这项研究是由卫生研究院资助R01-DE021683-01与健康研究资助R01-DE019434来MTL国家机构的国家机构的支持;霍华德·休斯医学研究所研究奖学金，以MTCDCW由ACS富兰克林·马丁教授研究奖学金，该Hagey实验室小儿再生医学和斯坦福大学儿童健康研究所教授学者奖的支持。

注:所有患者的样品与知情同意而获得的，和实验方案进行了审查并批准了斯坦福大学的机构审查委员会（议定书＃2188和＃9999）。 1.细胞分离培养: <ol><li>获得健康的女性患者在局部/全身麻醉进行腹部，胁肋，和/或大腿区域的选修lipoaspiration人类皮下脂肪组织。确保机构审查委员会（IRB）的批准已经从人体组织隔离携带者的协议获得的，并按照机构的安全防范措施，而这种材料的工作。 </li><li>以获得从lipoaspirated脂肪组织的SVF，先洗吸脂物三次1×无菌磷酸盐缓冲盐水等体积（PBS）中。小心吸弃底部水层。 </li><li>准备胶原酶消化缓冲液:0.075％I型胶原酶在汉克的平衡盐溶液（HBSS）中。制备FACS缓冲液:2％FBS，1％P188和1％青霉素 - 链霉素的PBS。过滤用市售0.22μm孔径快速流动聚醚砜过滤器这两种解决方案。 </li><li>消化洗涤脂肪组织，安全地在振荡水浴60分钟，在37℃（大约180摇动/分钟）添加的胶原酶消化缓冲等体积并放置消化容器中。 注:它是最好使用较大体积的消化容器比要求，因为这允许对最大消化振荡期间（ 即 ，加入250毫升的胶原酶消化缓冲，加入250毫升洗涤脂肪组织中的1L无菌烧瓶）。 </li><li>通过加入的FACS缓冲液等体积​​并允许坐在RT 5分钟中和酶的活性。接着，在离心机233×g离心20分钟，在4℃。 </li><li>小心吸弃上清，注意不要打扰高密度SVF颗粒。 </li><li>悬浮颗粒在5-10毫升RT红细胞裂解缓冲液，是根据粒料的大小。离开溶液坐在RT 5分钟，并离心在233×g离心5分钟，在室温。 </li><li>吸去上清液，并通过加入5-10毫升的传统的生长培养基的基础上，丸粒尺寸重悬沉淀（Dulbecco氏改良的Eagle培养基[DMEM] / 10％FBS / 1％青霉素 - 链霉素溶液〔笔链球菌]）。 </li><li>通过100微米的尼龙细胞过滤器过滤该悬浮液以去除细胞碎片。 </li><li>离心机在233×g离心5分钟，在4℃下，并弃去上清液而不扰乱沉淀。 </li><li>悬浮颗粒在5-10毫升的传统媒体增长的基础上，颗粒大小。 </li><li>放置2×10 6个细胞在15cm的标准培养皿，并建立原代培养物O / N在37℃/ 21％O 2，5％的CO 2。保持在亚汇合的水平，以防止自发分化，在37℃/ 21％O 2，5％CO 2中生长培养基。 注:在一个完全细胞密度汇合15厘米板是约4-6×10 6个细胞。 </li></ol> 2.染色<ol><li>染色前培养的ASCs在传统生长培养基，在37℃/ 21％O 2，5％的CO 2中 ，36小时/排序。 </li><li>从培养板使用的Accutase（按照制造商的协议）电梯携带者。 </li><li>一旦用FACS缓冲液（PBS，2％FBS和1％青霉素 - 链霉素）洗涤细胞。 </li><li>离心机在233×g离心5分钟，在4℃。 </li><li>弃去上清液而不扰乱沉淀。