Name: direct reprogramming of mouse fibroblasts into melanocytes
Uploaded: 2021-08-27T14:30:00
Description: Watch this Scientific Journal Video about Direct Reprogramming of Mouse Fibroblasts into Melanocytes at JoVE.com

本研究部分由国家自然科学基金（82070638和81770621）和江苏省自然科学基金（BK20180281）资助。

这项工作得到了江苏大学实验动物管理和使用委员会（UJS-IACUC-AP--20190305010）的批准。实验严格按照国际实验动物护理评估和认证协会（AAALAC International）制定的标准进行。没有涉及人类的实验，因此这项工作不需要人类研究伦理委员会的批准。有关试剂的详细信息，请参阅 材料表 。
1. 构建转录因子浓缩慢病毒包装系统
<ol><li>浓缩病毒的产生（?...

<ol>
	<li>Ezzedine, K., Eleftheriadou, V., Whitton, M., van Geel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitiligo.">Vitiligo.</a> Lancet. 386 (9988), 74-84 (2015).</li><li>Picardo, 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=Vitiligo.">Vitiligo.</a> Nature Reviews. Disease Primers. 1, 15011 (2015).</li><li>Speeckaert, R., van Geel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitiligo:+An+update+on+pathophysiology+and+treatment+options.">Vitiligo: An update on pathophysiology and treatment options.</a> American Journal of Clinical Dermatology. 18 (6), 733-744 (2017).</li><li>Cortelazzi, C., Pellacani, G., Raposio, E., Di Nuzzo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitiligo+management:+combination+of+surgical+treatment+and+phototherapy+under+reflectance+confocal+microscopy+monitoring.">Vitiligo management: combination of surgical treatment and phototherapy under reflectance confocal microscopy monitoring.</a> European Review for Medical and Pharmacological Sciences. 24 (13), 7366-7371 (2020).</li><li>Mohammad, T. F., Hamzavi, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+therapies+for+vitiligo.">Surgical therapies for vitiligo.</a> Dermatologic Clinics. 35 (2), 193-203 (2017).</li><li>Bishnoi, A., Parsad, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+molecular+aspects+of+vitiligo+treatments.">Clinical and molecular aspects of vitiligo treatments.</a> International journal of molecular sciences. 19 (5), 1509 (2018).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+transcription+factor-mediated+reprogramming+to+pluripotency.">A decade of transcription factor-mediated reprogramming to pluripotency.</a> Nature Reviews. Molecular Cell Biology. 17 (3), 183-193 (2016).</li><li>Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cell-based+cell+therapy-promise+and+challenges.">Pluripotent stem cell-based cell therapy-promise and challenges.</a> Cell Stem Cell. 27 (4), 523-531 (2020).</li><li>Xu, J., Du, Y., Deng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+lineage+reprogramming:+strategies,+mechanisms,+and+applications.">Direct lineage reprogramming: strategies, mechanisms, and applications.</a> Cell Stem Cell. 16 (2), 119-134 (2015).</li><li>Ieda, 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=Direct+reprogramming+of+fibroblasts+into+functional+cardiomyocytes+by+defined+factors.">Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.</a> Cell. 142 (3), 375-386 (2010).</li><li>Gasc&#243;n, S., Masserdotti, G., Russo, G. L., G&#246;tz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+neuronal+reprogramming:+achievements,+hurdles,+and+new+roads+to+success.">Direct neuronal reprogramming: achievements, hurdles, and new roads to success.</a> Cell Stem Cell. 21 (1), 18-34 (2017).</li><li>Atkinson, P. J., Kim, G. S., Cheng, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cellular+reprogramming+and+inner+ear+regeneration.">Direct cellular reprogramming and inner ear regeneration.</a> Expert Opinion on Biological Therapy. 19 (2), 129-139 (2019).</li><li>Kurita, 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=In+vivo+reprogramming+of+wound-resident+cells+generates+skin+epithelial+tissue.">In vivo reprogramming of wound-resident cells generates skin epithelial tissue.</a> Nature. 561 (7722), 243-247 (2018).</li><li>Yang, R., 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=Direct+conversion+of+mouse+and+human+fibroblasts+to+functional+melanocytes+by+defined+factors.">Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors.</a> Nature Communications. 5, 5807 (2014).</li><li>Fehrenbach, S., 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=Loss+of+tumorigenic+potential+upon+transdifferentiation+from+keratinocytic+into+melanocytic+lineage.">Loss of tumorigenic potential upon transdifferentiation from keratinocytic into melanocytic lineage.</a> Scientific Reports. 6, 28891 (2016).</li><li>Majumdar, G., Vera, S., Elam, M. B., Raghow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+streamlined+protocol+for+extracting+RNA+and+genomic+DNA+from+archived+human+blood+and+muscle.">A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.</a> Analytical Biochemistry. 474, 25-27 (2015).</li><li>Bachman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse-transcription+PCR+(RT-PCR).">Reverse-transcription PCR (RT-PCR).</a> Methods in Enzymology. 530, 67-74 (2013).</li><li>Donaldson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+staining.">Immunofluorescence staining.</a> Current Protocols in Cell Biology. 69 (1), 1-7 (2015).</li><li>Yin, 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=Non-viral+vectors+for+gene-based+therapy.">Non-viral vectors for gene-based therapy.</a> Nature Reviews. Genetics. 15 (8), 541-555 (2014).</li></ol>

