PBMC的人CD40激活的B细胞产生的协议分为两部分：A部分演示NIH/3T3细胞，将用于板约束的饲养层细胞表达CD40配体的准备。 B部分介绍了各种实际的CD40 - B的文化。 A.制备饲养细胞（N​​IH/3T3 tCD40L） tCD40L NIH/3T3壁小鼠成纤维细胞系，这不应该成为完全融合。因此，这些细胞被劈裂每周两次。超过6周以上的培养，不推荐。 <ol><li>从小学文化的老媒体取出，用无菌吸管和10 mL 1X PBS中洗细胞。吸的PBS洗涤后。 </li><li>添加4 mL的胰蛋白酶/ EDTA在75厘米的 2瓶5-10分钟，在37 ° C。使用温柔攻分离的细胞。 </li><li>野生型中添加10毫升，并轻轻旋转。 </li><li>转移到50 mL试管细胞悬液，用无菌吸管和旋转5分钟在225 XG细胞。 </li><li>去除上清，重悬沉淀在野生型中的10毫升。计数细胞悬液等分的细胞数，并准备适当数量的细胞50毫升管： <ol><li> 1.5 × 10 6细胞传代</li><li> 0.2 × 10 6细胞/孔的CD40 - B细胞培养用于照射</li><li>其余冻结（如需要）。 </li></ol></li><li>旋转细胞在225 5分钟XG。 </li><li>去除上清。 <ol><li>传代培养：重悬于1.5 × 10 6细胞10毫升，75厘米2细胞培养瓶中的野生型中（细胞密度为1.5 × 10 5细胞/ mL），加入的G - 418 [0.7毫克/毫升]和孵化细胞37℃，5％的CO 2 。每周两次分裂的细胞。 </li><li>对于CD40 - B细胞培养：您需要1.2 × 10 6 6孔板细胞。在野生型中重悬的细胞密度0.1 × 10 6细胞/ mL，他们在78 Gy的照射。板2毫升细胞悬液，每口井，并在37℃，5％的CO 2。使用B -细胞的刺激，这准备板块tCD40L NIH/3T3细胞是贴壁时（至少4个小时：坚持用显微镜检查;不要等到超过24 h开始的B细胞刺激）。 （继续与B） </li></ol></li></ol> B. CD 40 - B细胞培养一，制备外周血CD40的刺激（0天）： 请注意：在继续之前确定，饲养层细胞是贴壁。随时添加新的解决办法白细胞介素4和环孢素A培养基中，使用前。 <ol><li>取外周血，新鲜或适当解冻。悬浮外周血1X PBS毫升，两次把它们洗干净，和自旋向下第一时间为7分钟，第二次在7分钟190 XG删除其他细胞在265 XG。弃上清，并在20毫升的PBS重悬细胞。确定细胞的细胞悬液分装数。 </li><li>离心5分钟在225 XG所需的细胞量。对于6孔板4 × 10 6细胞/孔的需要，因此24 × 10 6细胞每盘。 </li><li>去除上清，重悬CD40 - B的文化与50 U /白细胞介素4毫升新鲜培养基中的外周血单个核细胞在1 × 10 6细胞/ mL，作为一种生长因子和0.63微克/毫升环孢素A到防止T细胞的产物（给定的浓度是指一毫升培养液）。 </li><li>去除上清从6孔板前培养与tCD40L NIH/3T3细胞。 </li><li>每孔2 mL的PBS第一和第二步在2毫升的CD40 - B的清洗介质，清洗附着tCD40L NIH/3T3细胞。 </li><li>轻轻添加4毫升的外周血单个核细胞悬液（1 × 10 6细胞/ mL），每孔6孔板。 </li><li>细胞在37 ° C的5％的CO 2。 </li><li>第7天，reculture细胞（3.2继续。） </li></ol>二。复垦的CD40的B细胞（7天，然后每隔3-4天）： <ol><li>从6孔板CD40 - B细胞10毫升到50毫升管的移液器和池他们resuspending收获集群。 </li><li>离心7分钟在225 XG，并完全取代CD40 - B的清洗介质的上清。虽然计数细胞数量的等分，5分钟旋转225 XG细胞。悬浮CD40的B细胞CD40 - B的培养基中，在浓度为1 × 10 6细胞/ mL 。 </li><li>在50 U / mL的浓度和0.63μg/ mL的环孢素A的中期添加新鲜的白细胞介素4的解决方案。 </li><li>从6孔板与tCD40L NIH/3T3细胞预孵育取出上清。 </li><li>每孔2 mL的PBS第一和第二步在2毫升的CD40 - B的清洗介质清洗贴壁细胞。 </li><l我&gt;轻轻每6孔板以及添加4毫升（1 × 10 6细胞/ ml）CD40 - B的悬挂。 </li><li>孵育板在37℃，5％的CO 2。 </li><li>再次传代培养细胞每隔3-4天，结束与高纯度的人CD40 B细胞激活后，共14天。 </li></ol> C.故障排除 - CD40 - B细胞，如果不增长？ <ol><li>你有检查饲养层细胞的CD40的配体表达？ </li><li>板势必给料机，用于刺激细胞不应超过24h以上吗？ </li><li>支原体可能是一个污染？ </li><li>白细胞介素4的解决方案，为补充新鲜解冻，并有适当的生物活性？ </li><li>环孢素A中添加正确的浓度？ </li></ol><img src="/files/ftp_upload/1373/1373fig1tmb.jpg" border="0" alt="图1" /> 图1，请<a href="https://www.jove.com/files/ftp_upload/1373/1373fig1.jpg">点击这里</a>为图1的放大版本。 <img src="/files/ftp_upload/1373/1373fig2tmb.jpg" border="0" alt="图2" /> 图2，请<a href="https://www.jove.com/files/ftp_upload/1373/1373fig2.jpg">点击这里</a>为图2的较大版本。

