作者要感谢 Jérôme Larghero 和 valériemarcel Vanneaux (Hôpital 圣路易斯, 巴黎, 法国) 在这里所描述的方法的建立过程中的投入。
这项工作得到了来自情报局的赠款的支持 [GPiPS: ANR-2010-RFCS005;SightREPAIR: ANR-16-CE17-008-02], 基金会倒 la 研究所 Médicale [生物工程项目 DBS20140930777] 和从 LABEX 复活 [ANR-10-LABX-73] 到奥利弗 Goureau 和 Christelle Monville。它得到了 NeurATRIS (Investissements d ' 艾文莉) 的支持, 为神经 [ANR-11-INBS-0011] 和 INGESTEM 的 biotherapies, 国家基础设施 (Investissements d ' 艾文莉) 工程为多潜能和分化的干细胞 [ANR-11-INBS-000] Christelle Monville。Barek 是由暗淡的 Stempole 和 LABEX 复兴 [ANR-10-LABX-73] 的研究金支持。Biotherapies 是法国反肌病 (AFM)-Téléthon 协会支持的罕见疾病研究所的一部分。

本议定书使用的所有人类材料都是按照欧洲联盟的规定使用的。本研究使用的人类 ES 细胞系来源于一个独特的胚胎。捐出胚胎的夫妇完全知情, 并同意匿名捐赠。一个临床级的人类 ES 细胞系来源于这个胚胎, 储存, 合格, 并正确记录的罗丝琳细胞 (英国)。在产妇剖宫产期间, 根据医院的指导原则 (APHP, Hôpital 圣路易斯) 签署了知情同意的胎盘捐献, 火腿是在无菌条件下采购的。
1、培养基和试剂的制备
<ol><li>
视网膜色素上皮细胞培养培养基的制备
	<ol>
<li>为了准备视网膜色素细胞培养培养基, 添加4% 血清替代品 (20 毫升的最终体积500毫升), 1,000x 稀释 2-巯基乙醇 (500 µL 为最终体积500毫升), 1% Eagle′s 最低基本培养基 (记忆体) 非必需氨基酸溶液 (5 毫升为最终体积500毫升) Dulbecco 的改良鹰的培养基 (DMEM) 高葡萄糖 (475 毫升的最终体积500毫升)。</li>
	</ol>
</li>
	<li>
运输/养护介质的制备
	<ol>
<li>要准备运输/保护培养基, 添加1% 青霉素-链霉素 (5 毫升的最终体积500毫升), 以 CO2独立的培养基 (495 毫升的最终体积500毫升), 并保持在4°c。</li>
	</ol>
</li>
	<li>
thermolysin 酶的泥沙
	<ol>
<li>thermolysin 库存液的制备<ol>
<li>要准备一个 thermolysin 的库存溶液, 解冻 thermolysin 粉, 在室温下储存在-20 摄氏度。</li>
			<li>由于酶活性可能是从批次到批次的变量, 计算此活动的基础上提供的分析证书与 thermolysin 粉的批次将使用。将 "活性中性蛋白酶计算 = ANPC" (在分析证明书中注明) 除以 181 (这是酪氨酸的分子量)。所得结果与所提供的 thermolysin 粉 (U/瓶) 的总酶活性有关。</li>
			<li>通过添加对应于总酶活性 (u/瓶) 的水量除以 200 (u/毫升), 在 200 U/毫升处准备一个库存溶液。</li>
			<li>用吹打的水来溶解酶。涡流解决方案三十年代, 并检查是否已完全暂停粉末。完全解散可能需要更多的吹打。</li>
			<li>整除库存解决方案, 并存储在-20 摄氏度。</li>
		</ol>
</li>
		<li>thermolysin 工作液的制备 注意: thermolysin 的解决方案必须在火腿治疗当天准备好。<ol>