病毒的质量对于该方案中直接重编程为黑素细胞的成功至关重要。该方案中病毒的包装和浓缩方法简单易重复，不依赖任何其他辅助浓缩试剂。在大多数实验室中可以成功遵循该方案。为保证浓缩病毒的质量，以下几点需要特别注意。一个是HEK-293T的电池状态。虽然HEK-293T细胞是永生化细胞，但用于制造浓缩病毒的细胞必须是10次传代内的健康细胞（滴度随着传代次数的增加而降低）。
<p class="j...

白癜风是一种皮肤病，严重影响受影响个体的身心健康。由于代谢异常，氧化应激，炎症介质的产生，细胞脱离和自身免疫反应等各种原因，功能性黑素细胞丢失，黑色素分泌停止，导致白癜风1，2的发展。这种情况发生得很广泛，在脸上尤其成问题。主要治疗方法是全身使用皮质类固醇和免疫调节剂。光疗可用于全身性或局部性疾病，并且有手术治疗，如穿孔皮肤移植和自体黑素细胞移植3，4，5。然而，使用药物治疗和光疗的患者容易复发，这些治疗的长期治疗效果较差。手术治疗是创伤性的，只有中等效果2，6。因此，白癜风需要一种新的有效治疗策略。
诱导多能干细胞（iPSCs）的重编程将这些细胞从其终末状态逆转为多能状态，这是由转录因子Oct4，Sox2，Klf4和c-Myc7介导的过程。然而，由于致瘤的可能性和较长的生产时间，该技术在应用于临床环境8时遭到了怀疑。直接重编程是一种技术，它使一种类型的终端单元转换为另一种类型的终端单元9。该过程是通过合适的转录因子实现的。各种细胞已经直接成功重编程，包括心肌细胞10，神经元11和耳蜗毛细胞12。一些研究人员甚至直接 原位重新编程皮肤组织，可用于伤口修复13。直接重编程的优点包括减少等待时间和成本，降低癌症风险，减少伦理问题，以及更好地了解细胞命运决定的潜在机制9。
虽然直接重编程法被广泛使用，但目前还没有确定的方法可以将皮肤细胞直接重编程为黑素细胞，特别是因为需要考虑许多转录因子14，15。转录因子Mitf，Sox10和Pax3已被用于将皮肤细胞直接重编程为黑色素细胞14。相比之下，在另一项研究15中，MITF，PAX3，SOX2和SOX9的组合也被用于将皮肤细胞直接重编程为人类黑素细胞。在该协议中，尽管使用了不同的筛选方法，但使用Mitf，Sox10和Pax3的组合获得了相同的结果，用于将皮肤细胞直接重编程为黑色素细胞，如前所述14。开发一种从其他皮肤细胞产生黑素细胞的系统可以提供将白癜风患者的其他皮肤细胞转化为黑素细胞的方案。因此，构建一种简单有效的方法对于这种直接重编程以成功产生黑素细胞至关重要。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-062</td>
			<td>Stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>0.45 &#956;M filter</td>
			<td>Millipore</td>
			<td>SLHVR33RB</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polystyrene round bottom tube</td>
			<td>Falcon</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>95%/100% ethanol</td>
			<td>LANBAO</td>
			<td>210106</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma</td>
			<td>A2786</td>
			<td>Stock concentration 40 mg/mL 
			Final concentration 24 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 Goat anti-Mouse IgG2a</td>
			<td>Invitrogen</td>
			<td>A21137</td>
			<td>Dilution of 1:500 to use</td>
		</tr>
		<tr>
			<td>Antibiotics(Pen/Strep)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Anti-TRP1/TYRP1 Antibody</td>
			<td>Millipore</td>
			<td>MABC592</td>
			<td>Host/Isotype: Mouse IgG2a 
			Species reactivity: Mouse/Human 
			Dilution of 1:200 to use</td>
		</tr>
		<tr>
			<td>Anti-TRP2/DCT Antibody</td>
			<td>Abcam</td>
			<td>ab74073</td>
			<td>Host/Isotype: Rabbit IgG 
			Species reactivity: Mouse/Human Dilution of 1:200 to use</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stemgent</td>
			<td>04-0004</td>
			<td>Stock concentration 10 mM 
			Final concentration 3 &#956;M</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td>Stock concentration 0.3 mg/mL 
			Final concentration 20 pM</td>
		</tr>
		<tr>
			<td>Cy3 Goat anti-Rabbit IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>111-165-144</td>
			<td>Dilution of 1:500 to use</td>
