这项工作得到了德国联邦教育和研究部 (FKZ 0312138A 和 FKZ 316159)、国家与欧盟结构基金 (ESF/IVWM-B34-0030/10 和 ESF/IVBM-B35-0010/12) 和 DFG (DA1296/2-1) 的联合。德国心脏基础 (F/01/12), BMBF (VIP + 00240) 和潮湿的基础。此外, 卡尔亨利和下午还得到了罗斯托克大学医疗中心的 FORUN 计划 (889001) 的支持。

胸骨人类细胞分离是从知情的捐赠者那里获得的, 他们书面同意使用他们的样本进行研究, 根据《赫尔辛基宣言》。罗斯托克大学的道德委员会批准了这项研究 (注册号: 2010 23, 延长于 2015年)。
1. 细胞制剂
注: 使用肝素钠 (250 毫升) 防止凝固的 bm 检查。
<ol><li>CD133+ SC 隔离
	<ol>
<li>准备所需的解决方案<ol>
<li>将996毫升 1x PBS 与4毫升 EDTA (0.5 米) 混合, 制备磷酸盐缓冲盐水 (PBS)/乙二胺二乙酸 (EDTA) 库存溶液 (2 毫米)。用0.45 µm 过滤器大力混合溶液和滤液。贮存在室温下 (RT)。</li>
			<li>将995毫升2毫米 PBS/EDTA 与5克牛血清白蛋白 (BSA) 混合, 制备 mac 缓冲液。用0.45 µm 过滤器大力混合溶液和滤液。存储在4摄氏度。</li>
			<li>在12.5 毫升的 PBS 中稀释500毫克胶原酶 b (从histolyticum 梭菌中), 制备胶原酶 b 库存溶液 (40 毫克/毫升)。大力搅拌解决方案, 使整除数350µL, 并存储在-20 摄氏度。</li>
			<li>准备脱氧核糖核酸酶 (DNAse) 我的股票溶液 (10 毫克/毫升) 稀释100毫克的 DNAse I (从牛胰腺) 在10毫升的 PBS。大力搅拌解决方案, 使整除数350µL, 并存储在-20 摄氏度。</li>
		</ol>
</li>
		<li>酶法消化人类 BM 的研究<ol>
<li>预热的人淋巴细胞培养基到 RT 和解冻酶 (1 整除每一个胶原酶 B 和 DNAse 每20毫升)。</li>
			<li>收集从注射器到50毫升锥形管, 并轻轻混合。丢弃任何现有的血块。 注: 将200µL 未稀释的 BM 转化为1.5 毫升管, 用于随后的流细胞分析。存储在4°c, 直到使用。</li>
			<li>将10毫升 BM 移植到一个新的50毫升圆锥管中, 加入6毫升的 PBS/EDTA (2 毫米), 20 毫升人淋巴细胞培养基, 175 µL 胶原酶 B (40 毫克/毫升) 和175µL DNAse I (10 毫克/毫升)。轻轻地混合溶液和孵化30分钟在 RT 上的振动筛。对剩余的 BM 样本重复。</li>
		</ol>
</li>
		<li>密度梯度离心<ol>
<li>50毫升密度梯度离心管, 应用15毫升的人淋巴细胞分离培养基成50毫升管。离心机在 1000 x g三十年代。</li>
			<li>小心地35毫升稀释的 BM 在密度梯度离心管过滤器之上和离心机在 445 x g在 RT 35 分钟。 注: 离心机设置为低加速度 (3), 无制动器 (1)。</li>
			<li>小心地从离心机上取下管子而不摇晃。小心地从上清溶液中丢弃 ~ 20 毫升, 而不直接触及过滤器顶部的混浊层。</li>
			<li>仔细转移多云层 (这是直接在过滤器的顶部, 并包含单个核细胞 (跨国公司)) 成一个新的50毫升圆锥管, 并填补了 PBS/EDTA 到最后的体积50毫升。 注: 将所有跨国公司从一个 BM 患者样本合并为一个新的管。</li>
			<li>通过将50毫升细胞悬浮液的10µL 转移到1.5 毫升管中, 对跨国公司进行计数。添加10µL 3% 乙酸与亚甲基蓝。轻轻地混合和应用10µL 到一个计数室。计算跨国公司的数量。</li>
