This work was supported in part by grants from NHRI (CS-104-PP-06 to B.L.Y.).

必须使用人体细胞获得批准的机构审查委员会患者的知情同意书。 人外周血单个核细胞1.密度梯度分离（外周血单个核细胞） <ol><li>加25 ml的肝素化全血到50ml管由25毫升吸管。 <ol><li>稀释细胞用25ml磷酸盐缓冲盐水（PBS）的。 </li></ol></li><li>新增15 ml的菲可 - 帕克密度梯度到一个新的50ml管中，并在倾斜的管，非常缓慢并小心地在25毫升的稀释的细胞悬浮液在密度梯度的添加使得存在没有混合的全血与密度梯度， 即 ，无干扰的血液密度梯度的接口。 </li><li>离心从步骤1.2以400×g的30至40分钟，在温度控制的吊桶式转子无制动器在20℃的悬浮液中。 注意:在离心后三个不同的层应当是显而易见:第Ë上层存在等离子体;底部的透明层作为菲可 - 帕克密度梯度;和薄的，中间层的细胞作为PBMC中</li><li>吸液和丢弃通过抽吸上层用巴氏吸管，小心不要打扰单核细胞（ 即，淋巴细胞，单核细胞和血小板）的相间层。 </li><li>仔细收集这种单核细胞层用10毫升吸管到一个新的50ml离心管中。 </li><li>填充用20毫升的PBS管，混的不好，离心300×g的10分钟，在20°C。离心后完全除去上清液。 </li><li>重悬细胞沉淀在20ml的PBS中并在离心机200×g下在20℃下10-15分钟。离心后完全除去上清液。 注:此步骤除去血小板，这是不必要的，内部的外周血单个核细胞;这个步骤可以重复，以确保完全除去血小板。 </li><li>如果没有选择特定的人群是需要的话，在白细胞完全培养基重悬细胞沉淀（10％胎牛血清（FBS），1％L-谷氨酰胺和1％青霉素/链霉素的RPMI-1640培养基）用于培养进行细胞计数16后，暂停1毫升每10 7 PBMC的介质在进行下一步骤之前。 </li></ol>白细胞种群2.磁标记（跳至步骤4如果没有选择特定人群是必需的） <ol><li>离心机的PBMC悬浮液在300×g的10分钟，吸出上清液完全。 </li><li>悬浮细胞沉淀在80微升PBS每10 7个细胞。 </li><li>加入20微升指定磁珠（即，CD14磁珠隔离单核细胞，CD4磁珠的T淋巴细胞隔离）每10 7个细胞。 </li><li>拌匀，孵育15分钟，在2〜8℃的冰箱里。 </li><li>加1-2毫升的PBS每10 7个细胞进行冲洗，并且离心分离机在300×g的10分钟。 </li><li>吸上清完全和悬浮高达10 8个细胞在500微升PBS。 </li></ol>白细胞亚群的3磁选注意:各种磁珠分离器和列可用于白细胞的特定人口的隔离通过细胞表面标记从许多制造商。隔离可以通过基于一个或几个正标记或由意外的种群耗尽后负选择阳性选择。进行正选择使用一个标记（ 即，CD4的T辅助淋巴细胞或CD14的单核细胞）的一般方法如下所述。 <ol><li>制备和素磁性分离器包括根据制造商的说明分离柱。 </li><li>应用含有标记的PBMC的样品管中。提供两个15毫升离心管中收集的积极标记和unlabel的编细胞级分，按照生产商的说明。 </li><li>计数细胞数15，重悬在白细胞完全培养基的阳性选择的白细胞（例如，CD4 + T淋巴细胞或CD14 +细胞），使用1毫升培养基每10 7个细胞。 </li></ol>白细胞4. CFSE染色增殖的评估注意:CFSE已被广泛用于在免疫学调查，对于在体内和体外研究 。我们的协议已经优化，MSC / PBMC（或其他白细胞）相互作用的体外研究。而参与实施CFSE在体外标记的一般步骤是相似的，有可能是在协议14的特定剂量和定时差异。这可能是由于多种因素，例如，所用的特定的细胞类型，细胞数目用后可容易影响CFSE荧光信号的强度。 <ol&gt; <li>稀释CFSE库存溶液（10mM）的PBS，以10μM的（CFSE-工作溶液）所需的工作浓度。制备10 7个细胞（即，外周血细胞或T细胞），用于与所述的CFSE工作溶液标记。 </li><li>离心机在300×g的10分钟以获得细胞沉淀并吸出上清液。 </li><li>悬浮细胞轻轻在1ml预热（37℃）的CFSE工作溶液，并在37℃下孵育细胞10分钟。 </li><li>洗去过量的CFSE，（体积）的预冷（4℃）的RPMI培养基含有10％FBS稀释的细胞悬浮液具有10。通过离心沉淀细胞，在300×g离心5分钟，丢弃上清液。洗以这种方式将细胞沉淀两次。 </li><li>通过离心重新沉淀细胞并计数细胞数目15。悬浮10 7个细胞（无论的PBMC或T细胞）在1ml新鲜预热的白细胞的完全培养基。 </li></ol>的M 5，共培养种姓与白细胞和白细胞的活化<ol><li>预暖的MSC完全培养基（10％FBS（预测试以获得最佳的MSC生长），1％L-谷氨酰胺和1％青霉素/链霉素的DMEM低葡萄糖培养基）至37℃不超过30分钟。 </li><li>种子的MSC以50,000细胞在1ml的MSC完全培养基中24孔板（MSC密度:25,000个细胞/ cm 2）的用于附着O / N在37℃培养箱，允许干细胞达到80％汇合。 </li><li>吸出培养基，并根据种子的MSC号，添加CFSE标记的外周血单个核细胞（先前标记-请参阅协议上述步骤4）到培养的MSC（接种前一天在24孔平板）的1ml白细胞完全培养基中的一1时10（气泡率）MSCs向的PBMC共培养比率。 </li><li>添加分裂素植物血凝素（PHA），非特异性白细胞活化剂，以10微克/ ml的终浓度在1毫升白细胞完全培养基的总体积每孔。 <ol><li>可选择性伊利，刺激对T淋巴细胞的激活具体有:使用α-CD3 / 28微珠，并加入到二细胞共培养，以获得1珠对T淋巴细胞:1的比例。 </li></ol></li><li>为阴性对照，板500000的PBMC /孔（或特异性效应白细胞种群;细胞密度:250000白细胞/厘米2）在一个24孔板用1ml白细胞完全只介质;对于阳性对照，在除了电镀的PBMC /相同数量均匀，加入的PHA到10微克/毫升的最终浓度。 </li><li>在第三和共培养实验的第5天，评估CFSE标记的白细胞的增殖（置于圆底管）通过流式细胞仪分析17具有适用于荧光素488纳米激发和发射滤光片。 注:细胞内细胞因子染色的流式细胞术分析，也可进行对这些CFSE标记的白细胞在这一点上，以评估作为莫变化白细胞的细胞因子表达谱通过干细胞dulated。因为CFSE评价与过滤器适合于荧光素，选择的抗体，以评估各种细胞因子需要被缀合到荧光比荧光素或类似的频谱其他（即，藻红蛋白 ，多甲藻素叶绿素蛋白复合物（PerCP））。 </li></ol> 6.变异:效应抑制测定磁珠选择的，MSC诱导的免疫调节白细胞对活性CFSE标记的效应CD4 + T细胞<ol><li>种子的MSCs在250,000个细胞在3ml的MSC完全培养基中6孔板（MSC密度:25,000个细胞/ cm 2）的用于附着O / N在37℃培养箱，允许干细胞达到80％汇合。至少3 6孔板是必要的，以确保足够的MSC-共培养的PBMC为亚群的选择。 </li><li>吸出培养基，并添加的PBMC分离按步骤1，但在6孔板以2.5×10 6个细胞/孔（细胞密度:250,000勒ukocytes /厘米2）用3ml白细胞完全培养基。共培养48-72小时在37℃培养箱。 </li><li>磁珠选择MSC诱导免疫调节白细胞（ 即 CD14 +细胞）根据第2-3特定人群。 注:细胞内细胞因子染色的流式细胞术分析可以在此时被执行以评估更改中白细胞的细胞因子表达谱， 即，表达白介素-10-如由干细胞调制。 </li><li>在添加CFSE标记在24孔板产生为每第2-4节异源CD4 + T细胞 1 ml的白细胞完全培养基（T细胞密度:250000个细胞/厘米2），以珠选择的MSC诱导的白细胞在各种比例即，1:10,1:5，1:2和1:1（细胞与细胞）的比例。 </li><li>刺激CD4 + T细胞，加入α-CD3 / 28偶联的微珠，以获得1珠-细胞:1的比例。 </li><li>对于阴性对照，板500000 CD4 + T细胞/孔在24孔板（T细胞密度:250000个细胞/ cm 2）的用1毫升白细胞完全只介质;对于阳性对照，在除了电镀CD4 + T细胞的数量相同/孔，加入α-CD3 / 28偶联的微珠，以获得1珠-细胞:1的比例。 </li><li>上共培养的第 3天，通过流式细胞仪分析16具有适用于荧光素488纳米激发和发射滤光片评估CFSE标记的CD4 + T细胞（置于圆底管）的增殖。 </li></ol>

