我们感谢三宅义子，早矢香Kanaami，奇哈纳藤田，美穗山口，夏子Henmi，和丽大野耐一从庆应义塾大学医学院提供技术援助。这部分工作是由R＆D Systems的支持计划，以加速实际使用的健康研究成果，以及高速公路项目为再生医学的实现提供资金。

1.准备激活人类T细胞<ol><li>收集肝素化的全血从供体通过静脉穿刺10毫升<ol><li>获得捐赠者的知情同意提前静脉穿刺。静脉穿刺必须由合格的，经过培训的人员来完成。 </li><li>处理的血液样本，用无菌设备及以下适当的生物安全准则。 </li></ol></li><li>稀释肝素化的全血用10ml D-PBS中的10毫升（ - ）。 </li><li>将15个毫升聚蔗糖溶液（菲可 - 帕克溢价）到一个新的50毫升锥形管。 </li><li>层在步骤1.2制得到在步骤1.3中所述的Ficoll溶液20毫升血液。使用电吸移管，让血液溶液制备步骤1.2运行慢慢沿管的内壁上。要小心，不要混用血液和聚蔗糖的解决方案。 </li><li>离心机在400×g下在室温30分钟。将离心减速速度"慢"。 注意:离心后，为白色薄层浮现的上部乳白色血浆层和下清晰的Ficoll层之间。这白色的薄薄的一层含有单核细胞。 </li><li>的单核细胞层转移到一个新的50毫升锥形管用1毫升吸移管。要小心，不要包括聚蔗糖的解决方案。 </li><li>添加D-PBS（ - ）与单核细胞，以10毫升的总体积，以及离心机在200×g下5分钟，在室温。 </li><li>弃去上清液。加入10 mL D-PBS的（ - ）对单核细胞，并在离心分离机200×g下5分钟，在室温。 </li><li>弃去上清液，加入1 ml的KBM502培养基向收集的单核细胞，并使用血细胞计数器计数细胞。超过5×10 6单核细胞可以从肝素化的全血与适当的分离用Ficoll 10 ml的获得。 </li><li>制备的抗人CD3抗体溶液以10微克/毫升在D-PBS的浓度（ - ）。抗人CD3抗体溶液添加至6孔板，浸泡冲浪每个王牌孔，孵育菜，在37℃在一个5％CO 2培养箱中至少30分钟。 </li><li>除去从各抗人CD3抗体溶液以及与用D-PBS洗一次（ - ）以接种细胞之前。 </li><li>分别以2.5×10 5个细胞/ cm 2的对抗人CD3抗体包被6孔板中KBM502介质的密度制备步骤1.9 ​​那种子的单核细胞。在5％-CO 2培养箱中3-7天在37℃的无介质改变，直到T细胞达到汇合。 T细胞在此期间都暂停，细胞粘附。 </li></ol> 2.感染人体T细胞SeV载体<ol><li>经过3-7天的培养，收集活化的T细胞，吹打现有的网上平台。它们转移到15毫升锥形管中并离心，在200×g下5分钟，在室温。 </li><li>去除上清，加入1毫升新鲜KBM502网上平台。计数细胞，和板1。5×10 6个细胞（在2ml新鲜培养基KBM502）插入抗CD3抗体包被的6孔板的每个孔中。 </li><li>冰上解冻OCT3 / 4-SeV载体/TSΔF，​​SOX2-的SeV /TSΔF，​​KLF4-的SeV /TSΔF，​​和c-MYC（HNL）-sev /TS15ΔF的SeV的解决方案。 </li><li>添加包含OCT3 / 4-SeV载体/TSΔF，​​SOX2-的SeV /TSΔF，​​KLF4-的SeV /TSΔF，​​和c-MYC（HNL）SeV的溶液-sev /TS15ΔF单独的井，每于感染复数（MOI） 10.