Name: isolation of mesenchymal stem cells from human alveolar periosteum and effects of vitamin d on osteogenic activity of periosteum-derived cells
Uploaded: 2018-05-04T12:30:00.000+00:00
Duration: 6 min 47 s
Description: Watch this Scientific Journal Video about Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells at JoVE.com

该项研究协议已获得长功夫纪念医院 (IRB99-1828B、100-3019C、99-3814B、102-1619C、101-4728B、103-4223C) 临床研究机构审查委员会批准。这项研究得到了长功夫纪念医院 (CMRPG392071、CMRPG3A1141、CMRPG3A1142 和 NMRPG3C0151) 的支持。这篇手稿是由华莱士的学术编辑编辑的。

该项研究协议获昌热心纪念医院机构评审委员会批准。所有与会者均提供书面知情同意。
1. 组织准备
<ol><li>在牙科手术中从病人身上收获骨膜组织 (图 1)。在局部麻醉下皮瓣反射后, 用骨膜分离器31从肺泡骨中取一块骨膜组织。</li>
	<li>收割后, 将5毫米 x 2 毫米的骨膜组织切片贮存在 Dulbecco 磷酸盐缓冲盐水 (DPBS) 中, 300 毫升青霉素和300毫克/毫升链霉素。在24小时内将组织转移到实验室。</li>
	<li>用手术刀将肺泡骨膜组织碎片切碎, 并保持0培养基中的样品 (由300毫升青霉素和300毫克/毫升的链霉素, α和5% 胎牛血清 [血清]) 在37摄氏度培养的孵化器湿润空气 (5% 二氧化碳)。在孵化器中, 准备一个水盆来保持湿度。</li>
	<li>3天后, 每周两次改变培养基。</li>
	<li>当细胞达到 subconfluence (80%), 释放黏附细胞与200µL 0.25% 胰蛋白酶在孵化器 (37 °c) 为3分钟, 然后使用4.5 毫升培养基终止反应。</li>
	<li>在新鲜的通道1培养基 (α, 10% 血清, 100 单位/毫升青霉素, 100 微克/毫升链霉素) 再次板块细胞。板是 5 x 103单元格 (按 hemocytometer 计数)。</li>
	<li>在单元格实现80% 汇流31之后, 执行每个后续通道。 注: 在开始时, 培养基将为橙色红色。大约三天后, 中等颜色应该变成黄色的阴影。细胞的加倍时间大约 30–40 h, 需要7天为3–5世代。</li>
	<li>培养分离的 PDCs 在α记忆补充与10% 个血清, 100 U/毫升青霉素和100毫克/毫升链霉素在孵化器 (37 °c, 5% CO2)。</li>
	<li>每天观察细胞生长, 每周两次更换生长培养基。使用第三到第五代细胞进行所有进一步的实验。</li>
</ol>2. 流式细胞仪
<ol><li>通过胰蛋白酶消化, 收获 1 x 106细胞:<ol>
<li>添加200µL 0.25% 胰蛋白酶。3分钟后, 使用4.5 毫升含有血清的培养基来终止胰蛋白酶的反应, 通过离心 (1500 rpm, 5 分钟, 22 °c) 来收集。</li>
		<li>用 1x DPBS 清洗细胞颗粒三次。并用重悬200µL 1x通透缓冲区中的单元格。用 hemocytometer 计数单元格。</li>
	</ol>
</li>
	<li>通过流式细胞术 (荧光活化细胞排序)32检查 PDCs 表达的表达表面标记。使用单克隆抗体对 CD19 (异硫氰酸荧光素 (FITC)), CD34 (FITC)、CD44 (藻红蛋白 [pe])、CD45 (FITC)、CD73 (pe)、CD90 (复方阿司匹林 [APC])、CD146 (pe)、STRO-1 (亚历克斯) 和 HLA-DR (FITC) 32。<ol>
