支持这项工作摆围早期平移第二研究奖的TR2-01821，国立卫生研究院/ NIDCR（了赠款R21的DE0177711和RO1的DE01607），加州大学发现格兰特07-10677，磨料水射流和RKS的T32培训奖学金奖（5T32DE007296-14），结果JNZ有摆围培训奖学金（TG2的-01169）。

1。血管周围的干细胞的分离这是在相邻的文章"从人类白色脂肪组织中的血管周围干细胞的分离纯化"详细阐述，由M. Corselli 等。 2。脚手架创作<ol><li>棚架每先前公布的协议（聚乳酸-乙醇酸）（PLGA的，Burmingham聚合物）与羟基磷灰石涂层4-6定制。磷灰石涂层PLGA支架的制作从85/15共聚物溶剂浇铸和微粒浸出过程。支架创建肌袋植入术，颅骨植入术，盘状的外形（直径4毫米）或圆柱（4毫米，直径6毫米的长度），股骨缺损的形状为球形（直径2毫米）。 </li><li>简言之，PLGA /氯仿溶液混合与蔗糖（聚合物/蔗糖比5/95，W / W）200-300微米直径铁氟龙模具创建定制的利弊投truct。冷冻干燥过夜后，支架从聚四氟乙烯模具，并沉浸在DDH 2解散蔗糖。棚架由70％的乙醇浸泡消毒30分钟，由三个DDH 2 O冲洗</li><li>磷灰石涂层，模拟体液（SBF）中的解决方案，准备按顺序溶于DDH 2 O 2氯化钙，氯化镁·6H 2 O 3，碳酸氢钠，和K 2 HPO 4•3H 2加入1M的盐酸，以增加溶解度，溶液的pH值降低到6。添加的Na 2 SO 4，氯化钾，氯化钠，最终的pH值调整到6.5（SBF中1）。 </li><li> Mg2 +和HCO 3 -免费模拟体液（SBF中2）准备加入CaCl 2和K 2 HPO 4•3H 2 O的 DDH 2，pH值降低到6。 KCl和NaCl添加和最终的pH值调整到6.8。所有的解决方案是无菌过滤通过0.22微米的PES membrANE（NALGENE）。紧接涂层工艺，干PLGA支架受到辉光放电氩等离子体刻蚀（Harrick科学），以提高润湿性和涂层均匀。 </li><li>蚀刻支架，然后在SBF 1孵育12小时，并改变对Mg2 +和HCO 3 -免费SBF的另外12小时在37°C下轻轻搅拌。涂层支架与DDH 2 O冲洗，以去除多余的负离子和冻干前进一步的研究。 </li></ol> 3。肌袋模型植入<ol><li> 100力晶暂停在PBS液（磷酸盐缓冲液）轻轻下降到球形共聚物为基础的种植立即植入前。细胞已被预标记的萤火虫荧光素酶的慢病毒插入，从而允许在体内植入跟踪后。细胞密度为2.5×10％植入5。 </li><li>用于免疫缺陷（严重联合免疫缺陷）小鼠6周龄。动物是anesthetized异氟醚吸入和躁动的丁丙诺啡（贝德福德实验室）。标准的优碘制剂后，在后肢的双边切口（纵向长度在2毫米）。 </li><li>口袋被切断股二头肌肌肉钝性分离肌纤维长轴平行。对于每个鼠标，插入，与产品分成合同为基础的PLGA植入和覆肌肉筋膜缝合用5-0 Vicryl（爱惜康）。 </li><li>接下来的皮肤封闭在皮下的模式与5-0 Vicryl。对待动物与丁丙诺啡术后48小时内和TMP / SMX为主（甲氧苄啶/磺胺甲恶唑; Qualitest）10天。 </li></ol> 4。颅骨缺损模型植入<ol><li> SCID小鼠（12-14周龄）的异氟醚麻醉后，头发被截断，每个协议的优碘消毒皮肤。 </li><li>一个8毫米的皮肤切口中矢状缝沿鼠标颅骨的。接下来，在calv宋体骨膜轻轻删除由棉条申请。 </li><li>接下来，用高速牙钻金刚石涂层的环钻位，4毫米的顶骨缺损的创建。缺陷是全厚度 - 然而，护理是采取不伤害底层的硬脑膜。 </li><li>定制栽种的产品分成合同为基础的共聚物植入然后轻轻地放置在缺损部位。细胞密度为2.5×10％植入5。最后，皮肤缝合用6-0 Vicryl。动物与丁丙诺啡治疗，术后48小时和TMP / SMX为主的10天。 </li></ol> 5。