<li>解冻的 thermolysin 的股票整除数在 200 U/毫升存储在-20 摄氏度。稀释在磷酸盐缓冲盐水 (PBS) 的库存溶液 200x, 以获得最终的酶活性的 1 U/毫升。准备40毫升治疗第1-4 火腿补丁。</li>
			<li>在使用前通过0.2 µm 过滤器筛选 thermolysin 解决方案。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
明胶泥沙
	<ol>
<li>20% 明胶溶液及块体的制备 注: 20% 明胶溶液及块可在使用前1周制备。<ol>
<li>温暖 CO2独立媒介到42°c 在水浴为30分钟 (至多 1 h)。在50毫升管, 加入10克明胶到40毫升温暖 CO2独立介质和漩涡的解决方案。</li>
			<li>将明胶 30–60 42 摄氏度溶解。涡旋每10分钟融汇溶液。</li>
			<li>一旦溶液是均匀的, 添加4毫升的20% 明胶溶液到四6厘米培养皿。避免气泡。添加一个塑料石蜡膜, 以保护菜肴, 并允许溶液凝固4摄氏度。</li>
		</ol>
</li>
		<li>8% 明胶溶液的制备 注: 8% 明胶溶液可在使用前一天制备, 贮存于4摄氏度。<ol>
<li>温暖 CO2独立媒介到42°c 为30分钟 (至多 1 h)。在50毫升管, 加入4克明胶到46毫升温暖 CO2独立介质和漩涡的解决方案。</li>
			<li>将明胶 30–60 42 摄氏度溶解。添加一个塑料石蜡膜, 以保护管, 并保持在水浴在37°c, 如果它将使用同一天, 或存储它在4°c, 如果它将使用后。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 人羊膜的 Thermolysin 治疗
<ol><li>
人羊膜洗膜
	<ol>
<li>有火腿供应作为小片断 (大约30毫米 x 30 毫米) 固定在尼龙脚手架, 要么已经在4°c 在 PBS 或冷冻。如果提供冷冻, 解冻的膜在37°c 在一个孵化器30分钟。</li>
		<li>洗膜:<ol>
<li>将第1-4 膜放置在含有80毫升 PBS 的250毫升瓶中。根据需要使用尽可能多的瓶子。</li>
			<li>以高速5分钟的速度在平板振动筛中摇动每瓶, 然后丢弃 PBS。添加80毫升 PBS 和重复。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Thermolysin 治疗
	<ol>
<li>添加40毫升的工作溶液的 thermolysin (1 U/毫升) 每瓶含膜 (第1-4 膜每瓶, 每次最多3瓶)。</li>
		<li>摇瓶5分钟, 在 450 rpm, 然后涡旋他们三十年代. 重复1x。</li>
		<li>丢弃 thermolysin 解决方案并添加80毫升的 PBS。</li>
		<li>在450转每分钟摇动瓶子5分钟。丢弃 pbs 并添加80毫升 pbs。重复3x。</li>
	</ol>
</li>
</ol>3. 人羊膜在培养物上的固定
<ol><li>使用长的无菌钳 (可以达到瓶子的底部) 一个一个地去除火腿, 并将每个火腿转移到含有10毫升 PBS 的10厘米培养皿中。</li>
	<li>在一个新的 10 cm 培养皿包含10毫升 PBS, 放置其中一个膜与尼龙朝下。将用于固定膜的四个剪辑中的两个分离到尼龙。</li>
	<li>在尼龙和膜之间插入文化插入的小环。</li>
	<li>确保膜完全覆盖小环。将区域性的第二部分插入到膜的顶部。火腿的基底膜正朝内的文化插入。分离最后两个剪辑。</li>
	<li>切, 用无菌剪刀, 多余的膜外的文化插入如有必要。使用镊子, 将固定在培养基中的膜转移到12井板上, 添加 PBS, 并将该板块存储在37摄氏度和 5% CO2的孵化器中。</li>