			<li>离心机的跨国公司悬浮在 300 x g 10 分钟. 弃上清。 注: 离心过程中, 冷却离心机从 RT 到4°c。</li>
		</ol>
</li>
		<li>人 CD133 抗体联用超顺磁性铁葡聚糖粒子 CD133+ SCs 的磁选 注意: 在这一步, 在冰上工作。存储解决方案, 直到使用4摄氏度。将 mac 永磁体、分离柱和预分离过滤器存放在摄氏4摄氏度。<ol>
<li>选择列大小。对于 < 1.2 x 108跨国公司, 使用 MS 列, 并为 > 1.2 x 108跨国公司, 使用 LS 列 (见材料表)。</li>
			<li>要准备 1 x 108总细胞数的磁选, 小心地并用重悬跨国公司在300µL mac 缓冲器 (4 °c)。添加100µL FcR 阻断试剂 (4 °c) 和100µL CD133 抗体挂钩超顺磁性铁葡聚糖颗粒 (4 °c)。</li>
			<li>轻轻地混合细胞悬浮和孵育30分钟在4摄氏度。在孵化期间轻轻摇动细胞悬浮液 (2–3x)。</li>
			<li>添加2毫升的 mac 缓冲器 (4 °c) 每 1 x 108全跨国公司. 在300摄氏度时, 将细胞悬浮液和离心机在 10 x g处轻轻混合, 4 分钟。</li>
			<li>设置 mac 磁铁持有人, 并附上 mac 永磁。安装 mac 列并在顶部应用预分离过滤器。</li>
			<li>平衡第一个 mac/LS 柱和预分离过滤器, 其0.5 毫升 (MS) 或3毫升 (LS) 的 mac 缓冲器 (4 °c)。 注: 不允许 mac 列在平衡后干燥。</li>
			<li>在500µL 的 mac 缓冲器 (4 °c) 中, 去掉上清和并用重悬的颗粒, 每 1 x 108个总细胞量。将电池悬浮液应用于预分离过滤器。</li>
			<li>清洗 mac 柱和预分离过滤器三次使用, 每洗, 0.5 毫升 (MS) 或3毫升 (LS) 的 mac 缓冲器 (4 °c)。 注: 在第三次洗涤步骤中, 将第二个 mac 列安装到 mac 永磁体中, 并平衡第二个 mac/ls 列, 其0.5 毫升 (ms) 或3毫升 (ls) mac 缓冲器 (4 °c)。</li>
			<li>丢弃预分离过滤器。</li>
			<li>将单元分数直接洗脱到第二个 mac 列上: 在第一个 mac 列上添加1毫升 (MS) 或5毫升 (LS) mac 缓冲器 (4 °c), 并从 mac 永磁体中取出 mac 列。立即将第一个 mac 列转移到第二个 mac 列上方, 然后使用所提供的柱塞将电池悬浮通过 mac 列。</li>
			<li>清洗 mac 柱三次, 每个洗涤0.5 毫升 (MS) 或3毫升 (LS) mac 缓冲器 (4 °c) 使用。</li>
			<li>洗脱将1毫升 (MS) 或5毫升 (LS) mac 缓冲器 (4 °c) 添加到第二个 mac 列上, 并从 mac 永磁体中移除 mac 列, 从而将单元格分数从该列中剔除。立即将第二个 mac 列转移到1.5 毫升管 (MS) 或15毫升圆锥管 (LS) 上, 然后使用所提供的柱塞将电池悬浮通过 mac 柱。</li>
			<li>离心机在 300 x g 10 分钟在4摄氏度。小心丢弃上清液, 并用重悬100µL 中获得的颗粒 (4 °c)。</li>
			<li>数 CD133+细胞: 将细胞悬浮液的6µL 转移到1.5 毫升管中。添加6µL 台盼蓝溶液 (0.4%)。轻轻地混合和应用10µL 到一个计数室。计算 CD133+单元格的数目。</li>
			<li>将 CD133+细胞储存在冰上直到播种。</li>
		</ol>
</li>
	</ol></li>
	<li>新鲜隔离 CD133 的表征 注: 以下工作应在一个房间内进行保护, 从明亮的灯光 (仅昏暗照明)。在冰上保留管子、缓冲器和抗体, 除非另有说明。<ol>
