine Gelatin</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">G1890</td>
			<td style="width:408px;">For dish coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Water</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">W3500</td>
			<td style="width:408px;">For solution preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">35 mm petri dishes</td>
			<td style="width:155px;">Sarstedt</td>
			<td style="width:119px;">83.39</td>
			<td style="width:408px;">For Fibroblast isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMEM, high glucose, pyruvate</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">41966052</td>
			<td style="width:408px;">For Fibroblast culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Fetal Bovine Serum</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">10500064</td>
			<td style="width:408px;">FBS; Component of Fibroblast and HP media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">L-Glutamine solution</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">G7513</td>
			<td style="width:408px;">Component of Fibroblast, HP and Pancreatic media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Trypsin-EDTA solution</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">T3924</td>
			<td style="width:408px;">For Fibroblast dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">KOVA GLASSTIC SLIDE 10 WITH GRIDS</td>
			<td style="width:155px;">Hycor Biomedical</td>
			<td style="width:119px;">87144</td>
			<td style="width:408px;">Cell counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">5-Azacytidine</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">A2385</td>
			<td style="width:408px;">5-aza-CR, for increrase cell plasticity in fibroblasts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Ham&#39;s F-10 Nutrient Mix</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">31550031</td>
			<td style="width:408px;">For HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMEM, low glucose, pyruvate</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">31885023</td>
			<td style="width:408px;">For HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">KnockOut Serum Replacement</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">10828028</td>
			<td style="width:408px;">Component of HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">MEM Non-Essential Amino Acids Solution</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">11140035</td>
			<td style="width:408px;">Component of HP and Pancreatic Basal media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">2-Mercaptoethanol</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">M7522</td>
			<td style="width:408px;">Component of HP and Pancreatic Basal media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Guanosine</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">G6264</td>
			<td style="width:408px;">Nucleoside mix stock component of HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Adenosine</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">A4036</td>
			<td style="width:408px;">Nucleoside mix stock component of HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Cytidine</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">C4654</td>
			<td style="width:408px;">Nucleoside mix stock component of HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Uridine</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">U3003</td>
			<td style="width:408px;">Nucleoside mix stock component of HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Thymidine</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">T1895</td>
			<td style="width:408px;">Nucleoside mix stock component of HP medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Millex-GS 0,22 &#181;m</td>
			<td style="width:155px;">Millipore</td>
			<td style="width:119px;">SLGS033SB</td>
			<td style="width:408px;">For sterilizing of solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">FGF-Basic (AA 1-155) Recombinant Human Protein</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">PHG0261</td>
