这项工作由Carraresi基金会和MiND FoodS Hub ID:1176436资助。所有作者都是COST Action CA16119体外3-D全细胞指导和健身（CellFit）的成员。

所有研究均由米兰大学伦理委员会审查和批准。所有动物实验均按照美国国立卫生研究院（NIH）发布的《实验动物护理和使用指南》进行。从健康的成年个体中分离人类细胞已获得米兰Ospedale Maggiore Policlinico伦理委员会的批准。我们研究中的所有方法都是根据批准的指南进行的。
1. 皮肤成纤维细胞分离
注意:下面描述的所有程序都可以应用于从不同哺乳动物物种（包括小鼠，猪和人类）中分离的成纤维细胞。从7周龄的雄性小鼠中分离出鼠细胞，并在当地屠宰场收集猪皮组织。在书面知情同意后，从成年患者中分离出人类细胞。
<ol><li>准备0.1%猪明胶溶液。<ol>
<li>称取0.1克猪明胶，将其溶解在100毫升水中。使用前通过高压灭菌对明胶溶液进行灭菌。</li>
		<li>通过加入1.5mL制备的溶液，用0.1%猪明胶涂覆35mm培养皿。在室温下孵育2小时。</li>
	</ol>
</li>
	<li>切下长约2-5厘米的哺乳动物（小鼠，猪和人类）皮肤活检，并将其置于含有2%抗生素抗真菌溶液的Dulbecco磷酸盐缓冲盐水（PBS）中。储存在+ 4°C直至使用。 注意:活检收集必须在同意的情况下进行，并在伦理委员会批准后，根据既定指南进行。</li>
	<li>在含有2%抗生素抗真菌剂溶液的新鲜无菌PBS中广泛洗涤收集的活检液三次。</li>
	<li>收集最后一次洗涤的活检，并将其放入无菌的100毫米培养皿中。使用无菌手术刀将它们切成约2毫米3 大小的碎片。</li>
	<li>在2小时孵育结束时，从35mm培养皿中除去多余的明胶溶液（在步骤1.1.2中描述），并使用无菌手术镊子立即将5-6个皮肤碎片放入每个预涂的培养皿中。</li>
	<li>通过在每个片段上加入100μL成纤维细胞分离培养基液滴（表1）来润湿片段。在37°C下在5%CO2 培养箱中培养。 注意:为防止培养基蒸发，请将35 mm培养皿置于含有无菌水的100 mm或更大的培养皿中。确保盖上两个培养皿。</li>
	<li>培养24小时后，检查35mm培养皿中培养基的量。如果需要，加入500μL成纤维细胞分离培养基以保持碎片湿润。</li>
	<li>小心地取出培养基，并使用移液器至少每2天培养一次。</li>
	<li>当成纤维细胞开始从放置在35mm培养皿（在步骤1.5中描述）的皮肤碎片中生长出来并开始形成细胞单层（通常为6天）时。使用无菌手术镊子去除皮片，并在2 mL成纤维细胞分离培养基中培养。</li>
	<li>继续在37°C下在5%CO2 培养箱中培养细胞单层，直到80%汇合，每隔一天刷新培养基。</li>
</ol>2. 成纤维细胞原代细胞系培养
<ol><li>当成纤维细胞达到80%汇合时，小心地取出成纤维细胞分离培养基，并用含有1%抗生素抗真菌溶液的3mLPBS洗涤细胞三次。</li>
	<li>对于细胞分离，在培养皿中加入600μL0.25%胰蛋白酶- EDTA溶液，并在37°C下孵育3-5分钟。</li>
	<li>当细胞开始从培养皿中分离时，加入5.4mL成纤维细胞培养基以中和胰蛋白酶（表1）。</li>
	<li>通过重复和温和的移液去除细胞。在新培养皿（不含明胶）中培养细胞，将传代比保持在1:2和1:4之间（取决于生长速率）。 注:无需离心。</li>
	<li>将细胞保持在培养物中，每2天更换一次培养基，直到它们达到80%汇合并传代。 注意:每周繁殖两次成纤维细胞，以保持剧烈生长。</li>
</ol>3. 成纤维细胞暴露于5-氮杂-CR
<ol><li>准备新鲜的1 mM 5-氮杂-CR储备溶液。<ol>
<li>称取2.44毫克5-氮杂-CR并将其溶解在10毫升DMEM高血糖中。通过涡旋重悬粉末。用0.22μm过滤器对溶液进行灭菌。 注意:5-氮杂-CR储备溶液必须在使用前立即制备。</li>
		<li>通过在1mL成纤维细胞培养基中稀释1μL5-氮杂-CR储备溶液（3.1.1.）来制备5-氮杂-CR工作溶液。 注:5-氮杂-CR工作溶液的浓度为1μM1，2，3，8，9。</li>
	</ol>
</li>
	<li>胰蛋白酶消化细胞如前所述（2.1.-2.3.）并通过重复和轻柔的移液来移出细胞。</li>
	<li>收集细胞悬浮液并将其转移到锥形管中。</li>