股骨缺损模型植入<ol><li> （12-14周龄）裸鼠大鼠在异氟醚吸入麻醉。股骨擦洗和准备每优碘标准协议（ 图1）。 </li><li>股骨外侧方面取得了一个-30-27毫米的纵向切口。然后暴露分离股纬度的股骨干eralis和股二头肌肌肉（ 图2）。 </li><li>为了最大限度地提高骨再生的一致性，覆股骨缺损骨膜完全切除股骨段删除。 </li><li>聚乙烯板（长度，23毫米，宽4毫米，高4毫米）放在股骨外侧表面。板包含六个预钻孔，以容纳直径为0.9毫米的螺纹克氏针（齐默）。以板为模板，6个螺纹克氏针钻板和两个皮质（ 图3）。 </li><li>接下来，用一个小的振荡锯片（斯瑞克，MI），创建中期骨干缺损6毫米。然后插入的PLGA已经充满了每个协议的细胞（ 图4）基于植入治疗节段性缺损。 </li><li>覆肌肉和筋膜4-0的Vicryl吸收缝线关闭到位，以确保植入物，与皮肤缝合。 </li&gt; </ol> 6。 在体内评估<ol><li>影像学评估在纵向的方式进行，由两个高分辨率的XR和高分辨率μCT（微型​​计算机断层扫描）分析。对于μCT分析（Skyscan 1172F），图像扫描19.73微米（100千伏和100毫安辐射光源，用0.5毫米的铝过滤器）的决议。使用DataViewer的，侦察，CTAN，和CTVol软件分析图像。 </li><li>生物发光成像也做了一个串行的方式来评估细胞植入，活力，增殖和排除植入部位的迁移。生物发光成像使用的IVIS Lumina的第二设备（卡尺生命科学版）。光输出被量化的生活图像软件（精诺真）。总的光输出记录在光子/秒/平方厘米/球面度。 </li><li>组织学和形态计量学分析进行抛尸。受聘的日常污渍，包括Masson染色，苯胺蓝，Pentachrome，天狼猩红。组织形态计量学的分析是很容易与任何苯胺蓝pentachrome的污渍，骨质深蓝色和黄色，分别出现在其中。在Adobe Photoshop中使用魔棒工具，每高功率场的像素计算。 </li></ol> 7。代表结果由于颅骨和股骨缺损的关键尺寸，预计应无显着性愈合，无生长因子或外源性干细胞治疗。 肌袋清扫手术演习的条款，应是沿筋膜飞机，从而减少出血，应遇到。即使肌袋模型进行双边，鼠标应走便于术后第1天。颅骨缺损，出血遇到，但可以用一个Q尖端浸泡。格外小心，应采取不损害底层硬脑膜 - 因为这会干扰与正常愈合。消防处的模型，护理是采取不损伤主要血管，以免引起出血过多或股神经，以防止神经系统的损害。克氏针钻与温和的压力，以免破坏过程中的骨皮质。 <img alt="图1。" src="/files/ftp_upload/2952/2952fig1.jpg" /> 图1。 （消防处）在athymic大鼠股骨缺损的术前准备 。 （12-14周龄）雄性大鼠在异氟醚吸入麻醉。股骨擦洗prepped每晚用优碘的标准协议。 <img alt="图2。" src="/files/ftp_upload/2952/2952fig2.jpg" /> 图2。手术暴露股骨缺损（FSD）的创造 。股骨外侧方面取得了一个27-30毫米的纵向切口。股骨干的侧面，然后分离股外侧暴露和股二头肌的肌肉。 <img alt="图3。" src="/files/ftp_upload/2952/2952fig3.jpg" /> 图3。内固定治疗股骨缺损（消防处）创建 。聚乙烯板（长度，23毫米，宽4毫米，高4毫米）放在股骨外侧表面。该板块包含6个前钻洞，以容纳直径为0.9毫米的螺纹克氏针。以板为模板，通过六个螺纹克氏针钻板和两个皮质。接下来，创建6毫米中旬骨干缺损。一旦执行，这是一个特制的棚架直接插入，这完全符合缺损部位（未显示）。 <img alt="图4。" src="/files/ftp_upload/2952/2952fig4.jpg" /> 图4。颅骨缺损手术的例子 。创建于裸鼠的右顶骨4毫米，圆形颅骨缺损。成像这里是一个缺损部位植入在8个星期术后的产品分成合同。注意内新骨缺损部位的存在。