孵育在37℃下在5％-CO 2培养箱中另外24小时。 </li></ol> 3.从人类T细胞SeV载体<ol><li> 24小时感染后，收集感染的细胞用吸管。如果有粘附细胞，他们可以很容易被拆卸吹打他们反复向上和向下。它们转移到15毫升锥形管中，并离心分离，在200×g下5分钟，在室温。 </li><li>删除包含SeV载体上清液，加2ml新鲜KBM502介质。 Replate细胞EAC小时井。在5％-CO 2培养箱在37℃下另外24小时。 </li></ol>在基底膜层4.重新设定种子细胞<ol><li>准备通过解冻基质凝胶基底膜基质溶液（请参阅材料清单用于本研究中使用的特定的品牌），在4℃和在10毫升的DMEM / F12培养基中溶解0.2毫升 注:基质胶（请参阅材料清单用于本研究中使用的特定的品牌）需要解冻O / N在4℃下被完全解冻。保持它在冰上使用前至。 </li><li>在RT种子细胞之前，每盘盘5毫升基底膜基质溶液为100毫米的菜肴，并留下了至少30分钟。都需要一个实验的至少两个100-mm培养皿中。 </li><li>收集的SeV感染的细胞通过移液，将它们在室温下转移到15毫升锥形管中，并离心分离，在200×g下5分钟。 </li><li>去除上清，加入1毫升新鲜KBM502网上平台。伯爵CE数量LLS使用血细胞计数器，删除在步骤4.2和板制备100-mm培养皿中的基底膜基质溶液1×10 5和1×10 6个细胞（在10ml新鲜mTeSR培养基）上的100毫米基底膜基质涂覆菜肴。最后，使用板，其菌落容易上手。 </li><li>孵育在5％-CO 2培养箱O / N的菜在37℃。 </li><li>介质更改至10ml充实mTeSR培养基隔日1次。 注意:在感染后约20-30天，菌落可以非常类似于胚胎干细胞（ESC）将出现。 </li></ol> 5.展开T细胞来源的iPS细胞<ol><li>准备通过解冻基质凝胶基底膜基质溶液（请参阅材料清单用于本研究中使用的特定的品牌），在4℃和在10毫升的DMEM / F12培养基中溶解0.2毫升</li><li>板1 ml的在6孔板每孔基底膜基质溶液中，并在室温离开在采摘菌落前至少30分钟。 </li><li>填的96孔平板与20μlmTeSR培养基中每孔。 </li><li>选择像用20微升移液管的体视显微镜下，胚胎干细胞的殖民地。 注意:胚胎干细胞和iPS细胞的集落是圆形和扁平，如图1B所示 。 </li><li>传输所选的菌落放入96孔板在步骤5.3充满mTeSR培养基。 </li><li>加入200μlmTeSR培养基到每个孔中，并仔细吸取上下打破菌落成小团块用200μl移液管的立体显微镜下。取下充分的准备基底膜基质解决方案5.2，并把每个菌落到基底膜基质包被6孔板2毫升mTeSR培养基中的一个孔，每孔。 </li><li>介质更改〜2毫升充实mTeSR培养基隔日1次。 </li></ol> 6.保持T细胞来源的iPS细胞 T细胞来源的iPS细胞可以保持并用作为人类的iPSC和人ESC相同的技术存储。一旦IPSC的殖民地长大，重复同样的程序扩展成更大的菜肴。 <ol><li>更改mTeSR培养基每1-2天。 </li><li>当菌落变得汇合每5-7天，通过用解离溶液用于人类的iPSC和根据制造商的说明人ESC细胞。 </li></ol>

