This research was supported by a grant of the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (grant number: HI14C2116) and by Research Fund of Seoul St. Mary's Hospital, The Catholic University of Korea.

该研究方案是根据美国国立卫生研究院的指导方针批准由韩国天主教大学（KC16EAME0162）的机构审查委员会和执行。与书面知情同意书，得到的所有组织。 1.离心重力加载和颗粒文化<ol><li>细胞培养和收获<ol><li>培养的ASC（P2-P3;见材料的列表）在补充有10％胎牛血清（FBS）和1％青霉素/链霉素（P / S）的Dulbecco改良的Eagle氏培养基，低葡萄糖（DMEM-LG）中于37℃下在含有5％CO 2的加湿培养箱。 </li><li>当细胞达到80％汇合时，丢弃培养基，用5ml 1×磷酸盐缓冲盐水的（PBS）洗涤细胞。 </li><li>添加1毫升的PBS含1mM EDTA的的并在含有5％CO 2的潮湿培养箱中于37℃孵育2分钟。 </li><li>轻轻敲击培养板中，添加4毫升新鲜培养基中，将细胞转移到有15毫升锥形管，并离心将细胞在250×g下在室温（RT）下2分钟。 </li></ol></li><li>离心重力加载<ol><li>离心（步骤1.1.4）后，除去上清而不会干扰沉淀并用10％的FBS重悬在10mL的DMEM-LG的沉淀。算使用血球细胞。 </li><li>对于CG装载，转移2.5×10 5个细胞到一个新的15毫升锥形管和离心机2400×g的15分钟。 </li></ol></li><li>团培养<ol><li>立即离心后，吸出上清液，并添加定义软骨分化培养基500μL的（CDM，高糖DMEM补充有1％FBS，1％ITS +预混料，100nM的地塞米松，1×MEM非必需氨基酸溶液，50微克/ mL的L-脯氨酸，和1％青霉素/链霉素）。在每次更换培养基添加新鲜制备的L-抗坏血酸-2-磷酸的50微克/毫升。作为阳性对照，诱导软骨differentiati在uncentrifuged细胞通过加入CDM含有10纳克/毫升TGF-β1。 </li><li>放置装有粒料松散封管在站立位置并在含有5％CO 2的潮湿培养箱中于37℃孵育。 </li><li>改变介质隔日3周。 </li></ol></li><li>微团培养<ol><li>离心（步骤1.2.2; 2400×g的15分钟）后，立即吸出上清液和悬浮颗粒在CDM的10微升。 </li><li>为了形成微团，将重悬的细胞在24孔板的孔的中心。 </li><li> 2小时后，小心地加入1毫升CDM向好，对板材的墙吸液，以免破坏微团。作为阳性对照，通过将含有10纳克/毫升TGF-β1的CDM执行与uncentrifuged细胞微团培养。 </li><li>孵育在37℃的微团在含5％CO 2的加湿培养箱。 </li><li>改变介质EVERY一天3周。 </li></ol></li></ol> 2.逆转录聚合酶链反应（RT-PCR）技术检测软骨分化标志转录上调<ol><li>在第14天，用一个移液管，以1×PBS中的球体粒料转移到一个新的1.5毫升管中并把它们洗干净。 </li><li>提取使用硫氰酸胍-苯酚-氯仿提取法9颗粒中的总RNA。 </li><li>从根据制造商的方案使用逆转录酶的总RNA的2微克合成cDNA（孵育在42℃进行1小时，然后使酶失活，在70℃，5分钟）。 </li><li>用特异于软骨细胞分化标志物10的引物进行PCR。 </li></ol> 3.染色检测软骨分化标志蛋白的表达<ol><li>石蜡包埋细胞团<ol><li>在第21天，用吸管收获球体粒料和用1×PBS洗涤。 </li><li>固定在4％低聚甲醛通过浸渍球体粒料24小时。 注意:多聚甲醛是高毒。