将细胞重悬于0.5-1毫升FACS缓冲液（取决于细胞数和抗体的期望的量）。 注:细胞悬浮液的体积，按照用FACS抗体生产商的建议，确定抗体浓度。然而，滴定抗体浓度，使用1起始浓度:100，通常建议，如果它是第一次使用的抗体。 </li><li>计数的总数目使用血球细胞。 </li><li>用于未染色样品与单色彩控制（根据所使用的荧光抗体的数量）在1.5ml的离心管储备100微升等分试样。 </li><li>添加适当的荧光标记的单克隆抗体（以预定的浓度，每制造商的说明），并孵育20-30分钟在冰上，避光。 <ol><li>标记成骨细胞亚群的稀释:抗CD90（APC抗 - 人CD90（Thy1肾炎））或抗CD105（FITC抗 - 人CD105（内皮糖蛋白））在FACS缓冲液以1:50的浓度。 </li><li>来标记其它的细胞群（ 例如 ，造血细胞和内皮细胞）的稀释:抗CD45（抗人CD45太平洋蓝），抗CD34（抗人CD34 APC），和/或抗CD31（抗人CD31 PE ）在FACS缓冲液以1:50的浓度。 </li><li>如果不使用直接标记的抗体，使用生物素化初级抗体用适当的链霉亲和共轭二进制抗体，如链霉亲和PE-Cy7的，在稀释度为1:100。 </li></ol></li><li>在4℃下填充管用FACS缓冲液，并离心在233×g离心5分钟，吸上清:温育后，洗细胞两次。 </li><li>通过一个70微米的尼龙细胞过滤悬浮细胞在400-500微升FACS缓冲和过滤样品以去除细胞团块。 </li><li>转移标记的细胞进入FACS管，带过滤器的顶部和保持样品在冰上进行分析的持续时间。 </li></ol> 3.荧光激活细胞分选注意:下面的步骤强制以前的知识在荧光激活细胞分选（FACS）或熟练的技术人员的协助。 <ol><li>使用适当的FACS软件，设计分析图检查前向散射（FSC），侧向散射（SSC），藻红蛋白（PE）-Texas红，蓝太平洋，异硫氰酸荧光素（FITC），别藻蓝蛋白（APC）以及其他任何使用荧光ð标记细胞。 </li><li>之前加载到细胞分选仪，简要地涡旋每个样品重悬细胞。 </li><li>与未染色样品的分析，以便确定门的总细胞群，以排除细胞碎片，并确定基线自发荧光展开。 </li><li>加入碘化丙啶（PI）至未染色样品，并将其进行分析，以可视化的总细胞群中活细胞的群体。构建解决这个人口大门。 </li><li>为了设置荧光补偿每个荧光染料的单色对照样品分析。 </li><li>染色样品设置门的位置进行排序分析。 </li><li>加入PI的染色样品，以染色死细胞。 </li><li>分类标记的细胞群。排序成为1.5毫升离心管中，或含有培养基锥形管（15ml）中。 </li><li>手动停止一旦已经获得了所需的细胞数量排序。 </li><li>我们荷兰国际集团的FACS，通过分析一小部分（ 例如 ，500个细胞）所获得的细胞群进行纯度检查。 注:此步骤确认排序细胞群的纯度。理想情况下，纯度应大于90％-95％。 </li><li>离心细胞，在233×g离心5分钟，在4℃下排序的完成和重悬后立即在1ml含有10％FBS的新鲜培养基中。 注意:使用介质的量可根据细胞沉淀的尺寸增大。 </li><li>板在明胶包被的板，以提高细胞生存力。取决于最终的细胞产量，板300000细胞每孔在6孔板中，每孔100,000个细胞在12孔板中。 </li></ol>