<li>添加抗体的样本和文化在黑暗中的4°c 30 分钟。</li>
		<li>用 1x DPBS 清洗细胞三次。</li>
		<li>用2% 甲醛固定细胞, 进行流式细胞分析32。</li>
	</ol>
</li>
</ol>3. 细胞附着和活性与成骨、脂肪和软骨分化
通过在六-井板上用特异分化培养基培养三通道的骨膜细胞, 诱导细胞分化成成骨、脂肪和软骨血统。
<ol><li>为成骨分化, 在六-井培养板上, 在5000个细胞的密度下培养α-记忆细胞。<ol>
<li>在达到80% 汇合, 培养细胞在媒介包含α记忆, 5% 血清, β甘油 (10 毫米), 10−7 M 地塞米松 (0.1 毫米) 和5毫升抗坏血酸 (100 uM)。培养由α和5% 血清组成的媒介中的阴性控制。</li>
		<li>每周更改两次媒体。</li>
		<li>4周后, 评估细胞的潜能, 通过使用冯科萨检测细胞来鉴别成骨性的血统, 这就区分了在文化33中钙化沉积的存在。<ol>
<li>加10% 福尔马林固定。在30分钟内, 加入5% 硝酸银, 在室温下用紫外线 (紫外线) 照射1小时。</li>
			<li>添加 5% Na2, 因此4四次, 每个反应允许3分钟。最后, 用蒸馏水冲洗细胞两次。 注: 冯科萨染色是一种沉淀反应, 银离子和磷酸盐在酸性物质存在的情况下反应;这种染色技术不特定于钙。在这项研究中, 当被调查的细胞用硝酸银溶液处理时, 钙--由强的紫外光减少-被银替换, 变得可看见作为金属银。</li>
		</ol>
</li>
	</ol>
</li>
	<li>对于脂肪分化, 培养细胞密度为5000细胞每井在六井培养板含有α。<ol>
<li>在实现80% 汇流时, 培养细胞的培养基中含有α, 5%, 0.5 毫米3异丁基1甲基黄嘌呤 (100 毫克/毫升), 1% 青霉素,10 −6 M 地塞米松 (1 毫米), 胰岛素 (5 毫克/毫升) 和消炎痛 (60 毫米)。</li>
		<li>培养由α和5% 血清组成的媒介中的阴性控制。</li>
		<li>6–9周后, 使用油红色 O 染色细胞和突出脂滴, 从而确定存在的脂肪分化33:<ol>
<li>加10% 福尔马林固定。在30分钟内, 用0.5% 油红色 O 在室温下染色10分钟, 然后每一次用60% 分异丙醇清洗细胞三次;最后, 用蒸馏水冲洗细胞。 注: 油红色 O 是一种 lysochrome (脂溶性) 重氮染料, 用于染色中性脂, 主要是三酰甘油, 脂蛋白和胆固醇酯。染料溶解于细胞中的脂滴, 使脂滴变红。</li>
		</ol>
</li>
	</ol>
</li>
	<li>对于软骨细胞分化, 在六井培养板上培养出每一个含5000细胞的井。<ol>
<li>添加含有α-记忆的分化培养基, 5% 血清, 10−7 M 地塞米松 (0.1 毫米), 丙酮酸钠 (100 µg/毫升), 胰岛素转铁蛋白-硒 a (1 x 其), 转化生长因子β (10 ng/毫升) 和5毫升抗坏血酸 (100 µM)。</li>
		<li>培养由α和5% 血清组成的媒介中的阴性控制。</li>
		<li>第4-5 周后, 使用阿利新蓝染色突出酸性多糖, 如多糖或某些类型的粘多糖, 在细胞的软骨, 以确定存在软骨素分化 33.<ol>
<li>加10% 福尔马林固定。在30分钟内, 加入3% 乙酸, 并允许2分钟的反应。随后, 用蒸馏水冲洗三次, 加1% 阿利新蓝8GX 孵化30分钟, 然后用蒸馏水冲洗。最后, 加入3% 醋酸, 清洗3分钟, 然后用蒸馏水冲洗完成。 注意: 染色后, 特定染色的细胞部分会变成蓝绿色, 被称为 "alcianophilic"。</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. 25-羟维生素 D 3 (125-(OH) 2 D 3) 对成骨作用的影响 
<ol><li>将 P3 P5 PDCs 分成六组, 并在6井板上培养不同的培养基:</li>
	<li>阴性组 (α和5% 血清);</li>