避免与眼睛，皮肤或粘膜接触。尽量减少暴露，并避免在制备过程中吸入。穿戴合适的个人防护装备。 </li><li>丢弃固定液和冲洗用去离子水（DW）的细胞沉淀。 </li><li>放置纱布一层到所述盒，并用移液管转移固定粒料。通过折叠的纱布覆盖颗粒并关闭盒盖。 </li><li>脱水粒料。 <ol><li>沉浸在70％乙醇（EtOH中）的粒料在室温下5分钟。 </li><li>沉浸在80％EtOH中的粒料在室温下5分钟。 </li><li>沉浸在95％EtOH中的粒料在室温下5分钟。 </li><li>浸泡在100％的乙醇的粒料在室温下5分钟。重复两次。 </li><li>浸泡在100％二甲苯粒料在室温15分钟。重复TWICE。 </li></ol></li><li>嵌入在石蜡中固定细胞沉淀在56℃下在模具中;粒料可以使用专门的自动组织处理系统被嵌入到石蜡中。 </li><li>从用旋转切片机的石蜡包埋细胞沉淀切成5微米厚的切片。 </li><li>浮动部分在50％EtOH中，然后用幻灯片将它们转移到50℃的水浴中。 </li><li>浮动部分放置到幻灯片。 </li></ol></li><li>番红O和阿尔新蓝染色<ol><li>再水合石蜡切片。 <ol><li>沉浸在二甲苯幻灯片15分钟。重复两次。 </li><li>浸泡在100％EtOH中的幻灯片5分钟。重复两次。 </li><li>沉浸在90％EtOH中的幻灯片5分钟。 </li><li>沉浸在80％EtOH中的幻灯片5分钟。 </li><li>浸入70％乙醇的幻灯片5分钟，然后把它们洗干净在1X PBS。 </li></ol></li><li>染色再水化切片用1％的番红O解决方案和3％阿尔新蓝为30分钟。 </li><li>丢弃染色液并用DW冲洗部分。 </li><li>染色用苏木精/曙红溶液（染液）中1分钟的部分。 </li><li>丢弃染色液并用DW冲洗部分。 </li><li>将安装介质上盖玻片下降消除任何残余的水后。小心地将含所述安装平台盖玻片滑动（细胞面朝下）。 </li><li>允许载片干燥，在室温2-3小时。 </li><li>使用明场显微镜（自动直立式显微镜，50倍和200倍）拍摄图像。 </li></ol></li><li>免疫荧光法<ol><li>补充水分的章节，如第3.2.1所述。 </li><li>沉浸在柠檬酸钠缓冲液的幻灯片（10mM柠檬酸钠，0.05％吐温20，pH 6.0）中，然后用微波在子沸点温度保持他们10分钟。 </li><li>酷幻灯片在室温下20分钟，然后用自来水清洗。 </li><li>孵育载玻片在3％的 H 2 O 2（在1×PBS）中15分钟，然后把它们洗干净用自来水15分钟11。 </li><li>封锁1小时封闭缓冲液（在PBS中的10％正常山羊血清）的幻灯片。 </li><li>吸出封闭溶液，然后应用，用5％正常山羊血清稀释到幻灯片的第一抗体。 </li><li>过夜孵育载玻片于4℃。 </li><li>洗玻片三次在1×PBS中，每次5分钟。 </li><li>孵育在黑暗用5％正常山羊血清在RT稀释1小时荧光标记的第二抗体的幻灯片。 </li><li>洗玻片三次在1×PBS中，在黑暗中，每次5分钟。 </li><li>孵育载玻片用DAPI（1微克/毫升）10分钟，然后把它们洗干净两次在1×PBS中，每次5分钟。 </li><li>将安装介质上盖玻片下降消除任何残余的水后。小心地将安装介质上的滑动（细胞面朝下）。 </li><li>允许的幻灯片干燥2-3在RT的黑暗小时。 </li><li>可视化使用荧光显微镜玻片（RHOD:594纳米，DAPI:340纳米; 60X和200倍）12。 </li></ol></li></ol>

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We declare that we have no conflicts of interest associated with this work.