<li>用于流细胞分析的 SCs 的制备<ol>
<li>使用两个样本 (每10µL) 的 BM 整除数和一个样本的新鲜隔离 CD133+ SCs (最小, 1 x 104细胞) 进行分析。把细胞转移到1.5 毫升的管子里。添加10µL 的 FcR 阻断试剂, 并填写与 mac 缓冲器 (4 °c) 的体积33µL。</li>
			<li>将下列抗体添加到各自管的内侧 (见表 1): anti-CD34-fluorescein 异硫氰酸酯 (FITC) (克隆: AC136), anti-CD133/2-phycoerythrin (PE) (克隆: 293C3), 同种控制小鼠 IgG 2 b-PE,anti-CD45-allophycocyanin (APC)-H7 (克隆: 2D1) 和 7-aminoactinomycin (反倾销)。在所有的抗体添加后, 摇下管的抗体侧。轻轻混合溶液 (最终体积50µL) 和孵化10分钟在4摄氏度。</li>
			<li>添加1毫升的1x 红细胞 (红细胞) 裂解缓冲液, 轻轻混合悬浮液, 在冰上孵育10分钟. 离心机悬浮在 300 x g在4°c 10 分钟。</li>
			<li>在100µL PBS (4 °c) 中丢弃上清和并用重悬得到的颗粒。储存在冰上直到流细胞分析。</li>
		</ol>
</li>
		<li>CD133 的流细胞分析<ol>
<li>为了检查 CD133+ SC 的细胞活力和纯度, 使用一个适应国际社会的 Hematotherapy 和接枝工程 (ISHAGE) 流式细胞术门策略41 (见图 1中的代表性描述)。使用以下顺序: 步骤 1: 单元格填充的选择 (图 1A) 步骤 2: 选择 CD45+单元格 (图 1B) 步骤 3: 选择可行的 CD45+单元格 (图 1C) 步骤 4: 选择可行的 CD45+/CD34+单元格 (图 1D) 步骤 5: 选择可行的 CD45+/CD34+/CD133+单元格 (图 1E) 根据制造商的说明运行流式细胞仪 (见材料表)。</li>
			<li>使用以下等式计算细胞的生存能力和纯度:</li>
		</ol>
</li>
	</ol></li>
</ol><img alt="Equation 1" src="/files/ftp_upload/57474/57474eq1v2.jpg">   等式1
<img alt="Equation 2" src="/files/ftp_upload/57474/57474eq2v2.jpg">   等式2
<ol start="3"><li>
新鲜分离 CD133 的细胞培养
	<ol>
<li>整除100x 重组人细胞因子补充在100µL 整除数和存储在-20 摄氏度。在培养基准备前解冻一100x 整除。</li>
		<li>制备 CD133+ SC 培养基, 无血清造血细胞扩张培养基, 辅以重组人细胞因子补充剂 (最终 1x) 和1% 青霉素/链霉素。</li>
		<li>种子 5 x 104新鲜隔离 CD133+ SCs 转染在24井板与500µL 培养基。孵育细胞在37°c, 5% CO2和 20% O2。 注: 种子 5 x 104新鲜隔离 CD133+ SCs 作为控制流细胞分析。</li>
	</ol>
</li>
</ol>2. 转染复合物的制备
注意: 在这一步, 保持整个工作空间核糖核酸 (RNAse) 免费使用 RNAse 净化解决方案, 并只使用无 RNAse 耗材。
<ol><li>
和平号的准备工作 注: 以下工作应在一个房间内进行保护, 从明亮的灯光 (仅昏暗照明)。 注: 对于和平号-标本: 使用菁3染料标记的前体和平号和未标记的前体和平号。<ol>
