Xeno-Free Reprogramming of Peripheral Blood Mononuclear Erythroblasts on Laminin-521.
Xeno-Free Reprogramming of Peripheral Blood Mononuclear Erythroblasts on Laminin-521.
复制标题
外周血单核成红细胞在层粘连蛋白 521 上的无异源重编程。
DOI：
10.1002/cpsc.103
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发表时间：
2020
影响因子：
--
通讯作者：
Schlaeger,ThorstenM
中科院分区：
文献类型：
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作者：
Skorik,Christian;Mullin,NathanielK;Shi,Michael;Zhang,Yosra;Hunter,Phoebe;Tang,Yang;Hilton,Brianna;Schlaeger,ThorstenM
将人诱导多能干细胞（hiPSC）衍生的细胞和组织转化到临床中需要临床级hiPSC的简化和可靠的生产。本文描述了一种完全不含动物成分的程序，仅使用自体患者材料和无异种试剂从供体外周血单核细胞（PBMC）可靠地衍生稳定的hiPSC细胞系。从全血捐献中分离PBMC，也从全血捐献中产生少量患者血清。然后在补充有自体患者血清的不含动物组分的成红细胞生长培养基中重编程之前扩增PBMC，从而消除对动物血清的需要。扩增后，使用cGMP级仙台病毒颗粒（CytoTune™ 2.1试剂盒）或附加型复制重编程质粒（Epi5™试剂盒）（两者均可商购获得）重编程成红细胞。在重组cGMP级人层粘连蛋白底物上扩增新生hiPSC与许多无异种或化学成分确定的培养基（一些可作为cGMP级试剂获得）相容，例如E8、Nutristem、Stemfit或mTeSR Plus。使用该方法衍生的hiPSC系显示预期的表面标志物和转录因子的表达、重编程剂衍生的核酸的损失、遗传稳定性和在体外稳健地分化成多个谱系的能力。© 2020 by John Wiley & Sons，Inc.基本方案1：使用CPT管分离外周血单核细胞支持方案1：从收集的自体血浆中去除凝血因子以产生血清基本方案1基本方案2：在含有自体血清的无动物成红细胞扩增培养基中进行PBMC扩增基本方案3：使用仙台病毒重编程颗粒对扩增的PBMC进行重编程替代方案：基本方案4：挑选、扩增和冷冻保存hiPSC克隆支持方案2：测试仙台病毒试剂盒重编程的hiPSC是否不存在仙台病毒RNA支持方案3：测试Epi5试剂盒重编程的hiPSC是否不存在附加体质粒DNA支持方案4：通过多色免疫荧光染色和共聚焦成像评估人多能干细胞培养物的未分化状态支持方案5：用细胞外基质包被板以支持hiPSC附着和扩增
Translating human induced pluripotent stem cell (hiPSC)–derived cells and tissues into the clinic requires streamlined and reliable production of clinical‐grade hiPSCs. This article describes an entirely animal component–free procedure for the reliable derivation of stable hiPSC lines from donor peripheral blood mononuclear cells (PBMCs) using only autologous patient materials and xeno‐free reagents. PBMCs are isolated from a whole blood donation, from which a small amount of patient serum is also generated. The PBMCs are then expanded prior to reprogramming in an animal component–free erythroblast growth medium supplemented with autologous patient serum, thereby eliminating the need for animal serum. After expansion, the erythroblasts are reprogrammed using either cGMP‐grade Sendai viral particles (CytoTune™ 2.1 kit) or episomally replicating reprogramming plasmids (Epi5™ kit), both commercially available. Expansion of emerging hiPSCs on a recombinant cGMP‐grade human laminin substrate is compatible with a number of xeno‐free or chemically defined media (some available as cGMP‐grade reagents), such as E8, Nutristem, Stemfit, or mTeSR Plus. hiPSC lines derived using this method display expression of expected surface markers and transcription factors, loss of the reprogramming agent–derived nucleic acids, genetic stability, and the ability to robustly differentiate in vitro to multiple lineages. © 2020 by John Wiley & Sons, Inc.Basic Protocol 1: Isolating peripheral blood mononuclear cells using CPT tubesSupport Protocol 1: Removal of clotting factors to produce serum from autologous plasma collected in Basic Protocol 1Basic Protocol 2: PBMC expansion in an animal‐free erythroblast expansion medium containing autologous serumBasic Protocol 3: Reprogramming of expanded PBMCs with Sendai viral reprogramming particlesAlternate Protocol: Reprogramming of expanded PBMCs with episomal plasmidsBasic Protocol 4: Picking, expanding, and cryopreserving hiPSC clonesSupport Protocol 2: Testing Sendai virus kit–reprogrammed hiPSC for absence of Sendai viral RNASupport Protocol 3: Testing Epi5 kit–reprogrammed hiPSC for absence of episomal plasmid DNASupport Protocol 4: Assessing the undifferentiated state of human pluripotent stem cell cultures by multi‐color immunofluorescent staining and confocal imagingSupport Protocol 5: Coating plates with extracellular matrices to support hiPSC attachment and expansion