	<li>维生素 c 组 (α, 5% 血清, 10 毫米β甘油, 10−7 M 地塞米松 + 100 uM 维生素 c);</li>
	<li>和 10−7 M 125-(OH)2D3组 (α记忆, 5% 血清, 10 毫米β甘油, 10−7 m 地塞米松 + 10−7 m 125-(oh)2D3)。</li>
	<li>在每个井中添加2毫升培养基到一个孵化器 (37 °c), 并每星期更换一次培养基两次。</li>
</ol>5. 反向转录/定量实时聚合酶链反应
<ol><li>在 7 d 的成骨细胞分化后, 使用商业试剂将总 RNA 分离到冰上 (参见材料表):<ol>
<li>每井加1毫升隔离剂。添加200µL 的 bromochloropropane 和离开10分钟产生分层 RNA, 然后离心机在 1万 rpm 15 分钟在4°c。</li>
		<li>离心后, 添加500µL 2 丙醇到500µL 的上清含 RNA。沉淀在冰上的 RNA, 并允许15分钟的反应。</li>
		<li>在做法以后, 离心机在1.2万转每分钟15分钟在4°c, 在之后 RNA 位于管子的底部。随后, 使用1毫升75% 醇洗涤和离心机, 在4摄氏度的8分钟内, 取出上清液。</li>
		<li>去掉上清, 用真空浓缩器从液体底部产生 RNA。用水恢复。</li>
	</ol>
</li>
	<li>用禽 myeloblastosis 病毒逆转录酶反向转录 RNA (1 微克)。设置聚合酶链反应 (PCR) 跑在25°c 为10分钟跟随50°c 为60分钟和然后85°c 为5分钟, 在最后维护在4°c 之前。</li>
	<li>合成一股互补 DNA (cDNA)。用 5 ul 1:10 稀释 cDNA 进行定量 PCR (qPCR)。用底漆为磷酸、BSP、OCN、CBFA1 和 collagen-1 进行定量 rt-pcr (qRT pcr)。 注意: 为了避免信号的 DNA 污染, 在不同的外显子上设计了每个引物的正向和反向序列。</li>
	<li>使用商业 PCR 主组合 (参见材料表) 按照制造商的说明执行 qPCR。设置热循环仪在50°c 为2分钟跟随95°c 10 分钟然后40个周期每个在95°c 十五年代跟随60°c 六十年代。 注: SYBR 协议还需要一个熔化曲线阶段, 这是运行在95°c 十五年代, 60 °c 六十年代, 然后95°c 十五年代。</li>
	<li>规范化的周期阈值的磷酸, BSP, CBFA1, collagen-1 和 OCN 的管家基因 GAPDH。34
</li>
	<li>底漆对在材料表中列出。</li>
</ol>6. 碱性磷酸酶活性
<ol><li>使用前面描述的技术35来评估细胞的碱性酶活性, 该方法将p-硝基苯基磷酸盐转换为p-硝基苯酚;p-硝基苯基磷酸酯是一种磷酸酶基质, 它在去磷酸化时呈黄色, 以 405 nm 波长确定。根据布拉德福德检测确定的总蛋白, 对形成的总的p-硝基苯酚进行规范化。</li>
	<li>比较磷酸酶活性的控制 (α, 5% 血清), 维生素 C (α-记忆, 5% 血清, 10 毫米β甘油, 10−7米地塞米松 + 100 uM 维生素 C), 125-(OH)2D3 (α记忆, 5% 血清, 10 毫米β甘油和 10−7 M地塞米松 + 10−8 M 125-(OH)2D3), 和维生素 c + 125-(OH)2D3 (α记忆, 5% 血清, 10 毫米β甘油, 10−7米地塞米松 + 100 uM 维生素 C + 125-(oh)2D3)组在1、2、3和4周的文化。</li>
	<li>在冰上执行蛋白质提取过程:<ol>
<li>从6井板上刮出细胞, 加入50µL 的溶解缓冲液。随后, 将细胞放在冰上30分钟, 以摧毁细胞并释放蛋白质。</li>
		<li>30分钟后, 离心机在 13000 rpm 4 摄氏度, 15 分钟。离心后, 提取上清液进行贮存和使用。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