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作者有没有竞争或利益冲突的披露。

We describe a protocol for generating iPSCs from human peripheral T cells in serum-free and feeder-free conditions using a combination of matrigel, mTeSR medium, and SeV vectors. For clinical applications of iPSCs, it is important to have a protocol for stably generating iPSCs and a less-invasive method for cell sampling. Although generating iPSCs with the combination of matrigel and mTeSR medium showed lower reprogramming efficiency than that with knockout serum replacement (KSR) medium and feeder-cells, this combination achieves stable generation of iPSCs from donors16. Stable iPSC generation and less-invasive cell sampling has the advantage of being able to increase the number of donors for iPSC generation. 
SeV vectors, a minus-strand RNA virus, is not integrated into the host genome. Therefore the risks of tumorigenesis can be avoided at the step of reprogramming factor induction20-24. Additionally, the feeder-free conditions, which use a combination of defined culture medium and matrigel instead of a feeder layer, make it possible to minimize the potential risks of exposure to unknown exogenous factors. The fusion of these techniques provides a less invasive and safer iPSC technology for regenerative medicine16.
Important steps in this protocol are the step of activating human T cells (Step 1) and infecting them with SeV vectors (Step 2). When no ESC-like colony is obtained in the culture dish at an optimal time after SeV infection, the following should be considered. First, the confluency of the mononuclear cells before T cell activation may not be appropriate because optimal activation of T cells is critical for SeV infection25,26. Too high a density of mononuclear cells leads to cell death, interfering with the proper activation of T cells. Too low a density of mononuclear cells also disturbs the proper activation and proliferation of T cells. Therefore, the confluency of mononuclear cells should be checked and adjusted accordingly. Second, the dosage of SeV vectors may not be sufficient. The induction efficiency of iPSC colonies depends on the dosage of the virus6. If no ESC-like colony is observed after SeV infection, the option of increasing the virus dosage up to an MOI of 15-20 should be considered. If signs of iPSC colony differentiation are observed, the frequency at which medium is changed may be increased up to every other day, or to every day when colonies are larger. 
The limitation in this protocol consists of this method not being virus free. Although SeV for cell reprogramming is commercially available with ease, users need to prepare equipment according to the appropriate biosafety level. As another method for generating integration-free iPSCs, episomal vectors have been used until now27-29. Although episomal vectors can be used with equipment with a lower level of biosafety, episomal vectors might insert into the host genome at extremely low rates. Therefore, additional checks are required to confirm the disappearance of transgenes, as when using SeV.
There is another limitation in that this technique is not absolutely free from animal-derived products. Some substrates, such as matrigel, anti-CD3 mAb, dissociation solution and SeV solution are derived from animal products and are therefore associated with a risk of transferring xenogeneic pathogens. However, the reduction of animal-derived substrates in the culture system is meaningful for the clinical application of iPSC technology due to the lower risk.

iPS细胞引起了极大关注，因为细胞在再生医学1-3一个突破性的来源。迄今为止，用于产生iPSCs的多样的方法已被报道4,5。其中，从人T细胞产生iPSCs的已经因为细胞采样6-8的较少创伤的方法的特别的兴趣。此外，从iPSCs的T细胞衍生的含有T细胞受体（TCR）基因重排，因而抗原特异性T细胞9,10的来源。因此，产生的T细胞衍生的iPS细胞安全是正在进行再生医学有用。 此方法是基于减少不可预知的致病性的风险的概念。为iPS细胞的临床应用安全是很重要的，以减少病原体11暴露的风险。先前在多能干细胞的许多培养系统，胎牛血清和饲养细胞已被用来作为基本试剂第 12条。然而，去除这两个从培养系统试剂是优选的iPSC产生，以减少不可预知的致病性的危险。 此外，此方法具有避免从患者和饲养细胞的制备费力侵袭性细胞采样的优点。因为T细胞衍生的iPSCs已经被成功地用于在疾病研究13,14，这种方法也适用和有益的，从患者产生疾病特异性的iPSC。 间T细胞重新编程的方法，使用仙台病毒（SeV载体）载体作为基因载体是能够产生iPSCs的高效率7,16的方法。此外，由于SeV载体是一个单链RNA病毒，并且不需要的DNA相对于复制，其在的iPSC代使用避免断宿主基因组17-19。因此，我们已经建立的方案，用于从人外周血T细胞的iPSC在无血清的FRee值，并使用基质胶，mTeSR培养基的组合无饲养条件下，和SeV载体。