<li>要准备和平号库存解决方案 (50 µM), 请在适当数量的无核酸酶水中稀释所需数量的和平号 (例如, 在100µL 水中稀释 5 nmol 和平号)。轻轻地混合解决方案, 整除和平号股票 (5 µL), 并存储在-20 摄氏度以下。</li>
	</ol>
</li>
	<li>
培贝的制备
	<ol>
<li>按照标准协议准备裴, 并注意裴库存浓度31,42 。</li>
	</ol>
</li>
	<li>
准备自然人流动
	<ol>
<li>按照标准协议准备 MNPs, 并注意自然人流动库存浓度31,42。</li>
	</ol>
</li>
	<li>
制备和平号/培配合物
	<ol>
<li>在无核酸酶的水中稀释 d-(+) 葡萄糖, 制备5% 葡萄糖溶液。轻轻地混合溶液, 整除的股票 (1.5 毫升), 并存储在4摄氏度。 注: 以下工作应在一个房间内进行保护, 从明亮的灯光 (仅昏暗照明)。</li>
		<li>使用 20 pmol 和平号每井24井板30。在5% 葡萄糖溶液中稀释和平号, 最终浓度为 0.25 pmol 和平号/µL。</li>
		<li>根据裴库存浓度 (步骤 2.2.1, 计算基于和平号 (20 pmol、步进 2.3.1) 和最佳氮气 (pei) 与磷酸 (N/P) 比值的 pei 所需量; 在这里, 7.5 的比率被使用了)30。根据等式稀释5% 葡萄糖溶液中的裴: <img alt="Equation 3" src="/files/ftp_upload/57474/57474eq3v2.jpg"> 重新排列以 <img alt="Equation 4" src="/files/ftp_upload/57474/57474eq4v2.jpg">    等式3 其中裴体积在µL 和 [裴] 是毫米, 和 N/P (为本协议优化) 是0.75。0.12 是一个特定于和平号的转换因子。</li>
		<li>将预稀释的裴加入预稀释的和平号和涡旋三十年代。</li>
		<li>在 RT 中孵化30分钟的和平号/裴复合体。</li>
	</ol>
</li>
	<li>
准备和平号/裴/自然人流动综合体 注: 以下工作应在一个房间内进行保护, 从明亮的灯光 (仅昏暗照明)。<ol>
<li>在孵化和平号/裴配合物时, 在 sonicating 的 sonicating 水浴中, 用 MNPs 20 分钟的时间准备 MNPs, 以确保粒子悬浮而不聚集。</li>
		<li>根据自然人流动库存解决方案 (步骤 2.3.1) 计算所需 MNPs (3 µg 铁/毫升) 的数量。</li>
		<li>将微气泡 MNPs 加入和平号/裴配合物和漩涡三十年代. 在 RT 中孵化30分钟的和平号/裴/自然人流动综合体。</li>
	</ol>
</li>
	<li>
基于 SIM 的四色标记转染复合物的制备 注: 使用未标记的前体和平号。<ol>
<li>按照制造商的说明, 使用菁5染料色标套件 (见材料表), 用菁5染料标记和平号。使用列出的分光光度计 (见材料表), 按照制造商 (核酸, RNA-40) 提供的预先设定量测量最终的和平号浓度。整除和平号 (5 µL) 和存储在-80 摄氏度保护免受光。</li>
		<li>根据制造商的说明, 使用合适的蛋白质标签套件 (见材料表) 将裴与明亮的绿色荧光染料贴上标签。使用茚检测 (步骤 2.2.1) 定义最终的裴浓度。整除 (在根据等式 3计算的容量) 和存放在4°c 保护免受光。</li>
		<li>用 1:1, 000 瓦特/w, 染料和自然人的比例来标记 MNPs 与生物素共轭的罗丹明染料。同时混合罗丹明染料和 MNPs, 同时配合和平号/裴复杂地层的制备, 在黑暗中孵化出30分钟的溶液。直接使用标记的 MNPs。</li>
	</ol>
</li>
</ol>3. CD133+ SCs 的转染
注意: 在这一步, 保持整个工作空间核糖核酸 (RNAse) 免费使用 RNAse 净化解决方案和使用仅 RNAse 耗材材料 注: 以下工作应在一个房间内进行保护, 从明亮的灯光 (仅昏暗照明)。