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We have nothing to disclose.

Increasingly, the immunomodulatory properties of MSCs are being translated into clinical use more rapidly than the multilineage capacity of these stem cells18-20. Thus, co-culture techniques of MSCs with leukocytes and assays to evaluate immune function are important to further delineate the specific mechanisms involved in these properties for optimizing effective therapeutic application. 
One of the most critical technical aspects for success in these assays is having adequate PBMC...

人间充质干细胞（MSCs）是体祖细胞可以分化成骨，软骨和脂肪组织1-4的近轴中胚层谱系，以及几extramesodermal谱系5。首先从成人骨髓中分离，这些多谱系祖细胞现已发现在多种组织和6-8，出乎意料的是，显示出具有出现高度适合于临床应用9-12强免疫调节性能。参与免疫调节作用的详细机制正在积极研究对特定病种有效的应用。其中一个最简单的方式来评估免疫调节是通过评估的效应白细胞增殖13的抑制。大多数效应白细胞，如T淋巴细胞和单核细胞增生不歇刺激或激活时。免疫调节功能，可以评估时抑制增殖证明。 传统上，效应白细胞增殖进行了评价通过检测的 [3 H]胸苷掺入DNA中。然而，这种方法具有缺点显著由于辐射和后期使用处置的关注，以及所需要的复杂的设备。虽然有非放射性测定法来评估细胞增殖，所述羧基琥珀酰亚胺酯（CFSE）测定具有其它优点，如允许识别特定细胞群，这是在涉及多种细胞类型的共培养实验中特别有用的。 CFSE是荧光染料的细胞可以通过流式细胞术分析来评估。作为细胞分裂时，这种细胞标记的强度成比例地降低;这不仅使得能够确定的总的细胞增殖，但也允许对细胞分裂的高达8分割的数目评估前的荧光变得难以DET等针对背景信号。此外，该荧光CFSE的稳定性允许在体内跟踪，使得细胞可以可视化长达很多个月14标记的细胞。 该测定也可以改变，以评估特定类型的效应子白细胞或特定人群的免疫调节功能的MSC诱导的免疫调节白细胞，如白介素-10（IL-10）产生的CD14 +单核细胞 15通过执行的磁珠表面标记选择之前或共培养适当的利息后的细胞群。我们的协议描述评估对效应白细胞MSCs的免疫调节作用（ 图 1中所示的流程图）的基本测定，并在此基础测定法对异源CD4 +效应 T淋巴细胞的MSC诱导的白细胞免疫调节评价的变型（图中所示的流程图在图4中）。