	<li>在室温下，在光学显微镜下使用计数室对细胞进行计数。计算重新悬浮细胞所需的培养基体积，以在补充有1μM 5-氮杂-CR的30μL成纤维细胞培养基中获得4×104 细胞（参见步骤3.1.2）。 注意:要使用的配方取决于腔室的具体类型。 <img alt="Equation 1" src="/files/ftp_upload/61655/61655equ01v3.jpg">
</li>
	<li>在室温下以150 ×g 离心细胞悬浮液5分钟。除去上清液并用补充有1μM 5-氮杂-CR的成纤维细胞培养基重悬沉淀（参见步骤3.1.2）。对于要使用的成纤维细胞培养基的体积，请参见步骤3.4。 注意:作为阴性对照，将细胞重悬于不含5-氮杂-CR的成纤维细胞培养基中相同浓度，并在PTFE粉末中进行细胞包封（步骤4.1.-4.13）。</li>
</ol>4. PTFE微生物反应器中的成纤维细胞封装
<ol><li>用聚四氟乙烯（PTFE）粉末填充35毫米培养皿以产生床（图1A）。 注意:使用35毫米细菌培养皿，以避免液体大理石粘附。为了获得薄的疏水性和多孔外壳，使用平均粒径为1μm的PTFE粉末，并以最大研磨量为2.0 NPIRI生产。这允许创建透气的液体大理石。此外，半透明涂层有助于实时观察细胞聚集过程 较大的粒径导致高多分散性，从而导致蒸发升高，变形和球形损失，以及微生物反应器的过早溶解。</li>
	<li>将含有4 x 104个细胞 的30μL单滴（参见步骤3.4.- 3.5.）分配到粉末床上（图1B）。</li>
	<li>以圆周运动轻轻旋转35毫米培养皿，以确保PFTE粉末完全覆盖液滴表面，形成液体大理石微生物反应器（图1C）。</li>
	<li>使用1，000μL移液器吸头拾取液体大理石微生物反应器，在边缘切割，以适应大理石的直径（图1D，E）。将液体大理石微生物反应器板到干净的细菌培养皿上以稳定其（图1F）。 注意:要产生摩擦力以抓住吸头内的大理石，请切割直径约略小于液体大理石直径的移液器吸头。</li>
	<li>将液体大理石微生物反应器从培养皿转移到96孔板（一个大理石/孔）中（图1G）。</li>
	<li>从孔的边缘慢慢加入100μL培养基。微生物反应器开始漂浮在培养基顶部（图1H）。 注:由于PTFE疏水性的破坏，微生物反应器在直接液体接触时破裂。作为替代方法，液体大理石微生物反应器可以单独放置在35mm细菌培养皿中。在这种情况下，为了防止液体大理石蒸发，必须将含有微生物反应器的35毫米培养皿插入100毫米培养皿中，事先用无菌水等分</li>
	<li>将液体大理石微生物反应器在37°C下在5%CO2培养箱中孵育18小时，2，3，8，9。 注意:1μm的PTFE粒径可以确保内部液体与周围环境之间的最佳气体交换。</li>
	<li>在5-aza-CR孵育18小时后，使用在边缘切割的1，000μL移液器尖端收集液体大理石微生物反应器（参见步骤4.5）。</li>
	<li>将微生物反应器置于新的35mm细菌培养皿中（图1D-F）。</li>
	<li>用针刺穿液体大理石并打破它。</li>
	<li>在立体显微镜下，用200μL移液器尖端回收形成的球体，在边缘切割（图1I，J）。 注意:包裹在PTFE中的表观遗传擦除的细胞形成3D球形结构（每个液体大理石中都有一个聚集体）。</li>
	<li>为了评估对5-aza-CR响应的多能态的获得，通过定性PCR检查多能性相关基因表达OCT4，NANOG，REX1和SOX2的发作（表2）。</li>
	<li>继续执行协议的第二步，如下所述。</li>
</ol>5. 在表观遗传学擦除细胞的PTFE微生物反应器中培养
<ol><li>准备新鲜的ESC培养基（表1）。</li>
	<li>在含有ESC培养基的培养皿中转移类器官以洗涤5-氮杂-CR残留物（参见步骤5.1.-5.2）。</li>
	<li>准备含有PTFE粉末床的新的35毫米细菌培养皿（另见步骤4.1.</li>
	<li>使用200μL移液器吸头将单个类器官在30μL ESC培养基的液滴中分配到粉末床上，在边缘切割（参见步骤4.9）。5.3.).</li>
	<li>以圆周运动轻轻旋转35mm培养皿以形成新的液体大理石微生物反应器，使用1，000μL移液器尖端拾取它，在边缘切割，并将新形成的微生物反应器放入96孔板（一个大理石/孔）的孔中（参见步骤4.3.-4.6）。</li>
	<li>要漂浮微生物反应器，从孔的边缘加入100μL培养基以缓慢沐浴大理石（见注4.7）。</li>
	<li>将液体大理石微生物反应器在37°C下在5%CO2 培养箱中培养所需的时间。按照5.3.-5.7中描述的步骤，每隔一天更换一次培养基。 注意:在本手稿中，提供了28天的类器官培养获得的结果。但是，如果需要，可以进行更长的培养期。</li>
</ol>