细胞的干性状态为SOX9的CG诱导过表达非常重要的。在我们的研究中，SOX9的表达可以通过CG在早期传代的ASC（2-3）引起的，而不是在后面通道的ASC。已经报道的是，在培养过程中，携带者包含CD34 +细胞，直至3代16。的ASCs往往失去的CD34的表达作为将细胞传代，从而产生至CG低响应。 离心重力，静水压力可以刺激CG过程中加载到细胞。因此，介质的体积可能是影?...

在关节软骨缺损不自然愈合。因此，干细胞移植已被提出作为削弱的软骨的修复一个有前途的方法。然而，该方法需要有足够数量的干细胞的同时获取和这些细胞的诱导来进行软骨细胞分化。骨髓（BM）已被广泛使用作为干细胞的来源，但是从骨髓细胞分离有两个主要的缺点:侵袭和产量不足。因为它易于获得的，脂肪组织是干细胞的一个优选来源。以往的研究表明，从脂肪组织中分离干细胞和诱导用细胞因子，如TGF-β11,2这些细胞软骨分化的可行性。这些方法是有效的，但价格昂贵。 作为一个成本较低的替代细胞因子，机械应力可以用来诱导软骨细胞分化。机械加载在维持关节软骨3的健康至关重要的作用，并且它可以诱导各种细胞软骨表型。例如，静水压经由MAP激酶/ JNK途径4诱导滑膜衍生的祖细胞的软骨形成的表型，和机械压缩通过上调软骨基因5诱导软骨在人类间质干细胞（MSCs）。此外，剪切应力有助于软骨相关的细胞外基质（ECM）的人MSC 6的表达。离心重力（CG），离心产生很容易地应用和控制的机械应力，可诱导细胞7差异表达基因。例如，在肺上皮癌细胞，白细胞介素（IL）-1b的表达通过离心8上调。 ŧherefore，作为实验诱导机械应力，CG可用于诱导干细胞软骨基因表达。然而，它的CG能否诱导干细胞的软骨细胞分化还不清楚。 在这项研究中，我们发现，CG诱导SOX9的上调，软骨的主要调控，在人类携带者，导致软骨细胞基因的过度表达。此外，我们比较的CG上与TGF-β1的软骨的影响，生长因子最常用于在体外诱导软骨中的干细胞。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Plasticware&lt;/strong&gt;</td></tr><tr><td>100 mm Dish</td><td>TPP</td><td>93100</td><td></td></tr><tr><td>60 mm Dish</td><td>TPP</td><td>93060</td><td></td></tr><tr><td>50 mL Cornical Tube</td><td>SPL</td><td>50050</td><td></td></tr><tr><td>15 mL Cornical Tube</td><td>SPL</td><td>50015</td><td></td></tr><tr><td>10 mL Disposable Pipette</td><td>Falcon</td><td>7551</td><td></td></tr><tr><td>5 mL Disposable Pipette</td><td>Falcon</td><td>7543</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;ASC Culture Media Materials&lt;/strong&gt;</td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>DMEM Low glucose</td><td>Life Technologies</td><td>11885-084</td><td>growth base media</td></tr><tr><td>Penicilin Streptomycin</td><td>Sigma Aldrich</td><td>P4333</td><td>1%</td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>16000-044</td><td>10%</td></tr><tr><td>PBS/1 mM EDTA</td><td>Life Technologies</td><td>12604-039</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Chondrogenic Differentiation Media Materials&lt;/strong&gt;</td></tr><tr><td>DMEM High glucose</td><td>Life Technologies</td><td>11995</td><td>chondrogenic differentiation base media</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100x)</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma Aldrich</td><td>D2915</td><td>100 nM</td></tr><tr><td>Penicilin Streptomycin</td><td>Life Technologies</td><td>P4333</td><td>1%</td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>16000-044</td><td>1%</td></tr><tr><td>Ascorbate-2-phosphate</td><td>Sigma Aldrich</td><td>A8960</td><td>50 &amp;#956;g/mL</td></tr><tr><td>L-proline</td><td>Sigma Aldrich</td><td>P5607</td><td>50 &amp;#956;g/mL</td></tr><tr><td>ITS</td><td>BD</td><td>354352</td><td>1%</td></tr><tr><td>Human TGF&amp;beta;1</td><td>Peprotech</td><td>100-21</td><td>10 ng/mL</td></tr><tr><td colspan="4">&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td>18 mm Cover Glass</td><td>Superior</td><td>HSU-0111580</td><td></td></tr><tr><td>4% Paraformaldyhyde</td><td>Tech &amp;amp; Innovation</td><td>BPP-9004</td><td></td></tr><tr><td>Tween 20</td><td>BIOSESANG</td><td>T1027</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Vector Lab</td><td>SP-5050</td><td></td></tr><tr><td>Anti-Collagen II antibody</td><td>abcam&amp;nbsp;</td><td>ab34712</td><td>1:100</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate</td><td>Molecular Probe</td><td>A-11037</td><td>1:200</td></tr><tr><td>DAPI</td><td>Molecular Probe</td><td>D1306</td><td></td></tr><tr><td>Prolong gold antifade reagent</td><td>Invitrogen</td><td>P36934</td><td></td></tr><tr><td>Slide Glass, Coated</td><td>Hyun Il Lab-Mate</td><td>HMA-S9914</td><td></td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596-018</td><td></td></tr><tr><td>Chloroform</td><td>Sigma Aldrich</td><td>366919</td><td></td></tr><tr><td>Isoprypylalcohol</td><td>Millipore</td><td>109634</td><td></td></tr><tr><td>Ethanol</td><td>Duksan</td><td>64-17-5</td><td></td></tr><tr><td>RevertAid First Strand cDNA Synthesis kit</td><td>Thermo Scientfic</td><td>K1622</td><td></td></tr><tr><td>i-Taq DNA Polymerase</td><td>iNtRON BIOTECH</td><td>25021</td><td></td></tr><tr><td>UltraPure 10x TBE Buffer</td><td>Life Technologies</td><td>15581-044</td><td></td></tr><tr><td>loading star</td><td>Dyne Bio</td><td>A750</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>9012-36-6</td><td></td></tr><tr><td>1 kb (+) DNA ladder marker</td><td>Enzynomics</td><td>DM003</td><td></td></tr><tr><td>Human adipose-derived stem cells (ASCs)</td><td>Catholic MASTER Cells</td><td></td><td></td></tr></tbody></table>