<ol>
	<li>Narsinh, K. H., 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=Single+cell+transcriptional+profiling+reveals+heterogeneity+of+human+induced+pluripotent+stem+cells.">Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells.</a> J Clin Invest. 121, 1217-1221 (2011).</li><li>Thorsen, T., Maerkl, S. J., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+large-scale+integration.">Microfluidic large-scale integration.</a> Science. 298, 580-584 (2002).</li><li>Warren, L., Bryder, D., Weissman, I. L., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+profiling+in+individual+hematopoietic+progenitors+by+digital+RT-PCR.">Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR.</a> Proc Natl Acad Sci U S A. 103, 17807-17812 (2006).</li><li>Warren, L. 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=Transcriptional+instability+is+not+a+universal+attribute+of+aging.">Transcriptional instability is not a universal attribute of aging.</a> Aging Cell. 6, 775-782 (2007).</li><li>Aalami, O. O., 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=Applications+of+a+mouse+model+of+calvarial+healing:+differences+in+regenerative+abilities+of+juveniles+and+adults.">Applications of a mouse model of calvarial healing: differences in regenerative abilities of juveniles and adults.</a> Plast Reconstr Surg. 114, 713-720 (2004).</li><li>Levi, B., 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=CD105+protein+depletion+enhances+human+adipose-derived+stromal+cell+osteogenesis+through+reduction+of+transforming+growth+factor+beta1+(TGF-beta1)+signaling.">CD105 protein depletion enhances human adipose-derived stromal cell osteogenesis through reduction of transforming growth factor beta1 (TGF-beta1) signaling.</a> The Journal of biological chemistry. 286, 39497-39509 (2011).</li><li>Chen, X. D., Qian, H. Y., Neff, L., Satomura, K., Horowitz, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thy-1+antigen+expression+by+cells+in+the+osteoblast+lineage.">Thy-1 antigen expression by cells in the osteoblast lineage.</a> J Bone Miner Res. 14, 362-375 (1999).</li><li>Malladi, P., Xu, Y., Yang, G. P., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+vitamin+D,+retinoic+acid,+and+dexamethasone+in+mouse+adipose-derived+mesenchymal+cells.">Functions of vitamin D, retinoic acid, and dexamethasone in mouse adipose-derived mesenchymal cells.</a> Tissue engineering. 12, 2031-2040 (2006).</li><li>Levi, B., 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=Depot-specific+variation+in+the+osteogenic+and+adipogenic+potential+of+human+adipose-derived+stromal+cells.">Depot-specific variation in the osteogenic and adipogenic potential of human adipose-derived stromal cells.</a> Plastic and reconstructive surgery. 126, 822-834 (2010).</li><li>James, A. W., 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=Estrogen/estrogen+receptor+alpha+signaling+in+mouse+posterofrontal+cranial+suture+fusion.">Estrogen/estrogen receptor alpha signaling in mouse posterofrontal cranial suture fusion.</a> PloS one. 4, e1720 (2009).</li><li>Locke, M., Feisst, V., Dunbar, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+human+adipose-derived+stem+cells:+separating+promise+from+clinical+need.">Concise review: human adipose-derived stem cells: separating promise from clinical need.</a> Stem Cells. 29, 404-411 (2011).</li><li>Glotzbach, J. P., 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=An+Information+Theoretic,+Microfluidic-Based+Single+Cell+Analysis+Permits+Identification+of+Subpopulations+among+Putatively+Homogeneous+Stem+Cells.">An Information Theoretic, Microfluidic-Based Single Cell Analysis Permits Identification of Subpopulations among Putatively Homogeneous Stem Cells.</a> PLoS One. 6, e21211 (2011).</li><li>Zhu, M., Heydarkhan-Hagvall, S., Hedrick, M., Benhaim, P., Zuk, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+isolation+of+adipose-derived+stem+cells+from+human+lipoaspirates.">Manual isolation of adipose-derived stem cells from human lipoaspirates.</a> J. Vis. Exp. , e50585 (2013).</li><li>Mitchell, J. B., 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=Immunophenotype+of+human+adipose-derived+cells:+temporal+changes+in+stromal-associated+and+stem+cell-associated+markers.">Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers.</a> Stem Cells. 24, 376-385 (2006).</li><li>McMurray, R. 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=Nanoscale+surfaces+for+the+long-term+maintenance+of+mesenchymal+stem+cell+phenotype+and+multipotency.">Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency.</a> Nat Mater. 10, 637-644 (2011).</li><li>Katz, A. J., Tholpady, A., Tholpady, S. S., Shang, H., Ogle, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+and+transcriptional+characterization+of+human+adipose-derived+adherent+stromal+(hADAS)+cells.">Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells.</a> Stem Cells. 23, 412-423 (2005).</li><li>McIntosh, K., 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=The+immunogenicity+of+human+adipose-derived+cells:+temporal+changes+in+vitro.">The immunogenicity of human adipose-derived cells: temporal changes in vitro.</a> Stem Cells. 24, 1246-1253 (2006).</li><li>Choudhery, M. S., Badowski, M., Muise, A., Pierce, J., Harris, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+age+negatively+impacts+adipose+tissue-derived+mesenchymal+stem+cell+expansion+and+differentiation.">Donor age negatively impacts adipose tissue-derived mesenchymal stem cell expansion and differentiation.</a> Journal of translational medicine. 12, 8 (2014).</li><li>Yoshimura, K., Suga, H., Eto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem/progenitor+cells:+roles+in+adipose+tissue+remodeling+and+potential+use+for+soft+tissue+augmentation.">Adipose-derived stem/progenitor cells: roles in adipose tissue remodeling and potential use for soft tissue augmentation.</a> Regenerative medicine. 4, 265-273 (2009).</li><li>Chung, M. T., 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=CD90+(Thy-1)+Positive+Selection+Enhances+Osteogenic+Capacity+of+Human+Adipose-Derived+Stromal+Cells.">CD90 (Thy-1) Positive Selection Enhances Osteogenic Capacity of Human Adipose-Derived Stromal Cells.</a> Tissue Eng Part A. , (2012).</li></ol>