最近发展起来的治疗方式, 即组织工程所涉及的 MSCs, 有许多优点。MSCs 是存在于几种组织类型中的多能细胞, 可分化成多种功能性胚层组织细胞37和其他细胞, 如成骨体。
骨膜充当祖细胞的利基, 作为骨骼的丰富的血管供应, 它包裹着38。在我们的研究中, 34 份调查样本中, 20 成功地取得了 MSC 的菌落, 成功的隔离率为58.8%。本研究根据其增殖和?...

虽然近年来已开发了许多相关技术, 但骨重建仍然受到多种限制因素的限制, 估计必要重建的程度往往是不可能的。除了获得良好的长期成功率外, 还需要硬组织增强来实现审美和功能目标。这种手术常用的方法包括自体和同种异体骨移植、xenografting 和异体骨移植。在各种类型的骨移植中, 自体骨移植被认为是最有效的。然而, 供体部位的发病率, 受损的血管, 和有限的组织可用性,1一直是主要的缺点, 自生骨移植。另外, 同种异体骨移植和异基因移植也与疾病传播有关。目前, 人工骨移植广泛用于解决这些问题。然而, 由于缺乏成骨潜能, 临床结局也有很大差异。纤维素等材料与体积波动、感染和强度缺乏有关。
用组织工程学进行骨隆增引起了相当大的兴趣。在这种技术中, 骨髓间充质干细胞 (MSCs) 最初用于促进成骨细胞分化, 然后移植到骨质丢失部位以实现骨修复。本程序目前已应用于细胞治疗。与其他方法相比, 提取有限数量的组织来实现骨重建更简单、更少侵入。
MSCs 作为一种用于牙科再生的细胞疗法的工具的潜在作用, 是各研究小组日益关注的问题之一。研究证实, MSCs 可以区别于以下类型的组织: 骨髓, 脂肪, 滑膜, 周细胞, 小梁骨, 人脐带和牙科组织 2, 3.MSCs 的常见来源包括骨髓、脂肪组织和牙科组织。与脂肪组织和骨髓间的干细胞相比, 牙干细胞的优点是容易获得, 收获后发病率较低。与胚胎干细胞相比, 来自牙科组织的 MSCs 出现 nonimmunogenic, 与复杂的伦理问题没有关联3。
在 2006年, 国际细胞治疗学会建议使用以下标准来识别 MSCs: 首先, mscs 必须能够附着在塑料上。其次, MSCs 必须是阳性的表面抗原 CD105, CD73, CD90 和阴性的标记, 单核, 巨噬细胞, 和 B 淋巴细胞除了造血抗原 CD45 和 CD344。作为最终标准, MSCs 必须能够在体外分化的标准条件下区分成以下三种类型的细胞: 成骨细胞、脂肪细胞和软骨组织4。迄今为止, 已有六种类型的人牙干细胞被分离和表征。第一种是从人牙髓组织中分离出来的, 称为产后牙髓干细胞5。随后, 三种其他类型的牙科干细胞被隔离并被描述为: 脱落的落叶牙齿的干细胞6、牙周韧带7和顶端的乳突8。最近, 牙科卵泡衍生的9, 牙龈组织衍生的10, 口腔芽干细胞 (DBSCs)11, 和根尖囊肿 mscs (hPCy-mscs)12也已确定。
Friedenstein 是第一个定义 MSCs13的。MSCs 具有很高的增殖潜能, 可以在移植前进行区分, 这表明它们是再生过程的理想候选者10。
尽管大多数研究都用骨髓作为干细胞的来源, 但最近14也使用了骨膜衍生细胞 (PDCs)。骨膜比骨髓更容易接近。因此, 在这项技术中, 我们使用肺泡骨膜, 以消除在手术中需要额外的切口和减少手术发病率的病人。骨膜是形成长骨骼外衬的结缔组织, 包括两个不同的层: 由成纤维细胞、胶原蛋白和弹性纤维组成的外层纤维层15, 直接接触的内富细胞形成层骨表面。形成层包含混合细胞种群, 主要是成纤维细胞16, 成骨细胞17, 毛细血管18, 以及一个关键亚群, 确定为 MSCs19,20,21。大多数研究报告说, PDCs 是可比的, 如果不是优越, 骨髓源干细胞的骨骼愈合和再生22,23,24。骨膜容易接近, 具有良好的再生效果。然而, 很少有研究聚焦于骨膜25、26、27。
在骨修复方面, 目前的临床实践涉及在支持性支架内扩增的骨膜祖细胞移植。最近的研究重点是获取有缺陷区域的干细胞, 并使用祖细胞进行组织再生20。牙医还预测牙周骨再生在牙周治疗和牙种植体中的应用前景。对于供体部位, 骨膜可以很容易地收获一般牙科外科医生。这与骨髓基质细胞比较有利, 因为在常规的口腔手术中可以访问骨膜。因此, 本研究的目的是建立一个捕捞 PDCs 的协议, 并评估人骨膜干细胞的形态、附着性、活力和增殖。
维生素 D 代谢物影响体内骨-矿物质动态平衡。一项研究报告说, 24 r、25-(OH)2D3活性维生素 D 对人骨髓间充质干细胞 (hMSCs)28的成骨细胞分化至关重要。骨骼稳态和修复由维生素 D3代谢产物网络调节, 其中 125-(OH)2D3 (骨化三醇) 是最具生物学活性和相关的骨骼健康的调节。维生素 D3对于钙化29至关重要。在一项使用2维昆明白鼠的研究中, 小鼠的胚体显示维生素 C 和维生素 d 补充剂有效促进了 ESC 型成骨细胞30的分化。在它的其他生物活动中, 125-(OH)2D3刺激体外hMSCs 与成骨细胞的分化, 可以根据碱性磷酸酶 (磷酸) 酶活性或 OCN 基因的增加来监测。表达。
很少有研究发现, 与维生素 C 和 125-(OH)2D3的联合治疗在人类 PDCs 中的剂量反应关系, 特别注重骨组织工程。因此, 在本研究中, 我们检查 125-(OH)2D3和维生素 C 的单一或联合治疗的最佳浓度, 以诱导人 PDCs 的成骨分化。本议定书的目的是确定从牙槽骨膜中分离出来的细胞是否包含有 MSC 表型的细胞, 以及这些细胞是否可以在培养中被扩展 (体外) 并分化形成所需的组织。.此外, 我们评估 PDCs 的能力, 以分化为骨, 软骨细胞和脂肪。研究的第二部分评估维生素 C 和 10−10的影响, 10−9, 10−8, 和 10−7 M 125-(OH)2D3 PDCs 的成骨活动。本研究的主要目的是评估维生素 C 和 125 (OH)2D3在 PDCs 的成骨细胞分化过程中的作用, 以及支持成骨细胞的基因, 如碱性磷酸酶、collagen-1、OCN、BSP 和 CBFA1。此外, 本研究根据这些结果确定了人类 PDCs 的最佳骨诱导条件。