	<li>重复这些操作 (从步骤3.2 到 3.5) 与其他膜。</li>
</ol>4. 人羊膜中视网膜色素上皮细胞的解冻和播种
<ol><li>
视网膜色素上皮细胞的解冻
	<ol>
<li>视网膜色素上皮细胞库被冷冻在100万细胞每低温小瓶在液氮。根据需要解冻尽可能多的低温瓶 (每膜35万个细胞)。</li>
		<li>准备一个15毫升管包含3毫升的视网膜色素介质每低温瓶。一旦瓶子解冻, 把细胞转移到每管。对其他瓶子重复此操作。</li>
		<li>融汇 (吸管向上和向下几次并用重悬细胞在 RPE 培养基), 并采取10µL 的细胞溶液和转移到1.5 毫升管。加入10µL 的台盼蓝。将此解决方案的15µL 放在一个计数室中, 然后在显微镜下用4X 目标计算活细胞的数量 (不是蓝色)。对其他瓶子重复此操作。</li>
		<li>同时, 离心机的15毫升管在 110 x g为5分钟. 丢弃上清液并将细胞并用重悬70万细胞/毫升。</li>
	</ol>
</li>
	<li>
视网膜色素上皮细胞在人羊膜中的播种
	<ol>
<li>从孵化器中取出含有膜的12井板。</li>
		<li>吸入 PBS添加500µL 的细胞悬浮液中的步骤4.1.4 在文化插入中心。添加1毫升的视网膜色素培养基外的文化插入 (井内)。重复其他膜。</li>
		<li>将含有膜的12井板放在孵化器中。</li>
	</ol>
</li>
</ol>5. 人羊膜视网膜色素上皮细胞培养的维持
<ol><li>每星期更新中等 2x, 通常在星期一和星期五, 直到至少30天的文化。</li>
	<li>吸入培养基内外的文化插入为每个井和更新它与新鲜的培养基 (1 毫升外的文化插入和500µL 内)。将盘子放在孵化器的37°c 和 5% CO2。</li>
</ol>6. 视网膜色素上皮修补术的制备
注: 从培养的30天开始, 组织就可以移植了。
<ol><li>将8% 的明胶溶液在37摄氏度的水浴中加热30分钟. 用冷 CO2独立介质填充 vibratome 的内腔 (4 °c)。</li>
	<li>
将20% 明胶块放入 vibratome。
	<ol>
<li>从冰箱里拿出6厘米的培养皿, 里面有20% 块明胶。使用手术刀, 切出一个5厘米 x 5 厘米块。用氰胶水或其等效物将块的顶部粘贴到支架上。</li>
		<li>在 vibratome 上放一把新刀片。</li>
		<li>调整块的水平, 直到它在刀片的同一水平。</li>
		<li>用新鲜 CO2-独立媒介在4°c 填满浴在支持附近。使用中等速度切割 vibratome 块, 直到块均匀切割, 从而使表面光滑。设置位置0。</li>
		<li>在凝胶块周围吸出培养基, 直到完全干燥, 然后从孵化器的37摄氏度将含有组织的12井培养皿吸入。</li>
		<li>使用镊子, 小心打开在明胶块顶部的文化插入。用剪刀切开, 不含细胞的细胞膜的所有部分 (细胞未培养的环外)。吸入整个残余介质。</li>
		<li>添加1毫升的液体8% 明胶溶液在37摄氏度, 以覆盖膜。小心地去除多余的。等5–8分钟, 让明胶凝固。</li>
		<li>添加新鲜 CO2-独立介质 (在4°c) 到浴缸, 直到膜覆盖。</li>
		<li>切割, 与 vibratome 在-100 µm 的位置, 以中等速度, 仍然知道如何该区块的行为。在该节的末尾, 要注意保持组织的方向。</li>
		<li>用手术刀, 切一个角落, 以确定该部分的方向。</li>
		<li>用刮刀将所嵌入的明胶中的膜收集起来, 将其放置在6井板中, 填充在4摄氏度的保护培养基上, 直到将其嫁接到接受者眼中。</li>
		<li>在移植时, 在手术显微镜下调整植入物的大小以接受者眼的大小 (1–3为大鼠, 10–15毫米2为非人类灵长类)。</li>
	</ol>
</li>
</ol>