没有一个作者有经济利益的任何产品，设备，还是在这个手稿中提到的药物。没有一个作家有任何竞争的财务权益报告。

目前，来自人体脂肪组织的SVF的ASC的均质亚群的分离仍然虽然期望目标的一个具有挑战性的。亲骨ASC亚隔离是特别理想的，因为这样的细胞可用于研究骨骼肌组织的形成和稳态。然而，脂肪组织的SVF窝藏显著异质对于干细胞的能力和分化潜能。11这种异质性的分子基础，不能从细胞的混合种群的理解，而是需要单细胞分析12用这种方法，表面标志物与先进的结构陶瓷的成骨分化能?...

The heterogeneous nature of stem cell populations is not yet fully understood and remains a major impediment to the development of clinically effective stem cell-based therapeutic applications. One of the most common ways to characterize a heterogeneous population of stem cells is to employ a cell sorting method, such as fluorescence-activated cell sorting (FACS), to separate cells based on their surface marker expression profiles. As sorting methods become more complex, it becomes possible to identify more distinct functional subpopulations of cells. Microfluidic-based technologies are becoming more and more frequently utilized in analysis of gene expression at the single cell level. Multiplexed quantitative polymerase chain reaction (qPCR) within a microfluidic chip allows for effective and reliable high-resolution, single cell transcriptional analysis.1-5
In a previous study using single cell transcriptional profiling of 48 genes, considerable transcriptional heterogeneity was observed among ASCs.6 However, the distribution of genes MSX2, BMP-5, BMP-7, ALP, OCN, RUNX2 exhibited a strong association with a cluster of cells possessing highly osteogenic transcriptional profiles. To isolate cells according to this osteogenic gene expression profile, surface antigen expression patterns were correlated with transcription patterns and surface marker expression of endoglin (CD105) was subsequently discovered to closely correlate with enhanced osteogenic differentiation potential of ASCs. Independent of CD105 expression, expression of surface receptor Thy-1 (CD90), a glycosyl-phosphatidylinositol-linked membrane protein previously shown by Chen et al. to be associated with osteoprogenitor cells, was also correlated with osteogenic gene expression.6,7 These findings provide the opportunity to prospectively isolate subpopulations within the larger heterogeneous pool of ASCs with increased osteogenic capacity for cell-based bone tissue engineering applications.