<ol><li>将准备好的 miRNA/裴/自然人流动的复合物直接放入适当的井中, 在培养基中含有新鲜隔离的 CD133+ SCs。</li>
	<li>轻轻地摇动盘子来回搅拌。孵育细胞为18小时在37°c, 5% CO2和 20% O2。</li>
</ol>4. 对和平号转染的分析
<ol><li>
对转染复合物的吸附效率和细胞毒性的检测 注: 对于和平号标本: 使用菁3染料标记的前体和平号的吸收评价和非转染 CD133+ SCs 为流式细胞术控制门。 注: 以下工作应在一个房间内进行保护, 从明亮的灯光 (仅昏暗照明)。 注意: 除非另有说明, 否则将管子、缓冲器和抗体放在冰上。<ol>
<li>在100毫升 PBS 中稀释4克粉煤灰, 并将溶液加热至80摄氏度, 以制备多聚甲醛溶液 (粉煤灰, 4%)。大力搅拌溶液, 将 pH 值调整为7.3。整除的粉煤灰 (1.5 毫升) 和存储在-20 摄氏度。</li>
		<li>18小时后转染, 收集每个井在一个单独的1.5 毫升管。用500µL 的 PBS 冲洗一次井, 将这个细胞悬浮液转移到同一管上。</li>
		<li>离心机在 300 x g 10 分钟在4摄氏度。在100µL 的4°c mac 缓冲器中, 丢弃上清和并用重悬得到的颗粒。</li>
		<li>添加0.5 µL 胺反应染料, 以区分活细胞和死体细胞, 轻轻混合悬浮, 并孵化10分钟4摄氏度。增加1毫升 PBS (4 °c) 和离心机在 300 x g 10 分钟在4°c。</li>
		<li>丢弃上清, 并用重悬在100µL PBS 中获得的颗粒, 加入33µL 粉煤灰 (4%), 并贮存在冰上或4摄氏度, 直至流细胞分析。</li>
		<li>根据图 2中的代表性描述安排浇口策略。根据制造商的说明, 在流式细胞仪上运行样品 (见材料表)。</li>
	</ol>
</li>
	<li>
和平号转染 CD133+ SCs 的表征 注: 使用未标记的前体和平号和非转染 CD133+ SCs 用于流式细胞术控制门。<ol>
<li>收集每个井在一个单独的1.5 毫升管和离心机在 300 x g 10 分钟在4摄氏度。</li>
		<li>按照步骤1.2 中的说明使用浇口策略。</li>
	</ol>
</li>
	<li>
模拟转染复合物细胞内分布的检测
	<ol>
<li>准备配合物并染2节和第3节所述的细胞。</li>
		<li>18小时后转染, 收集每个井在一个单独的1.5 毫升管。每次用500µL 的 PBS 冲洗一次, 然后把洗好的管子转移到井的各自管里。离心机在 300 x g 10 分钟在4摄氏度。</li>
		<li>在 PBS 中丢弃上清和并用重悬1毫升2% 胎牛血清 (血清) 中获得的颗粒, 轻轻混合悬浮液, 离心机在 300 x g处为10分钟, 4 摄氏度。</li>
		<li>放弃上清, 并用重悬100µL 中获得的颗粒, 并添加33µL 粉煤灰 (4%) 20 分钟。</li>
		<li>将细胞悬浮液转移到含有无菌玻璃盖玻片的新的24培养板中, 离心机在 300 x g处为10分钟, 4 摄氏度。</li>
		<li>丢弃上清, 并添加500µL 的 PBS。轻轻摇动盘子, 丢弃 PBS。</li>
		<li>将准备好的盖玻片放在显微幻灯片上, 使用水上安装培养基保存荧光, 并在 RT 上干燥至少1小时。</li>
		<li>使用100X 油浸泡目标获取图像。SIM 设置: 405、488、561和633毫微米激光线为励磁和 z 栈以16位深度在3个角度, 3 个阶段, 以平均 4, 网格每激光线:23 µm-405, 34 µm – 488, 42 µm-561, 51 µm-633。</li>
	</ol>
</li>
</ol>