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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=Human+RPE+stem+cells+grown+into+polarized+RPE+monolayers+on+a+polyester+matrix+are+maintained+after+grafting+into+rabbit+subretinal+space.">Human RPE stem cells grown into polarized RPE monolayers on a polyester matrix are maintained after grafting into rabbit subretinal space.</a> Stem Cell Reports. 2 (1), 64-77 (2014).</li><li>Ilmarinen, 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=Ultrathin+polyimide+membrane+as+cell+carrier+for+subretinal+transplantation+of+human+embryonic+stem+cell+derived+retinal+pigment+epithelium.">Ultrathin polyimide membrane as cell carrier for subretinal transplantation of human embryonic stem cell derived retinal pigment epithelium.</a> PloS One. 10 (11), e0143669 (2015).</li><li>Thumann, G., Schraermeyer, U., Bartz-Schmidt, K. U., Heimann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet's+membrane+as+membranous+support+in+RPE/IPE+transplantation.">Descemet's membrane as membranous support in RPE/IPE transplantation.</a> Current Eye Research. 16 (12), 1236-1238 (1997).</li><li>Kiilgaard, J. F., Scherfig, E., Prause, J. U., la Cour, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+amniotic+membrane+to+the+subretinal+space+in+pigs.">Transplantation of amniotic membrane to the subretinal space in pigs.</a> Stem Cells International. 2012, 716968 (2012).</li><li>Capeans, C., 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=Amniotic+membrane+as+support+for+human+retinal+pigment+epithelium+(RPE)+cell+growth.">Amniotic membrane as support for human retinal pigment epithelium (RPE) cell growth.</a> Acta Ophthalmologica Scandinavica. 81 (3), 271-277 (2003).</li><li>Ohno-Matsui, 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=The+effects+of+amniotic+membrane+on+retinal+pigment+epithelial+cell+differentiation.">The effects of amniotic membrane on retinal pigment epithelial cell differentiation.</a> Molecular Vision. 11, 1-10 (2005).</li><li>Paolin, A., 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=Amniotic+membranes+in+ophthalmology:+long+term+data+on+transplantation+outcomes.">Amniotic membranes in ophthalmology: long term data on transplantation outcomes.</a> Cell and Tissue Banking. 17 (1), 51-58 (2016).</li><li>Hu, Y., 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=A+novel+approach+for+subretinal+implantation+of+ultrathin+substrates+containing+stem+cell-derived+retinal+pigment+epithelium+monolayer.">A novel approach for subretinal implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium monolayer.</a> Ophthalmic Research. 48 (4), 186-191 (2012).</li><li>Pennington, B. O., Clegg, D. O. <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+therapies+in+combination+with+substrate+for+the+treatment+of+age-related+macular+degeneration.">Pluripotent stem cell-based therapies in combination with substrate for the treatment of age-related macular degeneration.</a> Journal of Ocular Pharmacology and Therapeutics: The Official Journal of the Association. 32 (5), 261-271 (2016).</li><li>Song, M. J., Bharti, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+into+the+future:+Using+induced+pluripotent+stem+cells+to+build+two+and+three+dimensional+ocular+tissue+for+cell+therapy+and+disease+modeling.">Looking into the future: Using induced pluripotent stem cells to build two and three dimensional ocular tissue for cell therapy and disease modeling.</a> Brain Research. 1638 (Pt A), 2-14 (2016).</li><li>Ramsden, C. 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=Stem+cells+in+retinal+regeneration:+Past,+present+and+future).">Stem cells in retinal regeneration: Past, present and future).</a> Development. 140 (12), 2576-2585 (2013).</li><li>da Cruz, 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=Phase+1+clinical+study+of+an+embryonic+stem+cell-derived+retinal+pigment+epithelium+patch+in+age-related+macular+degeneration.">Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration.</a> Nature Biotechnology. 36 (4), 328-337 (2018).</li><li>Kashani, A. 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=A+bioengineered+retinal+pigment+epithelial+monolayer+for+advanced,+dry+age-related+macular+degeneration.">A bioengineered retinal pigment epithelial monolayer for advanced, dry age-related macular degeneration.</a> Science Translational Medicine. 10 (435), (2018).</li><li>Binder, S., Stanzel, B. V., Krebs, I., Glittenberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+the+RPE+in+AMD.">Transplantation of the RPE in AMD.</a> Progress in Retinal and Eye Research. 26 (5), 516-554 (2007).</li><li>Dunn, K. C., Aotaki-Keen, A. E., Putkey, F. R., Hjelmeland, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARPE-19,+a+human+retinal+pigment+epithelial+cell+line+with+differentiated+properties.">ARPE-19, a human retinal pigment epithelial cell line with differentiated properties.</a> Experimental Eye Research. 62 (2), 155-169 (1996).</li><li>Salero, E., 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=Adult+human+RPE+can+be+activated+into+a+multipotent+stem+cell+that+produces+mesenchymal+derivatives.">Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives.</a> Cell Stem Cell. 10 (1), 88-95 (2012).</li></ol>