<table><tbody><tr><td>Ficoll-Paque PREMIUM</td><td>GE Healthcare</td><td>17-5442-02</td><td></td></tr><tr><td>Purified NA/LE mouse anti-human CD3</td><td>BD Pharmingen</td><td>555336</td><td></td></tr><tr><td>Bovine albumin fraction V solution</td><td>Gibco</td><td>15260-037</td><td></td></tr><tr><td>mTeSR1 medium kit</td><td>STEM CELL</td><td>5850</td><td>Warm at room temperature before use</td></tr><tr><td>Dissociation Solution</td><td>ReproCELL</td><td>RCHETP002</td><td></td></tr><tr><td>D-PBS(&amp;ndash;)</td><td>Wako</td><td>045-29795</td><td></td></tr><tr><td>SeV Vector kit CytoTune-iPS ver.1.0</td><td>DNAVEC</td><td>DV-0303c</td><td>Thaw on ice before use</td></tr><tr><td>100-mm tissue culture dish</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>96-well tissue culture plate</td><td>Falcon</td><td>353078</td><td></td></tr><tr><td>6-well tissue culture plate</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>15&amp;#160;ml Centrifuge Tube</td><td>Greiner Bio-One</td><td>188271</td><td></td></tr><tr><td>50&amp;#160;ml Centrifuge Tube</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr></tbody></table>

generation of induced pluripotent stem cells from human peripheral t cells using sendai virus in feeder-free conditions

最近，iPS细胞已经引起关注，因为细胞再生疗法的新来源。尽管用于产生iPSCs的初始方法依赖于通过侵入性活组织检查和转基因的逆转录病毒基因组的插入得到的皮肤成纤维细胞，已经出现了许多努力来避免这些缺点。人外周血T细胞是唯一的小区源用于产生iPSCs的。从T细胞衍生的iPSC含有T细胞受体（TCR）基因的重排，并且抗原特异性T细胞的来源。此外，在基因组中的T细胞受体的重排有标记单个细胞系和移植的和供体细胞区分的可能性。为iPS细胞的安全的临床应用中，它以最小化暴露新产生iPSCs的有害试剂的风险是重要的。虽然胎牛血清和饲养细胞已为多能干细胞培养必需的，最好是从培养系统中删除他们减少风险不可预知的致病性。为了解决这个问题，我们已经建立了一个协议，用于使用仙台病毒，以减少暴露的iPSC未定义的病原体的风险产生于人类外周T细胞的iPS细胞。虽然处理仙台病毒要求设备具备相应的生物安全水平，仙台病毒感染活化的T细胞无基因组插入，但效率很高。在这个协议中，我们展示了使用活化的T细胞培养和仙台病毒的组合从人外周血T细胞在无饲养条件iPS细胞的产生。

<html>使用此协议，用户能够生成从人外周血T细胞的iPSC稳定。 IPSC的一代，从T细胞的SeV和基底膜呈约0.002％ -细胞重编程的几个段落后，均未检出几个捐助情况和SeV载体的效率为0.005％，16 图1A显示源细胞的协议产生吨示意图iPS细胞使用基质胶和mTeSR中等无饲养条件。感染的SeV后约20至30天，iPSC的菌落通过菌落ESC样形态（图1B）识别。免疫荧光染色显示的典型的多能细胞标记物的表达（NANOG，OCT3 / 4，SSEA4，TRA-1-60，和TRA-1-81）中的T细胞来源的iPS细胞无饲养条件（图1C）下产生的。 "WIDTH ="700"/&gt; 图1（A）:无饲养条件本研究下的协议重新编程性T细胞的示意图。 （B）:在无饲养条件下采血后27天一个典型的ESC样IPSC的殖民地。 （℃）:ALP染色和免疫荧光染色为多能性和表面标记（NANOG，OCT3 / 4，SSEA4，TRA-1-60，和TRA-1-81）中的T细胞来源的iPS细胞无饲养条件下产生的。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53225/53225fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions at JoVE.com

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

从人外周T细胞无饲养条件下，使用仙台病毒诱导多能干细胞的生成

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

generation-induced-pluripotent-stem-cells-from-human-peripheral-t

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

11.9K 次观看.  Keio University School of Medicine. This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

视频: Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

科研

发育生物学

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

从血细胞使用仙台病毒和离心诱导多能干细胞代

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

由Stemgent人类TF慢病毒重新编程人类成纤维细胞生成诱导多能干细胞

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

生物学

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

从外周单核细胞生成诱导神经干细胞,分化至多巴胺能神经元前体,用于移植研究

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

人类心肌细胞的生成：从馈线人类诱导多能干细胞的分化协议

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

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 ...