Name: programming stem cells for therapeutic angiogenesis using biodegradable polymeric nanoparticles
Uploaded: 2013-09-27T16:00:00
Description: Watch this Scientific Journal Video about Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles at JoVE.com

作者要感谢美国心脏协会国家科学家开发格兰特（10SDG2600001），斯坦福大学Bio-X中心的跨学科倡议计划，以及斯坦福大学医学院的学者研究计划提供资金。

1。高分子合成<ol><li>在通风橱中，称出3,523毫克丁二醇酯（C），并转移到装有搅拌棒玻璃闪烁瓶中。 </li><li>预热5 - 氨基-1 - 戊醇（32）到90℃以溶解盐，然后在通风橱中，称出1533毫克32，并添加到含有C.这种方法的闪烁瓶中，将导致以摩尔比的C：32 = 1:1.2。 </li><li>马上含有这两种解决方案的小瓶放置到一个搅拌盘。以600rpm设定搅拌速度。 </li><li>在闪烁瓶中转移到烘箱设定在90℃。设定搅拌速度?...

<ol>
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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+may+be+uniquely+suited+for+combined+gene+therapy+and+cell+replacement:+Evidence+from+engraftment+of+Neurotrophin-3-expressing+stem+cells+in+hypoxic-ischemic+brain+injury.">Neural stem cells may be uniquely suited for combined gene therapy and cell replacement: Evidence from engraftment of Neurotrophin-3-expressing stem cells in hypoxic-ischemic brain injury.</a> Exp. Neurol. 199, 179-190 (2006).</li><li>Green, J. J., Langer, R., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combinatorial+polymer+library+approach+yields+insight+into+nonviral+gene+delivery.">A combinatorial polymer library approach yields insight into nonviral gene delivery.</a> Acc Chem Res. 41, 749-759 (2008).</li><li>Yang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+to+human+adult+and+embryonic+cell-derived+stem+cells+using+biodegradable+nanoparticulate+polymeric+vectors.">Gene delivery to human adult and embryonic cell-derived stem cells using biodegradable nanoparticulate polymeric vectors.</a> Gene Ther. 16, 533-546 (2009).</li><li>Niiyama, H., Huang, N. F., Rollins, M. D., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+hindlimb+ischemia.">Murine model of hindlimb ischemia.</a> J. Vis. Exp. , e1035 (2009).</li><li>Ceradini, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitor+cell+trafficking+is+regulated+by+hypoxic+gradients+through+HIF-1+induction+of+SDF-1.">Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1.</a> Nat. Med. 10, 858-864 (2004).</li><li>Kidd, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+of+mesenchymal+stem+cell+tropism+for+tumor+and+wounding+microenvironments+using+in+vivo+bioluminescent+imaging.">Direct evidence of mesenchymal stem cell tropism for tumor and wounding microenvironments using in vivo bioluminescent imaging.</a> Stem Cells. 27, 2614-2623 (2009).</li><li>Sunshine, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+end-groups+of+linear+polymer+determine+cell-type+gene-delivery+efficacy.">Small-molecule end-groups of linear polymer determine cell-type gene-delivery efficacy.</a> Adv. Mater. 21, 4947-4951 (2009).</li><li>Sunshine, J. C., Akanda, M. I., Li, D., Kozielski, K. L., Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+base+polymer+hydrophobicity+and+end-group+modification+on+polymeric+gene+delivery.">Effects of base polymer hydrophobicity and end-group modification on polymeric gene delivery.</a> Biomacromolecules. 12, 3592-3600 (2011).</li><li>Lynn, D. M., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradable+poly(&#946;-amino+esters):+Synthesis,+characterization,+and+self-assembly+with+plasmid+DNA.">Degradable poly(&#946;-amino esters): Synthesis, characterization, and self-assembly with plasmid DNA.</a> J. Am. Chem. Soc. 122, 10761-10768 (2000).</li><li>Eltoukhy, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+molecular+weight+of+amine+end-modified+poly(beta-amino+ester)s+on+gene+delivery+efficiency+and+toxicity.">Effect of molecular weight of amine end-modified poly(beta-amino ester)s on gene delivery efficiency and toxicity.</a> Biomaterials. 33, 3594-3603 (2012).</li><li>Glover, D. J., Lipps, H. J., Jans, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+safe,+non-viral+therapeutic+gene+expression+in+humans.">Towards safe, non-viral therapeutic gene expression in humans.</a> Nat. Rev. Genet. 6, 299-310 (2005).</li><li>Dave, U. P., Jenkins, N. A., Copeland, N. 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在这里，我们报告一个编程成体干细胞利用非病毒，可生物降解纳米粒子过度治疗因素的方法。此平台可用于治疗疾病，其中的干细胞可以自然的家，如局部缺血和癌症中特别有用。9-10此外，非病毒基因递送平台允许对治疗因素，这是适用于大多数组织再生和伤口瞬时表达愈合过程。转染过程取决于有效的DNA进入细胞时，一般在积极分裂的细胞类型，在非分裂细胞的效果很好，但不为好?...