<table><tbody><tr><td>Disposable 250 ml Conical Tubes</td><td>Corning (Thomas Scientific)</td><td>2602A43</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>DMEM, high glucose, GlutaMAX Supplement</td><td>Gibco</td><td>10566-016</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Betadine - Antiseptic Povidone/Iodine Solution</td><td>Purdue&amp;nbsp;</td><td>PFC-67618015017</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution, 1X</td><td>Cellgro</td><td>21-023-CV</td><td></td></tr><tr><td>Fetal Bovine Serum, Certified, US Origin</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Collagenase from&amp;nbsp;Clostridium histolyticum</td><td>Sigma-Aldrich</td><td>C0130-5G</td><td></td></tr><tr><td>ACCUTASE Cell Detachment Solution</td><td>Stem Cell Technologies</td><td>7920</td><td></td></tr><tr><td>APC Mouse Anti-Human CD90</td><td>BD Pharmingen</td><td>559869</td><td></td></tr><tr><td>FITC Mouse anti-Human CD105 (Endoglin)</td><td>BD Pharmingen</td><td>561443</td><td></td></tr><tr><td>Anti-Human CD45 eFluor 450 (Pacific Blue replacement)&amp;nbsp;</td><td>eBioscience</td><td>48-9459-41</td><td></td></tr><tr><td>Anti-Human CD34 APC</td><td>eBioscience</td><td>17-0349-41</td><td></td></tr><tr><td>Anti-Human CD31 (PECAM-1) PE</td><td>eBioscience</td><td>12-0319-41</td><td></td></tr><tr><td>Streptavidin PE-Cy7</td><td>eBioscience</td><td>25-4317-82</td><td></td></tr><tr><td>BD FACS Aria II instrument</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BD FACSDiva Software</td><td>BD Biosciences</td><td></td><td></td></tr></tbody></table>

isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis

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

使用CD90作为标记的细胞具有增强成骨结果在人的ASC（ 图1A，1B）的高度富集的群体的隔离。携带者沾满太平洋蓝偶联的抗人CD45，FITC缀合的抗人类CD105，和APC缀合的抗人CD90。排序后，纯度水平大于98％，如通过量化后的排序分析。 定义的基础上允许两种新型亚群的前瞻性孤立转录谱细胞群体。表征每个亚群有前途的（CD90 +和CD105 低 ）的成骨潜力，...

Watch this Scientific Journal Video about Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis at JoVE.com

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

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

隔离和人力富集脂肪基质细胞的成骨增强

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

isolation-enrichment-human-adipose-derived-stromal-cells-for-enhanced

Research

JoVE Journal

Developmental Biology

11.4K 次观看.  Stanford University School of Medicine. The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

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

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

隔离和脂肪干细胞分化的猪从皮下脂肪组织

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

科研

发育生物学

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

BMPR-IB +快速分离脂肪来源的使用基质细胞在颅骨缺损愈合模型

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

修复的临界大小的颅骨缺损模型脂肪间充质干细胞的收获Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

医学

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

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

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

生物工程

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

人网膜和皮下脂肪组织 CD34+CD31+ 内皮细胞的分离、扩增和脂肪诱导

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

免疫与感染

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

从小鼠腹内脂肪库分离成脂肪和纤维炎症基质细胞亚群

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

从人类Lipoaspirates脂肪来源干细胞的手动隔离

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

生物学

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

使用创新的无异种方法进行人类治疗，分离和培养人脂肪来源的干细胞

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...

脂肪组织来源的干细胞分离和表征

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

作者聚焦:分离和鉴定源自 Sprague Dawley 大鼠脂肪组织的间充质干细胞  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...