<table><tbody><tr><td>0.25% trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>2-phospho-L-ascorbic acidtrisodium salt</td><td>Sigma</td><td>49752</td><td></td></tr><tr><td>35-mm culture dishes</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>3-isobutyl-1-methylxanthine</td><td>Sigma</td><td>I5879</td><td></td></tr><tr><td>6&amp;nbsp; well plate</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>Alkaline phosphatase</td><td>ABI</td><td>Hs01029144_m1</td><td></td></tr><tr><td>Alkaline Phosphatase Activity Colorimetric Assay Kit</td><td>BioVision</td><td>K412-500</td><td></td></tr><tr><td>avian myeloblastosis virus reverse transcriptase</td><td>Roche</td><td>10109118001</td><td></td></tr><tr><td>CD146</td><td>BD</td><td>561013</td><td></td></tr><tr><td>CD19</td><td>BD</td><td>560994</td><td></td></tr><tr><td>CD34</td><td>BD</td><td>560942</td><td></td></tr><tr><td>CD44</td><td>BD</td><td>561858</td><td></td></tr><tr><td>CD45</td><td>BD</td><td>561088</td><td></td></tr><tr><td>CD73</td><td>BD</td><td>561014</td><td></td></tr><tr><td>CD90</td><td>BD</td><td>561974</td><td></td></tr><tr><td>Cell banker1</td><td>ZEAOAQ</td><td>11888</td><td></td></tr><tr><td>core binding factor alpha-1</td><td>ABI</td><td>Hs00231692_m1</td><td></td></tr><tr><td>dexamethasone</td><td>Sigma</td><td>D4902</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190250</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>26140-079</td><td></td></tr><tr><td>GAPDH</td><td>ABI</td><td>Hs99999905_m1</td><td></td></tr><tr><td>HLA-DR</td><td>BD</td><td>562008</td><td></td></tr><tr><td>indomethacin</td><td>Sigma</td><td>I7378</td><td></td></tr><tr><td>insulin</td><td>&lt;em&gt;sigma&lt;/em&gt;</td><td>91077C</td><td></td></tr><tr><td>insulin&amp;ndash;transferrin&amp;ndash;selenium-A</td><td>Sigma</td><td>I1884</td><td></td></tr><tr><td>MicroAmp Fast 96 well reaction plate(0.1ml)</td><td>Life</td><td>4346907</td><td></td></tr><tr><td>MicroAmp optical adhesive film</td><td>Life</td><td>4311971</td><td></td></tr><tr><td>Minimum Essential Medium 1X Alpha Modification</td><td>HyClone</td><td>SH30265.02</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Permeabilization buffer</td><td>eBioscience</td><td>00-8333-56</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>11360070</td><td></td></tr><tr><td>STRO-1</td><td>BioLegend</td><td>340103</td><td></td></tr><tr><td>SYBER Green PCR Master Mix</td><td>AppliedBiosystems</td><td>4309155</td><td></td></tr><tr><td>TaqMan Master Mix</td><td>Life</td><td>4304437</td><td></td></tr><tr><td>transforming growth factor-&amp;beta;</td><td>Sigma</td><td>T7039&amp;nbsp;</td><td></td></tr><tr><td>Trizol reagent (for RNA isolation)</td><td>Life</td><td>15596018</td><td></td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>Sigma</td><td>G9422</td><td></td></tr><tr><td>collagen-1</td><td>Invitrogen</td><td>forward primer 5&#039; CCTCAAGGGCTCCAACGAG-3&lt;br /&gt;reverse primer 5&#039;-TCAATCACTGTCTTGCCCCA-3&#039;</td><td></td></tr><tr><td>OCN</td><td>Invitrogen</td><td>forward primer 5&#039;-GTGCAGCCTTTGTGTCCAAG-3&#039;&lt;br /&gt;reverse primer 5&#039;-GTCAGCCAACTCGTCACAGT-3&#039;</td><td></td></tr><tr><td>BSP</td><td>Invitrogen</td><td>forward primer 5&#039; AAAGTGAGAACGGGGAACCT-3&#039;&lt;br /&gt;reverse primer 5&#039;-GATGCAAAGCCAGAATGGAT-3&#039;</td><td></td></tr><tr><td>Commercial ALP primers</td><td></td><td></td><td></td></tr><tr><td>Commercial CBFA1 primers</td><td></td><td></td><td></td></tr></tbody></table>