奥利弗. Goureau 是与人类多潜能干细胞产生视网膜细胞有关的未决专利的发明者。其他作者没有什么可透露的。

我们描述了一种生物支架上的 RPE 细胞培养方法及其在动物模型中的植入准备。该协议的关键步骤之一是保持火腿的方向一直沿着过程, 直到它纳入到明胶。事实上, 膜的本机上皮被去除, 其基底膜暴露9。视网膜色素上皮细胞必须在基底膜的顶端播种。在制备明胶时, 必须在规定的温度下与所有产品配合使用。事实上, 明胶的性质是刚性在4°c 和液体在体温 (37 °c)9。如果温度不受尊重, 明胶可以在不需要这种效果的步骤中凝固或液化。
一些生物支架已经提出, 像后弹力层的膜25或火腿26。特别是, 从剖宫产27的火腿, 被证明是良好的耐受性在视网膜下空间, 造成有限的炎症和减少脉络膜新生血管26。该膜成功地支持人类视网膜色素上皮细胞的培养9,28。此外, 这些膜也有悠久的历史, 在29诊所, 使他们的良好候选支架的视网膜色素细胞治疗。其他生物支持可以很容易地实现与本议定书。基于聚合物的合成脚手架已经是刚性的, 在植入13、30、31、32、33之前可能不需要凝胶嵌入。其他植入的系统最近已经开发, 以视网膜下分娩的人眼中的人类胚胎干细胞衍生的 RPE 单层在刚性聚酯脚手架34或在合成派瑞林基板设计模仿刷的膜35.尽管这些策略是有希望的, 但我们相信生物支架可以为视网膜色素组织工程提供最佳的平台。
本协议采用自发分化的方法, 从人类 ES 细胞中提取出种子视网膜色素上皮细胞。然而, 不同类型的 RPE 细胞可以使用, 无论是区别于人类诱导的多潜能干细胞或从从尸体获得的主要视网膜色素细胞, 甚至从视网膜色素细胞19,36,37, 38。此外, 如果使用多能干细胞, 在过去几年中开发了一些协议, 以获得基于自发分化的 RPE 细胞或使用小分子引导分化18,22。从这些不同的分化协议获得的 RPE 细胞也可以用这种方法进行组织工程。
这种方法的一个限制是在4°c 的嵌入组织的稳定性。由于它被包括在明胶刚刚装运到手术现场, 它必须保持在这个温度, 直到植入。在这方面, 手术应在48小时内进行。
这种方法可以很容易地转移到诊所。在2独立培养基中, 嵌入在明胶中的 RPE 组织保存在4摄氏度。如果需要, 此种媒介可以被美国食品和药物管理局 (FDA) 批准, 用于保护经验尸后获得的角膜, 并用于移植到人类体内。我们成功地证明了这一策略的有效性, 移植在一个概念验证的研究到啮齿类动物模型9, 我们目前正在验证的手术方法在非人类灵长类, 然后实施它的临床试验治疗特定形式的 RP 影响视网膜色素的患者。