<ol>
	<li>Pennarossa, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+demethylation+step+allows+the+conversion+of+adult+human+skin+fibroblasts+into+insulin-secreting+cells.">Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (22), 8948-8953 (2013).</li><li>Pennarossa, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+pig+dermal+fibroblast+into+insulin+secreting+cells+by+a+brief+exposure+to+5-aza-cytidine.">Reprogramming of pig dermal fibroblast into insulin secreting cells by a brief exposure to 5-aza-cytidine.</a> Stem Cell Reviews and Reports. 10 (1), 31-43 (2014).</li><li>Brevini, T. A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+molecular+changes+of+human+granulosa+cells+exposed+to+5-azacytidine+and+addressed+toward+muscular+differentiation.">Morphological and molecular changes of human granulosa cells exposed to 5-azacytidine and addressed toward muscular differentiation.</a> Stem Cell Reviews and Reports. 10 (5), 633-642 (2014).</li><li>Mirakhori, F., Zeynali, B., Kiani, S., Baharvand, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+azacytidine+step+allows+the+conversion+of+suspension+human+fibroblasts+into+neural+progenitor-like+cells.">Brief azacytidine step allows the conversion of suspension human fibroblasts into neural progenitor-like cells.</a> Cell Journal. 17 (1), 153-158 (2015).</li><li>Tan, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+tissue+engineering+and+regeneration+through+epigenetic+reprogramming+and+scaffold+manipulation.">Muscle tissue engineering and regeneration through epigenetic reprogramming and scaffold manipulation.</a> Scientific Reports. 5, 16333 (2015).</li><li>Brevini, T. A. L., Pennarossa, G., Acocella, F., Brizzola, S., Zenobi, A., Gandolfi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+conversion+of+adult+dog+skin+fibroblasts+into+insulin-secreting+cells.">Epigenetic conversion of adult dog skin fibroblasts into insulin-secreting cells.</a> Veterinary Journal. 211, 52-56 (2016).</li><li>Chandrakanthan, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDGF-AB+and+5-Azacytidine+induce+conversion+of+somatic+cells+into+tissue-regenerative+multipotent+stem+cells.">PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (16), 2306-2315 (2016).</li><li>Manzoni, E. F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-azacytidine+affects+TET2+and+histone+transcription+and+reshapes+morphology+of+human+skin+fibroblasts.">5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts.</a> Scientific Reports. 6, 37017 (2016).</li><li>Pennarossa, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+erasing+and+pancreatic+differentiation+of+dermal+fibroblasts+into+insulin-producing+cells+are+boosted+by+the+use+of+low-stiffness+substrate.">Epigenetic erasing and pancreatic differentiation of dermal fibroblasts into insulin-producing cells are boosted by the use of low-stiffness substrate.</a> Stem Cell reviews and reports. 14 (3), 398-411 (2018).</li><li>Pennarossa, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+PTFE+micro-bioreactor+to+promote+3D+cell+rearrangement+and+maintain+high+plasticity+in+epigenetically+erased+fibroblasts.">Use of a PTFE micro-bioreactor to promote 3D cell rearrangement and maintain high plasticity in epigenetically erased fibroblasts.</a> Stem Cell Reviews and Reports. 15 (1), 82-92 (2019).</li><li>Constantinides, P. G., Jones, P. A., Gevers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+striated+muscle+cells+from+non-myoblast+precursors+following+5-azacytidine+treatment.">Functional striated muscle cells from non-myoblast precursors following 5-azacytidine treatment.</a> Nature. 267 (5609), 364-366 (1977).</li><li>Taylor, S. M., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+new+phenotypes+induced+in+10T1/2+and+3T3+cells+treated+with+5-azacytidine.">Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine.</a> Cell. 17 (4), 771-779 (1979).</li><li>Taylor, S. M., Constantinides, P. A., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Azacytidine,+DNA+methylation,+and+differentiation.">5-Azacytidine, DNA methylation, and differentiation.</a> Current Topics in Microbiology and Immunology. 108, 115-127 (1984).</li><li>Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+5-azacytidine+and+its+2'-deoxyderivative+on+cell+differentiation+and+DNA+methylation.">Effects of 5-azacytidine and its 2'-deoxyderivative on cell differentiation and DNA methylation.</a> Pharmacology &amp; Therapeutics. 28 (1), 17-27 (1985).</li><li>Palii, S. S., Van Emburgh, B. O., Sankpal, U. T., Brown, K. D., Robertson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+inhibitor+5-Aza-2'-deoxycytidine+induces+reversible+genome-wide+DNA+damage+that+is+distinctly+influenced+by+DNA+methyltransferases+1+and+3B.">DNA methylation inhibitor 5-Aza-2'-deoxycytidine induces reversible genome-wide DNA damage that is distinctly influenced by DNA methyltransferases 1 and 3B.