<ol>
	<li>Schultze, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+human+B+cells:+an+alternative+source+of+highly+efficient+antigen+presenting+cells+to+generate+autologous+antigen-specific+T+cells+for+adoptive+immunotherapy.">CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.</a> J Clin Invest. 100, 2757-2765 (1997).</li><li>Schultze, J. L., Grabbe, S., vonBergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+and+CD40-activated+B+cells:+current+and+future+avenues+to+cellular+cancer+immunotherapy.">DCs and CD40-activated B cells: current and future avenues to cellular cancer immunotherapy.</a> Trends Immunol. 25, 659-664 (2004).</li><li>Bergwelt-Baildon, M. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+express+full+lymph+node+homing+triad+and+induce+T-cell+chemotaxis:+potential+as+cellular+adjuvants.">CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.</a> Blood. 107, 2786-2789 (2006).</li><li>Wiesner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+immortalization+of+human+B+cells+by+CD40+ligation.">Conditional immortalization of human B cells by CD40 ligation.</a> PLoS ONE. 3, 1464-14 (2008).</li><li>Urashima, M., Chauhan, D., Uchiyama, H., Freeman, G. J., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+ligand+triggered+interleukin-6+secretion+in+multiple+myeloma.">CD40 ligand triggered interleukin-6 secretion in multiple myeloma.</a> Blood. 85, 1903-1912 (1995).</li><li>Lapointe, R., Bellemare-Pelletier, A., Housseau, F., Thibodeau, J., Hwu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-stimulated+B+lymphocytes+pulsed+with+tumor+antigens+are+effective+antigen-presenting+cells+that+can+generate+specific+T+cells.">CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells.</a> Cancer Res. 63, 2836-2843 (2003).</li><li>von Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+primary+and+memory+cytotoxic+T+lymphocyte+responses+are+efficiently+induced+by+means+of+CD40-activated+B+cells+as+antigen-presenting+cells:+potential+for+clinical+application.">Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application.</a> Blood. 99, 3319-3325 (2002).</li><li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+can+be+generated+in+high+number+and+purity+in+cancer+patients:+analysis+of+immunogenicity+and+homing+potential.">CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential.</a> Clin Exp Immunol. 155, 249-256 (2009).</li><li>Mason, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-loaded+CD40-activated+B+cells+stimulate+antigen-specific+T-cell+responses+in+dogs+with+spontaneous+lymphoma.">RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma.</a> Gene Ther. 15, 955-965 (2008).</li></ol>