RPE 是至关重要的生存和稳态的感光细胞, 它是紧密关联1。一些病理状况改变其功能和/或生存, 包括 RP 和 AMD。
RP 是一组遗传性的基因突变, 影响感光细胞或视网膜色素的功能, 或2,3。据估计, 特别影响视网膜色素上皮细胞的突变占5% 的 RP2。AMD 是另一种情况下, 视网膜色素层被改变, 最终导致中心视力损失。AMD 是由遗传和环境因素复杂的相互作用引起的, 影响老年人4,5,6。根据预测, AMD 将是全球1亿9600万名患者的关注, 到 2020年7。对于这些疾病, 目前还没有有效的治疗方法, 建议的策略之一是移植新的视网膜色素细胞, 以弥补死/非功能性视网膜色素上皮细胞8。
最终产品的配方, 是必不可少的, 以确保最佳的功能效果。RPE 细胞作为细胞悬浮液注入, 尽管是一种简单而直接的分娩方法, 但对其生存、整合和功能9、10、11、12等问题提出了关注。,13. 科学家现在正在开发更复杂的配方, 以提供工程化的视网膜组织9、13、14、15、16。在这种情况下, 我们开发了一种原始的方法来生成体外视网膜色素组织, 可用于移植9。
从人类胚胎干细胞中提取的 RPE 细胞库用于本协议。然而, 不同细胞源 (人诱导的多潜能干细胞、原发性视网膜色素上皮细胞等) 的替代视网膜色素细胞库也适用于该协议。它包括定向分化协议使用细胞因子和/或小分子17,18,19,20,21,22。
要移植, 工程组织应在脚手架上准备。在过去的几年中, 不同的脚手架是基于聚合物或生物起源13,23,24的矩阵开发的。在这里, 使用的生物基质是火腿, 但其他基质, 如裸露的刷膜, 可以实施。此处描述的方法具有使用与 RPE 本地环境更相关的生物支架的优点。
人类 ES 细胞源性视网膜色素上皮细胞培养至少4周, 以充分组织为鹅卵石单层。在该阶段, 获得的上皮细胞功能和极化9。最后, 由于这种组织容易皱纹, 它嵌入在薄层的水凝胶载体, 使其更刚性和弹性, 并在注射过程中保护它。该产品然后存储在4°c, 直到嫁接。