			<td style="width:408px;">bFGF; Component of HP and Pancreatic Basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Bovine Serum Albumin</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">A3311</td>
			<td style="width:408px;">BSA; Component of Pancreatic Basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMEM/F-12</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">11320074</td>
			<td style="width:408px;">For Pancreatic Basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">B-27 Supplement Minus Vitamin A</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">12587010</td>
			<td style="width:408px;">Component of Pancreatic medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">N-2 Supplement</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">17502048</td>
			<td style="width:408px;">Component of Pancreatic Basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Activin A Recombinant Human Protein</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">PHG9014</td>
			<td style="width:408px;">For Pancreatic medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Retinoic Acid</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">R2625</td>
			<td style="width:408px;">For Pancreatic medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Dimethyl sulfoxide</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">D2650</td>
			<td style="width:408px;">DMSO; for Retinoic Acid stock preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Insulin-Transferrin-Selenium</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">41400045</td>
			<td style="width:408px;">ITS; for Pancreatic Final medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Anti-Vimentin antibody&#160;</td>
			<td style="width:155px;">Abcam</td>
			<td style="width:119px;">ab8069</td>
			<td>For immunocytochemical analisys. Working dilution 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">4&#8242;,6-Diamidino-2-phenylindole dihydrochloride</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">32670</td>
			<td>DAPI. For immunocytochemical analisys. Working dilution&#160; 1&#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">5-Methylcytidine</td>
			<td style="width:155px;">Eurogentec</td>
			<td style="width:119px;">MMS-900P-B</td>
			<td>For immunocytochemical analisys. Working dilution 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Anti-C Peptide antibody&#160;</td>
			<td style="width:155px;">Abcam</td>
			<td style="width:119px;">ab14181</td>
			<td>For immunocytochemical analisys. Working dilution 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Anti-PDX1 antibody&#160;</td>
			<td style="width:155px;">Abcam</td>
			<td style="width:119px;">ab47267</td>
			<td>For immunocytochemical analisys. Working dilution 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Mercodia Insulin ELISA</td>
			<td style="width:155px;">Mercodia</td>
			<td style="width:119px;">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成纤维细胞的图案之后18小时暴露于5-氮杂CR 以获得的成纤维细胞的一个成功的后生转化成胰岛素分泌细胞，我们使用脱甲基化剂，5-氮杂CR增加了它们的可塑性。人成纤维细胞以7.8×10 4的成纤维细胞/ cm 2的浓度接种于0.1％明胶预涂覆的菜肴。播种后二十四小时，将细胞瓦特ERE用1微米-5-氮杂 - CR 18小时温育。 在这个处理结束时，广泛的细胞表型的变化是可见的（邮5-氮杂CR， 图2A）。未处理的成纤维细胞（T0， 图2A）的典型的细长的形态，是由一圆形或椭圆形替代和单元尺寸变得相当小。细胞质是粒状，平坦化，细胞粘附于培养物表面。细胞核出现更大和空泡，作为宽松的染色质结构的结果。在我们的经验，这些形态变化的存在是必不可少的，必须仔细监控作为-5-氮杂 - CR处理的效率的指标。 后18小时暴露于5-氮杂CR，全球DNA甲基化的急剧下降也很明显，并通过5-甲基胞苷免疫染色的减弱强度清楚地证实（ <strong&gt;图2B）。 皮肤的后生转换过程中形态和功能改变成纤维细胞为胰岛素分泌细胞 为了诱导胰腺分化，5-氮杂CR处理的成纤维细胞暴露于胰腺感应一个三步协议中，3小时的恢复期之后。 在第一步骤中，细胞在7天在胰腺癌基础培养基补充有活化素A促进定形内胚层的承诺进行培养。在此间隔中，进一步展平以及逐渐细胞开始簇（第7天， 图3A）来组织。接着，胰腺谱系分化通过加入视黄酸的2天促进。响应于该处理，将细胞重新排列在一个网状图案和增长中清楚区分细胞聚集体（第10天， 图3A）。聚类形成process增加与时间和由第三和最后的步骤，其由细胞暴露于B27，bFGF和及其进一步的刺激。这导致了越来越多的细胞，在大型3D殖民地汇总（20日， 图3A）的招聘。大约36一天，这些殖民地如出现明显的圆形结构让人想起在体外典型的胰岛（36日， 图3A）的。 新EpiCC型的收购是伴随着回到了那些未经处理的成纤维细胞（5三菱商事36日图3B）所观察到的全球DNA甲基化水平的逐步提高。 后胰诱导36天，后生转换效率也被典型成熟胰腺标志物的表达，这是原本在未处理的成纤维细胞检测不到（T0证明， <stroNG&gt;图3B）。 C肽（C-PEP）与胰腺和十二指肠同源1（PDX1）的共定位证实EpiCC的善意作为产生胰岛素的那些（36日， 图3B）。此外，转换单元的功能表型可以通过向至20mM葡萄糖曝光，它代表了生理触发化合物反应的能力证实。更详细地说，EpiCC积极后D-葡萄糖刺激1小时分泌在培养基中的胰岛素。没有释放是暴露到L-葡萄糖等摩尔量的（ 图3C）后检测的。 <img alt="图1" src="/files/ftp_upload/53880/53880fig1.jpg" /> 图1:隔离和人体皮肤成纤维细胞的特性 （A）成纤维细胞生长出来的组织碎片的形象代表。 （B）的成纤维细胞显示均匀免疫阳性为其小号pecific标记波形蛋白（VIM）。细胞核用DAPI。 （比例尺，100微米）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53880/53880fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53880/53880fig2.jpg" /> 图2:人皮肤成纤维细胞的形态和甲基化的变化后-5-氮杂- CR处理 （A）未处理的成纤维细胞显示出细长形状（T 0）的代表性图像，以及5-氮杂CR处理的成纤维细胞显示圆形或椭圆形的形态，造粒。细胞质和放大和空泡核（邮5-氮杂CR）。 （比例尺，100微米）。 （B）的全球DNA甲基化的减少是可检测的5-氮杂CR处理后（后5-氮杂CR）。 （比例尺，50微米）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53880/53880fig2large.jpg" target="_blank">请CL</a>ICK这里查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53880/53880fig3.jpg" /> 图 3: 后生在转换过程中的形态和功能的变化 （A）的胰腺内分泌分化过程中发生的形态变化代表的照片。经过7天诱导，人类细胞逐渐簇（7天）组织。响应于加入视黄酸，它们重新排列在可区分的聚集体（10天）一网状图案和集群。这些编队与时代的进步，招募细胞和大型3D殖民地聚集（20日）。最后，集落变得倾向于分离并在培养基中，让人想起体外典型胰岛（36天）的自由浮动的球形结构。 （比例尺，100微米）。 （B）后在胰腺癌36天duction，人类EpiCC回报那些未经处理的成纤维细胞（5三菱商事36天），观测到的全球DNA甲基化水平。 （比例尺，50微米）。用C-PEP PDX1的共定位是在转换期间（36天）结束时检测的，而这些内分泌胰腺标志物是在未处理的成纤维细胞（T 0）完全不存在。 （比例尺，100微米）。 （℃）EpiCC胰岛素释放响应于20mM的D-葡萄糖和20mM的L-葡萄糖曝光。短线代表平均值±三个独立重复的SD。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53880/53880fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 材料/设备名称 </td><td> 量（体积/体积） </td><td> 评论/说明 </td></tr><tr><td>联队的F-10混合营养</td><td> 40％ </td><td></td></tr><tr><td> DMEM低血糖</td><td> 40％ </td><td></td></tr><tr><td>淘汰赛血清替代</td><td> 10％ </td><td></td></tr><tr><td> FBS </td><td> 5％ </td><td></td></tr><tr><td>抗生素抗真菌的解决方案</td><td> 1％ </td><td></td></tr><tr><td> L-谷氨酰胺溶液</td><td> 1％ </td><td></td></tr><tr><td> MEM非必需氨基酸溶液</td><td> 1％ </td><td></td></tr><tr><td> 2-巯基乙醇的库存</td><td> 1％ </td><td> 2-巯基乙醇备料:diluite 3.5微升2-巯基乙醇的5毫升无菌PBS中。避光在+4℃。注:2个星期内使用</td></tr><tr><td>核苷组合股票</td><td> 1％ </td><td>核苷混合备料:溶解0.042克鸟苷，0.040克腺苷，0.036克胞苷，0.036克尿苷，用50ml无菌水0.012克Timidine。熔化，在50℃以溶解。通过filtrat消毒离子并储存在+4℃， </td></tr><tr><td> ESGRO（LIF） </td><td> 0.1％ </td><td></td></tr><tr><td>重组人碱性FGF（bFGF）的股票</td><td> 0.1％ </td><td> bFGF的备料:加入5毫升0.1％牛血清白蛋白（BSA）的PBS在25微克的bFGF </td></tr></tbody></table> 表1:材料的解决方案。 <table fo:keep-together.within-page="1"><tbody><tr><td> 材料/设备名称 </td><td> 量（体积/体积） </td><td> 评论/说明 </td></tr><tr><td> DMEM / F-12 </td><td> 93％ </td><td></td></tr><tr><td> B-27负补充维生素A </td><td> 2％ </td><td></td></tr><tr><td> N-2补充</td><td> 1％ </td><td></td></tr><tr><td> MEM非必需氨基酸溶液</td><td> 1％ </td><td></td></tr><tr><td>抗生素抗真菌的解决方案</td><td> 1％ </td><td></td></tr><tr><td> 2-巯基乙醇的库存</td><td> 1％ </td><td> 2-巯基乙醇备料:diluite 3.5微升2-巯基乙醇的5毫升无菌PBS中。避光在+4℃。注:2个星期内使用</td></tr><tr><td> L-谷氨酰胺溶液</td><td> 1％ </td><td></td></tr><tr><td> BSA股票</td><td> 1％ </td><td> BSA股票的制备:溶解250毫克50毫升水。通过过滤和储存在+4℃下灭菌。 </td></tr></tbody></table> 表2:材料解决方案。

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

Nanyang Technological University

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

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

转染，选择和人类诱导多能干细胞集落采摘TALEN靶向用GFP基因进入AAVS1安全港

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

科研

发育生物学

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

一个有效的方法获取反分化脂肪细胞

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

协议小鼠胚胎成纤维细胞直接转化为滋养层干细胞

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid cooling in an &#34;open&#34; system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable &#181;S-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-&#181;m ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (&#181;S-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified &#181;S-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the &#181;S-VTF procedure has not affected its successful application, as high survival rates (&#62; 95%) and pregnancy rates continue today.

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

修改MicroSecure玻璃化:一种安全，简便，高效的冷冻程序人胚泡

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid ...

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

从非转化人成纤维细胞增生四倍体细胞的建立

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

成人皮肤角质形成细胞的生成与培养

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

一种简便有效的成人皮肤组织中人角质形成细胞的分离方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

成年小鼠数字截肢和再生:研究哺乳动物暴发体形成和内膜性细胞化的简单模型

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...