这项技术的总体目标是使用非病毒编程干细胞过表达治疗因素在缺血的部位，以促进治疗性血管新生。用在实验室中合成的可生物降解的纳米颗粒的干细胞进行了修改， 先体外后体内 ，然后再移植到后肢缺血的小鼠模型来验证他们的潜在增强血管生成和组织打捞。 控制血管生长是成功的组织再生的重要组成部分，以及用于治疗各种缺血性疾病，如中风，肢体局部缺血和心肌梗死。多种策略已经被开发，以促进血管生长，包括生长因子递送和基于细胞的疗法。1尽管在动物疾病模型中观察到的功效，这些方法仍然面临如需要超生理剂量的生长因子递送的限制，或没有足够的旁分泌单靠细胞释放。为克服上述限制的一个潜在策略是结合干细胞治疗和基因治疗，其中的干细胞是基因体内之前的前向移植编程以过表达所需的治疗因子。这种方法已被证明在多种疾病模型包括后肢缺血2，心脏疾病3，骨愈合4和神经损伤5， 等等 。然而，大多数的基因治疗技术依赖于病毒载体，这是与安全性问题，例如潜在的免疫原性和插入突变相关联。生物材料介导的非病毒基因递送可以克服这些限制，但通常从低转染效率受到影响。要加快新型生物材料的发现为有效的非病毒基因传递，最近的研究采用组合化学和高通量筛选方法。可生物降解聚合物的库，如聚（β-氨基ES字符）（PBAE）已开发和筛选，从而导致了与显着增强的转染效率比传统的聚合物载体同行领先的聚合物的发现。6-7 本文中，我们描述PBAE的合成和验证其转染脂肪来源的干细胞（ADSC中） 在体外能力，其次是基因修饰脂肪干细胞在后肢缺血的小鼠模型中过量表达血管内皮生长因子（VEGF）的后续的移植。该结果由追踪细胞命运利用生物发光成像，采用激光多普勒血流灌注成像（LDPI）评估组织灌注，并确定血管生成和组织打捞组织学评价。