<table>
	<tbody>
		<tr>
			<td>Sterile biosafety cabinet</td>
			<td>TechGen International</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid waste disposal system for aspiration</td>
			<td>Vacuubrand</td>
			<td>BVC 21</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2-controlled +37&#160;&#176;C cell incubator</td>
			<td>Thermo Electron Corporation</td>
			<td>BVC 21 NT</td>
			<td></td>
		</tr>
		<tr>
			<td>200&#160;&#181;L pipette: P200</td>
			<td>Gilson</td>
			<td>F144565</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#160;mL pipette: P1000</td>
			<td>Gilson</td>
			<td>F144566</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet aid</td>
			<td>Drummond</td>
			<td>75001</td>
			<td></td>
		</tr>
		<tr>
			<td>+4&#160;&#176;C refrigerator</td>
			<td>Liebherr</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>WPI</td>
			<td>501758</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (x2)</td>
			<td>WPI</td>
			<td>555227F</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Grant subaqua pro</td>
			<td>SUB6</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision balance</td>
			<td>Sartorius</td>
			<td>CP225D</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorff</td>
			<td>5804</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>SC30</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal Rocking Shaker</td>
			<td>IKA-WERKE</td>
			<td>IKA MTS 214D</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>VWR</td>
			<td>LAB DANCER S40</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel</td>
			<td>WPI</td>
			<td>500351</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic paraffin film</td>
			<td>VWR</td>
			<td>PM992</td>
			<td></td>
		</tr>
		<tr>
			<td>0.200 &#181;m single use syringe filter</td>
			<td>SARTORIUS</td>
			<td>16532</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe without needle 50 mL</td>
			<td>Dutscher</td>
			<td>50012</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottles 250mL</td>
			<td>Dutscher</td>
			<td>28024</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL sterile Falcon tubes</td>
			<td>Dutscher</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile Falcon tubes</td>
			<td>Dutscher</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>culture insert</td>
			<td>Scaffdex</td>
			<td>C00001N</td>
			<td></td>
		</tr>
		<tr>
			<td>60&#160;mm cell culture disches: B6</td>
			<td>Dutscher</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well cell culture plate</td>
			<td>Corning</td>
			<td>3512</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Ted Pella, Inc</td>
			<td>121-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue</td>
			<td>Castorama</td>
			<td>3178040670105</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X (500&#160;mL)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermolysine</td>
			<td>Roche</td>
			<td>5339880001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX</td>
			<td>Invitrogen</td>
			<td>61965-026</td>
			<td></td>
		</tr>
		<tr>
			<td>KSR CTS (KnockOut SR XenoFree CTS)</td>
			<td>Invitrogen</td>
			<td>12618-013</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM-NEAA (100X)</td>
			<td>Invitrogen</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>b-mercaptoethanol (50&#160;mM)</td>
			<td>Invitrogen</td>
			<td>31350-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2-independent medium</td>
			<td>GIBCO</td>
			<td>18045-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>MERCK</td>
			<td>104078</td>
			<td></td>
		</tr>
		<tr>
			<td>human amniotic membrane</td>
			<td>Tissue bank St Louis hospital (Paris, France)</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
	</tbody>
</table>

engineering transplantation-suitable retinal pigment epithelium tissue derived from human embryonic stem cells

眼睛的几个病理条件影响视网膜色素上皮 (RPE) 的功能和/或生存。这包括一些形式的视网膜色素变性 (RP) 和年龄相关的黄斑变性 (AMD)。细胞治疗是建议治疗这些疾病的最有希望的治疗策略之一, 在人类身上已经有了令人鼓舞的初步结果。然而, 移植物的制备方法对其在体内的功能结果有显著的影响。事实上, 作为细胞悬架移植的视网膜色素上皮细胞的功能比作为视网膜组织移植的细胞要少。在此, 我们描述了一个简单的和可重复的方法来设计视网膜色素组织和它的准备在体内植入。从人类多潜能干细胞中提取的视网膜色素细胞是在生物支持下播种的, 即人羊膜 (火腿)。与人工支架相比, 这种支持有一个基底膜接近刷的膜, 其中内源性视网膜色素细胞附加的优势。然而, 它的操作不容易, 我们制定了一些策略, 以适当的培养和准备移植在体内。