作者声明没有利益冲突。

近年来, CD133+ SCs 已成为一个有希望的细胞群体的 SC 为基础的治疗, 证明了几个阶段 I, II 和 III 临床试验43,44,45,46,47,48,49,50,51,52,</sup...

CD133是一种异种茎和祖细胞种群, 具有良好的再生医学潜力。他们的造血, 内皮和肌分化电位1,2,3使 CD133+细胞,例如, 通过分化促进血管新生过程通过分泌机制4,5,6,7进入新形成的血管和激活支持血管生成信号。
尽管在30多项经批准的临床试验 (ClinicalTrails.gov) 中表现出很高的潜力, 但其治疗结果仍处于争议性讨论4。事实上, 临床应用的 SCs 是阻碍了低滞留在器官的利益和大规模的初始细胞死亡5,8,9。额外的工程 CD133+ SCs 前移植可以帮助克服这些挑战。
有效细胞治疗的一个先决条件是减少大量的初始细胞死亡, 以提高治疗相关细胞的植入10。目前的研究表明, 在高度灌注的器官, 如大脑和心脏在第一 90–99%, 独立于移植细胞类型或应用路线11,12,13 , 巨大的细胞损失。,14,15,16,17,18,19,20,21。SC 标记使用磁性纳米粒子 (MNPs), 使一个创新的无创战略的目标细胞到感兴趣的地点22,23,24,25,26同时允许使用 MRI27和磁性粒子成像 (MPI) 进行细胞监测。最有效的体内研究应用磁化细胞靶向使用的细胞保留在地方管理以后倾向于细胞教导在静脉注射以后23,24,28.因此, 我们的小组设计了一个交付系统由超顺磁性氧化铁纳米粒子29组成。通过这种技术, CD133+ SCs 和人脐静脉内皮细胞 (血管内皮细胞) 可以有效地被靶向, 如在体外尝试30,31证明。
SC 疗法的另一个障碍是被影响的组织的敌对炎症环境在移植以后, 导致最初的细胞死亡32。除几项预适应研究外, 对治疗相关的大鹏湾的应用进行了33项试验;成功地证明, 抗凋亡的大鹏湾抑制体外凋亡, 增强细胞植入在体内33。这些由20–25核苷酸组成的小分子, 扮演信使 rna 转录后水平调制器 (基因) 的关键角色, 从而影响干细胞的命运和行为34。此外, 对大鹏湾的外源引入避免了不理想的稳定集成到宿主基因组34。
目前试图有效地引入核酸 (NAs) 到初级 SCs 主要是基于重组病毒8,35。尽管高转染效率, 重组病毒操作是一个主要的障碍, 对床边的翻译,例如, 无法控制的基因表达, 致病性, 免疫原学, 和插入诱变35 ,36。因此, 非病毒传递系统, 如聚合物基结构是至关重要的发展。其中, 聚乙烯亚胺 (裴) 代表了一个有效的运载工具, 为大鹏湾提供好处, 如 NA 冷凝, 以防止退化, 细胞摄取, 并通过细胞膜逃逸37,38的胞内释放。此外, 和平号配合物在临床试验中表现出高度的生物相容性39。因此, 我们的交付系统包括一个生物素化分支 25 kDa 裴绑定到一个链亲和素涂层的自然人-核心30,31,40。
在这篇手稿中, 我们提出了一个全面的协议, 描述 (i) 人工隔离 CD133+ sc 从人骨髓 (BM) 捐赠与 sc 产品的详细描述和 (ii) 一个有效和温和的转染策略的基于磁性非病毒聚合物的 CD133 遗传工程输送系统的应用.CD133+ SCs 是孤立和磁性丰富的人胸骨 BM 吸出物使用表面抗体为基础的磁性活化细胞分拣 (mac) 系统。然后利用流式细胞仪对细胞活力和细胞纯度进行分析。随后, 和平号/裴/自然人流动综合体已准备就绪, CD133+ SCs 被转染。18 h 转染后, 分析了其吸收效率和转染对 SC 标记表达和细胞活力的影响。此外, 利用四色标记和结构光照显微镜 (SIM) 对转染复合物的胞内分布进行了评价。