<ol>
	<li>Crisan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perivascular+origin+for+mesenchymal+stem+cells+in+multiple+human+organs.">A perivascular origin for mesenchymal stem cells in multiple human organs.</a> Cell Stem Cell. 3, 01-13 (2008).</li><li>Chen, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+multi-lineage+progenitor+cells+in+human+organs:+regenerative+units,+cytokine+sources+or+both.">Perivascular multi-lineage progenitor cells in human organs: regenerative units, cytokine sources or both.</a> Cytokine Growth Factor Rev. 20, 429-434 (2009).</li><li>Corselli, M., Chen, C. W., Crisan, M., Lazzari, L., Peault, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+ancestors+of+adult+multipotent+stem+cells.">Perivascular ancestors of adult multipotent stem cells.</a> Arterioscler. Thromb Vasc. Biol. 30, 1104-1104 (2010).</li><li>Zara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nell-1+enhances+bone+regeneration+in+a+rat+critical+sized+femoral+segmental+defect+model.">Nell-1 enhances bone regeneration in a rat critical sized femoral segmental defect model.</a> Plast. Reconstr. Surg. , (2010).</li><li>James, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deleterious+Effects+of+Freezing+on+Osteogenic+Differentiation+of+Human+Adipose-Derived+Stromal+Cells+In+Vitro+and+In+Vivo.">Deleterious Effects of Freezing on Osteogenic Differentiation of Human Adipose-Derived Stromal Cells In Vitro and In Vivo.</a> Stem Cells Dev. 20, 427-439 (2011).</li><li>Lee, M., Chen, T. T., Iruela-Arispeb, M. L., Wu, B. M., Dunn, J. C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+protein+delivery+from+modular+polymer+scaffolds.">Modulation of protein delivery from modular polymer scaffolds.</a> Biomaterials. 28, 1862-1870 (2007).</li><li>Aghaloo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+the+role+of+nell-1+gene+modified+goat+bone+marrow+stromal+cells+in+promoting+new+bone+formation.">A study of the role of nell-1 gene modified goat bone marrow stromal cells in promoting new bone formation.</a> Mol. Ther. 15, 1872-1880 (2007).</li><li>Levi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Adipose-Derived+Stromal+Cells+Stimulate+Autogenous+Skeletal+Repair+via+Paracrine+Hedgehog+Signaling+with+Calvarial+Osteoblasts.">Human Adipose-Derived Stromal Cells Stimulate Autogenous Skeletal Repair via Paracrine Hedgehog Signaling with Calvarial Osteoblasts.</a> Stem Cells Dev. 20, 243-257 (2011).</li><li>Leucht, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origin+and+Hox+status+determine+progenitor+cell+fate+during+adult+bone+regeneration.">Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration.</a> Development. 135, 2845-2854 (2008).</li><li>Rajashekhar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFATS+collection:+Adipose+stromal+cell+differentiation+is+reduced+by+endothelial+cell+contact+and+paracrine+communication:+role+of+canonical+Wnt+signaling.">IFATS collection: Adipose stromal cell differentiation is reduced by endothelial cell contact and paracrine communication: role of canonical Wnt signaling.</a> Stem Cells. 26, 2674-2681 (2008).</li><li>Zuk, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adipose+tissue+is+a+source+of+multipotent+stem+cells.">Human adipose tissue is a source of multipotent stem cells.</a> Mol Biol Cell. 13, 4279-4295 (2002).</li></ol>

KT，BP和CS是血管周围的干细胞相关专利申请从加州大学洛杉矶分校的发明者。博士。 KT的和CS是血管周围干细胞相关专利转让许可证从加州大学校董瘢痕实验室公司的创始人。嘉义洙博士也是瘢痕实验室，公司人员