火腿含有上皮层, 应在视网膜色素细胞播种前除去。用 thermolysin 在震动下对膜进行酶处理。为了不失去膜的极性 (上皮是在一侧), 它是固定在一个支持, 组成可能会有所不同取决于提供者 (图 1A)。在本步骤中检查膜的附着力, 并在必要时添加剪辑。在固定的文化插入时, 要小心地避免在膜上制造孔, 并保持其极性与基底膜朝上 (图 1B)。当细胞被播种在细胞膜上时, 它们可以从这些孔中逸出, 最终细胞浓度会减少。孔的存在可以可视化显微镜下或添加 pbs 内的文化插入和检查 pbs 是否泄漏。任何有孔的膜都应丢弃。
在对培养基进行固定后, 对相衬显微镜中残留细胞的存在进行评估 (图 1C和1D)。经典的, 一些死细胞可能留在膜表面, 但这些细胞将被淘汰时, 培养基改变 (图 1C)。如果没有细胞残留, 基底膜的纤维可以在更高的放大倍数 (图 1D) 中看到。如果情况并非如此, 则可以调整与 thermolysin 的孵化时间。
在视网膜色素细胞播种后的日子里, 检查细胞是否坚持。根据所使用的显微镜, 在最初的几周内很难清楚地看到细胞, 但是如果细胞不粘, 细胞就会在细胞培养基中漂浮。一旦上皮形成并开始成熟, 它就会变得更容易在显微镜下看到它 (图 2A、 2B和2C)。在3周, 细胞形成一个完整的单层上皮典型的视网膜色素 (鹅卵石组织)。4周后, 上皮细胞足够成熟, 为其植入准备 (图 2D)。然而, 由于后勤方面的原因, 它可以在文化中保留更多的星期。
图 3描述了移植物的制备方法。在膜同位语20% 明胶块后, 吸入所有多余细胞培养基。这一步是重要的, 因为任何剩余的培养基可以阻止8% 明胶与视网膜色素和膜的黏附。事实上, 培养基会在明胶层之间形成液体膜。
用于埋植植入物的明胶可以有不同的强度, 这取决于其开花指数 (即由供应商进行的质量控制测试, 在标准温度下评估凝胶的强度和浓度)。如果所用的明胶在嫁接过程中没有足够的刚性和松散, 则应将所用的明胶参照改成另一种强度较高的。在实验的基础上, 也可以改变明胶的浓度, 以调整其强度和弹性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58216/58216fig1.jpg"> 图 1: 在 thermolysin 治疗和固定后的火腿的代表性图像在一个文化插入.(a) 组织银行提供的膜的代表性图像。(B) 固定在文化插入物上的膜的代表性图像。(C) thermolysin 用低放大倍数处理的膜的代表性图像。(D) thermolysin 用高放大倍数处理的膜的代表性图像。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58216/58216fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58216/58216fig2.jpg"> 图 2: 在给视网膜色素细胞播种时火腿的代表性图像.(A) 播种前膜的代表性图像。(B和C) 在播种后3周的细胞膜上视网膜色素上皮细胞的代表性图像。膜可能不是完全平面的, 如面板C所示。缩放条 = 100 µm (C), 20 µm 放大倍数。(D) 在播种后30天的膜上视网膜色素上皮细胞的代表性图像, 对应于将其嵌入明胶的时间。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58216/58216fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58216/58216fig3.jpg"> 图 3: 描述了在明胶膜内的火腿内含有视网膜色素上皮细胞层的工程化视网膜组织的顺序步骤的方案.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58216/58216fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells at JoVE.com

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

从人胚胎干细胞中提取合适的视网膜色素上皮组织的工程移植

我们描述了一种由人类羊膜顶部培养的人多能干细胞的视网膜色素上皮细胞组成的视网膜组织的方法及其在动物模型中的移植准备。

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

engineering-transplantation-suitable-retinal-pigment-epithelium

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.6K 次观看.  Institut National de la Santé et de la Recherche Médicale (INSERM). 我们描述了一种由人类羊膜顶部培养的人多能干细胞的视网膜色素上皮细胞组成的视网膜组织的方法及其在动物模型中的移植准备。

视频: Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

从胚胎干细胞衍生心脏祖细胞

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

科研

发育生物学

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

视网膜色素上皮细胞的干细胞高效的推导

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

The objective of this report is to describe the protocols to derive the retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells using different sizes of embryoid bodies.

从诱导多能干得出视网膜色素上皮（RPE）（IPS），由胚体大小不同的细胞

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

为内胚层细胞从人类胚胎干细胞系生成的分离纯化一种快速有效的方法

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

多能干细胞衍生的心肌修复心肌细胞

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

分化软骨细胞与外周血源性人诱导多能干细胞

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

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 ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...