</a> Molecular and Cellular Biology. 28 (2), 752-771 (2008).</li><li>Vining, K. H., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+forces+direct+stem+cell+behaviour+in+development+and+regeneration.">Mechanical forces direct stem cell behaviour in development and regeneration.</a> Nature Reviews. Molecular Cell Biology. 18 (12), 728 (2017).</li><li>Matamoro-Vidal, A., Levayer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+influences+of+mechanical+forces+on+cell+competition.">Multiple influences of mechanical forces on cell competition.</a> Current Biology. 29 (15), 762-774 (2019).</li><li>Yim, E. K., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-dependent+cell+signaling+in+stem+cell+differentiation.">Force-dependent cell signaling in stem cell differentiation.</a> Stem Cell Research &amp; Therapy. 3 (5), 41 (2012).</li><li>Kumar, A., Placone, J. K., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+extracellular+forces+that+determine+cell+fate+and+maintenance.">Understanding the extracellular forces that determine cell fate and maintenance.</a> Development. 144 (23), 4261-4270 (2017).</li><li>Bissell, M. J., Rizki, A., Mian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture:+the+ultimate+regulator+of+breast+epithelial+function.">Tissue architecture: the ultimate regulator of breast epithelial function.</a> Current Opinion in Cell Biology. 15 (6), 753-762 (2003).</li><li>Streuli, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+mediates+tissue-specific+gene+expression+in+mammary+epithelia.">Laminin mediates tissue-specific gene expression in mammary epithelia.</a> The Journal of Cell Biology. 129 (3), 591-603 (1995).</li><li>Christman, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Azacytidine+and+5-aza-2[prime]-deoxycytidine+as+inhibitors+of+DNA+methylation:+mechanistic+studies+and+their+implications+for+cancer+therapy.">5-Azacytidine and 5-aza-2[prime]-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy.</a> Oncogene. 21, 5483-5495 (2002).</li><li>Stresemann, C., Lyko, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modes+of+action+of+the+DNA+methyltransferase+inhibitors+azacytidine+and+decitabine.">Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine.</a> International Journal of Cancer. 123 (1), 8-13 (2008).</li><li>Ledda, S., Idda, A., Kelly, J., Ariu, F., Bogliolo, L., Bebbere, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+in+vitro+maturation+of+sheep+oocytes+in+a+liquid+marble+microbioreactor.">A novel technique for in vitro maturation of sheep oocytes in a liquid marble microbioreactor.</a> Journal of Assisted Reproduction and Genetics. 33 (4), 513-518 (2016).</li><li>Vadivelu, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+three-dimensional+multiple+spheroid+model+of+olfactory+ensheathing+cells+using+floating+liquid+marbles.">Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles.</a> Scientific Reports. 5, 15083 (2015).</li><li>Vadivelu, R. K., Kamble, H., Munaz, A., Nguyen, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Marble+as+bioreactor+for+engineering+three-dimensional+toroid+tissues.">Liquid Marble as bioreactor for engineering three-dimensional toroid tissues.</a> Scientific Reports. 7 (1), 12388 (2017).</li><li>Vadivelu, R. K., Kamble, H., Munaz, A., Nguyen, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Marbles+as+bioreactors+for+the+study+of+three-dimensional+cell+interactions.">Liquid Marbles as bioreactors for the study of three-dimensional cell interactions.</a> Biomedical Microdevices. 19 (2), 31 (2017).</li><li>Varelas, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAZ+controls+Smad+nucleocytoplasmic+shuttling+and+regulates+human+embryonic+stem-cell+self-renewal.">TAZ controls Smad nucleocytoplasmic shuttling and regulates human embryonic stem-cell self-renewal.</a> Nature Cell Biology. 10 (7), 837-848 (2008).</li><li>Panciera, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Expandable+Tissue-Specific+Stem/Progenitor+Cells+through+Transient+Expression+of+YAP/TAZ.">Induction of Expandable Tissue-Specific Stem/Progenitor Cells through Transient Expression of YAP/TAZ.</a> Cell Stem Cell. 19 (6), 725-737 (2016).</li><li>Pennarossa, G., Paffoni, A., Ragni, G., Gandolfi, F., Brevini, T. A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+signaling-directed+YAP/TAZ+regulation+encourages+3D+spheroid+colony+formation+and+boosts+plasticity+of+parthenogenetic+stem+cells.">Rho signaling-directed YAP/TAZ regulation encourages 3D spheroid colony formation and boosts plasticity of parthenogenetic stem cells.</a> Advances in Experimental Medicine and Biology. 1237, 49-60 (2020).</li></ol>