		</tr>
		<tr>
			<td>DMEM (High glucose)</td>
			<td>HyClone</td>
			<td>SH30243.01</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>DMSO&#160;</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>Stored at -20 &#176;C 
			Heat-inactivated before use</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G9391</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>GFP-PURO plasmids (Mitf, Sox10, Pax3, Sox2, Sox9 and Snai2)</td>
			<td>Hanheng Biological Technology Co., Ltd.</td>
			<td>pHBLPm003198 pHBLPm001143 pHBLPm002968 pHBLPm002981 pHBLPm004348 pHBLPm000325</td>
			<td>Stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Abcam</td>
			<td>ab220365</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>Human EDN3</td>
			<td>American-Peptide</td>
			<td>88-5-10A</td>
			<td>Stock concentration 100 &#956;M 
			Final concentration 0.1 &#956;M</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>H0888</td>
			<td>Stock concentration 100 &#181;g/mL 
			Final concentration 0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>L-DOPA</td>
			<td>Sigma</td>
			<td>D9628</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Transfection reagent, stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Masson-Fontana staining kit</td>
			<td>Solarbio</td>
			<td>G2032</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Solarbio</td>
			<td>G8590</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>PBS (-)</td>
			<td>Gibco</td>
			<td>C10010500BT</td>
			<td>Stored at RT</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (TPA)</td>
			<td>Sigma</td>
			<td>P8139</td>
			<td>Stock concentration 1 mM 
			Final concentration 200 nM</td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td>cationic polymeric transfection reagent; Stock concentration 8 &#956;g/&#181;L 
			Final concentration 4 ng/&#181;L</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Gibco</td>
			<td>A11138-03</td>
			<td>Stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Recombinant human bFGF</td>
			<td>Invitrogen</td>
			<td>13256-029</td>
			<td>Stock concentration 4 &#956;g/mL 
			Final concentration 10 ng/mL</td>
		</tr>
		<tr>
			<td>Recombinant human insulin</td>
			<td>Sigma&#160;</td>
			<td>I3536</td>
			<td>Stock concentration 10 mg/mL 
			Final concentration 5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Recombinant human SCF</td>
			<td>R&#38;D</td>
			<td>255-SC-010</td>
			<td>Stock concentration 200 &#956;g/mL 
			Final concentration 100 ng/mL</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma</td>
			<td>1330-20-7</td>
			<td>Stored at RT</td>
		</tr>
	</tbody>
</table>