<table><tbody><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare</td><td>71-7167-00 AG</td><td>Density grandient for isolation of peripheral blood mononuclear cells (PBMCs)</td></tr><tr><td>Vibrant CFDA-SE Cell Tracer Kit (CFSE)</td><td>Life Technologies</td><td>V12883</td><td>Cellular label for detection of cell division</td></tr><tr><td>Phytoagglutinin (PHA)</td><td>Sigma</td><td>L8902</td><td>Activation of human PBMCs</td></tr><tr><td>Dynabeads Human T-Activator CD3/28</td><td>Life Technologies</td><td>111.32D</td><td>Activation of human T lymphocytes, &lt;em&gt;e.g.&lt;/em&gt; CD4&lt;sup&gt;+&lt;/sup&gt; T cells, CD8&lt;sup&gt;+&lt;/sup&gt; T cells, &lt;em&gt;etc.&lt;/em&gt;</td></tr><tr><td>autoMACS&amp;trade; Separator</td><td>Miltenyi Biotec</td><td>autoMACS&amp;trade; Separator</td><td>Magnetic based cell separator</td></tr><tr><td>autoMACS&amp;reg; Columns</td><td>Miltenyi Biotec</td><td>130-021-101</td><td>separation columns</td></tr><tr><td>CD14 microbeads, human&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-050-201</td><td>For positive selection of CD14&lt;sup&gt;+&lt;/sup&gt; human monocytes and macrophages from PBMCs</td></tr><tr><td>CD4 microbeads, human&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-045-101</td><td>For positive selection of CD4&lt;sup&gt;+&lt;/sup&gt; human T lymphocytes from PBMCs</td></tr><tr><td>RPMI 1640 Medium</td><td>Life Technologies</td><td>11875</td><td>Human PBMC/leukocyte culture medium</td></tr><tr><td>DMEM, Low glucose, pyruvate</td><td>Life Technologies</td><td>11885</td><td>Human mesenchymal stem cell (MSC) culture medium</td></tr><tr><td>L-glutamine</td><td>Life Technologies</td><td>25030-081</td><td>Supplementation for MSC complete medium</td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15070-063</td><td>Supplementation for PBMC/leukocyte and MSC complete medium</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>1) Hyclone, for MSC culture&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations)</td><td>1) SH30070.03M 2) 10091-148</td><td>Pre-test lots for support of MSC &lt;em&gt;in vitro&lt;/em&gt; culture</td></tr><tr><td>24-well cell culture plate</td><td>Corning</td><td>COR3524</td><td>Co-culture plate</td></tr><tr><td>50 ml centrifuge tube</td><td>Corning</td><td>430291</td><td>Isolation PBMCs from whole blood by Ficoll-Paque PLUS</td></tr><tr><td>15 ml centrifuge tube&amp;nbsp;</td><td>Corning</td><td>430766</td><td>Collection of the labeled and unlabeled cell fractions</td></tr><tr><td>Round-bottom tubes</td><td>BD Falcon&amp;nbsp;</td><td>352008</td><td>Collection of cells for flow cytometric analysis</td></tr></tbody></table>

assessment of the immunomodulatory properties of human mesenchymal stem cells (mscs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

图1表示了实验的总体架构，而图2展示了可视化作为用相差倒置显微镜的各种细胞培养条件的外观。的MSC是贴壁细胞与成纤维细胞，纺锤形的形态，而PBMC和白细胞小圆非贴壁细胞。这两个形态不同的细胞类型，可以清楚地看出，在共培养。在测定结束时，当PBMC中（或leuckotyes）吸出流式细胞仪分析，即使粘附的MSCs无意中包含（即，由于附接不良或移位通过剧烈抽吸），应?...