A.媒体的制备： <table border="1"cellspacing="0"><tbody><tr><td colspan="2"> 直属细胞野生型中 </td><td colspan="2"> 馈线小区选择培养基 </td></tr><tr><td> 试剂 </td><td> 集中 </td><td> 试剂 </td><td> 集中 </td></tr><tr><td> DMEM - HAM / F12 </td><td></td><td> DMEM - HAM / F12 </td><td></td></tr><tr><td> L -谷氨酰胺</td><td> 365微克/毫升</td><td> L -谷氨酰胺</td><td> 365微克/毫升</td></tr><tr><td>胎牛血清</td><td> 10％ </td><td>胎牛血清</td><td> 10％ </td></tr><tr><td> HEPES </td><td> 10毫米</td><td> HEPES </td><td> 10毫米</td></tr><tr><td>庆大霉素</td><td> 15微克/毫升</td><td>庆大霉素</td><td> 15微克/毫升</td></tr><tr><td></td><td></td><td> G - 418 </td><td> 0.7毫克/毫升</td></tr></table><table cellspacing="0" border="1"><tbody><tr><td colspan="2"> CD40 - B的清洗介质 </td><td colspan="2"> CD40 - B的培养基 </td></tr><tr><td> 试剂 </td><td> 集中 </td><td> 试剂 </td><td> 集中 </td></tr><tr></tr></tbody><tbody><tr><td> IMDM </td><td> </td><td> IMDM </td><td> </td></tr></tbody><tbody><tr><td> L -谷氨酰胺</td><td> 584微克/毫升</td><td> L -谷氨酰胺</td><td> 584微克/毫升</td></tr></tbody><tbody><tr><td> HEPES </td><td> 25毫米</td><td> HEPES </td><td> 25毫米</td></tr></tbody><tbody><tr><td>庆大霉素</td><td> 15微克/毫升</td><td>庆大霉素</td><td> 15微克/毫升</td></tr></tbody><tbody><tr><td> </td><td> </td><td> RH转</td><td> 50微克/毫升</td></tr></tbody><tbody><tr><td> </td><td> </td><td> RH胰岛素</td><td> 5微克/毫升</td></tr></tbody><tbody><tr><td> </td><td> </td><td> AB - Humanserum </td><td> 10％ </td></tr></tbody></table> B.其它试剂： <table cellspacing="0" border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 订单号 </td></tr><tr></tr><tr><td> DMEM / HAM的F12 </td><td>临时机场管理局</td><td>猫编号：E15 - 813 </td></tr><tr><td> IMDM </td><td> Invitrogen公司Gibco公司® </td><td>楼盘21980-032 </td></tr><tr><td>贝科的PBS（10X） </td><td>临时机场管理局</td><td>猫没有H15 - 011 </td></tr><tr><td> AB人血清</td><td> Invitrogen公司</td><td> CAT无34005100 </td></tr><tr><td>全息转人类</td><td>西格玛</td><td> CAT没有T0665 </td></tr><tr><td>胰岛素人类</td><td>西格玛</td><td> CAT没有I2643 </td></tr><tr><td> Gencin ® </td><td>三角洲选择</td><td>货号7395800 </td></tr><tr><td>重组人白细胞介素- 4 </td><td> Immunotools </td><td> CAT无11130045 </td></tr><tr><td>环孢素A </td><td>诺华公司</td><td>猫没有NDC 0078-0109-01 </td></tr><tr><td>胎牛血清</td><td>龙沙</td><td>猫没有DE14 - 802C </td></tr><tr><td> HEPES缓冲液</td><td>临时机场管理局</td><td>猫没有S11 - 001 </td></tr><tr><td> G - 418硫酸盐</td><td>临时机场管理局</td><td>猫没有P02 - 012 </td></tr><tr><td>胰蛋白酶/ EDTA（10X） </td><td> Invitrogen公司Gibco公司® </td><td>楼盘15400-054 </td></tr></tbody></table> C.杂项用品： <table cellspacing="0" border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 订单号 </td></tr><tr></tr><tr><td>无菌移液器</td><td> Sarstedt </td><td></td></tr><tr><td> 6孔板</td><td> NUNC </td><td> CAT没有140675 </td></tr><tr><td> 50毫升锥形管</td><td> BD猎鹰™ </td><td> CAT没有352070 </td></tr><tr><td>组织培养瓶</td><td> SARSTEDT </td><td> CAT没有83.1813.002 </td></tr></tbody></table>