direct reprogramming of mouse fibroblasts into melanocytes

黑素细胞功能的丧失导致白癜风，严重影响受影响个体的身心健康。目前，白癜风没有有效的长期治疗。因此，必须开发一种方便有效的白癜风治疗方法。将皮肤细胞直接重编程为黑素细胞的再生医学技术似乎是一种有前途的白癜风新疗法。这涉及将患者的皮肤细胞直接重编程为功能性黑素细胞，以帮助改善白癜风患者黑素细胞的丢失。然而，这种方法需要首先在小鼠身上进行测试。虽然直接重编程被广泛使用，但没有明确的方案可以直接重编程为黑素细胞。此外，可用转录因子的数量是压倒性的。 
在这里，提出了一种浓缩的慢病毒包装系统方案，以产生用于将皮肤细胞重编程为黑素细胞的转录因子，包括Sox10，Mitf，Pax3，Sox2，Sox9和Snai2。小鼠胚胎成纤维细胞（MEFs）感染了所有这些转录因子的浓缩慢病毒，用于 在体外将MEF直接重编程为诱导黑素细胞（iMels）。此外，筛选了这些转录因子，并优化了系统以直接重编程为黑素细胞。iMels中黑色素特征标记物在基因或蛋白质水平上的表达显著增加。这些结果表明，将成纤维细胞直接重编程为黑素细胞可能是白癜风的成功新治疗策略，并证实了黑素细胞发育的机制，这将为成纤维细胞在 体内进一步直接重编程为黑素细胞提供基础。

本文包括浓缩慢病毒包装系统的方案，用于生产转录因子慢病毒，用于将成纤维细胞直接重编程为黑素细胞，以及用于筛选转录因子和直接从MEF重编程黑素细胞的方案。
通过观察GFP的荧光强度（图1A）或流式细胞术（图1B）在用未浓缩慢病毒（1x）和浓缩慢病毒（100x）感染HEK-293T细胞48小时后，评估浓缩慢病毒生产的成功。浓缩病毒...

Watch this Scientific Journal Video about Direct Reprogramming of Mouse Fibroblasts into Melanocytes at JoVE.com

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

在这里，我们描述了一种针对黑素细胞的优化直接重编程系统和一种高效、浓缩的病毒包装系统，可确保顺利的直接重编程。

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, ...