PSCS隔离是很好的描述其他地方1-3，其中包括一个单独提交的朱庇特的出版物，特别是解决PSC的隔离协议和方法。这篇文章的具体目的是描述和演示3 PSC的协议， 在体内骨形成/再生的应用。 SCID小鼠肌袋是常用描述模型异位人骨形成7。异位和原位（缺陷）的模型，包括旁互动与宿主骨形成细胞8，以及丰富的成骨细胞信号的因素目前在骨骼缺陷微骨之间存在重要的差异。两个缺陷在这里，的颅骨defect8和股骨缺损4。两者都记录是至关重要的大小（即不会自行愈合）。 有趣的差异之间存在颅骨和股骨缺损。首先，细胞的:裸鼠PSC和内源性细胞的细胞之间的相互作用是非常不同的。产品分成合同的颅骨缺损，互动与底层的硬脑膜（最外层的脑膜），以及那些成骨细胞和骨膜细胞circumscribing缺损部位。重要的是，和周围的成骨细胞植入的细胞8，或植入的细胞和基础硬脑膜（列维等。新闻）之间的相互作用是正常干细胞的关键介导的骨进行。在股骨缺损（消防处），裸鼠的产品分成合同暴露在一个非常不同的细胞和细胞因子的环境。例如，消防处的网站是由骨髓和相应的间充质干细胞，以及内膜，骨膜及长骨成骨细胞。从理论上讲，每个单元有伤害自己的反应，每个人都可能有细胞:与PSC移植瘤细胞的相互作用。 其他明显的差异之间存在颅骨股骨缺损。颅骨骨骼初步形成通过膜内骨化，而通过中间软骨（软骨内骨化）长骨形成。此外，损伤后的修复过程也模仿这些发展的起源。消防处后，被发现，而没有软骨中间形成内顶骨缺损软骨骨痂形成。最后，可能会有所不同胚胎起源的头骨长骨。头骨（ -周细胞-包括血管周围细胞在整个头部区域）大部分是来自神经嵴（mesectoderm），而四肢骨骼的近轴中胚层衍生9。所有这些差异可能会导致在在力晶介导的骨修复方面的显着性差异。 使用产品分成合同已超过传统的脂肪来源的基质细胞（ASCs的）有几个好处。物业服务公司并不需要文化和纯化细胞人口WHICH不包括其他不参加-甚至可以负调控的基质细胞-内皮细胞，如10成骨细胞分化。与此相反，例如，ASCs的克隆分析表明，只有一个亚群是能够接受成骨细胞分化的体外 11。最终，骨组织工程的努力可能会纳入osteocompetent干细胞与外源性生长因子（如物业服务公司）和骨传导支架（如在本方法中使用的HA-PLGA的），以便以最好的治疗骨缺损。

use of human perivascular stem cells for bone regeneration

分离出人体血管周围干细胞（PSCS）可以从多个组织足够数量的1-3骨组织工程的目的。物业服务公司是一个&#39;周细胞流式细胞仪排序人口（CD146 + CD34-CD45 - ）和"外膜细胞（CD146的CD34 + CD45-），其中每一个我们曾报告有间质干细胞的特性。产品分成合同，间充质干细胞一样，是能够接受的成骨细胞分化，以及分泌亲成骨细胞因子1,2。在本议定书中，我们证明在多种动物模型，包括在SCID肌袋植入osteogenicity产品分成合同（严重联合免疫缺陷）小鼠，SCID小鼠颅骨缺损和股骨缺损在athymic大鼠（消防处）。大腿肌肉袋模型，用于评估异位骨形成。颅骨缺损集中在顶骨和标准直径为4毫米（批判地中型）8。 FSDS是bicortical的的和稳定与聚乙烯栏的K-线4。消防处的描述，也是一个关键尺寸的缺陷，这并不显着医治自己的4。相反，如果添加到缺损部位的干细胞或生长因子，可以显着的骨再生赞赏。 PSC的异种移植的总体目标是证明这种细胞类型的成骨能力，在异位和原位骨再生模型。

Watch this Scientific Journal Video about Use of Human Perivascular Stem Cells for Bone Regeneration at JoVE.com

Use of Human Perivascular Stem Cells for Bone Regeneration

人体血管周围干细胞（PSCS）是一种新型的干细胞间充质干细胞（MSCs）相似的骨骼组织再生类。物业服务公司可以通过流式细胞仪（荧光激活细胞分选）在标准吸脂程序采购的脂肪组织中分离，然后结合骨修复支架，以达到骨形成<em&gt;在体内</em&gt;。

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

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20.6K 次观看.  School of Dentistry, UCLA. 人体血管周围干细胞（PSCS）是一种新型的干细胞间充质干细胞（MSCs）相似的骨骼组织再生类。物业服务公司可以通过流式细胞仪（荧光激活细胞分选）在标准吸脂程序采购的脂肪组织中分离，然后结合骨修复支架，以达到骨形成在体内。

视频: Use of Human Perivascular Stem Cells for Bone Regeneration

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

科研

生物工程

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

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

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

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

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

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

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D磁干细胞聚集和生物反应器为成熟软骨再生

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

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

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

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 ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

骨再生生物材料的生物相容性曲线

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

人骨髓间充质干细胞吸收含有法卡林多醇的新型脂质包覆纳米颗粒

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

维持静止人类造血干细胞的文化方法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...