isolation of mesenchymal stem cells from human alveolar periosteum and effects of vitamin d on osteogenic activity of periosteum-derived cells

间充质干细胞 (MSCs) 存在于各种组织中, 可分化为多种细胞类型, 包括成骨细胞。在骨髓间充质干细胞的牙科来源中, 骨膜是一种易于接触的组织, 被确定为包含层间的 mscs。然而, 这个来源还没有得到广泛的研究。
维生素 D3和 125-(OH)2D3已被证明可以刺激体外诱导干细胞分化为成骨细胞。此外, 维生素 C 有助于胶原蛋白的形成和骨骼细胞的生长。然而, 还没有研究维生素 D3和维生素 C 对 MSCs 的影响。
本文提出了一种从人肺泡骨膜中分离 MSCs 的方法, 并对 125-(OH)2D3可能对这些细胞施加骨诱导影响的假说进行了研究。我们还研究了骨髓间充质干细胞在人肺泡骨膜中的存在, 并评估了干细胞黏附和增殖。要评估维生素 C (作为控制) 和各种浓度的 125-(OH)2D3 (10−10) 的能力, 10−9, 10−8, 10−7 M) 改变关键 mRNA 生物标志物采用实时聚合酶链反应 (rt-pcr) 测定了碱性磷酸酶、骨涎蛋白 (BSP)、核结合因子 alpha-1 (CBFA1)、collagen-1 和骨钙素 (OCN) 的分离 MSCs mRNA 表达。

对于所有的定量分析, 这些数据被显示为平均的标准偏差 (SD)。所有统计分析都是使用学生的t测试进行的。共获得34份样本, 平均参加者年龄为 48.1, 12.3 y 十一的样本是从男性患者和23从女性患者获得的。从磨牙区和六的前区获得二十八份样品;26从上颌骨和8从下颌骨获得。平均期限在牙科做法和文化之间是 0.5, 0.1 h。在34份调查样本中, 20 成功地取得了 MSC 的菌落, 成功?...