作者没有什么可透露的。

在过去的几十年里，一些研究的重点是制定策略，使终末分化的细胞恢复到不那么投入和更高的宽容状态。这里描述的方案允许从成体成熟的终末分化细胞开始产生和长期维持多能细胞。该方法结合了两个独立的步骤，涉及诱导高允许状态，这是通过化学表观遗传擦除实现的，并使用3D培养系统确保其后续维护。
在PTFE包封的细胞中观察到的3D球体结构的形成（...

在过去的几十年里，被广泛接受的单向进展到细胞承诺和分化的概念被完全修改。已经证明，细胞规格可以颠倒过来，并且使用不同的方法可以将终末分化的细胞推向不那么投入和更高的宽容状态。
在提出的几种方法中，最有前途的方法之一涉及使用化合物诱导细胞进入所谓的化学诱导多能性。这种方法中使用的小分子能够相互作用并修饰成年成熟细胞的表观遗传特征，从而避免了对任何转基因和/或病毒载体1，2，3，4，5，6，7，8，9，10的需求。.最近的许多研究表明，通过提供诱导高甲基化基因11，12，13，14，15再激活的特定生化和生物刺激，可以将细胞从一种表型切换到另一种表型。这些去甲基化事件允许将终末分化的细胞转化为原始祖细胞，多能或高可塑性/多能细胞1，2，3，4，5，6，7，8，9，10。
同时，许多研究最近都集中在理解机械检测相关线索上，更具体地说，是使用机械力直接影响细胞可塑性和/或分化的可能性16，17，18，19。事实上，已经清楚地表明，细胞外基质（ECM）在控制细胞命运中起着关键作用。特别是，ECM产生的生物力学和生物物理信号直接调节分子机制和信号通路，影响细胞行为和功能20，21。这些最近的数据为开发新型3D培养系统铺平了道路，这些系统更接近于模仿体内细胞微环境，复制驱动细胞行为的机械和物理刺激。
我们在这里描述了一个两步方案，它将化学表观遗传擦除与机械衰老相关线索相结合，以产生哺乳动物多能细胞。在第一步中，将细胞与去甲基化分子5-氮杂胞苷（5-氮杂-CR）一起孵育。该试剂能够通过直接的10-11易位2（TET2）介导的作用8，10 和间接抑制DNA甲基转移酶（DNMT）22，23的组合作用来诱导显着的全局DNA去甲基化。该步骤诱导表观遗传阻滞的去除，随后重新激活多能性相关基因表达，因此产生高可塑性细胞1，2，3，8，10，以下称为"表观遗传擦除细胞"。在第二步中，将细胞封装在3D培养系统中。为此，将非反应性疏水性合成化合物聚四氟乙烯（PTFE;粒径为1μm）用作微生物反应器，其允许创建通过使用传统2D培养系统无法实现的细胞微环境10。PTFE粉末颗粒粘附在液滴的表面，其中细胞被重新悬浮并将液核与支撑表面隔离开来，同时允许内部液体和周围环境24之间的气体交换。由此获得的"PTFE微生物反应器"，也称为"液体大理石"，鼓励细胞彼此自由相互作用，促进3D细胞重排25，26，27，并通过生物机械测量相关线索10延伸并稳定地维持获得的高可塑性状态。