直接重编程以产生黑素细胞已有报道。然而，不同的研究中使用了各种转录因子。在该协议中，我们验证并减少了转录因子数，仍然具有更高的重编程效率。我们建立了一个直接重编程系统，仅使用三种转录因子Mitf，Sox10和PAX3来生成黑素细胞，并修改了培养系统以使其更加稳定和稳健。如何再生功能性黑素细胞是一个重要的问题，可能有助于解决白癜风等色素沉着疾病的治疗限制。正确的重编程提供了前瞻性 我们的技术具有方便、可操作性强、稳定性好等特点，更易于推广和重复。要开始生产浓缩的慢病毒，将1.5倍10倍的6个HEK-293T细胞放入60毫米培养皿中，并在37摄氏度下用正常培养基培养细胞在加湿培养箱中用5%二氧化碳培养。孵育24小时后，当HEK-293T细胞达到80%至90%汇合时，用3.5mL DMEM替换培养基，然后将细胞在37摄氏度下在加湿培养箱中用5%二氧化碳孵育两小时。从培养箱中取出细胞后，用含有2%FBS的DMEM替换培养基，并以滴剂方式将新鲜制备的转染复合物混合物加入细胞中。轻轻混合液体混合物。在完成8小时后，用3.5 mL正常培养基更换细胞培养基。在24和48小时收集病毒上清液。接下来，将两个不同时间点收集的病毒上清液混合，并在4摄氏度下以200 xg离心混合物5分钟。然后将收集的上清液通过0.45微米过滤器，将滤液收集在50mL无菌锥形管中。将过滤后的病毒上清液在4摄氏度下以6， 000 xg离心浓缩过夜。完成后，确保病毒沉淀在锥形管的底部可见。慢慢倒出上清液。用于将病毒沉淀溶解在具有100倍*体积的病毒上清液的正常培养基中。使用P1000微量移液管轻轻上下移液，直到获得均匀的100x浓缩病毒混合物。将浓缩的病毒分成微量离心管，以80摄氏度储存。将1次10至第5个HEK-293T细胞放入一孔六孔板中。在装有5%二氧化碳的加湿培养箱中，用37摄氏度的正常培养基培养这些细胞。24小时后，向每个孔中加入0.1至0.2微升的100倍荧光浓缩病毒。随后向每个孔中加入每微升四纳克阳离子聚合物转染试剂。感染病毒后约8至12小时，用正常培养基替换培养基。感染后四十八小时，用1毫升无菌PBS清洗培养皿以除去死细胞。然后在室温下使用每孔六孔板的250微升0.05%胰蛋白酶-EDTA溶液对细胞进行胰蛋白酶消化。将板以200 xg在四摄氏度下离心五分钟。除去上清液后，将细胞沉淀重新悬浮在一mLPBS中，并将细胞悬浮液加入五mL聚苯乙烯圆底管中。然后将管放入流式细胞仪中分析样品。对于成纤维细胞的直接重编程，在室温下用1 mL 0.1%明胶溶液涂覆一孔六孔细胞培养板。15~30分钟后，当孔完全被明胶覆盖时，吸出0.1%明胶溶液。接下来，将5次10次到第4只小鼠胚胎成纤维细胞（MEF）中，放入一孔涂有0.1%明胶的六孔板中，并用37摄氏度的正常培养基在具有5%二氧化碳的加湿培养箱中培养细胞过夜。24小时后，当MEF达到40%至50%汇合度时，用普通培养基替换培养基。接下来，在冰上融化冷冻的浓缩病毒。使用等式计算要添加的病毒的体积，然后根据计算的体积将六个转录因子的浓缩病毒添加到每个孔中。向孔中加入每毫升四微克的阳离子聚合物转染剂。将其视为慢病毒感染的第零天。在第一天，感染8至12小时后，用新鲜的正常培养基替换含有病毒的培养基，同时每毫升加入0.5微克嘌呤霉素以筛选稳定的感染细胞系。在感染后第二天，48小时，用重编程培养基逐渐替换上清液培养基。在添加三个微摩尔CHIR99021之前，请改变总培养基体积的四分之一。从第三天到第七天，根据细胞的状况，通过逐渐用更高比例的重编程培养基替换培养基来改变培养基，以便在五天内切换到完全重编程培养基。接下来，用500微升0.05%胰蛋白酶-EDTA消化酶在室温下分离细胞三分钟。当大约百分之六十的细胞漂浮起来时，通过添加正常培养基（消化酶体积的两倍）来停止消化。将细胞悬浮液收集在15mL无菌锥形管中，以200 xg在4摄氏度下离心5分钟。除去上清液，然后用重编程培养基重新悬浮细胞沉淀。完成后，将重新悬浮的细胞接种在60毫米无菌培养皿中。在37摄氏度下在加湿的5%二氧化碳培养箱中培养细胞。在HEK-293T细胞中慢病毒感染48小时后，通过观察GFP的荧光强度或流式细胞术来评估浓缩慢病毒生产的成功。发现浓缩病毒的滴度在10至第8个TU / mL时很高。在成纤维细胞的直接重编程过程中，细胞形态逐渐变为细长的细胞突触和增大的细胞核。然而，细胞在第二十天后逐渐老化。去除转录因子Mitf，Pax3或Sox10导致黑素细胞基因Tyr，Tyrp1和Mlana表达的沉默，表明对成纤维细胞转化为黑素细胞的影响最大。因此，与六种转录因子相比，使用三种转录因子直接编程诱导的黑素细胞基因的表达更高。为了鉴定MEF诱导的黑素细胞或iMels，使用免疫荧光染色检测黑素细胞标志物TYRP1和DCT的表达。此外，黑色素特异性染色方法如DOPA和Masson-Fontana染色显示出积极的结果。293T细胞应均匀分布。此外，必须轻轻地向细胞中添加转染混合物，以防止细胞漂浮。该方案稳定实用，可用于重新编程其他类型的细胞。有望为未来医学中黑素细胞再生的体内直接重编程提供参考和策略。

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Anatomy and Physiology

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Unit 1

The Integumentary System

SCIENTISTS IN ACTION

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

科研

发育生物学

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

方法细胞或DNA的精确本地化转移到着床后早期小鼠胚胎

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

体细胞重编程的动力学测量和实时可视化

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

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

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 ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Establishment of Proliferative Tetraploid Cells from Nontransformed 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 ...

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

小儿食管上皮细胞重编程的条件在组织工程和疾病使用调查

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

无异条件下多原代羊水和膜细胞重新编程

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...