<table><tbody><tr><td>7-AAD</td><td>BD Biosciences</td><td>559925</td><td></td></tr><tr><td>Acetic Acid with Methylene Blue</td><td>Stemcell Technologies</td><td>7060</td><td>3%</td></tr><tr><td>anti-CD133/2-PE (clone: 293C3)</td><td>Miltenyi Biotec GmbH</td><td>130-090-853</td><td></td></tr><tr><td>anti-CD34-FITC (clone: AC136)</td><td>Miltenyi Biotec GmbH</td><td>130-081-001</td><td></td></tr><tr><td>anti-CD45-APC-H7 (clone: 2D1)</td><td>BD Biosciences</td><td>560178</td><td></td></tr><tr><td>rhodamine dye; Atto 565 dye conjugated to biotin</td><td>ATTO-TEC GmbH</td><td>AD 565-71</td><td></td></tr><tr><td>BD FACS LSRII flow cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BD FACSDiva Software 6.1.2</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich GmbH</td><td>A7906</td><td></td></tr><tr><td>CD133 antibody-linked superparamagnetic iron dextran particles; CD133 MicroBead Kit</td><td>Miltenyi Biotec GmbH</td><td>130-097-049</td><td></td></tr><tr><td>collagenase B</td><td>Roche Diagnostics GmbH</td><td>11088831001</td><td></td></tr><tr><td>counting chamber</td><td>Paul Marienfeld GmbH &amp;amp; Co. KG</td><td></td><td></td></tr><tr><td>Cyanine 3 dye labelled precursor miR; Cy3 Dye-Labeled Pre-miR Negative Control #1</td><td>Ambion</td><td>AM17120</td><td></td></tr><tr><td>Cyanine 5 dye miR labelling kit; Cy5 dye Label IT miRNA Labeling Kit</td><td>Mirus Bio</td><td>MIR 9650</td><td></td></tr><tr><td>DNAse I</td><td>Roche Diagnostics GmbH</td><td>10104159001</td><td>(100 U/mL)</td></tr><tr><td>ELYRA PS.1 LSM 780 confocal microscope</td><td>Carl Zeiss Jena GmbH</td><td></td><td></td></tr><tr><td>FcR Blocking Reagent, human</td><td>Miltenyi Biotec GmbH</td><td>130-059-901</td><td></td></tr><tr><td>bright green protein labeling kit; Oregon Green 488 Protein Labeling Kit</td><td>Thermo Fisher Scientific</td><td>O10241</td><td></td></tr><tr><td>aqueous mounting medium; Fluoroshield</td><td>Sigma-Aldrich GmbH</td><td>F6182</td><td></td></tr><tr><td>density gradient centrifugation tube; Leukosep Centrifuge Tube</td><td>Greiner Bio-One</td><td>89048-932</td><td></td></tr><tr><td>MACS magnet holder; MACS MultiStand</td><td>Miltenyi Biotec GmbH</td><td>130-042-303</td><td></td></tr><tr><td>MACS pre-separation filter</td><td>Miltenyi Biotec GmbH</td><td>130-041-407</td><td>30 &amp;micro;m</td></tr><tr><td>MACS separation column (MS / LS)</td><td>Miltenyi Biotec GmbH</td><td>130-042-201 / 130-042-401</td><td></td></tr><tr><td>MACS permanent magnet; MACS Separator</td><td>Miltenyi Biotec GmbH</td><td>130-042-302</td><td></td></tr><tr><td>Millex-HV PVDF Filter</td><td>Merck</td><td>SLHV013SL</td><td>0.45 &amp;mu;m</td></tr><tr><td>mouse IgG 2b-PE</td><td>Miltenyi Biotec GmbH</td><td>130-092-215</td><td></td></tr><tr><td>amine reactive dye; Near-IR LIVE/DEAD Fixable Dead Cell Stain Kit</td><td>Thermo Fisher Scientific</td><td>L10119</td><td></td></tr><tr><td>human lymphocyte separating medium; Pancoll</td><td>Pan Biotech GmbH</td><td>P04-60500</td><td>density: 1.077 g/mL</td></tr><tr><td>PBS</td><td>Pan Biotech GmbH</td><td>P04-53500</td><td>without Ca and Mg</td></tr><tr><td>PEI</td><td>Sigma-Aldrich GmbH</td><td>408727</td><td>branched; 25 kDa</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>100 U/mL, 100 &amp;mu;g/mL</td></tr><tr><td>PFA</td><td>Merck Schuchardt OHG</td><td>1040051000</td><td></td></tr><tr><td>unlabelled precursor miR; Pre-miR miRNA Precursor Negative Control #1</td><td>Ambion</td><td>AM17110</td><td></td></tr><tr><td>RBC lysis buffer</td><td>eBioscience</td><td>00-4333-57</td><td></td></tr><tr><td>RNAse decontamination solution; RNaseZap</td><td>Thermo Fisher Scientific</td><td>AM9780</td><td></td></tr><tr><td>human lymphocyte medium; Roswell Park Memorial Institute (RPMI) 1640 medium</td><td>Pan Biotech GmbH</td><td>P04-16500</td><td></td></tr><tr><td>recombinant human cytokine supplement; StemSpan CC100</td><td>Stemcell Technologies</td><td>2690</td><td></td></tr><tr><td>serum-free haematopoietic cell expansion medium; StemSpan H3000</td><td>Stemcell Technologies</td><td>9800</td><td></td></tr><tr><td>Streptavidin MagneSphere Paramagnetic Particles</td><td>Promega Corporation</td><td>Z5481</td><td></td></tr><tr><td>Trypan Blue solution</td><td>Sigma-Aldrich GmbH</td><td>T8154</td><td>0.4 %</td></tr><tr><td>UltraPure EDTA</td><td>Thermo Fisher Scientific</td><td>15575020</td><td>0.5 M; pH 8.0</td></tr><tr><td>ZEN2011 software</td><td>Carl Zeiss Jena GmbH</td><td></td><td></td></tr><tr><td>NanoDrop 1000 Spectrophotometer</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Sonorex RK 100 SH sonicating water bath</td><td>Bandelin electronic</td><td></td><td>Ultrasonic nominal output: 80 W; Ultrasonic frequency: 35 kHz</td></tr></tbody></table>

protocol for microrna transfer into adult bone marrow-derived hematopoietic stem cells to enable cell engineering combined with magnetic targeting