<table><tbody><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M7522</td><td>Component of ESC medium</td></tr><tr><td>5-Azacytidine</td><td>Sigma-Aldrich</td><td>A2385</td><td>5-aza-CR, for fibroblast epigenetic erasing</td></tr><tr><td>Adenosine</td><td>Sigma-Aldrich</td><td>A4036</td><td>Component of nucleoside mix for ESC medium</td></tr><tr><td>Antibiotic Antimycotic Solution (100&amp;times;)</td><td>Sigma-Aldrich</td><td>A5955</td><td>Component of fibroblast and ESC media</td></tr><tr><td>CFX96 Real-Time PCR</td><td>Bio-Rad Laboratories</td><td>NA</td><td>Thermal cycler for quantitative PCR</td></tr><tr><td>Cytidine</td><td>Sigma-Aldrich</td><td>C4654</td><td>Component of nucleoside mix for ESC medium</td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Thermo Fisher Scientific</td><td>41966052</td><td>For fibroblast isolation and culture medium</td></tr><tr><td>DMEM, low glucose, pyruvate</td><td>Thermo Fisher Scientific</td><td>31885023</td><td>For ESC medium</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D5652</td><td>PBS; for biopsy and cell wash and for solution preparation</td></tr><tr><td>Dynabeads mRNA DIRECT Micro Purification Kit</td><td>Thermo Fisher Scientific</td><td>61021</td><td>mRNA estraction</td></tr><tr><td>ESGRO Recombinant Mouse LIF Protein</td><td>Sigma-Aldrich</td><td>ESG1106</td><td>Component of ESC medium</td></tr><tr><td>Fetal Bovine Serum, qualified, heat inactivated</td><td>Thermo Fisher Scientific</td><td>10500064</td><td>Component of fibroblast and ESC media</td></tr><tr><td>FGF-Basic (AA10-155) Recombinant Human Protein</td><td>Thermo Fisher Scientific</td><td>PHG0024</td><td>Component of ESC medium</td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma-Aldrich</td><td>G1890</td><td>For dish coating</td></tr><tr><td>GeneAmp PCR System 2700</td><td>Applied Biosystems</td><td>NA</td><td>Thermal cycler for qualitative PCR</td></tr><tr><td>Global DNA Methylation ELISA Kit</td><td>CELL BIOLABS</td><td>STA-380</td><td>Methylation study</td></tr><tr><td>GoTaq G2 Flexi DNA Polymerase</td><td>Promega</td><td>M7801</td><td>Qualitative PCR</td></tr><tr><td>Guanosine</td><td>Sigma-Aldrich</td><td>G6264</td><td>Component of nucleoside mix for ESC medium</td></tr><tr><td>Ham&#039;s F-10 Nutrient Mix</td><td>Thermo Fisher Scientific</td><td>31550031</td><td>For ESC medium</td></tr><tr><td>KnockOut Serum Replacement</td><td>Thermo Fisher Scientific</td><td>10828028</td><td>Component of ESC medium</td></tr><tr><td>KOVA glasstic slide 10 with grids</td><td>Hycor Biomedical</td><td>87144</td><td>For