这项工作是从国立关节炎和肌肉骨骼皮肤疾病，美国国立卫生研究院RO1AR054458赠款的支持。

1。与Endorem标记的hMSCs <ol><li>细胞增长到80％汇合至少18小时前标签。 </li><li>在这段时间内，标签媒体准备加入Endorem剂量100微克铁/ ml的无血清培养基样品。 </li><li>培养基细胞生长至汇合后，吸气和细胞用PBS或无血清培养基洗1倍。 </li><li>接下来，冲洗液被吸入，加入先前准备的标签媒体。细胞，然后在37℃孵育℃，5％CO 2 4小时。 </li><li>经过4个小时的潜伏期，标签媒体是吸气和细胞，用PBS漂洗和往常一样胰酶消化。 </li><li>胰酶消化后，细胞被冲3倍，用PBS 400rcf离心5分钟。 </li><li>这种洗涤步骤后，重新悬浮细胞，计数，可行性评估，并用实验需要。 </li></ol> 2。准备琼脂糖<ol><li>后的细胞都被贴上了，现在是时候准备琼脂糖溶液。 </li><li>罚款的规模，琼脂糖粉80毫克称重，并添加到PBS2毫升。 </li><li>该解决方案，然后轻轻地动摇和高压灭菌。 </li><li>约40分钟后，可以取出液的琼脂糖溶液，高压灭菌器。测量体积应与预热PBS相应调整。 </li><li>然后溶液冷却至42 ° C </li></ol> 3。创建MASI <ol><li>当琼脂糖溶液已达到所需的温度，细胞计数，并为最后一次在1.5毫升Eppendorf管离心。 </li><li>离心后，取上清液丢弃，细胞的DMEM混合浓度达到30 × 10 6细胞/ ml。 </li><li>现在，42℃的温暖琼脂糖混合细胞。 </li><li>一个寒冷的枪头，可能导致琼脂糖，以巩固内部的提示，因此它应预热尖吹打热无菌PBS向上和向下</li><li>现在，媒体和混合介质与细胞悬浮彻底，以便创建一个同质的悬挂是相同体积的琼脂糖。 </li><li>保持室内预热水浴Eppendorf管中，边搅拌，使琼脂糖不冷静下来，足以巩固。 </li><li>当悬浮液是均匀的，被植入的细胞中的缺陷。 </li></ol><img src="/files/ftp_upload/1793/1793fig1.jpg" alt="图1" /> 图1。冠状T2加权SE图像与髌骨标本植入软骨缺损的hMSCs（TR 4000毫秒/ TE 18.27 MS）。

<ol>
	<li>Mauck, R. L., Yuan, X., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenic+differentiation+and+functional+maturation+of+bovine+mesenchymal+stem+cells+in+long-term+agarose+culture.">Chondrogenic differentiation and functional maturation of bovine mesenchymal stem cells in long-term agarose culture.</a> Osteoarthritis Cartilage. 14, 179-189 (2006).</li><li>Pittenger, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+potential+of+adult+human+mesenchymal+stem+cells.">Multilineage potential of adult human mesenchymal stem cells.</a> Science. 284, 143-147 (1999).</li><li>Rahfoth, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+allograft+chondrocytes+embedded+in+agarose+gel+into+cartilage+defects+of+rabbits.">Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits.</a> Osteoarthritis Cartilage. 6, 50-65 (1998).</li><li>Henning, T. D., Boddington, S., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+hESCs+and+hMSCs+with+iron+oxide+nanoparticles+for+non-invasive+in+vivo+tracking+with+MR+imaging.">Labeling hESCs and hMSCs with iron oxide nanoparticles for non-invasive in vivo tracking with MR imaging.</a> J Vis Exp. , (2008).</li></ol>

所描述的协议提供了一个可重复的氧化铁纳米粒子标记的干细胞，这些标记的MSCs植入软骨缺损的方法。这种技术允许与磁共振成像，这使得MASI故障的早期检测软骨缺损的非侵入性干细胞移植的写照。一个错位或标记的细胞外排，可以诊断的基础上从MR图像的移植部位的标签消失。对这些事件的早期诊断是可取的，将防止不必要的替代侵入性诊断程序的病人。所述的非侵入性诊断MASI成果方法，可?...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>D-MEM High Glucose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D5648</td>
<td>&nbsp;</td>
<tr>
<td>PBS (Ca++, Mg++ free)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14190-144</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA 0.05%</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25399-120</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
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<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140-122</td>
<td>&nbsp;</td>
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<td>Ferumoxides (Endorem)</td>
<td>
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<td>Guerbet</td>
<td>&nbsp;</td>
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<td>Agarose Typ VII</td>
<td>&nbsp;</td>
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<td>A9045</td>
<td>Dilute Agarose in PBS to final concentration of 4%</td>
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implantation of ferumoxides labeled human mesenchymal stem cells in cartilage defects

组织工程领域的集成工程，细胞生物学和医学对特定的细胞和组织功能再生的原则。矩阵相关的干细胞植入（MASI）的目标是再生软骨缺损，因关节炎或外伤关节损伤。成人间质干细胞（MSCs）分化为软骨细胞谱系细胞的能力，并已显示出可喜的成果细胞为基础的关节软骨修复技术。自体干细胞可以从各种组织中分离，在细胞培养，可以扩大，而不会失去它们的分化潜能，并表现出软骨细胞分化，在 体外和体内的1，2。 为了提供地方留成和软骨缺损移植干细胞的可行性，脚手架是必要的，这也支持了随后的分化和增殖。脚手架指导组织形成的架构，允许细胞外基质，由干细胞产生，扩大。以前的调查表明，2％琼脂糖支架可以支持稳定的透明软骨的发展并不会诱发免疫反应的3。 MASI移植的干细胞的长期保存是软骨再生的关键。氧化铁纳米粒子标记的MSCs可在体内长期跟踪与非侵入性磁共振成像技术4。 本演示将展示标签MSCs的氧化铁纳米粒子，成软骨缺损的细胞琼脂糖结构和这些结构的植入代技术。标记的结构可以跟踪非侵入性磁共振成像。

Watch this Scientific Journal Video about Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects at JoVE.com

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

演示的目的是展示一个高度重复性的方法来生成矩阵相关联的干细胞植入软骨缺损，可与磁共振成像的可视化。干细胞标记与美国FDA批准的Ferumoxides用琼脂糖混合，植入软骨缺损，并与7T磁共振扫描仪成像。

Ferumoxides植入人类间质干细胞在软骨缺损的标记

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

implantation-ferumoxides-labeled-human-mesenchymal-stem-cells

Research

JoVE Journal

Biology

10.5K 次观看.  Medical Center, University of California San Francisco. 演示的目的是展示一个高度重复性的方法来生成矩阵相关联的干细胞植入软骨缺损，可与磁共振成像的可视化。干细胞标记与美国FDA批准的Ferumoxides用琼脂糖混合，植入软骨缺损，并与7T磁共振扫描仪成像。

视频: Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

人类神经干细胞计数

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

科研

生物学

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

从噬菌体抗体库的肽调节间质细胞的基因表达和使可能在Unicortical骨缺损的成骨

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs&#39; size was 30-200 nm, with a particle concentration of 7.75 &#215; 1010 particles/mL and a protein concentration of 80 &#181;g/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells&#39; small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

一种从人间充质干细胞中分离小细胞外囊泡的简单台式过滤方法

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...