<table border="1">
	<tbody>
		<tr>
			<td>
				Name of the Reagent</td>
			<td>
				Company</td>
			<td>
				Catalogue Number</td>
			<td>
				Comments (optional)</td>
		</tr>
		<tr>
			<td>
				DMEM</td>
			<td>
				Invitrogen</td>
			<td>
				11965</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Fetal Bovine Serum</td>
			<td>
				Invitrogen</td>
			<td>
				10082</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Penicillin/Streptomycin</td>
			<td>
				Invitrogen</td>
			<td>
				15070</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Basic Fibroblast Growth Factor</td>
			<td>
				Peprotech</td>
			<td>
				100-18B</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				1,4-Butanediol Diacrylate (90%)</td>
			<td>
				Sigma Aldrich</td>
			<td>
				411744</td>
			<td>
				Acronym: C</td>
		</tr>
		<tr>
			<td>
				5-amino-1-pentanol (97%)</td>
			<td>
				Alfa Aesar</td>
			<td>
				2508-29-4</td>
			<td>
				Acronym: 32</td>
		</tr>
		<tr>
			<td>
				Tetraethyleneglycoldiamine &gt;99%)</td>
			<td>
				Molecular Biosciences</td>
			<td>
				17774</td>
			<td>
				Acronym: 122</td>
		</tr>
		<tr>
			<td>
				Sodium Acetate</td>
			<td>
				G-Biosciences</td>
			<td>
				R010</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Phosphate Buffered Saline</td>
			<td>
				Invitrogen</td>
			<td>
				14190-144</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Tetrahyofuran Anhydrous (&gt;99.9%)</td>
			<td>
				Sigma Aldrich</td>
			<td>
				401757</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Diethyl Ether Anhydrous (&gt;99%)</td>
			<td>
				Fisher Scientific</td>
			<td>
				E138-4</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				DMSO Anhydrous (&gt;99.9%)</td>
			<td>
				Sigma Aldrich</td>
			<td>
				276855</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Gelatin</td>
			<td>
				Sigma Aldrich</td>
			<td>
				G9391</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Trypsin-EDTA</td>
			<td>
				Invitrogen</td>
			<td>
				25200</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				D-luciferin</td>
			<td>
				GoldBio</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Optimal Cutting Temperature (O.C.T)</td>
			<td>
				Tissue-Tek</td>
			<td>
				4583</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Rat anti-Mouse CD31</td>
			<td>
				BD Pharmingen</td>
			<td>
				550274</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Alexa Fluor 594 anti-rat IgG</td>
			<td>
				Invitrogen</td>
			<td>
				A11007</td>
			<td>
				&nbsp;</td>
		</tr>
	</tbody>
</table>

	&nbsp;

programming stem cells for therapeutic angiogenesis using biodegradable polymeric nanoparticles

控制血管生长是成功的组织再生和伤口愈合，以及用于治疗局部缺血性疾病，如中风，心脏发作，或外周动脉疾病的关键。直接输送的血管生成生长因子具有刺激新血管生长的潜力，但通常与诸如缺乏靶向和半衰期短的体内的局限性有关。基因治疗提供了一种替代的方法通过提供基因编码血管生成因子，但常常需要使用病毒，并通过安全的担忧限制。在这里，我们描述了最近开发的战略，刺激血管生长，通过编程的干细胞使用可生物降解的聚合物纳米粒子以过度血管生成因子原位 。特别是我们的战略，利用其迁移往体内缺血组织能力优势利用干细胞作为运载工具。采用优化的聚合载体，脂肪来源干细胞被修饰而过表达的血管生成的基因，编码血管内皮生长因子（VEGF）。我们描述了用于聚合物的合成中，纳米粒子的形成， 在体外转染的干细胞，以及用于验证的VEGF表达干细胞的功效在鼠后肢缺血模型中促进血管生成的方法的过程。

在混合在一起时，带正电荷的聚合物（C32-122）和带负电的DNA质粒自组装成纳米颗粒。纳米粒子的形成可通过电泳分析得到证实，即 C32-122和质粒DNA之间的络合作用将防止在电泳过程中动员的DNA。该聚合物可作为转染试剂，以促进DNA的增强吸收到靶细胞和编码蛋 ​​白质的后续表达（ 图2）。细胞可被转染的任何治疗性基因或报道DNA，例如绿色荧光蛋白（GFP），以方便使用荧光?...

Watch this Scientific Journal Video about Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles at JoVE.com

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

我们描述了编程干细胞的方法，使用可生物降解的聚合物纳米颗粒以过表达的治疗因子的血管生成。所述工艺包括聚合物的合成，转染脂肪来源的干细胞在体外和验证编程的干细胞的功效，以促进血管生成的小鼠后肢缺血模型。

编程干细胞的治疗性血管新生使用可生物降解高分子纳米微球

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Research

JoVE Journal

Bioengineering

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

制定通过Nanoprecipitation技术嵌段聚合物纳米粒子

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

科研

生物工程

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

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

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

订货单细胞和单胚胎的三维约束：高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Polymeric Microneedle Array Fabrication by Photolithography

聚合物微针阵列制造用光刻

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

制造超疏水高分子材料生物医学应用

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

闪速纳米沉淀法包聚物纳米粒子中疏水亲水化合物的作用

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

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

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 ...