cell counting</td></tr><tr><td>Leica MZ APO Stereo Microscope</td><td>Leica</td><td>NA</td><td>For organoid observation</td></tr><tr><td>L-Glutamine solution</td><td>Sigma-Aldrich</td><td>G7513</td><td>Component of fibroblast and ESC media</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100X)</td><td>Thermo Fisher Scientific</td><td>11140035</td><td>Component of ESC medium</td></tr><tr><td>Millex-GS 0.22 &amp;micro;m pore filters</td><td>Millipore</td><td>SLGS033SB</td><td>For solution sterilization</td></tr><tr><td>M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant</td><td>Promega</td><td>M3681</td><td>mRNA reverse transcription</td></tr><tr><td>Multiskan FC Microplate Photometer</td><td>Thermo Fisher Scientific</td><td>51119000</td><td>For ELISA plate reading</td></tr><tr><td>Nikon Eclipse TE300 Inverted Phase Contrast Microscope</td><td>Nikon</td><td>NA</td><td>For cell observation</td></tr><tr><td>Perkin Elmer Thermal Cycler 480</td><td>Perkin Elmer</td><td>NA</td><td>Thermal cycler for reverse transcription</td></tr><tr><td>Poly(tetrafluoroethylene) 1 &amp;mu;m particle size</td><td>Sigma-Aldrich</td><td>430935</td><td>For generating micro-bioreactor</td></tr><tr><td>PureLink Genomic DNA Mini Kit</td><td>Thermo Fisher Scientific</td><td>K182001</td><td>Genomic DNA estraction</td></tr><tr><td>TaqMan Gene Expression Cells-to-CT Kit</td><td>Thermo Fisher Scientific</td><td>AM1728</td><td>Quantitative PCR</td></tr><tr><td>Thymidine</td><td>Sigma-Aldrich</td><td>T1895</td><td>Component of nucleoside mix for ESC medium</td></tr><tr><td>Tissue Culture Dish 100X20 mm, Standard</td><td>Sarstedt</td><td>833902</td><td>For fibroblast isolation</td></tr><tr><td>Tissue Culture Dish 35X10 mm, Standard</td><td>Sarstedt</td><td>833900</td><td>For Fibroblast isolation</td></tr><tr><td>Tissue Culture Dish 35X10 mm, Suspension</td><td>Sarstedt</td><td>833900500</td><td>Bacteriology petri dish for liquid marble culture</td></tr><tr><td>Tissue Culture Plate 96 Well,Standard,F</td><td>Sarstedt</td><td>833924005</td><td>For liquid marble culture</td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma-Aldrich</td><td>T3924</td><td>For fibroblast dissociation</td></tr><tr><td>Tube 15ml, 120x17mm, PS</td><td>Sarstedt</td><td>62553041</td><td>For cell suspension centrifugation</td></tr><tr><td>Uridine</td><td>Sigma-Aldrich</td><td>U3003</td><td>Component of nucleoside mix for ESC medium</td></tr></tbody></table>

a two-step strategy that combines epigenetic modification and biomechanical cues to generate mammalian pluripotent cells