Watch this Scientific Journal Video about Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells at JoVE.com

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

本文提出了一种研究维生素 c (维生素 c) 和125二羟基维生素 D [125-(OH)2d3] 诱导的骨膜衍生细胞 (PDCs) mRNA 表达标志物的协议。此外, 我们评估 PDCs 的能力, 以分化为骨, 软骨细胞和脂肪。

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

isolation-mesenchymal-stem-cells-from-human-alveolar-periosteum

Research

JoVE Journal

Bioengineering

9.3K 次观看.  Chang Gung University. 本文提出了一种研究维生素 c (维生素 c) 和125二羟基维生素 D [125-(OH)2d3] 诱导的骨膜衍生细胞 (PDCs) mRNA 表达标志物的协议。此外, 我们评估 PDCs 的能力, 以分化为骨, 软骨细胞和脂肪。

视频: Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

人类骨髓间充质干细胞的分离及其培养的多​​孔骨基质

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

科研

发育生物学

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

隔离和人力富集脂肪基质细胞的成骨增强

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

酶法从凝块骨髓样本拯救间充质干细胞

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

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

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

医学

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

分离和富集大鼠间质干细胞（MSCS）及衍生的干细胞分离单细胞集落

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

生物学

Isolation and Culture of Bone Marrow Mesenchymal Stem Cells from the Human Mandible

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various origins have been found to obtain hMSCs recently, while bone marrow was still considered the main source. Bone marrow-derived MSCs (BMSCs) from different donor bone sites have distinct characteristics due to microenvironmental factors. Studies have shown that BMSCs from maxillofacial bone may have greater proliferative and osteogenic capacities than BMSCs from long bones or the iliac crest. And maxillofacial BMSCs were considered more suitable for stem cell therapy in the maxillofacial tissues. The mandible, especially the ascending ramal area with sufficient marrow, was a feasible donor site for harvesting BMSCs. This study described a protocol for harvesting, isolating, and culturing human mandibular bone marrow-derived MSCs (hmBMSCs). Furthermore, immunophenotyping of hmBMSCs, proliferation assays, and in vitro induction of osteogenic, adipogenic, and chondrogenic differentiation was performed to identify the cultured cells. Applying this protocol can help the researchers successfully obtain enough high-quality hmBMSCs, which is necessary for further studies of the biological function, microenvironmental effects, and clinical applications.

The present protocol describes an efficient procedure for isolating and culturing of human mandibular bone marrow-derived mesenchymal stem cells using the whole bone marrow adherence method. The cultured cells were identified by cell proliferation assays, flow cytometry, and multilineage differentiation induction.

Human mesenchymal stem cells (hMSCs) have shown great potential in bone regeneration, immune modulation, and treating refractory chronic diseases. Various ...

生物工程

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

大鼠骨髓骨髓的隔离和培养

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

大鼠颌骨髓间充质干细胞的基于生态位的分离

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

作者聚焦：高纯度颚骨骨髓间充质干细胞在组织工程中的应用

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

大鼠颌骨骨髓间充质干细胞的分离和培养技术

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

摘要

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此视频中的章节

人类骨髓间充质干细胞的分离及其培养的多孔骨基质

隔离和人力富集脂肪基质细胞的成骨增强

酶法从凝块骨髓样本拯救间充质干细胞

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

人牙小泡干细胞的分离、鉴定及微 rna 遗传修饰

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

分离和富集大鼠间质干细胞（MSCS）及衍生的干细胞分离单细胞集落

Isolation and Culture of Bone Marrow Mesenchymal Stem Cells from the Human Mandible

大鼠骨髓骨髓的隔离和培养

大鼠颌骨骨髓间充质干细胞的分离和培养技术

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

摘要

探索更多视频

此视频中的章节

人类骨髓间充质干细胞的分离及其培养的多​​孔骨基质

隔离和人力富集脂肪基质细胞的成骨增强

酶法从凝块骨髓样本拯救间充质干细胞

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

人牙小泡干细胞的分离、鉴定及微 rna 遗传修饰

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

分离和富集大鼠间质干细胞（MSCS）及衍生的干细胞分离单细胞集落

Isolation and Culture of Bone Marrow Mesenchymal Stem Cells from the Human Mandible

大鼠骨髓骨髓的隔离和培养

大鼠颌骨骨髓间充质干细胞的分离和培养技术

人类骨髓间充质干细胞的分离及其培养的多孔骨基质