虽然 CD133+造血干细胞 (SCs) 已被证明在再生医学领域提供了很高的潜力, 他们注射到受伤的组织后保持率低, 以及观察到的大量细胞死亡率导致非常限制治疗效果。为了克服这些限制, 我们寻求在其管理之前为合适的细胞工程学建立一个非病毒基础的协议。microRNA (和平号) 加载的磁性 polyplexes 对人体 CD133+表达 SCs 的修饰涉及吸收效率和安全性, 以及细胞的靶向电位。依靠我们的协议, 我们可以达到高和平号的80–90% 吸收率, 而 CD133+干细胞的性质仍然不受影响。此外, 这些修饰的细胞提供了磁性靶向的选择。我们在这里描述一个安全和高效的程序, 以修改 CD133+ SCs。我们期望这种方法提供一个标准的技术, 以优化治疗干细胞效应, 并通过磁共振成像 (MRI) 监测所管理的细胞产品。

提出的协议描述了人工隔离和磁性丰富的人 BM 衍生 CD133+ SCs 与随后的病毒独立细胞工程战略, 作为一种非侵入性技术的体外细胞操作和在体内监测工具。
这三步隔离技术允许跨国公司从预消化的胸骨 BM 通过密度梯度离心分离。然后, 使用适当的隔离技术, CD133+细胞分数可以被磁性丰富。这允许丰?...

Watch this Scientific Journal Video about Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting at JoVE.com

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

该协议说明了一个安全和有效的程序, 以修改 CD133+造血干细胞。所提出的非病毒, 磁性 polyplex 公司的方法可以为优化治疗干细胞效应, 并通过磁共振成像监测所管理的细胞产品提供了依据。

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

protocol-for-microrna-transfer-into-adult-bone-marrow-derived

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

6.6K 次观看.  Rostock University Medical Center. 该协议说明了一个安全和有效的程序, 以修改 CD133+造血干细胞。所提出的非病毒, 磁性 polyplex 公司的方法可以为优化治疗干细胞效应, 并通过磁共振成像监测所管理的细胞产品提供了依据。

视频: Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

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

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

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 ...

科研

发育生物学

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

人牙小泡干细胞的分离、鉴定及微 rna 遗传修饰

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

遗传学

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h.

非人类灵长类动物造血干细胞和祖细胞的制备及基因修饰

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...

原代骨髓来源的巨噬细胞中精确基因破坏的指南

High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes

Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the physiology of macrophages in vivo more closely than immortalized macrophage cell lines and can be derived from mice already carrying defined genetic changes. However, disrupting gene function in BMDMs remains technically challenging. Here, we provide a protocol for efficient CRISPR/Cas9 genome editing in BMDMs, which allows for the introduction of small insertions and deletions (indels) that result in frameshift mutations that disrupt gene function. The protocol describes how to synthesize single-guide RNAs (sgRNA-Cas9) and form purified sgRNA-Cas9 ribonucleoprotein complexes (RNPs) that can be delivered by electroporation. It also provides an efficient method for monitoring editing efficiency using routine Sanger sequencing and a freely available online analysis program. The protocol can be performed within 1 week and does not require plasmid construction; it typically results in 85% to 95% editing efficiency.

作者聚焦：在骨髓来源的巨噬细胞中进行高效的 CRISPR/Cas9 基因组编辑以实现精确的基因破坏

This protocol describes the procedure for genome editing in mouse bone marrow-derived macrophages using Cas9-sgRNA ribonucleoprotein complexes assembled in vitro and delivered by electroporation.

使用电穿孔Cas9-sgRNA复合物对原代骨髓来源的巨噬细胞进行高效基因破坏

Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

细胞标记和超顺磁性氧化铁纳米粒子靶向

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

生物工程

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

工程人造血干细胞和祖细胞中的致癌杂合功能获得突变

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional ...

癌症研究

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

摘要

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重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

人牙小泡干细胞的分离、鉴定及微 rna 遗传修饰

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

非人类灵长类动物造血干细胞和祖细胞的制备及基因修饰

使用电穿孔Cas9-sgRNA复合物对原代骨髓来源的巨噬细胞进行高效基因破坏

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使用电穿孔Cas9-sgRNA复合物对原代骨髓来源的巨噬细胞进行高效基因破坏

细胞标记和超顺磁性氧化铁纳米粒子靶向

工程人造血干细胞和祖细胞中的致癌杂合功能获得突变

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离