细胞表型可以用不同的方法逆转或修饰，具有每种技术特有的优点和局限性。在这里，我们描述了一种新策略，该策略将化学表观遗传擦除与机械检测相关线索相结合，以产生哺乳动物多能细胞。需要两个主要步骤。在第一步中，成体成熟（终末分化）细胞暴露于表观遗传橡皮擦5-氮杂胞苷以驱动它们进入多能状态。该方案的这一部分是基于对控制细胞命运和分化的表观遗传机制的日益了解而开发的，并且涉及使用表观遗传修饰剂来消除细胞分化状态，然后驱动进入瞬时高可塑性窗口。
在第二步中，将擦除的细胞封装在聚四氟乙烯（PTFE）微生物反应器（也称为Liquid Marbles）中，以促进3D细胞重排以扩展并稳定地保持获得的高可塑性。PTFE是一种非反应性疏水性合成化合物，其使用允许产生细胞微环境，这在传统的2D培养系统中无法实现。该系统通过生物机械检测相关线索鼓励和促进多能性的维持。
这里描述的技术程序是允许在成人体细胞中诱导和维持高可塑性状态的简单策略。该协议允许在所有测试的哺乳动物物种中衍生出高可塑性细胞。由于它不涉及基因转染的使用，并且没有病毒载体，因此它可能代表了转化医学应用的显着技术进步。此外，微生物反应器系统通过体外重新创建特定的微环境，允许长期培养高可塑性细胞，即ESC，iPSCs，表观遗传擦除细胞和MSC，从而为干细胞类器官技术提供了显着的进步。

本方案描述了从成体细胞生成和稳定维持哺乳动物多能细胞的所有步骤。这种方法已经成功地从不同的哺乳动物物种（即小鼠，猪和人类）中分离出成纤维细胞。这里报道的代表性结果是从所有细胞系获得的，无论来源物种如何。
形态学分析表明，在与去甲基化剂5-氮杂-CR孵育18小时后，成纤维细胞封装在PTFE微生物反应器（3D Post 5-aza-CR）聚集体中并形成3D球形结构，显示出均...

Watch this Scientific Journal Video about A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells at JoVE.com

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

我们在这里提出了一种将化学表观遗传擦除与机械检测相关线索相结合的方法，以有效地产生哺乳动物多能细胞，而无需基因转染或逆转录病毒载体。因此，这种策略对转化医学很有希望，并且代表了干细胞类器官技术的显着进步。

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 ...

a-two-step-strategy-that-combines-epigenetic-modification

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

2.2K 次观看.  Università degli Studi di Milano. 我们在这里提出了一种将化学表观遗传擦除与机械检测相关线索相结合的方法，以有效地产生哺乳动物多能细胞，而无需基因转染或逆转录病毒载体。因此，这种策略对转化医学很有希望，并且代表了干细胞类器官技术的显着进步。

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

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

科研

发育生物学

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

从MEFS iPS细胞生成通过强制SOX - 2的表达，10月4，c - Myc基因和Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

生物学

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

生物工程

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

使用无缝基因组编辑将点突变引入人类多能干细胞

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

遗传学

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

的iPS细胞从中间体可重编程小鼠模型的细胞表面标志物介导的净化

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

从血液高效的iPS细胞生成使用附加体和HDAC抑制剂

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Induced Pluripotency

Induced pluripotent stem cells (iPSCs) are somatic cells that have been genetically reprogrammed to form undifferentiated stem cells. Like embryonic stem cells, iPSCs can be grown in culture conditions that promote differentiation into different cell types. Thus, iPSCs may provide a potentially unlimited source of any human cell type, which is a major breakthrough in the field of regenerative medicine. However, more research into the derivation and differentiation of iPSCs is still needed to actually use these cells in clinical practice.
This video first introduces the fundamental principles behind cellular reprogramming, and then demonstrates a protocol for the generation of iPSCs from differentiated mouse embryonic fibroblasts. Finally, it will discuss several experiments in which scientists are improving or applying iPSC generation techniques.

Induced Pluripotency and Yamanaka Factors

诱导多能性

Induced pluripotent stem cells (iPSCs) are somatic cells that have been genetically reprogrammed to form undifferentiated stem cells. Like embryonic stem ...

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

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此视频中的章节

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

从MEFS iPS细胞生成通过强制SOX - 2的表达，10月4，c - Myc基因和Klf4

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

使用无缝基因组编辑将点突变引入人类多能干细胞

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

诱导多能干细胞来源于小鼠的生成

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

的iPS细胞从中间体可重编程小鼠模型的细胞表面标志物介导的净化

从血液高效的iPS细胞生成使用附加体和HDAC抑制剂

诱导多能性