Watch this Scientific Journal Video about Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs at JoVE.com

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

人类骨髓间充质干细胞（MSC）的免疫调节属性显示越来越重要的临床应用。用MSC和外周血白细胞的共同培养系统的预染色的荧光染料羧基琥珀酰亚胺酯（CFSE），我们描述对效应白细胞增殖和特定亚群的体外评估的MSC免疫调节的。

人骨髓间充质干细胞（干细胞）的免疫调节性能评估

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

assessment-immunomodulatory-properties-human-mesenchymal-stem-cells

Research

JoVE Journal

Immunology and Infection

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

12.2K 次观看.  National Health Research Institutes (NHRI). 人类骨髓间充质干细胞（MSC）的免疫调节属性显示越来越重要的临床应用。用MSC和外周血白细胞的共同培养系统的预染色的荧光染料羧基琥珀酰亚胺酯（CFSE），我们描述对效应白细胞增殖和特定亚群的体外评估的MSC免疫调节的。 

视频: Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

生产和治疗间充质干行政/基质细胞（MSC）球体底漆在三维培养下无异物条件

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

科研

发育生物学

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

在体外人骨髓间充质干细胞分化为心肌样细胞功能

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

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

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

超声引导下人骨髓间充质干细胞在实验性肠炎小鼠模型中的应用

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

医学

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

间充质干细胞调节巨噬细胞吞噬作用;定量和成像

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

免疫与感染

<meta charset="utf8"></head><body>BAMBI高MFGE8高 UC-MSC 亚群的功能分析

Isolation of Human BAMBIhighMFGE8high Umbilical Cord-Derived Mesenchymal Stromal Cells

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various diseases. Human UC-MSCs are a heterogeneous population consisting of three main subpopulations with different cell shapes, proliferation rates, differentiation abilities, and immune regulatory functions. Previously, BAMBIhighMFGE8high UC-MSCs, the first subgroup successfully isolated from UC-MSCs were found to fail to alleviate lupus nephritis. Hence, the function and underlying mechanism of this subgroup in MSC therapy for diseases remains unknown. It is necessary to isolate and further investigate BAMBIhighMFGE8high UC-MSCs in terms of their phenotype, metabolism, and function to completely understand the nature of this MSC subgroup. In this protocol, we describe a detailed method for isolating the BAMBIhighMFGE8high subpopulation from human UC-MSCs. The subpopulation of UC-MSCs is labeled with two surface markers, BAMBI and MFGE8, by flow cytometry sorting. The isolated cells are cultured and verified by flow cytometry analysis. The specific genes expressed in the BAMBIhighMFGE8high UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBIhighMFGE8high UC-MSCs.

<meta charset="utf8"></head><body>人 BAMBI高MFGE8高 脐带来源的间充质基质细胞的分离

The isolation of BAMBIhighMFGE8high MSCs, one of the three main subgroups constituting heterogeneous human UC-MSCs, is helpful for fully understanding the characteristics and functions of this subtype for its future application to improve clinical efficacy in specific diseases. Here, we present a method for sorting BAMBIhighMFGE8high UC-MSCs.

人 BAMBI高MFGE8高 脐带来源的间充质基质细胞的分离

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various ...

表征 hiPSC 驱动 MSC 中的表面抗原和增殖能力

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

作者聚焦:iPSC 和遗传病研究的进展 

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

人诱导多能干细胞分化为间充质基质细胞的两种代表性方法比较

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...

人骨髓间充质干细胞（干细胞）的免疫调节性能评估

摘要

探索更多视频

此视频中的章节

生产和治疗间充质干行政/基质细胞（MSC）球体底漆在三维培养下无异物条件

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

在体外人骨髓间充质干细胞分化为心肌样细胞功能

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

超声引导下人骨髓间充质干细胞在实验性肠炎小鼠模型中的应用

间充质干细胞调节巨噬细胞吞噬作用;定量和成像

人 BAMBI^高MFGE8^高脐带来源的间充质基质细胞的分离

人 BAMBI^高MFGE8^高脐带来源的间充质基质细胞的分离

人 BAMBI^高MFGE8^高脐带来源的间充质基质细胞的分离

人诱导多能干细胞分化为间充质基质细胞的两种代表性方法比较

人骨髓间充质干细胞（干细胞）的免疫调节性能评估

摘要

探索更多视频

此视频中的章节

生产和治疗间充质干行政/基质细胞（MSC）球体底漆在三维培养下无异物条件

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

在体外人骨髓间充质干细胞分化为心肌样细胞功能

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