generation of human cd40-activated b cells

CD40激活的B细胞（CD40 - B细胞）已被确定为肿瘤免疫治疗的免疫刺激抗原提呈细胞（APC）1-3的替代来源。树突状细胞（DCs），最好的APC，CD40 - B细胞有几个不同的生物和技术性能相比。与区议会，B细胞的MHC和共刺激分子（图1b）的表达增加，表现出强烈的的迁徙能力和目前的T细胞抗原提呈效率，刺激白细胞介素4和CD40配体（CD40L）。然而，在不成熟或成熟的DCS相比，CD40 - B细胞表达完整的淋巴结归巢CD62L，CCR7/CXCR4组成的黑社会，和白细胞功能抗原-1（LFA1，CD11a/CD18），归巢到次级淋巴器官所必需的（图1a）3。 CD40 - B细胞可以产生而没有从非常小，它可以进一步扩大在体外大量高纯度CD40的B细胞（&gt; 10 9细胞每名患者）从健康的捐赠者以及癌症金额外周血困难患者（图1c，d）项1,4。 在这个协议中，我们演示了如何获取充分激活的人PBMC CD40 - B从细胞。细胞培养的关键分子CD40配体，白细胞介素4（IL - 4）和环孢素A（CsA），这是在3-4天养殖周期的补充。 CD40的刺激对于实验室的目的是提供NIH/3T3细胞表达重组人CD40配体（tCD40L NIH/3T3）5。为了避免污染与非转染的细胞，表达人CD40配体的转染，定期进行检查（图）。 CD40 - B细胞培养，经过14天，包括95％以上的纯B细胞和CD40 - B细胞超过65天的扩张是没有任何损失的功能：1，4经常可能。 CD40 - B细胞的有效占用，过程和目前T细胞抗原6。他们不只有总理naϊve，而且还扩大记忆性T细胞7,8。 CD40激活的B细胞可用于研究B细胞的活化，分化和功能。此外，他们代表一个有前途的治疗或预防接种抗肿瘤9工具。

Watch this Scientific Journal Video about Generation of Human CD40-activated B cells at JoVE.com

Generation of Human CD40-activated B cells

在这个视频中，我们目前的<em&gt;体外</em&gt;的产生和扩大人CD40激活的B细胞（CD40 - B）从外周血单个核细胞（PBMC）的刺激与CD40配体和IL - 4。

人CD40激活的B细胞的生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

generation-of-human-cd40-activated-b-cells

Research

JoVE Journal

Biology

15.1K 次观看.  University Hospital of Cologne, Department I of Internal Medicine. 在这个视频中，我们目前的体外的产生和扩大人CD40激活的B细胞（CD40 - B）从外周血单个核细胞（PBMC）的刺激与CD40配体和IL - 4。

视频: Generation of Human CD40-activated B cells

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

为核型分析人类胚胎干细胞的染色体蔓延制备

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

科研

生物学

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 ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

肝细胞样细胞从人类胚胎干细胞群的鲁棒代

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

来自人类脐带血内皮细胞集落形成细胞的表型和功能特性

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

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 Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

的分散Presomitic中胚层细胞培养的斑马鱼分割时钟在单细胞成像代

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

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 ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

人外周血代文化血液的产物内皮细胞

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...