chondrogenic differentiation induction of adipose-derived stem cells by centrifugal gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

离心重力诱导的脂肪来源的干细胞的软骨形成分化标记物的表达。 以确定的离心力重力是适于诱导软骨细胞分化的程度，携带者用15分钟不同程度的CG（0，300，600，1200，2400 XG）刺激。刺激之后，将携带者进行了重新接种到培养板和培养24小时。如在图1A中所示，SOX9 mRNA表达显著2400 xg离心增加;它是约2400 XG比（携带?...

Watch this Scientific Journal Video about Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity at JoVE.com

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

脂肪干细胞的软骨细胞分化诱导离心重力

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

chondrogenic-differentiation-induction-adipose-derived-stem-cells

Research

JoVE Journal

Developmental Biology

9.4K 次观看.  The Catholic University of Korea. Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

视频: Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

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

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 ...

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

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

分化软骨细胞与外周血源性人诱导多能干细胞

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

脐带血诱导的多能干细胞的软骨形成颗粒形成

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

神经冠状原脂肪衍生干细胞的分离、培养和异位诱导，来自围阴脂肪组织

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

基于状的不均匀 Nanopatterns 对局部控制表面粘附的研究--一种直接软骨分化的方法

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

生物工程

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

在重力和自制微型生物反应器下使用低粘附条件从各种软骨细胞生成球状体

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

人主动脉周脂肪祖细胞的分化能力

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

生物学

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 ...